Increased anti-SARS-CoV-2 antibody titers in plasma-derived intravenous immunoglobulin collected in Southern China

Summary

Although the COVID-19 pandemic is no longer considered an emergency, the ongoing spread of the virus poses a persistent threat to public health. COVID-19 poses a challenge to the system due to the risk of saturation and the associated economic burden. To assess changes in the anti-SARS-CoV-2 antibody profile, we analyzed nine batches of intravenous immunoglobulin (IVIG) prepared from plasma samples collected between November 2020 and June 2023. Antibody changes against SARS-CoV-2 spike, nucleocapsid, and membrane proteins, and neutralizing antibodies were measured. Antibody titers against wild-type SARS-CoV-2 and the three variants consistently increased. Furthermore, different IVIG batches demonstrated that antibody-dependent cellular cytotoxicity activity was correlated with anti-spike and neutralizing antibody titers. The anti-SARS-CoV-2 antibody titers fluctuated according to the progress of the pandemic and changes in COVID-19 vaccine coverage. Our research shows that immune cell-mediated virus elimination and the neutralizing effects of anti-SARS-CoV-2 antibodies underlie the therapeutic effects of IVIG.

Graphical abstract

Highlights

• •SARS-CoV-2 antibody (Ab) titers tested in 9 IVIG batches made from pooled plasma
• •Binding/neutralizing Ab titers against wild-type and 3 variants increased over time
• •Anti-spike/-nucleocapsid binding Ab titers correlated with neutralizing Ab titers
• •IVIG exerts anti-COVID-19 effects via synergism between Ab blockade and ADCC

Health sciences

Introduction

SARS-CoV-2 emerged in late 2019 and rapidly spread worldwide. Consequently, on January 30, 2020, the World Health Organization (WHO) declared the epidemic a Public Health Emergency of International Concern (PHEIC).¹ SARS-CoV-2 is a positive-sense, single-stranded RNA virus. Its main structural proteins include the S protein (SP), N protein (NP), M protein (MP), and E protein. During SARS-CoV-2 infection, the SP of the virus interacts with angiotensin-converting enzyme 2 (ACE2) on the cell membrane, facilitating endocytic entry into the host cell for replication.² The NP, a key structural protein of SARS-CoV-2 that is crucial for viral replication,³ is highly conserved and has a low mutation rate.⁴ The MP primarily facilitates viral envelope assembly and structural maintenance.⁵ The EP is a small, multi-transmembrane protein primarily responsible for maintaining the morphological stability of viral particles and assisting in the budding process during viral maturation.⁶

Various SARS-CoV-2 variants emerged during the pandemic. Among these, the Omicron variant, initially identified in South Africa in November 2021, attracted attention owing to its ability to evade the immune response established against the original SARS-CoV-2 strain and previous variants, leading to global spread.⁷ A key feature of the Omicron BA.4/5 subvariant is a mutation of three amino acids (L452R/F486V/R493Q) in the SP, which changes its immune evasion capabilities and transmissibility.⁸ The XBB.1 lineage, a recombinant of the Omicron subvariants BA.2.75 and BJ.1, that exhibits greater immune evasion capabilities than that of the original Omicron variant, became prevalent in China in 2023.⁹ The Omicron variant is more transmissible but has lower pathogenicity than the wild type (WT) of SARS-CoV-2 and earlier variants.¹⁰ Consequently, following the global spread of XBB.1, as herd immunity developed and the virulence of SARS-CoV-2 decreased, the WHO ended its designation of COVID-19 as a PHEIC on May 5, 2023.¹¹ JN.1 a subvariant of Omicron BA.2.86, was first discovered in Luxembourg in September 2023 and replaced XBB as the dominant SARS-CoV-2 variant in China in early 2024.¹² During the PHEIC, a range of COVID-19 vaccines, including inactivated, mRNA, and adenovirus-vector vaccines, were rapidly developed and introduced globally following expedited approval.¹³ In December 2020, China authorized the emergency use of the WT inactivated COVID-19 vaccine and initiated extensive vaccination campaigns in early 2021.¹⁴ The cumulative number of COVID-19 vaccine doses administered in China reached 3.4 billion by 2023.¹⁵

Intravenous immunoglobulin (IVIG) is a biologically active immunoglobulin derived from the pooled plasma of healthy donors,¹⁶ and reflects humoral immunity at the population level at the time of plasma donation.¹⁷ Ongoing mutations in SARS-CoV-2 and evolution of population immunity have led to the dynamic enrichment of neutralizing antibodies in IVIG.¹⁸ resulting in the formation of a diverse multi-epitope “antibody cluster” targeting various SARS-CoV-2 variants. Results from studies in the United States and Europe on commercial IVIG preparations prepared from fractionated plasma showed that SARS-CoV-2 antibody (Ab) titers increased with the age of the IVIG preparations.^19,20 However, there have been no similar cases in China.

