항체 피드백은 SARS 이후 면역 기억을 조절합니다

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항체에 의한 체액성 면역의 피드백 억제는 19091년에 처음으로 기록되었습니다. 후속 연구에서는 상황에 따라 항체가 면역 반응을 강화하거나 억제할 수 있음이 밝혀졌습니다2,3. 그러나 기존 항체가 기억 B 세포의 발달에 어떤 영향을 미치는지에 대해서는 알려진 바가 거의 없습니다. 여기에서 우리는 2개의 고친화도 항-SARS-CoV-2 단클론 항체와 이후 2회의 mRNA 백신을 접종받은 개인의 기억 B 세포 반응을 조사했습니다4,5,6,7,8. 우리는 단클론 항체를 받은 사람들이 대조군에 비해 극히 낮은 수준의 항원 결합 및 중화 역가를 생산한다는 사실을 발견했습니다. 그러나 단클론 항체를 투여받은 개인의 기억 B 세포는 적은 수의 체세포 돌연변이를 운반하고 변경된 수용체 결합 도메인(RBD) 표적 특이성을 나타내는 저친화도 IgM 항체를 주로 발현한다는 점에서 대조 개체의 기억 B 세포와 달랐습니다. 에피토프 마스킹으로. 더욱이, 테스트된 항-RBD 기억 항체 77개 중 1개만이 바이러스를 중화시켰습니다. 이러한 발견의 기초가 되는 메커니즘은 동일한 항체의 존재 하에 형성된 배중심이 저친화도 B 세포에 의해 지배된다는 것을 보여주는 생쥐 실험에서 조사되었습니다. 우리의 결과는 기존의 고친화성 항체가 두 가지 메커니즘을 통해 배중심과 기억 B 세포 선택을 편향한다는 것을 나타냅니다. (1) B 세포의 활성화 임계값을 낮춤으로써 풍부한 저친화성 클론이 면역 반응에 참여할 수 있도록 합니다. (2) 동족 에피토프의 직접적인 마스킹을 통해. 이는 추가 백신 접종에 의해 유발된 기억 항체의 표적 프로필이 변화하는 것을 부분적으로 설명할 수 있습니다9.

단클론 항체의 수동적 투여가 인간의 백신 접종에 대한 후속 체액 반응에 어떻게 영향을 미칠 수 있는지 조사하기 위해 우리는 SARS-CoV-2에 대한 두 가지 지속성 단클론 항체의 조합을 단회 투여받은 18명의 건강한 지원자 그룹을 연구했습니다. SARS-CoV-2 mRNA 백신 2회 접종(그림 1a). 2개의 항체(C144-LS 및 C135-LS)는 SARS-CoV-2 스파이크(S) 단백질의 RBD에 있는 클래스 2 및 3 에피토프에 높은 친화력(해리 상수(Kd) = 18nM 및 Kd = 6)으로 결합합니다. nM) 및 각각 2.55 및 2.98 ng ml-1의 절반 최대 억제 농도(IC50) 값으로 바이러스를 중화합니다5,8.

a, 첫 번째 백신 투여 시점을 기준으로 몇 주를 나타내는 마커가 포함된 연구 설계의 도식. mAb, 단클론 항체. b, 시간 경과에 따른 C135-LS(상단, 파란색) 및 C144-LS(하단, 빨간색)의 혈청 수준이 표시됩니다. 굵은 점선은 단클론항체 수혜자(n = 18)의 평균 혈청 농도를 나타내고, 가는 점선 검정색 선은 개별 참가자를 나타냅니다. 두 개의 실선 수직선은 중앙값을 나타내고 회색 음영 영역은 단일클론항체 투여부터 백신 접종까지의 시간 범위를 나타냅니다. c-f, 단클론 항체 수혜자(n = 18, 녹색) 및 대조군(n = 26, 파란색). 각 점은 한 개인을 나타냅니다. 점선으로 표시된 수평선은 음성 대조군으로 사용된 건강한 개인의 전염병 전 혈장 샘플의 중간 결합 활성을 나타냅니다. c, d, IgM (c) 및 IgG (d) WT RBD에 대한 결합 역가. e, R346S/E484K(왼쪽) 및 N440K/E484K RBD에 결합하는 IgG(확장 데이터 그림 1). f, NTD에 결합하는 IgG. g–i, SARS-CoV-2 WT S로 위형화된 HIV-1에 대한 단클론 항체 수용자(n = 18, 녹색) 및 대조군(n = 26, 파란색)에 대한 혈장 반최대 중화 역가(NT50) 값(g) , R346S/Q493K 돌연변이 S(h) 및 R346S/N440K/E484K 돌연변이 S(i)(확장 데이터 그림 2). g-i의 슈도바이러스의 S 단백질에는 R683G 치환이 포함되어 있습니다. c–i의 빨간색 가로 막대와 g–i의 빨간색 숫자는 중앙값을 나타냅니다. c – i의 통계적 유의성은 단일 클론 항체 수용자와 각 시점에 대한 대조군 간의 차이를 독립적으로 비교하는 양측 Mann-Whitney U 테스트를 사용하여 결정되었습니다. P 값은 플롯 위에 표시됩니다. 모든 실험은 적어도 이중으로 수행되었습니다.

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>