Oral administration of a recombinant modified RBD antigen of SARS-CoV-2 as a possible immunostimulant for the care of COVID-19

Abstract

Background: Developing effective vaccines against SARS-CoV-2 that consider manufacturing limitations, equitable access, and acceptance is necessary for developing platforms to produce antigens that can be efficiently presented for generating neutralizing antibodies and as a model for new vaccines.

Results: This work presents the development of an applicable technology through the oral administration of the SARS-CoV-2 RBD antigen fused with a peptide to improve its antigenic presentation. We focused on the development and production of the recombinant receptor binding domain (RBD) produced in E. coli modified with the addition of amino acids extension designed to improve antigen presentation. The production was carried out in shake flask and bioreactor cultures, obtaining around 200 mg/L of the antigen. The peptide-fused RBD and peptide-free RBD proteins were characterized and compared using SDS-PAGE gel, high-performance chromatography, and circular dichroism. The peptide-fused RBD was formulated in an oil-in-water emulsion for oral mice immunization. The peptide-fused RBD, compared to RBD, induced robust IgG production in mice, capable of recognizing the recombinant RBD in Enzyme-linked immunosorbent assays. In addition, the peptide-fused RBD generated neutralizing antibodies in the sera of the dosed mice. The formulation showed no reactive episodes and no changes in temperature or vomiting.

Conclusions: Our study demonstrated the effectiveness of the designed peptide added to the RBD to improve antigen immunostimulation by oral administration.

Fig. 1

Scheme of Spike (S) protein architecture of the SARS-CoV-2 (A): NTD N-terminal domain, RBD receptor-binding domain (330–525 WA1/2020), RBD-P receptor-binding domain fused to 40AV peptide, RBM receptor binding motif, SD1 subdomain 1, SD2 subdomain 2, S1/S2 protease cleavage site, S2' protease cleavage site, FP fusion peptide, HR1 heptad repeat 1, CH central helix, HR2 heptad repeat 2, TM transmembrane domain. Representative scheme of the peptide fused RBD and peptide free RBD (B)

Fig. 2

Kinetics of biomass growth (circles) and recombinant protein production (triangles) of RBD-P (filled symbols) and RBD (open symbols) from SARS-CoV-2 by E. coli BL21 (DE3), in shake flasks (A) and in bioreactors (B). Data presents the average and standard deviation of the cultures carried out at least in triplicate. Induction with IPTG started after 6 h of culture. Inset figures are logarithmic biomass growth. Glucose consumption was also presented for shake flasks (C) and bioreactors (D) cultures

Fig. 3

Kinetic comparison in SDS-PAGE (12%) of total protein (TP) produced in cultures of E. coli BL21 (DE3) grow up in shake flasks (A, B) and bioreactors (C, D). RBD-P producing cultures are shown in (A) and (C) and RBD in (B) and (D). In all gels, lanes 1 and 6 correspond to PT before induction. Lanes 2, 3, and 4 (7,8, and 9) show samples taken from cultures at 1, 5, and 10 h after induction, respectively. Lane 5 corresponds to the molecular weight marker (MWM). Lanes 6 to 9 are samples from replica experiments. Recombinant proteins are marked by a black arrow

Fig. 4

Coomassie blue stained 12% acrylamide gel electrophoresis of the IBs obtained at the end of E. coli BL21 (DE3) cultures producers of RBD-P and RBD. IBs from cultures in shake flasks (A); lane 1: molecular weight marker (MWM), lane 2: IBs of RBD-P, and lane 3: IBs of RBD. IBs from two independent bioreactor cultures (B); lanes 1 and 4 are IBs from cultures producers of RBD-P, lane 3 is the MWM, and lanes 2 and 5 are IBs from cultures producers of RBD. Immunodetection by Western Blot of purified RBD-P and RBD (C) recombinantly produced in E. coli BL21 (DE3) in bioreactor and detected with anti-Spike antibody (Sino Biological 40,591-MM43); lane 1: RBD-P, and lane 2: RBD, and lanes 3 and 4: molecular weight marker (MWM)