In the present study, we investigated the titers of binding and neutralizing antibodies against four SARS-CoV-2 strains in IVIG products derived from plasma collected in China between November 2020 and June 2023 and analyzed the correlation between binding and neutralizing antibodies. The analysis focused on assessing changes in the affinity of IVIG for different structural proteins of SARS-CoV-2 and the cytotoxic activity of IVIG against the original wild-type strain of SARS-CoV-2 to provide a theoretical basis for the expanded use of IVIG products in COVID-19 prevention, management, and surveillance.

Results

Fluctuations in anti-SARS-CoV-2 antibody titers in nine commercial IVIG preparations over time

Analysis of SP Ab titers in batches of IVIG using enzyme-linked immunosorbent assay (ELISA) revealed a consistent increase in SP-binding Ab titers against the four SARS-CoV-2 strains between November 2020 and June 2023. Specifically, the titers rose between October-December 2021 and January-March 2022, peaked in January-March 2022, and then showed a downward trend until November-December 2022. By June 2023, the titers of SP-binding antibodies were 60–80 times higher than those in the plasma collected in November–December 2020 (Figure 1A).

Figure 1.

Changes in binding antibody titers of various proteins in IVIG over time between November-December 2020 and June 2023

(A) Antibody titers against SP of the four SARS-CoV-2 strains.

(B) Antibody titers against NP and MP in the WT strain. The titer is presented as the initial dilution concentration of IVIG at 50,000 μg/mL/EC₅₀. Abbreviations: IVIG, intravenous immunoglobulin; SP, spike protein; MP, membrane glycoprotein; NP, nucleocapsid protein; WT, wild type; 2020end, November–December 2020; 2021early January–February 2021; 2021mid, June 2021; 2021end, October–December 2021; 2022early, January–March 2022; 2022mid, June 2022; 2022end, November–December 2022; 2023early, January–March 2023; 2023mid, June 2023.

Between November 2020 and June 2023, the increase in plasma NP Ab levels was similar to that of SP Ab levels. However, by June 2023, the Ab titer for SP in the WT strain exceeded that for NP by over 14-fold. Compared to the levels in November–December 2020, NP Ab titers surged 23-fold in the population (Figure 1B). Notably, the MP Ab titer remained relatively stable between November 2020 and June 2023. The dose-response curves for determining the Ab titers against each protein in the samples are shown in Figure S1.

Changes in the titers of SARS-CoV-2 neutralizing antibodies in IVIG preparations over time

We used a pseudovirus model to assess the neutralizing Ab titers of nine batches of IVIG products against four SARS-CoV-2 strains. This involved substituting the virulence gene of the vesicular stomatitis virus with a luciferase reporter gene to create a foundational vector. Subsequently, the SPs of the four strains were individually affixed to the vector surface, mimicking the interaction of each strain’s SP with ACE2 receptors on host cell surfaces and their subsequent cellular invasion by the virus. The neutralizing and protective effects of IVIG against infection by the four SARS-CoV-2 strains were assessed by analyzing the correlation between the sample concentration and fluorescence signal intensity.

The inhibition rate curves for determining the effects of neutralizing antibodies in IVIG preparations against SARS-CoV-2 strains are shown in Figures 2A–2D. From 2020 to 2021, WT strains predominated, and comprehensive immune defense against this strain has not yet been established. Neutralizing antibodies generated by the WT strain exhibited limited cross-protection against subsequent variants, resulting in minimal neutralizing activity against the four strains analyzed in this study (Figure 2E). By 2022, following widespread vaccination and natural infections, the neutralizing Ab titers directed against the four strains increased 30- to 80-fold. Between November-December 2022 and January-March 2023, a surge in second SARS-CoV-2 infections caused by variant strains widely activated immune memory, leading to a substantial simultaneous elevation in neutralizing Ab titers against all strains. Relative to the baseline measurement of plasma collected in November–December 2020, the neutralizing Ab titers surged by 427-fold for the WT strain, 128-fold for Omicron BA.4/5, 7-fold for XBB.1, and 335-fold for JN.1. By June 2023, distinctive trends in neutralizing Ab titers had emerged among the different strains. Ab titers against the WT strain declined notably after an initial increase, whereas those against Omicron BA.4/5 stabilized after their respective surges. Neutralizing Ab titers against JN.1 remained lower than those against Omicron BA.4/5. Conversely, the neutralizing Ab titers against XBB.1 increased continuously.

Figure 2.

Changes in neutralizing antibody titers against various SARS-CoV-2 strains in IVIG

(A–D) Dose–response curves for determining neutralizing antibody titers in IVIG against WT (A) (n = 3), Omicron BA.4/BA.5 (B), XBB.1 (C), and JN.1 (D) (n = 2) at different time points of plasma collection. Data are presented as mean ± SD.

