At multivariate analysis, only treatment with MMF  and active neoplasia  were independent predictors of seroconversion failure. These findings suggest that MMF dose reduction or suspension may be required to optimize vaccine response in these patients.Vaccines are the most effective means to prevent the potentially deadly effects of SARS-CoV-2 infection, but not all vaccinated individuals gain the same degree of protection. Patients undergoing chronic immunosuppressive therapy due to autoimmune diseases or liver transplants, for example, may show impaired anti-SARS-CoV-2 antibody response after vaccination. We performed a prospective observational study with parallel arms, aiming to (a) evaluate seroconversion after anti-SARS-CoV-2 mRNA vaccine administration in different subgroups of patients receiving immunosuppressive treatment for rheumatological or autoimmune diseases or to prevent organ rejection after liver transplantation and (b) identify negative predictors of IgG anti-SARS-CoV-2 development. Out of 437 eligible patients, 183 individuals were enrolled at the Rheumatology and Hepatology Tertiary Units of “Maggiore della Carità” University Hospital in Novara: of those, 52 were healthy subjects, while among the remaining 131 patients, 30 had a diagnosis of spondyloarthritis, 25 had autoimmune hepatitis, 10 were liver transplantation recipients, 23 suffered from connective tissue diseases (including 10 cases that overlapped with other diseases), 40 were treated for rheumatoid arthritis, and 5 had vasculitis. Moreover, all patients were receiving chronic immunosuppressive therapy. The immunogenicity of mRNA COVID-19 vaccines was evaluated by measuring IgG anti-SARS-CoV-2 antibody titers before vaccination and after 10, 30, and 90 days since the first dose administration. Of the selected cohort of patients, 24.0% did not develop any detectable anti-SARS-CoV-2 IgG after a complete mRNA-based two doses primary vaccination cycle. At univariate analysis, independent predictors of an absent antibody response to vaccine were a history of liver transplantation (OR 11.5, 95% CI 2.5–53.7,  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a coronavirus responsible for coronavirus disease 2019 (COVID-19), a very heterogeneous disease ranging from a nearly asymptomatic state to a life-threatening condition with high in-hospital mortality, with the highest toll among the elderly or those with chronic diseases and cancer ,4. COVIDSince 2020, more than 50 vaccines against COVID-19 have been developed and tested in phase II and III trials ,9,10,11.Patients affected by chronic immune-mediated inflammatory diseases have high rates of morbidity and mortality due to infections that rely on immunodeficiency, related to either the autoimmune diseases themselves or to the immunosuppressive drugs used to treat these illnesses . Thus, tTo date, few data are available on the efficacy of anti-COVID-19 vaccination in patients with inflammatory autoimmune diseases or in transplanted patients receiving immunosuppressive treatments. To fill this gap, we performed a prospective observational study to assess seroconversion rates after vaccine administration in different subgroups of patients undergoing chronic immunosuppressive treatment due to autoimmune diseases or liver transplantation.We performed a prospective observational study (acronym RIVALSA) with parallel arms including healthy subjects and autoimmune/liver-transplanted patients receiving immunosuppressive drugs with planned COVID-19 vaccination between March and July 2021. Inclusion and exclusion criteria are reported in The study protocol was approved by the local ethical committee (CE 72/21), and the study was conducted in strict accordance with the Declaration of Helsinki. Among all the patients attending either the Rheumatology or Hepatology Unit of “Maggiore della Carità” University Hospital in Novara , eligible subjects (patients receiving immunosuppressive drugs to treat autoimmune diseases or to prevent organ rejection after liver transplantation) were asked to participate in the study; from the 437 subjects screened, 131 patients were included in the study together with 52 healthy subjects selected among the healthcare professionals of these units and/or their relatives. (1)Assessment of the IgG anti-SARS-CoV-2 titer in response to vaccination in patients who completed the primary vaccination cycle receiving immunosuppressive therapy due to inflammatory autoimmune diseases or organ rejection prevention after liver transplantation and proportion of the failure of IgG seroconversion relative to healthy subjects in patients who completed the primary vaccination cycle, 90 days after the first vaccine dose;(2)Identification of predictors of the absence of an IgG anti-SARS-CoV-2 seroconversion 90 days after the first vaccine dose in patients who completed the primary vaccination cycle.The predefined endpoints of the cohort study were as follows:All individuals enrolled in the study underwent blood sampling before anti-SARS-CoV-2 vaccination (t0) and after 10, 30, and 90 days from the first vaccination dose . Blood samples were collected using EDTA as an anticoagulant, and blood fractions were immediately separated via centrifugation and stored at −80 °C until the time of analysis.IgG anti-spike protein antibodies were determined by performing an ELISA test, using a CE-IVD commercial kit (EUROIMMUN Medizinische Labordiagnostika AG Anti-SARS-CoV-2 QuantiVac ELISA (IgG), Lübeck, Germany) ,26, foll2 test or Fisher’s exact test when appropriate, while continuous variables were compared with the Mann-Whitney U test. Odds ratios (ORs) with confidence intervals (CIs) were calculated at univariate analysis. A set of variables associated with seroconversion failure at univariate analysis were used to build multivariate stepwise logistic regression models. The statistical significance threshold was set at 0.05 (two-tailed). Statistical analyses were performed with Statistica for Windows release 12  and MedCalc® Statistical Software version 20.014 .The relevant clinical and experimental data of each subject  were collected from clinical records or during an ad hoc interview and stored in a dedicated database (REDCap). Categorical variables were expressed as frequencies (percentage), while the measures of central tendency and dispersion of continuous variables were, respectively, expressed as medians and interquartile range (IQR). Categorical variables were compared with Pearson’s χAmong the 131 patients, 69.5% were women with a median age of 58 years (IQR 49–67). Healthy controls had a median age of 32 years (IQR 29–45) and were mainly women (65.4%). Most of them were healthcare professionals and had no comorbidities.The baseline population features (patients and controls) are shown in Comparing the two groups, we observed a significant difference in the median antibody titers between healthy subjects and patients at 10, 30, and 90 days since the first vaccine dose .This difference was also confirmed after the exclusion of those subjects with baseline antibody positivity for any time with the exception of the assessment at 90 days after vaccination .p = 0.0010).Interestingly, 50 out of the 50 healthy individuals with complete follow-up (100%) developed anti-SARS-CoV-2 IgG antibodies; by contrast, only 100 out of 119 patients with complete follow-up (84.0%) showed a detectable antibody response to vaccination at 90 days  was built. Treatment with MMF and active cancer as comorbidity were independent predictors of the absence of vaccine response in this model  nor with b-DMARDs  was associated with the absence of IgG anti-spike antibody detection. These results are even more interesting when compared with the existing literature about DMARDs and immune response to vaccines. Indeed, previous studies assessing antirheumatic drug effects on vaccine immunogenicity were mainly focused on seasonal influenza and pneumococcal vaccines. These showed reduced humoral responses in methotrexate-, abatacept-, and rituximab-treated patients and a minimal or absent response in patients treated with anti-cytokine drugs. Such data thus raise the question about the effectiveness of anti-SARS-CoV-2 vaccination in DMARD-treated patients and foster new studies aimed to evaluate immunomodulatory drug effect on COVID-19 vaccination in rheumatologic patients ,33,34,35+ T cells in a third of patients treated with methotrexate [With the aim to evaluate the immunogenicity of a single dose BNT162b2 mRNA vaccine, Bugatti et al., studied 140 chronic inflammatory arthritis patients and observed that anti-cytokine drugs had a low impact on vaccination (>80% response), while the treatment with methotrexate and/or glucocorticoids had a greater effect in reducing the IgG seroconversion, even after methotrexate withholding before and after vaccination . In a laotrexate . On the otrexate , a resulInterestingly, studying a cohort of 404 rheumatic patients, Ruddy et al., showed that anti-TNF-α therapy did not influence antibody response (100%), while only 82% of patients receiving glucocorticoids and 73% receiving MMF had IgG seroconversion . In our Notably, MMF and calcineurin inhibitors impaired immunogenicity to vaccination in our population as well. Indeed, the present study provided evidence of a perdurable absence of antibody response up to 3 months after the first dose administration and the following second one, suggesting a definitive absence of