Seasonal influenza virus epidemics are consistent sources of morbidity and mortality, with an estimated 3-5 million infections and 290,000-650,000 deaths annually, worldwide [1]. Consequently, significant resources are both lost due to infection and spent in attempts to mitigate and prevent infection, all with limited success. Current influenza vaccines exhibit suboptimal efficacy, ranging from 10% to 60% [[2], [3], [4], [5], [6]], and significant logistical efforts are required to supply cold-chain dependent vaccines [[7], [8], [9]]. All these aspects combine to underscore an urgent need for “universal” influenza vaccines with high efficacy, ideally with improved storage and stability profiles.

Efforts to develop universal influenza A virus (IAV) vaccines have largely focused on generating neutralizing antibody responses. However, these approaches have encountered limitations in generating broad-based, neutralizing antibody responses against genetically diverse IAV strains [10,11]. However, antibody-focused approaches neglect to evaluate CD4+ and/or CD8+ T cell responses, which are critical in controlling and resolving IAV infections [12,13]. Thus, protective immunity induced by “universal” influenza vaccine candidates will likely contain components that induce both humoral and cell-mediated immunity.

Herein, we report on the use of a unique influenza A hemagglutinin derived from A/equine/1/KY/91 (H3N8) virus that elicits broadly neutralizing antibodies to a wide range of circulating influenza virus strains [14]. Furthermore, nucleoprotein derived from A/HK/1/68 (H3N2) virus was selected as a second immunogen to induce T cell responses due to the highly conserved nature of nucleoprotein across IAV strains [15,16].

To stabilize and deliver these potentially universal immunogens and address gaps in inactivated virus-based vaccination strategies, biomaterials-based particle vaccines utilizing 1,6-bis(p-carboxyphenoxy)hexane (CPH) and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) as a 20:80 CPTEG:CPH polyanhydride copolymer and Pluronic F127® based pentablock copolymer micelles were designed. These platforms have been previously shown to be biocompatible and biodegradable and to induce robust local and systemic humoral and cell-mediated immune responses when used to deliver IAV antigens [[17], [18], [19], [20], [21]]. The polyanhydride particles are capable of encapsulating, stabilizing, protecting, transporting, and releasing immunogenic payloads in a controlled manner [[22], [23], [24]]. Furthermore, polyanhydride particles and pentablock copolymer micelles can simultaneously deliver multiple immunogens and adjuvants to immune cells. In this work, we studied two adjuvants. CpG 1668 has been previously shown to be an effective pulmonary adjuvant [[25], [26], [27]], and as a TLR 9 agonist, it is well suited for intranasal delivery by polyanhydride particles where it will be released and detected in endosomal compartments [28,29]. Pentablock copolymer micelles have been shown to be effective transmembrane carriers allowing for efficient cytosolic delivery [30]. Therefore, cyclic di-GMP (CDN) [31], a cytosolic STING agonist, which has been previously used as an adjuvant for subcutaneous polymeric particle-based vaccines [20,21], was selected.

In this work, we build upon previous studies that demonstrated the ability of these vaccine platforms to stabilize and provide sustainable delivery of IAV antigens [23], to induce protective immunity IAV [20,21,32], including newly discovered, potentially universal IAV antigens [14]. We describe the preparation, characterization, induction of immune response, and efficacy of two polymeric particle-based vaccines delivered via intranasal or subcutaneous immunization. Furthermore, we investigated the roles in protective immunity played by serum antibody and CD8+ T cells using passive transfer of antisera and T cell depletion studies, respectively [33,34].