Polyanhydride nanovaccine against swine influenza virus in pigs

Abstract

We have recently demonstrated the effectiveness of an influenza A virus (IAV) subunit vaccine based on biodegradable polyanhydride nanoparticles delivery in mice. In the present study, we evaluated the efficacy of ∼200 nm polyanhydride nanoparticles encapsulating inactivated swine influenza A virus (SwIAV) as a vaccine to induce protective immunity against a heterologous IAV challenge in pigs. Nursery pigs were vaccinated intranasally twice with inactivated SwIAV H1N2 (KAg) or polyanhydride nanoparticle-encapsulated KAg (KAg nanovaccine), and efficacy was evaluated against a heterologous zoonotic virulent SwIAV H1N1 challenge. Pigs were monitored for fever daily. Local and systemic antibody responses, antigen-specific proliferation of peripheral blood mononuclear cells, gross and microscopic lung lesions, and virus load in the respiratory tract were compared among the groups of animals. Our pre-challenge results indicated that KAg nanovaccine induced virus-specific lymphocyte proliferation and increased the frequency of CD4⁺CD8αα⁺ T helper and CD8⁺ cytotoxic T cells in peripheral blood mononuclear cells. KAg nanovaccine-immunized pigs were protected from fever following SwIAV challenge. In addition, pigs immunized with the KAg nanovaccine presented with lower viral antigens in lung sections and had 6 to 8-fold reduction in nasal shedding of SwIAV four days post-challenge compared to control animals. Immunologically, increased IFN-γ secreting T lymphocyte populations against both the vaccine and challenge viruses were detected in KAg nanovaccine-immunized pigs compared to the animals immunized with KAg alone. However, in the KAg nanovaccine-immunized pigs, hemagglutination inhibition, IgG and IgA antibody responses, and virus neutralization titers were comparable to that in the animals immunized with KAg alone. Overall, our data indicated that intranasal delivery of polyanhydride-based SwIAV nanovaccine augmented antigen-specific cellular immune response in pigs, with promise to induce cross-protective immunity.

Introduction

Swine influenza A virus (SwIAV) causes considerable economic losses in the pig industry worldwide [1]. Currently, multiple antigenically diverse strains of three major SwIAV subtypes H1N1, H1N2 and H3N2 are circulating in pig populations. Since pigs serve as a mixing vessel for human and avian IAV, numerous distinct SwIAV strains are frequently generated, and some of these have zoonotic potential [2]. An effective vaccination strategy can prevent economic losses in the pig industry and limit zoonotic transmission of SwIAVs to humans. Vaccination against SwIAV is frequently practiced on pig farms using either bivalent or multivalent whole virus inactivated (WIV) vaccines which protect against homologous virus but are ineffective against heterologous strains [3], [4], [5], [6]. Since SwIAV undergoes frequent mutation with antigenic drift and shift, there is an urgent need to develop broadly cross-protective vaccines. Moreover, WIV vaccines do not elicit high levels of antigen-specific secretory IgA antibody response in the respiratory tract where the disease is localized. It is also known that strong mucosal immunity can correlate with cross-protective efficacy against influenza [7], [8]. Recently, WIV vaccine formulations were reported to enhance the severity of lung lesions in pigs infected with heterologous IAV, raising concerns over judicious selection and use of vaccines [3], [4], [6]. To overcome these limitations, a novel vaccine delivery platform is needed for prevention and control of influenza in pigs.

Biodegradable and biocompatible polyanhydrides have been widely used for vaccine antigen delivery due to safety [9], [10], [11] and their adjuvant properties [12]. The most well-studied polyanhydride copolymers are based on sebacic acid (SA), 1,6-bis(p-carboxyphenoxy)hexane (CPH), and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaocatane (CPTEG) monomers. Polyanhydrides are surface eroding polymers, which minimize the exposure of encapsulated antigen to moisture providing a better microenvironment for the encapsulated vaccine antigen(s) [12], [13]. Polyanhydride nanoparticles retain the structural and biological activity of released vaccine antigens [14], [15], [16], [17], [18], [19] and also have pathogen mimicking properties to activate dendritic cells and enhance innate immune response [19], [20], [21], [22]. Recent studies have shown induction of high virus neutralizing antibody titer and enhanced cell-mediated immune responses against IAV in mice vaccinated with a hemagglutinin-based polyanhydride nanovaccine [23], [24]. In this study, we analyzed the immunogenicity and protective efficacy of 20:80 CPTEG:CPH nanoparticles encapsulating whole inactivated SwIAV vaccine against a heterologous and virulent zoonotic SwIAV H1N1 challenge in pigs. Our results indicated that nanovaccine encapsulation of SwIAV augmented the virus specific cell-mediated immune response and reduced the virus load and fever in pigs.