key: cord-0960334-samuy90i
authors: Díez, José María; Romero, Carolina; Cruz, María; Vandeberg, Peter; Merritt, W Keither; Pradenas, Edwards; Trinité, Benjamin; Blanco, Julià; Clotet, Bonaventura; Willis, Todd; Gajardo, Rodrigo
title: Anti-SARS-CoV-2 hyperimmune globulin demonstrates potent neutralization and antibody-dependent cellular cytotoxicity and phagocytosis through N and S proteins
date: 2021-10-25
journal: J Infect Dis
DOI: 10.1093/infdis/jiab540
sha: 78c07da45b07a79b92a9f4e374bcfacbafe631c9
doc_id: 960334
cord_uid: samuy90i

BACKGROUND: Although COVID-19 vaccinations have provided a significant reduction in infections, effective COVID-19 treatments remain an urgent need. METHODS: Functional characterization of anti-SARS-CoV-2 hyperimmune immunoglobulin (hIG) from human convalescent plasma was performed by different virus neutralization methodologies (plaque reduction, virus induced cytotoxicity, TCID(50) reduction and immunofluorimetry) at different laboratories using geographically different SARS-CoV-2 isolates (USA (1), Italy (1), Spain (2): 2 containing the D614G mutation). Neutralization capacity against the original Wuhan SARS-CoV-2 strain and variants (D614G mutant, B.1.1.7, P.1 and B.1.351) was evaluated using a pseudovirus expressing the corresponding spike (S) protein. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) was also evaluated. RESULTS: All SARS-CoV-2 isolates were potently neutralized by hIG as shown by all four methodologies. Wild-type SARS-CoV-2 and variants were effectively neutralized using the pseudovirus. hIG induced ADCC and ADCP against SARS-CoV-2 N and S proteins but not E protein. Very low concentrations (25-100 µg IgG/mL) were required. A potent effect was triggered by antibodies in hIG solutions against the SARS-CoV-2 S and N proteins. CONCLUSIONS: Beyond neutralization, IgG Fc-dependent pathways may play a role in combatting SARS-CoV-2 infections using COVID-19 hIG. This could be especially relevant for the treatment of more neutralization-resistant SARS-CoV-2 variants.

Currently, there is no effective standardized treatment for COVID-19, although multiple therapeutic options are available [1] . Among the available therapeutic strategies, passive immunization using COVID-19 convalescent plasma (CCP), monoclonal antibodies (mAb) or hyperimmune globulin (hIG: IgG enriched with anti-SARS-CoV-2 antibodies) is of particular relevance [2] . As the SARS-CoV-2 pandemic spreads and vaccination progresses, commercial IgG products derived from healthy plasma donors become gradually enriched in anti-SARS-CoV-2 antibodies [3] . To date, the antibody levels in the general population are still low [3] , therefore the plasma collected and the products produced cannot yet be considered hyperimmune.

Anti-SARS-CoV-2 hyperimmune immunoglobulin (hIG) is typically prepared from pools of 100-1000 liters from CCP donors. hIG products have a high titer of neutralizing antibodies against SARS-CoV-2 in a standardized and concentrated product [4] . This represents an advantage over treatment with CCP. Moreover, in contrast to mAb, hIG are polyclonal antibodies which recognize different epitopes of the virus. Key targets of anti-SARS-CoV-2 antibodies include: the S protein [5] , responsible for viral entry through recognition of the primary host cellular receptor angiotensin-converting enzyme-2 (ACE2); and the N protein [6] , which makes up the helical nucleocapsid. The E protein, a small polypeptide, and the M protein, embedded in the envelope [7] , have been less studied as potential immune targets.