seroconversion. Such interesting results are in agreement not only with those of Furer et al., on autoimmune inflammatory rheumatic patients  but alsoIn univariate analysis, being affected by SLE, undergoing liver transplantation, and being treated with MMF or calcineurin inhibitors were all predictors of the lack of response to vaccination. When all of these predictors were analyzed together in a comprehensive multivariate model, only MMF treatment remained associated with an increased risk of vaccination failure. Such observation is of great interest, as MMF is a common treatment used in either SLE or liver transplantation and may account for the principal lack of immunogenicity observed in these patients. The observed impaired humoral response to mRNA-based vaccines in patients treated with MMF and calcineurin inhibitors could be explained by their pharmacological mechanism of action. Both classes of drugs, in fact, are known to impair T-cell proliferation and activation processes, especially by suppressing follicular T helper cells, thus reducing B-cell-mediated antibody production ,46,47,48As an additional point of interest, we observed that the concomitant diagnosis of neoplasia was a negative predictor of antibody response in all the analyses. This result is not surprising if we consider that, due to their therapeutic regimen, these patients show an increased risk to develop severe COVID-19 manifestations. To date, oncological patients have been generally excluded from anti-SARS-CoV-2 efficacy clinical trials, and studies assessing vaccine immunogenicity in these individuals are still few. The emerging literature on this topic highlights that seroconversion in oncological patients is significantly reduced compared with a healthy population ,50,51.Recently, Mitchell et al.  observedDue to the ongoing pandemic situation, and according to recent lines of evidence highlighting that vaccine efficacy decreases over time ,54, manyAs the pandemic situation evolved, different SARS-CoV-2 variants emerged, acquiring new mutations that could account for immune escape properties. Such lines of evidence foster studies aimed to investigate the ability of vaccine-elicited immunity to neutralize these new threats. According to the “Istituto Superiore di Sanità” , at the time of the present study, the prevalent variant in Italy was the lineage B.1.1.7 (Alpha variant). Different research groups investigated the ability of post-immunization sera to neutralize circulating variants, highlighting that the Alpha variant is unlikely to escape neutralization through both BNT162b2- and mRNA-1273-elicited antibodies and to increase reinfection risk ,60,61,62The present study has some limitations. First of all, the sample size was relatively small, and the population of patients was heterogeneous in terms of diseases, limiting the possibility to identify predictors of vaccination failure with smaller effects than those evidenced due to eventual biases. Additionally, control cohort heterogeneity could, to some extent, limit the possibility to elaborate conclusions stronger than those proposed. Furthermore, we focused only on people receiving mRNA-based vaccines, as only few of our patients received adenoviral vaccines, thus precluding the evaluation of this kind of vaccine immunogenicity. In addition, the chosen assay detected only IgG and not IgM or IgA against SARS-CoV-2 spike protein. Therefore, we cannot exclude that other antibodies targeted to other viral antigens may be present. Moreover, we did not evaluate T-cell-mediated response; thus, a deeper investigation of the cell-mediated response to viral infection would be necessary to better elucidate the role of MMF in vaccination failure. Due to the local COVID-19 infection recording protocol, not establishing a routine identification of viral variants, it was not possible to draw any conclusion about the possible role of the timely prevalent variants of concern in determining vaccination failure. Furthermore, the adopted study setting and the healthcare data availability did not allow a deeper investigation of vaccine efficacy in protecting against symptomatic infection. Finally, the monocentric design of the study may have led to some bias due to the clinical practice of our center.In this prospective, observational study, we showed that a limited but relevant proportion of patients receiving immunosuppressive therapy for inflammatory autoimmune diseases or prevention of solid organ rejection after liver transplantation did not develop detectable anti-SARS-CoV-2 IgG three months after a complete, two-dose mRNA vaccine schedule. The principal predictors of the lack of seroconversion were ongoing treatment with MMF and the compresence of active neoplasia as comorbidity. Individuals under treatment with calcineurin inhibitors or those affected by SLE or undergoing liver transplantation also had a high risk of a failure of vaccine response, but the risk may be biased by concomitant MMF treatment. Our study suggests that MMF treatment modification, its temporary suspension, or a different vaccine regimen may be required in these populations to enhance immune responses.


Patients with end-stage renal disease (ESRD) requiring hemodialysis (HD) constitute a high-risk group in terms of susceptibility to and severity of infection. High mortality rates have been observed in this population group with SARS-CoV-2 infection, and thus strategies to halt viral transmission are deemed crucial. Vaccination with an mRNA-based vaccine has been shown to decrease the risk of severe disease and substantially reduce mortality, both in healthy individuals and ESRD patients. The cellular and humoral immune responses elicited in HD patients, however, are diminished when compared to healthy controls, with the waning immunity that ensues placing them at high risk for reinfection. Several variables have been found to be associated with this suboptimal response to vaccination, with immunosuppression, older age, and underlying comorbidities playing a central role. Lately, the effects of booster dose administration have been studied in HD patients, with results indicating an enhanced immune response capable of inducing a protective immune profile in this group.The outbreak of SARS-CoV-2 has raised considerable concern about the detrimental effects it can induce in public health, with the interest of the scientific community being focused on the development of preventive and therapeutic approaches. Patients with end-stage renal disease (ESRD) are amongst vulnerable populations for critical illness owing to the presence of other comorbidities, their defective immune system, and their inability of self-isolation. To date, vaccination constitutes the most promising method to manage viral dispersion. Therefore, it is particularly important to investigate the effectiveness of available vaccines against SARS-CoV-2 in this risk group. Here, we summarize initial experience regarding the humoral and cellular immune responses elicited in dialysis patients after completion of the recommended vaccination regimen, as well as after booster dose administration, with one of the two mRNA vaccines, namely, BNT162b2 and mRNA-1273. In conclusion, a significantly diminished and delayed immune pattern was observed in ESRD patients compared to healthy population, with a peak in antibody titers occurring 3–5 weeks after the second dose. A booster dose significantly augmented the immune response in dialysis patients with either mRNA-based vaccine. Variables adversely correlating with the weak immunogenicity observed in dialysis patients include immunosuppressive therapy, older age, comorbidities, longer time in hemodialysis treatment, and higher body mass index. On the contrary, previous COVID-19 infection and administration of the mRNA-1273 vaccine are deemed to induce a more favorable immune response. Further investigation is needed to thoroughly understand the efficacy of mRNA-based vaccines in hemodialysis patients and define predictive factors that can influence it. The COVID-19 pandemic poses a considerable threat to public health , with caHowever, severe concerns have been raised about the immunization status and safety of dialysis patients after vaccination due to their distinct clinical condition . End staThe aim of this review is to investigate the cellular and humoral immune response patterns induced in dialysis patients after administration of the BNT162b2 or mRNA-1273 vaccines.The adequate laboratory examination of humoral response levels plays a highly significant role in assessing vaccination efficacy. Serological protocols relying on the quantification of specific anti-viral antibodies are considered as reliable biomarkers . As immuOverall, case-control studies have demonstrated a defect in the humoral immunity elicited by HDPs compared to healthy participants after two doses of the mRNA vaccine, regardless of the diagnostic test used ,22,23,24In conclusion, evidence from analyzed vaccine studies evaluating both the cellular and humoral responses indicates a significantly lower and delayed humoral response in HDPs after vaccination, compared to healthy individuals, that reached a peak in antibody titers 3–5 weeks after the second dose. Markedly impaired anti-BNT162b2 humoral responses were also observed by case-control studies evaluating only the antibody status of vaccinated patients. More specifically, according to Rincon-Arevalo et al. , a