Intranasal COVID vaccine that works against variants in animals

Abstract

Current parenteral coronavirus disease 2019 (Covid-19) vaccines inadequately protect against infection of the upper respiratory tract. Additionally, antibodies generated by wild type (WT) spike-based vaccines poorly neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. To address the need for a second-generation vaccine, we have initiated a preclinical program to produce and evaluate a potential candidate. Our vaccine consists of recombinant Beta spike protein coadministered with synthetic CpG adjuvant. Both components are encapsulated within artificial cell membrane (ACM) polymersomes, synthetic nanovesicles efficiently internalized by antigen presenting cells, including dendritic cells, enabling targeted delivery of cargo for enhanced immune responses. ACM vaccine is immunogenic in C57BL/6 mice and Golden Syrian hamsters, evoking high serum IgG and neutralizing responses. Compared to an ACM-WT spike vaccine that generates predominantly WT-neutralizing antibodies, the ACM-Beta spike vaccine induces antibodies that neutralize WT and Beta viruses equally. Intramuscular (IM)-immunized hamsters are strongly protected from weight loss and other clinical symptoms after the Beta challenge but show delayed viral clearance in the upper airway. With intranasal (IN) immunization, however, neutralizing antibodies are generated in the upper airway concomitant with rapid and potent reduction of viral load. Moreover, antibodies are cross-neutralizing and show good activity against Omicron. Safety is evaluated in New Zealand white rabbits in a repeated dose toxicological study under Good Laboratory Practice (GLP) conditions. Three doses, IM or IN, at two-week intervals do not induce an adverse effect or systemic toxicity. Cumulatively, these results support the application for a Phase 1 clinical trial of ACM-polymersome-based Covid-19 vaccine (ClinicalTrials.gov identifier: NCT05385991).

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease 2019 (Covid-19), an ongoing global public health emergency. Despite the availability of safe and efficacious vaccines, achieving herd immunity remains a challenge due in part to rapid viral evolution. Multiple variants of concern (VOCs) have emerged, the latest being the heavily mutated Omicron, which exhibits the highest resistance to neutralizing antibodies from past vaccination or infection. Currently approved vaccines generate robust systemic immunity, yet poor immunity at the respiratory tract. We have demonstrated that a polymersome-based protein subunit vaccine with wild type (WT) spike protein and CpG adjuvant induces robust systemic immunity (humoral and T cell responses) in mice. Both antigen and adjuvant are encapsulated in artificial cell membrane (ACM) polymersomes – synthetic, nanoscale vesicles that substantially enhance the immune response through efficient delivery to dendritic cells. In the present study, we have formulated a vaccine candidate with the spike protein from Beta variant and assessed its immunogenicity in golden Syrian hamsters. Two doses of ACM-Beta spike vaccine administered via intramuscular (IM) injection evoke modest serum neutralizing titers that are equally efficacious towards WT and Beta viruses. In contrast, the ACM-WT spike vaccine induces a predominantly WT-specific serum neutralizing response with pronounced reduction in potency towards the Beta variant. Remarkably, immunogenicity of the ACM-Beta spike vaccine is greatly enhanced through intranasal (IN) administration. Following IN challenge with the Beta variant, IM-immunized hamsters are fully protected from disease but not infection, displaying similar peak viral RNA loads in oral swabs as non-vaccinated controls. In contrast, hamsters IN vaccinated with ACM-Beta spike vaccine are protected from disease and infection, exhibiting a ∼100-fold drop in total and subgenomic RNA load as early as day 2 post challenge. We further demonstrate that nasal washes from IN-but not IM-immunized animals possess virus neutralizing activity that is broadly efficacious towards Delta and Omicron variants. Altogether, our results show IN administration of ACM-Beta spike vaccine to evoke systemic and mucosal antibodies that cross-neutralize multiple SARS-CoV-2 VOCs. Our work supports IN administration of ACM-Beta spike vaccine for a next-generation vaccination strategy that not only protects against disease but also an infection of the respiratory tract, thus potentially preventing asymptomatic transmission.

Competing Interest Statement

J.H.L., D.S., T.W.C., S.L.C., S.V., G.S., T.Y.A., S.W. and M.N. are employees of ACM Biolabs Pte Ltd, Singapore. P.V is acting Chief Medical Officer of ACM Biosciences AG, Basel, Singapore.

Abstract

Multiple successful vaccines against SARS-CoV-2 are urgently needed to address the ongoing Covid-19 pandemic. In the present work, we describe a subunit vaccine based on the SARS-CoV-2 spike protein co-administered with CpG adjuvant. To enhance the immunogenicity of our formulation, both antigen and adjuvant were encapsulated with our proprietary artificial cell membrane (ACM) polymersome technology. Structurally, ACM polymersomes are self-assembling nanoscale vesicles made up of an amphiphilic block copolymer comprising of polybutadiene-b-polyethylene glycol and a cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane. Functionally, ACM polymersomes serve as delivery vehicles that are efficiently taken up by dendritic cells, which are key initiators of the adaptive immune response. Two doses of our formulation elicit robust neutralizing titers in C57BL/6 mice that persist at least 40 days. Furthermore, we confirm the presence of memory CD4⁺ and CD8⁺ T cells that produce Th1 cytokines. This study is an important step towards the development of an efficacious vaccine in humans.