Importantly, IgG possess other antiviral properties, beyond neutralization, which have been described for CCP. These include antigen-dependent Fc functions, e.g. antibody dependent cellular phagocytosis (ADCP) [8] , antibody dependent cellular cytotoxicity (ADCC) [9] and complementmediated cytotoxicity [10] . These well-described effector functions of antibodies (mediated by the interaction of immunoglobulin Fc with cellular Fc receptors) may add to neutralizing activity and may enable non-neutralizing antibodies or antibodies with poor-neutralizing capacity to block or clear infection. hIG efficacy is being tested in ongoing randomized clinical trials in inpatients (IV administration) [11] , and outpatients (SC administration) [12] .

Apart from their SARS-CoV-2 neutralizing capacity, no further antiviral capacity of hIG has been experimentally demonstrated. In this study, we report an extensive functional characterization of a well-characterized hIG product [4] . We performed neutralization assays on several virus isolates and on a pseudovirus expressing the most relevant S variants to date, and, for the first time, we evaluated the capacity of hIG to trigger antigen-dependent IgG Fc functions.

The anti-SARS-CoV-2 hyperimmune globulin 10% (hIG) (Grifols, Barcelona, Spain) prepared from CCP [4] was functionally characterized in vitro. Neutralization of four geographically diverse isolates of SARS-CoV-2 were assessed by four different methodologies (plaque reduction, protection from virusinduced cytotoxicity, TCID 50 reduction and immunofluorimetry-based methodology) at four different laboratories. The capacity of the hIG to neutralize SARS-CoV-2 variants was also evaluated using a M a n u s c r i p t 5 pseudovirus test platform expressing the S proteins of the relevant variants. Finally, the capacity of hIG to induce antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) on the same samples and the viral protein responsible of eliciting these responses were evaluated. Positive (CCP) and negative single-donation plasmas were used for comparison.

At NIH, Vero cells were acquired from the American Type Culture Collection (ATCC #CCL-81; Manassas, VA, USA). At CReSA-IRTA, Vero cells were obtained from the ATCC (ATCC CRL-1586). At CNB-CSIC, Vero cell lines were kindly provided by Dr E Snjider (University of Leiden Medical Center, The Netherlands). At Texcell, Vero cells provided by Pasteur Institut were used. At IrsiCaixa, HEK293T cells overexpressing WT human ACE-2 (Integral Molecular, USA) were used for pseudovirus neutralization assays. Culture conditions for these cell lines are detailed in the supplemental information following this article.

Stock viruses were prepared by collecting the supernatant from Vero cells, as previously described [13] . At NIH, SARS-CoV-2 (GenBank #MT020880) was provided by the U.S. Centers for Disease Control and Prevention (Washington isolate, CDC; Atlanta, GA, USA), isolated from the first US COVID-19 patient [14] . At IRTA-CReSA, SARS-CoV-2 was isolated from nasopharyngeal swab from an 89-year-old male patient from Badalona (Spain) in March 2020 (accession ID EPI ISL 418268 at GISAID repository: http://gisaid.org) with the Spike mutations D614G, NSP12, and P323L. At CNB-CSIC, SARS-CoV-2MAD6 was isolated from nasopharyngeal swab from a 69-year-old male patient from Hospital "12 de Octubre" in Madrid (Spain). Full-length virus genome was identical to SARS-CoV-2 reference sequence (Wuhan-Hu-1 isolate, GenBank MN908947), except for the presence of a silent mutation C3037>T, and two mutations leading to aa changes: C14408>T (in nsp12) and A23403>G (D614G in S protein). At Texcell, 2019-nCoV strain 2019-nCoV/Italy-INMI1 (https://www.ncbi.nlm.nih.gov/nuccore/MT066156) isolated from the first case of COVID-19 in Italy was used [15] .

SARS-CoV-2 antibody-positive plasmas were collected by plasmapheresis from CCP donors (single donation) at Grifols US plasma collection centers (Biomat USA Inc., Interstate Blood Bank Inc., Talecris Plasma Resources, Inc.). CCP was collected during the first half 2020 from donors with different degrees of COVID-19 severity (mild to hospitalized).