delayThe suboptimal humoral response after a two-dose vaccine regimen and the need for continuous and robust antibody responses in dialysis patients necessitates modifications and/or enhancements to the primary vaccination regimen. Although vaccination with two doses of an mRNA-based vaccine prevents severe SARS-CoV-2 infection to a significant extent in dialysis patients, the waning humoral response that ensues about 4 months after the last dose increases the risk of reinfection without excluding the possibility of severe disease . A boostAlthough antibodies are considered to play a significant preventive role against COVID-19 infection, T-cell effects also complement this protective purpose, with the BNT162b2 vaccine eliciting robust CD8+ and CD4+ RBD-specific T-cell responses . A promip < 0.0001). In addition to IFNγ responses, interleukin (IL)-2 levels also increased between the second and the third vaccine dose. Consistent findings were demonstrated by Strengert et al. [In line with humoral immunogenicity, the T-cell-mediated IFNγ-response was significantly diminished in HDPs after completion of the recommended vaccination regimen, regardless of the days elapsed from the last injection. Schrezenmeier et al.  and Stumt et al. , althougt et al. . Nonethet et al.  recomment et al.  suggestet et al. . Inconsit et al. . Duni ett et al.  also stuGenerally, a considerable lack of information on cellular immunity in HDPs following completion of any type of available COVID-19 vaccine scheme was identified in the literature, highlighting the urgent need for further investigation. p = 0.96). Additionally, there was a statistically significant decline in the T-cell response between 3 weeks and 3 months after the third dose (p < 0.001). This observation needs to be interpreted cautiously, since it may reflect normal T-cell physiology rather than failure of the third dose to achieve a sustained cellular response. Becker et al. [p = 0.564). When comparing the T-cell response after the third and after the fourth dose, however, a statistically significant difference was observed (p < 0.0005). The cellular response after a booster vaccine dose has been less extensively analyzed compared to the vaccine-elicited humoral response, but results of associated studies provide insight into the potential protective role of T-cell-mediated immune responses against SARS-CoV-2 infection among dialysis patients. Melin et al.  analyzedr et al.  assessedThe results of the above studies highlight the need to conduct more research regarding the effect of booster dose administration on the T-cell response, since published results, although promising, are scarce, and thus definite conclusions are hard to make. Among the factors associated with poor immune responses after vaccination completion in HDPs, the presence of an immunosuppressive regimen was the most reported ,23,24,55Other predictors of the observed weak immunogenicity among dialysis patients are associated with the clinical characteristics of the analyzed population. Patients with kidney failure are characterized by decreased lymphocyte count, systemic inflammation, malnutrition, and a defective clearance of uremic toxins, with all these parameters suggested to affect vaccination outcomes . The ureHowever, reviewed studies indicate that increasing the viral exposure of this vulnerable cohort may circumvent their immune dysfunctions, as previous COVID-19 infection was associated with favorable antibody responses ,21. In aAn optimal approach to strengthen the immune response to SARS-CoV-2 vaccination in dialysis patients requires adherence to a vaccination regimen. This could be accomplished via a meaningful interaction between the healthcare provider and the patient and should aim to reinforce the positive outcomes following vaccination. Results from published studies support the administration of an mRNA-based vaccine in dialysis patients, both as part of a primary vaccine series and also during booster dose administration. Vaccines with a different mechanism of action have been shown to confer lower cellular and humoral immune response rates when compared to an mRNA-based vaccine, although it needs to be noted that studies contemplating a non-mRNA-based vaccine regimen in dialysis patients are limited. Regarding the optimal mRNA-vaccine that should be used, studies have found that the mRNA-1273 vaccine is associated with higher antibody titers when compared to the BNT162b2 vaccine, although antibody neutralization capacity and cellular responses between these two vaccines have not been studied extensively, making conclusions about the most appropriate mRNA-based vaccine in dialysis patients problematic ,62. As bESRD patients in HD treatment are shown to develop a promising but impaired humoral and cellular immune response, compared to the healthy population, after completion of the vaccination regimen with the available mRNA anti-SARS-CoV-2 vaccines, BNT162b2 and mRNA-1273. Taking into consideration the risk factors associated with this vulnerable group, adaptation of vaccination protocols should be a health priority. Booster dose administration with mRNA-based vaccines seems to augment both the humoral and cellular responses in DPs, and implementation of this strategy may result in sustained immunological responses in this high-risk group. Additionally, further research regarding the lifespan and the effectiveness of observed responses are urgently needed, with great attention to patients receiving immunosuppressive therapy, older individuals, and those with comorbidities, conditions deemed to adversely contribute to immunization outcomes.


Further, we demonstrate a decline in antibodies over six months in responders and controls, and question if second vaccination non-responders benefit from of a third vaccination.Here we confirm lower humoral and cellular responses in hematological patients compared with controls and highlight the response in risk-groups such as CAR-T-cell recipients. The main risk factor for a poor humoral response was anti-CD20 therapy. CD19p = 0.001). Risk factors for a poor serological response were age (<65a), history of anti-CD20 therapy within the year preceding vaccination, CD19+ B-cells < 110/µL, and CD4+ T-cells > 310/µL. The magnitude of T-cell response was higher in patients <65a and with CD19+ B-cells < 110/µL. Patients and healthy controls demonstrated a significant decrease in SARS-CoV-2 S antibody levels over the period of six months (p < 0.001). A third vaccination demonstrated a strong serological response in patients who had responded to the previous doses (p < 0.001). The third vaccination yielded seroconversion in three out of 19 patients in those without serological response. We conclude that both humoral and cellular responses after SARS-CoV-2 immunization are impaired in patients with hematological malignancies. A third vaccination enhanced B-cell response in patients who previously responded to the second vaccination but may be of limited benefit in patients without prior seroconversion.Here we analyzed SARS-CoV-2-specific antibodies and T-cell responses after two coronavirus disease 2019 vaccinations over a six-month period in patients with hematological malignancies and assessed the effect of a third vaccination in a subgroup. Sixty-six patients and 66 healthy controls were included. After two vaccinations seroconversion was seen in 52% and a T-cell-specific response in 59% of patients compared with 100% in controls ( The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2), poses a major challenge to the health care systems around the world ,4.Concerns regarding the serological response to vaccines in patients with hematological malignancies are based either on the direct impact of the underlying disease or the administered therapy ,6,7,8,9.In addition, hematological patients are at an increased risk of breakthrough infection, especially in terms of accelerated waning of antibody levels over time and increasing immune-evasion capabilities of emergent variants ,15. A thIn the present study, we aim to evaluate serological and T-cell responses after two doses of mRNA COVID-19 vaccination and describe the efficacy of a third vaccine dose in patients not responding to the first two immunizations.The study is part of a prospective cohort study performed at the Medical University of Vienna, Vienna, Austria, “Characterization of the responsiveness after mRNA SARS-CoV-2 vaccination in patients with immunodeficiency or immunosuppressive therapy”; Eudra CT Nr. 2021-000291-11. Ethical approval for this study was granted by the local ethics committee of the Medical University of Vienna, Austria (EK Nr. 1073/2021).Patients with hematologic malignancies older than 18 years, who were treated at the Clinical Division of Hematology and Hemostaseology, were enrolled in the study. Further, 66 age- and sex-matched healthy controls (HCs) were included. All patients and HCs were vaccinated as participants of the Austrian vaccination program. Antibodies against the SARS-CoV-2 receptor-binding domain and the nucleocapsid protein were determined before vaccination as well as 10–20 days after the first, 12–28 days, and 5–6 months after the second vaccination. T-cell responses were evaluated in 32 patients 12–28 days after their second vaccination and in 10 patients 28 days after their third vaccination. At each visit, medication history, disease activity, leukocyte subtypes , the possibility