**4. Significance of convalescent plasma therapy during SARS-CoV and MERS-CoV outbreaks**

Earlier, CP therapy was used to treat similar coronavirus outbreaks, i.e., SARS and the MERS in the last two decades (**Table 2**).

#### **4.1 SARS-CoV outbreak**

The first outbreak of the twentieth century that surfaced from China in 2002 led to severe respiratory illness and pneumonia-like symptoms in patients. A case study published in 2003 reports that CP administration as an adjunctive treatment alongwith ribavirin and prednisolone in a 57-year-old SARS patient on Day 14 reduces the viral load and fever within a few days. Further, a better resolution of a basal lung infiltrate was seen in chest X-ray post- convalescent plasma treatment [40]. A group of Hong-Kong-based researchers, Soo et al. (2004) and Cheng et al. (2005), first reported using convalescent plasma as an emergency therapy to contain this outbreak. They found that patients treated with steroids and CP had low mortality and got early discharge from the hospitals compared to patients treated with steroids alone [15, 37, 41].

Further, they have found that patients with early administration of convalescent plasma (before 14 days of symptoms onset) have shorter hospital stay and have less mortality (p = 0.08) than those who received convalescent plasma after 14 days [15]. A similar study by Yeh et al*.* reports about the beneficial effect of CP administration in SARS infected Health-workers who were severely ill and showed no response to available antiviral treatments. The CP administration in them resulted in a reduced viral load and fever within a day, followed by resolution of pulmonary infiltrates and a time-dependent increase in anti-SARS-CoV IgM and IgG antibodies [36]. In conclusion, early administration of convalescent plasma led to better patient outcomes in terms of fast recovery, short hospital stays, reduction in viral load, and improvement in clinical symptoms.

#### **4.2 MERS-CoV outbreak**

This outbreak took place due to another strain of respiratory illness causing coronaviruses, referred to as MERS, pointing to its origin from central-east Asia (Arabian countries). From the prior experience of using CP during SARS-CoV outbreak, passive immunotherapy by administering NAb in patients serves as a vital tool in battle with this disease. Chan et al. in 2012, found that convalescent sera of previously recovered SARS patients have cross-reactive NAb that can work effectively against novel coronavirus strains [42]. Corti et al. in 2015, based on anecdotal evidence, studied the prevalence of MERS specific antibodies in dromedary camels from the middle east (Oman) and European countries (Netherland and Chile). They found that all Omanian camels in their study group had MERS-CoV-specific NAb in their serum compared to European animals,


#### **Table 2.**

*List of studies describing the significance of CP therapy during SARS-CoV and MERS-CoV outbreaks.*

highlighting the importance of passive immunotherapy upon successfully detecting the transmission source. These antibodies of CP have the potential to neutralize the MERS-CoV if administered in the patients [38]. The effectiveness of CP was also reported by van Dorelman et al. in 2017, in common marmosets infected with MERS-CoV. They found that marmosets treated with hyperimmune plasma and m336 (monoclonal antibodies) showed a reduction in viral load and overall severity [39]. However, the use of MERS-CoV antibodies is very challenging, as some studies report that they are produced at a low level and are short-lived in mild infections [43, 44].

#### **5. Mechanism of action of convalescent plasma**

Convalescent plasma to be donated contains specific antibodies for particular infectious diseases or pathogens. These antibodies possess neutralization activity. This activity is achieved in different ways: either by impeding the binding of viral particles with the endosomes, by hindering the viral protein proteolytic cleavage, by blocking the release of viral progeny, or by inhibiting the viral surface glycoproteins from invading the human cells [45]. A report published by Lu *et al.* suggests that when a neutralizing antibody (NAb-3BNC117) was passively administered on mice model, it helped block NAb helped block the new infection, enhanced clearance of the infected cells, and accelerated the HIV-1 infected cell clearance [46].

The other mode of action includes antibody-dependent-cellular-phagocytosis (ADCP), complement system activation, and antibody-dependent cellularmediated cytotoxicity (ADCC). CP antibodies induce clearance of virusinfected cells by ADCP. A cross-talk is established among the CP antibodies, which helps in eliciting the Fc-dependent effector functions. The activated complement system helps eliminate the virus by two means. First, by direct means i.e., through complement dependent cytotoxicity. Second, indirect means, i.e., by phagocytosis, help clear the associated complement targets. In the case of nCoV-2019, the recovered patients may develop serum antibody response (IgG) against various virus epitopes [1]. This IgG competes with the angiotensin-converting enzyme-2 receptor (ACE-2) to bind with the virus receptor-binding domain (RBD) and may neutralize the nCoV-2019 infection. Therefore, in this case, the binding domain acts as both, i.e., the epitope for antibody and a binding site for the receptor enzyme [2]. Literature suggests that when CP therapy is administered at an early stage of nCoV-2019 infection, the therapeutic effect may be more effective [47]. In most viral infections, the peak of viremia starts appearing in the early first week of the illness. However, between days 10–14 or early in some cases, the host primary immune response starts exerting its potential effect [47].

