Nature volume 613, pages 735-742 (2023)Citer cet article
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La rétro-inhibition de l’immunité humorale par les anticorps a été documentée pour la première fois en 19091. Des études ultérieures ont montré que, selon le contexte, les anticorps peuvent renforcer ou inhiber les réponses immunitaires2,3. Cependant, on sait peu de choses sur la manière dont les anticorps préexistants influencent le développement des cellules B mémoire. Ici, nous avons examiné la réponse des lymphocytes B mémoire chez des individus ayant reçu deux anticorps monoclonaux anti-SARS-CoV-2 de haute affinité, puis deux doses d'un vaccin à ARNm4,5,6,7,8. Nous avons constaté que les receveurs d’anticorps monoclonaux produisaient des titres de liaison à l’antigène et neutralisants qui n’étaient que légèrement inférieurs à ceux des individus témoins. Cependant, les cellules B mémoire des individus ayant reçu les anticorps monoclonaux différaient de celles des individus témoins dans la mesure où elles exprimaient principalement des anticorps IgM de faible affinité portant un petit nombre de mutations somatiques et présentaient une spécificité cible du domaine de liaison au récepteur (RBD) altérée, ce qui est cohérent. avec masquage épitopique. De plus, seul 1 des 77 anticorps anti-mémoire anti-RBD testés ont neutralisé le virus. Le mécanisme à la base de ces découvertes a été examiné dans des expériences sur des souris qui ont montré que les centres germinatifs formés en présence des mêmes anticorps étaient dominés par des cellules B de faible affinité. Nos résultats indiquent que les anticorps préexistants de haute affinité biaisent la sélection du centre germinal et des cellules B mémoire par deux mécanismes distincts : (1) en abaissant le seuil d'activation des cellules B, permettant ainsi à de nombreux clones de faible affinité de participer à la réponse immunitaire ; et (2) par masquage direct de leurs épitopes apparentés. Cela peut expliquer en partie le changement du profil cible des anticorps mémoire provoqués par les vaccinations de rappel9.
Pour examiner comment l'administration passive d'anticorps monoclonaux pourrait influencer les réponses humorales ultérieures à la vaccination chez l'homme, nous avons étudié un groupe de 18 volontaires sains qui ont reçu une dose unique d'une combinaison de deux anticorps monoclonaux à action prolongée contre le SRAS-CoV-2 et ont ensuite reçu deux doses d'un vaccin à ARNm du SRAS-CoV-2 (Fig. 1a). Les 2 anticorps – C144-LS et C135-LS – se lient aux épitopes de classe 2 et 3 sur le RBD de la protéine Spike (S) du SRAS-CoV-2 avec une affinité élevée (constante de dissociation (Kd) = 18 nM et Kd = 6 nM, respectivement) et neutralisent le virus avec des valeurs de concentration inhibitrice demi-maximale (CI50) de 2,55 et 2,98 ng ml−1, respectivement5,8.
a, Schéma de la conception de l'étude, avec des marqueurs indiquant les semaines par rapport au moment de la première dose de vaccin. mAb, anticorps monoclonal. b, les niveaux sériques de C135-LS (en haut, bleu) et de C144-LS (en bas, rouge) au fil du temps sont affichés. Les lignes pointillées épaisses de couleur indiquent les concentrations sériques médianes parmi les receveurs d’anticorps monoclonaux (n = 18), et les fines lignes noires pointillées représentent les participants individuels. Les deux lignes verticales pleines indiquent la valeur médiane et les zones grisées indiquent l'intervalle de temps entre l'administration d'anticorps monoclonaux et la vaccination. c – f, titre de liaison plasmatique semi-maximal (BT50) au RBD après une (vax 1) et deux doses (vax 2) de vaccination à ARNm pour les receveurs d'anticorps monoclonaux (n = 18, vert) et les témoins (n = 26, bleu). Chaque point représente un individu. Les lignes horizontales pointillées représentent l’activité de liaison médiane des échantillons de plasma pré-pandémiques provenant d’individus en bonne santé qui ont été utilisés comme contrôles négatifs. titres de liaison c, d, IgM (c) et IgG (d) au WT RBD. e, IgG se liant aux RBD R346S/E484K (à gauche) et N440K/E484K (Extended Data Fig. 1). f, liaison des IgG au NTD. g – i, valeurs du titre neutralisant plasmatique semi-maximal (NT50) pour les receveurs d'anticorps monoclonaux (n = 18, vert) et les témoins (n = 26, bleu) contre le VIH-1 pseudotypé avec le SRAS-CoV-2 WT S (g) , le mutant R346S/Q493K S (h) et le mutant R346S/N440K/E484K S (i) (données étendues, figure 2). La protéine S des pseudovirus de g – i contenait une substitution R683G. Les barres horizontales rouges en c-i et les chiffres rouges en g-i représentent les valeurs médianes. La signification statistique de c – i a été déterminée à l'aide de tests U bilatéraux de Mann – Whitney comparant indépendamment les différences entre les receveurs d'anticorps monoclonaux et les témoins pour chaque point temporel; Les valeurs P sont affichées au-dessus des tracés. Toutes les expériences ont été réalisées au moins en double.
