Potent Anti-SARS-CoV-2 Efficacy of COVID-19 Hyperimmune Globulin from Vaccine-Immunized Plasma

Abstract

Coronavirus disease 2019 (COVID-19) remains a global public health threat. Hence, more effective and specific antivirals are urgently needed. Here, COVID-19 hyperimmune globulin (COVID-HIG), a passive immunotherapy, is prepared from the plasma of healthy donors vaccinated with BBIBP-CorV (Sinopharm COVID-19 vaccine). COVID-HIG shows high-affinity binding to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein, the receptor-binding domain (RBD), the N-terminal domain of the S protein, and the nucleocapsid protein; and blocks RBD binding to human angiotensin-converting enzyme 2 (hACE2). Pseudotyped and authentic virus-based assays show that COVID-HIG displays broad-spectrum neutralization effects on a wide variety of SARS-CoV-2 variants, including D614G, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B.1.617.2), and Omicron (B.1.1.529) in vitro. However, a significant reduction in the neutralization titer is detected against Beta, Delta, and Omicron variants. Additionally, assessments of the prophylactic and treatment efficacy of COVID-HIG in an Adv5-hACE2-transduced IFNAR^-/- mouse model of SARS-CoV-2 infection show significantly reduced weight loss, lung viral loads, and lung pathological injury. Moreover, COVID-HIG exhibits neutralization potency similar to that of anti-SARS-CoV-2 hyperimmune globulin from pooled convalescent plasma. Overall, the results demonstrate the potential of COVID-HIG against SARS-CoV-2 infection and provide reference for subsequent clinical trials.

Keywords: COVID-19 hyperimmune globulin; SARS-CoV-2 variant; passive immunotherapy; sinopharm COVID-19 vaccine.

Figure 1

Workflow for COVID‐HIG production. Plasma containing anti‐SARS‐CoV‐2 antibodies was collected from immunized healthy donors who were vaccinated with BBIBP‐CorV (two‐dose). The plasma samples were combined to obtain pooled BBIBP‐CorV plasma. Then, pooled BBIBP‐CorV plasma was fractionated and purified to prepare COVID‐HIG by commercial cold ethanol fractionation. M, maltose. COVID‐HIG, COVID‐19 hyperimmune globulin.

Figure 2

In vitro affinity and competition assays of COVID‐HIG. a) Fluorescence intensity of different concentrations of COVID‐HIG (COVID‐HIG‐001, ‐002, and ‐003) and IVIG respond to CHO‐K1/Wuhan‐1 S cells. IVIG was used as a control. Values are presented as the mean ± standard deviation (SD) of three technical replicates (n = 3). b) COVID‐HIG showed high‐affinity binding to the SARS‐CoV‐2 WIV04 S protein, the NTD, and NP; and c) WIV04, Beta, and Delta RBDs in biolayer interferometry (BLI) assays. We performed BLI experiments for twice and one representative result data is shown. d) Competitive enzyme‐linked immunosorbent assays (ELISAs) were conducted to determine the blocking potency of COVID‐HIG in the RBD (WIV04, Beta, and Delta)‐hACE2 interactions. IVIG was used as a control. Values are presented as the mean ± standard error (SEM) of three technical replicates (n = 3). Wuhan‐1, SARS‐CoV‐2 Wuhan‐Hu‐1 strain. WIV04, nCoV‐2019BetaCoV/Wuhan/WIV04/2019 strain. NTD, N‐terminal domain of S protein. NP, nucleocapsid protein. RBD, receptor‐binding domain of S protein. K _D, equilibrium dissociation constant. k _on, association rate constant. k _dis, dissociation rate constant. To be noted, since COVID‐HIG consists of multiple antibodies, the K _D value does not represent a real affinity. The average affinity of the minority of specific antibodies with many epitope specificities remains unknown. hACE2, human angiotensin‐converting enzyme 2. IVIG, human immune globulin intravenous. IC₅₀, 50% inhibitory concentration.

