The efficacy of a 2,4-diaminoquinazoline compound as an intranasal vaccine adjuvant to protect against influenza A virus infection in vivo

Abstract

Adjuvants are substances added to vaccines to enhance antigen-specific immune responses or to protect antigens from rapid elimination. As pattern recognition receptors, Toll-like receptors 7 (TLR7) and 8 (TLR8) activate the innate immune system by sensing endosomal single-stranded RNA of RNA viruses. Here, we investigated if a 2,4-diaminoquinazoline-based TLR7/8 agonist, ( S )-3-((2-amino-8-fluoroquinazolin-4-yl)amino)hexan-1-ol (named compound 31), could be used as an adjuvant to enhance the serological and mucosal immunity of an inactivated influenza A virus vaccine. The compound induced the production of proinflammatory cytokines in macrophages. In a dose-response analysis, intranasal administration of 1 µg compound 31 together with an inactivated vaccine (0.5 µg) to mice not only enhanced virus-specific IgG and IgA production but also neutralized influenza A virus with statistical significance. Notably, in a virus-challenge model, the combination of the vaccine and compound 31 alleviated viral infection-mediated loss of body weight and increased survival rates by 40% compared with vaccine only-treated mice. We suggest that compound 31 is a promising lead compound for developing mucosal vaccine adjuvants to protect against respiratory RNA viruses such as influenza viruses and potentially coronaviruses.

Design, Synthesis, and Biological Evaluation of New 2,6,7-Substituted Purine Derivatives as Toll-like Receptor 7 Agonists for Intranasal Vaccine Adjuvants

“…The assay was conducted following the procedures outlined in our previous report . Specifically, PR8 or HK virus (ATCC), diluted in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6), was immobilized onto immunoplate plates (SPL Life Sciences) at an amount of 50 μg of total protein per well overnight at 4 °C.…”

“…For the preparation of an influenza A virus vaccine, PR8 (A/H1N1) was inactivated with 0.02% formalin and purified by 20% sucrose gradient ultracentrifugation according to our previous report. 41 Female BALB/c mice (6−7 weeks of age; Orient Bio) were administered with the inactivated vaccine (0.5 μg per mouse, based on total protein concentration) either alone or in combination with increasing amounts of 27b (0.01, 0.1, and 1 μg per mouse) or with 1 μg of GS-9620 via the intranasal route. Three weeks after immunization, mice were infected with maPR8 virus (a gift from Prof. H. J. Kim at Chung Ang University) or with maHK virus (adapted to mouse in-house) at a dose of 10 × MLD 50 intranasally.…”

Evaluation of Antiviral Activity of Gemcitabine Derivatives against Influenza Virus and Severe Acute Respiratory Syndrome Coronavirus 2

“…Total RNA was prepared using Trizol (Invitrogen) according to the manufacturer’s instructions. After cDNA synthesis with an oligo­(dT) primer, viral mRNA was quantified using the NS gene-specific primer by real-time RT-PCR and normalized to the mouse GAPDH mRNA level …”

“…After cDNA synthesis with an oligo(dT) primer, viral mRNA was quantified using the NS gene-specific primer by real-time RT-PCR and normalized to the mouse GAPDH mRNA level. 38 …”

“…After cDNA synthesis with an oligo(dT) primer, viral mRNA was quantified using the NS gene-specific primer by real-time RT-PCR and normalized to the mouse GAPDH mRNA level. 38 Cell-Based Viral Polymerase Assay. Polymerase activity of influenza A virus was examined as previously described with the following modifications.…”