Novel protocol for selection of SARS-CoV2 convalenscent plasma

Introduction. SARS-CoV2 2019 infection represent global problem. At this
 moment there is no vaccine or efficient treatment of infected patients.
 Treatment with blood plasma rich with anti SARS-CoV2 specific antibodies
 represents rare safe and effective treatment of Covid19 patients. Patients
 and Material. A total number of 950 patients were analyzed in this study,
 whose samples were collected in time interval from 01.05. till 15.08.2020.
 Patients were enrolled in study from Covid-19 hospitals, out-clinics and as
 family members of SARS-CoV2 infected patients. Original ELISA tests were
 developed to measure the concentration of anti-S1S2 Spike and
 anti-Nucleoprotein (IgG, IgA, IgM) SARS-CoV2 antibodies. Blood convalescent
 plasma was selectively collected from recovered patients according to
 specific antibodies concentration. Results. The highest concentrations of
 anti-S1S2 Spike or anti-Nucleoprotein specific IgG antibodies were detected
 in patients with the moderate/heavy clinical form of infection as well as in
 group of family members of SARS-CoV2 infected patients. Extremely high
 concentration of anti S1S2 Spike IgG and anti-Nucleoprotein IgG was
 demonstrated in 3% and 6% (respectively) of patients recovered from severe
 Covid19. From our hospitalized patients 63% and 51% had modest antibody
 levels (anti S1S2 Spike and anti-Nucleoprotein, respectively). After 60 days,
 in our selected donors? concentration of anti S1S2 Spike IgG antibodies
 increased in 67%, paralleled with increase of anti-Nucleoprotein IgG
 antibodies in 58% of donors. Conclusion. Originally developed ELISA tests
 enable novel protocol for selection of convalescent blood plasma donor.


INTRODUCTION
To date, there are no drugs approved by the US Food and Drug Administration (FDA) for the treatment of patients with COVID-19. Current clinical research includes measures for infection prevention and control and, also, supportive care, including oxygen and mechanical ventilation support when necessary. Myriad of drugs that have been approved for other indications, are used, as well as a variety of new drugs whose effects are being studied in several hundreds of clinical trials that are underway around the world.
Scientists around the world are working tirelessly, and information about the mechanisms of transmission, the clinical spectrum of the disease, new diagnostics and strategies for prevention and therapy is spreading rapidly. In general, there are many unknowns regarding the virus-host interaction, the development of the epidemic, the possibilities and success of treatment, and any research in this field is extremely important.
The significance of the corona virus disease pandemic 2019 (COVID-19), due to the new severe acute respiratory syndrome of corona virus 2 (SARS-CoV-2), caused a sudden and significant increase in the number of hospitalizations in the world due to pneumonia with multiorgan disease. SARS-CoV-2 is spread primarily by respiratory droplets -close face-to-face contact. The infection can be spread by asymptomatic, presymptomatic and symptomatic carriers. The average time from exposure to the virus to the onset of symptoms is 5 days, and 97.5% of people develop symptoms within 11.5 days. The most common symptoms are high fever, dry cough and difficulty of breathing. Radiographic and laboratory abnormalities, such as lymphopenia and elevated lactate dehydrogenase, are common but nonspecific. The diagnosis is made by detecting SARS-CoV-2 by PCR (polymerase chain reaction), although false-negative test results can occur in 20% to 67% of patients, depending on the quality and timing of the test. The disease may have an asymptomatic or fulminant course, characterized by sepsis and acute respiratory failure. Approximately 5% of patients with COVID-19, ie. 20% of those hospitalized have serious symptoms that require intensive care. More than 75% of patients hospitalized with COVID-19 require additional oxygen, which includes the best therapeutic measures for the treatment of acute hypoxic respiratory failure (1).
In current trials, antiviral therapy, immune modulators, and anticoagulants are being tested.
The death rate from COVID-19 varies significantly depending on age, ranging from 0.3 deaths per 1,000 patients (age 5 to 17 years) to 304.9 deaths per 1,000 cases in patients aged 85 years or older in the United States. Among patients hospitalized in the intensive care unit, the mortality rate is up to 40%. At least 120 SARS-CoV-2 vaccines are being prepared. Until an effective vaccine is available, the primary methods for reducing the spread are face masks, social distancing as well as the application of all therapeutic modalities (1).
Since some 10% of patients fail to cope with this disease despite the applied measures, However, a subsequent multicenter, randomized clinical trial in China of 103 patients with severe COVID-19 found no statistical significance in the time to clinical improvement within 28 days in patients receiving convalescent plasma compared to standard treatment (51.9% compared to 43.1%) (4). Since the study was discontinued, this limited the possibility of detecting a clinically important difference. Alternative approaches include the use of convalescent hyperimmune globulin produced from plasma and monoclonal antibodies directed to SARS-CoV-2 (5-6).
The virulence of a particular virus is often considered related with the immune response it encounters in the human body. For Covid-19, the immune response is divided into two phases. The initial phase is thought to involve the development of a specific adaptive and immune response needed to eliminate the virus and stop the disease from progressing. It is therefore important to provide treatments that have previously stimulated an immune response, such as antibodies and immunomodulators (2,6); there may be a loss of 20% of its own antibodies, that will resume a few days later. Immune response, however, weakens with age, making the elderly particularly vulnerable. If the immune response is weak or damaged due to other complications such as cardiovascular disease and diabetes, the virus multiplies and can lead to tissue damage. The second phase of the immune response leads to damage of the cells that caused the pneumonia. Such infection is very dangerous, since pneumonia causes respiratory disorders, making it difficult for individuals to breathe on their own. Different therapies are tested in different phases of the disease, making it important to identify exact phase of the patient's disease, before starting treatment (1).
The World Health Organization warns that there is no evidence that the presence of antibodies means that you are protected from reinfection with Covid-19. The level of immunity and how long the immunity lasts are still unknown. Ongoing studies will eventually reveal more data on this.
Due to all of the above, any research related to Covid-19 therapy is crucial, including the collection and administration of convalescent's plasma to patients.
Patients with resolved viral infection will develop an immune response with polyclonal antibodies to various CoV-2019 viral antigens, and some of these polyclonal antibodies, if the patient has them in high titers, will probably neutralize the virus and prevent more severe forms of infection. Doctors are trying to use this fact in a therapeutic sense, and that is why the idea of collecting plasma (rich in antibodies) from the convalescents after Covid came about (1-13).
The potential danger of using such plasma in terms of side effects in recipients, including but not limited to allergic reactions, acute lung injury, and circulatory overload in patients with cardiac disorders, must also be mentioned here (14)(15)(16).
Our institution also worked on testing the antibody titers in patients who overcame Covod-19 and developed its own protocol for collecting plasma by manual technique using a system of multiple bags. Since plasma transfusion is a routine medical procedure, no new medical approvals are required to perform it. In fact, the same basic concept was used to treat several Ebola patients with convalescent serum during the 2014-15 epidemic.

