Since its outbreak, Covid-19 has been responsible for more than 6 000 000 deaths.¹ Cancer is one of the most important risk factors for severe disease and death; hematological malignancies (HMs), specifically, have been associated with an estimated mortality rate of 37%.²

At present, the anti-SARS-CoV-2 vaccination represents the most effective strategy to reduce the incidence and severity of Covid-19.

Here, we present the results of a prospective, cohort study aimed to evaluate the humoral and cell-mediated immune response and the clinical efficacy of anti-SARS-CoV-2 vaccination in adult patients with HMs.

The study included consecutive adult patients with HMs who had completed the first cycle of anti-SARS-CoV-2 vaccination. At enrollment, information was collected regarding patient demographics, HM characteristics, last HM therapy, anti-SARS-CoV-2 vaccination, and previous Covid-19. Active disease was defined as being at the time of diagnosis or having a progressive disease. Active treatment was defined as any ongoing therapy, or any therapy discontinued in the 6 months prior to vaccination, or stem cell transplant (SCT) performed within 3 months from vaccination. Humoral and cellular immunity testing was planned 4 weeks after the completion of the first vaccination cycle. Seronegative patients underwent additional serology testing 4 weeks after the administration of a booster dose of vaccine. During follow-up, all patients were monitored for SARS-CoV-2 breakthrough infections through telephone interviews, starting 7 days after the completion of the first cycle of vaccination. The DiaSorin's LIAISON® SARS-CoV-2 TrimericS IgG assay was used to test humoral immunity. The assay's range was increased from 4.81 - 2080 BAU/ml to 4.81–41 600 BAU/ml for a more accurate determination of Ab titer. Technical details of the assay are summarized in Supplementary Table 1. The anti-spike T-cell-mediated immune response was tested by quantifying spike-specific IFNγ-producing T-cells by enzyme-linked immunosorbent spot (ELISpot) assay and by characterizing different subpopulations of T-cells through multicolor fluorescence-activated cell sorting (FACS). Both analyses were performed ex vivo using whole thawed peripheral blood mononuclear cells (PBMC). To evaluate IFNγ ELISpot response, spot forming cells (SFC) were counted by a stereomicroscope and values were defined as positive if there was at least a two-fold increase from the negative value and above a threshold of 4 SFC per well. Flow cytometry analysis was used to differentiate T-cell subpopulations based on the expression of CD4+, CD8+, CD4 + IFNγ+, and CD8 + IFNγ+. Then, the following subsets of spike-specific CD4+ and CD8+ memory T-cells were characterized: effector memory T-cells (T_EM; CD45RA-CCR7−), effector memory T-cells re-expressing CD45RA (T_EMRA; CD45RA + CCR7−), central memory T-cells (T_CM; CD45RA-CCR7+). Each T-cell subpopulation was also characterized based on the production of either IFNγ or IL-17A. Cells were analyzed by using a BD FACS Fortessa x20 for T-cell subset analysis and for IFNγ and IL-17A production. Flow data were analyzed using the FlowJo v10 software (TreeStar).

All cases of SARS-CoV-2 breakthrough infection in our cohort were registered, with details regarding disease severity, duration, and outcome. We considered December 22, 2021, to be the cutoff date after which the SARS-CoV-2 variant of concern (VOC) Omicron became prevalent in Italy, based on the national epidemiological data.^{3, 4} To estimate the vaccination's efficacy in reducing the severity of Covid-19, we compared our results with those collected in a group of patients with HMs, treated at our Institution, who had been affected by Covid-19 during the pre-vaccination period of the pandemic. A detailed description of ELISpot assay, FACS and statistical analysis can be found in Supplemental Material S1. The study was approved by the Institutional Review Board (protocol No. 84–2021) and conducted in accordance with the Helsinki Declaration of 1975 as revised in 2013. All patients signed a written informed consent. The study was registered at ClinicalTrials.gov (identifier, NCT04878822).