COVID-19 specific antibody levels in the CCP were classified as high (positive at ≥ 1/10000 dilution), medium (positive at 1/1000), and low (positive at 1/100) as determined by anti-SARS-CoV-2 S ELISA methods: human anti-SARS-CoV-2 virus spike 1 [S1] IgG ELISA Kit (Alpha Diagnostic Intl., Inc.), against S1 subunit spike protein; EI-2606-9601-G, Anti-SARS-CoV-2 IgG ELISA Kit (Euroimmun AG, Luebeck, Germany), against structural protein (S1 domain); DEIASL019, SARS-CoV-2 IgG ELISA Kit (Creative Diagnostics), against virus lysate. M a n u s c r i p t 6 SARS-CoV-2 antibody-negative plasma (pre-pandemic collection during 2019) was used as a negative control. CCP was used to compare responses between positive CCP samples of different positivity grades (low, medium, and high) with negative plasma controls and hIG.

A previously described cell-based immunofluorescence assay (CBIFA) was used at NIH. The details of this assay are included in the supplemental information. Data are reported based on a 4-parameter regression curve (using a constrained fit) as a 50% neutralization titer (half maximal inhibitory dilution : ID 50 ) [4, 16] .

A cytopathic-cytotoxicity luminometry assay (CCLA) was used at IRTA-CReSA. ID 50 values were determined from the fitted neutralization curves as the plasma dilutions that produced 50% neutralization. A summary of the assay is included in the supplemental information and details of the technique are available elsewhere [17, 18] .

Plaque forming units (PFU)-based neutralization assay was used at CNB-CSIC. Details of the assay are included as supplemental information. The neutralization potency of the hIG product (ID 50 value) was expressed as plaque reduction neutralization test (PRNT 50 ) value, calculated as the -log 10 of the reciprocal of the highest hIG dilution to reduce the number of plaques by 50% compared with the number of plaques without IVIG [18] .

Median Tissue Culture Infectious Dose (TCID 50 )-based microneutralization assay was used at Texcell. A brief summary of the assay is included as supplemental information. The viral titer is expressed in "dose infecting 50% of tissue cultures per mL" with a confidence interval of 95%. For neutralization plate, the ID 50 value was expressed as the Neutralization Titer 50 (NT 50 ) value, calculated as the antibody titer neutralizing sample according to the Spearman-Kärber formula. The NT 50 corresponds to the dilution of sample which prevent the cells from CPE (no lysis) in 50% of the replicates. The criteria for the validation of the run were: back titration of the virus in the TCID 50 criteria; integrity of the uninfected cells (medium control only); and absence of cell layer or presence of CPE in infected wells (virus control only).

SARS-CoV-2.SctΔ19 Wuhan, B.1.1.7, P.1 and B.1.351 variants were generated (GeneArt) from the full protein sequence of the respective spike sequences, with a deletion of the last 19 amino acids in Cterminal [19] . Sequences were human-codon optimized and inserted into pcDNA3.1(+). The G614 spike mutant was generated by site-directed mutagenesis as previously described [20] . A summary of the methodology is included in the supplemental information.

In the experiments performed at IrsiCaixa AIDS Research Institute, HIV reporter pseudovirus expressing SARS-CoV-2 S protein and Luciferase were generated using a plasmid coding for a nonreplicative HIV reporter pNL4-3.Luc.R-.E-obtained from the NIH AIDS Reagent Program [21] and the M a n u s c r i p t 7 spike expression plasmids (as described above and in the supplemental information). The methodology is summarized in the supplemental information. The neutralization assay has been previously validated in a large subset of samples [22] . Neutralization assays were performed in duplicate as previously described [22] . The neutralization assay is briefly described in the supplemental information. The values were normalized, and the ID 50 (the reciprocal dilution inhibiting 50% of the infection) was calculated by plotting and fitting the log of plasma dilution vs. response to a 4-parameters equation in Prism 8.4.3 (GraphPad Software, USA).

