STING agonist-containing microparticles improve seasonal influenza vaccine efficacy and durability in ferrets over standard adjuvant

Abstract

The current pandemic highlights the need for effective vaccines against respiratory viruses. An ideal vaccine should induce robust and long-lasting responses with high manufacturing scalability. We use an adjuvant comprised of a Stimulator of Interferon Genes (STING) agonist incorporated in a scalable microparticle platform to achieve durable protection against the influenza virus. This formulation overcomes the challenges presented by the cytosolic localization of STING and the hydrophilicity of its agonists. We evaluated a monoaxial formulation of polymeric acetalated dextran microparticles (MPs) to deliver the STING agonist cyclic GMP-AMP (cGAMP) which achieved >10× dose-sparing effects compared to other published work. Efficacy was evaluated in ferrets, a larger animal model of choice for influenza vaccines. cGAMP MPs with recombinant hemagglutinin reduced viral shedding and improved vaccine outcomes compared to a seasonal influenza vaccine. Importantly, sustained protection against a lethal influenza infection was detected a year after a single dose of the vaccine-adjuvant.

Introduction

The present SARS-CoV-2 pandemic underscores the high probability and impact of a respiratory infection worldwide. It draws global attention to the importance of vaccines against these respiratory infections and emphasizes the need for continuous improvement to vaccine platforms. Influenza viruses remain a significant burden to society, with up to 650,000 deaths attributed to the virus each year worldwide [1], and an annual economic impact of $90 billion in the U.S. alone [2]. While vaccination is one of the greatest preventative measures against infection, the influenza vaccine has a poor estimated effectiveness ranging from 13 to 60% [3]. This low efficacy is often due to antigenic mismatch resulting from antigenic drift that occurs during the lengthy production process and flu season [3,4], as well as strain adaptation due to growth in eggs [5,6]. Additionally, protection provided by the current seasonal influenza vaccine approach is relatively short lived, on the order of three to four months, while the flu season spans six months or more [7]. This highlights the need to generate influenza vaccines with more durable responses.

The subunit influenza vaccine Flublok, the only FDA-approved influenza vaccine consisting of recombinant protein antigens, may address some of these concerns, but still has limitations. For instance, due to its lack of immunogenicity it requires hemagglutinin (HA) protein doses that are three-fold higher than what is used in conventional flu shots (45 versus 15 μg). Moreover, Flublok has induced lower antibody titers in adults aged 18–49 (its recommended age group) compared to conventional inactivated viral influenza vaccines [8].

An effective way to improve the durability of a vaccine response is to include an adjuvant in the formulation. Squalene-based emulsions (e.g., microfluidization 59 [MF59], adjuvant system 3 [AS03]) are used clinically with inactivated influenza viruses as a vaccine, but they have a limited half-life in vivo [9] and alone do not drive strong type 1 helper T cell (Th1)-biased cellular responses [10]. Similarly, traditional aluminum salt (alum) adjuvants are unable to drive strong cellular immune responses required for protection against influenza [11]. Pathogen-associated molecular patterns (PAMPs), which stimulate pattern recognition receptors (PRRs) on antigen-presenting cells (APCs), are an exciting alternative because they drive a balanced humoral and cellular immune response [12,13]. Existing FDA-approved vaccine adjuvants target PRRs on either plasma or endosomal membranes [14], and include monophosphoryl lipid A (MPL) with alum, MPL with liposomal QS-21, or cytosine phosphoguanine (CpG). In contrast, there is still a significant need to improve agonists for cytosolic PRRs such as Stimulator of Interferon Genes (STING), whose stimulation can contribute significantly to antiviral responses [15]. The difficulty in reaching cytosolic PRRs typically forces the need for high agonist doses that increase the potential for toxicity (e.g., 3000 μg CpG in the hepatitis B vaccine Heplisav-B).

STING agonist delivery can be enhanced using formulations such as polymeric particles, micelles, or liposomes [[16], [17], [18], [19], [20], [21], [22], [23], [24], [25]]. Polymeric particles have advantages over liposomes because of their extended shelf-life and enhanced stability [26,27]. STING's cytosolic location, however, requires use of polymers that can enhance intracellular release of its agonists. Poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) are not sensitive for degradation in the acidic endosomal environment, limiting the extent of cytosolic delivery. Additionally, the polymer hydrolyzes into acidic byproducts that can be detrimental to vaccine components. Another biopolymer, acetalated dextran (Ace-DEX), also degrades through hydrolysis but into pH-neutral biocompatible byproducts dextran, acetone, and ethanol. Additionally, Ace-DEX degrades more rapidly in the acidic endosomal environment (~pH 5.0) of APCs than at neutral pH [[28], [29], [30], [31]]. This leads to triggered degradation of Ace-DEX MPs once endocytosed by APCs, resulting in release of the MP cargo. The targeted intracellular release leads to enhanced adjuvant innate signaling as demonstrated by the higher bioactivity of the STING agonist cyclic GMP-AMP (cGAMP) when delivered in Ace-DEX MPs compared to PLGA MPs or liposomes [22,32,33]. Another added advantage of Ace-DEX compared to PLGA MPs is their stability outside the cold-chain [34]. We have previously reported the use of coaxial electrospray for the encapsulation cGAMP into Ace-DEX MPs as a vaccine adjuvant [22]. We have recently improved the technology by using monoaxial electrospray [35]. A simpler method that addresses the issue of ease of scalability, but has not been validated for vaccine use. In the current work, the monoaxial electrospray method for encapsulation of cGAMP into Ace-DEX MPs was first validated biological efficacy as compared to the previously published coaxial method. Furthermore, subunit recombinant HA influenza vaccine adjuvanted with monoaxial electrospray cGAMP-loaded Ace-DEX MPs (cGAMP MPs) were evaluated against state-of-the-art influenza vaccine formulations in multiple ferret studies. Ferrets were studied because they represent the animal model of choice for assessing influenza vaccine efficacy [[36], [37], [38]] and exhibit a similar disease pathogenesis as in humans. Furthermore, upon vaccination, relevant parameters such as body weight, body temperature and presence of replicating virus in lung tissue and the airway can be assayed. This report shows that the monoaxial cGAMP Ace-DEX MPs used induced a durable and robust response over the span of a year, and achieved a > 10× dose sparing of cGAMP compared to a recent paper using a different particle formulation [39]. Together these studies show that in the context of subunit vaccination, monoaxial cGAMP MPs are a potent and well-tolerated adjuvant that elicits robust protective antiviral immunity against influenza respiratory challenge.