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SARS-CoV-2 vaccine response and rate of breakthrough infection in patients with hematological disorders

Background The clinical efficacy of SARS-CoV-2 vaccines according to antibody response in immunosuppressed patients such as hematological patients has not yet been established. Patients and methods A prospective multicenter registry-based cohort study conducted from December 2020 to December 2021 by the Spanish transplant and cell therapy group was used to analyze the relationship of antibody response at 3–6 weeks after full vaccination (2 doses) with breakthrough SARS-CoV-2 infection in 1394 patients with hematological disorders. Results At a median follow-up of 165 days after complete immunization, 37 out of 1394 (2.6%) developed breakthrough SARS-CoV-2 infection at median of 77 days (range 7–195) after full vaccination. The incidence rate was 6.39 per 100 persons-year. Most patients were asymptomatic (19/37, 51.4%), whereas only 19% developed pneumonia. The mortality rate was 8%. Lack of detectable antibodies at 3–6 weeks after full vaccination was the only variable associated with breakthrough infection in multivariate logistic regression analysis (Odds Ratio 2.35, 95% confidence interval 1.2–4.6, p = 0.012). Median antibody titers were lower in cases than in non-cases [1.83 binding antibody units (BAU)/mL (range 0–4854.93) vs 730.81 BAU/mL (range 0–56,800), respectively (p = 0.007)]. We identified 250 BAU/mL as a cutoff above which incidence and severity of the infection were significantly lower. Conclusions Our study highlights the benefit of developing an antibody response in these highly immunosuppressed patients. Level of antibody titers at 3 to 6 weeks after 2-dose vaccination links with protection against both breakthrough infection and severe disease for non-Omicron SARS-CoV-2 variants. Supplementary Information The online version contains supplementary material available at 10.1186/s13045-022-01275-7.

The current study analyzes the clinical benefit of full SARS-CoV-2 vaccination (2 doses) through an observational registry conducted by the Spanish Hematopoietic Stem Cell Transplantation and Cell Therapy Group (GETH-TC) aimed at monitoring the response to SARS-CoV-2 vaccine and the breakthrough SARS-CoV-2 infections over time in a large cohort of 1394 patients with hematological disorders.

Study population
This is a prospective multicenter registry-based cohort study conducted by the Infectious Complications Subcommittee (GRUCINI) of the GETH-TC in collaboration with the Spanish Society of Hematology and Hemotherapy (SEHH). Details of this registry have been previously described elsewhere [20,21]. In brief, the registry included consecutive adult patients with a prior history of hematological disorders who were fully vaccinated against SARS-CoV-2 between December 30, 2020, and June 30, 2021, in 21 participating Spanish centers. Patients were followed and monitored for development of SARS-CoV-2-reactive IgG antibodies (SCoV2-R-A) and breakthrough SARS-CoV-2 infection at different time points (3-6 weeks, 3, 6 and 12 months) after the complete vaccination schedule (defined as two vaccine doses). The status of all included patients was updated on December 1, 2021. All patients included in this registry gave their signed informed consent in accordance with the declaration of Helsinki. The local research ethical committee of the Hospital Clínico Universitario of Valencia approved the registry and study protocol (reference code 35.21).

Inclusion criteria and cohort selection
As of December 1, 2021, the GETH-TC registry included 1683 patients with different hematological disorders who had been fully vaccinated against COVID-19. With the aim of assessing the risk of breakthrough SARS-CoV-2 infection according to antibody detection at 3-6 weeks after the second vaccine dose, the current study focused on patients with available serological testing 3 to 6 weeks after the second vaccine dose. We excluded 289 patients from 3 centers, initially included with limited data (only filiation data), that did not obtain the institutional approval for serological testing and 1394 hematological patients were included in the final study analysis.

