Affinity of anti-spike antibodies to three major SARS-CoV-2 variants in recipients of three major vaccines.

Background: Measuring anti-viral antibody affinity in blood plasma or serum is a rational quantitative approach to assess humoral immune response and acquired protection. Three common vaccines against SARS-CoV-2-Comirnaty developed by Pfizer/BioNTech, Spikevax developed by Moderna/NIAID, and Jcovden (previously Janssen COVID-19 Vaccine) developed by Johnson & Johnson/Janssen (J&J)-induce antibodies to a variety of immunogenic epitopes including the epitopes located in the ACE2 receptor-binding domain (RBD) of the spike protein. Blocking RBD with antibodies interferes with the binding of the virus to ACE2 thus protecting against infection.
Methods: We perform measurements in the serum of the recipients of Pfizer, Moderna, and J&J vaccines, and we compare the apparent affinities of vaccine-induced antibodies against the RBD of the ancestral SARS-CoV-2 virus and the Delta and Omicron variants. We use our recently published method to determine the apparent affinity of anti-spike protein antibodies directly in human serum. This involves probing antibody-antigen equilibria with a small number of antigen-coated magnetic microparticles and imaging them on a fluorescence microscope.
Results: Recipients of two-dose Pfizer and Moderna vaccines, as well as recipients of the single-dose J&J vaccine, develop high-affinity antibodies toward RBD derived from ancestral SARS-CoV-2. Affinities of these antibodies to Delta-RBD are approximately 10 times weaker, and even more drastically reduced (∼1000-fold) toward Omicron-RBD. Conclusions: Vaccine-induced antibodies against ancestral SARS-CoV-2 RBD demonstrate ~10-fold and ~1000-fold weaker affinities toward Delta- and Omicron-RBD, respectively. Our approach offers a direct means for evaluating vaccine-induced adaptive immunity and can be helpful in designing or updating vaccines.

Introduction

Measuring anti-viral antibody affinity in blood plasma or serum is a rational, quantitative approach to assessing humoral immune responses and adaptive immunity. Three common vaccines against SARS-CoV-2, developed by Pfizer/BioNTech, Moderna/NIAID, and Johnson & Johnson/Janssen (J&J), induce antibodies to a variety of immunogenic epitopes, including the epitopes located in the receptor-binding domain (RBD) of the spike protein. Spike RBD mediates the binding with membrane-bound angiotensin-converting enzyme 2, commonly known as the ACE2 receptor, which is the precursor for the entry of the SARS-CoV-2 virus into human cells. ACE2 is located on the surface of various human epithelial cells and serves as the anchoring point for the virus to breach the membrane[1-3]. Antibodies directed toward the receptor-binding domain of viral spike protein S1 can interfere with the binding of the virus to host cell ACE2 and thus prevent infection[4-6]. Additionally, studies have shown that nearly 90% of neutralizing antibody activity in SARS-CoV-2 patients is related to spike RBD[7,8]. Therefore, assessing the affinities of anti-RBD antibodies, generated in response to vaccination against SARS-CoV-2, can serve as a quantitative measure of the humoral immune response and acquired anti-viral protection against ancestral SARS-CoV-2[9] and its Delta and Omicron variants[10,11].

It was recently found that the neutralizing effect of monoclonal antibodies developed against ancestral SARS-CoV-2 is not as effective against the mutated variants[12,13]. Reductions in neutralization efficacy have also been observed for vaccine-induced antibodies[14,15]. However, the reported neutralizing antibody titers encompass two primary parameters that drive the RBD and anti-RBD binding reaction: antibody affinity and antibody concentration, which should be quantified independently.

In our previous publication[16], we presented a simple imaging-based method for measuring antibody affinities independently of concentration. This was done directly in human blood serum or plasma without purification or labeling and used to follow the development of the humoral immune response in convalescent COVID-19 patients. We also showed that high antibody affinities can be achieved faster by vaccination than from infection.

In this work, we extend this method to directly compare the antibody affinities to RBD from the ancestral SARS-CoV-2 strain and its Delta and Omicron variants. We show that recipients of Pfizer, Johnson & Johnson, and Moderna vaccines develop high-affinity antibodies to RBD derived from the ancestral SARS-CoV-2. The affinity of these antibodies is ~10-times weaker to the Delta-RBD, and ∼1000-fold weaker to the Omicron-RBD.