Figure 3

Antiviral activity of COVID‐HIG against SARS‐CoV‐2 pseudotyped viruses in vitro. a) All three batches of COVID‐HIG have potent neutralization potency against the eight pseudotyped SARS‐CoV‐2 virus strains. Pseudotyped viruses were preincubated with serial dilutions of COVID‐HIG at different concentrations for 1 h at 37 °C. Next, Huh‐7 cells were incubated with the pseudotyped viruses for 24 h. Luciferase was detected to assess infection. The y‐axis represents percent inhibition. Data are shown as mean ± SD of three independent experiments (n = 3). b) Comparison of the IC₅₀ for the pseudotyped SARS‐CoV‐2 variants from the pseudotyped Wuhan‐1 strain. The IC₅₀ (mg mL⁻¹) of COVID‐HIG against Wuhan‐1 or seven spike variants of SARS‐CoV‐2 is shown and marked on top of each group and lined with SD shown as error bars. c) Summary of the fold‐change in neutralization potency and P‐value of the IC₅₀ for the pseudotyped SARS‐CoV‐2 variants in relation to the pseudotyped Wuhan‐1 strain. The light red background indicates significantly decreased neutralization potency of COVID‐HIG against pseudotyped SARS‐CoV‐2 variants compared with that of the pseudotyped Wuhan‐1 strain. Statistical significance was determined using one‐way ANOVA.

Figure 4

Neutralization of COVID‐HIG against SARS‐CoV‐2 WIV04, Beta, and Delta strains in vitro. a) PRNTs showed that COVID‐HIG‐001, ‐002, and ‐003 significantly inhibited infection by SARS‐CoV‐2 WIV04, Beta, and Delta strains in Vero E6 cells. Viruses were incubated with COVID‐HIG at 37 °C for 1 h. Next, Vero E6 cells were infected with WIV04, Beta, and Delta strains and stained with hematoxylin/eosin at 48 h (Beta and Delta variants) or 72 h (WIV04 strain) postinfection. The y‐axis represents percent inhibition. The mean from two independent replicates is shown (n = 2). b) Comparison of the PRNT₅₀ of the variants and WIV04; statistical significance was analyzed using one‐way ANOVA. *P < 0.05. c) Microneutralization assays showed that COVID‐HIG‐001, ‐002, and ‐003 significantly inhibited the SARS‐CoV‐2 WIV04, Beta, and Delta strains in Vero E6 cells. Viruses were incubated with COVID‐HIG at 37 °C for 1 h. Next, Vero E6 cells were infected with the WIV04, Beta, and Delta strains. After 24 h, the infected cell supernatant was analyzed using real‐time reverse transcription‐PCR (qRT‐PCR). The y‐axis represents percent inhibition. The mean from two independent replicates is shown (n = 2). d) Comparison of the IC₅₀ of the variants and WIV04; statistical significance was analyzed using one‐way ANOVA. *P < 0.05. PRNT, plaque reduction neutralization tests.

Figure 5

Prophylactic treatment with COVID‐HIG protects mice from SARS‐CoV‐2 infection. a) Experimental design. Adv5‐hACE2 was intranasally inoculated into IFNAR^−/− C57BL/6 mice (n = 8 per group). Five days after transduction, Adv5‐hACE2‐transduced mice were intraperitoneally injected with 300 mg kg⁻¹ COVID‐HIG 24 h before (prophylactic treatment) infection or with 200 µL 10% maltose 2 h after SARS‐CoV‐2 infection (control, shared with the therapeutic treatment group). b) Daily body weight changes in COVID‐HIG prophylactic‐treated or maltose‐treated mice. Data are shown as the mean ± SEM of n = 8 animals per group. Statistical significance was determined using two‐way ANOVA. **P < 0.01. c) Viral RNA levels in the lung tissues of COVID‐HIG prophylactic‐treated or maltose‐treated mice were determined using qRT‐PCR at 6 day‐ post‐infection. Data are represented as mean ± SEM of n = 8 animals per group. Statistical significance was determined using an independent t‐test. *P < 0.05. d) Histopathological analyses of COVID‐HIG‐treated or untreated mice challenged with SARS‐CoV‐2. Representative images of lung sections stained with hematoxylin and eosin at 6 day‐post‐challenge. Blue arrows indicate pathological changes in the alveolar wall and alveolar cavity. Yellow arrows indicate bronchiole lesions. Green arrows indicate pathological changes to blood vessels. The image in the lower panel is an enlarged view of the black dotted box in the image in the upper panel.