1) somatic hypermutation, and the encircled numbers indicate the number of sequences analysed for all cells irrespective of isotype (f), and for IgM and IgG analysed independently (g). The red horizontal bars and numbers in f and g indicate the mean values. Statistical significance was determined using two-tailed Mann–Whitney U-tests (a–c and f), Kruskal–Wallis tests with subsequent Dunn’s correction for multiple comparisons (g) and two-sided Fisher’s exact tests to compare fractions (f and g)./p> 0.99 and P = 0.40 for IgM and IgG, respectively). Thus, IgM- and IgG-expressing B cells in vaccinated individuals who had received C144-LS and C135-LS carry normal numbers of somatic mutations, but the relative ratio of the two memory cell types is reversed, which accounts for the overall lower level of mutation in their memory compartment. Finally, in contrast to the controls, there was no enrichment for the VH3-53, VH1-69, VH1-46 and VH3-66 heavy chains, which often target class 1 and 2 epitopes. Instead, there was relative enrichment for the VH3-9, VH5-51, VH4-39 and VH1-8 genes (Extended Data Fig. 4f). The limited number of cells sequenced precludes definitive conclusions about the precise clonotype distribution in this population, but the relative change in VH gene use frequency implies that B cell recruitment into the memory compartment of monoclonal antibody recipients is altered. In summary, the data suggest that pre-existing antibodies can alter the cellular and molecular composition of the RBD-specific MBC compartment that develops in response to mRNA vaccination./p>10 µg ml−1; the solid black lines are antibodies that were below or equal to the negative control anti-HIV1 antibody 3BNC117 (thick yellow dashed line). C144 (thick, red dashed line) was used as a positive control. b, EC50 values derived from a for monoclonal antibody recipients (green) and controls (blue) for all antibodies, irrespective of isotype. c, EC50 values as in b, but IgM and IgG were analysed independently. The grey shaded area between the horizontal dotted lines indicates antibodies with EC50 > 10 µg ml−1 (poor binding) and non-binding antibodies, arbitrarily grouped at 10 and 20 µg ml−1, respectively. The ring plots summarize the fraction of all antibodies tested for the respective groups (encircled number). d, IC50 values for all monoclonal antibodies isolated from vaccinated monoclonal antibody recipients (green) or control individuals (blue). The ring plots illustrate the fraction of non-neutralizing (non-neut.; IC50 > 1,000 ng ml−1) antibodies (black slices) among all antibodies tested for the respective group (encircled number). e, IC50 values as described in d, but IgM and IgG antibodies were analysed independently. f–l, Monoclonal antibody binding to monomeric and multimerized antigen by BLI. f, Schematic of monomeric binding measurements in which IgG was immobilized onto the biosensor chip and subsequently exposed to monomeric RBD (top), and multimeric binding using 6P-stabilized WT SARS-CoV-2 S protein trimers that had been tetramerized using streptavidin (bottom). g, BLI traces obtained under monovalent conditions as shown in f (top). Each curve represents one antibody. The coloured solid lines denote binding above the background represented by polyreactive antibody ED3835 (dotted black line) and anti-HIV-1 antibody 3BNC117 (dashed black line). The grey lines show non-binding antibodies. C144 (thick, red dashed line) was used as a positive control. h, BLI traces as described in g for antibodies that showed no measurable binding in g and were subsequently tested for binding under polyvalent conditions as illustrated in f (bottom). i, The percentage of binding antibodies under monovalent conditions for all antibodies and by isotype. The values below the bars indicate the number of antibodies tested. j, The percentage of binding antibodies as described in i for the antibodies shown in h. k, Kd values derived under monomeric binding conditions in g for monoclonal