Technical considerations and definitions
Antibody detection or seropositivity was defined when SARS-CoV-2-reactive IgG antibodies (SCoV2-R-A) were detected at any level above the lower limit of detection level for each tests used. We assessed seropositivity using serological ELISA or chemiluminescence immunoassay following manufacturer instructions according to their availability at the microbiology services of each participating center. As recommended by the SEHH, in vaccinated individuals serological testing included the detection of IgG against both the nucleocapsid (N) and surface (S) proteins (anti-N and anti-S IgG, respectively) [7]. Of the 1394 patients included, 1244 (89%) received quantitative assessment, whereas the remainder was assessed through qualitative testing. Antibody levels were normalized according to the WHO standard, and results were reported as SCoV2-R-A binding antibody units per milliliter (BAU/mL). Additional file 1: Table S1 summarizes the technical characteristics of the serological tests used and normalization of antibody titers to BAU/mL according to WHO standards.
Pre-vaccination SARS-CoV-2 infection was defined as patients with prior history of PCR-proven COVID-19 and/or positive SARS-CoV-2 serostatus (IgG and/or IgM) before the first vaccine dose.
Patients with respiratory symptoms underwent PCR screening for the development of SARS-CoV-2 according to the treating physician criteria. Breakthrough SARS-CoV-2 infection was defined as molecular (PCR test) or humoral (anti-N seroconversion between two  Table S2.

Endpoints and statistical analysis
The primary objective of the study was to assess the occurrence of breakthrough SARS-CoV-2 infection and its correlation with qualitative and quantitative humoral response at 3-6 weeks after full COVID-19 vaccination.
We also analyzed the effect of different cutoff values of quantitative SCoV2-R-A titers on development and severity of breakthrough SARS-CoV-2 infection. The main patient characteristics were reported by descriptive statistics on the total available information: Medians and ranges were used for continuous variables, while absolute and percentage frequencies were used for categorical variables. For comparisons, Fisher exact test or Mann-Whitney's U test was used when appropriate.
Univariate and multivariate analyses were tested using logistic regression models. Variables with a p value ≤ 0.1 in the univariate model were included in the multivariate analysis. A p value < 0.05 was considered statistically significant. All p values are two-sided. A median test subanalysis to check the protective effect of the amount of SCoV2-R-A was carried out in patients with available quantitative SCoV2-R-A titers normalized to BAU/mL. All analyses were performed using the statistical software SPSS v. 25(IBM SPSS Statistics, Armonk, New York, USA).
Overall, the SCoV2-R-A detection rate at 3-6 weeks after the complete vaccination was 78.2%. Among those with quantitative antibody testing, the median SCoV2-R-A titer was 720.26 BAU/mL (range 0-58,600). We compared SCoV2-R-A titers at 3-6 weeks after full vaccination in patients with and without SARS-CoV-2 infection prior to vaccination (excluding 7 patients with breakthrough SARS-CoV-2 infection after the second vaccine dose and before the first serological testing) and found higher titers in those with (median 2550 BAU/mL, range 0-10,400) vs those without (median 493.6 BAU/ mL, range 0-6338.6) (p < 0.0001) infection (Fig. 1).

Breakthrough SARS-CoV-2 infection
We identified 37 patients (2.6%) with breakthrough SARS-CoV-2 infection at median of 77 days (range 7-195) after the second vaccine dose. The overall incidence rate of breakthrough SARS-CoV-2 infection was 6.39 per 100 persons-year. The main clinical and SARS-CoV-2 breakthrough infection characteristics are detailed in Table 2. Most patients were diagnosed through molecular PCR testing (n = 22, 60%), whereas those remaining (n = 15, 40%) were diagnosed by seroconversion of anti-N between two consecutive serological tests. Seven patients (19%) developed SARS-CoV-2 infection between the second vaccine dose and SCoV2-R-A testing at 3-6 weeks, with an incidence rate of 6.14 per 100 persons-year. Twelve patients (32%) developed the infection between 3-6 weeks and 3 months after the complete vaccination with an estimated incidence rate of 6.9 per 100 persons-year. Finally, 18 patients had the infection between 3 and 7 months after the complete vaccination with an estimated incidence rate of 14.09 per 100 persons-year. Eighteen patients had COVID-19 (48.6%), whereas 19 (51.4%) were asymptomatic. SARS-CoV-2 was detected by PCR in 4 asymptomatic patients during screening performed before hospital admission for scheduled procedures/treatments. Pneumonia was documented in 7 cases (19%), whereas the SARS-CoV-2 infection-related hospital admission rate was 32% (n = 12). There were 3 COVID-19-related deaths (8%) at a median of 26 days (range 7-67) after SARS-CoV-2 detection.

