key: cord-0279804-ubrtwbqa
authors: Fiorino, F.; Sicuranza, A.; Ciabattini, A.; Santoni, A.; Pastore, G.; Simoncelli, M.; Polvere, J.; Galimberti, S.; Auddino, S.; Barate', C.; Montagnani, F.; Sammartano, V.; Bocchia, M.; Medaglini, D.
title: The slower antibody response in myelofibrosis patients after two doses of mRNA SARS-CoV-2 vaccine calls for a third dose
date: 2021-09-23
journal: nan
DOI: 10.1101/2021.09.21.21263627
sha: b3e673b056426d7ab558feefed0db87669041ed4
doc_id: 279804
cord_uid: ubrtwbqa

Immunization with mRNA SARS-CoV-2 vaccines has been highly recommended and prioritized in fragile categories with higher risk of mortality after COVID-19 disease compared to healthy people, including patients with myelofibrosis (MF). Available data on the vaccine immune re-sponse developed by MF patients, and the impact of the treatment with the inhibitor of JAK-STAT signaling ruxolitimib, are still fragmented to support an informed decision for a third dose for this category of subjects. Here, we show that 76% of MF patients develop spike-specific IgG after the second vaccine dose, but the response has a slower kinetic compared to healthy subjects, suggesting a reduced capability of their immune system to promptly react to vaccina-tion. A reduced ACE2/RBD inhibition binding activity of spike-specific antibodies was also ob-served, especially in ruxolitimib treated patients. Our results contribute to answer the open question on the induction of the antibody responses in MF patients following vaccination with COVID-19 mRNA vaccines, showing a slow kinetic that support the need for a third dose of SARS-CoV-2 mRNA vaccines.

Onco-hematologic patients have paid a great price to COVID-19 disease caused by pandemic SARS-CoV-2 infection with a mortality rate reaching 34% of infected patients [1, 2] . Immunization with mRNA SARS-CoV-2 vaccines has been highly recommended and prioritized in fragile categories including patients with hematologic malignancies (https://www.cdc.gov/coronavirus/2019ncov/vaccines/recommendations/immuno.html), however consolidated data regarding the efficacy of anti-COVID-19 vaccines in this setting and the need of a third vaccine dose are still lacking.

Patients with myelofibrosis (MF), a clonal hematopoiesis stem cells disorder belonging to the Philadelphia-negative myeloproliferative neoplasms (MPN), have a considerable higher risk of mortality after COVID-19 disease compared to the general population [1] . In addition, SARS-CoV-2 infection elicits an impaired antibody response in this subgroup of MPNs [3] , similarly to other neoplastic hematologic disorders. In many MF patients the myeloproliferative disease can be controlled by the JAK1/JAK2 inhibitor ruxolitinib, a powerful inhibitor of JAK-STAT signaling that deeply reduces inflammatory cytokine production and impairs to some extent cellular immune responses [4] . Indeed, ruxolitinib has been successfully employed in dampening the cytokine storm responsible of fatal acute respiratory distress syndrome following severe COVID-19 infection, as documented by several studies [5, 6] . On the other hand, the sudden interruption of ruxolitinib in MF COVID-19 infected patients has been followed by much higher death rate, probably due to the cytokine rebound following the withdraw of the drug [7] .

Not enough is known about groups that might need a third vaccine dose, such as those with a compromised immune system [8] . The Centers for Disease Control and Prevention (CDC) recommends the third dose for immunocompromised subjects including blood cancer patients under treatment (https://www.cdc.gov/coronavirus/2019-ncov/vaccines/recommendations/immuno.html), while the World Health Organization (WHO) called for a moratorium on boosters until at least the end of the year (https://www.who.int/director-general/speeches/detail/who-director-general-sopening-remarks-at-the-media-briefing-on-covid-19---8-september-2021 and https://www.who.int/news/item/10-08-2021-interim-statement-on-covid-19-vaccine-booster-doses).

Several countries have or are in the process to start the third dose administration in fragile subjects despite the vaccine immune response of each category of fragile subjects has not been fully elucidated. The need and priority of a third vaccine dose in different categories of fragile patients, such as in MF patients, is a burning question, however currently available data on the vaccine All rights reserved. No reuse allowed without permission.

perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in Based on these premises, it is of sure interest to investigate the immune response against SARS-CoV-2 elicited by vaccination in MF patients and the potential active role of ruxolitinib in influencing this response. Up to date, preliminary data of mRNA SARS-CoV-2 vaccination observed in small groups of MPN patients are still controversial. In fact, ruxolitinib was found to interfere negatively with the generation of the humoral immune response after a single dose of mRNA vaccines in 30 MPNs patients (13 with MF) as compared to healthy subjects [9] while in another report ruxolitinib was shown not impairing the serological immune response measured after a not specified distance from second dose of BNT162b2 mRNA vaccine in 10 MF patients [10] . Different antibody responses have been reported among 42 MPN patients, with lower response to mRNA vaccine in patients with MF (10 subjects) compared to subjects affected by polycythemia vera and essential thrombocythemia [11] , while another study conducted on 20 MPN subject reported that patients with a diagnosis of MF (n = 9) had significantly higher post-first dose anti-spike IgG and neutralising antibody titres compared to patients with other MPN subtypes [12] .

