Artificial Cell Membrane Polymersome-Based Intranasal Beta Spike Formulation as a Second Generation Covid-19 Vaccine

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).

Keywords: ACM; Beta spike; Covid-19; intranasal; polymersome; vaccine.

Conflict of interest statement

The authors declare the following competing financial interest(s): J.H.L., D.S., T.W.C., S.L.C., S.V., G.S., T.Y.A., and S.W. are employees of ACM Biolabs Pte Ltd., Singapore. M.N. is the Chief Executive Officer of ACM Biolabs Pte Ltd., Singapore. P.V. is the acting Chief Medical Officer of ACM Biosciences AG, Basel, Switzerland.

Figures

Figure 1

ACM-Covid-19 vaccine preclinical development program. The vaccine consisted of insect cell-produced recombinant spike protein and synthetic CpG adjuvant separately encapsulated in ACM polymersomes for coadministration. Immunogenicity was assessed in mice and hamsters after IM or IN administration. Protection against a live virus challenge was examined in hamsters. Key efficacy readouts are indicated. Safety was evaluated in a repeated dose GLP toxicological study in rabbits. Vaccine-related adverse effects were not detected.

Figure 2

Serum antibody response to ACM-Covid-19 vaccines. (a) Immunization, sample collection, and live virus challenge schedule. Golden Syrian hamsters (n = 8) were IM administered one of the following: (i) PBS; (ii) fS1S2(WT) + fCpG; (iii) ACM-S1S2(WT) + ACM-CpG; (iv) ACM-S1S2(Beta) + ACM-CpG. (b, c) WT and Beta spike-specific serum IgG titers, respectively. The bar graph represents mean ± SD. Two-way repeated measures ANOVA with Tukey’s or Šídák’s multiple comparisons was performed. Significant differences between Day 20 and Day 34 IgG titers, between placebo and vaccinated animals, and between free S1S2(WT)-vaccinated and ACM-vaccinated animals are indicated for each time point. *: P ≤ 0.05; **: P≤ 0.01; ***: P ≤ 0.001; ****: P≤ 0.0001; ns: not significant; NA: not applicable. (d–f) Neutralizing potency toward WT virus or Beta variant. Geometric mean titers (GMTs) are indicated at the top of each graph; fold-change in GMT with respect to WT virus is indicated at the bottom. Lower limit of detection (1:20 serum dilution) is indicated by the horizontal dashed line. A two-tailed paired t test was performed.

Figure 3

Live SARS-CoV-2 Beta variant challenge. Hamsters (n = 8) were inoculated with Beta variant via the intranasal route. (a) Changes in body weight over 14 days relative to the initial weight (horizontal dashed line). Mean ± SEM is shown. (b) Area under the curve (AUC) analysis for changes in body weight. Bar graph represents mean ± SD. Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons was performed. Only significant differences are shown. *:P ≤ 0.05; **: P ≤ 0.01; ***:P ≤ 0.001. (c) Peak COVID scores of hamsters within 14 days after the virus challenge. Hamsters were observed daily for signs of disease (mild ruffled or ruffled fur, hunched back, labored/heavy breathing, and lethargy) or were noted as normal. Individual COVID scores were assigned (0 normal; ≤2 mild disease; 3 moderate disease; ≥4 severe disease). Plotted are individual peak COVID scores with average scores per group, gender depicted as bar graphs, and range depicted as whiskers. One-way ANOVA with Tukey’s multiple comparisons was performed. Statistical significance with respect to gender-matched placebo controls is indicated above the bar graph. ***: P ≤ 0.001; ****:P ≤ 0.001; ns: not significant. (d) Histopathological analysis of SARS-CoV-2-related microscopic findings in lungs Day 14 post-challenge. Lungs of male (top) and female (bottom) hamsters were collected, fixed, processed to H&E-stained sections, and evaluated by a board-certified pathologist. Microscopic findings (hyperplasia, inflammation, and fibrosis) were scored according to severity and size of involved tissue or noted as unremarkable. (e, f) Viral RNA loads in oral swabs as determined by qPCR. Copy numbers (mean ± SEM) of total and subgenomic RNA (sgRNA) are shown, respectively. Horizontal dashed lines represent lower limits of detection (62 and 31 RNA copies/mL, respectively). Two-way repeated measures ANOVA with Tukey’s multiple comparisons was performed. Only significant differences with respect to placebo controls are shown. *: P ≤ 0.05; **:P ≤ 0.01; ***: P ≤ 0.001; ****:P ≤ 0.0001.