Risk factors (including antibody level titers) for breakthrough SARS-CoV-2 infection
After excluding 7 patients with breakthrough SARS-CoV-2 infection before the first serological testing, we performed a univariate and multivariate regression model to assess predictors of breakthrough SARS-CoV-2 infection ( Table 3). None of the 109 patients who developed COVID-19 before vaccination developed breakthrough infection (p = 0.1 in univariate analysis). The only variable that was found to have an impact on the risk of breakthrough SARS-CoV-2 infection in univariate and multivariate analyses was absence of SCoV2-R-A Regarding the impact of the magnitude of SCoV2-R-A load, we restricted the analysis to 27 patients, since 7 cases became infected before the first antibody determination after vaccination and another 3 cases only had qualitative SCoV2-R-A testing. Overall, 1234 patients were evaluable for antibody levels normalized as BAU/ mL. The median SCoV2-R-A levels at 3-6 weeks after the full vaccination were significantly lower in the 27 patients with breakthrough SARS-CoV-2 infection as compared to those without [1.83 BAU/mL (range    (Fig. 2). SCoV2-R-A levels were classified as "low" (< 250 BAU/mL) in 501 patients (41%) and as "high" (> 250 BAU/mL) in 542 (44%) or "very high" (> 4900 BAU/mL) in 191 (15%) cases.
Although the median SCoV2-R-A levels were higher in asymptomatic breakthrough SARS-CoV-2 infection (n = 13) than in those with COVID-19 (n = 14), the difference was not statistically significant [792.57 BAU/ mL (range 0-2550) vs 0 BAU/mL (range 0-4854.93), p = 0.44]. Additionally, median SCoV2-R-A levels were lower, although not statistically significant, in those requiring hospital admission vs those who did not [0 BAU/mL (range 0-195.15) vs 309.57 BAU/mL (range 0-4854.93), respectively, p = 0.2]. However, breakthrough SARS-CoV-2 infection was more common in the group with "low" SCoV2-R-A levels, as was COVID-19, pneumonia, hospital admission and oxygen requirement (p ≤ 0.05 for all comparisons) ( Table 4). Breakthrough SARS-CoV-2 infection severity did not show significant differences according to the timing after the second vaccine dose (after 2nd dose and first serological testing vs the first testing and 3rd months vs after the 3rd months).

