Background: SARS-CoV-2 or (CoV2) utilizes the receptor binding domain (RBD) of its surface SPIKE protein to infect human cells that express ACE2 receptor. Infected persons can be asymptomatic or progress to a potentially fatal pneumonia-associated illness. Antibodies that prevent SPIKE/RBD/ACE2 interaction can result from infection or vaccination. However, their quantity, function, and cross-reactive protection requires serological testing. We obtained 50 deidentified serum samples, and were blinded to CoV2 vaccination (Vax+; Vax-) or CoV2 positive test (CoV2+; CoV2-). Serum IgG antibodies to SPIKE and RBD were quantified and efficacy in preventing CoV2 infection in vitro determined. We hypothesized that high titer RBD IgG would be efficient at preventing viral infection and be effective against a later CoV2 variant.

Methods: ELISA was used to quantify SPIKE and RBD antibodies in sera. Neutralizing capability of antibodies was determined by mixing sera with a firefly luciferase lentivirus psuedotyped with Wuhan CoV2 SPIKE. The mixture was applied to ACE2 expressing cells and luciferase activity used to calculate infection rates. Samples were unblinded to determine correlation between in vitro results and CoV2 exposure or vaccination. Cross-protection was determined by repeating neutralization assays with the Omicron BA.2 variant.

Results Thirteen participants reported no CoV2 vaccine or CoV2 positive test (Vax-/CoV2-) yet 7 had anti-SPIKE and -RBD titers and neutralizing function suggesting prior asymptomatic CoV2 infection. The remaining 37 participants reported Vax-/CoV2+ (n=8), Vax+/CoV2- (n=27), or Vax+/CoV2+ (n=2). Titers to RBD were comparatively lower than titers to SPIKE in most patient samples. Average RBD antibody titers were higher for the Vax+/CoV2- group (1:2600) versus Vax-/CoV2+ cohort (1:900). Highest RBD antibody titers were found in Vax+/CoV2+ patients (1:5400). Finally, sera with RBD titer ≥1:2700 and neutralization capacity had equal neutralizing function on Omicron BA.2, a variant with 28 amino acid changes in SPIKE and 16 in the critical RBD region.

Conclusion High-titer RBD sera correlated with greater viral neutralization activity highlighting the functional importance of this domain in infection. High-titer anti-RBD IgG to the initial Wuhan CoV2 virus also provided potent neutralization activity against the BA.2 variant despite 16 amino acid changes in its RBD region. This information could be used in future vaccination efforts and passive immunization, as well as provide a greater level of confidence to healthcare workers, first responders, and other high-risk populations regardless of the CoV2 strain. Serial immune studies in high risk populations could provide indications for booster vaccines.

Functional Serologic Testing and Cross Reactivity of Serum Antibodies to SARS-CoV-2 SPIKE Protein

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