A novel definition and treatment of hyperinflammation in COVID-19 based on purinergic signalling.

Hyperinflammation plays an important role in severe and critical COVID-19. Using inconsistent criteria, many researchers define hyperinflammation as a form of very severe inflammation with cytokine storm. Therefore, COVID-19 patients are treated with anti-inflammatory drugs. These drugs appear to be less efficacious than expected and are sometimes accompanied by serious adverse effects. SARS-CoV-2 promotes cellular ATP release. Increased levels of extracellular ATP activate the purinergic receptors of the immune cells initiating the physiologic pro-inflammatory immune response. Persisting viral infection drives the ATP release even further leading to the activation of the P2X7 purinergic receptors (P2X7Rs) and a severe yet physiologic inflammation. Disease progression promotes prolonged vigorous activation of the P2X7R causing cell death and uncontrolled ATP release leading to cytokine storm and desensitisation of all other purinergic receptors of the immune cells. This results in immune paralysis with co-infections or secondary infections. We refer to this pathologic condition as hyperinflammation. The readily available and affordable P2X7R antagonist lidocaine can abrogate hyperinflammation and restore the normal immune function. The issue is that the half-maximal effective concentration for P2X7R inhibition of lidocaine is much higher than the maximal tolerable plasma concentration where adverse effects start to develop. To overcome this, we selectively inhibit the P2X7Rs of the immune cells of the lymphatic system inducing clonal expansion of Tregs in local lymph nodes. Subsequently, these Tregs migrate throughout the body exerting anti-inflammatory activities suppressing systemic and (distant) local hyperinflammation. We illustrate this with six critically ill COVID-19 patients treated with lidocaine.

An adjuvant strategy enabled by modulation of the physical properties of microbial ligands expands antigen immunogenicity.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.

Vascular Damage, Thromboinflammation, Plasmablast Activation, T-Cell Dysregulation and Pathological Histiocytic Response in Pulmonary Draining Lymph Nodes of COVID-19.

Although initial immunophenotypical studies on peripheral blood and bronchoalveolar lavage samples have provided a glimpse into the immunopathology of COVID-19, analyses of pulmonary draining lymph nodes are currently scarce. 22 lethal COVID-19 cases and 28 controls were enrolled in this study. Pulmonary draining lymph nodes (mediastinal, tracheal, peribronchial) were collected at autopsy. Control lymph nodes were selected from a range of histomorphological sequelae [unremarkable histology, infectious mononucleosis, follicular hyperplasia, non-SARS related HLH, extrafollicular plasmablast activation, non-SARS related diffuse alveolar damage (DAD), pneumonia]. Samples were mounted on a tissue microarray and underwent immunohistochemical staining for a selection of immunological markers and in-situ hybridization for Epstein Barr Virus (EBV) and SARS-CoV-2. Gene expression profiling was performed using the HTG EdgeSeq Immune Response Panel. Characteristic patterns of a dysregulated immune response were detected in COVID-19: 1. An accumulation of extrafollicular plasmablasts with a relative paucity or depletion of germinal centers. 2. Evidence of T-cell dysregulation demonstrated by immunohistochemical paucity of FOXP3+, Tbet+ and LEF1+ positive T-cells and a downregulation of key genes responsible for T-cell crosstalk, maturation and migration as well as a reactivation of herpes viruses in 6 COVID-19 lymph nodes (EBV, HSV). 3. Macrophage activation by a M2-polarized, CD163+ phenotype and increased incidence of hemophagocytic activity. 4. Microvascular dysfunction, evidenced by an upregulation of hemostatic (CD36, PROCR, VWF) and proangiogenic (FLT1, TEK) genes and an increase of fibrin microthrombi and CD105+ microvessels. Taken together, these findings imply widespread dysregulation of both innate and adoptive pathways with concordant microvascular dysfunction in severe COVID-19.

Temporal metabolic response to mRNA COVID-19 vaccinations in oncology patients.

