“Early transfusion of convalescent plasma in older patients with COVID-19 to prevent disease progression: A structured summary of a study protocol for a randomised controlled trial”

Objectives The primary objective is to demonstrate that COVID-19 convalescent plasma (CCP) prevents progression to severe pneumonia in elderly COVID-19 pneumonia patients with chronic comorbidities. Secondary objectives are to demonstrate that CCP decreases the viral load in nasopharyngeal swabs and increases the anti-SARS-CoV-2 antibody titre in recipients. Trial design This is a randomized, open-label, parallel group, phase II/III study with a superiority framework. The trial starts with a screening phase II designed with two-tailed alpha=0.2. In case of positive results, the trial will proceed in a formally comparative phase III (alpha=0.05). Participants Adult patients with confirmed or suspected COVID-19 who are at risk according to CDC definition are eligible. Inclusion criteria are all the following: age ≥ 65; pneumonia at CT scan; PaO2/FiO2 ≥300 mmHg; presence of one or more comorbidities; signed informed consent. Exclusion criteria are one of the following: age < 65; PaO2/FiO2 < 300 mmHg; pending cardiopulmonary arrest; refusal to blood product transfusions; severe IgA deficiency; any life-threatening comorbidity or any other medical condition which, in the opinion of the investigator, makes the patient unsuitable for inclusion. The trial is being conducted at three reference COVID-19 centres in the middle of Italy. Intervention and comparator Intervention: COVID-19 Convalescent Plasma (CCP) in addition to standard therapy. Patients receive three doses (200 ml/day on 3 consecutive days) of ABO matched CCP. Comparator: Standard therapy Main outcomes A. Primary outcome for Phase II: Proportion of patients without progression in severity of pulmonary disease, defined as worsening of 2 points in the ordinal scale of WHO by day 14. B. Primary outcome for Phase III: Proportion of patients without progression in severity of pulmonary disease, defined as worsening of 2 points in the ordinal scale of WHO by day 14. Secondary outcomes for Phase III: Decreased viral load on nasopharyngeal swab at days 6, 9 and 14; Decreased viremia at days 6 and 9; Increased antibody titer against SARS-CoV2 at days 30 and 60; Proportion of patients with negative of SARS-CoV2 nasopharyngeal swab at day 30; Length of hospital stay; Mortality rate at day 28; Total plasma related adverse event (day 60); Total non-plasma related adverse events (day 60); Severe adverse events (SAE) (day 60). Randomisation Treatment allocation is randomized with a ratio 1:1 in both phase II and phase III. Randomization sequences will be generated at Fondazione Policlinico Gemelli IRCCS through the RedCap web application. Randomized stratification is performed according to age (under/over 80 years), and sex. Blinding (masking) None, this is an open-label trial. Numbers to be randomised (sample size) Phase II: 114 patients (57 per arm). Phase III: 182 patients (91 per arm) Trial Status The trial recruitment started on May 27, 2020. The anticipated date of recruitment completion is April 30, 2021. The protocol version is 2 (May 10, 2020). Trial registration The trial has been registered on ClinicalTrials.gov (May 5, 2020). The Identifier number is NCT04374526 Full protocol The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. Supplementary information Supplementary information accompanies this paper at 10.1186/s13063-020-04821-1.


BACKGROUND.
Blood products collected from convalescent donors have been frequently adopted to provide passive immunization to patients with life threatening diseases. In general, this approach is used when there are no specific vaccines or drugs available for emerging infection-related diseases, namely due to viruses. 1 Different types of blood products may be used to convey passive immunity, including whole blood, plasma, or serum. 2 In addition, immunoglobulins and high-titer immunoglobulins can be obtained from further plasma processing. 2

