Ambient Temperature Stable, Scalable COVID-19 Vaccines

There is an urgent unmet need for a safe vaccine available worldwide to prevent human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in order to halt the global pandemic that has already caused >247 million infections and >5 million deaths.

Despite the fact that there are more than 180 vaccine development projects (42 in clinical trials) internationally and more than ten approved vaccine uses in case urgently, there is a demand for vaccines that enable rapid global distribution and induce long-lasting, transmission-blocking immunity. 2] Another key issue is adaptability to emerging viral variants.

Vaccine design is currently hampered by our limited understanding of the complex immune response required to prevent infection and induce long-lasting immunity.[3-6] large-scale manufacturability. These challenges are further exacerbated by the risk of vaccine candidates inducing antibody-dependent enhancement (ADE) of infection and/or immunopathology due to induction of a “cytokine storm” and associated inflammation,[7-9] and vaccine-induced thrombosis with thrombocytopenia (VITT).

Cytokine storm is the leading cause of severe cases of coronavirus disease 2019 (COVID-19) and d increase in mortality. Therefore, the effective vaccine must be precisely designed to induce the desired immune responses and long-lasting immunity while avoiding ADE/immunopathology/VITT in combination with robust large-scale manufacturability.

Vaccines and vaccine candidates currently approved for emergency use in clinical evaluation include viral vectors (replicating, non-replicating), virus-like particles (VLPs), protein subunits, DNA or RNA vaccines and inactivated virus vaccines, primarily considering the SARS-CoV-2 spike glycoprotein (S) as a major vaccine candidate antigen.

Targeted immune responses are neutralizing antibodies that bind to the receptor binding domain (RBD) located in the S1 subunit of S and block virus attachment to human angiotensin converting enzyme 2 (ACE2 ) and thus prevent viral infection. However, even in convalescent patients (CP), these antibodies do not last more than a few months, i.e. the immunity is not long-lasting.[12] This suggests that successful SARS-CoV-2 vaccines require higher immunogenicity and durability than natural infection for long-lasting immunity.

Unlike mRNA and adenovirus vector vaccine types, currently only approved for emergency use as COVID-19 vaccines, protein subunit vaccines against various infectious diseases have a long history of use. approved demonstrating their safety and efficacy.[13-15] RBD and S1 have recently been shown to induce neutralizing antibodies and include T-cell epitopes proposed to contribute to cell-mediated immunity.

-19 CP, respectively. The SARS-CoV-2 nucleocapsid (N) protein contributed 11% to 27% of the CD4+ T cell response in COVID-19 PCs, while peak and M CD4+ T cell responses ( membrane protein) were significant and correlated with anti-SARS-CoV-2 IgG and IgA titers.[19] CD8+ T cells mainly recognized S and M. PCs also showed high titers of antibodies against N. [20] Therefore, we decided to study the immunogenicity of S1, RBD, epitopes of S1 and M in the presence or absence of N.

To improve immunogenicity, we attached these antigens to biopolymer (BP) particles with a size of ≈200-500 nm whose core is composed of biocompatible polyhydroxybutyrate (PHB) surrounded by covalently linked PHB synthase used as domain anchor for antigens. 15, 21-23] BP presentation of protein antigens has been shown to further enhance immunogenicity by facilitating antigen uptake and processing by antigen-presenting cells. Immune responses were specific to the displayed antigens.

Additionally, we have recently shown that bacterial or viral antigens displayed on BPs were highly immunogenic inducing both strong cell-mediated and long-lasting humoral immune responses that protected mice from infection by the respective pathogen. [26-28] Therefore, the BP platform was designed to be suitable for the development of vaccines against SARS-CoV-2. and self-assembly inside modified bacteria or using modified BPs that