(E) Changes in neutralizing antibody titers against the four strains over time. The titer is presented as the initial dilution concentration of IVIG at 50,000 μg/mL/IC₅₀, and the values below 4 (first dilution ratio) were set to 1. Abbreviations: IVIG, intravenous immunoglobulin; WT, wild type; nAb, neutralizing antibody; 2020end, November–December 2020; 2021early January–February 2021; 2021mid, June 2021; 2021end, October–December 2021; 2022early, January–March 2022; 2022mid, June 2022; 2022end, November–December 2022; 2023early, January–March 2023; 2023mid, June 2023.

Correlation between binding and neutralizing antibody titers

The correlation between SP-binding and neutralizing Ab titers was assessed separately for the four SARS-CoV-2 strains across nine batches of IVIG products. Pearson’s correlation analysis revealed that the SP-binding and neutralizing Ab titers for each strain were significantly and positively correlated (p < 0.05). The correlation coefficients (r values) for WT, BA.4/5, and JN.1 were all >0.9, whereas the correlation coefficient for XBB.1 was weaker (r = 0.731) (Figure 3). Notably, for the WT strain, NP-binding and neutralizing Ab titers were significantly and strongly correlated (r = 0.922, p = 0.0004), whereas the correlation between MP-binding and neutralizing Ab titers was the lowest and not statistically significant for the WT strain (r = 0.392, p > 0.05).

Figure 3.

Correlation between the titers of binding and neutralizing antibodies

(A–D) Correlation between SP-binding and neutralizing antibody titers of the WT (A), Omicron BA.4/BA.5 (B), XBB.1 (C), and JN.1 strains (D).

(E and F) Correlation between neutralizing antibodies in the WT strain and NP-binding antibodies (E) and MP-binding antibodies (F). Abbreviations: MP, membrane protein; NP, nucleocapsid protein; SP, spike protein; WT, wild type; nAb, neutralizing antibody.

Affinity of IVIG for antigen and FcγR

In this study, we used surface plasmon resonance (SPR) technology to assess the binding affinity of one batch of IVIG to different WT antigens and FcγR. Specifically, the interactions between the IVIG samples obtained in June 2023 and the SP, NP, and MP of the WT strain were examined. The IVIG derived from plasma collected in June 2023 exhibited a high affinity for SP (KD = 1.03 × 10⁻⁷ M) and NP (KD = 1.18 × 10⁻⁷ M) and a weak affinity for MP (KD = 1.53 × 10⁻⁶ M) of the WT strain (Figures 4A–4C). The data for the SP, NP, and MP were analyzed using a 1:1 steady-state affinity model.

Figure 4.

Binding affinity of IVIG to various structural proteins and Fc receptors

(A–C) Affinity of IVIG products for the WT strain SP (A), NP (B), and MP (C) of IVIG from 2023mid (June 2023).

(D and E) Affinity of IVIG collected from 2023mid (June 2023) toward human CD16a (FcγRIIIa) (D) and human CD32a (FcγRIIa) (E). Abbreviations: IVIG, intravenous immunoglobulin; MP, membrane protein; NP, nucleocapsid protein; SP, spike protein; WT, wild type. KD, equilibrium dissociation constant.

Human CD16a (FcγRIIIa) and CD32a (FcγRIIa) are Fc receptors expressed on the surface of immune cells, facilitating the binding of the Fc fragment of IgG and subsequent mediation of Ab-dependent cellular immune responses.²¹ The IVIG derived from plasma collected in June 2023 exhibited an affinity of 1.78 × 10⁻⁶ M for human CD16a (FcγRIIIa) and an affinity of 5.94 × 10⁻⁶ M for human CD32a (FcγRIIa) (Figures 4D and 4E). The data for CD16a and CD32a were analyzed using a 1:1 kinetic binding model.

Cytotoxic effects of IVIG on cell lines expressing the spike protein

CHO-K1 cells overexpressing the WT strain SP on their membrane surface were used as target cells for Ab-dependent cellular cytotoxicity (ADCC) and Ab-dependent cellular phagocytosis (ADCP). Genetically modified Jurkat T cells served as effector cells for assessing ADCC activity to mimic NK cell functionality, with the stable expression of CD16a on their membrane surface and transfection of the firefly luciferase gene driven by the nuclear factor of activated T cells (NFAT) response element. THP-1 cells naturally express CD32a (FcγRIIA) and CD64 (FcγRI)²²; and were transfected with NF-κB response elements along with the firefly luciferase gene, which were used as effector cells for assessing ADCP activity. Quantitative assessment of luciferase activity through bioluminescence enabled the characterization of ADCC and ADCP efficacy (Figures 5A and 5B).

Figure 5.

Killing response of IVIG against the WT strain via ADCC and ADCP

(A and B) Schematic diagram of the mechanisms underlying ADCC (A) and ADCP (B).

(C and D) Immune effect assessment results of ADCC (C) and ADCP (D) against the WT strain in the IVIG samples prepared at different plasma collection times (n = 3). Data are presented as mean ± SD. Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; ADCP, antibody-dependent cellular phagocytosis; EC₅₀, half-maximal effective concentration; IVIG, intravenous immunoglobulin; WT, wild type; SP, spike protein; 2020end, November–December 2020; 2022early, January–March 2022; 2023mid, June 2023.

