Evaluation of dual pathogen recognition receptor agonists as adjuvants for respiratory syncytial virus -virus-like particles for pulmonary delivery

Ahmedali S Mandviwala ¹ Komal Liman ¹ Anke Huckriede ² Vidya Avinash Arankalle ¹ Harshad P Patil ^1*

• ¹ Interactive Research School for Health Affairs, Bharati Vidyapeeth Deemed University, Pune, India
• ² Department of Medical Microbiology and Infection Prevention, University Medical Center Groningen, Groningen, Netherlands

Introduction: Respiratory syncytial virus (RSV) remains a significant global health concern, particularly for infants and young children in developing countries. Despite extensive research, no effective RSV vaccine is widely available. Dual pattern recognition receptor (PRR) agonists as adjuvants enhance immune responses, but separate agonists may not target the same cell.. This study evaluates the efficacy of two different dual PRR binding chimeric molecules CL413 (TLR2/TLR7 agonist) and CL429 (TLR2/NOD2 agonist) as adjuvants for RSV virus-like particles (VLPs) delivered via the pulmonary route in mice for induction of mucosal and systemic immunity.Methods: BALB/c mice were immunized twice with RSV-VLPs alone or adjuvanted with CL413, CL429, or mixtures of single PRR agonists (Pam3CSK4+L18-MDP or Pam3CSK4+imiquimod) via the pulmonary route. The single PRR agonist mixtures served as controls.. Immune responses were evaluated by measuring antibody levels in sera and respiratory tract; cytokine production, B and T cell responses in the lungs and spleen.Results: Pulmonary immunization with CL413-adjuvanted VLPs induced robust nasal IgA responses against RSV F and G proteins, enhanced serum IgG levels, and promoted a balanced Th1/Th2 response. CL413 elicited strong pro-inflammatory responses in the lungs, including elevated levels of IFN-γ, TNF-α, IL-6, and IL-17A. Flow cytometry analysis revealed increased numbers of tissue-resident class-switched B cells in the lungs of mice immunized with VLPs adjuvanted with CL413 and CL429. CD4+ and CD8+ T cell responses were also enhanced in both lungs and spleens of mice receiving VLPs adjuvanted with chimeric molecules to various extents. Mice immunized with formalin-inactivated RSV (FI-RSV), used as a positive control for vaccine-induced pathology, developed alveolitis and perivascular infiltration. In contrast, all adjuvanted VLP groups were protected from lung pathology after RSV challenge.Discussion: The lack of pathology, combined with robust mucosal and systemic immune responses, suggests that pulmonary delivery of adjuvanted RSV-VLPs may provide effective protection without the risk of vaccine-enhanced disease. The study also demonstrates that the chimeric TLR2/TLR7 agonist CL413 is a promising adjuvant for RSV-VLPs to induce mucosal and systemic immune response , warranting further investigations in more advanced preclinical models.