of meanwhile SARS-CoV-2-infection , and vaccination associated adverse events were reported. ® Anti-SARS-CoV-2 assay [® e801 analyzer  at the Department of Laboratory Medicine, the Medical University of Vienna (certified acc. to ISO 9001:2015 and accredited acc. to ISO 15189:2012).Humoral immunity was quantified by assessing antibodies to the receptor-binding domain of the viral spike protein using the Elecsys anti-SARS-CoV-2 S immunoassay . The ran-2 assay . Antibod5 cells per well were incubated with SARS-CoV-2 peptides , AIM-V medium , or PHA  in 96-well plates coated with 1.5 μg anti-IFN-γ  for 24 h. After washing, spots were developed with 0.1 μg biotin-conjugated anti-IFN-γ , streptavidin-coupled alkaline phosphatase , and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma). Spots were counted using a Bio-Sys Bioreader 5000 Pro-S/BR177  and Bioreader software generation 10. T-cell responses were considered positive when mean spot counts were at least threefold higher than the mean spot counts of the unstimulated wells.Cellular immunity was assessed by a T-cell stimulation assay. PepMix™ SARS-CoV-2 peptide pools were purchased from JPT . The pools comprised 15-mer peptides overlapping by 11 amino acids (aa) covering the entire sequences of the SARS-CoV-2 spike protein. The spike peptides were split into two sub-pools, S1 (aa 1-643) and S2 (aa 633-1273). Peptides were dissolved in dimethyl sulfoxide and diluted in an AIM-V medium for use in the ELISpot assays. For ex vivo ELISpot assays, PBMCs were thawed. A total of 1–2 × 10The statistical analysis was performed using SPSS Statistics 27 , and figures were produced in the R packages “ggplot2”, “ggpubr”, and “viridis”. According to the distribution, continuous variables are represented as the median with 25% quartile (Q1) and 75% quartile (Q3). Differences in unpaired groups were assessed using the Wilcoxon rank-sum test. For multiple testing, Bonferroni correction was applied when indicated. Categorical variables are represented as numbers and rates in percent. Differences in categorical variables of unpaired groups were compared using Fisher’s exact test. The association of relevant variables with seroconversion was described with univariate and a multivariable logistic regression analysis adjusting for age and sex.Overall, 66 patients suffering from hematologic malignant diseases, as well as 66 healthy controls (HCs), were included in the study. The median age of our cohort was 62  years, and 26 (39%) were female. The median age of the HCs was 50 , and 26 (39%) were female. Patient’s baseline demographics are highlighted in p < 0.001). Additionally, SARS-CoV-2 S antibody levels were significantly lower in patients  compared to HCs .At the baseline, two patients and three healthy controls had detectable antibodies in the nucleocapsid-based chemiluminescence assay. After the first mRNA SARS-CoV-2 vaccination fewer patients  showed antibody response in comparison to healthy controls . The hematological malignancies associated with the lowest response were aggressive non-Hodgkin lymphomas (NHL) , followed by indolent NHL , CLL , multiple myeloma , and myeloid diseases . One patient with Langerhans cell histiocytosis did seroconvert. One patient each with ALL and Hodgkin’s lymphoma did not seroconvert. Several variables such as age < and >65a, sex, CD8+ T-cells < 280/µL and CD19+ B-cells < 110/µL, anti-CD20 therapy, and chemotherapy were analysed for association with the rate of seroconversion. The presence of Anti-CD20 therapy within the last year , the number of CD19+ B-cells < 110/µL , and the number of CD4+ T-cells < 310/µL  were associated with lower rates of seroconversion. Age > 65a correlated with an increased rate of seroconversion  (After the second mRNA SARS-CoV-2 vaccination seroconversion was evident in 52% (34/66) of the patients and in 100% (66/66) of the HCs (= 0.046) . Multiva= 0.046) .p = 0.1). It was highest in patients who received allogeneic stem cell transplantation , followed by a patient who received autologous stem cell transplantation . Six patients received anti-CD19 CAR T-cells, of which only one patient (17%) showed seroconversion after four weeks.Obinutuzumab was exclusively administered in patients <65a  and patients <65a suffered numerically more often from aggressive NHL, associated with the poorest seroconversion rate . The overall response rate in bone marrow transplant recipients was similar to the other patients in this cohort . Antibody titers were lower in patients aged <65a , history of anti-CD-20 therapy within the last 12 months , CD19+ B-cells < 110/µL , and CD4+ T-cells < 310/µL   in comparison to HCs  .+ B-cells (p < 0.001). CD4+ T-cell levels correlated with the seroconversion rate of the different hematological malignancies (p = 0.037). Additionally, CD4+ T-cell levels were lowest in patients with aggressive NHL , followed by indolent NHL  and CLL . CD4+ T-cell levels did not correlate with the history of anti-CD20 therapy within the last 12 months (p = 0.14).The history of anti-CD-20 therapy within the last 12 months correlated with lower CD19SARS-CoV-2 S antibody levels were reassessed 202 days  after the second vaccination in 53 patients and 58 HCs. For further analysis, the patients were divided into two groups in dependence of seroconversion four weeks after the second vaccination . p < 0.001; HCs four weeks after vaccination, . Interestingly, 10 of 32 (31%) of the 2VR demonstrated a delayed increase of SARS-CoV-2 S antibodies between the four-week visit and the six-month visit, compared to 7 of 58 (12%) of the HCs. Further, SARS-CoV-2 S antibodies of patients responding to the second vaccination (2VR) did differ significantly compared to HCs six months after the second vaccination .SARS-CoV-2 S antibodies of second vaccination responders and HCs significantly decreased over the course of six months after the second vaccination . The third vaccination was performed in 33 patients  at a median of 176  days after the second vaccination. The vaccination was performed in 17 patients (51.5%) with BNT162b2, eight patients (224.24%) with mRNA-1273, and in eight patients (24.24%) with AZD1222. Only 3 of the 19 2VNR experienced seroconversion after the third vaccination (16%). The 2VR demonstrated a significant increase in SARS-CoV-2 S antibody level . A logistic regression model did not find an association for sex, anti-CD20 therapy within the last year, CD8+ T-cells < 280/µL or CD19+ B-cells < 110/µL, or frequency of T-cell responses  compared to patients >65a , and CD19+ B-cells < 110/µL , compared to patients with CD19+ B-cells within the normal range  at 4 weeks after the second vaccine dose. After the third dose, T-cell responses were reassessed in 10 of 22 patients. Five of those ten patients had already mounted T-cell-specific responses after the second vaccination, which increased to 7 out of 10 patients after the third vaccination (SARS-CoV-2-specific T-cell responses were determined in 32 (49%) patients after the second vaccination and 16 HCs to assess if vaccination induced a cellular immune response. Interestingly, 19 out of 32 patients (59%) demonstrated a T-cell response compared to 100% (16/16) of the HCs  are significantly correlated with anti-SARS-CoV-2 S IgG levels after the second vaccination. CD4+ T-cell levels were particularly low in patients with aggressive NHL, the disease with the lowest humoral response. An analysis of T-cell responses in humans with COVID-19 disease and unexposed individuals demonstrated that virus-specific CD4+ T-cells are present in 100% of convalescent patients and that CD4+ T cell levels correlated with the magnitude of anti-SARS-CoV-2 S IgG [+ T-cell responses guide the adaptive immune response to the second vaccination [We demonstrated that levels of CD4-2 S IgG . This iscination .Another risk factor for seroconversion we found was age <65a. This is somewhat in contrast to previous literature, but the uneven distribution of obinutuzumab/rituximab therapy and the numerical higher number of aggressive lymphomas in patients aged <65a might contribute to this finding .+ T-cells was shown [+ B-cells within the normal range. We did not find a gender-associated difference in the SARS-CoV-2-specific T-cells response [In addition to the antibody response to mRNA SARS-CoV-2 vaccination, T-cell responses contributed to vaccine efficacy in healthy controls ,25,27. Fas shown . In our response . Furtherresponse .A special subgroup in our cohort comprises six anti-CD19 CAR T-cells recipients. Only 1 of 6 patients demonstrated seroconversion. Given the mechanism of action with T-cells eliminating CD19-expressing malignant and normal cells, this finding is not surprising .Another aim of this study was to assess long-term responses after mRNA SARS-CoV-2 vaccinations in hematological patients. In both HCs and patients, the visit after six months demonstrated a significant reduction of anti-SARS-CoV-2 S IgG levels. However, patients responding to the second vaccination more often demonstrated a delayed increase of SARS-CoV-2 S antibodies in comparison to HCs.Previously, the lack of immunogenicity of mRNA SARS-CoV-2 vaccination has led to evaluations of third vaccine immunization in different COVID-19 high-risk cohorts. Recent reports of transplant recipients demonstrated that a third dose of mRNA vaccine had substantially higher immunogenicity compared to the placebo ,30. FirsWe report several limitations. In this cohort, hematological patients were older than HCs, as HCs consisted mainly of healthcare professionals working at our center. Hence, the demonstrated difference between patients and HCs could be overestimated, as older age was shown to be a major factor for weaker serological responses . Further+ 19 B-cell and CD4+ T-cell levels were shown to be additional predictive markers for seroconversion. T-cell responses were reported in 59% of the patients and were higher in patients with low CD19+ B-cell counts. Whether the preserved T-cell response in patients with anti-CD20 therapy and low CD19+ B cell counts contribute to some degree of protection requires further evaluation. Over a period of six months, vaccine responders and HCs demonstrated a significant decline in antibodies. In those patients, the third vaccination was demonstrated to be effective in booster B-cell response. In vaccine non-responders, a third vaccination yielded seroconversion in a minority of the patients, suggesting a limited benefit from a third vaccination in terms of seroconversion in this group.In conclusion, our findings demonstrate that the full immunization schedule was able to induce anti-SARS-CoV-2 S IgG responses in 52% of patients. B-cell depleting therapy was the major factor for a poor serological response. CD