The ADCC activity of plasma against SARS-CoV-2 increased over time, with the EC₅₀ decreasing from 0.58 to 0.024 mg/mL, a 24-fold increase (Figure 5C). In contrast, ADCP activity did not show a discernible temporal trend, with the EC₅₀ fluctuating within the range of 0.69–1.34 mg/mL (Figure 5D).

Discussion

In the present study, we monitored the temporal fluctuations of SARS-CoV-2 antibodies in IVIG products derived from plasma collected in China between November-December 2020 and June 2023 (Table 1). By analyzing the binding activity, neutralizing capacity, molecular affinity, and functional effects of IVIG products from nine different batches of IVIG, this study elucidated the evolution of population immunity against SARS-CoV-2 WT and three different variants, providing valuable insights into population immune surveillance and the potential medical utilization of IVIG products.

Table 1.

Information on plasma collection time and corresponding codes

Code	Time period for plasma collection	The main circulating strains	Vaccine coverage percentage
2020end	November–December 2020	There were no localized outbreaks of COVID-19	Sinopharm BIBP vaccine has been approved through the conditional marketing authorization process (2020.12.31)
2021early	January–February 2021		More than 22.000 million doses of COVID-19 vaccine administered
2021mid	June 2021		Over 1 billion vaccine doses have been administered
2021end	October–December 2021	Delta B.1.617.2	The full vaccination rate for the entire population has exceeded 70%
2022early	January–March 2022	Omicron BA.2	/
2022mid	June 2022	There were no localized outbreaks of COVID-19	The full vaccination rate for the entire population is approaching 90%
2022end	November–December 2022	Omicron BA.5.2, and BF.7	The full vaccination rate for the entire population has reached 90.6%
2023early	January–March 2023	Omicron XBB	/
2023mid	June 2023	Omicron XBB and its subvariants (XBB.1.5, XBB.1.9.1, XBB.1.16)	/

The predominant circulating strains and vaccine coverage percentage data were sourced from the official websites of the Chinese Center for Disease Control and Prevention and the National Health Commission of the People’s Republic of China, as well as from related reports. Since the plasma was sourced from donors in Jiangxi and Guizhou provinces, the information regarding the circulating viral strains pertains only to these two provinces.

The trends in SP-binding and neutralizing Ab titers were consistent over time. The SP is the protein that SARS-CoV-2 uses to infect cells; therefore, after vaccination or infection, the body easily produces a strong humoral immune response against this structural protein. The SP titers of the four SARS-CoV-2 strains in IVIG samples gradually increased throughout the study period, consistent with the timeline of COVID-19 cases and vaccination in the population. The Chinese National Medical Products Administration authorized the world’s first inactivated COVID-19 vaccine in December 2020.²³ Although binding antibodies were present in IVIG collected in November–December 2020 and 2021, the promotion of vaccines was still in the initial stage at this time; therefore, the neutralization activity was minimal. Vaccination primarily induces low-affinity binding antibodies targeting non-critical epitopes,²⁴ but the production of neutralizing antibodies requires the maturation and differentiation of B cells.²⁵ By 2021, Ab titers in IVIG prepared from pooled plasma showed a moderate increase compared to those in November–December 2020. By the end of 2021, the vaccination coverage rate was 77.6%. Ab titers increased further by January–March 2022 owing to the widespread availability of free vaccinations. Higher vaccine coverage further enhanced the neutralization capacity of the population. The levels of vaccine-induced SP-specific antibodies increased in the population, promoting increased neutralization activity of IVIG derived from the pooled plasma. The decline observed in June 2022 was attributable to three key factors: the decreased spread of the WT strain, the natural waning of antibodies in the population against previously prevalent strains, and the rapid emergence of new variants, such as Omicron BA.5, which reduced the cross-binding effectiveness of pre-existing antibodies.²⁶ This phenomenon also indirectly demonstrates two critical trends: the continuous enhancement of the immune evasion ability of the virus and the dynamic fragility of the population’s immune barrier. Following a decrease in pathogenicity of SARS-CoV-2 and a decrease in COVID-19 mortality,²⁷ the Chinese National Health Commission officially reclassified COVID-19 from a Class A to a Class B infectious disease in December 2022. After reclassification, COVID-19 prevention and control measures were relaxed, and new SARS-CoV-2 variants emerged. BF.7, the first dominant epidemic strain that emerged after the relaxation of control measures, overcame the level of immunity in the population and led to a new surge in infections.²⁸ This led to an increase in Ab titers in IVIG prepared from pooled plasma collected in November–December 2022 and January–March 2023. The varying neutralization capacities of IVIG in 2023 highlight the capacity of variants to mount immune evasion: neutralizing Ab titers against the WT strain fluctuated as it was replaced, and Omicron BA.4/5 was stabilized. Despite the high vaccine coverage rate, the neutralizing Ab titer against XBB.1 in IVIG remained low, indicating that this variant has achieved substantial immune escape, consistent with the reported resistance of the XBB variant to therapeutic monoclonal antibodies.²⁹ Neutralizing titers against JN.1, a new strain, continued to increase concomitantly with population immunity, suggesting recognition by memory immune cells and targeted immune responses. The trend of NP Ab titers in IVIG mirrors that of SP antibodies, but the magnitude is lower because NP antibodies are more conserved; they function solely as viral structural proteins and do not participate in the physiological processes of infection. In China, the inactivated whole-virus vaccines that are widely used among the population elicit measurable levels of NP-binding antibodies in the plasma of recipients. The MP, a transmembrane structural protein, showed stable Ab titers over the course of the study owing to its limited involvement in viral invasion, fewer exposed antigenic epitopes, and a conserved structure.