Between April 14 and July 26, 2021, we enrolled 414 patients with HMs who had been administered anti-SARS-CoV-2 vaccination. At data cutoff (February 21, 2022), 365 patients vaccinated with the double-dose regimen of mRNA vaccines entered the analysis (Supplementary Figure 1). Patients' characteristics at the time of the first vaccination cycle are reported in Supplementary Table 2. Overall, 298 out of 365 patients (82%) developed a humoral immune response. Lower seroconversion rates were observed in patients receiving anti-CD20-based therapies (4%), BTK inhibitors (42%), JAK2 inhibitors (68%), and daratumumab-based therapies (69%). Detailed results of serology testing are reported in Supplementary Table 3 and Supplementary Figure 2. Multivariate analysis showed that diagnosis of lymphoma (RR 3.01, 95% CI 1.53–5.93; p = .0014), immunotherapy (RR 9.42, 95% CI 2.66–33.33; p = .0005), treatment with biologics (RR 4.05, 95% CI 1.29–12.71; p = .0166), having SCT as last treatment (RR 5.59, 95% CI 1.16–26.92; p = .0319), and being on active treatment (RR 8.09, 95% CI 2.93–22.31; p < .0001) were all significantly associated with negative serology testing (Supplementary Table 4). The overall GM titer of Abs in the subset of seropositive patients was 778 BAU/ml (95% CI 658–920). Supplementary Figure 3 shows that advancing age, active disease, and being on active treatment were significantly associated with a lower GM titer.

Between September 19 and October 15, 2021, 57 out of 67 patients who were seronegative after the first vaccination cycle received a booster dose. All patients were vaccinated with homologous booster vaccines (49 with BNT162b2 and 8 with mRNA-1273). Demographic and clinical characteristics of this subgroup are summarized in Supplementary Table 5.

The administration of a booster dose of vaccine led to seroconversion in 21 (37%).

Notably, in the subset of patients who seroconverted after the booster dose the GM titer of Abs was 258 BAU/ml (95% CI 141–473), significantly lower than the one we found after the first vaccination cycle (778 BAU/ml; p = .0009). Results of serology testing after booster vaccination are summarized in Supplementary Figure 4. The ELISpot analysis was performed on 107 patients; among those, 63 were seronegative and 44 were seropositive after double-dose vaccination. Results stratified by demographic and clinical characteristics are reported in Supplementary Table 6. The multivariate analysis showed active disease status to be significantly associated with a lower probability of a positive response to ELISpot assay (RR 0.39, 95% CI 0.19–0.80; p = .0103) (Supplementary Table 7). Among the 107 patients who underwent ELISpot analysis, FACS was also performed in a subgroup of 92 to characterize different subpopulations of T-cells. As summarized in Supplementary Figures 5–7, anti-SARS-CoV-2 vaccination stimulates the development of spike-specific memory T-cells across both seropositive and seronegative patients, although generally lower in the latter.

During a median follow-up of 269 days (min-max, 13–356), 29 patients (8%) developed a SARS-CoV-2 breakthrough infection, with a rate of 2.98 per 10 000 person-days (Figure 1A).

Anti-SARS-CoV-2 vaccination seems to be less effective in preventing infection by the VOC Omicron, as the incidence of breakthrough infections has risen from 1.17 per 10 000 person-days to 9.82 per 10 000 person-days after the spread of Omicron (p < .0001). Overall, infection occurred at a median time of 105 days (min-max, 26–257) from the last vaccine dose. This time did not differ significantly before and after the spread of Omicron (149 vs. 103 days; p = .9627). The majority of patients (26 out of 29) developed non-severe Covid-19. Seropositive status after vaccination was associated with a lower cumulative risk of breakthrough infection as compared to seronegative status (p < .0001) (Figure 1B). This observation was confirmed by a Cox model, which found a lower risk of post-vaccination Covid-19 in seropositive patients (HR 0.11, 95% CI 0.03–0.43; p = .0017). Conversely, cellular immunity evaluated by ELISpot did not appear to correlate with the risk of breakthrough infection. By comparing clinical characteristics of Covid-19 diagnosed prior to vaccination versus after vaccination in patients with HMs, we found that the rate of severe or critical disease (10% vs. 33%; p = .0242), the rate of hospitalization (17% vs. 50%; p = .0024), and the median duration of disease (16 days vs. 22 days; p = .0094) were all significantly lower in vaccinated patients. As shown in Supplementary Table 8, the two subgroups of patients affected by Covid-19 (pre-vaccination vs. post-vaccination) are similar in terms of demographic and clinical characteristics.

