In the SARS-CoV-2 Pandora Pandemic: Can the Stance of Premorbid Intestinal Innate Immune System as Measured by Fecal Adnab-9 Binding of p87:Blood Ferritin, Yielding the FERAD Ratio, Predict COVID-19 Susceptibility and Survival in a Prospective Population Database?

Abstract

SARS-CoV-2 severity predictions are feasible, though individual susceptibility is not. The latter prediction allows for planning vaccination strategies and the quarantine of vulnerable targets. Ironically, the innate immune response (InImS) is both an antiviral defense and the potential cause of adverse immune outcomes. The competition for iron has been recognized between both the immune system and invading pathogens and expressed in a ratio of ferritin divided by p87 (as defined by the Adnab-9 ELISA stool-binding optical density, minus the background), known as the FERAD ratio. Associations with the FERAD ratio may allow predictive modeling for the susceptibility and severity of disease. We evaluated other potential COVID-19 biomarkers prospectively. Patients with PCR+ COVID-19 tests (Group 1; n = 28) were compared to three other groups. In Group 2 (n = 36), and 13 patients displayed COVID-19-like symptoms but had negative PCR or negative antibody tests. Group 3 (n = 90) had no symptoms and were negative when routinely PCR-tested before medical procedures. Group 4 (n = 2129) comprised a pool of patients who had stool tests and symptoms, but their COVID-19 diagnoses were unknown; therefore, they were chosen to represent the general population. Twenty percent of the Group 4 patients (n = 432) had sufficient data to calculate their FERAD ratios, which were inversely correlated with the risk of COVID-19 in the future. In a case report of a neonate, we studied three biomarkers implicated in COVID-19, including p87, Src (cellular-p60-sarcoma antigen), and Abl (ABL-proto-oncogene 2). The InImS of the first two were positively correlated. An inverse correlation was found between ferritin and lysozyme in serum (p < 0.05), suggesting that iron could have impaired an important innate immune system anti-viral effector and could partially explain future COVID-19 susceptibility.

Keywords: ACE inhibitors; Abl; Adnab-9; COVID-19; Src; adaptive immune system; diabetes mellitus; innate immune system; lysozyme; organ specific neoantigen; p87; prognostic biomarker; race; sex.

Figure 1

Flow diagram of patients who submitted stool cards and had ferritin levels for a FERAD calculation. Less than 50% of patients turned in cards, and only 507 of those had ferritin concentration levels determined opportunistically and available from the computerized patient record system (CPRS). FERAD ratios in symptomatic (Sx) and asymptomatic (asym) patients totaled 36 and 461, where the results of a PCR COVID-19 test were unknown (unk). The proportion of stool cards delivered by postal mail was not substantially different from the known return proportion of 44% for occult blood card return, as determined for that time period at the Detroit VAMC.

Figure 2

Scattergram of comparable clinical parameters in the current COVID-19 cumulative cohort versus the same parameters in the first wave of COVID-19 patients from the same medical center.

Figure 3

Correlation of paired ferritin levels, initial and last drawn in 56 patients (a), and a scattergram of 27 pairs (b) of initial ferritin, plotted against Stool bound Adnab-9 binding > 0.01 showing a positive correlation between stool p87 and initial ferritin values.

Figure 4

A bar diagram depicting pre-morbid FERAD values of COVID-19-positive patients, as compared to controls and COVID-19-negative test patients.

Figure 5

Significant correlation between lysozyme and ferritin in the serum.

Figure 6

Bar diagram of overall survival by FERAD ratio quartile. Abbreviations: AA—African American; Cau—Caucasian; Eff—Effluent; WB—Western blot.

Figure 7

Scattergram of an inverse correlation of p87 stool bands and overall survival in Group 2 patients.

Figure 8

Photomicrograph ofAdnab-9 staining by immunohistochemistry in normal-appearing colonic mucosa. This photomicrograph with a particular magnification of 50×, in a normal colon biopsy section, showed the strong specific reddish-brown staining in the cytoplasm.

Figure 9

Bar Diagram showing reduced fixed p87 antigen in the ceca of AA, as compared to Caucasians.

Figure 10

Scattergram of stool p87 negatively correlated with ferritin.

Figure 11

Relationship of outcomes in patients with positive fecal p87 ELISA (p87 ELISA stool+) and those with negative stool ELISA but detectable p87 bands in stool (Bands+).

Figure 12

Western blot of stool with specific p87 bands and Molecular Weight Markers. Monoclonal antibody Adnab-9 that recognized p87 reacted in a Western blot of fecal samples. The blot showed positive bands with the relative mobility (M) molecular weight standards designated on the right. “Wet” represented a sample that had been freshly obtained and processed less than 24 h, being left at room temperature. D1, D3, and D6 were the sample stored at room temperatures for 1, 3, and 6 days, respectively. Paradoxically, while D6 appeared to have stronger bands, as compared to D1 and D3, the conditions of prolonged changes in the temperature and humidity could have caused these changes in the protein bands.

Figure 13

Mean FERAD ratios increased with the severity of COVID-19.

Figure 14

Direct significant correlation between sets of NLR obtained at time of initial stool collection and COVID-19.

Figure 15

Inverse correlation between sets of NLR obtained at time of initial stool collection and during time of non-COVID-19 related symptoms.

Figure 16

Depiction of 3 biomarkers expressed in the stools of a neonate during the first months of life as determined by ELISA.

Figure 17

A Scattergram showing a positive correlation of Src and Adnab-9-defined p87 in the infant stool.

Figure 18

The p87 immunohistochemical labeling of lung adenocarcinoma tissue at intermediate power of magnification (25×). The photomicrograph shows the adenocarcinoma diffusely involving the lung, but some normal bronchioles were observed (arrows) that exhibited a deep reddish-brown substrate, indicating the presence of p87. The dark brown p87 was observed in the cytoplasm of clearly malignant adenocarcinoma, shown on the left (smaller arrow), and in normal cells located in the upper center (large arrow).

Figure 19

A bar diagram showing significant differences between the patients with DM and controls.