SARS-CoV-2 spike protein conjugated to a polymeric glyco-adjuvant induces potent neutralizing antibodies and cellular responses

Laura Taylor Gray 1*, Michal M. Raczy 1*, Priscilla Briquez 1*, Tiffany M. Marchell 2*, Aaron T. Alpar 1, Rachel P. Wallace 1, Lisa R. Volpatti 1, Maristella Sasso 1, Mindy Nguyen 1, Aslan Mansurov 1, Shijie Cao 1, Erica Budina 1, Elyse A. Watkins 1, Nikolaos Mitrousis 1, Ruyi Wang 1, Ani Solanki 3, Joseph W. Reda 1, Andrew C. Tremain 2, Marcin Kwissa 1, Melody A. Swartz 1,2,4, Jeffrey A. Hubbell 1,2

1 Pritzker School of Molecular Engineering, University of Chicago

2 Committee on Immunology, University of Chicago

3 Animal Resources Center, University of Chicago

4 Ben May Department of Cancer Research, University of Chicago

*These authors contributed equally

Here, we report the creation of a prophylactic subunit vaccine for COVID-19 consisting of two components: (1) a mannosylated, toll-like receptor 7 (TLR7)-agonizing polymer, p(Man-TLR7), and (2) the SARS-CoV-2 spike protein. Previously, we have shown that vaccination with p(Man-TLR7) induces potent humoral and cellular responses in multiple vaccine models. As such, we hypothesized that this polymeric glyco-adjuvant would also be a potent vaccine against SARS-CoV-2. Following a prime (day 0)-boost (day 21) regimen in C57BL/6 mice, we observed high titers of anti-spike IgGs induced by spike-p(Man-TLR7) at day 28, and these antibodies were found to neutralize SARS-CoV-2 to a greater extent than human convalescent serum in in vitro assays. Furthermore, with the addition of alum to our vaccine, we observed an added improvement in neutralization, such that neutralization matched that of a clinically relevant control of spike adjuvanted with MPLA and alum. Antibodies against the spike protein were observed to persist for at least eight weeks following the initial priming of the mice. Additionally, antibody responses were compared across mice ranging in age from 8 weeks to greater than 64 weeks old, with high antibody levels observed in all ages of mice assessed. These high IgG titers in spike-p(Man-TLR7) vaccinated mice were accompanied by increased numbers of Tfh cells in the spleen. Moreover, a skewing towards a Th1 response was observed in mice vaccinated with spike-p(Man-TLR7) as compared to spike alone or spike with MPLA and alum, as evidenced by an increase in the ratio of IgG2b to IgG1 levels, as well as an increase in IFNg secretion upon spike protein restimulation of the draining lymph nodes and spleen. Furthermore, strong CD4+ T cell responses were observed in both the draining lymph nodes and spleens of mice vaccinated with spike-p(Man-TLR7) upon restimulation with spike peptide pools. In the draining lymph nodes, increases in IFNg+ CD4+ T cells were observed in mice vaccinated with spike-p(Man-TLR7), particularly when alum was added to our vaccine, as compared to mice vaccinated with spike alone and the clinically relevant controls of spike with MPLA plus alum and spike with an AS03-like adjuvant. In the spleen, increases in single cytokine-positive (IFNg+, TNFa+, and IL-2+) CD4+ T cells, as well as in double cytokine-positive polyfunctional CD4+ T cells, were observed in mice vaccinated with spike-p(Man-TLR7), especially when combined with alum. In conclusion, vaccination with spike-p(Man-TLR7) results in a long-lasting humoral response, as well as a potent cellular response.