This study has some limitations, such as the absence of a baseline immune evaluation. However, this was balanced out by a careful anamnestic evaluation at enrollment, which led to the exclusion of 7% of all patients enrolled because of previous SARS-CoV-2 infection. This rate was comparable to the rate of local cumulative Covid-19 cases during the enrollment phase (10%).⁵ As previously reported,⁶ our study showed that anti-SARS-CoV-2 vaccination is associated with lower immunogenicity in patients with HMs. The seroconversion rate after full vaccination was 82%; the seroconversion rate in 57 seronegative patients who underwent booster vaccination was 37%. Patients undergoing active treatment, especially with anti-CD20 and anti-CD38 monoclonal antibodies, BTK inhibitors, and JAK2 inhibitors are at high risk of seroconversion failure. Cell-mediated immunity showed positive responses across seropositive and seronegative patients. The incidence of Covid-19 was 2.98 per 10 000 person-days, lower in seropositive patients. The rate of severe/critical Covid-19, of hospitalization, and the median duration of disease were all significantly lower in vaccinated patients as compared to non-vaccinated patients.

自爆发以来，Covid-19 已导致超过 600 万人死亡。¹癌症是导致严重疾病和死亡的最重要危险因素之一；具体而言，血液系统恶性肿瘤 (HMs) 与 37% 的估计死亡率有关。²

目前，抗 SARS-CoV-2 疫苗接种是降低 Covid-19 发病率和严重程度的最有效策略。

在这里，我们展示了一项前瞻性队列研究的结果，该研究旨在评估体液和细胞介导的免疫反应以及抗 SARS-CoV-2 疫苗接种在成年 HM 患者中的临床疗效。

该研究包括连续完成第一轮抗 SARS-CoV-2 疫苗接种的 HMs 成年患者。入组时，收集了有关患者人口统计数据、HM 特征、最后一次 HM 治疗、抗 SARS-CoV-2 疫苗接种和之前的 Covid-19 的信息。活动性疾病被定义为在诊断时或患有进行性疾病。积极治疗被定义为任何正在进行的治疗，或在接种疫苗前 6 个月内停止的任何治疗，或在接种疫苗后 3 个月内进行的干细胞移植 (SCT)。计划在第一个疫苗接种周期完成后 4 周进行体液和细胞免疫测试。血清阴性患者在接种加强剂量疫苗 4 周后接受了额外的血清学检测。随访期间，从完成第一个疫苗接种周期后的 7 天开始，通过电话采访对所有患者进行 SARS-CoV-2 突破性感染监测。DiaSorin 的 LIAISON® SARS-CoV-2 TrimericS IgG 测定用于测试体液免疫。检测范围从 4.81 - 2080 BAU/ml 增加到 4.81-41 600 BAU/ml，以更准确地测定 Ab 滴度。该测定的技术细节总结在补充表 1 中。通过酶联免疫吸附点 (ELISpot) 测定法量化尖峰特异性产生 IFNγ 的 T 细胞并通过表征不同的细胞来测试抗尖峰 T 细胞介导的免疫反应通过多色荧光激活细胞分选 (FACS) 筛选 T 细胞亚群。两种分析均使用解冻的全外周血单核细胞 (PBMC) 离体进行。为了评估 IFNγ ELISpot 反应，通过立体显微镜对斑点形成细胞 (SFC) 进行计数，如果从负值至少增加两倍且高于每孔 4 个 SFC 的阈值，则将值定义为阳性。流式细胞术分析用于根据 CD4+、CD8+、CD4+IFNγ+ 和 CD8+IFNγ+ 的表达来区分 T 细胞亚群。然后，对以下尖峰特异性 CD4+ 和 CD8+ 记忆 T 细胞亚群进行了表征：效应记忆 T 细胞（T 和 CD8 + IFNγ+。然后，对以下尖峰特异性 CD4+ 和 CD8+ 记忆 T 细胞亚群进行了表征：效应记忆 T 细胞（T 和 CD8 + IFNγ+。然后，对以下尖峰特异性 CD4+ 和 CD8+ 记忆 T 细胞亚群进行了表征：效应记忆 T 细胞（T_电磁; CD45RA-CCR7-)、重新表达 CD45RA 的效应记忆 T 细胞 (T _EMRA ; CD45RA + CCR7-)、中央记忆 T 细胞 (T _CM ; CD45RA-CCR7+)。每个 T 细胞亚群的特征还基于 IFNγ 或 IL-17A 的产生。通过使用 BD FACS Fortessa x20 分析细胞以进行 T 细胞亚群分析以及 IFNγ 和 IL-17A 的产生。使用 FlowJo v10 软件 (TreeStar) 分析流量数据。

