Development and validation of cashew patches.
To identify the optimal excipient for patch manufacturing, lyophilized
cashew protein extract was solubilized in either PBS 1X, NaCl 0.45% or
phosphate buffer 0.1M and deposited on epicutaneous patches. Deposits
were dried and then resolubilized using distilled water and analyzed by
ELISA or SDS PAGE (Figure S2 ). Cashew-specific IgG1 or IgE
generated in orally sensitized mice reacted with all extract deposits,
demonstrating that their immunogenicity was preserved (Figure
S2A ). Moreover, the electrophoretic profiles of all deposits were
comparable to that of the initial protein extract (Figure S2B ).
However, the best protein integrity was obtained with phosphate buffer,
as demonstrated by the absence of aggregated proteins in the pellet,
that was selected as the preferred excipient. Patches loaded with
fluorescent cashew proteins were applied to cashew-sensitized mice for
48 hours to evaluate the capacity of cashew patches to deliver allergens
to skin dendritic cells (DCs). To control for the degree of
sensitization, cashew-specific IgG1, IgG2a and IgE were quantified from
plasma collected before patches were applied. All mice presented similar
antibody titers that were consistent with previous experiments (data not
shown). The number of fluorescent DCs was then measured from BLNs
(Figure 3 ). A significant increase of cashew-positive DCs was
observed in mice that received cashew patches compared to those
receiving excipient patches. Of note, this increase was more pronounced
in cDC2 dermal DCs and Langerhans cells than in cDC1 dermal DCs.
Overall, these data demonstrate that cashew patches can be produced
while maintaining allergen integrity and that they are able to deliver
allergens to skin DCs, especially Langerhans cells and cDC2.
EPIT with cashew patches increased cashew-specific IgG2a in
sensitized mice .
Mice were sensitized orally to cashew as described above and received
cashew patches for up to 16 weeks, at a rate of 1 patch per week for 48
hours per application (EPIT). Plasma was collected every two weeks
during treatment to measure the evolution of cashew-specific IgE, IgG1
and IgG2a (Figure 4 ). A transient increase of cashew-specific
IgE was observed in EPIT mice compared to the sham group, with a peak
level at week 12 post-treatment followed by a significant decrease
(Figure 4A ). Similarly, an increase of cashew-specific IgG1 was
observed in EPIT mice, with a peak level at week 10 post-treatment
followed by a significant decrease (Figure 4B ). Finally, a
progressive and continuous increase in cashew specific IgG2a was
observed in treated mice compared to the sham group (Figure
4C ). Overall, these data indicate that EPIT to cashew can strongly
modulate cashew-specific antibody responses.
EPIT with cashew patches protects sensitized mice against
IgE-mediated anaphylaxis .
Mice were sensitized and treated as described above. At the end of the
treatment period (i.e. 8, 12 or 16 weeks), mice were challenged
orally to cashew (Figure 5 ). A clear protective effect was
observed in EPIT mice compared to the sham group and this protective
effect was further improved with increasing length of treatment. This
protection was characterized by a decrease in both temperature drop
(Figures 5A, 5C and 5E ) and clinical symptoms (Figures
5B, 5D and 5F ). To evaluate the capacity of EPIT to protect against
mast-cell activation, plasma was collected immediately after oral
challenge to measure the level of mMCP-1 and mMCP-7 (Figure 6 ).
A sharp and significant decrease of both mMCP-1 and mMCP-7 was observed
in EPIT mice compared to the sham group. Of note, the prolongation of
the treatment period does not induce further decrease in the plasmatic
concentration of these two proteases. Overall, these data indicate that
EPIT to cashew can efficiently protect mice against IgE-mediated
anaphylaxis induced by oral challenge.