### **6. Use of CP therapy on nCoV-2019 patients**

In the absence of any specific treatment of nCoV-2019 and vaccines with proven long-term results, CP administration comes to the rescue for an effective treatment for critically ill nCoV-2019 patients (**Tables 3** and **4)**. This is a kind of passive immunization method where CP from a recovered nCoV-2019 individual is obtained and uses them on diseased individuals to resolve the symptoms and reduce disease course by suppressing the viremia [18]. The use of passive immunotherapy is a widely used approach to combat various infectious diseases, which consisted of various formulations such as whole blood, pooled human sera containing immunoglobulin and convalescent plasma. Plasma collected through apheresis with subsequent CP transfusion is the most commonly used passive immunotherapy approach to battle against pandemics happened earlier [24]. Theoretically, a person who got infected from an infectious disease, upon recovery, blood is screened for NAb specific to the causative pathogen suffered earlier. The convalescent plasma containing a high-titer NAb is used as a therapy to maximize the capacity to neutralize an infecting pathogen (**Figure 1**) [56].

Studies from the current nCoV-2019 pandemic suggested that it elicits a robust immune response resulting in cytokine storm which generates high levels of IgM and IgG mostly that persists for months even after the symptom disappears. Thus, a large window period and maximum chances of successful extraction of high titer anti-SARS-CoV-2 immunoglobulins act as a boon to utilize it as passive immunotherapy [57, 58]. Further studies have elaborated on NAb response. More severe disease may lead to higher antibody response levels [58], and antibody level decreases considerably within the first 90 days after symptom start among individuals suffered from the mild disease [59].













**Table 3.**

*List of completed CP based Clinical trials registered on ClinicalTrial.gov nCoV-2019 patients.*



**Table 4.** *Status of mortality rate after CP administration in hospitalized nCoV-19 patients (studies with sample size >100).*

**Figure 1.**

*Diagrammatic representation of the use of CP in nCoV-2019 patients.*

#### **6.1 Source/donor requirement**

In order to overcome various challenges for enrolment of successful plasma donors during the outbreak different strategies such as social distancing, travel restrictions, and imposed lockdowns have been implemented. To recruit possible plasma contributors, this includes donor self-identification, social awareness utilizing social/formal/e-media outlets and clinician referral of individuals got previous exposure to the infection [60]. NAb titres can be examined in possible donor or CP units through ELISA/chemiluminescence assay or pseudovirus neutralization assays which are known as indirect methods or directly under biosafety level 3 conditions by using live SARS-CoV-2 neutralization assays. USFDA has permitted the use of CP therapy under the clause of EUA for hospitalised nCoV-2019 infected patients [61]. CP units were categorized as lower or higher antibody titre based on qualitative chemiluminescent immunoassay for detection of neutralizing IgG against SARS-CoV2 spike protein [60]. The collected plasma is treated for pathogen inactivation to avoid the risk of transfusion transmitted infections. A donor can donate CP weekly for several months till the antibody titers are high. There are several factors that restrict the donation, including the individuals who already received the CP for their nCoV-2019 treatment (minimum of 90 days) are not allowed to donate blood products.

#### **6.2 Who can donate CP**


### **6.3 Plasma donation**

Convalescent plasma donors may be identified during national disease-specific cohorts, during hospitalization of the patient, by the practitioners treating outpatients, and through various specific online/social helpline-networks.

#### *6.3.1 FDA guidelines*

The FDA recommends three approaches for the administration of CP. First is directed for the treatment of patients of nCoV-2019 through EUA. Second, patients with severe nCoV-2019 illness who are unable to participate in RCT through expanded access protocol. The third one involves clinical trials where clinicians are advised to enroll patients in the trials to examine the effectiveness of CP therapy in nCoV-2019 [63].

#### **6.4 Dosage**

Various dosage regimens were utilized in various hospital setups for the management of SARS-CoV2 infected patients. Universally 200 ml of convalescent plasma in 1 or 2 doses with an infusion rate of 100 to 200 ml/h are administered with an interval of 12 hr. apart. The dosage regimen is decided according to body weight and antibody titer [64]. Standard hospital procedures and recommendations should be followed for thawing and transfusion of plasma through a peripheral or central venous catheter.

#### **6.5 Follow up**

CP therapy is still an experimental model. For future scientific investigations and correlations, the blood products of the recipient should be stored (prior and after transfusion). As per published trials, the response post CP therapy is mainly assessed i.e., PaO2/FiO2 ratio clinically or through Ct scan or X-ray (radiological) of the infected organ. However, elicitation of nCoV-2019 antibody titer or increased ALC in recipients, as well as a decline in SARS-CoV2 viral load either in terms of absolute quantification or increase of cycle threshold (Ct) value, could be considered as surrogate endpoints [65].

#### **7. Risk associated with the use of CP therapy**

Major adverse events associated with CP transfusion are not much evident so far. However, risk assessment before/after the transfusion is important. Several studies/clinical trials have shown that the use of CP therapy in severely ill nCoV-2019

patients is safe and early administration with adequate anti-SARS-CoV-2-NAb titer is helpful in faster recovery and survival of nCoV-2019 patients [18, 66]. CP therapy is contraindicated in certain individuals such as those who are allergic to plasma protein or sodium citrate, patients with selective IgA deficiency (70 mg/ dl in patients four years old or older), or the ones who received treatment with immunoglobulins in the last 30 days as it could lead to the development of serum sickness [67].

However, large U.S. national registry, through its interim report, showed that among over 100,000 hospitalized adults that had nCoV-2019 infection, low incidences of transfusion reactions were documented in the first 5,000, and 20,000 patients transfused with nCoV-2019 CP therapy, which is suggestive of the fact that transfusion of convalescent plasma is safe and poses no additional risk of complications among hospitalized patients with nCoV-2019 [52, 68]. An RCT compared the safety of convalescent plasma transfusion with fresh frozen plasma transfusion documented a comparatively less rates between the controls (7%) and CP (4%) group of patients and highlighted the safety profile of CP transfusion [69].
