Current status of mucosal vaccines against SARS-CoV2: a hope for protective immunity.

INTRODUCTION: The current vaccines used to fight against COVID-19 are effective, however the induction of protective immunity is a pending goal required to prevent viral transmission, prevent the generation of new variants, and ultimately eradicate SARS-CoV-2. Mucosal immunization stands as a promising approach to achieve protective immunity against SARS-CoV-2; therefore, it is imperative to innovate the current vaccines by developing mucosal candidates, focusing not only on their ability to prevent severe COVID-19 but to neutralize the virus before invasion of the respiratory system and other mucosal compartments. AREAS COVERED: This review covers the current advances on the development of anti-COVID-19 mucosal vaccines. Biomedical literature, including PubMed and clinicaltrials.gov website, was analyzed to identify the state of the art for this field. The achievements in preclinical and clinical evaluations are presented and critically analyzed. EXPERT OPINION: There is a significant advance on the development of mucosal vaccines against SARSCoV-2, which is a promise to increase the efficacy of immunization against this pathogen. Both preclinical and clinical evaluation for several candidates have been performed. The challenges in this road (e.g. low immunogenicity, a reduced number of adjuvants available, and inaccurate dosage) are identified and also critical perspectives for the field are provided.

An Algae-Made RBD from SARS-CoV-2 Is Immunogenic in Mice.

Despite the current advances in global vaccination against SARS-CoV-2, boosting is still required to sustain immunity in the population, and the induction of sterilizing immunity remains as a pending goal. Low-cost oral immunogens could be used as the basis for the design of affordable and easy-to-administer booster vaccines. Algae stand as promising platforms to produce immunogens at low cost, and it is possible to use them as oral delivery carriers since they are edible (not requiring complex purification and formulation processes). Herein, a Chlamydomonas-made SARS-CoV-2 RBD was evaluated as an oral immunogen in mice to explore the feasibility of developing an oral algae-based vaccine. The test immunogen was stable in freeze-dried algae biomass and able to induce, by the oral route, systemic and mucosal humoral responses against the spike protein at a similar magnitude to those induced by injected antigen plus alum adjuvant. IgG subclass analysis revealed a Th2-bias response which lasted over 4 months after the last immunization. The induced antibodies showed a similar reactivity against either Delta or Omicron variants. This study represents a step forward in the development of oral vaccines that could accelerate massive immunization.

Production and characterization of chimeric SARS-CoV-2 antigens based on the capsid protein of cowpea chlorotic mottle virus.

The COVID-19 pandemic has highlighted the need for new vaccine platforms to rapidly develop solutions against emerging pathogens. In particular, some plant viruses offer several advantages for developing subunit vaccines, such as high expression rates in E. coli, high immunogenicity and safety, and absence of pre-immunity that could interfere with the vaccine's efficacy. Cowpea chlorotic mottle virus (CCMV) is a model system that has been extensively characterized, with key advantages for its use as an epitope carrier. In the present study, three relevant epitopes from the SARS-CoV-2 Spike protein were genetically inserted into the CCMV CP and expressed in E. coli cultures, resulting in the CCMV1, CCMV2, and CCMV3 chimeras. The recombinant CP mutants were purified from the formed inclusion bodies and refolded, and their immunogenicity as a subunit vaccine was assessed in BALB/c mice. The three mutants are immunogenic as they induce high IgG antibody titers that recognize the recombinant full-length S protein. This study supports the application of CCMV CP as an attractive carrier for the clinical evaluation of vaccine candidates against SARS-CoV-2. Furthermore, it suggests that VLPs assembled from these chimeric proteins could result in antigens with better immunogenicity.

Production and Purification of LTB-RBD: A Potential Antigen for Mucosal Vaccine Development against SARS-CoV-2.

Most of the current SARS-CoV-2 vaccines are based on parenteral immunization targeting the S protein. Although protective, such vaccines could be optimized by inducing effective immune responses (neutralizing IgA responses) at the mucosal surfaces, allowing them to block the virus at the earliest stage of the infectious cycle. Herein a recombinant chimeric antigen called LTB-RBD is described, which comprises the B subunit of the heat-labile enterotoxin from E. coli and a segment of the RBD from SARS-CoV-2 (aa 439-504, carrying B and T cell epitopes) from the Wuhan sequence and the variant of concern (VOC)-delta. Since LTB is a mucosal adjuvant, targeting the GM1 receptor at the surface and facilitating antigen translocation to the submucosa, this candidate will help in designing mucosal vaccines (i.e., oral or intranasal formulations). LTB-RBD was produced in E. coli and purified to homogeneity by IMAC and IMAC-anionic exchange chromatography. The yields in terms of pure LTB-RBD were 1.2 mg per liter of culture for the Wuhan sequence and 3.5 mg per liter for the delta variant. The E. coli-made LTB-RBD induced seric IgG responses and IgA responses in the mouth and feces of mice when subcutaneously administered and intestinal and mouth IgA responses when administered nasally. The expression and purification protocols developed for LTB-RBD constitute a robust system to produce vaccine candidates against SARS-CoV-2 and its variants, offering a low-cost production system with no tags and with ease of adaptation to new variants. The E. coli-made LTB-RBD will be the basis for developing mucosal vaccine candidates capable of inducing sterilizing immunity against SARS-CoV-2.

Nanoclays: Promising Materials for Vaccinology.

Clay materials and nanoclays have gained recent popularity in the vaccinology field, with biocompatibility, simple functionalization, low toxicity, and low-cost as their main attributes. As elements of nanovaccines, halloysite nanotubes (natural), layered double hydroxides and hectorite (synthetic) are the nanoclays that have advanced into the vaccinology field. Until now, only physisorption has been used to modify the surface of nanoclays with antigens, adjuvants, and/or ligands to create nanovaccines. Protocols to covalently attach these molecules have not been developed with nanoclays, only procedures to develop adsorbents based on nanoclays that could be extended to develop nanovaccine conjugates. In this review, we describe the approaches evaluated on different nanovaccine candidates reported in articles, the immunological results obtained with them and the most advanced approaches in the preclinical field, while describing the nanomaterial itself. In addition, complex systems that use nanoclays were included and described. The safety of nanoclays as carriers is an important key fact to determine their true potential as nanovaccine candidates in humans. Here, we present the evaluations reported in this field. Finally, we point out the perspectives in the development of vaccine prototypes using nanoclays as antigen carriers.