The antibody response decreased 12 weeks after the primary vaccination. After the booster, humoral and cellular responses significantly improved, with comparable seroconversion rates between the heterologous and homologous groups. Positive sVNT against the wild type occurred in >90% of LT patients, with only 12.3% positive against the Omicron variant. Conclusions: ChAdOx1/BNT162b2 evoked a significantly higher immunological response than ChAdOx1/ChAdOx1 in LT recipients. The booster strategy substantially induced robust immunity against wild type in most patients but was less effective against the Omicron strain.Background: Heterologous prime-boost vaccination potentially augments the immune response against SARS-CoV-2 in liver transplant (LT) recipients. We investigated immunogenicity induced by different primary prime-boost vaccination protocols and the subsequent response to the booster vaccine among LT recipients. Methods: LT recipients, who received primary immunisation with ChAdOx1/ChAdOx1 or ChAdOx1/BNT162b2, were administered the third dose of mRNA-1273 three months following the primary vaccination. Blood samples were collected before and after primary vaccination and post-booster. The levels of receptor binding domain antibody (anti-RBD) and neutralising antibody (sVNT) and spike-specific T-cell responses were assessed. Results: Among the 89 LT recipients, patients receiving ChAdOx1/BNT162b2 had significantly higher anti-RBD titres, sVNT, and cellular response after primary vaccination than those receiving ChAdOx1/ChAdOx1 ( The rapid spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has become a global public health concern and has been declared a pandemic since December 2019 ,4. PatieAlthough considered safe and effective in minimising disease severity and mortality, several reports have revealed inadequate immune responses in LT recipients after a standard two-dose SARS-CoV-2 vaccination, with serological response rates ranging from 47–79% ,9,10. HoTo explore these issues among LT recipients, we conducted this study with three main aims: (1) to evaluate SARS-CoV-2 specific humoral and cellular immune responses after primary vaccination with heterologous versus homologous prime-boost protocols, (2) to explore immune dynamics after primary immunisation, and (3) to assess the subsequent response to the booster vaccine following different primary vaccine series.This prospective, longitudinal observational study was conducted between June 2021 and February 2022 at a single liver transplant centre . SARS-CoV-2 naïve LT recipients aged ≥ 18 years were enrolled in the study. All LT patients were immunised with either homologous [ChAdOx1 /ChAdOx1] or heterologous [ChAdOx1/BNT162b2 ] as their primary vaccine protocol. Vaccination schemes were determined by the national policies of the Ministry of Health of Thailand according to the availability of vaccines. All participants received an additional dose of mRNA-1273  three months following the standard two-dose vaccine series. The exclusion criteria were LT patients with a history of confirmed SARS-CoV-2 infection, prior immunisation with any SARS-CoV-2 vaccine, acute cellular rejection, or pregnancy. Blood samples were collected immediately before the first vaccination, four weeks after the second vaccination, twelve weeks after the second vaccination, and four weeks after the booster vaccine.Demographic, clinical, and biochemical data were obtained from the electronic medical records at the time of the first vaccination. All LT recipients were screened for active respiratory tract infection and SARS-CoV-2 exposure by questionnaire at every visit. Adverse events after vaccination were assessed using a questionnaire and a phone interview.® anti-SARS-CoV-2 S using Cobas e411 immunoassay analysers . The sensitivity and specificity of this assay were 93.9% and 99.6%, respectively  (p = 0.32)  U/mL vs. 152.1 (13.6–678.8) U/mL, (= 0.02)] . However = 0.32) .p = 0.09, 43.8% reduction rate), whereas the median anti-RBD IgG in patients receiving ChAdOx1/BNT162b2 significantly decreased to 638.5 (142.5–1245.5) U/mL . Nevertheless, the SARS-CoV-2 spike total antibodies remained significantly higher in the heterologous prime-boost group than in the homologous prime-boost group (p < 0.001).Twelve weeks after the primary vaccination, a decline in anti-RBD antibodies was observed in both primary vaccine platforms . The medp < 0.001). Seroconversion occurred in 81.3% of LT recipients receiving ChAdOx1/ChAdOx1/mRNA-1273 and in 94.7% of those receiving ChAdOx1/BNT162b2/mRNA-1273. Although the additional vaccine yielded strikingly high antibody levels, no significant difference was observed in the anti-RBD titre and seroconversion rate between the two groups after the booster  (The booster vaccine significantly improved immune response . The medctively) .p = 0.025). Owing to the high seroconversion rate after booster vaccination, we could not identify significant predictors of the immune response following the third vaccine.Potential variables as predictors of poor humoral response after primary vaccination were evaluated using logistic regression analysis . In univp = 0.01] (p < 0.001)  was investigated at four weeks following primary immunisation and four weeks after the booster vaccination. Following primary immunisation, participants receiving ChAdOx1/BNT162b2 had significantly higher median sVNT levels than those receiving ChAdOx1/ChAdOx1  . ChAdOx1< 0.001) .p = 0.06) (p = 0.04). Notably, the sVNT level demonstrated a significant correlation with the anti-RBG titre after primary vaccination  and post-booster .After an additional dose of mRNA-1273, the median sVNT level to wild type increased to 97.6% (96.8–97.8%) in patients vaccinated with ChAdOx1/BNT162b2 and 97.3% (69.2–97.6%) in those immunised with ChAdOx1/ChAdOx1; the difference was not statistically significant ( = 0.06) . HoweverNeutralising antibodies to the Omicron variant were assessed in blood samples after booster vaccination. Notably, almost all LT patients had a substantially low level of sVNT to the Omicron strain . Only 10p > 0.05) (6 PBMC cells of LT patients receiving the heterologous primary vaccine protocol was significantly higher than those receiving the homologous protocol (p = 0.02) or sVNT  and spike-specific T-cell responses.The T-cell response was evaluated in 58 randomly selected LT patients: 41 in the ChAdOx1/BNT162b2 group and 17 in the ChAdOx1/ChAdOx1 group. The baseline clinical and laboratory characteristics were comparable between the two groups ( > 0.05) . The resprotocol . A signiOverall, most patients experienced minor adverse events after injection of the primary vaccination and booster . Both vaIn this prospective study, we evaluated the immunogenicity and dynamics of SARS-CoV-2 specific immune response as well as the subsequent benefit of the booster among LT recipients who received homologous versus heterologous primary vaccination. Following primary immunisation, the heterologous protocol (ChAdOx1/BNT162b2) induced a more robust immune response than that with the homologous protocol (ChAdOx1/ChAdOx1). The antibody level eventually decreased three months later, regardless of the vaccine platform. The additional third dose significantly improved humoral and cellular immune responses in both the primary vaccine groups. Most LT recipients had strong immunity against the wild-type strains. Nevertheless, the neutralising activity against Omicron variants was substantially attenuated in both groups, even after booster vaccination.n = 11). In addition, Mendizabal et al. reported that heterologous adenoviral vector/mRNA improved the humoral response in Argentinian LT patients [At the beginning of the pandemic, the mRNA vaccine was unavailable in most low- and middle-income countries. In Thailand, inactivated and adenoviral-vectored vaccines were the first vaccines to be inoculated in the general population during the outbreak before the arrival of mRNA vaccines . The usepatients . In our patients ,21. We aWe also evaluated cellular immune responses, which may contribute to immune longevity . Our finNotably, the decrease