Introduction
Since the outbreak of the global pandemic caused by SARS-CoV-2, WHO has reported ~170 million confirmed cases on June third 2021 including ∼3.5 million deaths (WHO, June 3rd 2021). The pandemic has put a heavy toll on public health systems and world’s economy. To limit the damage, efforts have been directed towards vaccine development. On 12th May 2021 around one billion doses of different vaccines have been administered worldwide (WHO, 12th May 2021). Although SARS-CoV-2 causes mostly mild symptoms such as coughing, fever and breathlessness, symptoms may become much more severe, in particular in elderly people and people with chronic diseases which may develop severe pneumonia and other symptoms including organ failure and death (1, 2). In comparison to SARS-CoV-1and MERS-CoV, SARS-CoV-2 causes less morbidity; however, it transmits much more readily, mostly because non-symptomatic and pre-symptomatic individuals can spread the virus. Thus, while MERS-CoV and SARS-CoV outbreaks have been sporadic and geographically restricted, SARS-CoV2 has rapidly spread around the world (3, 4).
The positive sense ssRNA SARS-CoV-2 virus has a genome of about 29,700 nucleotides with 79.5% identity to SARS-CoV-1. Its genome encodes four main structural proteins; spike (S) protein, membrane (M) protein, nucleocapsid (N) protein as well as the envelope (E) protein (5, 6). SARS-CoV-2 binds to angiotensin converting enzyme 2 (ACE2) via the RBD domain of its S protein that protrudes from the viral envelope. Interaction of RBD with ACE2 is the first step in a cascade of event leading to viral entry and ultimately replication (7). Neutralizing antibodies against SARS-CoV-2 are mostly targeting the receptor binding domain (RBD) of the S protein. Within RBD, the receptor binding motif (RBM) is of particular importance as it directly interacts with ACE2 (8). Compared to SARS-CoV-1, the affinity to ACE2 of the S protein expressed by the original variant of SARS-CoV-2 is about 4-fold higher, offering an explanation for the increased infectivity of the latter (9). Interestingly, RBM shows no glycosylation or other post-translational modifications and therefore is well suited for production in bacterial expression systems (10).
Vaccines are the most reliable, cost-effective and efficient strategy to prevent infectious diseases. Vaccine candidates must induce sufficient quantities of high affinity antibodies to neutralize the invading virus. Since the initiation of the pandemic, a full spectrum of vaccine types has been tested in preclinical and clinical trials. Vaccine platforms employed mRNA, DNA, viral vectors, inactivated or live-attenuated virus (9, 11) and recombinant proteins. The full-length of S protein, the RBD domain, S1 subunit, fusion protein (FP) as well as the N-terminal domain (NTD) of the S protein have been targeted by vaccines that are licensed or undergoing development (3, 12).
Virus-like particles (VLPs) represent one of the conventional vaccine platforms in the sense that there are globally marketed products (e.g., HBV and HPV vaccines) that that have demonstrated the clinical usefulness of this modality. VLPs consist of viral structural proteins that upon recombinant expression, self-assemble into particles with a mostly icosahedrons and rarely helical symmetry (13). Recently, we have developed an immunologically optimized VLP platform based on the cucumber mosaic virus (CuMVTT-VLPs) (14). CuMVTT VLPs incorporate a universal T cell epitope derived from tetanus toxin (TT) utilizing the pre-existing T cell memory response induced by vaccination against tetanus toxin (15). The newly developed platform enhances the interaction between T helper cells and B cells, and is expected to improve responses in elderly individuals who are often less reactive to vaccines. This is supported by the fact that pre-existing immunity to the chosen TT epitope is very broad in humans (and animals) as the peptide binds to essentially all HLA-DR molecules and most people have been immunized many times against TT. In addition, the CuMVTT-VLPs are packaged with bacterial RNA which is a ligand for toll like receptor (TLR) 7 and 8 and serves as a potent adjuvant (15, 16). By displaying antigens on CuMVTT-VLPs, it was possible to induce high levels of antigen-specific antibodies in mice, rats, cats, dogs and horses and treat diseases such as atopic dermatitis in dogs or insect bite hypersensitivity in horses (17, 18).
In the current study, we have designed and developed a scalable and immunogenic VLP-based COVID-19 vaccine by genetically fusing the RBM domain of the S protein from SARS-CoV-2 into CuMVTT-VLPs. The data shows that this vaccine is highly immunogenic inducing both RBD-specific IgG and IgA antibodies as well as a strong viral neutralizing antibody response. Furthermore, the vaccine production process is highly scalable, potentially allowing the production of millions of doses in a single 1000L fermenter run.