Utilizing the K18-hACE2 mouse model to develop protective COVID-19 vaccines

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic is caused by the respiratory virus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Similar to other respiratory viruses, SARS-CoV-2 is transmitted through inhalation of respiratory droplets and aerosols from infected individuals. Once inhaled, SARS-CoV-2 utilizes the receptor binding domain (RBD) on the spike protein to bind to human Angiotensin Converting Enzyme 2 (hACE2) receptor to gain entrance into host cells to begin viral replication. SARS-CoV-2 infection can result in mild to severe cases of COVID-19 ranging from asymptomatic infections, cold or flu like symptoms to respiratory failure. The onset of the pandemic in 2019 triggered a push to develop vaccines and therapeutics to prevent and treat SARS-CoV-2 infections. At the end of 2020, companies such as Moderna and Pfizer began to administer the first COVID-19 mRNA vaccines, and now in 2022, there are ten World Health Organization (WHO) approved vaccines with many more vaccines in clinical trials and pre-clinical development. At this time, approximately 12 billion doses have been administered worldwide accounting for 61% of the global population being fully vaccinated. However, with the continual emergence of SARS-CoV-2 variants of concern (VOC), each harboring new mutations that can negatively impact vaccine efficacy, there is a need to study and develop new vaccine approaches to improve immunity against VOC. Here, we devised three approaches to help improve vaccine efficacy against SARS-CoV-2 using the pre-clinical Keratin promoter 18-human Angiotensin Converting Enzyme 2 (K18-hACE2) mouse model. First, we evaluated the pathogenesis and response of VOC against human convalescent plasma (HCP) obtained from patients infected with the ancestral strain of SARS-CoV-2. Second, we assessed the vaccine efficacy of four adjuvanted Beta VOC or ancestral strain derived RBD Virus-like particle (VLP) vaccines against Alpha and Beta VOC challenge. Third, we evaluated intranasal administration of a RBD carrier protein-based vaccine adjuvanted with a lipid A mimetic. K18-hACE2 challenge models were used to establish SARS-CoV-2 VOC lethal challenge doses for Alpha, Beta, and Delta. Once a lethal viral dose was determined for each VOC, we evaluated the VOC response against polyclonal antibodies obtained from high titer HCP in a passive immunization study. The objective of the study was to assess the efficacy of antibodies derived from the ancestral strain on emerging VOC since binding and neutralizing antibodies against SARS-CoV-2 are the main correlates of protection for measuring immunity against SARS-CoV-2. Passive immunization of HCP and challenge using ancestral strain, Alpha, Beta or Delta resulted in protection against ancestral strain (100% survival), partial protection against Alpha (60%), and no protection against Beta or Delta challenge (0% survival). Survival outcomes of passive immunization and VOC challenge were also reflected on disease outcomes, viral RNA levels in the lung, brain, and nasal wash (Delta challenge only), and lung pathology. Despite poor outcomes, human RBD and nucleocapsid IgG levels remained stable in the serum and lung in the HCP treated and VOC challenged animals. Therefore, the VOC challenge mouse model established in this study was further used to study vaccine efficacy. Additionally, the HCP passive immunization study demonstrated to us that antibodies generated against the ancestral strain may not protect against VOC. Therefore, to better improve vaccine efficacy against VOC, Beta specific RBD antigens were utilized to study the efficacy of a VLP delivery approach in a murine challenge model. In this study, vaccines were formulated with RBD from either the ancestral strain (Wu) or Beta VOC conjugated to Hepatitis B surface antigen (HBsAg) VLP and adjuvanted with Aluminum hydroxide (Alum) or Squalene-in-water emulsion (SWE) and compared against Pfizer mRNA vaccine. Overall, all RBD-VLP vaccines generated RBD binding antibodies against multiple VOC RBD, broadly neutralizing antibodies against VOC RBD, decreased viral burden in the lung and brain, and lowered inflammation in the lung similar to Pfizer mRNA. However, only Beta and Wu RBD VLP adjuvanted with Alum, and Beta RBD VLP adjuvanted with SWE were able to protect mice (100% survival) against both Alpha and Beta challenge. Next, we evaluated intranasal (IN) vaccination as an approach to improve vaccine efficacy against SARS-CoV-2. We developed a prototype RBD vaccine conjugated to a diphtheria toxoid carrier protein Economical CRM197 (EcoCRM) and adjuvanted with a toll-like receptor agonist 4 (TRL4), Bacterial Enzymatic Combinatorial Chemistry (BECC) called BReC-CoV-2 (BECC+ RBD-EcoCRM COVID-19 vaccine). Overall, IN immunization with BReC-CoV-2 resulted in protection against SARS-CoV-2, decreased viral burden in the lung, brain and nasal wash, generated high levels of RBD IgG in the serum and lung that were capable of neutralizing VOC RBD, as well as induced mucosal IgA in the lung and nasal wash compared to intramuscular (IM) vaccination of BReC-CoV-2. Furthermore, heterologous IN prime and IM boost strategy with BReC-CoV-2 resulted in protection (100% survival) against a lethal Delta challenge. Altogether, the three approaches to improve vaccine efficacy demonstrated that the addition of VOC vaccine antigens accompanied with immunostimulatory adjuvants can improve vaccine responses to VOC and intranasal immunization can enhance vaccine protection by inducing mucosal antibody responses at the site of infection. Together, these vaccine approaches can help improve vaccine efficacy against emerging VOC in future COVID-19 vaccines