Immunoglobulin germline gene polymorphisms influence the function of SARS-CoV-2 neutralizing antibodies

The human immunoglobulin heavy-chain (IGH) locus is exceptionally polymorphic, with high levels of allelic and structural variation. Thus, germline IGH genotypes are personal, which may influence responses to infection and vaccination. For an improved understanding of inter-individual differences in antibody responses, we isolated SARS-CoV-2 spike-specific monoclonal antibodies from convalescent health care workers, focusing on the IGHV1-69 gene, which has the highest level of allelic variation of all IGHV genes. The IGHV1-69∗20-using CAB-I47 antibody and two similar antibodies isolated from an independent donor were critically dependent on allele usage. Neutralization was retained when reverting the V region to the germline IGHV1-69∗20 allele but lost when reverting to other IGHV1-69 alleles. Structural data confirmed that two germline-encoded polymorphisms, R50 and F55, in the IGHV1-69 gene were required for high-affinity receptor-binding domain interaction. These results demonstrate that polymorphisms in IGH genes can influence the function of SARS-CoV-2 neutralizing antibodies

A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo

Experiments with replication-competent SARS-CoV-2 were performed in the Biomedicum BSL3 core facility, Karolinska Institutet. We thank Jonas Klingström for providing Calu-3 cells and sharing the Swedish SARS-CoV-2 isolate, and Alex Sigal from the Africa Health Research Institute for providing the beta variant (B.1.351/501Y.V2) isolate. We thank Penny Moore and the NICD (South Africa) for providing the B.1.351/beta variant spike plasmid, which was generated using funding from the South African Medical Research Council. We gratefully acknowledge the G2P-UK National Virology consortium funded by MRC/UKRI (grant ref: MR/W005611/1.) and the Barclay Lab at Imperial College for providing the B.1.617.2 spike plasmid. All cryo-EM data were collected in the Karolinska Institutet’s 3D-EM facility. We thank Agustin Ure for assistance with figure generation and Tomas Nyman (Protein Science Facility at KI) for providing access to SPR instruments. L.H. was supported by the David och Astrid Hageléns stiftelse, the Clas Groschinskys Minnesfond and a Jonas Söderquist’s scholarship. This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 101003653 (CoroNAb), to B.M. and G.M.M. B.M.H. is supported by the Knut and Alice Wallenberg Foundation (KAW 2017.0080 and KAW 2018.0080). The work was supported by project grants from the Swedish Research Council to E.S. (2020-02682), B.M.H. (2017-6702 and 2018-3808), B.M. (2018-02381) and to G.M.M. (2018-03914 and 2018-03843). E.S. is supported by Karolinska Institutet Foundation Grants, National Molecular Medicine Program Grants, and the grants from the SciLifeLab National COVID-19 Research Program, financed by the Knut and Alice Wallenberg Foundation. We thank National Microscopy Infrastructure, NMI (VR-RFI 2016-00968).N

Virulence mechanisms of Moraxella in the pathogenesis of infection.

PURPOSE OF REVIEW: Moraxella catarrhalis is an emerging human-specific pathogen responsible for upper and lower respiratory tract infections. Understanding the events in the complex pathogenesis and underlying mechanisms during M. catarrhalis infection is a key to the development of novel therapeutics and vaccines. RECENT FINDINGS: Several novel findings have been reported on Moraxella pathogenesis and, in parts, explain how the species stands as a commensal in preschool children and survives in the host. Molecular structures for different adhesins in addition to target ligands with respect to signalling and invasion have been defined. Evasion of the complement system allows Moraxella to survive in the mucosa and by neutralizing [alpha]1-antichymotrypsin the protease activity is increased, resulting in tissue destruction and thus promotion of bacterial attachment. Moraxella-dependent cell activation via immunoglobulin D in addition to toll-like receptors and specific epithelial cell inhibition by cross-linking of carcinoembryonic antigen-related cell adhesion molecule-1 in the early innate immune response and, finally, the ability of M. catarrhalis to form biofilms are other specific research areas of interest. SUMMARY: Recent advances have allowed a more detailed picture of the processes involved in bacteria-host cell interactions, the cause of inflammatory processes and specific host defense responses against the intriguing species Moraxella

Cholesterol-rich domains are involved in Bordetella pertussis phagocytosis and intracellular survival in neutrophils

Bordetella pertussis-specific antibodies protect against whooping cough by facilitating host defense mechanisms such as phagocytosis However. the mechanism involved in the phagocytosis of the bacteria under non-opsonic conditions is still poorly characterized. We report here that B. pertussis binding and internalization is cholesterol dependent. Furthermore, WC found Cholesterol to be implicated in B. pertussis survival upon interaction with human neutrophils. Pre-treatment of PMN With cholesterol sequestering drugs like nystatin or methyl-beta-cyclodextrin (M beta CD) resulted in a drastic decrease of uptake of non-opsonized B. pertussis. Conversely. phagocytosis of opsonized bacteria was not affected by these drugs. showing that cholesterol depletion affects neither the viability of PMN nor the route of entry of opsonized B. pertussis. Additionally, intracellular survival rate of non-opsonized bacteria was significantly decreased in cholesterol-depleted PMN. Accordingly. confocal laser microscopy studies showed that non-opsonized B. pertussis co-localized with lysosomal markers only in cholesterol-depleted IIMN but not ill normal PMN. Our results indicate that B. pertussis docks to molecules that eventually prevent cellular bactericidal activity. (C) 2008 Elsevier Ltd. All rights reserved

Superantigen- and TLR-dependent activation of tonsillar B cells after receptor-mediated endocytosis.