Here, we prospectively profiled the spike-specific antibody response and the impact of the treatment with ruxolitinib in 42 consecutive MF patients, referring to our Hematology Units, at two time points were recruited at the Hematology Unit, Azienda Ospedaliera Universitaria Senese (Siena, Italy).

A

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ACE2/RBD inhibition was tested with a SARS-CoV 2 surrogate virus neutralization test (sVNT) kit 

Kruskal-Wallis test, followed by Dunn's post test for multiple comparisons, was used to assess the statistical differences of ELISA titers and ACE2/RBD inhibition percentages among at different All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 23, 2021. ; https://doi.org/10.1101/2021.09.21.21263627 doi: medRxiv preprint groups of subjects for each time point post vaccination. A P-value ≤ 0.05 was considered significant.

Analyses were performed using GraphPad Prism v9 (GraphPad Software, USA).

The antibody response after two doses of mRNA SARS-CoV-2 vaccine was studyed in a cohort of 42 MF patientsand 40 healthy volunteers as controls (HC). 16 Patients received the second dose of mRNA anti SARS-Cov-2 vaccine, 3-4 weeks apart from the first dose, and plasma samples were collected after 7 and 30 days (Fig. 1 ). Plasma samples were tested for IgG against the SARS-CoV-2 spike protein [13] by ELISA and for ACE-2/RDB inhibition activity using a SARS-CoV-2 surrogate virus neutralization test (sVNT). While the spike-specific IgG titres in HC were slightly declining a month after the booster dose (Fig.   2B ), the antibodies elicited in MF patients were still increasing, thus showing a different kinetics of the secondary response, with a slower development of the humoral response in MF patients. The antibody response dynamic observed in these patients suggests a lower immunostimulating effect elicited by the first vaccine dose, as it was reported by two recent publications [9, 11] . In particular, Pimpinelli et al, observed that MF subjects developed a lower humoral response compared to MNP patients with an undetectable primary immune response [11] . All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 23, 2021. ; https://doi.org/10.1101/2021.09.21.21263627 doi: medRxiv preprint ACE2-RBD antibody binding inhibition, a surrogate SARS-CoV-2 neutralization test that qualitatively detects antibodies capable of inhibiting the interaction between the receptor binding domain (RBD) of the viral spike glycoprotein and the angiotensin-converting enzyme 2 (ACE2) protein [14, 15] , was evaluated in plasma collected 7 and 30 days after the second vaccine dose (Fig.   2C ). The percentage of healthy subjects that developed a positive inhibition activity (> 30%) already 7 days after the booster dose was around 98% (39/40), and it was stably maintained at day 30 (Fig.   2D ). Among MF patients, a positive inhibition activity was observed in 14/26 (54%) subjects without ruxolitinib treatment at both time points, while in ruxolitinib-treated MF patients the percentage of subjects developing antibodies with positive inhibitory activity was 18% (2/11) at day 7 and 40% (6/15) one month after the second vaccine dose (Fig. 2D) ..In MF patients, expecially under ruxolitinib treatement,was therefore observed, a reduced ACE2-RBD binding inhibition activity of plasma antibodies compared to HC, even though the percentage of ruxolitinib-treated patients with a positive value increased over time. Taken after two vaccine doses, but the development of the secondary antibody response in these patients showed a slower evolution compared to HC, in contrast with the characteristics of rapidity and intensity typically associated with secondary responses to booster doses, suggesting a reduced capability of their immune system to react to vaccination. This observation is particularly relevant for defining the vaccination schedule more fitting for these patients, suggesting a possible positive role for a third vaccine dose.

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perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in in the different groups. Kruskal-Wallis test, followed by Dunn's post test for multiple comparisons, was used for assessing statistical differences between groups. *P≤ 0.05; ***P ≤ 0.001. Healthy control (HC), myelofibrosis (MF), ruxolitinib (Ruxo).

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in Our results, obtained in a not negligible number of MF patients prospectively studied after two doses of mRNA vaccinations, therefore shed a light on the controversial preliminary observations [9] [10] [11] [12] .