BACKGROUND: mRNA COVID-19 vaccines are known to provide an immune response seen on FDG PET studies. However, the time course of this metabolic response is unknown. We here present a temporal metabolic response to mRNA COVID-19 vaccination in oncology patients undergoing standard of care FDG PET. METHODS: 262 oncology patients undergoing standard of care FDG PET were included in the analysis. 231 patients had at least one dose of mRNA COVID-19 vaccine while 31 patients had not been vaccinated. The SUVmax of the lymph nodes ipsilateral to the vaccination was compared to the contralateral to obtain an absolute change in SUVmax (ΔSUVmax). RESULTS: ΔSUVmax was more significant at shorter times between FDG PET imaging and COVID-19 mRNA vaccination, with a median ΔSUVmax of 2.6 (0-7 days), 0.8 (8-14 days), and 0.3 (> 14 days), respectively. CONCLUSION: Consideration should be given to performing FDG PET at least 2 weeks after the COVID-19 vaccine.

Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs.

Nucleoside-modified messenger RNA (mRNA)-lipid nanoparticles (LNPs) are the basis for the first two EUA (Emergency Use Authorization) COVID-19 vaccines. The use of nucleoside-modified mRNA as a pharmacological agent opens immense opportunities for therapeutic, prophylactic and diagnostic molecular interventions. In particular, mRNA-based drugs may specifically modulate immune cells, such as T lymphocytes, for immunotherapy of oncologic, infectious and other conditions. The key challenge, however, is that T cells are notoriously resistant to transfection by exogenous mRNA. Here, we report that conjugating CD4 antibody to LNPs enables specific targeting and mRNA interventions to CD4+ cells, including T cells. After systemic injection in mice, CD4-targeted radiolabeled mRNA-LNPs accumulated in spleen, providing ∼30-fold higher signal of reporter mRNA in T cells isolated from spleen as compared with non-targeted mRNA-LNPs. Intravenous injection of CD4-targeted LNPs loaded with Cre recombinase-encoding mRNA provided specific dose-dependent loxP-mediated genetic recombination, resulting in reporter gene expression in about 60% and 40% of CD4+ T cells in spleen and lymph nodes, respectively. T cell phenotyping showed uniform transfection of T cell subpopulations, with no variability in uptake of CD4-targeted mRNA-LNPs in naive, central memory, and effector cells. The specific and efficient targeting and transfection of mRNA to T cells established in this study provides a platform technology for immunotherapy of devastating conditions and HIV cure.

SARS-CoV-2 Induces Lymphocytopenia by Promoting Inflammation and Decimates Secondary Lymphoid Organs.

While lymphocytopenia is a common characteristic of coronavirus disease 2019 (COVID-19), the mechanisms responsible for this lymphocyte depletion are unclear. Here, we retrospectively reviewed the clinical and immunological data from 18 fatal COVID-19 cases, results showed that these patients had severe lymphocytopenia, together with high serum levels of inflammatory cytokines (IL-6, IL-8 and IL-10), and elevation of many other mediators in routine laboratory tests, including C-reactive protein, lactate dehydrogenase, α-hydroxybutyrate dehydrogenase and natriuretic peptide type B. The spleens and hilar lymph nodes (LNs) from six additional COVID-19 patients with post-mortem examinations were also collected, histopathologic detection showed that both organs manifested severe tissue damage and lymphocyte apoptosis in these six cases. In situ hybridization assays illustrated that SARS-CoV-2 viral RNA accumulates in these tissues, and transmission electronic microscopy confirmed that coronavirus-like particles were visible in the LNs. SARS-CoV-2 Spike and Nucleocapsid protein (NP) accumulated in the spleens and LNs, and the NP antigen restricted in angiotensin-converting enzyme 2 (ACE2) positive macrophages and dendritic cells (DCs). Furthermore, SARS-CoV-2 triggered the transcription of Il6, Il8 and Il1b genes in infected primary macrophages and DCs in vitro, and SARS-CoV-2-NP+ macrophages and DCs also manifested high levels of IL-6 and IL-1ß, which might directly decimate human spleens and LNs and subsequently lead to lymphocytopenia in vivo. Collectively, these results demonstrated that SARS-CoV-2 induced lymphocytopenia by promoting systemic inflammation and direct neutralization in human spleen and LNs.