ADCC and ADCP specific mechanisms were assayed using bioluminescent reporter assays for quantifying ADCC/ADCP pathway activation by several therapeutic antibody drugs: ADCC Reporter Bioassay, Core Kit, Promega (ADCC Reporter Bioassays, FcγRIIIa V158 variant (high affinity), Catalog number G7010, G7018, Promega Corporation) and FcγRIIa-H (high affinity) ADCP Bioassay, Core kit, Promega (ADCP FcγRIIa-H Reporter Bioassay, Core Kit, Promega Corporation Catalog number: G9995). The assays were performed following the manufacturer's guidelines and are summarized in the supplemental information. ADCC and ADCP induction was expressed as induction ratio (IR), which corresponds to the detected signal versus the 1U/mL kit calibrator. HEK293T cells expressing SARS-CoV-2 spike glycoprotein as a transmembrane protein (Innoprot, Reference: P30908) were then used to verify these functionalities (ADCC/ADCP) for SARS-CoV-2 S antigen. A SARS-CoV-2 spike glycoprotein cell line was stably developed transfecting the HEK293T cell line with a SARS-CoV-2 spike glycoprotein expression plasmid (Innoprot, Derio, Basque Country, Spain).

In these experiments, samples were assayed at increasing concentrations in order to perform a kinetic curve (concentration/response curve) for the dynamic evaluation of ADCC and ADCP functionalities in all sample types.

Plasma samples with high, medium, low, and null positivity for COVID-19 infection (determined by anti-SARS-CoV-2 S ELISA methods) were used as comparators.

M a n u s c r i p t 8

Neutralization titers were calculated using GraphPad Prism 8 version 8.4.3 nonlinear regression curve fit as half-maximal inhibitory dilution (ID 50 ). The titers obtained for different batches (n≥3) are expressed as the mean value ± standard deviation (SD).

All the methods tested demonstrated neutralization of infectivity by hIG in the four SARS-CoV-2 isolates (USA (1), Italy (1), and Spain (2)). ID 50 results are shown in Table 1 . Differences in ID 50 are ascribed to differences in the methodologies employed reflecting their differential sensitivities. The D614G mutation was present in the isolates from Spain.

The neutralization assays with pseudovirus demonstrated the neutralization capability against wildtype (original Wuhan virus) spike and all variants: D614G, B.1.1.7 UK, P.1 Brazilian and B.1.351 South African (Table 2 and Figure 1 ). The levels of neutralization were very similar for Wuhan D614G and B.1.1.7 spikes and lower for the P1 and B.1.351 spikes, but still showed consistent neutralization capacity. The negative control (normal IgG IVIG -pre-pandemic) showed no detectable neutralization.

Strong ADCC and ADCP induction ratios (IR) by hIG were observed on plates coated with SARS-CoV-2 N antigen (IR of 6 or higher for ADCC and an IR of 10 or higher for ADCP) at low hIG concentrations (µg IgG/mL) but not with the E and S antigens. Some ADCP induction by the S antigen was observed at higher concentrations of hIG (IR around 2 at 5 mg IgG/mL) (Supplementary information Figures S1 and S2). ADCC and ADCP induction response for pre-pandemic plasma and pre-pandemic IVIG samples (negative controls) were very weak as expected (IR around 1) at any concentration. These results are summarized in Figures 2a and 2b. ADCC induction ratio by HEK293T cells expressing SARS-CoV-2 S glycoprotein was above negative control value for all batches (n=9; data analyzed at 100 µg/mL) (Figure 3a ). High and medium IgG ELISA SARS-CoV-2 antibody-positive single-donation plasmas were also above the negative control value, but the low SARS-CoV-2 positive and negative SARS-CoV-2 plasmas were not above the negative control. Regarding ADCP induction ratio, the seven hIG batches and the high SARS-CoV-2 positive plasma were above the IR of the negative samples, whereas medium SARS-CoV-2 positive and negative SARS-CoV-2 plasma were not (Figure 3b ).

