Resistance of Omicron subvariants BA.2.75.2, BA.4.6 and BQ.1.1 to neutralizing antibodies

Convergent evolution of SARS-CoV-2 Omicron BA.2, BA.4 and BA.5 lineages has led to the emergence of several new subvariants, including BA.2.75.2, BA.4.6. and BQ.1.1. The subvariants BA.2.75.2 and BQ.1.1 are expected to become predominant in many countries in November 2022. They carry an additional and often redundant set of mutations in the spike, likely responsible for increased transmissibility and immune evasion. Here, we established a viral amplification procedure to easily isolate Omicron strains. We examined their sensitivity to 6 therapeutic monoclonal antibodies (mAbs) and to 72 sera from Pfizer BNT162b2-vaccinated individuals, with or without BA.1/BA.2 or BA.5 breakthrough infection. Ronapreve (Casirivimab and Imdevimab) and Evusheld (Cilgavimab and Tixagevimab) lost any antiviral efficacy against BA.2.75.2 and BQ.1.1, whereas Xevudy (Sotrovimab) remained weakly active. BQ.1.1 was also resistant to Bebtelovimab. Neutralizing titers in triply vaccinated individuals were low to undetectable against BQ.1.1 and BA.2.75.2, 4 months after boosting. A BA.1/BA.2 breakthrough infection increased these titers, which remained about 18-fold lower against BA.2.75.2 and BQ.1.1, than against BA.1. Reciprocally, a BA.5 breakthrough infection increased more efficiently neutralization against BA.5 and BQ.1.1 than against BA.2.75.2. Thus, the evolution trajectory of novel Omicron subvariants facilitated their spread in immunized populations and raises concerns about the efficacy of most currently available mAbs.N

Easy pan-detection of human IgA immunoglobulins

International audienceIgA antibodies are key immune effectors against invading pathogens but also possess essential immunoregulatory functions. Detecting and quantifying human IgA+ B-cell subsets and secreted IgA molecules is needed for investigating the protective, modulatory and pathophysiologic roles of IgAs. Here, we produced a recombinant tagged trimeric form of the streptococcal IgA-binding peptide (SAP) by transient transfection-based eukaryotic expression system. The trimeric SAP (tSAP) probe had a higher production yield and apparent binding affinity to human IgA1 and IgA2 immunoglobulins when compared to the dimeric SAP molecule classically used to purify IgAs. tSAP bound both monomeric and dimeric IgAs, and allowed immunoblot detection and ELISA quantification of serum IgA antibodies in humans and non-human primates. Fluorescently labeled tSAP also permitted an accurate quantification of circulating human blood IgA-expressing memory B cells by flow-cytometric analyses. Thus, the easy-to-produce high affinity recombinant tSAP probe we developed is a versatile and valuable tool to quantify secreted and membrane-bound human but also primate IgA immunoglobulins

Influence de la polyréactivité naturelle et induite des immunoglobulines solubles et des récepteurs de lymphocytes B sur la modulation de la réponse immunitaire