Classical B lymphocyte activation is dependent on BCR cross-linking in combination with physical interaction with Th cells. Other B cell molecules that contribute to the activation are complement, cytokine, and TLRs recognizing specific pathogen-associated molecular patterns. Moraxella (Branhamella) catarrhalis is a common Gram-negative respiratory pathogen that induces proliferation in human IgD-expressing B cells independently of T cell help. The activation is initiated by the B cell superantigen Moraxella IgD-binding protein (MID) through a nonimmune cross-linking of IgD. However, IgD cross-linking alone is not sufficient to induce proliferation. In this study, we characterized the significance of TLRs in superantigen-dependent B cell activation using whole bacteria or rMID in the presence or absence of TLR ligands. IgD cross-linking by MID sensitized B cells obtained from children with tonsillar hyperplasia for mainly TLR9, whereas TLRs 1, 2, 6, and 7 were less important. The Moraxella-induced activation was inhibited when a dominant-negative TLR9 ligand was added. Interestingly, BCR-mediated endocytosis of whole Moraxella and degradation of live bacteria in naive B cells were observed with fluorescence, confocal, and transmission electron microscopy. This unique observation proved the strong intracellular TLR9 response as well as highlighted the Ag-presenting function of B cells. In conclusion, our findings suggest an important role of TLRs in the adaptive immune response and reveal novel insights into the T cell-independent B cell activation induced by bacteria

Junín virus induces autophagy in human A549 cells

Autophagy, a highly regulated degradative process that promotes cellular homeostasis, is increasingly recognised as a fundamental component of the cellular response against viral infection. In this study, we investigated the role of autophagy during Junín virus (JUNV) multiplication using human A549 cells. We found that JUNV infection induces an increment of the LC3-II/LC3-I ratio, an accumulation of punctate pattern in RFP-LC3-transfected cells and the colocalisation of viral nucleoprotein and LC3 protein, suggesting autophagosome formation. JUNV infection also induced the degradation of the autophagy receptor p62, suggesting that complete autophagic flux was triggered. In addition, we showed that inhibition of autophagy with bafilomycin A1 or 3-methyladenine significantly reduces viral multiplication. Moreover, viral yield was increased when autophagy was induced using rapamycin. Furthermore, JUNV infection induced the colocalisation of p62, ATG16, RAB5, RAB7A and LAMP1 with the autophagosomal LC3 protein. That suggests that phagosomes undergo the maturation process during viral infection. Finally, we demonstrated that siRNA experiments targeting essential autophagy genes (ATG5, ATG7 and Beclin 1) reduce viral protein synthesis and viral yield. Overall, our results indicate that JUNV activates host autophagy machinery enhancing its multiplication.Fil: Perez Vidakovics, Maria Laura Anabella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Ure, Agustin Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Arrías, Paula Nazarena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Romanowski, Victor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Gomez, Ricardo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

DNA-launched RNA replicon vaccines induce potent anti-SARS-CoV-2 immune responses in mice

Abstract The outbreak of the SARS-CoV-2 virus and its rapid spread into a global pandemic made the urgent development of scalable vaccines to prevent coronavirus disease (COVID-19) a global health and economic imperative. Here, we characterized and compared the immunogenicity of two alphavirus-based DNA-launched self-replicating (DREP) vaccine candidates encoding either SARS-CoV-2 spike glycoprotein (DREP-S) or a spike ectodomain trimer stabilized in prefusion conformation (DREP-Secto). We observed that the two DREP constructs were immunogenic in mice inducing both binding and neutralizing antibodies as well as T cell responses. Interestingly, the DREP coding for the unmodified spike turned out to be more potent vaccine candidate, eliciting high titers of SARS-CoV-2 specific IgG antibodies that were able to efficiently neutralize pseudotyped virus after a single immunization. In addition, both DREP constructs were able to efficiently prime responses that could be boosted with a heterologous spike protein immunization. These data provide important novel insights into SARS-CoV-2 vaccine design using a rapid response DNA vaccine platform. Moreover, they encourage the use of mixed vaccine modalities as a strategy to combat SARS-CoV-2

B cell activation by outer membrane vesicles--a novel virulence mechanism.

Secretion of outer membrane vesicles (OMV) is an intriguing phenomenon of Gram-negative bacteria and has been suggested to play a role as virulence factors. The respiratory pathogens Moraxella catarrhalis reside in tonsils adjacent to B cells, and we have previously shown that M. catarrhalis induce a T cell independent B cell response by the immunoglobulin (Ig) D-binding superantigen MID. Here we demonstrate that Moraxella are endocytosed and killed by human tonsillar B cells, whereas OMV have the potential to interact and activate B cells leading to bacterial rescue. The B cell response induced by OMV begins with IgD B cell receptor (BCR) clustering and Ca(2+) mobilization followed by BCR internalization. In addition to IgD BCR, TLR9 and TLR2 were found to colocalize in lipid raft motifs after exposure to OMV. Two components of the OMV, i.e., MID and unmethylated CpG-DNA motifs, were found to be critical for B cell activation. OMV containing MID bound to and activated tonsillar CD19(+) IgD(+) lymphocytes resulting in IL-6 and IgM production in addition to increased surface marker density (HLA-DR, CD45, CD64, and CD86), whereas MID-deficient OMV failed to induce B cell activation. DNA associated with OMV induced full B cell activation by signaling through TLR9. Importantly, this concept was verified in vivo, as OMV equipped with MID and DNA were found in a 9-year old patient suffering from Moraxella sinusitis. In conclusion, Moraxella avoid direct interaction with host B cells by redirecting the adaptive humoral immune response using its superantigen-bearing OMV as decoys