Correlation analysis revealed different associations between SP-binding and neutralizing antibodies against different strains. Neutralizing antibodies against WT, BA.4/5, and JN.1 predominantly targeted the SP, which was also detected using binding Ab assays. Thus, in contrast to the more complex pseudovirus neutralization assay, ELISA can be used as a rapid and cost-effective method for assessing immune protection against these strains, facilitating large-scale surveillance. For other structural proteins, such as NP and MP, NP-binding and neutralizing antibodies were strongly correlated with the WT strain, highlighting the importance of NP antibodies in early immunity. However, the correlation for MP was weaker, indicating that binding Ab titers are not reliable indicators of neutralizing Ab activity against the WT strain.

The results of the SPR assays and functional experiments elucidated the impact of IVIG binding to viral proteins and Fc receptors on the immune response. By June 2023, IVIG demonstrated a notably higher affinity for SP and NP than for MP, which aligns with the variations in the immunogenicity of these proteins. Moreover, IVIG enhanced the interaction between SARS-CoV-2 SP and CD16a through a bridging mechanism, promoting the effective elimination of the virus by innate immune cells. However, the ADCP activity of IVIG did not exhibit a discernible temporal trend. To verify the correlation between the ADCC activity of IVIG against the WT strain and the titers of SP-binding and neutralizing antibodies across the 2020 end, 2022 early, and 2023 mid batches of IVIG, we performed a correlation analysis. The results demonstrated a strong positive correlation between ADCC activity and both SP-binding Ab titers (r = 0.983) and neutralizing Ab titers (r = 0.975) against the WT strain; however, these correlations did not reach statistical significance (p > 0.05), likely due to the small sample size (N = 3; Figure S2).

Due to the promotion of inactivated whole-virus vaccines in China, IVIG production pools diverse antibodies against SARS-CoV-2 and capitalizes on individual immune memory resulting from vaccination and natural infection by pooling plasma from multiple donors into a readily available resource.¹⁷ IVIG offers crucial passive immune protection to individuals with compromised immune systems and inadequate vaccine response. Moreover, IVIG serves as a dynamic biomarker that reflects herd immunity levels in the population. Unlike monoclonal antibodies that target specific sites, IVIG binds to various epitopes on the viral SP. This ability enables IVIG to block the virus from binding to the ACE2 receptor on cell surfaces through steric hindrance.³⁰ In contrast, adenovirus-vectored vaccines, recombinant protein vaccines, and other vaccines targeting only the SP primarily elicit SP-specific antibodies, whereas IVIG derived from populations immunized with whole inactivated virus vaccines provides broader Ab coverage, thereby enhancing resistance to the virus, offering a multifaceted mechanism for neutralizing the virus and reducing the risk of viral evasion.³¹

This study provides a systematic exploration of the time-dependent anti-SARS-CoV-2 properties of IVIG produced in China. This confirms that the underlying immune status of individual plasma donors is the primary driver of the dynamic alterations observed in the spectrum of IVIG antibodies. Given its capacity for immune resource conversion, IVIG can contribute to the management of COVID-19 and other epidemic diseases by taking advantage of vaccine-mediated immunity and the history of infection in the population. Consequently, monitoring changes in the composition, concentration, and functional efficacy of IVIG antiviral antibodies is of scientific and clinical value and warrants consideration.

Limitations of the study

This study had some limitations. The plasma collected from the nine batches of IVIG products was mainly concentrated in Guizhou and Jiangxi provinces, and ignored the differences in immunity levels between different regions, gender, and sex when used to represent the entire population of China (Table 1). Owing to variability in plasma collection intervals and quantities across different batches of IVIG, it was impossible to eliminate batch-to-batch differences. The findings of this study can only indicate general trends of Ab changes in the population plasma from November 2020 to June 2023. Moreover, because this study did not include an in vivo research component, the results of the in vitro viral titer and ADCC assays can only serve as a reference for the effectiveness of IVIG treatment for COVID-19 but cannot be directly correlated. Finally, it should be noted that the conclusions of this study regarding the trends of binding antibodies, neutralizing antibodies, as well as ADCC and ADCP effects are solely derived from the analysis of observed data trends and have not been statistically validated.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yan Li (liyan@scidc.org.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •All data generated or analyzed during this study are included in this published article and supplemental information.
• •This article does not report the original code.
• •Any additional information required to reanalyze the data reported in this article is available from the lead contact upon request.