Afin de faire face à l extraordinaire diversité des antigènes auxquels l organisme est exposé, des mécanismes élaborés de génération de diversité des récepteurs des lymphocytes T et B sont apparus au cours de l évolution. Toutefois, ces mécanismes aléatoires permettent l émergence de récepteurs potentiellement délétères dirigés contre des antigènes du Soi. Aussi les effecteurs cellulaires subissent-ils une éducation leur permettant de discriminer le Soi du non-Soi. Pourtant, une fraction non-négligeable des anticorps circulants est capable de reconnaître plusieurs antigènes structurellement différents et notamment des antigènes du Soi. En outre, des travaux récents ont démontré que certaines immunoglobulines monoréactives deviennent polyréactives après exposition à différents agents pro-oxydants. Lors de mon travail de thèse, j ai étudié la polyréactivité des lymphocytes B naïfs murins et j ai observé que la faible stimulation du BcR confère un phénotype régulateur caractérisé par la production d IL-10 et la polarisation des lymphocytes T CD4+ vers un profil Tr1/Th2. En parallèle, j ai observé différents degrés de sensibilité des immunoglobulines solubles à l induction de polyréactivité par différents agents chaotropiques et pro-oxydants. Les résultats de mon travail soulèvent la question de l importance des lymphocytes B poly/autoréactifs naturels comme source de cellules régulatrices dans les conditions physiologiques. Par ailleurs, mes résultats mettent en exergue l importance des molécules libérées dans le microenvironnements inflammatoires sur les fonctions des immunoglobulines et des lymphocytes BIn order to counter the enormous antigen diversity, elaborated mechanisms for the generation of diversity of the T and B-cell receptors appeared along the evolution. However, such random mechanisms enable the appearance of broadly-reactive receptors that could be potentially deleterious. Thus, the cellular effectors are educated along their development in order to discriminate self from non-self. The education of the immune system implies several mechanisms of negative selection of self-reactive T and B cells during their ontogeny. However, a non-negligible fraction of circulating immunoglobulins is able to bind to various structurally unrelated antigens including self-antigens. Besides, recent studies have demonstrated that certain monoreactive immunoglobulins become polyreactive following exposure to various chaotropic agents and pro-oxidative molecules. During my PhD work, I studied the natural polyreactivity of B lymphocytes from naïve mice and I observed that a weak BcR-stimulation confers them regulatory properties characterised by the production of IL-10 and the polarization of CD4+ T cells into Tr1/Th2 cells. I also observed different degrees of sensibility of soluble immunoglobulins to the induction of polyreactivity depending on the nature of the pro-oxidative molecules. The results of my work emphasize the question of the importance of the naturally poly/self-reactive B cells as a source of regulatory cells under physiological conditions. Besides, my results highlight the importance of molecules present at the site of inflammation and their functions on soluble immunoglobulins and circulating B cellsPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Binding mechanisms of therapeutic antibodies to human CD20

International audienceMonoclonal antibodies (mAbs) targeting human antigen CD20 (cluster of differentiation 20) constitute important immunotherapies for the treatment of B cell malignancies and autoimmune diseases. Type I and II therapeutic mAbs differ in B cell binding properties and cytotoxic effects, reflecting differential interaction mechanisms with CD20. Here we present 3.7- to 4.7-angstrom cryo–electron microscopy structures of full-length CD20 in complexes with prototypical type I rituximab and ofatumumab and type II obinutuzumab. The structures and binding thermodynamics demonstrate that upon binding to CD20, type II mAbs form terminal complexes that preclude recruitment of additional mAbs and complement components, whereas type I complexes act as molecular seeds to increase mAb local concentration for efficient complement activation. Among type I mAbs, ofatumumab complexes display optimal geometry for complement recruitment. The uncovered mechanisms should aid rational design of next-generation immunotherapies targeting CD20

Oxidized hemoglobin triggers polyreactivity and autoreactivity of human IgG via transfer of heme

International audienceIntravascular hemolysis occurs in diverse pathological conditions. Extracellular hemoglobin and heme have strong pro-oxidative and pro-inflammatory potentials that can contribute to the pathology of hemolytic diseases. However, many of the effects of extracellular hemoglobin and heme in hemolytic diseases are still not well understood. Here we demonstrate that oxidized hemoglobin (methemoglobin) can modify the antigen-binding characteristics of human immunoglobulins. Thus, incubation of polyclonal or some monoclonal human IgG in the presence of methemoglobin results in an appearance of binding reactivities towards distinct unrelated self-proteins, including the protein constituent of hemoglobin i.e., globin. We demonstrate that a transfer of heme from methemoglobin to IgG is indispensable for this acquisition of antibody polyreactivity. Our data also show that only oxidized form of hemoglobin have the capacity to induce polyreactivity of antibodies. Site-directed mutagenesis of a heme-sensitive human monoclonal IgG1 reveals details about the mechanism of methemoglobin-induced antigen-binding polyreactivity. Further here we assess the kinetics and thermodynamics of interaction of a heme-induced polyreactive human antibody with hemoglobin and myoglobin. Taken together presented data contribute to a better understanding of the functions of extracellular hemoglobin in the context of hemolytic diseases