Convalescent plasma in past outbreaks.
A meta-analysis of Spanish influenza A (H1N1) cases suggested that patients with pneumonia who received influenza-convalescent human blood products seemed to have a reduction in the risk for death. 3 9 In the latter condition a multicenter, prospective cohort study, showed that patients receiving convalescent plasma from recovered donors within 5 days of symptom onset had lower viral load and reduced mortality 9 Regarding Ebola, after preliminary positive results, WHO endorsed in 2014 the use of convalescent plasma or serum under blood regulatory authorities. 10,11 The prospective controlled study carried out by the Ebola-Tx Consortium reported that transfusing up to 500 ml of convalescent plasma with unknown levels of neutralizing antibodies in 84 Ebola virus patients was not associated with a significant reduction of mortality. 12 A subsequent study sought to determine whether the level of neutralizing antibodies in different plasma donations could have influenced the response and survival of treated patients. 13 Although more than 90% of donations contained high IgG titers (> 1:1000) at enzyme-linked immunosorbent assay (ELISA), a titer of 1:160 at neutralizing test was found in only 5% of donations. 13 Therefore, in this study, neither viral load or mortality were associated with the dose of neutralizing antibodies received. 13 Positive results in two critically ill patients affected from Middle East respiratory syndrome coronavirus (MERS-CoV) were related to the presence of high neutralizing antibody titers in the donor plasma. 14 A recent meta-analysis gathering data from 32 studies, suggested that the administration of convalescent plasma or serum is safe and able to reduce mortality in SARS of viral etiology H1N1 and H5N1). 15

Experiences in COVID-19 pandemia.
Three studies have so far reported the use of convalescent plasma in critical COVID-19 patients. 16,17 The first case-series included 5 patients (1 with pre-existing cardiovascular comorbidities) in mechanical ventilation, with a median interval between admission and plasma transfusion of 20 days. All patients had previously received antiviral drugs and methylprednisolone. The titer of neutralizing anti SARS-CoV-2 in donated plasma ranged from 1:80 to 1:480 and the titer of specific anti-SARS-CoV-2 antibodies (anti receptor binding domain IgG and IgM) was higher than 1:1000. Each patient received 400 ml of fresh plasma from a single donor. All treated patients were viremic at the time of plasma infusion and the viral load declined thereafter, along with the clinical recovery. 16 The second study reports 4 cases of COVID-19 in critically ill patients. Three of them had comorbidities (cardiovascular disease, chronic broncopneumopathy, chronic renal failure), and one patient was pregnant. All 4 patients were intubated, received anti-infectious agents for documented bacterial and/or fungal coinfections, and had been treated with additional therapies before plasma infusion (antivirals including interferon, immunoglobulins and/or methylprednisolone). The convalescent plasma was given at variable doses (from 200 ml to 2400 ml). In all patients a progressive decline of viremia was documented after plasma infusion. 17 The third study is under review and is unpublished so far (it is available at the MedRxiv website, a free online archive for complete unpublished and not peer reviewed manuscripts in the medical, clinical, and related health sciences). 18  and exaggerated pro-inflammatory and anti-inflammatory cytokines (IL-2R, IL-6,TNF-α and IL-10) suggest that disease severity and outcome of COVID-19 is due to the SARS-CoV-2-induced cytokine storm. 36 Sars-CoV-2 seems able to induce a functional exhaustion of specified T and NK lymphocyte subpopulations, breaking down antiviral immunity. 37 One possible explanation is that the immune system of elderly people, exposed to chronic stimulation associated with comorbidities, might be more susceptible to this Sars-CoV-2 effect. 37 As a result, in these patients, the activation of the innate immune system might fail to produce an adequate adaptive response (i.e., virus-specific CD8+ T-cells). 38 This could result in a persistent self-induced inflammation that then causes mortality. 38 We therefore hypothesize that transfusing the convalescent plasma containing neutralizing antibodies at an early phase of disease could prevent or switch-off the persistent inflammatory response elicited by the virus, before that cytokine storm exerts devastating effects. As a proof of concept that CCP has an activity on the infection, in enrolled patients we will assess the viral load in blood and nasopharyngeal swab before and plasma infusion.

INTERVENTIONS Treatment
Patients will be randomized 1: 1 to receive one of the following treatments

Stopping rule
The study will be discontinued if 6 (10%) trial participants in the arm A will have a severe adverse event connected with plasma infusion (Appendix B).

Concomitant therapies
Patients enrolled in this study receive standard therapies in addition to CCP. The enrollment of participants in other interventional trials is not allowed.

Phase II Primary outcome
 Proportion of patients without progression in severity of pulmonary disease defined as worsening of 2 points in the ordinal scale of WHO within day 14.
The WHO ordinal scale to assess the disease progression is provided in Appendix C.

Secondary outcome
 Decreased viral load on nasopharyngeal swab (days 6 and 9) Phase III Phase III of the study will start soon after completing the analysis of data relative to phase II and if the primary outcome of phase II is met.

Primary outcome
 Proportion of patients without progression in severity of pulmonary disease defined as worsening of 2 points in the ordinal scale of WHO within day 14.