Acknowledgments

This work was supported by the Sichuan Institute for Drug Control (2024-KYYL-010) and Chengdu Rongsheng Pharmaceuticals Co. Ltd, and did not receive any specific grants from funding agencies in the public or not-for-profit sectors.

Author contributions

Conceptualization, H.L. and Y.L.; methodology: C.Y.S. and J.Z.W.; investigation: C.Y.S., J. Z.W., Y.L.D., K.R.C., H.S.Y., J.M., and Y.F.J.; formal analysis, C.Y.S., J. Z.W., Y.L.D., K.R.C., J.M., and Y.F.J.; writing – original draft, C.Y.S.; writing – review and editing, J.Z.W.; project administration, Y.L.D., H.S.Y., H.L., and Y.L.; supervision, H.L. and Y.L.

Declaration of interests

J.Z.W., Y.L.D., H.S.Y., and H.L. are employees of Chengdu Rongsheng Pharmaceuticals Co., Ltd H.L. is an employee of Beijing Tiantan Biological Products Co., Ltd. Y.F.J. is an employee of Sinopharm Shanghai Plasma-derived Biotherapies Co., Ltd. Chengdu Rongsheng Pharmaceuticals Co., Ltd. filed patents for the production methods of ADCC and ADCP with the China National Intellectual Property Administration. All other authors declare that they have no competing interests. It is important to note that the opinions expressed in this work should not be construed as endorsements of particular products or entities.

Declaration of generative AI and AI-assisted technologies in the writing process

No AI was used in the preparation of this article.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
anti-human IgG (H&L) (goat) antibody peroxidase conjugate	Rockland	Cat: 609-103-123; RRID: AB_219575
Bacterial and viral strains
SARS-CoV-2 Pseudovirus (WT_VSV)	Vazyme	DD1502
SARS-CoV-2 Pseudovirus (BA.4/BA.5)	Vazyme	DD1576
SARS-CoV-2 Pseudovirus (XBB.1)	Vazyme	DD1596
SARS-CoV-2 Pseudovirus (JN.1)	Vazyme	DD15110
Chemicals, peptides, and recombinant proteins
SARS-CoV-2 S protein, Super stable trimer (WT)	Acro	SPN-C52H9
SARS-CoV-2 Spike Trimer Protein (BA.4/Omicron)	Acro	SPN-C5229
SARS-CoV-2 Spike Trimer Protein (XBB.1/Omicron)	Acro	SPN-C522t
SARS-CoV-2 Spike Trimer Protein (JN.1/Omicron)	Acro	SPN-C5221
SARS-CoV-2 (COVID-19) Nucleocapsid protein	Acro	NUN-C5227
Recombinant SARS-CoV-2 Membrane Glycoprotein, C-terminal	RayBiotech	230-01124
human CD16a (176F)	Acro	10389-H08H
human CD32a (167R)	Acro	10374-H08H
Experimental models: Cell lines
GS-E5/angiotensin-converting enzyme 2 (ACE2) clone8	ProBio	RD00825
CHO-K1/Spike_SARS2	ProBio	RD00819
GS-J2C/CD16A 158V	ProBio	RD00830
THP-1/NF-κB-Luc	ProBio	RD00941
Software and algorithms
Prism 10	GraphPad Software	www.graphpad.com/feature
Other
TMB two-Component Substrate solution	ACMEC Biochemical	AC13935
Bio-Lite Luciferase Assay	Vazyme	DD1201-01
Fire-Lumi luciferase detection kit	GenScript	L00877C-100
His Capture Kit	Cytiva	28-9950-56
10 x HBS-EP+	Cytiva	BR-1006-69

Method details

Intravenous immunoglobulin preparations

Nine batches of commercial IVIG preparations from Sinopharm Shanghai Plasma-derived Biotherapies Co., Ltd. (Shanghai, China) prepared from sequential pools of blood collected at different times were included in this study (each batch sourced from >4,000 plasma donations). The collected plasma was concentrated in the Guizhou and Jiangxi provinces. We selected different IVIG preparations according to the plasma collection time corresponding to the IVIG (Table 1). In this study, we used commercially available IVIG preparations, therefore, ethical approval was not required. The IVIG preparations contained 5% immunoglobulin G (IgG) (50 mg/mL), and all IVIG preparations were commercial products whose quality met the release requirements.