Figure 4

IN administration enhanced the immunogenicity of ACM-S1S2(Beta) + ACM-CpG and induced neutralizing antibodies in the upper airway. Hamsters (n = 8) were IM or IN administered an identical dose of ACM-S1S2(Beta) + ACM-CpG on Days 0 and 21. (a, b) WT and Beta spike-specific serum IgG titers, respectively. Bar graph represents mean ± SD. Two-way repeated measures ANOVA with Šídák’s multiple comparisons was performed. *:P ≤ 0.05; **: P ≤ 0.01; ***:P ≤ 0.001; ****: P ≤ 0.0001; ns: not significant. (c) Neutralizing potency toward WT virus or Beta variant. Bar graph represents mean ± SD. Geometric mean titers (GMTs) are shown on top of the bar graphs. Lower limit of detection (1:20 serum dilution) is indicated by the horizontal dashed line. A two-tailed paired t test was performed. (d) Changes in body weight following the intranasal challenge with live SARS-CoV-2 Beta variant. Animals were monitored for 14 days. Initial body weight is indicated by the horizontal dashed line. Mean ± SEM is shown. (e) Area under the curve (AUC) analysis for changes in body weight. Bar graph represents mean ± SD. Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons was performed. (f) Peak mean COVID scores of hamsters within 14 days after the virus challenge. Hamsters were observed daily for signs of disease such as mild ruffled or ruffled fur, hunched back, labored/heavy breathing, and lethargy or were noted as normal. Individual COVID scores based on clinical observation were assigned (0 normal, ≤2 mild disease, 3 moderate disease, ≥4 severe disease). Plotted are individual peak COVID scores (individual data points) and mean peak COVID scores per group and gender (bar) with the range (whiskers). One-way ANOVA with Tukey’s multiple comparisons was performed. A significant difference with respect to gender-matched placebo controls is indicated, where present. (g) Histopathological analysis of SARS-CoV-2-related microscopic findings in hamster lungs Day 14 post-challenge. Lungs of male (top) and female (bottom) hamsters were collected, fixed, processed to H&E-stained sections, and evaluated by a board-certified pathologist. Microscopic findings (hyperplasia, inflammation, and fibrosis) were scored according to the severity and size of the involved tissue. (h, i) Viral RNA loads in oral swabs as determined by qPCR. Copy numbers (mean ± SEM) of total and subgenomic RNA (sgRNA) are shown, respectively. Horizontal dashed lines represent lower limits of detection (62 and 31 RNA copies/mL, respectively). Two-way repeated measures ANOVA with Tukey’s multiple comparisons was performed. *: P ≤ 0.05; **: P≤ 0.01; ***: P ≤ 0.001; ****: P≤ 0.0001; ns: not significant. (j, k) Neutralizing activity of nasal washes from hamsters IM or IN immunized, respectively. Neutralization of RBD from WT, Delta, or Omicron virus was determined using a cPass kit (% inhibition indicated above the bar graphs). To overcome their highly dilute nature, nasal washes from each group were pooled and concentrated 40-fold.

Figure 5

Body weight changes in rabbits IM or IN administered ACM-S1S2(Beta) + ACM-CpG. (a) Immunization and sample collection schedule. New Zealand white rabbits were administered 0.5 mL of 20 μg of ACM-S1S2(Beta) + 100 μg of ACM-CpG 7909 on Days 0, 14, and 28. IM injection was performed on the left quadriceps muscle on Days 0 and 28 and on the right on Day 14. IN was performed with 0.25 mL per nostril. Rabbits were segregated into a main study group (five males and five females) and a recovery group (two males and two females). (b, f) Changes in body weight of the main study group over 28 days after IM or IN administration, respectively. Male and females were analyzed separately. Initial body weight is indicated by the horizontal dashed line. Mean ± SEM is shown. (c, g) Area under the curve (AUC) analysis for changes in body weight after IM or IN administration, respectively. Bar graph represents mean ± SD. Two-way ANOVA with Šídák’s multiple comparisons was performed. ns: not significant. (d, h) Changes in body weight of the recovery group over 57 days after IM or IN administration, respectively. Males and females were combined for analysis. (e, i) AUC analysis for changes in body weight after IM or IN administration, respectively.

Figure 6

Serum C-reactive protein (CRP) levels and histopathological findings at the sites of administration. Rabbits were IM (a–c) or IN (d–f) administered placebo or ACM-S1S2(Beta) + ACM-CpG. (a, d) Serum CRP levels on Days 0, 2, 30, and 57. Data from Days 0, 2, and 30 was derived from the main study group (n = 10) and segregated on the basis of gender; data from Day 57 was derived from the recovery group (n = 4) and was not segregated. Two-way ANOVA with Šídák’s multiple comparisons was performed. *:P ≤ 0.05; ns: not significant. (b, c, e, f) Histopathological examination of the iliac lymph node, quadriceps muscle, and nose. Tissues were fixed, processed to H&E-stained sections, and evaluated by two pathologists. Microscopic findings (increased cellularity, hemorrhage, inflammatory infiltrate and cell debris) were scored according to severity. Analysis of the main study group on Day 30 (b, e) was segregated on the basis of gender, whereas the recovery group on Day 57 (c, f) was analyzed irrespective of gender.