Competing Interest Statement

The authors declare the following competing financial interests: D.E.A. and Y.J.T. developed the cPass kit; J.H.L, A.K.K., T.A.C., T.W.C., W.W.W.Y., and M.N. are employees of ACM Biolabs Pte Ltd; F.G. is part of the ACM SAB. The authors declare no non-financial competing interests.

Abstract

Multiple successful vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed to address the ongoing coronavirus disease 2019 (Covid-19) pandemic. In the present work, we describe a subunit vaccine based on the SARS-CoV-2 spike protein coadministered with CpG adjuvant. To enhance the immunogenicity of our formulation, both antigen and adjuvant were encapsulated with our proprietary artificial cell membrane (ACM) polymersome technology. Structurally, ACM polymersomes are self-assembling nanoscale vesicles made up of an amphiphilic block copolymer comprising poly(butadiene)-b-poly(ethylene glycol) and a cationic lipid, 1,2-dioleoyl-3-trimethylammonium-propane. Functionally, ACM polymersomes serve as delivery vehicles that are efficiently taken up by dendritic cells (DC1 and DC2), which are key initiators of the adaptive immune response. Two doses of our formulation elicit robust neutralizing antibody titers in C57BL/6 mice that persist at least 40 days. Furthermore, we confirm the presence of functional memory CD4⁺ and CD8⁺ T cells that produce T helper type 1 cytokines. This study is an important step toward the development of an efficacious vaccine in humans.

Keywords: ACM; Covid-19; neutralizing antibody; polymersome; spike; vaccine.

Conflict of interest statement

The authors declare the following competing financial interest(s): D.E.A. developed the cPass kit; J.H.L, A.K.K., T.A.C., T.W.C., W.W.W.Y., S.V., and M.N. are employees of ACM Biolabs Pte Ltd; F.G. is part of the ACM SAB.

Abstract

Multicompartmentalized polymersomes are formed using block co-polymers PMOXA–PDMS–PMOXA and PS-PIAT, and are subsequently proven to be capable of selective encapsulation of biomacromolecules. This architecture mimics the compartmentalization found in cells and may serve as a simple, albeit robust, model system.

Abstract

G-protein coupled receptors (GPCRs) play a key role in physiological processes and are attractive drug targets. Their biophysical characterization is, however, highly challenging because of their innate instability outside a stabilizing membrane and the difficulty of finding a suitable expression system. We here show the cell-free expression of a GPCR, CXCR4, and its direct embedding in diblock copolymer membranes. The polymer-stabilized CXCR4 is readily immobilized onto biosensor chips for label-free binding analysis. Kinetic characterization using a conformationally sensitive antibody shows the receptor to exist in the correctly folded conformation, showing binding behaviour that is commensurate with heterologously expressed CXCR4.

Abstract

Compartmentalization, as a design principle, is a prerequisite for the functioning of eukaryotic cells. Although cell mimics in the form of single vesicular compartments such as liposomes or polymersomes have been tremendously successful, investigations of the corresponding higher-order architectures, in particular bilayer-based multicompartment vesicles, have only recently gained attention. We hereby demonstrate a multicompartment cell-mimetic nanocontainer, built-up from fully synthetic membranes, which features an inner compartment equipped with a channel protein and a semi-permeable outer compartment that allows passive diffusion of small molecules. The functionality of this multicompartment architecture is demonstrated by a cascade reaction between enzymes that are segregated in separate compartments. The unique architecture of polymersomes, which combines stability with a cell-membrane-mimetic environment, and their assembly into higher-order architectures could serve as a design principle for new generation drug-delivery vehicles, biosensors, and protocell models.

Abstract

The dopamine receptor D2 (DRD2), a G-protein coupled receptor is expressed into PBd₂₂-PEO₁₃ and PMOXA₂₀-PDMS₅₄-PMOXA₂₀ block copolymer vesicles (see scheme). The conformational integrity of the receptor is confirmed by antibody- and ligand-binding assays. Replacement of bound dopamine is demonstrated on surface-immobilized polymersomes, thus making this a promising platform for drug screening.

Abstract

To improve the stability of cell membrane mimics, there has been growing interest in the use of block copolymers. Here, we present an easy approach to create an array of planar polymeric matrices capable of hosting membrane proteins. The array of polymeric matrices was formed by the selective deposition of triblock copolymers onto an array of hydrophilic islands situated within a hydrophobic background. The thickness of these matrices corresponds to the length of a single polymer chain. These polymeric matrices were used to host cell-free expressed membrane proteins, and offers a prototype from which a membrane protein array can be created for diagnostics or drug discovery purposes.