Fetomaternal immune cross talk modifies T-cell priming through sustained changes to DC function.

BACKGROUND: Prenatal exposure to infections can modify immune development. These environmental disturbances during early life potentially alter the incidence of inflammatory disorders as well as priming of immune responses. Infection with the helminth Schistosoma mansoni is widely studied for its ability to alter immune responsiveness and is associated with variations in coinfection, allergy, and vaccine efficacy in endemic populations. OBJECTIVE: Exposure to maternal schistosomiasis during early life, even without transmission of infection, can result in priming effects on offspring immune responses to bystander antigenic challenges as related to allergic responsiveness and vaccination, with this article seeking to further clarify the effects and underlying immunologic imprinting. METHODS: Here, we have combined a model of chronic maternal schistosomiasis infection with a thorough analysis of subsequent offspring immune responses to allergy and vaccination models, including viral challenge and steady-state changes to immune cell compartments. RESULTS: We have demonstrated that maternal schistosomiasis alters CD4+ responses during allergic sensitization and challenge in a skewed IL-4/B-cell-dominant response to antigenic challenge associated with limited inflammatory response. Beyond that, we have uncovered previously unidentified alterations to CD8+ T-cell responses during immunization that are dependent on vaccine formulation and have functional impact on the efficacy of vaccination against viral infection in a murine hepatitis B virus model. CONCLUSION: In addition to steady-state modifications to CD4+ T-cell polarization and B-cell priming, we have traced these modified CD8+ responses to an altered dendritic cell phenotype sustained into adulthood, providing evidence for complex priming effects imparted by infection via fetomaternal cross talk.

Shared B cell memory to coronaviruses and other pathogens varies in human age groups and tissues.

Vaccination and infection promote the formation, tissue distribution, and clonal evolution of B cells, which encode humoral immune memory. We evaluated pediatric and adult blood and deceased adult organ donor tissues to determine convergent antigen-specific antibody genes of similar sequences shared between individuals. B cell memory varied for different pathogens. Polysaccharide antigen-specific clones were not exclusive to the spleen. Adults had higher clone frequencies and greater class switching in lymphoid tissues than blood, while pediatric blood had abundant class-switched convergent clones. Consistent with reported serology, prepandemic children had class-switched convergent clones to severe acute respiratory syndrome coronavirus 2 with weak cross-reactivity to other coronaviruses, while adult blood or tissues showed few such clones. These results highlight the prominence of early childhood B cell clonal expansions and cross-reactivity for future responses to novel pathogens.

Axillary Lymph Nodes Hypermetabolism After BNT162b2 mRNA COVID-19 Vaccination in Cancer Patients Undergoing 18F-FDG PET/CT: A Cohort Study.

