Natura volume 613, pagine 735–742 (2023) Citare questo articolo
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L'inibizione del feedback dell'immunità umorale da parte degli anticorpi è stata documentata per la prima volta nel 19091. Studi successivi hanno dimostrato che, a seconda del contesto, gli anticorpi possono potenziare o inibire le risposte immunitarie2,3. Tuttavia, si sa poco su come gli anticorpi preesistenti influenzino lo sviluppo delle cellule B della memoria. Qui abbiamo esaminato la risposta delle cellule B della memoria in individui che hanno ricevuto due anticorpi monoclonali anti-SARS-CoV-2 ad alta affinità e successivamente due dosi di un vaccino mRNA4,5,6,7,8. Abbiamo scoperto che i destinatari degli anticorpi monoclonali producevano titoli leganti e neutralizzanti l'antigene che erano solo leggermente inferiori rispetto a quelli degli individui di controllo. Tuttavia, le cellule B della memoria degli individui che hanno ricevuto gli anticorpi monoclonali differivano da quelle degli individui di controllo in quanto esprimevano prevalentemente anticorpi IgM a bassa affinità che portavano un piccolo numero di mutazioni somatiche e mostravano una specificità target alterata del dominio di legame del recettore (RBD), coerente con mascheramento epitopico. Inoltre, solo 1 su 77 anticorpi anti-RBD della memoria testati ha neutralizzato il virus. Il meccanismo alla base di questi risultati è stato esaminato in esperimenti sui topi che hanno dimostrato che i centri germinali formati in presenza degli stessi anticorpi erano dominati da cellule B a bassa affinità. I nostri risultati indicano che gli anticorpi preesistenti ad alta affinità influenzano la selezione delle cellule B del centro germinale e della memoria attraverso due meccanismi distinti: (1) abbassando la soglia di attivazione per le cellule B, consentendo così ad abbondanti cloni ad affinità inferiore di partecipare alla risposta immunitaria; e (2) attraverso il mascheramento diretto dei loro epitopi affini. Ciò potrebbe in parte spiegare lo spostamento del profilo target degli anticorpi della memoria indotto dalle vaccinazioni di richiamo9.
Per esaminare come la somministrazione passiva di anticorpi monoclonali potrebbe influenzare le successive risposte umorali alla vaccinazione negli esseri umani, abbiamo studiato un gruppo di 18 volontari sani che hanno ricevuto una singola dose di una combinazione di due anticorpi monoclonali a lunga durata d’azione contro SARS-CoV-2 e successivamente hanno ricevuto due dosi di un vaccino a mRNA SARS-CoV-2 (Fig. 1a). I 2 anticorpi, C144-LS e C135-LS, si legano agli epitopi di classe 2 e 3 sull'RBD della proteina spike (S) di SARS-CoV-2 con elevata affinità (costante di dissociazione (Kd) = 18 nM e Kd = 6 nM, rispettivamente) e neutralizzare il virus con valori di concentrazione inibitoria semimassima (IC50) rispettivamente di 2,55 e 2,98 ng ml−15,8.
a, Schema del disegno dello studio, con marcatori che indicano settimane relative al momento della prima dose di vaccino. mAb, anticorpo monoclonale. b, vengono mostrati i livelli sierici di C135-LS (in alto, blu) e C144-LS (in basso, rosso) nel tempo. Le linee tratteggiate colorate spesse indicano le concentrazioni sieriche mediane tra i destinatari dell'anticorpo monoclonale (n = 18) e le linee nere tratteggiate sottili rappresentano i singoli partecipanti. Le due linee verticali continue indicano il valore mediano e le aree ombreggiate in grigio indicano l'intervallo di tempo dalla somministrazione dell'anticorpo monoclonale alla vaccinazione. c-f, Il titolo di legame plasmatico semimassimo (BT50) con RBD dopo una (vax 1) e due dosi (vax 2) di vaccinazione con mRNA per i destinatari di anticorpi monoclonali (n = 18, verde) e controlli (n = 26, blu). Ogni punto rappresenta un individuo. Le linee orizzontali tratteggiate rappresentano l'attività di legame mediana dei campioni di plasma pre-pandemia provenienti da individui sani utilizzati come controlli negativi. titoli di legame c,d, IgM (c) e IgG (d) con WT RBD. e, legame delle IgG agli RBD R346S/E484K (a sinistra) e N440K/E484K (dati estesi, Fig. 1). f, legame delle IgG al NTD. g–i, valori del titolo neutralizzante metà massimo del plasma (NT50) per i destinatari di anticorpi monoclonali (n = 18, verde) e controlli (n = 26, blu) contro HIV-1 pseudotipizzato con SARS-CoV-2 WT S (g) , mutante R346S/Q493K S (h) e mutante R346S/N440K/E484K S (i) (Dati estesi Fig. 2). La proteina S negli pseudovirus in g–i conteneva una sostituzione R683G. Le barre orizzontali rosse in c–i e i numeri rossi in g–i rappresentano i valori mediani. La significatività statistica in c-i è stata determinata utilizzando i test U di Mann-Whitney a due code confrontando le differenze tra i destinatari dell'anticorpo monoclonale e i controlli per ciascun punto temporale in modo indipendente; I valori P sono mostrati sopra i grafici. Tutti gli esperimenti sono stati eseguiti almeno in duplicato.
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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