in total anti-spike antibodies was observed in both vaccine protocols three months after the primary vaccination. Immune waning may diminish the protective effect against SARS-CoV-2, especially in patients in the homologous group, wherein less than half of those demonstrated detectable antibodies. Antibody kinetics were consistent with the results observed in a recent study in LT recipients .Regarding the diminished immune response in LT recipients after primary immunisation ,24, the The emergence of variants of concern, for example the Omicron variant (B.1/B.1.1.529), has raised concerns about vaccine efficacy. With highly prevalent mutations in the spike protein resulting in the potential to escape the host immune response, it is anticipated that a markedly low humoral immune response against the Omicron strain will be observed . DespiteThe strength of our study is that it was a longitudinal prospective study that provided comprehensive information regarding SARS-CoV-2-specific immune response, including anti-spike antibodies, neutralising activities, and T-cell responses, from baseline to post-booster in LT recipients. We investigated different types of primary vaccine regimens based on previous studies conducted in the Western region. Therefore, our results could be helpful in settings where the mRNA vaccine was not the predominant platform for primary immunisation or experienced vaccine supply issues, especially in developing countries.Our study had some limitations. First, although we present clinical evidence in a real-world setting, the study was conducted in a single transplant centre, precluding generalisation to a broader population of LT recipients. Second, the study was limited by the absence of randomisation. We observed a relatively lower number of participants in the homologous vaccine group than in the heterologous vaccine regimen. However, the baseline characteristics were comparable between patients immunised with the two different vaccine platforms. Third, our study included only LT patients receiving homologous primary vaccination with ChAdOx1/ChAdOx1 and thus does not reflect whether ChAdOx1/BNT162b is superior to homologous BNT162b/BNT162b protocol. Lastly, there were no comparative data among healthy populations for immune responses. In a state of immunosuppression, weaker immune responses were expected among LT recipients in several studies ,8,9. DesPrimary vaccination with the heterologous regimen induced better SARS-CoV-2 specific immune response among LT patients than the homologous protocol. Waning immunity was observed after the primary immunisation, regardless of the vaccine regimen. The booster strategy produced a more robust immune reaction in most LT recipients. Nevertheless, protective efficacy against emerging virulent strains and immune sustainability after booster vaccination should be assessed in future studies.


Effective SARS-CoV-2 vaccination in patients receiving treatment with B-cell depleting agents is challenging. Information on vaccination responses in these patients are a valuable tool to develop efficient vaccination regimens.In this single-center retrospective observational study, we report the humoral and cellular response in 34 patients receiving anti-CD20 antibody treatment for renal immune disease.After base immunization with SARS-CoV-2-vaccines, 92.0% developed a cellular, 32.4% a humoral response. Humoral immunity correlated with B-cell counts and the timespan between anti-CD20 antibody treatment and vaccination. All patients with > 21/µl B-cells, or > 197 days after treatment showed humoral response.Adequate timing of SARS-CoV-2-vaccinations after anti-CD20 antibody treatment and CD19 measurements are crucial to generate immunity. Awaiting partial B-cell recovery by postponing regularly scheduled anti-CD20 treatment should be considered in patients with stable immune disease.Trial registration: This study has been retrospectively registered in the German Clinical Trials Register (DRKS00027049) on 29/10/2021.The online version contains supplementary material available at 10.1186/s12879-022-07722-7. Successful vaccination of risk groups is essential to reduce morbidity and mortality in the COVID-19 (coronavirus disease 2019) pandemic. Vaccine-induced immunity relies on antibody-driven , and T-cell mediated (cellular) immune response .Effective vaccination of immunocompromised patients is especially challenging as many immunosuppressive agents impair the response to vaccines, increasing the risk for severe COVID-19, prolonged infection, and viral evolution , 5.Several immune-mediated and hematologic diseases are treated with monoclonal anti-CD20 antibodies. These drugs deplete circulating B-cells, leading to impaired maturation of memory B-cells and reduced numbers of antibody-producing plasma cells. ConsequChronic kidney disease is a further independent risk factor known to impair vaccination responses . Hence, Here, we report the humoral and cellular immune response to mRNA- and vector-based SARS-CoV-2 vaccines  in patients receiving the monoclonal anti-CD20 antibody Rituximab for renal autoimmune disease. Our results help to optimize vaccination strategies by taking prior therapies and cellular parameters into account.In this single-center retrospective observational study, we report data from the University of Freiburg Medical Center, Germany. Inclusion criteria were age ≥ 18 years, current treatment with the anti-CD20 antibody Rituximab for renal autoimmune disease, and completed base-immunization with either mRNA-based , or vector-based  SARS-CoV-2 vaccines. In vaccination regimens requiring two doses for base immunization, the second dose was given between 2 and 12 weeks after the first vaccination. Exclusion criteria were prior SARS-CoV-2 infections, and administration of antimetabolite drugs  within two months before vaccination.Demographic data, past medical history, treatment details and laboratory findings  were extracted from electronic patient records of outpatient visits between April and September 2021. B cell counts were measured in between 5 weeks prior, to 12 weeks after vaccination by flow cytometry , and ≥ 200 mIU/ml (for IFN-γ negative control > 100 mIU/ml), using a double-cut-off strategy integrating the result of background stimulation according to in-house standard procedure  received mRNA vaccines (BNT162b2: n = 25 [73.5%]), mRNA-1273: n = 1 [2.9%]). Six patients (17.6%) received vector-based vaccines . Two patients (5.9%) received a first dose of the vector-based vaccine ChAdOx1, followed by a mRNA-based vaccine .Humoral response was detected in 11 (32.4%) patients. Cellular response was detected in 23 (92%) patients. One individual had neither response, while an isolated response was observed in another individual. Kidney function, proteinuria, or hematuria, as well as timespans between first and second vaccinations and the timepoint of testing after vaccination were not altered between response groups (Table Kidney function, proteinuria and hematuria did not correlate with SARS-CoV-2-IgGs or IFN-γ-release (Additional file B-cell counts at vaccination correlated with the time from last anti-CD20-antibody treatment to vaccination and were significantly higher in patients with a humoral response Table A. SigmoiPatients receiving additional corticosteroids are overrepresented in humoral and cellular non-responder groups Table . A subgrFinally, there were no significant differences in humoral, or cellular response in patients receiving different vaccination regimens. However, in line with previous reports , we obseAnti-CD20 treatment impairs humoral response to vaccinations. Data on SARS-CoV-2 vaccines is still limited, but previous studies have shown negative effects on antibody production after the BNT162b2 vaccine in patients receiving B-cell depleting agents , 14.This study confirms impaired humoral response to SARS-CoV-2 vaccinations in patients receiving anti-CD20 treatment for renal autoimmune diseases. We show that the humoral response strongly correlates with B-cell counts and the interval between last anti-CD20 antibody administration and vaccination. Both, time from Rituximab to humoral response and CD19 counts necessary for vaccination response, are similar to results reported in parallel studies –20. ThisOur study shows that the cellular response to SARS-CoV-2 vaccines is independent of CD19 depletion, confirming other recent reports , 21. TheIn line with other recent studies –24, we oWe do not observe reduced kidney function to be a statistically significant risk factor for impaired vaccination response. However, parallel studies report reduced response to mRNA SARS-CoV-2 vaccines in both subjects with chronic and end stage renal disease , 26.This study has several limitations, the major being the small study population, the retrospective character, and the lack of a standardized follow-up. The multitude of anti-CD20 antibody treatment regimens further complicate the interpretation. However, our study provides valuable information on how to optimize the humoral vaccination response, and how to reduce the risk for COVID-19 in an especially vulnerable patient cohort.Based on our data, awaiting partial B-cell recovery, and thus postponing regularly scheduled anti-CD20 treatment, should be considered in patients with stable renal disease. We and others  observedAdditional file 1. Supplemental Figure S1, Supplemental Tables S1–S5, Supplemental Methods.