Methods

Serum was highly diluted to bring anti-spike antibody to similarly low levels for all samples, on the order of tens of picomolar (∼5 µg/L). Maintaining the antibody concentration well below the expected dissociation constant (KD) makes the measured affinity independent of antibody concentration. Twenty-four aliquots of diluted serum (patient antibody) were incubated with serially titrated RBD antigen to achieve equilibrium. Subsequently, a small number of magnetic microparticles, coated with wild-type RBD, were added to the reaction solutions to probe the amount of free antibody (not bound to antigen) without noticeably perturbing the equilibria. The microparticles carrying captured antibodies were washed, reacted with fluorescently labeled anti-human IgG conjugate, washed again, and imaged on a fluorescence microscope. Using the zero-RBD titration point values as the maximum fluorescence intensity, it is possible to quantify the fraction of antibody prevented from microparticle capture due to the prior formation of antibody-RBD complex. Plotting the fraction of blocked antibody (% inhibition) as a function of RBD concentration generates a binding curve. Fitting the curve yields an apparent affinity of the polyclonal antibodies.

Fluorescence imaging

Fluorescently labeled magnetic microparticles were imaged in optical-bottom 96-well plates on an IX83 inverted microscope (Olympus, Tokyo, Japan). Epifluorescence excitation was produced using an X-Cite LED illumination system (Excelitas, Wheeling, IL). An automated stage was employed to obtain 9 acquisitions per well using a pco.panda camera (PCO, Kelheim, Germany) and a 20x air objective (UPlanXApo, NA = 0.80, Olympus). Each acquisition consisted of a brightfield image to locate the microparticles and a fluorescent image (Cy3 filter cube, Edmond Optics, Barrington, NJ) to record the fluorescence intensity. Imaging analysis, including large fluorescent aggregate removal (e.g., dust/hair), calculation of the total microparticle pixel area and total microparticle fluorescence, was performed in Metamorph Advanced software (Molecular Devices, San Jose, CA), as previously described[17].

Reagents

ARCHITECT wash buffer (phosphate buffer containing detergent, Abbott Laboratories, Abbott Park, IL) served as the primary diluent for all experiments. Three variants of recombinant receptor-binding domain (RBD) of the S1 subunit spike protein served as antigens. CHO-cell expressed, wild-type RBD (residues 319–541) was produced in-house. SARS-CoV-2 Delta (B.1.617.2) and Omicron (B.1.1.529) variant RBD proteins were purchased from GenScript (Piscataway, NJ). The Delta-RBD construct consists of residues 319–591 and includes the L452R and T478K mutations, and the Omicron-RBD (residues 319–541) has 15 mutations. Magnetic microparticles (4.7 µm, Agilent, Santa Clara, CA), coated with the same recombinant WT-RBD, were used to capture anti-spike antibody in serum samples. Captured antibodies were detected using Cy3-labeled donkey anti-human IgG antibody conjugate (Jackson ImmunoResearch, West Grove, PA).

Determining serum dilutions

To determine antibody affinities which are independent of antibody concentrations, the antibody concentration in the titration experiments must be close to or below the binding affinity dissociation constant (on the close order of 1 nM). To provide an initial estimate of the anti-spike antibody levels in the patient serum samples, a SARS-CoV-2 IgG II Quant ARCHITECT assay was performed using a commercially available assay kit, according to the package instructions (link to the package insert: https://www.corelaboratory.abbott/us/en/offerings/segments/infectious-disease). Subsequently, this data was used to create the initial estimated serum dilutions. A single aliquot of diluted serum from each patient was run through the experimental protocol below to determine the fluorescence intensity level, which is proportional to the antibody level. An intensity signal of ∼2000 counts was targeted, being the optimal balance between minimizing antibody concentration and maintaining a clearly detectable signal. The results of these single patient aliquot experiments were used to further refine individual patient dilution factors to bring all samples to approximately the same low antibody level.

Identifying presumably pre-infected individuals

As mentioned above, we employed a SARS-CoV-2 IgG II Quant ARCHITECT assay to quantitate IgG antibodies against the spike receptor-binding domain (RBD) of SARS-CoV-2. These assays were performed on the post-vaccination serum samples as described above, but also on the pre-vaccination samples associated with each individual. The assay cut-off value for detecting the presence of anti-RBD IgG is 50 AU/mL, as per the assay insert. Pre-vaccination sample IgG levels above this value are presumed to indicate that the individual experienced a prior SARS-CoV-2 infection.

