Mucosal TLR2-activating protein-based vaccination induces potent pulmonary immunity and protection against SARS-CoV-2 in mice.

Current vaccines against SARS-CoV-2 substantially reduce mortality, but protection against infection is less effective. Enhancing immunity in the respiratory tract, via mucosal vaccination, may provide protection against infection and minimise viral spread. Here, we report testing of a subunit vaccine in mice, consisting of SARS-CoV-2 Spike protein with a TLR2-stimulating adjuvant (Pam2Cys), delivered to mice parenterally or mucosally. Both routes of vaccination induce substantial neutralising antibody (nAb) titres, however, mucosal vaccination uniquely generates anti-Spike IgA, increases nAb in the serum and airways, and increases lung CD4+ T-cell responses. TLR2 is expressed by respiratory epithelia and immune cells. Using TLR2 deficient chimeric mice, we determine that TLR2 expression in either compartment facilitates early innate responses to mucosal vaccination. By contrast, TLR2 on hematopoietic cells is essential for optimal lung-localised, antigen-specific responses. In K18-hACE2 mice, vaccination provides complete protection against disease and sterilising lung immunity against SARS-CoV-2, with a short-term non-specific protective effect from mucosal Pam2Cys alone. These data support mucosal vaccination as a strategy to improve protection in the respiratory tract against SARS-CoV-2 and other respiratory viruses.

Cell-free expression of natively folded hydrophobins.

Hydrophobins are a family of cysteine-rich proteins unique to filamentous fungi. The proteins are produced in a soluble form but self-assemble into organised amphipathic layers at hydrophilic:hydrophobic interfaces. These layers contribute to transitions between wet and dry environments, spore dispersal and attachment to surfaces for growth and infection. Hydrophobins are characterised by four disulphide bonds that are critical to their structure and function. Thus, obtaining correctly folded, soluble and functional hydrophobins directly from bacterial recombinant expression is challenging and in most cases, initial denaturation from inclusion bodies followed by oxidative refolding are required to obtain folded proteins. Here, we report the use of cell-free expression with E. coli cell lysate to directly obtain natively folded hydrophobins. All six of the hydrophobins tested could be expressed after optimisation of redox conditions. For some hydrophobins, the inclusion of the disulfide isomerase DsbC further enhanced expression levels. We are able to achieve a yield of up to 1 mg of natively folded hydrophobin per mL of reaction. This has allowed the confirmation of the correct folding of hydrophobins with the use of 15N-cysteine and 15N-1H nuclear magnetic resonance experiments within 24 h of starting from plasmid stocks.