MATERIAL AND METHODS
Specific antibody detection. Specific anti-S1S2 SARS CoV2 antibodies and anti-Nucleoprotein SARS CoV2 antibodies were quantified with home-based ELISA test.

Average anti SARS-CoV2 antibody concentration in investigated groups.
As previously explained, we have quantified specific anti S1S2 Spike and anti-Nucleoprotein antibodies in Covid-19 patients with heavy or mild clinical picture. All our hospitalized patients demonstrated heavy (but not critical) clinical presentation, with average 11 days of hospital treatment. As expected, sera from hospitalized patients contained significantly more anti S1S2 Spike specific IgG (Figure 1A.)

Selection of donor's convalescent plasma according to concentration of SARS-CoV2 specific antibodies.
Widely accepted criteria for selection of convalescent plasma donors are levels of specific anti SARS-CoV2 IgG antibodies, most frequently antibodies to envelope antigens. In our group of hospitalized patients 225 had detectable anti S1S2 Spike specific IgG antibodies, while 202 had detectable anti-Nucleoprotein IgG antibodies. According to our data, from group that presented severe Covid19, 3% of patients demonstrated extremely high concentration of anti S1S2 Spike IgG and 6% demonstrated extremely high concentration of anti-Nucleoprotein IgG antibodies, that measured more than 1000 EU/ml (Table 1.).
Further 8% had high anti S1S2 Spike response and 15% had high anti-Nucleoprotein IgG levels, that measured in hundreds EU/ml. Specific anti SARS-CoV2 antibody response reflected in levels between 10-100 was detected in 26% and 28% (anti S1S2 Spike and anti-Nucleoprotein, respectively). Finally, 63% and 51% of our hospitalized Covid19 patients had modest antibody levels, ranging from 5-10 EU/ml (anti S1S2 Spike and anti-Nucleoprotein, respectively). Patients that demonstrated level of specific anti SARS-CoV2 antibodies of IgG class in hundreds or thousands were selected as convalescent plasma donors. Several patients with high concentration of specific anti SARS-CoV2 antibodies had to be excluded as potential donors according to medical indications (cardiovascular disease, hemophilia).

