The ST sequence was inserted in the ADDomer gene in a region coding for the exposed flexible loop called the variable loop (Figure?1A). response against the antigen displayed on its surface. This plug and play vaccine platform represents a promising new highly versatile tool to combat emergent pathogens. Key words: Virus-like particle, Antigen display, Vaccine platform, Adenovirus, COVID-19, SARS-CoV-2, Receptor-binding domain, Neutralizing antibodies Graphical abstract Open in a separate window In this study, Chevillard and colleagues report a non-infectious adenovirus-inspired self-assembling particle designed to spontaneously and covalently display up to 60 SARS-CoV-2 glycoantigens. Immunization of mice with this pseudoviral mimic generates a?strong neutralizing antibody response capable of neutralizing SARS-CoV-2 in the long term. A new tool for pandemic preparedness. Introduction Although genomic vaccines have recently demonstrated their capacity to elicit protective immune responses, 1 protein-based vaccines remain highly attractive due to their lower cost, and ease of transport and storage, crucial to reach developing countries and remote locations, as well as better social acceptance, as recently highlighted by hesitancy regarding COVID-19 RNA vaccines. Protein immunogenicity is highly increased through presentation to the immune system in a multimeric manner, and a number of vaccine platforms have been developed to this aim, with various advantages and limitations.2 We have previously reported that a non-infectious virus-like particle (VLP) derived from human adenovirus of type 3 and consisting of 60 identical penton base monomers could Zapalog be exploited to display epitopes of interest on its surface.3, 4, 5 In this vaccine platform, named ADDomer, exposed loops of the penton base protein were engineered to allow insertion of foreign peptides such as a linear neutralizing epitope from Chikungunya virus. However, this design did not permit the insertion of structurally complex antigens. To overcome this limitation while keeping the immunogenicity advantage of ADDomer, we describe here a redesigned platform Zapalog offering a highly versatile capacity to display large and structurally complex antigens with potential post-translational modifications. In order to decorate the adenovirus-based VLPs with large antigens, the SpyTag/SpyCatcher system6, 7, 8, 9 was combined with the ADDomer technology. In this system, derived from an aspartate residue from the 13 amino acid SpyTag peptide (ST) can spontaneously create a covalent bond with a lysine residue encompassed in the complementary Lamin A (phospho-Ser22) antibody SpyCatcher module (SC). We thus genetically inserted the sequence coding for the ST peptide into the variable loop of the ADDomer that was previously used for small antigen insertion, yielding a VLP with 60 potential attachment sites for complex antigens engineered with an SC anchor. In order to assess the newly developed platform, we decided to first test it as a potential candidate vaccine to elicit neutralizing antibodies (NAbs) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is an enveloped positive strand RNA virus belonging to the beta-coronavirus genus from the Coronaviridae family. It is the etiological agent of coronavirus disease 2019 (COVID-19), and at the origin of the pandemic that started in December 2019, leading to 5,542,359 deaths as of January Zapalog 19, 2022 (https://covid19.who.int/), and vast socio-economic consequences.10 Protection against SARS-CoV-2 infection and COVID-19 can be mediated by neutralizing Abs targeting the envelope trimeric glycoprotein spike (S) exposed at the surface of the virus.11,12 Accordingly, spike-based vaccine approaches eliciting SARS-CoV-2 Zapalog NAb responses have been successful at preventing COVID-19.13, 14, 15 The ectodomain of the S protein is divided into the S1 and S2 domains. The Spike protein binds to the host angiotensin-converting enzyme 2 (ACE2), which serves as an entry receptor, as previously reported with SARS-CoV, responsible for the 2002C2004 SARS outbreak.16, 17, 18, 19 A subdomain of S1, named receptor binding domain (RBD), is the contact interface between the virus and the ACE2 receptor. Numerous studies have shown that the RBD comprises multiple distinct antigenic sites and is a prime target for NAbs in COVID-19 convalescent patients.20, 21, 22, 23 These NAbs24 strongly inhibit cell infection by SARS-CoV-2 by competing with the RBD-ACE2 interaction. Moreover, anti-RBD NAbs have been shown to confer protection against SARS-CoV-2 challenge in animal models of COVID-19, as well as to prevent COVID-19 in humans, thus confirming the interest of using RBD as a vaccine immunogen.25, 26, 27 This prompted us to use the RBD as the antigen.