antibody recipients (green) and controls (blue) irrespective of isotype. The ring plots illustrate the fraction of antibodies tested for the respective group (encircled number) that measurably bound to monomeric RBD (binding, white) and those for which a Kd value could not be established (no Kd, black). l, Kd values as described in k were analysed independently for IgM and IgG. m, Schematic of the BLI competition experiment: (1) the capture antibody of known epitope specificity (class-reference antibody) was bound to the biosensor chip; (2) exposed to antigen; and (3) the antibody of interest was added to the chip. n, The distribution of the epitopes targeted. The number in the centre is the number of antibodies tested. Slices coloured in shades of red and blue represent class 1, 2 and 3 or combined epitopes, and shades of grey represent class-4-containing epitopes or epitopes that could not be classified. For b–e, k and l, the red horizontal bars and numbers represent the median values. ND, not determined. Statistical significance was determined using two-tailed Mann–Whitney U-tests (b, d and k), Kruskal–Wallis tests with subsequent Dunn’s correction for multiple comparisons (c, e and l), two-sided Fisher’s exact tests (d, e, k and l) and the two-sided χ2 contingency statistic (b, c and n)./p> 0; top = experiment-specific upper plateau of the normalizer control antibody or plasma sample reaching saturation for at least 3 consecutive dilution steps. The curve fit was constrained to an upper limit that corresponds to the maximal optical density achieved by the known normalizer control to limit interplate/interexperiment variability (batch effects). Pentameric IgM BT50 values were established using previously measured IgG antibodies as normalizer controls. Pre-pandemic plasma samples from healthy donors and isotype control monoclonal antibodies were used as negative controls as indicated and were used for validation5. All of the reported EC50 and BT50 values are the average of at least two independent experiments./p>10 µg ml−1 (poor binding) and non-binding antibodies arbitrarily grouped at 10 and 20 µg ml−1, respectively. b, Plots show IC50s of 2 IgM-derived control antibodies (covering a wide range of neutralizing activity) in blue and 15 IgM-derived monoclonal antibodies from mAb recipients (as in a) in green, expressed as human IgG1 (IgG) or pentameric IgM (IgM5). For both panels (a, b), ring plots summarize the fraction of antibodies in the indicated category among all tested (encircled number). Red horizontal bars and numbers indicate median values. For panel a, statistical significance was determined using the two-tailed Wilcoxon matched-pairs rank test to compare differences between the same monoclonal antibodies expressed as IgG or pentameric IgM, and the Chi-squared contingency statistic was used to compare categorical distributions from ring plots./p> 1) SHM among all sequences analysed (encircled number) for the respective group. f, Percentage of sequences belonging to clones, defined as 2 or more sequences with the same IGHV and IGLV genes and with highly similar CDR3s, among all sequences obtained from the respective animal (as in Fig. 4d). Each dot represents one individual mouse from the anti-RBD mAb (n = 6, green) or control group (n = 6, blue). g, Affinity constants (Kd) of germinal centre B-cell-derived Fabs for WT SARS-CoV-2 RBD, as established from the monovalent interaction of Fabs with RBD monomers by BLI (also see Fig. 4f–i, Supplementary Table 6 and methods). Each dot represents a single Fab from the anti-RBD mAb (n = 8, green) or control group (n = 22, blue). Red horizontal bars (c-g) and numbers (e, g) indicate median (c, d, f, g) and mean (e) values. Statistical significance was determined using the two-tailed Mann-Whitney test for c-g d, and the two-sided Fisher’s exact test was used to test the relative contribution of mutated and unmutated sequences in e./p>
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