Evolutionary trajectory of receptor binding specificity and promiscuity of the spike protein of SARS-CoV-2

International audienceSARS-CoV-2 infects cells by attachment to its receptor—the angiotensin converting enzyme 2 (ACE2). Regardless of the wealth of structural data, little is known about the physicochemical mechanism of interactions of the viral spike (S) protein with ACE2 and how this mechanism has evolved during the pandemic. Here, we applied experimental and computational approaches to characterize the molecular interaction of S proteins from SARS-CoV-2 variants of concern (VOC). Data on kinetics, activation-, and equilibrium thermodynamics of binding of the receptor binding domain (RBD) from VOC with ACE2 as well as data from computational protein electrostatics revealed a profound remodeling of the physicochemical characteristics of the interaction during the evolution. Thus, as compared to RBDs from Wuhan strain and other VOC, Omicron RBD presented as a unique protein in terms of conformational dynamics and types of non-covalent forces driving the complex formation with ACE2. Viral evolution resulted in a restriction of the RBD structural dynamics, and a shift to a major role of polar forces for ACE2 binding. Further, we investigated how the reshaping of the physicochemical characteristics of interaction affects the binding specificity of S proteins. Data from various binding assays revealed that SARS-CoV-2 Wuhan and Omicron RBDs manifest capacity for promiscuous recognition of unrelated human proteins, but they harbor distinct reactivity patterns. These findings might contribute for mechanistic understanding of the viral tropism and capacity to evade immune responses during evolution

Antibody-mediated catalysis: Induction and therapeutic relevance

Abzymes are immunoglobulins endowed with enzymatic activities. The catalytic activity of an abzyme resides in the variable domain of the antibody, which is constituted by the close spatial arrangement of amino acid residues involved in catalysis. The origin of abzymes is conferred by the innate diversity of the immunoglobulin gene repertoire. Under deregulated immune conditions, as in autoimmune diseases, the generation of abzymes to self-antigens could be deleterious. Technical advancement in the ability to generate monoclonal antibodies has been exploited in the generation of abzymes with defined specificities and activities. Therapeutic applications of abzymes are being investigated with the generation of monoclonal abzymes against several pathogenesis-associated antigens. Here, we review the different contexts in which abzymes are generated, and we discuss the relevance of monoclonal abzymes for the treatment of human diseases

NK cell spatial dynamics and IgA responses in gut-associated lymphoid tissues during SIV infections

International audienceAbstract HIV infection induces tissue damage including lymph node (LN) fibrosis and intestinal epithelial barrier disruption leading to bacterial translocation and systemic inflammation. Natural hosts of SIV, such as African Green Monkeys (AGM), do not display tissue damage despite high viral load in blood and intestinal mucosa. AGM mount a NK cell-mediated control of SIVagm replication in peripheral LN. We analyzed if NK cells also control SIVagm in mesenteric (mes) LN and if this has an impact on gut humoral responses and the production of IgA known for their anti-inflammatory role in the gut. We show that CXCR5 + NK cell frequencies increase in mesLN upon SIVagm infection and that NK cells migrate into and control viral replication in B cell follicles (BCF) of mesLN. The proportion of IgA+ memory B cells were increased in mesLN during SIVagm infection in contrast to SIVmac infection. Total IgA levels in gut remained normal during SIVagm infection, while strongly decreased in intestine of chronically SIVmac-infected macaques. Our data suggest an indirect impact of NK cell-mediated viral control in mesLN during SIVagm infection on preserved BCF function and IgA production in intestinal tissues