Intranasal AAV Vaccination of SARS-CoV-2 Induce Strong and Sustained Neutralizing Antibodies in Mice

Abstract

The COVID-19 pandemic continues to pose significant health challenges, despite existing vaccines. This study evaluates the immunogenicity of recombinant adeno-associated viruses (AAV) expressing SARS-CoV-2 spike proteins, administered intramuscularly and intranasally in mice. Both delivery methods of AAV5-spike, AAV5-spike stabilized trimer as well as AAV44.9-spike elicited robust serum anti-spike antibodies within 8-12 weeks, with high levels of anti-spike antibodies sustained for over a year. Comparison of mouse serum antibodies 16 weeks post intramuscular or intranasal AAV5 administration demonstrated similar SARS-CoV-2 spike binding neutralizing activity in vitro. Analysis of changes in cellular immunity by ELISpot at 12 weeks post-AAV spike transduction revealed interferon-γ induction in response to peptide challenge. Despite a decline in AAV vector DNA at the injection site, the persistence of anti-spike antibodies demonstrated that AAV-vectors can elicit lasting immune responses, highlighting nasal AAV administration as a potential strategy to block respiratory virus infections.

Figure 1.. SARS-CoV-2 Spike Antibody Levels by LIPS in Treated Mice at Different Times post IM and IN After AAVs Administration.

Each line represents a value from each serum from mice over time. As control, untreated serum mice were also quantified and shown individually. Antibody levels are expressed in relative light units (LU) on a log scale. The dashed line represents the cut-off value for determining spike antibody seropositivity.

Figure 2.. Neutralizing Activity of Mouse Serum Anti-Spike Antibody After 16 Weeks Post-Administration.

Mouse sera collected from control or mice transduced with AAV-spike by the IM or IN route were tested for in vitro neutralizing activity in blocking interactions between spike-Ace proteins as described in the material and methods. Each data point represents an individual mouse serum from each of the five groups of mice tested. Statistical analysis by Mann Whitney U test revealed highly statistical (P ≤0.001) neutralizing activity in the mice receiving AAV-spike by IM and IN administration.

Figure 3.. Levels of IFN-γ in Splenocytes Obtained from Mice Transduced with AAV5-GFP or AAV5-spike and Challenged with a Mixture of Spike peptides.

ELISpot assay was used with splenocytes extracted from animals 16 weeks after vaccination and stimulated with SARS-CoV-2 spike peptides. The levels of IFN-γ positive PBMCs from the AAV-GFP (GFP CTRL) and AAV5-Spike (Spike) transduced mice. As shown on the left side of the figure, splenocytes were treated with ConA (+ ConA) as positive control for T-cell activation. The horizontal bars represent the median value in each group.

Figure 4.. Longitudinal Analysis of the Amount of Detectable AAV5-Spike DNA at the Site of IM inject.

Mice administered IM AAV5-Spike were monitored over time to determine the turnover of the AAV5 at the site of muscle injection. Biopsied material was then subjected to qPCR as described in the material and methods.