# Safety Assessment of a Sublingual Vaccine Formulated with Poly(I:C) Adjuvant and Influenza HA Antigen in Mice and Macaque Monkeys: Comparison with Intranasal Vaccine

## TL;DR

This study compares the safety of sublingual and intranasal vaccines in mice and macaques, finding that sublingual vaccination avoids harmful brain inflammation linked to intranasal delivery.

## Contribution

The study introduces a novel sublingual vaccine formulation and demonstrates its safety advantage over intranasal delivery in terms of brain inflammation.

## Key findings

- Intranasal vaccination upregulated inflammation-related genes in the olfactory bulb and pons of mice and macaques.
- Sublingual vaccination did not cause adverse effects in the brain regions tested in either species.
- Intranasal vaccine effects in the lungs were transient and not observed at 7 days post-vaccination.

## Abstract

A sublingual vaccine comprising the Poly(I:C) adjuvant and influenza HA antigen was evaluated for safety in both mice and macaque monkeys relative to its intranasal counterpart. Safety was assessed in terms of harmful effects corresponding to the upregulation of the inflammation-associated genes Saa3, Tnf, IL6, IL1b, Ccl2, Timp1, C2, Ifi47, Aif1, Omp, Nos2, and/or Gzmb in mice and SAA2, TNF, IL6, IL1B, CCL2, TIMP, C2, AIF1, and GZMB in macaques. Quantitative gene expression analyses were performed using RT-qPCR with RNA samples from four tissue types, the olfactory bulb, pons, lung, tongue, and lymph node, from both mice and macaques. In mice, the intranasally delivered vaccine markedly upregulated the inflammation-related genes in the olfactory bulb 1 day and 7 days after vaccination. The adverse effects of intranasal vaccination were also observed in macaques, albeit to a lesser extent than in mice. The intranasal vaccination also upregulated these genes in the pons of both mice and macaques. In contrast, the sublingual vaccine did not adversely affect the olfactory bulb or pons in either mice or macaques. The intranasally administered vaccine significantly upregulated these genes in the lungs only 1 day after vaccination, but not 7 days later, in both mice and macaques. We conclude that intranasal vaccination results in unfavorable side effects corresponding to upregulated inflammatory genes in the brain (olfactory bulb and pons). Sublingual vaccination, however, did not induce these side effects in either mice or macaques and was hence evaluated as safe.