Enzyme-linked immunosorbent assay (ELISA)

SARS-CoV-2 SP (WT, BA.4/5, XBB.1, JN.1), NP, and MP (Acro) were diluted to 1.0 μg/mL in a coating buffer (1× phosphate-buffered saline [PBS]) to serve as antigens for the assay. Subsequently, 50 μL of the diluted antigen was added to each well of a 96-well microplate, which was then incubated at 4°C overnight (for 16 h). After discarding the coating buffer, the microplate was washed once for 10 s with washing buffer (1× PBS containing 0.05% Tween 20) using an automatic plate washer and then gently dried. Next, 150 μL of blocking buffer (1× PBS containing 1% bovine serum albumin [BSA]) was added to each well, and the plate was incubated at 37°C for 1 h before discarding the blocking buffer. Starting from a concentration of 5000 μg/mL, 3-fold serial dilution was used to prepare test samples at 11 concentrations. An aliquot of 100 μL of each concentration was added to the corresponding well, the buffer was diluted in parallel as a negative control, and one well was left with only dilution buffer as a blank control. The plates were incubated at 37°C for 1 h. The solutions in the wells were discarded, and the plate was washed three times with wash buffer for 10 s each. Subsequently, 100 μL of anti-human IgG (H&L) (goat) antibody peroxidase conjugate (Rockland), diluted to 25.0 ng/mL with dilution buffer, was added to each well. The plate was incubated at 37°C for 30 min, the solutions in the wells were discarded, and the plate was washed three times with washing buffer for 10 s each time. The plate was then dried, and 100 μL of TMB two-Component Substrate solution (ACMEC Biochemical) was added, followed by incubation at room temperature (20–25°C) for 15 min. The reaction was stopped by adding 50 μL of 1M HCl, and the absorbance was measured at 450 nm using a microplate reader (MD SpectraMax iD5). The half-maximal effective concentration (EC₅₀) value was automatically calculated using the software (SoftMax Pro 7.1.1). The stock concentration of the test sample was 50,000 μg/mL, and the dilution factor was determined to be 50,000/EC₅₀.

Pseudovirus-based neutralization assay

Starting with a concentration of 12,500 μg /mL as the sample working solution, six concentrations were generated via 4-fold serial dilutions for the Omicron BA.4/BA.5, XBB.1, JN.1 strains; and eight concentration were generated via 3-fold serial dilutions for the WT strain. Aliquots of 100 μL of the sample working solution at six different concentrations were dispensed into the designated wells on the detection plate. Blank and negative control wells were filled with 100 μL of Dulbecco’s modified Eagle medium (DMEM) (Gibco). SARS-CoV-2 pseudoviruses were purchased from Nanjing Vazyme Biotech Co., Ltd. (Table S1) were rapidly thawed in a 37°C water bath, transferred to a 15 mL centrifuge tube, and diluted in complete DMEM to achieve 650 50% tissue culture infectious dose (TCID₅₀) per well using complete DMEM. Aliquots of 50 μL of each sample concentration and the negative control (VC) were added to the wells. For the blank control (CC), 50 μL of complete DMEM was added to each well. The samples were then incubated at 37°C for 1 h. GS-E5/ACE2 clone8 target cells (ProBio, RD00825) were prepared for digestion, transferred to a 15 mL centrifuge tube, centrifuged at 300 × g for 5 min, and the supernatant was discarded. The cells were resuspended in complete DMEM and adjusted to 40,000 cells/well. After incubation, 100 μL of the cell suspension was added to each well. The suspensions were gently mixed and cultured at 37°C with 5% CO₂ for 24 h. After incubation, the Bio-Lite Luciferase Assay (Vazyme) detection reagent was prepared. Supernatant volumes of 170 μL were removed from each well on the culture plate, 80 μL of the detection reagent was added to each well, and the samples were incubated at room temperature (20–25°C) for 3–5 min. The luciferase signal in each well was measured using a microplate reader (MD SpectraMax iD5). The inhibition rate was calculated using the following equation:

(1 – [mean RLU of sample − mean RLU of CC] / [mean RLU of VC − mean RLU of CC])× 100%, where RLU stands for relative light unit. The highest serum dilution ratio with a 50% neutralization rate (IC₅₀) of each sample was determined using the Reed–Muench method based on the inhibition rate results, and a dilution titer over the first dilution (4) was considered positive, while below the first dilution was set to 1. Data were analyzed using GraphPad Prism 10.1.0 (GraphPad Software, Boston, MA, USA). The mutations in the pseudovirus S protein are listed in Table S1.

Measurement of antibody-dependent cellular cytotoxicity (ADCC)

To measure ADCC, CHO-K1/Spike_SARS2 cells that overexpressed the WT strain SP on the membrane surface (ProBio, RD00819) were digested using 2 mL of Accutase (Gibco). The cells were pelleted by centrifugation and subsequently resuspended in 5 mL ADCC assay buffer (RPMI 1640 supplemented with 10% fetal bovine serum [FBS], Gibco). The cell density was adjusted to 10,000 cells/well, and 40 μL of the suspension was dispensed into each well of a 96-well cell culture plate. Working solutions of the sample and negative control were prepared using the ADCC assay buffer (sample formulation buffer with a concentration of 12 μg/mL polysorbate 80 + 12 μg/mL maltose). To generate a gradient of test sample solutions with eight concentrations, the sample was diluted using a 3-fold serial dilution, starting from a concentration of 6 mg/mL. The test sample solution, control working solution, or ADCC assay buffer was dispensed into the respective wells of a 96-well plate in 20 μL aliquots. The assay plate was incubated at room temperature (20–25°C) for 30 min. Effector cells GS-J2C/CD16A 158V (ProBio, RD00830) were harvested and resuspended in the ADCC assay buffer. Aliquots of 40 μL were dispensed into each well of a 96-well plate, and the cell density was adjusted to 60,000 cells/well. The assay plate was incubated in a cell culture incubator at 37°C with 5% CO₂ for 6 h. After incubation, the 96-well assay plate was removed from the incubator, and 80 μL/well of Fire-Lumi luciferase detection kit (GenScript) working solution was added to each well. The plates were incubated for 5–10 min. Chemiluminescence was measured at room temperature (20–25°C) using a PHERAstar FSX high-throughput screening plate reader (BMG Labtech).