Pooled hIG and high SARS-CoV-2 positive plasma showed ADCC induction by HEK293T cells expressing SARS-CoV-2 S glycoprotein ( Figure 4 ). Activity correlated with increasing concentrations. Maximal induction ratio in kinetics studies was observed at 150 µg/mL. Higher concentrations of hIG M a n u s c r i p t 9 interfered with the read-out systems (data not shown). Pre-pandemic IVIG samples and other plasmas did not show relevant activity.

Here we report the SARS-CoV-2 neutralization capacity of hIG products for different virus isolates from several regions of the world and for the most relevant SARS-CoV-2 variants. To our knowledge, this is the first time that several hIG product batches are assayed to evaluate these functionalities. Moreover, the robustness of the anti-SARS-CoV-2 activity of hIG was demonstrated for the first time in different immune effector mechanisms (ADCC, ADCP), with different methodologies, and identifying the viral proteins involved.

Four viral infectivity neutralization methods (CBIFA, CCLA, PFU, TCID 50 ) showed strong neutralization of SARS-CoV-2. ID 50 values varied since they are method-dependent. Our results using CBIFA were consistent with those reported in the hIG manufacturing characterization (ID 50 325±76) [4] . Other neutralization methodologies allowed the detection and reporting of more potent neutralization activity, i.e., higher ID 50 .

Since high neutralization capacity was shown employing multiple virus isolates in several virus-cell systems with different methodologies, we can describe the neutralization capacity of hIG as robust and likely to be reproducible under normal physiological conditions after administration to patients. The study of the neutralization capacity with a pseudovirus expressing S glycoproteins from the most relevant SARS-CoV-2 variants (D614G, B.1.1.7, P.1, and B1.351) is especially important given the current situation in UK, Brazil and South Africa. In these regions, the predominant variants have been recently classified by the CDC/WHO as variants of great concern [23] . In this study, some reduction of pseudovirus neutralization for P.1 and B1.351 has been shown (preliminary results for some CCP and derived products [24] ). Effective neutralization of emergent variants is relevant because the plasma used to produce the hIG was collected prior to detection of these variants. However, these hyperimmune products were demonstrated to neutralize these new variants consistently, although this capability has not been observed elsewhere [25] .

Beyond neutralization there are other IgG Fc-dependent functionalities of hIG that may play a role in the protection from and/or resolution of SARS-CoV-2 infection, especially when differences in neutralization activity have been detected for some variants [25, 26] .

In studies of antigen-dependent Fc function with SARS-CoV-2 antigens, only hIG showed relevant ADCC and ADCP activity for the N protein. This is the most abundant protein in coronaviruses. It is highly conserved and is highly immunogenic [27] . This finding could be particularly relevant for variants capable of escaping anti-S neutralization. In fact, S glycoprotein is one of the most important targets for COVID-19 vaccine and therapeutic research [28] .

However, no apparent activity against E and S proteins was observed in Fc function experiments with antigen-coated plates. While E protein is the smallest of all the structural proteins of SARS-CoV-2, S protein is structurally complex [29] . In both cases, the possibility that the antigen attached to the plate acquired an inadequate conformation to be detected by the test, must be considered. Sprotein associated ADCC in COVID-19 patients has been recently reported [30] . Although E protein M a n u s c r i p t 10 has recently being considered as a potential therapeutic target [31] , we further explored Fc functionality related to the more relevant S protein, using HEK293T cells expressing SARS-CoV-2 S glycoprotein.

In HEK293T S cells, all hIG batches induced considerable ADCC and ADCP activity comparable to high titer CCP. This result confirmed that hIG possesses activity against the S protein that was not detected using S antigen-coated plates. Moreover, the ADCC induction ratio was concentrationdependent, with activity at concentrations as low as 25 µg/mL. It is important to remember that the IR value of the positive plasma corresponds to a single SARS-CoV-2 positive donor with a high antibody titer, while the hyperimmune IR corresponds to multiple donors with variable titers. ADCC and ADCP are mechanisms for antigen-dependent antibodies through which virus-infected or otherwise diseased cells are targeted for destruction or elimination. This occurs through multiple components of the cell-mediated immune system, primarily through FcγRIIIa expressed on natural killer cells (for ADCC), and by monocytes-macrophages, neutrophils and dendritic cells via FcγRIIa (CD32a), FcγRI (CD64) and FcγRIIIa (CD16a) for ADCP. The role of antibodies against SARS-CoV-2 N protein in these mechanisms could be a determining factor in resolving SARS-CoV-2 infections through an S-protein independent mechanism. This should be further investigated since, theoretically, protein N is not accessible to antibodies in an intact virus or infected cell.