Apparent affinity titration experiments

Patient sample was diluted in ARCHITECT wash buffer to achieve low antibody concentrations (∼60 pM), well below the expected dissociation constant of the target binding reactions, and was aliquoted into 24 wells of two 96-well plates. Different SARS-CoV-2 variants of RBD were titrated into the patient sample wells starting at 5 µM RBD, next at 1 µM RBD and then in two-fold serial dilutions down to 2 pM, plus additional wells containing no RBD (buffer only) as zero titration point controls. All assay steps were carried out on a KingFisher magnetic microparticle plate processor (Thermo Fisher Scientific, Waltham, MA) maintained at 37 °C. The patient aliquots with titrated RBD were incubated for 30 min to equilibrate the antibody-antigen binding reaction. Subsequently, 2 µL of RBD-coated microparticles (0.1% solids) were added to the reactions and incubated for 5 min to sample the amount of patient antibody not already bound to RBD. The microparticles with captured antibody were washed with ARCHITECT wash buffer, incubated for 10 min with 80 µL of the Cy3-labeled anti-human conjugate (3.6 nM), washed again, and imaged on a fluorescence microscope. Two pseudo-data points at extremely high concentrations (100% inhibition at 1 mM and 10 mM RBD, see Apparent affinity calculations below) were appended to the experimental data, and the resulting binding curves were fit to a standard four-parameter logistic model in Origin 2016 (OriginLab, Northampton, MA). The recovered RBD concentration at 50% inhibition is an apparent dissociation constant of the binding reaction since the patient produces a polyclonal antibody response. The inverse of this value is the apparent affinity of the polyclonal antibodies. Considering the probable steric hindrance effect caused by the relative sizes of the RBD antigen (~30 kDa) and the antibodies interacting in solution, we do not expect a large fraction of RBD molecules to bind to more than one antibody. As such, the apparent affinity should be a measure of largely one-on-one interactions between the antigen and antibody.

Apparent affinity calculations

The highest RBD concentration employed in the titration experiments for all three variants was 5 µM. As the observed antibody affinities to Omicron-RBD are quite weak, this means that in most cases the Omicron binding curves only barely reach 50% inhibition and none achieve 100% inhibition. It is impractical to use RBD concentrations that are much higher since protein aggregation and nonspecific background noise begins to compromise the results. As such, the data did not justify extrapolating a 50% binding point beyond 5 µM (or 5000 nM). Thus, any Omicron affinities weaker than 0.0002 × 109 M−1 (i.e., 1/5000 nM) were fixed to 0.0002, nor was any value lower than 0.0002 used in calculating the relative affinities. Although, for the purposes of this work, 0.0002 represents a lower bound, many of those samples likely have affinities to Omicron-RBD that are still weaker. It is also important to note that, with regards to the weaker affinities to Delta- and especially to Omicron-RBD, we have made a further assumption in fitting the data. WT-RBD curves saturate at a high WT-RBD concentration which establishes 100% inhibition (i.e., all of the patient’s antibody was bound to RBD in solution) and defines the top of the binding curve. At this saturation point, the measured fluorescence signal comes from nonspecific binding of other, non-SARS-CoV-2 antibodies from that patient. As this value is independent of the RBD variant used to bind the anti-spike antibodies, it can be taken from the WT-RBD experiments and applied to those of the Delta- and Omicron-RBD curves, pinning the endpoint of the binding curve fits to the same value for all three variants. In practice, this was accomplished by appending pseudo-data points (non-experimental) for 100% inhibition at extreme concentration values, 1 mM and 10 mM, for all three variants.

SARS-CoV-2 patient samples

Serum samples from random, unidentified individuals who received either two doses of the Pfizer/BioNTech vaccine, one dose of the Johnson & Johnson/Janssen vaccine, or two doses of the Moderna vaccine were purchased from Access Biologicals, LLC (Vista, CA). Additional Pfizer and Johnson & Johnson vaccine panels were purchased from Precision for Medicine (Norton, MA). For each vaccine panel, the serum samples used in this study were drawn approximately 35–60 days after the individual received their first dose of vaccine. Total numbers of patients measured in this study were as follows: Pfizer: 26 individuals, Moderna: 29 individuals, and J&J: 19 individuals. Six additional patients were not included in this study as they were deemed not measurable due to very low antibodies or very high background (Pfizer: 3, Moderna: 1, J&J: 2).

Ethics

Patient serum samples used for this study were purchased from Access Biologicals, LLC and Precision for Medicine and collected with informed consent. The samples purchased from Access Biologicals, LLC were collected under an IRB protocol approved by Ballad Health System IRB (IRB #00003204). The samples purchased from Precision for Medicine were collected under an IRB protocol approved by IntegReview IRB, LLC (IRB00008463).

Statistics and reproducibility

Fluorescence signals and apparent affinity values are reported as the mean ±  Fluorescence signal uncertainties were obtained from multiple images of the same sample (n = 9), as described above. Box plots display interquartile range (Q1–Q3), median, and mean, along with whiskers to min/max values. One-way ANOVA was performed to determine if there was a statistically significant difference in affinity among the three vaccinated groups—Pfizer, J&J, or Moderna. Two-way ANOVA was performed to determine if there was a statistically significant difference in relative affinity across the three vaccine groups and two variants. A One-way ANOVA was performed to determine if there was a statistically significant difference in anti-spike antibody concentration levels among the three vaccinated groups. If there was a significant difference among the three groups, a pairwise comparison was performed using Tukey’s Honest Significant Differences (HSD) Test to determine which vaccinated groups were significantly different. Tukey’s HSD test was performed with a 95% confidence level. Analyses performed in JUMP 16.0 (SAS, Cary, NC).