Measurement of antibody-dependent cellular phagocytosis (ADCP)

To measure ADCP, CHO-K1/Spike_SARS2 cells (ProBio, RD00819) were digested using 2 mL Accutase (Gibco), followed by centrifugation to isolate the target cells. The cells were resuspended in 5 mL of ADCP assay buffer (RPMI 1640 with 10% FBS; Gibco). Aliquots (40 μL) of the suspension were dispensed into a 96-well cell culture plate at a cell concentration of 20,000 cells/well. Working solutions of the sample and negative control were prepared using the ADCP assay buffer (sample formulation buffer with a concentration of 20 μg/mL polysorbate 80 + 20 mg/mL maltose). Starting from a concentration of 10 mg/mL, a gradient of eight concentrations of the test sample solution was prepared using a 3-fold serial dilution. Aliquots (20 μL) of the test sample solution, control working solution, or ADCP assay buffer were added to the corresponding wells of a 96-well plate. The assay plate was incubated at room temperature (20–25°C) for 30 min. THP-1/NF-κB-Luc (ProBio, RD00941) effector cells were prepared by resuspending them in the ADCP assay buffer. The cell concentration was adjusted to 30,000 cells/well, and 40 μL of cell suspension was added to the corresponding wells of a 96-well plate. The assay plate was incubated in a cell culture incubator at 37°C with 5% CO₂ for 4 h. After incubation, the 96-well assay plate was removed from the incubator, and 80 μL of Fire-Lumi luciferase detection kit (GenScript) working solution was added to each well. The cell culture was incubated for 5–10 min, and chemiluminescence values were measured at room temperature (20–25°C) using a PHERAstar FSX high-throughput screening plate reader (BMG Labtech).

Surface plasmon resonance (SPR)

SPR data were analyzed using Biacore 8K (Cytiva) evaluation software version 4.0 with a CM5 chip. SARS-CoV-2 WT SP, NP, and MP, human CD16a (176F), and CD32a (167R) proteins were obtained from Acro. These proteins were covalently immobilized in pairs on CM5 chips in flow cells 1 and 2 via amino coupling at concentrations of 2.5, 3, 20, 5, and 5 μg/mL for SARS-CoV-2 WT SP, NP, and MP, and human CD16a (176F), and CD32a (167R) proteins, respectively. For SP and NP detection, samples were loaded in 2-fold serial dilutions starting at 10,000 nM, whereas for MP detection, loading began at 20,000 nM. Human CD16a (176F) and CD32a (167R) were detected by loading with 2-fold serial dilutions, starting at 800 nM. Subsequently, the samples flowed over the surfaces of flow cells 1 and 2 at 30 mL/min, and the responses were recorded in real time. In each detection cycle, the signal values resulting from flow cell-1 injection and running buffer injection were used for the double subtraction of resonance units. The strength of these molecular interactions was quantified using the equilibrium dissociation constant (KD), calculated by dividing the dissociation rate constant (Kd) by the association rate constant (Ka): KD = Kd/Ka.

Quantification and statistical analysis

Data were analyzed using GraphPad Prism 10.1.0 software (GraphPad Software, Boston, MA, USA). For in vitro plate-based assays (Figures 2 A–D, 5 C, D, and S1), data are presented as mean ± standard deviation (SD), where the exact value of n represents the number of technical replicates per sample. Specific n values are indicated in the respective figure legends. For correlation analyses (Figures 3 and S2), antibody titer data were log10-transformed to normalize the distribution prior to analysis. The normality of the log-transformed data was verified using the Shapiro–Wilk test. The association between variables was evaluated using Pearson’s correlation analysis (via simple linear regression), where N represents the number of independent IVIG batches analyzed (N = 9 for Figures 3; N = 3 for Figure S2). Although the limited sample size for Figure S2 (N = 3) precluded formal normality testing, Pearson’s correlation was applied to maintain methodological consistency with the main datasets. Results with P-values less than 0.05 were considered statistically significant.

Additional resources

Invention patent

The ADCC assay used in this study was authorized by the China National Intellectual Property Administration on September 19, 2025 (CN 120249435 B).

Published: March 13, 2026