The slower antibody response observed in MF patients, regardless ruxolitinib treatment, may explain the apparent impairment of anti-Spike and neutralizing antibodies production claimed by Guglielmelli et al in MPN patients on ruxolitinib [9] , and measured after only one dose of mRNA vaccines. Given our results, the JAK1/2 inhibitor treatment slightly reduces the spike-specific antibody titers elicited by two vaccine doses (Fig. 2B) , but impacts on their functionality, as observed by the reduced percentage of patients with a positive ACE2-RBD binding activity (Fig. 2D ). To note, none of the 42 MF patients has developed COVID-19 infection after the vaccination so far. On this regard it should be taken into consideration that MF patients "per se" experience a much higher infection rate than other MPNs patients [17] and that on the other hand, ruxolitinib seems to restore impaired monocyte functionality [18] , critical for triggering the adaptive immune responses. Ongoing studies focused on the detection of Spike-specific B memory cells will further shape both the natural and vaccine-mediated anti-COVID response in this subset of fragile patients and will better clarify the grade and persistence of the immune response against SARS-CoV-2 we may expect in mRNA vaccinated MF patients.

Our data show that MF patients develop a humoral response after two doses of mRNA SARS-CoV-2 vaccine even if with a slower kinetics compared to healthy subjects and that a reduced antibody inhibition binding activity is observed expecially with ruxolitimib treatment. Our results therefore contribute to answer the open question on the induction of the antibody responses in MF patients following vaccination with COVID-19 mRNA vaccines and on the impact of ruxolitimib treatment.

This knowledge is of critical importance to assess the need for repeated booster doses of SARS-CoV-2 mRNA vaccines and guide vaccination policies tailored for MF patients, making the case for a third vaccine dose administration. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 23, 2021. ; https://doi.org/10.1101/2021.09.21.21263627 doi: medRxiv preprint

Clinical Characteristics and Risk Factors Associated with COVID-19 Severity in Patients with Haematological Malignancies in Italy: A Retrospective, Multicentre, Cohort Study

Outcomes of Patients with Hematologic Malignancies and COVID-19: A Systematic Review and Meta-Analysis of 3377 Patients

COVID-19 Elicits an Impaired Antibody Response against SARS-CoV-2 in Patients with Haematological Malignancies

Immunological Consequences of JAK Inhibition: Friend or Foe?

Ruxolitinib Rapidly Reduces Acute Respiratory Distress Syndrome in COVID-19 Disease. Analysis of Data Collection From RESPIRE Protocol

The Janus Kinase 1/2 Inhibitor Ruxolitinib in COVID-19 with Severe Systemic Hyperinflammation

High Mortality Rate in COVID-19 Patients with Myeloproliferative Neoplasms after Abrupt Withdrawal of Ruxolitinib

The Most Important Questions

Impaired Response to First SARS-CoV-2 Dose Vaccination in Myeloproliferative Neoplasm Patients Receiving Ruxolitinib

Vaccination with BNT162b2 MRNA COVID-19 Vaccine in Patients with Myelofibrosis

Lower Response to BNT162b2 Vaccine in Patients with Myelofibrosis Compared to Polycythemia Vera and Essential Thrombocythemia

Single Dose of BNT162b2 MRNA Vaccine All rights reserved

doi: medRxiv preprint against SARS-CoV-2 Induces High Frequency of Neutralising Antibody and Polyfunctional T-Cell Responses in Patients with Myeloproliferative Neoplasms

Evidence of SARS-Cov-2-Specific Memory B Cells Six Months after Vaccination with BNT162b2 mRNA Vaccine

Humoral Immunity to SARS-CoV-2 BNT162b2 Vaccine in Kidney Transplant Recipients and Dialysis Patients

A Simple Protein-Based Surrogate Neutralization Assay for SARS-CoV-2

Antibody Responses to SARS-CoV-2 in Patients with COVID-19

Risk of Infections in Patients with Myeloproliferative Neoplasms-a Population-Based Cohort Study of 8363 Patients

The Role of Circulating Monocytes and JAK Inhibition in the Infectious-Driven Inflammatory Response of Myelofibrosis

We would like to thank all the volunteers who participated to the study, the Ematology Unit Nursing staff who chose to cooperate for blood withdrawal. We thank Ludovica Soldateschi, Roberta Vanni and Serena Vastola for the technical support and Gianni Pozzi for critical discussion of the data.

The authors declare no conflict of interest.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 23, 2021. ; https://doi.org/10.1101/2021.09.21.21263627 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 23, 2021. ; https://doi.org/10.1101/2021.09.21.21263627 doi: medRxiv preprint