MENSA, a Media Enriched with Newly Synthesized Antibodies, to Identify SARS-CoV-2 Persistence and Latent Viral Reactivation in Long-COVID

Abstract

Post-acute sequelae of SARS-CoV-2 (SARS2) infection (PASC) is a heterogeneous condition, but the main viral drivers are unknown. Here, we use MENSA, Media Enriched with Newly Synthesized Antibodies, secreted exclusively from circulating human plasmablasts, to provide an immune snapshot that defines the underlying viral triggers. We provide proof-of-concept testing that the MENSA technology can capture the new host immune response to accurately diagnose acute primary and breakthrough infections when known SARS2 virus or proteins are present. It is also positive after vaccination when spike proteins elicit an acute immune response. Applying the same principles for long-COVID patients, MENSA is positive for SARS2 in 40% of PASC vs none of the COVID recovered (CR) patients without any sequelae demonstrating ongoing SARS2 viral inflammation only in PASC. Additionally, in PASC patients, MENSAs are also positive for Epstein-Barr Virus (EBV) in 37%, Human Cytomegalovirus (CMV) in 23%, and herpes simplex virus 2 (HSV2) in 15% compared to 17%, 4%, and 4% in CR controls respectively. Combined, a total of 60% of PASC patients have a positive MENSA for SARS2, EBV, CMV, and/or HSV2. MENSA offers a unique antibody snapshot to reveal the underlying viral drivers in long-COVID thus demonstrating the persistence of SARS2 and reactivation of viral herpes in 60% of PASC patients.

Figure 1.. Primary wild type COVID-19 infection responses in MENSA and serum.

Dot plots show IgG antibody reactivity against S1-RBD in the MENSA (A) and serum (B) of patients experiencing Wild Type SAR-CoV-2 infections in 2020. Similar results are also shown for anti-Nucleocapsid IgG in the MENSA (C) and serum (D). Samples from patients with acute Mild/Moderate (59 samples from 54 patients; blue dots) and Severe/Critical (60 samples from 56 patients; red dots) infections were collected less than 30 DPSO (acute) and/or after 60 DPSO (convalescence). Early pandemic healthy controls, with no prior exposure to SARS2 (n=60), are shown as black dots on the left of each panel. All units are represented as Median Fluorescent Intensity minus background (Net MFI). Dashed lines indicate the C₀ threshold of positivity for each sample type and antigen. Serum C₀s were calculated as the average Net MFI plus five standard deviations of the 60 healthy controls (RBD: 1724; N: 682). For MENSA C0s, a subset of the convalescent patients was identified as a confirmed COVID Recovered (CR) population (no sequelae; N=19) and was used as a contemporary control group to calculate the average Net MFI plus 3 standard deviations (RBD: 570; N: 441). Pair-wise comparisons were performed using the Kruskal-Wallis test in GraphPad Prism (unpaired, nonparametric test; ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).

Figure 2.. Primary vaccination responses in MENSA and serum.

Dot plots show IgG antibody reactivity against S1-RBD in the MENSA (A) and serum (B) of subjects receiving their primary COVID-19 mRNA vaccination, with no prior infection. Similar results are also shown for anti-nucleocapsid IgG in the MENSA (C) and serum (D). Samples were collected from 11 healthy adults prior to vaccination (Baseline; n=7), after Dose 1 Peak (9–20 DPV; n=8), Dose 2 Peak (6–12 DPV; n=8), Dose 3 Baseline (>80 DPV dose 2 through 0 DPV dose 3; n=6), and Dose 3 Peak (4–12 DPV dose 3; n=7). All values are reported as average Net MFI (Median Fluorescent Intensity – Background). Dashed lines indicate the C₀ threshold of positivity for each sample type and antigen as determined in Figure 1. Pair-wise comparisons were performed using the Kruskal-Wallis test in GraphPad Prism (unpaired, nonparametric test; ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).

Figure 3.. Kinetics of MENSA and serum after SARS2 infection and multiple vaccine doses.

Line graphs show MENSA (dark blue) and serum (light blue) IgG antibody responses to S1-RBD (A,B) and Nucleocapsid (C,D) over time for a single patient starting with an initial primary SARS2 infection in 2020, through three doses of Pfizer COVID-19 mRNA vaccination in 2021, and a breakthrough Omicron infection in 2022. All values are reported as average Net MFI (Median Fluorescent Intensity – Background). Red vertical dashed lines represent a new exposure event. The primary and breakthrough infection events are symbolized as virions. Each vaccination dose event is symbolized as a syringe. Horizontal dashed black lines represent the C₀ threshold of positivity for each sample and antigen combination as determined from Figure 1.

Figure 4.. Percentage of PASC patients with symptoms.

Sixty PASC patients recruited in the Emory Long-COVID clinic with self-reported symptom questionnaires at enrollment. Percentages of each symptom is shown to the right of each bar. The twelve-symptom PASC scores based on Thaweethai et al JAMA 2023 are shown in black with point scores in parentheses after each symptom.

Figure 5.. Prolonged, elevated MENSA IgG responses for SARS2 in a subset of PASC patients.

Dot plots show MENSA and serum IgG antibody responses to S1-RBD (A,B) and Nucleocapsid (C,D) in samples collected between 60–279 DPSO since initial COVID-19 Wild Type infection from patients who completely recovered from their acute illness (CR; n=19) and patients who suffer PASC (n=39). Blue dots represent a Mild/Moderate acute disease severity. Red dots represent a Severe/Critical acute disease severity. All values are reported as average Net MFI (Median Fluorescent Intensity – Background). Dashed lines indicate the C₀ threshold of positivity for each sample type and antigen combination as determined from Figure 1. Pair-wise comparisons were performed using the Mann-Whitney test in GraphPad Prism (unpaired, nonparametric test; ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).

Figure 6.. Phage immunoprecipitation sequencing (PhIP-Seq) analysis of MENSA of PASC and CR patients for discovery.

PhIP-seq analysis determines the level of binding of antibodies to 149,259 peptides tiling all protein-coding sequences from viruses with human hosts in MENSA samples from three CR (left column) and 10 PASC patients (middle column). Data are presented as z-scores of the anti-viral antibodies detected. Each row represents a linear peptide of viruses that were differentially bound in PASC and CR groups. Color code identifies virus species (right column).

Figure 7.. MENSA and serum in PASC, CR, and healthy adults for SARS2, EBV, CMV, and HSV2.

MENSA (A) and serum (B) samples were collected from healthy donors prior to SARS2 exposure (top), COVID recovered (middle), and PASC (bottom) patients, tested for IgG reactivity against SARS2, EBV, CMV, and HSV2 antigens, and presented in a heat map. Green cells represent Net MFI values ≥ the C₀ thresholds calculated for each sample type and antigen combination, while white or grey cells represent values below the C₀. In MENSA, a C₀ was calculated as the average Net MFI of 22/23 CR samples plus three standard deviations for each antigen. Each SARS2 Serum C₀ was calculated as the average plus three standard deviations of the 16 Healthy Donor samples collected prior to SARS2 exposure. For each of the remaining viruses, three clinically confirmed negative serum samples were obtained as virus specific negative controls and the average Net MFI plus three standard deviations were used to calculate C₀ for each antigen.