For other viruses such as influenza A, the effects of non-neutralizing antibodies against internal and more conserved virus proteins suggest that these antibodies play an important role in viral immunity [32] . They reduce virus titers and ameliorate disease via ADCC [33, 34] . Anti-NP antibodies can facilitate particle and antigen uptake and presentation, leading to reduced viral titers and morbidity [34] [35] [36] .

MAb and vaccine efficacy against SARS-CoV-2 are based on anti-S neutralizing activity. Here we demonstrated the activity of hIG COVID19 through other proteins (N) and through mechanisms involving host immune system cells (ADCC and ADCP). This opens the door to combine therapeutic and prophylactic strategies by using these products in combination to increase effectiveness. However, it has been reported that the treatment with CCP has little effect on the outcome of the disease in hospitalized patients [37] [38] [39] [40] . Similarly, mAb has limited efficacy once patients are hospitalized, but there is clinical benefit when mAb are administered early in the course of disease [41] [42] [43] [44] . Preliminary results suggest that hIG may act in a similar way, i.e., by preventing patient hospitalization [45] . In addition, for certain groups of patients such as those with primary or acquired antibody deficiency, the use of SARS-CoV-2 hIG might be much more beneficial as compared to typical COVID-19 patients. Nevertheless, direct comparison of different products evaluated with different technologies and/or methodologies is always difficult, if not futile. Precise knowledge of the mechanism of action of hIG will help to predict effectiveness and save time and effort in the selection of the target patient population. In addition, hyperimmune IgG product has anti-viral activities beyond neutralization that when combined with neutralization have the potential to provide a more robust treatment against new infectious threats. M a n u s c r i p t 11 Conclusions hIG solutions had strong neutralization capacity against SARS-CoV-2, not only against viruses that plasma donors were exposed to, but also against the new SARS-CoV-2 emerging variants. Under our experimental conditions, viral N and S proteins induced antigen-dependent Fc functions, such as ADCC and ADCP even at low concentrations. The fact that similar results were obtained with multiple experimental approaches suggest that hIG treatment is a promising therapeutic option for SARS-CoV-2 therapy. M a n u s c r i p t 18 M a n u s c r i p t 19 . Six hyperimmune samples were assayed and showed high ADCC activity (100 µg/mL) when using S expressing HEK293 cells. SARS-CoV-2 positive plasma samples (100 µg/mL) showed also this functionality. Non-statistical differences in ADCP induction ratios among hyperimmune batches are attributable to inter-assay variability. Fig. 4 . Antibody-dependent cellular cytotoxicity (ADCC) induction ratio kinetic curve in hyperimmune samples, prepandemic immunoglobulins, SARS-CoV-2 positive (high, medium and low SARS-CoV-2 IgG titers) and SARS-CoV-2 IgG negative single-donation plasmas, in SARS-CoV-2 Spike Glycoprotein expressing HEK293T cells (Innoprot). Hyperimmune batches demonstrate high ADCC functionality at incremental concentrations, with a peak at 150 µg/mL. Non-statistical differences in ADCP induction ratios among hyperimmune batches are attributable to inter-assay variability.

