Synthetic delivery vector STxB induces mucosal immunity against viral antigens

In a recent study posted to the bioRxiv* preprint server, researchers describe a mucosal vector vaccine for targeting dendritic cells.

Study: A synthetic delivery vector for mucosal vaccination. Image Credit: blckptchstudio /

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

The need for mucosal vaccines

One of the most significant scientific advancements made during the coronavirus disease 2019 (COVID-19) pandemic was the rapid development and deployment of effective messenger ribonucleic acid (mRNA) vaccines. Current mRNA-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines successfully prevented severe COVID-19; however, they are often unable to protect vaccinated individuals against infection, particularly following exposure to recently identified SARS-CoV-2 variants of concern (VOCs).

Moreover, mRNA vaccines do not generate mucosal immunity in the ear-nose-throat region, which is necessary to prevent viral transmission. Thus, novel mucosal vaccines are urgently needed, as they could stimulate the production of immunoglobulin A (IgA) and mucosal CD8+ T-cells, including resident memory CD8+ T lymphocytes (TRM).

About the study

The fluorenylmethyloxycarbonyl (Fmoc) synthesis strategy was used to generate Shiga toxin B (STxB), which is a protein-based antigen delivery vehicle that can be used to target dendritic cells specifically. Previously applications of STxB as an antigen carrier have shown that this protein successfully induces both cellular and humoral immunity against infectious diseases and certain tumors.

For the current study, the researchers selected STxB for its low immunogenicity, high stability in circulation, and ability to cross mucosal barriers. As compared to previous studies that have produced STxB from bacteria, which is often a time-consuming and expensive process, the researchers of the current study utilized the Fmoc strategy, which is a solid-phase peptide synthesis approach that relies on Fmoc to protect amino acid side chains.

Initially, Fmoc synthesis had a yield of 16.5%; however, the subsequent incorporation of pseudoprolines into the STxB monomers prevented peptide chain aggregation, thereby increasing the yield to 26%.

STxB monomers were then folded into functional homopentamers through an in vitro approach, during which six molar (M) guanidine hydrochloride was initially used to dissolve the protein. The protein was then diluted into a refolding buffer, incubated at 4 °C overnight, and subjected to dialysis before purification using a HisTrap column. Notably, this refolding process led to an efficiency of over 90%, which ensured the immunogenicity, biocompatibility, and targeting capabilities of STxB.

Subsequently, several antigens originating from mucosal SARS-CoV-2 and type 16 human papillomavirus (HPV) were chemically coupled with STxB. These antigen conjugates were then intranasally administered to mice twice at days 0 and 14. On day 21, mice were sacrificed, and bronchoalveolar lumen (BAL) fluid and lungs were obtained for analysis.

Study findings

Compared to recombinant STxB, synthetic STxB (sSTxB) homopentamers exhibited a secondary structure that was extremely similar to the recombinant protein. Furthermore, dynamic light scattering (DLS) revealed that both recombinant and sSTxB had hydrodynamic radii between 2.5 and 2.9 nanometers (nm). Aside from a slight difference of 1.1 °C  in the melting temperature (Tm) of sSTxB as compared to the recombinant form of this protein, these proteins exhibited highly similar biophysical properties.

Following the intranasal administration of sSTxB(70C) conjugated with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, mice exhibited functional CD8+ T-cells against the viral RBD in their lungs and a significantly greater frequency of interferon γ (IFNγ)-producing CD8+ T-cells as compared to control mice. In addition, intranasal administration of sSTxB(70C)-RBD also induced the production of IgA and IgG in BAL fluid at rates that were seven- and eight times greater than controls, respectively.

Additional sSTxB variants were constructed to compare their efficacy to that of recombinant STxB(70C) when conjugated with the SL8 peptide from ovalbumin and the G15F peptide from the HPV16 E7 protein. Treatment with sSTxB(70C)-G15F and sSTxB(70C)-SL8 induced the production of specific CD8+ T-cells, with sSTxB(70C)-G15F administration inducing significantly high levels of CD8+ T-cells against the E749-57 viral protein.


The current study provided evidence that a linear synthesis of STxB without liquid chromatography (LC) is a highly efficient process that produces various sSTxB variants, which, when coupled with different viral peptides and full-sized proteins, are capable of inducing mucosal immunity in mice. More specifically, these vaccines were found to induce the production of functional CD8+ T-cells with resident memory phenotype and specific mucosal IgA.

Future biomedical applications of this technology could allow various handles to be introduced into STxB for the development of urgently needed mucosal vaccines.

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Preliminary scientific report. Billet, A., Hadjerci, J., Tran, T., et al. (2023). A synthetic delivery vector for mucosal vaccination. bioRxiv. doi:10.1101/2023.05.15.540756.

Posted in: Device / Technology News | Medical Science News | Medical Research News | Pharmaceutical News

Tags: Amino Acid, Antigen, Assay, Bacteria, Cell, Chromatography, Conjugation, Coronavirus, covid-19, Dialysis, Dynamic Light Scattering, Ear, Efficacy, Fluorescence, Frequency, Guanidine, immunity, Immunoglobulin, in vitro, Infectious Diseases, Interferon, Liquid Chromatography, Lungs, Pandemic, Peptides, Phenotype, Protein, Receptor, Respiratory, Ribonucleic Acid, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Spike Protein, Syndrome, Technology, Throat, Toxin, Vaccine

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Written by

Bhavana Kunkalikar

Bhavana Kunkalikar is a medical writer based in Goa, India. Her academic background is in Pharmaceutical sciences and she holds a Bachelor's degree in Pharmacy. Her educational background allowed her to foster an interest in anatomical and physiological sciences. Her college project work based on ‘The manifestations and causes of sickle cell anemia’ formed the stepping stone to a life-long fascination with human pathophysiology.

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