Figure 6

Therapeutic treatment with COVID‐HIG protects mice from SARS‐CoV‐2 infection. a) Experimental design. Adv5‐hACE2‐transduced mice were intraperitoneally injected once with 600, 300, or 100 mg kg⁻¹ COVID‐HIG 2 h after SARS‐CoV‐2 infection (n = 8 per group). Another group was injected with 300 mg kg⁻¹ COVID‐HIG 0, 1, and 2 d after SARS‐CoV‐2 infection (therapeutic treatment, n = 8 per group). The control group was injected with 200 µL 10% maltose 2 h after (control) SARS‐CoV‐2 infection (n = 8 per group). b) Daily body weight changes of COVID‐HIG therapeutic group or maltose‐treated mice. Data are shown as the mean ± SEM of n = 8 animals per group. Statistical significance was determined using two‐way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. c) Viral RNA levels in the lung tissues of the COVID‐HIG therapeutic group or maltose‐treated mice were determined using qRT‐PCR at 6 day‐post‐infection. Data are represented as the mean ± SEM of n = 8 animals per group. Statistical significance was determined using one‐way ANOVA. **P < 0.01, ***P < 0.001. d) Histopathological analyses of COVID‐HIG‐treated or untreated mice challenged with SARS‐CoV‐2. Representative images of lung sections stained with hematoxylin and eosin at 6 day‐post‐challenge. Blue arrows indicate pathological changes to the alveolar wall and alveolar cavity. Yellow arrows indicate bronchiole lesions. Green arrows indicate pathological changes to the blood vessels. The image in the lower panel is an enlarged view of the black dotted box in the image in the upper panel.

Figure 7

Comparison of RBD‐IgG titer and neutralization potency between COVID‐HIG and PCP‐HIG. Three batches of PCP‐HIG— a) PCP‐HIG‐001, b) PCP‐HIG‐002, and c) PCP‐HIG‐003 —have potent neutralizing activities against the pseudotyped SARS‐CoV‐2 Wuhan‐1 strain. Data are shown as mean ± SD from three independent experiments (n = 3). d) RBD‐IgG titers of COVID‐HIG and PCP‐HIG were determined using SARS‐CoV‐2 RBD ELISA. The mean ± SD from three independent experiments (n = 3) of COVID‐HIG (COVID‐HIG‐001, ‐002, and ‐003) or PCP‐HIG (PCP‐HIG‐001, ‐002, and ‐003) is shown. Statistical significance was determined using t‐test. **P < 0.01. e) Neutralization potency of COVID‐HIG and PCP‐HIG against the pseudotyped SARS‐CoV‐2 Wuhan‐1 strain. The mean ± SD of COVID‐HIG (COVID‐HIG‐001, ‐002, and ‐003) or PCP‐HIG (PCP‐HIG‐001, ‐002, and ‐003) is shown. Statistical significance was determined using t‐test. ns, no significance (P > 0.05). PCP‐HIG, anti‐SARS‐CoV‐2 hyperimmune globulin from pooled convalescent plasma of donors who had recently recovered from COVID‐19. ELISA, enzyme‐linked immunosorbent assay. PBNAs, pseudotyped virus‐based neutralization assays.