Fig. 5

Secondary structure characterization of RBD forms by CD spectroscopy. The spectra were recorded in pure water A RBD and B RBD-P and in a 50% (v/v) TFE aqueous mixture, C RBD and D RBD-P. Secondary structure contents (αH, α helix; βS, β strand; O, other) were calculated from a deconvolution analysis of the CD spectra with the BeStSel webserver. Black circles are experimental spectra, lines correspond to the best-fit spectra, and residuals between calculated and experimental data are shown with asterisks. High-resolution chromatography of the RBD-P and RBD antigens on an Xbridge Protein BEH C4 reverse phase column (E). The interest components were eluted at 39.6 min, in a gradient from 0 to 60% acetonitrile in 60 min, with a flow of 0.8 mL/min, and injected 25 μg

Fig. 6

Recognition of recombinant RBD and RBD-P by human IgG polyclonal antibodies. The ELISA was performed using sera samples from no hospitalized individuals convalescing from COVID-19 (median age around 40 years). Also, twelve human sera obtained before the COVID-19 pandemic were used as control. Infection with SARS-CoV-2 was confirmed by RT-PCR from nasopharyngeal swabs performed in clinics. In boxplots, each data point is the mean and its deviation of replica per serum. Also, each boxplot presents the mean in a dotted line, the median as the middle line, the interquartile range as box limits, and the 2.5th and 97.5th percentiles as the whiskers. For all variables with the same letter, the difference between the means is not statistically significant. If two variables have different letters, they are significantly different (p < 0.05)

Fig. 7

Micrographs by TEM of the oil-in-water emulsion without recombinant proteins (A, B), oil-in-water emulsion with RBD-P (C, D) and oil-in-water emulsion with recombinant RBD (E, F) developed for oral administration. Scale bars are found inside the micrographs

Fig. 8

Immunization scheme of BALB/c mice with RBD-P and RBD (A). Analysis of antigen recognition with polyclonal antibodies of murine serum immunized with recombinant RBD and RBD-P orally (B) and intramuscularly (C). The Y axis shows the absorbance value measured at 450 nm Serum was placed at a dilution 1:200 for intramuscularly groups, and at a dilution 1:100 for orally groups. In boxplots, each data point is the mean of two wells per mouse serum. Also, each boxplot presents the mean as an X, the median as the middle line, the interquartile range as box limits, and the 2.5th and 97.5th percentiles as the whiskers. For all variables with the same letter, the difference between the means is not statistically significant. If two variables have different letters, they are significantly different (p < 0.05). The immunization scheme was prepared using Biorender

Fig. 9

Indirect ELISA titration against recombinant RBP from E. coli of pre-immune (black) and hyperimmune female (left) and male (right) mice sera using RBD (grey) and RBD-P (light grey) antigens administrated orally (A, B) and intramuscularly (C, D). Results are presented as the mean and standard deviation of at least triplicates. There is a significant difference (p < 0.05) in all cases when the results using RBD (grey) and RBD-P (light grey) are compared for each dilution evaluated

Fig. 10

Neutralization assay of the hyperimmune sera obtained 30 days post-immunization of BALB/c mice with RBD-P and RBD orally (A) and intramuscularly (B). The y-axis corresponds to the observed percentage of the inhibition of HRP-conjugated RBD interaction with hACE2. Dashed lines represent manufacturers’ cutoff values (30%). The neutralization assay was performed in duplicate for each mouse serum, and per group. C+ is the positive control. Each boxplot presents the mean as an X, the median as the middle line, the interquartile range as box limits, and the 2.5th and 97.5th percentiles as the whiskers. For all variables with the same letter, the difference between the means is not statistically significant. If two variables have different letters, they are significantly different (p < 0.05). When there are two letters in a data set, this is not significantly different from the data sets with those letters