Although mRNA-based vaccines against SARS-CoV-2 induce a robust immune response and prevent infections and hospitalizations, there are limited data on the antibody response in individuals with humoral immunodeficiency. The aim of this study was to evaluate the humoral immune response after two vaccine doses with BNT162b2 or mRNA-1273 in patients with humoral immunodeficiency disease.This cross-sectional study assessed 39 individuals with hypogammaglobulinemia under immunoglobulin replacement therapy. IgG anti-SARS-CoV-2 spike protein antibodies (anti-S) were measured 4 weeks to 4 months after two doses of an mRNA vaccine against SARS-CoV-2. The proportion of patients, who developed a humoral immune response to the spike protein were evaluated and compared to 19 healthy controls.After vaccination with two vaccine doses, 26/39 patients (66.7%) with humoral immunodeficiency disease and all healthy controls developed anti-S. In subjects with baseline IgG <3 g/l, only 1/5 (20%) showed a humoral immune response. 10 out of 26 with CVID (38.5%) and 7/9 under immunosuppressive drugs (77.8%) developed no immune response (13 subjects with no response) compared to 0/19 in healthy controls. Subgroup analysis in patients without immunosuppressive drugs revealed lower anti-S in patients with moderate to severe humoral immunodeficiency disease: baseline IgG <3 g/l: 12.0 AU/ml (95%CI 12.0–125.0), baseline IgG 3–5 g/l: 99.9 AU/ml (95%CI 14.4–400.0), baseline IgG >5 g/l: 151.5 AU/ml (95%CI 109.0–400.0), healthy controls 250.0 AU/ml (95%CI 209.0–358.0), p = 0.007.In most patients with mild to moderate humoral immunodeficiency we found only slightly lower anti-S antibodies compared with healthy controls after two vaccine doses with BNT162b2 and mRNA-1273. However, in patients with a decreased baseline IgG below 3 g/l and/or under immunosuppressive drugs, we found severely impaired humoral immune responses. Following the global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), vaccinations have been introduced since the end of 2020 to control the pandemic. In particular, mRNA-based vaccines are considered highly effective in prevention of infection and hospitalization, even against variants of concern ,2. To daWe studied the humoral immune response against the S1/S2 spike protein of SARS-CoV-2 in patients with humoral immunodeficiency disease. Subjects with a primary or secondary hypogammaglobulinemia  and treated by intravenous (IVIG) or subcutaneous immunoglobulin (SCIG) replacement therapy in our outpatient clinic were included if they had received two mRNA Covid-19 vaccine doses  or mRNA-1273 ) 4–6 weeks apart between January and June 2021. CVID was defined with the following characteristics: significant reduced total IgG, reduced IgA or IgM values, poor vaccine response, recurrent infections and absent T-cell impairment . As the All data on disease, treatment, and vaccinations were collected by the attending physicians based on the medical records. All individual vaccination dates and the vaccines administered were obtained from the original vaccination records in each study participant. Healthy volunteers were recruited among the private and professional surroundings of the investigators without performing a matching procedure. Single serum samples for analysis of vaccine antibodies were collected 2 weeks to 4 months after the second Covid-19 vaccination in all study participants and healthy controls.Blood samples were run on an Abbott ARCHITECT i2000 instrument using the Abbott SARS-CoV-2 nucleoprotein assay and the DiaSorin LIAISON® SARS-CoV-2 S1/S2 IgG assay following the manufacturer’s instructions. The Abbott assay is a chemiluminescent microparticle immunoassay for qualitative detection of IgG in human serum or plasma against the SARS-CoV-2 nucleoprotein. The Liaison SARS-CoV-2 S1/S2 is a chemiluminescence assay, that uses paramagnetic microparticles coated with S1 and S2 fragments of the viral surface spike protein (positive cut-off of >12 AU/ml). According to the manufacturer’s information, the Liaison SARS-CoV-2 S1/S2 IgG assay of >15 AU/ml reached a plaque-reduction neutralization test (PRNT) titer of 1:40 in 17/18 patients and a PRNT titer of >1:160 if SARS-CoV-2 S1/S2 IgG of 80 AU/ml were analysed . PresencThis study was approved by the local ethics committee . All subjects included signed informed consent.Statistical analysis were performed using Graphpad Prism 9 . Patient characteristics are summarized with descriptive statistics. Values are median and interquartile ranges (IQR) for continuous variables, categorical variables reported as n (%). The 95% confidence intervals (95%CI) from the proportions of patients with positive anti-S values in a specific group were calculated as Wilson/Brown interval. Anti-S titers were compared by Kruskal-Wallis or Mann-Whitney test, the median 95%CI was calculated for all groups.47 patients with a humoral immunodeficiency treated by immunoglobulin replacement therapy and vaccinated twice with one of the Covid-19 mRNA vaccines were identified in a single center. Eight subjects were excluded . Of the 39 patients included, 28 were female (71.8%) compared to 15/19 healthy controls (79.0%). The median age was 61 years  in the study group and 53 years  in the control group . CharactAfter two vaccine doses, we found detectable anti-S levels in 26/39 subjects (66.7% [95%CI 51.0–79.4%]) with a humoral immunodeficiency disease, and in all healthy controls . In CVID, the proportion of patients with presence of anti-S was 16/26 (61.5% [95%CI 42.5–77.6%]), and thus lower than in non-CVID patients  and in the healthy control group , p = 0.002. All individuals with a humoral non-response to the vaccines (n = 13) were either under immunosuppressive drugs (including anti-CD20 therapy in the past)  and/or had a diagnosis of CVID . Two patients treated by immunosuppressive treatment showed a low anti-S response . Median In a post-hoc analysis, we obtained anti-S levels from 22 patients after the third vaccination . A third26/39 of our patients with humoral immunodeficiency showed a detectable immune response against SARS-CoV-2 after mRNA vaccination. After the second Covid-19 vaccine, the majority of individuals with a mild to moderate IgG deficiency had a specific anti-S concentration that was slightly below the level of healthy controls. Even in most patients with CVID an immune response was detected after a second dose of the Covid-19 mRNA vaccine. Our findings are in line with recent studies, demonstrating an increase of anti-S levels in most individuals with humoral immunodeficiency without immunosuppressive drugs after two Covid-19 vaccinations –22. In aTherefore, anti-S antibody determination might be useful to detect non-responders after completion of a two Covid-19 vaccination schedule in patients at risk, to enable a timely third vaccination. In patients under immunosuppressive drugs or humoral immunodeficiency, a full vaccination schedule should include three Covid-19 mRNA vaccinations followed by a booster vaccine . If a deOur study has limitations: Although both mRNA vaccines seem to be effective in a majority of patients with humoral immunodeficiencies, we have no long-time data since the observational period was very short. Blood samples were taken over a wide time interval (4 weeks to 4 months) in both study groups. Since antibody titers decrease over time, it is therefore likely, that the measured antibody levels underestimate the peak levels in these patients. Furthermore, it has to be assumed that patients with a humoral immunodeficiency have a faster decline of the IgG-anti-S titer than healthy controls. In addition, it is unclear whether the anti-S antibodies we measured are neutralizing in nature. We cannot make any statement about the T-cell response, which certainly plays a major role. Specific T-cell responses seem to be defective in about one third of CVID patients . Lastly,Although individuals with humoral immunodeficiencies have impaired responses to vaccines, BNT162b2 and mRNA-1273 mRNA vaccines produce humoral immune responses in the majority of these patients. Most individuals with mild to moderate humoral immunodeficiency disease without immunosuppressive drugs did not have significantly reduced anti-S values compared to healthy individuals. Treatment with immunosuppressive drugs and a diagnosis of CVID are risk factors for reduced or even absent humoral response. In these individuals, a full vaccination schedule should include three Covid-19 mRNA vaccinations as primary series followed by a booster vaccine.