PURPOSE: Recognition of the pattern of FDG uptake in hypermetabolic axillary lymph nodes (HALs) and association with recent messenger RNA (mRNA) vaccination are important to prevent patient anxiety and further needless examinations or costly biopsies in cancer patients. MATERIALS AND METHODS: This study was a retrospective cohort study in a single tertiary care institution. We investigate the occurrence and pattern of HAL on FDG PET/CT scans from 650 consecutive cancer patients with recent BNT162b2 mRNA COVID-19 vaccination. RESULTS: Between December 20, 2020, and February 8, 2021, 650 patients (351 female patients [54%]; mean age, 68.9 years) had recent mRNA COVID-19 vaccination and an FDG PET/CT scan. HALs were found in 57 (14.5%) of 394 patients (95% confidence interval [CI], 10.9%-18.7%) 12.3 ± 5.9 (1-22) days after dose 1 and in 111 (43.3%) of 256 patients (95% CI, 35.3%-52.2%; P < 0.0001) after 7.5 ± 5.4 (1-22) days after dose 2. There was no difference between dose 1 and dose 2 concerning SUVmax (3.7 ± 1.8 [1.3-11.3] and 4.5 ± 3.9 [1.4-26.3], P = 0.13, respectively), SUVmean (2.1 ± 1.0 [0.7-6.5] and 2.7 ± 2.4 [0.8-17], P = 0.08, respectively), and reactogenicity volume (2.7 ± 2.3 [0.2-11.6] cm3 and 2.7 ± 2.4 [0.2-15.5] cm3, P = 0.98, respectively). There was no difference in number and in size of positive lymph nodes between dose 1 and dose 2: 3.2 ± 2.2 (1-10) and 3.7 ± 2.4 (1-12) (P = 0.18), and 1.4 ± 0.4 cm (0.7-2.5 cm) and 1.5 ± 0.4 cm (0.6-3.2 cm) (P = 0.75), respectively. CONCLUSIONS: A cluster pattern of hypermetabolic ipsilateral small axillary lymph nodes is common after mRNA COVID-19 vaccination, mainly after the second injection.

18F-FDG-Avid Lymph Nodes After COVID-19 Vaccination on 18F-FDG PET/CT.

ABSTRACT: A 68-year-old man with right cheek melanoma after resection underwent 18F-FDG PET/CT, which was unremarkable except for multiple FDG-avid subcentimeter but rounded lymph nodes in the left axilla. The patient had undergone a COVID-19 vaccination in the left arm 3 weeks prior. As under vaccinations have been documented to cause reactive FDG-avid lymph nodes, the nodes in our patient were considered benign, reactive to the COVID-19 vaccination. Although FDG-avid benign, reactive nodes have been an uncommon finding in the past, the upcoming surge in COVID-19 vaccinations makes this an important finding for the interpreting physician to consider and recognize.

COVID-19 Vaccine-Related Local FDG Uptake.

ABSTRACT: We present a case of increased FDG uptake in the lymph nodes after COVID-19 vaccine administration. Restaging PET/CT scan of a 70-year-old woman with a history of multiple relapsed Hodgkin lymphoma showed muscle activity in the left upper arm laterally, which is in the deep musculature of the left deltoid muscle. There was also increased activity in several normal-sized left axillary nodes as well. On further review of the patient's history, she had received her second shot of the Pfizer-BioNTech COVID-19 vaccine approximately 2 days before the restaging PET/CT scan.

COVID-19 Vaccination Manifesting as Incidental Lymph Nodal Uptake on 18F-FDG PET/CT.

ABSTRACT: Benign uptake on 18F-FDG PET can be seen with inflammatory conditions. We report a case of an 86-year-old woman with successfully treated nasal melanoma who underwent routine follow-up 18F-FDG PET, day 6 after the second dose of Pfizer-BioNTech COVID-19 vaccine inoculated in the left deltoid muscle. 18F-FDG PET showed increase tracer uptake in the left deltoid muscle and in 2 normal-sized left subpectoral nodes. These findings were considered secondary to vaccination. With the current drive of global COVID-19 immunization, this case highlights the importance of documenting vaccination history at the time of scanning to avoid false-positive results.

Eliciting B cell immunity against infectious diseases using nanovaccines.

Infectious diseases, including the coronavirus disease 2019 (COVID-19) pandemic that has brought the world to a standstill, are emerging at an unprecedented rate with a substantial impact on public health and global economies. For many life-threatening global infectious diseases, such as human immunodeficiency virus (HIV) infection, malaria and influenza, effective vaccinations are still lacking. There are numerous roadblocks to developing new vaccines, including a limited understanding of immune correlates of protection to these global infections. To induce a reproducible, strong immune response against difficult pathogens, sophisticated nanovaccine technologies are under investigation. In contrast to conventional vaccines, nanovaccines provide improved access to lymph nodes, optimal packing and presentation of antigens, and induction of a persistent immune response. This Review provides a perspective on the global trends in emerging nanoscale vaccines for infectious diseases and describes the biological, experimental and logistical problems associated with their development, and how immunoengineering can be leveraged to overcome these challenges.