Discussion
The current study highlights the influence of qualitative and quantitative humoral response monitoring early after full SARS-CoV-2 vaccination in predicting the risk of breakthrough SARS-CoV-2 infection in hematological patients. Patients lacking SCoV2-R-A at 3-6 weeks after vaccination had an increased risk of breakthrough SARS-CoV-2 infection. In addition, higher levels of SCoV2-R-A early after complete vaccination were linked to a lower risk of breakthrough SARS-CoV-2 infection and lower disease severity.
Hematological patients are historically characterized by a low humoral response rate with any vaccinepreventable disease [22,23]. However, development of mRNA vaccines during the SARS-CoV-2 pandemic has overcome the poor serological response rates in this particular population (> 70%) [14,19,20,24,25]. The median 720.26 BAU/mL found in our cohort was similar to the results reported in other large series of patients with diverse hematological conditions (median values < 1 × 10 3 BAU/mL) and significantly lower than those found in healthy individuals (> 1 × 10 3 BAU/m) [26]. Although the clinical benefit of mounting a serological response is currently lacking in this scenario, to the best of our knowledge this is the first report providing evidence of its link with clinical efficacy. Notably, although prior SARS-CoV-2 infection was not significantly associated with lower risk of breakthrough SARS-CoV-2 infection in our series, none of these patients were infected after vaccination. We previously reported a higher rate of detectable SCoV2-R-A in this patient subset [21], whereas in the current study we were able to demonstrate higher SCoV2-R-A titers compared to SARS-CoV-2-naïve patients. It is likely that the higher antibody titers along with natural immunity may confer strong protection in these patients. The design of our registry (prospective longitudinal with several SCoV2-R-A determinations over time) enabled us to capture occurring breakthrough SARS-COV-2 infections through PCR screening in symptomatic patients or in asymptomatic patients before planned treatments but also by monitoring anti-N seroconversion in asymptomatic patients at different pre-specified time points. SARS-CoV-2 infection in hematological patients mirrored national epidemiological data [1,27]. The monitoring period during which our study was conducted (mostly
Although hematological malignancies showed a higher risk of breakthrough infection compared to solid tumors [29], the rate observed in our cohort (2.6%) was similar to other series with comparable median follow-ups which included hematological patients (2.3%) [26] or health care workers (2.3%) [30]. However, we observed that the risk of breakthrough infection increased with longer follow-up. This fact could suggest either that a decrease in antibody titters over time may reduce protection (which formed the rationale behind boosters), or that the risk of being infected increases with longer exposure time in an ongoing pandemic. The protective threshold levels of anti-SARS-CoV-2 antibodies below which the humoral defense against different SARS-CoV-2 variants is suboptimal have not yet been established. However, both binding and neutralizing antibodies are thought to be potential correlates of protection against COVID-19 and are correlated with each other [31,32]. In fact, recent data in the general population suggest that higher levels of binding and neutralizing antibodies after the second dose correlate with a reduced risk of symptomatic infection [33,34]. In line with these observations, our findings support that SCoV2-R-A titration early after vaccination could be an accurate strategy to predict breakthrough infection risk and could be useful in counseling additional vaccine doses or anti-SARS-CoV-2 monoclonal antibodies in this immunosuppressed population. This assumption is supported by the fact that patients without detectable antibodies had higher risk of breakthrough infection, patients with breakthrough infection had lower SCoV2-R-A titers and those with higher antibody titers showed lower disease severity.
A vaccine efficacy of 80% against symptomatic infection in the general population was achieved with 264 BAU/ ml [33]. In the COVE trial, this threshold shows > 90% vaccine efficacy [34]. Based on these data, we empirically selected the cutoff of 250 BAU/mL to segregate our cohort into poor and good responders. The cutoff of 250 BAU/mL in our series predicted risk of breakthrough infection and disease severity. No patients with SCoV2-R-A > 250 BAU/mL were admitted to hospital due to SARS-CoV-2 in our cohort. Nevertheless, the kinetics wane of SCoV2-R-A waning after vaccination in this scenario remains to be determined in order to establish the best time points for serological monitoring and the optimal moment to administer further vaccine doses.
Finally, we observed an important reduction in COVID-19 severity, exemplified by reduced overall pneumonia (19%), symptomatic SARS-CoV-2 infection (48.6%) and mortality (8%) rates. Rates of these outcomes in hematological patients with COVID-19 during the first and second COVID-19 waves were as high as > 70%, > 90% and > 25%, respectively [1]. Our findings are in line with real world data, where both mRNA vaccines were shown to reduce symptomatic SARS-CoV-2 infection, COVID-19-related symptoms, hospital admissions and mortality in adults [35][36][37][38]. Although the severity of COVID-19 seems lower after vaccination, severely immunosuppressed patients still develop life-threatening disease. In these vulnerable patients, preventive transmission measures (hand washing, social distancing, wearing mask, etc.) are still highly recommended. However, the largest reduction in the incidence of respiratory virus infections in immunocompromised patients has been observed when preventive transmission measures have been instituted globally [39] as compared to when such measures are applied only to immunosuppressed patients and their caregivers; in fact, in these latter scenarios the time of onset and incidence of different respiratory virus infections (including SARS-CoV-2) in HSCT recipients strongly mirror those in the patients' communities [1,19].
The limitations of this study comprise the use of different serological tests, absence of neutralizing antibody testing, absence of cellular immune response analyses and the lack of molecular data regarding the SARS-CoV-2 variants in patients with breakthrough infections. However, most SARS-CoV-2 infections reported in our cohort occurred when the Delta variant was dominant in Spain. The performance of antibody titration with the omicron variant remains to be evaluated.

Conclusion
We provide evidence that serological monitoring after SARS-CoV-2 vaccination could be useful in identifying hematological patients at high risk of breakthrough SARS-CoV-2 infection. SCoV2-R-A levels link with protection in this vulnerable population being 250 BAU/  Lymphocyte count < 1.0 × 10 9 /L 1.5 (0.7-3.3) 0.27 to the invaluable microbiology services for their commitment in SARS-CoV-2-reactive IgG antibody monitoring in these highly immunosuppressed patients from all participating centers, in particular to Santiago Muñoz Criado from Microbiological Division of Hospital Clínico Universitario of Salamanca and to Tamar Talaván from microbiological Division of Hospital Infanta Leonor of Madrid. Special thanks to all hematology units from participating centers for their commitment in the current study. Finally, we also want to thank patients, nurses and study coordinators for their foremost contributions in this study.
Author contributions JLP, RM, DN and AC were responsible for the conception and the design of the study. JLP, RM, PR-B and DN performed the data analysis and generated the