Secondary outcomes
 Decreased viral load on nasopharyngeal swab (days 6, 9 and 14)  Decreased viremia at day 6 and 9  Increased antibody titre against SARS-CoV2 (days 30 and 60) Appendix C provides the definitions for adverse events and severe adverse events.

Timeline
The study timeline is shown in the Gantt diagram (Appendix D)

Procedures and visits
A study flow-chart is shown in Appendix E. Plasma administration: days 1,2, 3. All patients will sign the informed consent to receive blood products in use at each center. is a secure, web-based application designed to support data capture for research studies, providing 1) an intuitive interface for validated data entry; 2) audit trails for tracking data manipulation and export procedures; 3) automated export procedures for seamless data downloads to common statistical packages; and 4) procedures for importing data from Data management Each participating site must maintain appropriate medical and research records for this trial and regulatory/institutional requirements for the protection of confidentiality of study subjects. The Principal Investigator is responsible for assuring that the data collected are complete, accurate, and recorded in a timely manner.

Statistical methods Primary endpoint:
Based on the above-mentioned design, the comparison of proportion of progression free patients will be made between A vs B through the Fisher exact test at the completion of phase II and phase III.

Secondary endopoints
Descriptive results will be presented as means ± standard deviation (SD), medians with interquartile range (IQR), and percentages with 95% confidence intervals (CI). Chi-square or Fisher's exact test will be used to compare categorical variables. ANOVA and Student's t-test will be used for continuous variables unless they will be not normally distributed, in which case the Kruskal-Wallis or Mann-Whitney test will be used to compare continuous parameters. Kaplan-Meier product-limit estimates will be used to calculate time to different secondary endpoints. The logrank test will be used to assess the difference between the survival curves among arms. Changes in some biomarkers respect to baseline values will be compared using paired Wilcoxon test. The association between primary outcome and transfusion dose of CCP expressed as ml/kg of the recipient body weight, adjusted for anti-Sars-CoV-2 neutralizing and ELISA antibody concentrations, will be investigated through regression analysis. All tests will be two-sided and a P value inferior to 0.05 will be regarded as significant.

Analysis settings
Analysis will be carried out in the "intention to treat" set.

Data monitoring
Each study site agrees to allow monitors from Monitoring Unit of Fondazione Policlinico Gemelli (FPG) IRCCS direct access to the study records and medical records from those patients enrolled in the clinical study. In accordance with the applicable regulations and good clinical practice (GCP), the monitor shall periodically contact the center. The duration, nature and frequency of such visits/contacts shall depend on the rate of recruitment, the quality of the documents in the possession of the center, and its adherence to the protocol.
Through these contacts, the monitor must: control and evaluate the progress of the study, examine the collected data, conduct Source Document Verification (SDV), identify every problem and find solutions.
The aims of the monitoring activity are to verify that: the rights and wellbeing of the subject are respected, the study data are accurate, complete and verifiable by original documents and the study is conducted in accordance with the protocol and any approved amendments, GCP and the applicable regulations.

Research ethics approval
The study will be conducted with the approval of the Ethics Committee,

after verification of compliance with the European Union Clinical Practice
Standards and in accordance with ICH Good Clinical Practice (GCP) and the ethical principles expressed in Declaration of Helsinki. The study will be carried out adhering to local legal requirements and the applicable national law, whichever represents the greater protection for the individual. Study protocol, patient information and informed consent will be submitted to the appropriate Ethical Committee for approval. Will inform the Ethical Committee about any changes in the study protocol which could interfere with the patient's safety.

Protocol amendments
Any protocol amendments will be communicated (e.g., changes to eligibility criteria, outcomes, analyses) to investigators, EC/IRBs, trial participants, trial registries, and regulators.

Informed consent for patients
The participant, adequately informed in clear, simple and understandable words of the technical terms used, will be invited to provide written informed consent. The participant will be provided with a description of the general aims of the research, the methodology and procedures used, the indication of any benefits or possible risks and adverse effects. In addition to the consent to participate to the study, all patients will sign the consent to receive blood products in use at each center. The physicians treating the hospitalized patient are responsible for information of the patient and obtaining of the Informed Consent. The consent will be expressed orally: two different witnesses will state that the patient was properly informed and fully understood study aim and procedures, and will sign the consent. In the event that the interested party revokes consent to the processing of data for research purposes, the biological sample taken for such purposes would also be destroyed. Blood samples will be collected during routinely performed samples during the investigations necessary for the pathology in progress. Participation in the research will not entail any additional costs for the participant. Should a medical problem arise due to the study, the participant will be provided with the most appropriate treatment. In accordance with the law on the The informed consent for patients is provided in Appendix F.