Therapeutic options for the 2019 novel coronavirus (2019-nCoV)

Convalescent plasma and hyperimmune globulin therapy in COVID-19

Anti-SARS-CoV-2 antibodies in healthy donor plasma pools and IVIG products

Production of anti-SARS-CoV-2 hyperimmune globulin from convalescent plasma

SARS-CoV-2 spike protein: a key target for eliciting persistent neutralizing antibodies

Role of IgG against N-protein of SARS-CoV2 in COVID19 clinical outcomes

Antibody-Dependent Cellular Phagocytosis in Antiviral Immune Responses

Presence of antibody-dependent cellular cytotoxicity (ADCC) against SARS-CoV-2 in COVID-19 plasma

Antibodies in Convalescent Plasma

Inpatient Treatment With Anti-Coronavirus Immunoglobulin (ITAC)

A Study to Evaluate the Safety and Efficacy of C19-IG 20% in SARS-CoV-2 Infected Asymptomatic Ambulatory Outpatients (COVID-19)

Antigenic cross-reactivity between severe acute respiratory syndrome-associated coronavirus and human coronaviruses 229E and OC43

Isolation and characterization of SARS-CoV-2 from the first US COVID-19 patient

Molecular characterization of SARS-CoV-2 from the first case of COVID-19 in Italy

Micro-Neutralization Assay for Qualitative Assessment of SARS-CoV-2 (COVID-19) Virus-Neutralizing Antibodies in Human Clinical Samples

Construction of a severe acute respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus RNA synthesis

Cross-neutralization activity against SARS-CoV-2 is present in currently available intravenous immunoglobulins

Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

An efficient one-step site-directed and site-saturation mutagenesis protocol

Vpr Is Required for Efficient Replication of Human Immunodeficiency Virus Type-1 in Mononuclear Phagocytes

SARS-CoV-2 infection elicits a rapid neutralizing antibody response that correlates with disease severity

SARS-CoV-2 Variant Classifications and Definitions

Reduced neutralization of SARS-CoV-2 variants by convalescent plasma and hyperimmune intravenous immunoglobulins for treatment of COVID-19

Resistance of SARS-CoV-2 variants to neutralization by monoclonal and serum-derived polyclonal antibodies

Emerging SARS-CoV-2 variants reduce neutralization sensitivity to convalescent sera and monoclonal antibodies

The Nucleocapsid Protein of SARS-CoV-2: a Target for Vaccine Development

SARS-CoV-2 SPIKE PROTEIN: an optimal immunological target for vaccines

Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19

Longitudinal analysis of humoral immunity against SARS-CoV-2 Spike in convalescent individuals up to eight months post-symptom onset

Coronavirus envelope protein: current knowledge

Influenza A Virus Vaccination: Immunity, Protection, and Recent Advances Toward A Universal Vaccine

Immunological assessment of influenza vaccines and immune correlates of protection

Prophylactic and therapeutic activity of fully human monoclonal antibodies directed against Influenza A M2 protein

A Novel Role for Non-Neutralizing Antibodies against Nucleoprotein in Facilitating Resistance to Influenza Virus

Human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza A viruses

Effects of treatment of COVID-19 with convalescent plasma in 25 B-cell depleted patients

A randomized double-blind controlled trial of convalescent plasma in adults with severe COVID-19

Severe Acute Respiratory Syndrome Coronavirus 2 Convalescent Plasma Versus Standard Plasma in Coronavirus Disease

Infected Hospitalized Patients in New York: A Double-Blind Randomized Trial

Effects of potent neutralizing antibodies from convalescent plasma in patients hospitalized for severe SARS-CoV-2 infection

Learning from past failures: Challenges with monoclonal antibody therapies for COVID-19

The effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis

CONVALESCENT plasma for COVID-19: A meta-analysis of clinical trials and real-world evidence

Tackling COVID-19 with neutralizing monoclonal antibodies

INSIGHT-013) evaluating hyperimmune globulins as a treatment for hospitalized patients with COVID-19

A cell-to-cell HIV transfer assay identifies humoral responses with broad neutralization activity

The authors acknowledge the expert technical assistance of the Immunotherapies Unit personnel (D. 

A c c e p t e d M a n u s c r i p t 12 A c c e p t e d M a n u s c r i p t