**4. Convalescent plasma (CP)**

The first and foremost important criteria for the convalescent plasma immunotherapy is that recovered SARS-CoV-2 donor should have high neutralizing antibody (nAb) titer and specific to the virus [28]. It is proposed that SARS-CoV-2 specific nAbs may reduce the viral load, severity of diseases and further also increase the nAb titer level of COVID-19 patients. CP may be a potentially effective strategy and first line of defense against the current wreaked havoc SARS-CoV-2 viral pandemic.

### **4.1 Clinical outcomes of CP**

A Single dose of CP with a high concentration of nAbs rapidly reduced the viral load and eventually increased clinical outcomes of ten severe adult SARS-CoV-2 patient from china by successful use of the CP immunotherapy. Single dose of 200 mL of inactivated CP with high neutralization titer of >1:640 was transfused according to the WHO blood transfusion protocol. Interestingly this recent study showed significant increase in nAb titer level, disappearance of SARS-CoV-2 RNA at an undetectable level, reduction of pulmonary lesions and amelioration of laboratory parameters in all patients after CP transfusion. The clinical symptoms were improved within 3 days and the viraemia was also disappeared on 7 days of CP immunotherapy (**Figure 5**) [28].

The recent study from China over 175 patients recovered from SARS-CoV-2 clearly showed the production of SARS-CoV-2-specific neutralizing antibodies after 10–15 days of infection. Interestingly this study had several findings that convalescent plasma recovered from SARS-CoV-2 patients specifically inhibited SARS-CoV-2 alone, but not the SARS-CoV infection, the peak of neutralizing antibodies were detected in all patients after 10 days of infection and variations in nAb titers

#### **Figure 5.**

*Comparison of serum neutralizing antibody titer before and after CP immunotherapy. [CP, Convalescent plasma]. Source adapted from Duan et al. [28].*

were observed as 30% of infected patients corresponds to low nAb titer (< 500), 17% to medium-low titers (500–999), 39% to medium-high titers (1000–2500) and 14% to high nAb titers (>2500). Moreover, the elderly and middle-aged patients had substantially higher plasma nAb titers and spike-binding antibodies than young aged SARS-CoV-2 infected patients [29]. Further this study showed the noteworthy findings of CP immunotherapy can be specifically used for the prevention and treatment of SARS-CoV-2.

Five critical patients from china with SARS-CoV-2 and acute respiratory distress syndrome (ARDS) had eventually recovered after CP immunotherapy. Clinically significant improvement of four out of five were reported promptly. This recent study showed increase in nAb titer level (**Figure 6**) and titers of IgG and IgM in the sera got increased in a time-dependent manner after convalescent plasma transfusion. It was observed that the Ct value of all patients seems to be negative after 12 days of transfer of CP immunotherapy [30].

Ye et al. reported an increase in anti-SARS-CoV-2 nAb titers and further increase in IgG and IgM antibodies. CP transfusion resulted in the resolution of ground glass opacities and consolidation in patients lung. The clinically significant outcome of this study is that all the six SARS-CoV-2 infected patients were found negative in throat swab analysis by real-time PCR assay due to reduction in the viral load after CP immunotherapy [31].

SARS-CoV-2 infected patients over the age of sixty years received CP treatment had a significantly prolonged recovery time estimated by viral clearance (10 to 29 days) compared to younger patients, who recovered from the disease in less than a week after receiving CP immunotherapy [32].

The level of specific neutralizing antibody against SARS-CoV peaked at 4 months and gradually disappearing to an undetectable level of 25.8% (IgG) and 16.1% (nAbs) in serum after 3 years of recovery [33]. Similarly, MERS-CoV infected patients showed a low prevalence of 2.7% IgG seroreactivity and the antibodies titer dropped rapidly within 3 months of recovery [26].

Even though the level of specific nAb titers in sera were decreasing gradually, it is also possible to isolate potent neutralizing human monoclonal antibodies (mAbs) from memory B cell repertoire of convalescent patients against SARS-CoV [34] and SARS-CoV-2 [35]. The detailed possible FDA approved protocol for convalescent plasma immunotherapy transfusion was discussed in this recent studies for SARS-CoV-2 [36] and Ebola virus [37]. The possible protocol of CP immunotherapy transfusion for COVID-19 patients is depicted in **Figure 7**.

#### **Figure 6.**

*Comparison of serum neutralizing antibody titer before and after CP immunotherapy. [CP, Convalescent plasma]. Source adapted from Shen et al. [30].*

#### **Figure 7.**

*Schematic representation of CP immunotherapy for SARS-CoV-2 patients highlighting the major steps.*

### **4.2 Immunomodulatory effects**

In a mouse model study, SARS-CoV pathogenesis is directly regulated by complement and its absence showed significantly reduced respiratory disease, decreased neutrophilia in their lungs, reduced systemic inflammation and viral load remains unchanged in a complement-deficient mice. Since SARS-CoV pathogenesis was mainly immune-driven, inhibiting the complement signaling pathway after SARS-CoV infection is also an effective immune therapeutic strategy [38]. By scavenging complement fragments of C3a and C5a, intravenous immunoglobulin prevents immune damage and restricts the development of immune complexes [39]. Similarly, passive antibody transfer may limit the cellular damage induced by the activation of complement cascade in an excessive inflammatory area.

#### **Figure 8.**

*A brief overview of possible mechanisms of action of CP immunotherapy. [nAb, neutralizing antibody titer; ADCC, antibody-dependent cellular cytotoxicity; ADCP, antibody-dependent cell mediated phagocytosis; CDC, complement-dependent cytotoxicity; ADE, antibody-dependent enhancement.*

IgG therapy controls the cytotoxic effect of T-cells by modulating the balance of Th17/Treg and decreasing CD8+ T cells and Th17 cells [40]. The possible mechanisms of action of CP immunotherapy are summarized in **Figure 8**.

Feline coronaviruses, HIV and dengue viruses use an antibody-dependent enhancement (ADE) phenomenon to take advantage of pre-existing poorly nAbs to effectively infect host target cells in order to combat anti-viral humoral immune response [41].

In vitro assays with human promonocyte cell lines HL-CZ demonstrated that ADE was primarily mediated by highly diluted antibodies against spike proteins, significantly increasing the rate of apoptosis of SARS-CoV infected cells [42].