Informed consent for donors
An informed consent, asking for their availability to be contacted by the medical staff of transfusion centers, will be provided to patients not enrolled in this study who are discharged from hospital after COVID-19.
These patients will be informed about the possibility to donate their plasma and modalities of plasma donation. If they agree to participate to the study, they will be contacted after discharge and plasma donation will be arranged if their donor suitability will be confirmed.
The informed consent for patients is provided in Appendix G.

Confidentiality
All subject related information including Case Report Forms, laboratory specimens, evaluation forms, reports, etc. will be kept strictly confidential.
All records will be kept in a secure, locked location and only research staff will have access to the records. Subjects will be identified only by means of a coded number specific to each subject. All computerized databases will identify subjects by numeric codes only, and will be password protected.
Upon request, subject records will be made available to the study audit, monitoring representatives of the study promoter, or representatives of regulatory agencies (CNS).

Access to data
Only people officially registered as study investigators or data managers will receive a user login to access the REDCap web platform and enter/manage data. Source documentation should support the data collected on the CRF's. Source documents include all recordings of observations or notations of clinical activities and all reports and records necessary for the evaluation and reconstruction of the clinical

Insurance
After study approval by EC, the sponsor will stipulate an insurance policy with Lloyd's Insurance Company S.A to cover all risks connected with plasma therapy.

Dissemination policy
Investigators and sponsor will communicate trial results to participants, healthcare professionals, and the public through scientific publications.
Authorship eligibility will be defined according to ICMJE guidelines. Adverse events include:

APPENDICES
• The exacerbation of a pre-existing pathology; • An increase in the frequency or intensity of an episodic event or preexisting condition; • A condition occurring or diagnosed after the administration of the study drug, even if current before the start of the study; • Persistent diseases/symptoms at the baseline visit that worsen after the start of the study.
Adverse events do NOT include: • Medical or surgical procedures (e.g. surgery, endoscopy, tooth extraction, transfusions), but the condition requiring the procedure is an adverse event • Diseases or conditions present at the beginning of the study that have not worsened, but remained stable during the course of the study.
• Situations in which no unexpected adverse event has occurred (e.g. hospital admission for elective cosmetic surgery/social problems).
• An overdose of antiviral agents or concomitant drugs without onset of symptoms or associated signs.
• Laboratory abnormalities deemed by the investigator to be of no clinical significance.
Adverse events will be graded according with CTCAE v5.0. (Appendix B) Grade 1 and Grade 2 events are not considered adverse events, but details of these events must be documented in detail in the subject's study files.
Stable chronic conditions which are present prior to clinical trial entry and do not worsen are not considered adverse events and will be accounted for in the subject's medical history. Although an adverse drug event may rate only as "possibly related" soon after discovery, it can be flagged as requiring more information and later be upgraded to "probably related"

Serious Adverse Events (SAE's
or "definitely related", as appropriate. Notification deadlines: • if, in addition to being serious and unexpected, a SAE is also fatal or lifethreatening, a preliminary SAE report must be completed as soon as possible and, in any case, within 24 hours after being informed about the event.
• For all other serious and unexpected adverse events, the investigator must complete a SAE report as soon as possible after the manifestation of the event and, in any case, no later than 15 days after becoming aware of the event.
Monitoring of AE'S/SAE'S. Any AE that occurs between the times a study participant signs the informed consent form and the time s/he departs the study at the end of the final follow-up visit (or at the time of early discontinuation of the subject from the study for any reason) will be captured and recorded. At each contact with the subject, the investigator (or designate) must seek information on adverse events by specific questioning and, as appropriate, by examination.
All AEs and SAEs must be followed up: • until their complete resolution Unexpected' adverse reaction -Definition: Article 2(p) of Directive 2001/20/EC defines 'unexpected adverse reaction' as follows: 'an adverse reaction, the nature or severity of which is not consistent with the applicable product information (e.g. investigator's brochure for an unauthorised investigational product or summary of product characteristics for an authorised product)'.
The term 'severity' is used here to describe the intensity of a specific event. This has to be distinguished from the term 'serious'. Reports which add significant information on the specificity, increase of occurrence, or severity of a known, already documented serious adverse reaction constitute unexpected events