A lymph node-targeted Amphiphile vaccine induces potent cellular and humoral immunity to SARS-CoV-2.

The profound consequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mandate urgent development of effective vaccines. Here, we evaluated an Amphiphile (AMP) vaccine adjuvant, AMP-CpG, composed of diacyl lipid-modified CpG, admixed with the SARS-CoV-2 Spike-2 receptor binding domain protein as a candidate vaccine (ELI-005) in mice. AMP modification efficiently delivers CpG to lymph nodes, where innate and adaptive immune responses are generated. Compared to alum, immunization with AMP-CpG induced >25-fold higher antigen-specific T cells that produced multiple T helper 1 (TH1) cytokines and trafficked into lung parenchyma. Antibody responses favored TH1 isotypes (IgG2c and IgG3) and potently neutralized Spike-2-ACE2 receptor binding, with titers 265-fold higher than natural convalescent patient COVID-19 responses; T cell and antibody responses were maintained despite 10-fold dose reduction in Spike antigen. Both cellular and humoral immune responses were preserved in aged mice. These advantages merit clinical translation to SARS-CoV-2 and other protein subunit vaccines.

Screening and development of monoclonal antibodies for identification of ferret T follicular helper cells.

The ferret is a key animal model for investigating the pathogenicity and transmissibility of important human viruses, and for the pre-clinical assessment of vaccines. However, relatively little is known about the ferret immune system, due in part to a paucity of ferret-reactive reagents. In particular, T follicular helper (Tfh) cells are critical in the generation of effective humoral responses in humans, mice and other animal models but to date it has not been possible to identify Tfh in ferrets. Here, we describe the screening and development of ferret-reactive BCL6, CXCR5 and PD-1 monoclonal antibodies. We found two commercial anti-BCL6 antibodies (clone K112-91 and clone IG191E/A8) had cross-reactivity with lymph node cells from influenza-infected ferrets. We next developed two murine monoclonal antibodies against ferret CXCR5 (clone feX5-C05) and PD-1 (clone fePD-CL1) using a single B cell PCR-based method. We were able to clearly identify Tfh cells in lymph nodes from influenza infected ferrets using these antibodies. The development of ferret Tfh marker antibodies and the identification of ferret Tfh cells will assist the evaluation of vaccine-induced Tfh responses in the ferret model and the design of novel vaccines against the infection of influenza and other viruses, including SARS-CoV2.

Lipid-based vaccine nanoparticles for induction of humoral immune responses against HIV-1 and SARS-CoV-2.

The current health crisis of corona virus disease 2019 (COVID-19) highlights the urgent need for vaccine systems that can generate potent and protective immune responses. Protein vaccines are safe, but conventional approaches for protein-based vaccines often fail to elicit potent and long-lasting immune responses. Nanoparticle vaccines designed to co-deliver protein antigens and adjuvants can promote their delivery to antigen-presenting cells and improve immunogenicity. However, it remains challenging to develop vaccine nanoparticles that can preserve and present conformational epitopes of protein antigens for induction of neutralizing antibody responses. Here, we have designed a new lipid-based nanoparticle vaccine platform (NVP) that presents viral proteins (HIV-1 and SARS-CoV-2 antigens) in a conformational manner for induction of antigen-specific antibody responses. We show that NVP was readily taken up by dendritic cells (DCs) and promoted DC maturation and antigen presentation. NVP loaded with BG505.SOSIP.664 (SOSIP) or SARS-CoV-2 receptor-binding domain (RBD) was readily recognized by neutralizing antibodies, indicating the conformational display of antigens on the surfaces of NVP. Rabbits immunized with SOSIP-NVP elicited strong neutralizing antibody responses against HIV-1. Furthermore, mice immunized with RBD-NVP induced robust and long-lasting antibody responses against RBD from SARS-CoV-2. These results suggest that NVP is a promising platform technology for vaccination against infectious pathogens.