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SARS後の免疫記憶を制御する抗体フィードバック
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SARS後の免疫記憶を制御する抗体フィードバック

Jul 11, 2023

Nature volume 613、pages 735–742 (2023)この記事を引用

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抗体による体液性免疫のフィードバック阻害は 1909 年に初めて記録されました 1。その後の研究では、状況に応じて、抗体が免疫応答を増強または阻害できることが示されました 2,3。 しかし、既存の抗体が記憶 B 細胞の発達にどのような影響を与えるかについてはほとんどわかっていません。 ここで我々は、2 つの高親和性抗 SARS-CoV-2 モノクローナル抗体とその後 2 回の mRNA ワクチンを受けた個人における記憶 B 細胞の応答を調べました4、5、6、7、8。 我々は、モノクローナル抗体のレシピエントが生成した抗原結合力価および中和力価が、対照個体と比較してほんのわずかに低いことを発見しました。 しかし、モノクローナル抗体を投与された個体の記憶 B 細胞は、少数の体細胞変異を保有し、一貫して変化した受容体結合ドメイン (RBD) 標的特異性を示す低親和性 IgM 抗体を主に発現するという点で、対照個体の記憶 B 細胞とは異なりました。エピトープマスキングあり。 さらに、検査した77個の抗RBDメモリー抗体のうち、ウイルスを中和したのは1個だけでした。 これらの発見の根底にあるメカニズムは、同じ抗体の存在下で形成される胚中心が低親和性 B 細胞によって支配されることを示すマウスの実験で調べられました。 我々の結果は、既存の高親和性抗体が、次の 2 つの異なるメカニズムを通じて胚中心とメモリー B 細胞の選択にバイアスをかけていることを示しています。(1) B 細胞の活性化閾値を低下させ、それによって豊富な低親和性クローンが免疫応答に参加できるようにすることによって。 (2) 同族エピトープの直接マスキングによる。 これは、追加ワクチン接種によって誘発される記憶抗体の標的プロファイルの変化を部分的に説明する可能性がある9。

モノクローナル抗体の受動投与がヒトのワクチン接種に対するその後の体液性反応にどのような影響を与えるかを調べるために、我々は、SARS-CoV-2に対する2つの長時間作用型モノクローナル抗体の組み合わせを単回投与され、その後ワクチン接種を受けた18人の健康なボランティアのグループを研究した。 SARS-CoV-2 mRNAワクチンの2回投与（図1a）。 2 つの抗体 (C144-LS および C135-LS) は、SARS-CoV-2 スパイク (S) タンパク質の RBD 上のクラス 2 およびクラス 3 エピトープに高い親和性で結合します (解離定数 (Kd) = 18 nM および Kd = 6)。それぞれnM）、それぞれ2.55および2.98 ng ml-1の最大阻害濃度の半分（IC50）値でウイルスを中和します5、8。

a. 研究計画の概略図。マーカーは最初のワクチン投与時からの週数を示します。 mAb、モノクローナル抗体。 b、C135-LS (上、青) および C144-LS (下、赤) の血清レベルの経時的な変化を示します。 太い色の破線はモノクローナル抗体レシピエント (n = 18) の血清濃度の中央値を示し、細い黒い点線は個々の参加者を表します。 2 本の縦の実線は中央値を示し、灰色の影付き領域はモノクローナル抗体投与からワクチン接種までの時間の範囲を示します。 c〜f、モノクローナル抗体レシピエント（n = 18、緑色）および対照（n = 26、n = 26、青）。 それぞれの点は 1 人の個人を表します。 水平破線は、陰性対照として使用された健康な個人からのパンデミック前の血漿サンプルの結合活性の中央値を表します。 c、d、WT RBDに対するIgM (c) およびIgG (d) 結合力価。 e、R346S/E484K (左) および N440K/E484K RBD への IgG 結合 (拡張データ図 1)。 f、NTDに結合するIgG。 g–i、SARS-CoV-2 WT S でシュードタイプ化された HIV-1 に対するモノクローナル抗体レシピエント (n = 18、緑色) およびコントロール (n = 26、青色) の血漿最大半値中和力価 (NT50) 値 (g) 、R346S/Q493K 変異体 S (h) および R346S/N440K/E484K 変異体 S (i) (拡張データ図 2)。 g-i のシュードウイルスの S タンパク質には R683G 置換が含まれていました。 c-i の赤い水平バーと g-i の赤い数字は中央値を表します。 c-i の統計的有意性は、各時点でのモノクローナル抗体レシピエントとコントロールの差を個別に比較する両側マン・ホイットニー U 検定を使用して決定されました。 P 値はプロットの上に表示されます。 すべての実験は少なくとも二重に実行されました。

 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>