Plasmodium infection induces cross-reactive antibodies to carbohydrate epitopes on the SARS-CoV-2 Spike protein

Abstract

Individuals with acute malaria infection generated high levels of antibodies that cross-react with the SARS-CoV-2 Spike protein. Cross-reactive antibodies specifically recognized the sialic acid moiety on N-linked glycans of the Spike protein and do not neutralize in vitro SARS-CoV-2. Sero-surveillance is critical for monitoring and projecting disease burden and risk during the pandemic; however, routine use of Spike protein-based assays may overestimate SARS-CoV-2 exposure and population-level immunity in malaria-endemic countries.

Figure 1.. High frequency of cross-reactive antibodies to SARS-CoV-2 Spike protein from Plasmodium-infected individuals.

In A. and B., Violin plots showing normalized IgG and IgM responses among patients from different cohorts. A. Among all subjects, those in non-malaria endemic areas had significantly lower IgG and IgM than others (t-test IgG p-value<0.0001 and IgM p-value<0.0001.) Subjects with acute malaria infection (symptomatic and asymptomatic) had significantly higher IgG and IgM than uninfected subjects living in malaria endemic areas (t-test p-value<0.0001 for both IgG and IgM). B. Normalized IgG was significantly higher among subjects with P. falciparum, P. malariae, and mixed infections than among negative controls (Welch Two Sample t-test p-value<0.0001 for P. falciparum, p-value= 0.044 for P. malariae and p-value=0.008 for mixed infections), and normalized IgM was significantly higher among subjects with P. falciparum and mixed infections but not P. malariae than among negative controls (Welch Two Sample t-test p-value<0.0001 for P. falciparum, p-value= 0.106 for P. malariae, and p-value=0.018 for mixed infections). C. Normalized IgG and IgM over time in 21 subjects with P. falciparum monoinfection on Day 0. Both IgG and IgM peaked between Day 0 and Week 4 for all subjects. Reinfection, shown by red circles, boosted IgG response in 1 of 4 subjects and IgM response in 2 of 4 subjects. Bold trend line based on local regression (LOESS). In A. B. and C., normalized IgG or IgM calculated by IgG or IgM OD divided by IgG or IgM of positive control (camelid monoclonal chimeric nanobody VHH72 antibody was IgG control, and pooled convalescent serum from SARS-CoV-2 patients was IgM control). Black dashed lines represent cutoffs for positivity, calculated from normalized IgG and IgM values from 80 RT-qPCR negative HCWs (mean + 3 SDs).

Figure 2.. Cross-reactive IgG targets sialic acid on N-linked glycans and is not due to cross-reactivity with other coronaviruses.

A. Cross-reactivity to human coronaviruses is only observed for NL63 and differed between S1 positive and S1 negative subjects (S1 positives on the left side of the vertical white line and S1 negatives on the right side) in REAP testing using a REAP score cutoff of 1.5. B. S1 protein was subjected to three conditions to modify the structure: denaturing; treating with PNGase F to remove all N-linked glycans; and treating with Neuraminidase to remove sialic acid. ELISA was performed on each protein condition and the figure shows percent of S1 Native OD under each condition for 3 controls (pooled convalescent serum from SARS-CoV-2 patients, mouse monoclonal M122 antibody, and camelid monoclonal chimeric nanobody VHH72 antibody, all outlined in red) and 20 subjects (outlined in black). Neuraminidase treatment decreased samples to 23.0% (95% CI: 1.1, 44.9) of OD of S1 native, significantly more than SARS-CoV-2 convalescent serum (decreased to 63% of OD of S1 native), suggesting cross-reactivity was due to reactions with terminal sialic from glycosylated sites. C. In a neutralizing assay using pseudotyped viruses (VSV-Renilla luciferase pseudotyped with Spike), 20 samples (red circles) with high S1 Spike IgG ELISA reactivity showed no neutralization. Bold trend line based on local regression (LOESS) of samples. Control (serum from a COVID-19 positive inpatient, in blue) shows neutralization at dilutions of 1/40 and less.