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Corona mortis, can lead to death in the worst-case scenario if injured. Despite being well-known, exact anthropometric data are lacking. The purpose of this study was to determine diameters of the regular obturator artery, the Corona mortis and the inferior epigastric artery. In addition, the level of origin of the Corona mortis was quantified. The obturator artery and its norm variants were dissected bilaterally in 75 specimens  and measured using two different methods. The Corona mortis was present in 36 of the 150 hemipelves (24%), presenting in one third of all cases bilaterally. Its level of origin measured from the commencement of the inferior epigastric artery was subject to high variability (4.4–28.3 mm). The mean diameters of the Corona mortis  and the regular obturator artery  were similar for both methods. There were no significant sex nor side differences. The diameter of the inferior epigastric artery was significantly smaller distal to the origin of the Corona mortis. The high incidence, non-predictable level of origin of the Corona mortis and its size similar to the regular obturator artery support its clinical relevance even to date. Clinicians should always be aware of an additional arterial vessel close to the pelvic brim.An enlarged anastomosis connecting the vascular territory of the external iliac and the obturator artery may replace most or all of the latter. This relatively common vascular variation, known as  Corona mortis, what means crown of death. The first author, who described the course of this replaced obturator artery was the anatomist Haller . While alive, all individuals bequested their post mortem bodies for their use in medical education and research. In addition to their informed consent, institutional approval was obtained from the Ethics Committee of the Medical University of Vienna . All specimens were embalmed by perfusion and immersion with a diluted solution of formalin (1.6%) and phenol (4%). Bodies with malignant tumours or vascular surgical interventions in the pelvic and femoral regions were excluded.Corona mortis was determined overall, and regarding sex and body side. As generally accepted . To minimize the probability of a methodological error, two different methods of measurements were performed. With the first one, the diameter of the vessels was determined without any compression. This method was named “unfolded” , IBM SPSS Statistics 26 , and GraphPad Prism 9.3.1 . To prove a possible correlation between the incidence of a α = 0.05. In the text, mean values ± standard deviations are given.The significance level was set at Corona mortis was observed in 27 individuals (36.0%). It was present in 16 of the 38 males (42.1%) and in 11 of the 37 females (29.7%). In relation to the pelvic halves, a Corona mortis occurred in 36 of 150 cases (24%). Thereby it was present in 19 male (25%) and 17 female hemipelves (23%).In the 27 individuals with a Corona mortis, it was present on only one side in 18 cases (66.7%). In the remaining nine individuals (33.3%) the Corona mortis was observed bilaterally. This was twice as much the case in females than in males . In males, the unilateral cases were present in nine left and only four right sides. In females, the unilateral cases were more balanced (two left vs. three right sides). For detailed information, please refer to Table A Corona mortis with sex (p = 0.771) or with body side (p = 0.444).There was no correlation between the occurrence of a Corona mortis were coincidentally present in only two left male hemipelves   distal to the commencement of the inferior epigastric artery from the external iliac artery. Although it originated more distally in women and on the left side on average, there was no difference between sexes (p = 0.210) nor sides (p > 0.999). In the four cases with Corona mortis origin from the external iliac artery, this was highly variable, ranging from 15.0 to 27.7 mm proximal to the commencement of the inferior epigastric artery. Due to the small number of cases, no further statistical analyses were conducted.The 9%) Fig. . In only9%) Fig. . The levCorona mortis was 2.5 ± 0.5 mm (“unfolded”), and 2.1 ± 0.4 mm (“Hillen”). The maximum diameter of the Corona mortis was 3.7 mm (“unfolded”) and 3.1 mm (“Hillen”). However, these values were measured in two different male individuals. The minimum diameter was determined in one female individual with 1.3 mm (“unfolded”) and 0.9 mm (“Hillen”). In cases with bilateral Coronae mortis the diameters were mostly the same on both sides and showed differences of 0.0 mm to at most 0.6 mm (both measurements methods). Only in one bilateral case, the difference amounts 1.2 mm (“unfolded”). For further details regarding sex and body side, please refer to Fig. Corona mortis, neither for the method “unfolded” (p = 0.947) nor for the method “Hillen” (p = 0.880). There were also no statistically significant differences in the diameters of the Corona mortis between sexes or body sides for both methods.The diameter of the regular obturator artery averaged 2.4 ± 0.6 mm (“unfolded”) and 2.0 ± 0.5 mm (“Hillen”). This vessel originated in 79 cases from the ventral and in 35 cases from the dorsal trunk of the internal iliac artery. In two cases, it was impossible to identify a clear trunk-formation of the branches of the internal iliac artery. The average diameter of the Corona mortis was 3.4 ± 0.7 mm (“unfolded”) and 2.8 ± 0.5 mm (“Hillen”). Distally to the commencement of the Corona mortis it was reduced to 2.8 ± 0.5 mm (“unfolded”) and 2.3 ± 0.4 mm (“Hillen”), respectively  as well as for the method “Hillen” (p < 0.001).The average diameter of the inferior epigastric artery proximally to the origin of the Corona mortis measured with the method “Hillen” were significantly smaller than those measured with the method “unfolded” (p < 0.001). The mean difference was − 0.4 ± 0.2 mm .Diameters of the regular obturator artery and the  mm Fig. d. HoweveCorona mortis, the incidence was calculated from 32 former anatomical, surgical, and radiological studies including 8.257 hemipelves  are also based on case numbers far below 100. Among others, Japanese authors observed by trend a lower occurrence of a Corona mortis , and 2.1 ± 0.4 mm (“Hillen”). It was slightly higher in male than in female bodies, but was almost equally concerning the side of the body. The mean diameters measured by other authors ranged from 1.63 to 4.0 mm (Hong et al. In the present study, the mean diameter of the Corona mortis but also the diameter of the regular obturator artery was measured for comparison. The difference between their two mean diameters was only 0.1 mm for both measurements methods. Therefore, the Coronae mortis observed here are likely to have almost completely replaced the non-existent regular obturator artery. In practical terms, this means that injury to the Corona mortis may cause a similar amount of blood loss as an injury to the regular obturator artery.In this study, for the first time, not only the diameter of the Corona mortis. As expected, there was a significant reduction in diameter after commencement of the Corona mortis. On the other hand, this means that in the case of a Corona mortis, the inferior epigastric artery in its area of origin, is considerably stronger than usual. In practice, this means that in the case of a Corona mortis, not only is there an increased risk of bleeding from it. Injury to the inferior epigastric artery proximal to the origin of the Corona mortis may also result into higher amount of blood loss than in cases with a regular obturator artery. In the case of arterial occlusive disease, a Corona mortis may also become an important link between the circulation territories of the external and internal iliac arteries. People with this vascular variant may be better able to compensate for occlusions in this area and may not become symptomatic or may become symptomatic later.This study was also the first to compare the diameter of the inferior epigastric artery proximal and distal to the origin of the To minimize the probability of a systematic error, the measurement of diameters was accomplished in two different ways. The determined values of both methods were statistically significantly different from each other but the measured values correlated highly between the two methods. The results of both methods are affected by different factors. The measurement described by Hillen  may be iCorona mortis. However, it was quantified for the first time that the level of origin of the Corona mortis is highly variable in relation to the origin of the inferior epigastric artery. In addition, it was demonstrated for the first time that the diameter of a Corona mortis is nearly identical to that of a regular obturator artery. It was also shown that the diameter of the inferior epigastric artery decreased significantly after the commencement of the Corona mortis. All these results support the clinical relevance of this common vascular variant: It can be expected in about a quarter of all hemipelves. One cannot predict at which level the Corona mortis originates from the inferior epigastric artery or the external iliac artery. Therefore, clinicians should always be aware of the possibility of an additional arterial vessel in this area, which can lead to severe arterial bleeding in the event of an injury.The current study confirms previous data on the incidence of a