Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Jul 17:19:7201-7214.
doi: 10.2147/IJN.S463546. eCollection 2024.

An Immunoreceptor-Targeting Strategy with Minimalistic C3b Peptide Fusion Enhances SARS-CoV-2 RBD mRNA Vaccine Immunogenicity

Chun-Ta Chiu  1 , Hsiao-Han Tsai  2 , Jing-Yuan Chen  1   3 , Che-Ming Jack Hu  2 , Hui-Wen Chen  1   3
Affiliations

An Immunoreceptor-Targeting Strategy with Minimalistic C3b Peptide Fusion Enhances SARS-CoV-2 RBD mRNA Vaccine Immunogenicity

Chun-Ta Chiu et al. Int J Nanomedicine. .

Abstract

Introduction: The clinical success of mRNA vaccine during the COVID-19 pandemic has inspired emerging approaches to elevate mRNA vaccine immunogenicity. Among them, antigen fusion protein designs for improved immune cell targeting have been shown to augment humoral immunity against small antigen targets.

Methods: This research demonstrates that SARS-CoV-2 receptor binding domain (RBD) fusion with a minimalistic peptide segment of complement component 3b (C3b, residues 727-767) ligand can improve mRNA vaccine immunogenicity through antigen targeting to complement receptor 1 (CR1). We affirm vaccines' antigenicity and targeting ability towards specific receptors through Western blot and immunofluorescence assay. Furthermore, mice immunization studies help the investigation of the antibody responses.

Results: Using SARS-CoV-2 Omicron RBD antigen, we compare mRNA vaccine formulations expressing RBD fusion protein with mouse C3b peptide (RBD-mC3), RBD fusion protein with mouse Fc (RBD-Fc), and wild-type RBD. Our results confirm the proper antigenicity and normal functionality of RBD-mC3. Upon validating comparable antigen expression by the different vaccine formulations, receptor-targeting capability of the fusion antigens is further confirmed. In mouse immunization studies, we show that while both RBD-mC3 and RBD-Fc elevate vaccine immunogenicity, RBD-mC3 leads to more sustained RBD-specific titers over the RBD-Fc design, presumably due to reduced antigenic diversion by the minimalistic targeting ligand.

Conclusion: The study demonstrates a novel C3b-based antigen design strategy for immune cell targeting and mRNA vaccine enhancement.

Keywords: complement component 3b; complement receptor 1; follicular dendritic cells; immune cell targeting; nanotechnology; vaccinology.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflicts of interest in this research.

Figures

None
Graphical abstract
Figure 1
Figure 1
Design and production of immune cell targeting mRNA-LNP vaccines. (A) Designs and sketches of mRNAs and immunogens: RBD, RBD-mC3, and RBD-mFc. The mRNA was composed of a Kozak sequence (Kz), a signal peptide (SP), a SARS-CoV-2 Omicron RBD with or without a targeting ligand, and a 120-adenosine segmented polyA tail. (B) The in vitro transcription of three mRNAs. The mRNAs were presented in the denaturing single-stranded RNA electrophoresis. (C) The cryo-EM image of lipid nanoparticles (LNPs).
Figure 2
Figure 2
Validations of vaccine antigenicity via Western blot and immunofluorescence assay (IFA). (A) Schematic of antigenicity validation. The antigens were expressed via lipofectamine transfection into 293T cells and were analyzed by Western blot. The mRNA-LNP vaccines were directly transfected into 293T cells and examined by IFA. (B) In Western blot, the expressed RBD-mC3, RBD-mFc, and RBD were detected by anti-RBD polyclonal antibodies. Homologous SARS-CoV-2 Omicron RBD recombinant protein was the positive control (PC). GAPDH was the loading control. (C) In IFA, antigens were labeled by green fluorescence (FITC). Cell nuclei were stained by DAPI and presented blue fluorescence. Cells without any transfection were the negative control. (D) Expression levels of three antigens were presented as fluorescence integrated density. Data are presented as median with SD and analyzed through one-way ANOVA Tukey’s multiple comparison test. Non-specific (ns) P > 0.05.
Figure 3
Figure 3
Confirmation of targeting proficiency for mC3 and mFc ligands via immunofluorescence assay (IFA). (A) Illustration of the ligands-receptors interactions that were demonstrated by IFA. Following mRNA transfection into 293T cells, receptors were incubated with the cells. Then, RBD-mC3 bound CR1 while RBD-mFc bound FcγR. The RBD-mC3 and RBD-mFc were labeled by anti-RBD monoclonal antibodies. The CR1 and FcγR were marked using anti-CR1 polyclonal antibodies and anti-FcγR monoclonal antibodies, respectively. (B) Antibodies targeting CR1 (FITC, green) and RBD-mC3 (Cy5, purple) depicted the respective distributions of CR1 and RBD-mC3. (C) Colocalization analysis of RBD-mC3 and CR1. (D) Antibodies against FcγR (green) and RBD-mFc (purple) showed the distributions of FcγR and RBD-mFc respectively. The blue color (DAPI) represented the cell nuclei. (E) Colocalization analysis of RBD-mFc and FcγR. Three regions of interest (ROI) were analyzed. Pearson’s coefficient (R) was calculated in colocalization analysis. R > 0.8 suggests a very strong correlation.
Figure 4
Figure 4
Immunogenicity study of immune cell targeting mRNA vaccines in mice with low dosages. (A) Immunization schedule and vaccination groups. Mice were administrated with three doses of vaccines (1 µg or 10 µg) via the intramuscular route. Mice in the negative control group (NC) were injected with PBS. (B) Comparison of anti-RBD IgG titers from 10 µg groups. (C) Comparison of anti-RBD IgG titers from 1 µg groups. ELISA was conducted to assess the antibody levels in the sera using plates coated with recombinant homologous RBD proteins. Data are presented as median with SD and analyzed through two-way ANOVA Tukey’s multiple comparison test. Orange asterisk displayed the significance between RBD-mC3 and RBD vaccines. Blue asterisk and ns showed the analysis between RBD-mFc and RBD vaccines. Non-specific (ns) P > 0.05, *P < 0.05, **P < 0.01.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Fang E, Liu X, Li M, et al. Advances in COVID-19 mRNA vaccine development. Signal Transduct Target Ther. 2022;7(1):94. doi:10.1038/s41392-022-00950-y - DOI - PMC - PubMed
    1. Drobysh M, Ramanaviciene A, Viter R, et al. Biosensors for the determination of SARS-CoV-2 virus and diagnosis of COVID-19 infection. Int J Mol Sci. 2022;23(2):666. - PMC - PubMed
    1. Drobysh M, Ramanaviciene A, Viter R, Ramanavicius A. Affinity sensors for the diagnosis of COVID-19. Micromachines. 2021;12(4):390. - PMC - PubMed
    1. Chaudhary N, Weissman D, Whitehead KA. mRNA vaccines for infectious diseases: principles, delivery and clinical translation. Nat Rev Drug Discov. 2021;20(11):817–838. doi:10.1038/s41573-021-00283-5 - DOI - PMC - PubMed
    1. Anand P, Stahel VP. Correction to: the safety of Covid-19 mRNA vaccines: a review. Patient Saf Surg. 2021;15(1):22. doi:10.1186/s13037-021-00296-4 - DOI - PMC - PubMed

MeSH terms

Grants and funding

The authors thank the funding from National Science and Technology Council, Taiwan (MOST109-2327-B-002-009, MOST111-2321-B-002-017, MOST111-2124-M-001-007, NSTC112-2124-M-001-010, NSTC113-2124-M-001-018) and National Taiwan University (113L892501).