我们队列中的所有 SARS-CoV-2 突破性感染病例均已登记，并提供了有关疾病严重程度、持续时间和结果的详细信息。根据国家流行病学数据，我们认为 2021 年 12 月 22 日是 SARS-CoV-2 关注变体 (VOC) Omicron 在意大利流行的截止日期。^3、4为了估计疫苗接种在降低 Covid-19 严重程度方面的功效，我们将我们的结果与在我们机构治疗的一组 HM 患者中收集的结果进行了比较，这些患者在疫苗接种前期间受到 Covid-19 的影响大流行。ELISpot 测定、FACS 和统计分析的详细描述可在补充材料 S1 中找到。该研究得到了机构审查委员会（第 84-2021 号协议）的批准，并根据 2013 年修订的 1975 年赫尔辛基宣言进行。所有患者都签署了书面知情同意书。该研究在 ClinicalTrials.gov 上注册（标识符，NCT04878822）。

在 2021 年 4 月 14 日至 7 月 26 日期间，我们招募了 414 名接受过抗 SARS-CoV-2 疫苗接种的 HM 患者。在数据截止时（2022 年 2 月 21 日），365 名接种了双剂量 mRNA 疫苗方案的患者进入分析（补充图 1）。补充表 2 报告了第一个疫苗接种周期时的患者特征。总体而言，365 名患者中有 298 名（82%）产生了体液免疫反应。在接受基于抗 CD20 的疗法 (4%)、BTK 抑制剂 (42%)、JAK2 抑制剂 (68%) 和基于 daratumumab 的疗法 (69%) 的患者中观察到较低的血清转化率。血清学检测的详细结果见补充表 3 和补充图 2。多变量分析显示淋巴瘤的诊断 (RR 3.01, 95% CI 1.53–5.93; p = .0014)，免疫治疗 (RR 9.42, 95% CI 2.66–33.33; p =  .0005)，生物制剂治疗 (RR 4.05, 95% CI 1.29–12.71; p =  .0166)，SCT 作为最后治疗 (RR 5.59 , 95% CI 1.16–26.92; p =  .0319) 和积极治疗 (RR 8.09, 95% CI 2.93–22.31; p  < .0001) 都与阴性血清学检测显着相关（补充表 4）。血清阳性患者亚组中 Abs 的总体 GM 滴度为 778 BAU/ml (95% CI 658–920)。补充图 3 显示，年龄增长、活动性疾病和积极治疗与较低的 GM 滴度显着相关。

在 2021 年 9 月 19 日至 10 月 15 日期间，在第一个疫苗接种周期后呈血清反应阴性的 67 名患者中，有 57 名接受了加强剂量。所有患者均接种了同源加强疫苗（49 人接种 BNT162b2，8 人接种 mRNA-1273）。该亚组的人口统计学和临床​​特征总结在补充表 5 中。