Time related concentration change of SARS-CoV2 specific antibodies in samples of convalescent plasma donors.
Until the end of August we have selected and collected plasma rich with specific antibodies from 12 people (12/225, 5.3% of patients cured from severe Covid19). All of these patients had at least two points of specific antibody measurements, with time interval no less than 60 days. Interestingly, concentration of anti S1S2 Spike IgG antibodies increased in 67% of our plasma donors, paralleled with increase of anti-Nucleoprotein IgG antibodies in 58% of donors (Table 2.). Concentration of anti S1S2 Spike and anti-Nucleoprotein of IgA class increased in 50% of all donors. Interestingly, while anti S1S2 Spike IgM concentration decreased in donors, concentration of anti-Nucleoprotein IgM antibodies again was increased in our plasma donors.

DISCUSSION
Convalescent plasma has a strong historical advantage and good biological value. Although this therapeutic approach is promising, it has not yet been shown to be safe in the treatment of COVID-19. Data after transfusion of ABO-compatible human convalescent plasma COVID-19 to 5,000 hospitalized adults with severe or life-threatening COVID-19, 66% of which in the intensive care unit were analyzed. The incidence of all serious adverse events, including mortality (0.3%), in the first 4 hours after transfusion was <1%. Of the 36 reported, 25 were convalescent plasma related, including mortality (n = 4), circulatory overload associated with transfusion (n = 7), acute lung injury associated with transfusion (n = 11), and severe allergic reaction after transfusion (n = 3). However, physicians estimate that only 2 of 36 reactions are definitely associated with convalescent plasma transfusion. Mortality rate after 7 th day was 14.9%. Given the lethal nature of COVID-19 and the large population of critically ill patients included in these analyzes, the mortality rate does not appear to be too high. These early indicators suggest that convalescent plasma transfusion is safe in hospitalized patients with COVID-19 (13)(14)(15)(16).
In addition to the antiviral mechanisms of neutralizing antibodies, the immunomodulatory effects of plasma components may be beneficial. Several small and large studies have shown the effects of convalescent plasma for the treatment of severe viral illness. Plasma transfusion can cause minor adverse events such as fever, nausea, allergic reactions, bloodborne pathogens transmission and some serious adverse events such as acute lung injury (TRALI), transfusion-associated circulatory overload (TACO) and antibody-dependent enhancement (ADE (13)(14)(15)(16)(17)(18)(19).
In June 2020, the U.S. Department of Defense began an action to collect plasma units from patients who had fully recovered from COVID-19, in order to support the development of effective treatment. The goal is to collect 10,000 units of this plasma until 10.09. 2020 (20).
There are currently no licensed vaccines or targeted therapies against the virus itself.
Plasma with anti-SARS CoV-2 antibodies, obtained from recovered individuals in whom COVID-19 has been confirmed, have begun to be collected using apheresis devices and stored in blood banks in some countries to be administered to patients with COVID-19 in order to reduce the need for intensive care and a lower mortality rate. Therefore, it is necessary to point out some important issues related to convalescent plasma and its use in the treatment of patients as a form of anti-viral therapy. The protective effect can last for weeks and months. After the donor's assessment, 200-600 mL of plasma can be collected with apheresis devices (which are used in the world and in our country). The donation interval may vary between countries. Hence the necessity of testing antibody titer values.
Although limited published studies are not prospective or random, until vaccination or targeted antiviral therapy is approved, plasma therapy appears to be a safe and likely effective treatment for critically ill patients with COVID-19. It can also be used for prophylactic purposes, but the safety and efficacy of this approach should be tested in randomized clinical trials and a conclusion reached (21).
Eligibility criteria for plasma donors may vary from country to country, but certainly include, above all, a safe procedure, health and antibody titers checks, and consent to the procedure. The antibody titer will vary according to the duration between the time of collection and the onset of infection. In previous studies, it has been observed that seroconversion occurs between 8 and 21 days after the onset of symptoms. In clinical trials, initially one plasma unit was given (200 mL) and repeted after 12 hours. The duration of antibody efficacy is not known, but it is estimated that it will last for weeks to several months (21)(22)(23)(24)(25)(26)(27). On August 23, 2020, the Prospective, well-controlled, adequately initiated randomized trials are needed to determine whether convalescent plasma is effective and safe for the treatment of COVID-19.
All this confirms the fact that it is necessary to collect and store certain quantities of convalescent plasma in order to provide reserves, which encouraged us to provide plasma reserves for the treatment of COVID-19 patients with modest funds and without additional costs.

CONCLUSION
Originally developed ELISA tests enable novel protocol for selection of convalescent blood plasma donor. According to our data, it is necessary to regrute and test a large number of patients recovered frome severe Covid19 in order to have sufficient number of appropriate convalescent plasma donors.