Nature volume 613, páginas 735–742 (2023)Cite este artigo
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A inibição por feedback da imunidade humoral por anticorpos foi documentada pela primeira vez em 19091. Estudos subsequentes mostraram que, dependendo do contexto, os anticorpos podem aumentar ou inibir as respostas imunes2,3. No entanto, pouco se sabe sobre como os anticorpos pré-existentes influenciam o desenvolvimento das células B de memória. Aqui examinamos a resposta das células B de memória em indivíduos que receberam dois anticorpos monoclonais anti-SARS-CoV-2 de alta afinidade e subsequentemente duas doses de uma vacina de mRNA4,5,6,7,8. Descobrimos que os receptores dos anticorpos monoclonais produziram títulos de ligação ao antígeno e de neutralização que foram apenas fracionariamente mais baixos em comparação com os indivíduos de controlo. No entanto, as células B de memória dos indivíduos que receberam os anticorpos monoclonais diferiram daquelas dos indivíduos controle, pois expressavam predominantemente anticorpos IgM de baixa afinidade que carregavam um pequeno número de mutações somáticas e apresentavam especificidade alterada do alvo do domínio de ligação ao receptor (RBD), consistente. com mascaramento de epítopo. Além disso, apenas 1 dos 77 anticorpos de memória anti-RBD testados neutralizou o vírus. O mecanismo subjacente a estas descobertas foi examinado em experiências em ratos que mostraram que os centros germinativos formados na presença dos mesmos anticorpos eram dominados por células B de baixa afinidade. Nossos resultados indicam que anticorpos pré-existentes de alta afinidade influenciam o centro germinativo e a seleção de células B de memória através de dois mecanismos distintos: (1) diminuindo o limiar de ativação para células B, permitindo assim que clones abundantes de menor afinidade participem da resposta imune; e (2) através de mascaramento direto dos seus epítopos cognatos. Isto pode explicar em parte a mudança no perfil alvo dos anticorpos de memória provocados pelas vacinações de reforço9.
Para examinar como a administração passiva de anticorpos monoclonais pode influenciar as respostas humorais subsequentes à vacinação em humanos, estudamos um grupo de 18 voluntários saudáveis que receberam uma dose única de uma combinação de dois anticorpos monoclonais de ação prolongada contra SARS-CoV-2 e posteriormente receberam duas doses de uma vacina de mRNA SARS-CoV-2 (Fig. 1a). Os 2 anticorpos – C144-LS e C135-LS – ligam-se aos epítopos de classe 2 e 3 no RBD da proteína spike (S) do SARS-CoV-2 com alta afinidade (constante de dissociação (Kd) = 18 nM e Kd = 6 nM, respectivamente) e neutralizar o vírus com valores de meia concentração inibitória máxima (IC50) de 2,55 e 2,98 ng ml-1, respectivamente5,8.
a, Esquema do desenho do estudo, com marcadores indicando semanas relativas ao momento da primeira dose da vacina. mAb, anticorpo monoclonal. b, São mostrados os níveis séricos de C135-LS (parte superior, azul) e C144-LS (parte inferior, vermelho) ao longo do tempo. As linhas tracejadas coloridas grossas indicam as concentrações séricas medianas entre os receptores de anticorpos monoclonais (n = 18), e as linhas pretas pontilhadas finas representam participantes individuais. As duas linhas verticais sólidas indicam o valor mediano e as áreas sombreadas em cinza indicam o intervalo de tempo desde a administração do anticorpo monoclonal até a vacinação. c – f, A metade do título máximo de ligação plasmática (BT50) ao RBD após uma (vax 1) e duas doses (vax 2) de vacinação com mRNA para receptores de anticorpos monoclonais (n = 18, verde) e controles (n = 26, azul). Cada ponto representa um indivíduo. As linhas horizontais tracejadas representam a atividade de ligação mediana de amostras de plasma pré-pandémicas de indivíduos saudáveis que foram utilizadas como controlos negativos. c, d, títulos de ligação de IgM (c) e IgG (d) a WT RBD. e, ligação de IgG a RBDs R346S/E484K (esquerda) e N440K/E484K (Extended Data Fig. 1). f, ligação de IgG ao NTD. g – i, valores do título neutralizante semimáximo plasmático (NT50) para receptores de anticorpos monoclonais (n = 18, verde) e controles (n = 26, azul) contra HIV-1 pseudotipado com SARS-CoV-2 WT S (g) , mutante R346S/Q493K S (h) e mutante R346S/N440K/E484K S (i) (Dados Estendidos Fig. 2). A proteína S nos pseudovírus em g –i continha uma substituição R683G. As barras horizontais vermelhas em c – i e os números vermelhos em g – i representam os valores medianos. A significância estatística em c – i foi determinada usando testes U de Mann-Whitney bicaudais, comparando as diferenças entre receptores de anticorpos monoclonais e controles para cada ponto de tempo independentemente; Os valores de P são mostrados acima dos gráficos. Todas as experiências foram realizadas pelo menos em duplicado.
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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