21 人 (37%) 接种了加强剂量的疫苗导致血清转化。

值得注意的是，在加强剂量后血清转化的患者亚组中，Abs 的 GM 滴度为 258 BAU/ml（95% CI 141-473），显着低于我们在第一个疫苗接种周期后发现的值（778 BAU/ml；p  = .0009）。补充图 4 总结了加强疫苗接种后的血清学检测结果。对 107 名患者进行了 ELISpot 分析。其中，63人血清学阴性，44人双次接种后血清学阳性。补充表 6 报告了按人口统计学和临床​​特征分层的结果。多变量分析显示，活动性疾病状态与 ELISpot 检测阳性反应的可能性较低显着相关（RR 0.39，95% CI 0.19-0.80；p = .0103)（补充表 7）。在接受 ELISpot 分析的 107 名患者中，还在 92 名亚组中进行了 FACS，以表征不同的 T 细胞亚群。正如补充图 5-7 中总结的那样，抗 SARS-CoV-2 疫苗接种刺激了血清阳性和血清阴性患者中尖峰特异性记忆 T 细胞的发育，尽管后者通常较低。

在 269 天（最小-最大，13-356）的中位随访期间，29 名患者（8%）发生 SARS-CoV-2 突破性感染，发病率为 2.98/10 000 人日（图 1A） .

抗 SARS-CoV-2 疫苗在预防 VOC Omicron 感染方面似乎不太有效，因为在 Omicron 传播后，突破性感染的发病率已从每 10 000 人日 1.17 例上升至每 10 000 人日 9.82 例( p  < .0001)。总体而言，从最后一剂疫苗开始，感染发生的中位时间为 105 天（最小-最大，26-257）。该时间在 Omicron 传播前后没有显着差异（149 天对 103 天；p  = .9627）。大多数患者（29 人中的 26 人）发展为非严重的 Covid-19。与血清阴性状态相比，疫苗接种后的血清阳性状态与较低的突破性感染累积风险相关（p < .0001)（图 1B）。Cox 模型证实了这一观察结果，该模型发现血清阳性患者接种疫苗后 Covid-19 的风险较低（HR 0.11，95% CI 0.03–0.43；p  = .0017）。相反，由 ELISpot 评估的细胞免疫似乎与突破性感染的风险无关。通过比较 HMs 患者接种疫苗前与疫苗接种后诊断的 Covid-19 的临床特征，我们发现重症或危重疾病的发生率（10% 对 33%；p  = .0242）、住院率（17 % 与 50%；p  = .0024）和中位病程（16 天与 22 天；p = .0094）在接种疫苗的患者中均显着降低。如补充表 8 所示，受 Covid-19 影响的两个患者亚组（接种前与接种后）在人口统计学和临床​​特征方面相似。

这项研究有一些局限性，例如缺乏基线免疫评估。然而，这被入组时仔细的回忆评估所抵消，这导致由于先前的 SARS-CoV-2 感染而排除了所有入组患者的 7%。这一比率与注册阶段本地累积 Covid-19 病例的比率 (10%) 相当。⁵如前所述，⁶我们的研究表明，抗 SARS-CoV-2 疫苗接种与 HM 患者较低的免疫原性有关。完全接种后的血清转化率为82%；57 名血清阴性患者接受加强疫苗接种后，血清转化率为 37%。接受积极治疗的患者，尤其是抗 CD20 和抗 CD38 单克隆抗体、BTK 抑制剂和 JAK2 抑制剂的患者血清转换失败的风险很高。细胞介导的免疫在血清阳性和血清阴性患者中显示出阳性反应。Covid-19 的发病率为 2.98/10 000 人日，血清阳性患者的发病率较低。与未接种疫苗的患者相比，接种疫苗的患者的重症/危重症 Covid-19、住院率和中位病程均显着降低。