A liposome-displayed hemagglutinin vaccine platform protects mice and ferrets from heterologous influenza virus challenge

Recombinant influenza virus vaccines based on hemagglutinin (HA) hold the potential to accelerate production timelines and improve efficacy relative to traditional egg-based platforms. Here, we assess a vaccine adjuvant system comprised of immunogenic liposomes that spontaneously convert soluble antigens into a particle format, displayed on the bilayer surface. When trimeric H3 HA was presented on liposomes, antigen delivery to macrophages was improved in vitro, and strong functional antibody responses were induced following intramuscular immunization of mice. Protection was conferred against challenge with a heterologous strain of H3N2 virus, and naive mice were also protected following passive serum transfer. When admixed with the particle-forming liposomes, immunization reduced viral infection severity at vaccine doses as low as 2 ng HA, highlighting dose-sparing potential. In ferrets, immunization induced neutralizing antibodies that reduced the upper respiratory viral load upon challenge with a more modern, heterologous H3N2 viral strain. To demonstrate the flexibility and modular nature of the liposome system, 10 recombinant surface antigens representing distinct influenza virus strains were bound simultaneously to generate a highly multivalent protein particle that with 5 ng individual antigen dosing induced antibodies in mice that specifically recognized the constituent immunogens and conferred protection against heterologous H5N1 influenza virus challenge. Taken together, these results show that stable presentation of recombinant HA on immunogenic liposome surfaces in an arrayed fashion enhances functional immune responses and warrants further attention for the development of broadly protective influenza virus vaccines.

Influenza virus is a persistent global health concern, mainly because the efficacy of current vaccines is suboptimal. The narrow breadth of protection and rapid waning of vaccination-induced antibodies along with the circulation of variant viruses necessitates annual reformulation and readministration of seasonal influenza virus vaccines. Seasonal influenza epidemics result in a global total of 3 to 5 million severe infections each year, with 290,000 to 650,000 deaths (1). The viral envelope contains two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), of which there are many antigenically distinct subtypes (H1-18, N1-11) (2, 3). Both proteins are subject to antigenic drift; the selection of advantageous mutations that alters antigenicity and promotes escape from preexisting immunity. In addition to seasonal epidemics, novel forms of the virus have arisen throughout history to cause pandemics with high morbidity and mortality rates. These strains generally arise by antigenic shift, a process through which two different influenza virus strains reassort their genomes within host cells. This results in an influenza virus strain to which the human population is largely naive (4, 5). The influenza virus''s capacity to circumvent immune recognition through antigenic change has been a significant barrier to the development of long-lasting immunity and highly efficacious vaccines.Currently, seasonal influenza vaccination relies on a global viral surveillance system and predictive models that determine which strains will be incorporated into the seasonal vaccine each year. However, seasonal vaccine efficacy varies significantly depending upon the degree of matching between the predicted and circulating strains and does not provide significant protection when variant seasonal or pandemic strains emerge. Most seasonal influenza vaccines are still produced in embryonated chicken eggs, which necessitates a protracted vaccine production timeline and can result in egg-adapted vaccine strains that are antigenically dissimilar from circulating strains (6, 7). To address this, future vaccine strategies should be developed that can either yield a more rapid production process or protect against a more extensive repertoire of influenza virus strains, ideally including strains that are yet to arise. Both approaches favor the application of recombinant influenza virus antigens, with molecular structures that can be precisely controlled and modified. Recombinant antigens have proven safe and effective in the Flublok vaccine (8), and several studies have assessed epitope-based vaccine strategies based on conserved protein sequences in the stalk region (9, 10) and head region (11) of recombinant HA antigens. However, for many experimental recombinant vaccines to achieve a sufficient degree of immunogenicity, recombinant antigens must be administered in conjunction with an adjuvant to increase the magnitude of the immune response that they elicit. While regulatory agencies have approved several adjuvanted influenza vaccines for clinical use (12, 13), existing adjuvants may not be optimum for recombinant proteins. New adjuvants are being developed including nanoparticle-based carriers for recombinant antigens (14, 15). Liposomes, in particular, have been developed as vaccine adjuvants (16) and have been assessed with recombinant HA in preclinical studies (17–19). In this study, to enhance liposome immunogenicity, a synthetic monophosphoryl lipid A (MPLA) variant, phosphorylated hexaacyl disaccharide (PHAD), was incorporated into the lipid membrane. MPLA is used as a component of AS01, a liposomal adjuvant currently included in the Shingrix vaccine for Herpes Zoster (20, 21).We have developed an adjuvant system based on liposomes containing cobalt-porphyrin phospholipid (CoPoP). CoPoP provides a methodology that results in biostable binding due to the sequestration of His-tagged antigens directly within the bilayer (22). The CoPoP contained in the liposomal membranes results in rapid binding of His-tag modified recombinant antigens at room temperature (RT) with straightforward coincubation. When used in this way for immunization, CoPoP particles have been shown to enhance antibody responses to recombinant antigens derived from several pathogens (23–26). CoPoP has entered human clinical trials as a component of a SARS-CoV-2 vaccine (ClinicalTrials.gov Identifier: {"type":"clinical-trial","attrs":{"text":"NCT04783311","term_id":"NCT04783311"}}NCT04783311). In this study, we assess whether CoPoP can contribute to a platform for influenza virus vaccines. Recombinant HA trimers with trimerizing foldon domains from T4 bacteriophage fibritin were used to generate antigen associated with CoPoP liposome surfaces with conformation putatively replicating the trimers formed by native HA complexes (27, 28).