Broad-spectrum Delta-BA.2 tandem-fused heterodimer mRNA vaccine delivered by lipopolyplex

Abstract

Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, continues to mutate and generates new variants with increasingly severe immune escape, urging the upgrade of COVID-19 vaccines. Here, based on a similar dimeric RBD design as our previous ZF2001 vaccine, we developed a novel broad-spectrum COVID-19 mRNA vaccine, SWIM516, with chimeric Delta-BA.2 RBD dimer delivered by lipopolyplex (LPP). Unlike the popular lipid nanoparticle (LNP), this LPP-delivered mRNA expresses only in the injection site, which avoids potential toxicity to the liver. We demonstrated the broad-spectrum humoral and cellular immunogenicity of this vaccine to Delta and Omicron sub-variants in naïve mice and as booster shots. When challenged with Delta or Omicron live virus, vaccinated human angiotensin-converting enzyme (hACE2) transgenic mice and rhesus macaques were both protected, displaying significantly reduced viral loads and markedly relieved pathological damages. We believe the SWIM516 vaccine qualifies as a candidate for the next-generation broad-spectrum COVID-19 vaccine.

Fig 1. Design and in vitro characterization of the SWIM516 mRNA vaccine.

(a) Schematic of the design of the Delta-BA.2 mRNA vaccine (SWIM516). The amino acid substitutions above the column indicate the mutations in Delta and BA.2 RBDs compared with PT RBD. (b) Particle size distributions of LPP characterized by dynamic light scattering. The number indicates z-average. (c) Cryo-electron microscopy image of lipopolyplex (LPP). Scale bar, 50 nm. (d) Zeta potential for LPP at pH 4.3 and 7.2 in phosphate saline buffer (PBS). (e) Naked or LPP-encapsulated mRNA was transfected into HEK293T cells. The expression of Delta-BA.2 in the supernatant was analyzed by Western blot. (f) Locations of LNP- and LPP-delivered luciferase expression in vivo. mRNAs encoding luciferase were encapsulated by LNP or LPP before injection in both legs of BALB/c mice. The bioluminescence images of mice were captured at the indicated time points post-injection.

Fig 2. Evaluation of the immunogenicity of the SWIM516 vaccine.

(a) Mice immunization and sample collection schedule for evaluating the immunogenicity of SWIM516 vaccine. (b) Titers of IgG specific to the RBDs of the indicated SARS-CoV-2 variants on day 28. Numbers on top indicate the average IgG titers of each group. Dashed line indicates starting dilution (20 folds). Data are shown as geometric mean titer (GMT) ± 95% confidence interval (CI). (c) NT₅₀ of neutralizing antibodies against the pseudotyped viruses (pVirus) of indicated SARS-CoV-2 variants. Numbers on top indicate the GMT of each group. Radar chart was drawn based on GMT. Dashed line indicates starting dilution (60 folds). Data are shown as GMT ± 95% CI. (d) ELISpot assay quantifying the IFNγ-secreting splenocytes after re-stimulation by RBD peptide pool of indicated SARS-CoV-2 variants. Data are shown as means ± SD (standard deviation). Numbers on top indicate average SFU/10⁵ splenocytes. (e) ICS assays quantifying the proportions of IFNγ- and TNFα-secreting CD8⁺ and CD4⁺ T cells stimulated by BA.2 RBD peptide pool. Data are shown as means ± SD. ELISA and pseudovirus neutralization assays were repeated twice. All statistical significances were calculated by Mann-Whitney test (*, p<0.05; **, p<0.01; ***, p<0.001, ****, p<0.0001).

Fig 3. Evaluation of the immunogenicity of SWIM516 as boosters.

(a) Mice immunization and sample collection schedule for evaluating the immunogenicity of SWIM516 vaccine as the third or fourth shot. (b) The low and high dosages of inactivated vaccine (IV), protein subunit vaccine (PV) and mRNA vaccine (RV) used in the prime-boost immunization protocols. (c) The prime-boost immunization protocols of three-dose (left) or four-dose (right) groups. Underlined numbers on top indicate group numbers. The numbers in groups names of (d) and (e) correspond with the group numbers in (c), letter L and H indicate low-dose and high-dose groups, respectively. (d) NT₅₀ of neutralizing antibody against Delta or BA.2 pseudotyped viruses in three-dose (left) or four-dose (right) groups. Numbers on top indicate GMT. Dashed line indicates starting dilution (60 folds). Data are shown as GMT ± 95% CI. Pseudovirus neutralization assays were repeated twice. (e) ELISpot assay quantifying the IFNγ-secreting splenocytes after re-stimulation by Delta or BA.2 RBD peptide viruses in three-dose (left) or four-dose (right) groups. Data are shown as means ± SD. Numbers on top indicate average SFU/10⁵ splenocytes. All statistical significances were calculated by Mann-Whitney test (*, p<0.05; **, p<0.01; ***, p<0.001).

Fig 4. Evaluation of the protective efficacy of SWIM516 in mice.

(a) Mice immunization, challenge and sample collection schedule for evaluating the protective efficacy of the SWIM516 vaccine. (b) Titration of viral loads in lung, trachea or turbinal after Delta (top) or BA.1 (bottom) challenge of transient Ad5-hACE2 mice. (c) Titration of viral loads in lung and trachea after Delta (top) or BA.1 (bottom) challenge of hACE2 transgenic mice. Data in (b) and (c) are shown as means ± SD. (d) Pathological analysis of lung tissues of mice after Delta or BA.1 challenge of hACE2 transgenic mice. Uninfected indicate mice without viral challenge. Black bars represent 100 μm. All statistical significances were calculated by Student’s T test (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001).

Fig 5. Evaluation of the protective efficacy of SWIM516 in rhesus macaques.

(a) Rhesus macaques immunization, challenge and sample collection schedule for evaluating the protective efficacy of SWIM516 vaccine. (b) PRNT₅₀ of neutralizing antibody against BA.1. Numbers on top indicate GMT, data are shown as GMT ± 95% CI. (c) Diagram of samples collected in different lobes of rhesus macaque lungs after BA.1 challenge. Legend indicated the lobe of lungs (UL: upper left; ML: middle left; LL: lower left; UR: upper right; MR: middle right; LR: lower right). (d) Quantitation of viral load in rhesus macaque lung samples, data are shown as mean ± SD. (e) Pooled analyses of pathological scores for lung tissues of macaques after BA.1 challenge, data are shown as GMT ± 95% CI. Statistical significances were calculated by Mann-Whitney test (*, p<0.05; ****, p<0.00001). (f) Pathological analysis of lung tissues of rhesus macaque after BA.1 challenge. Black bars represent 100 μm.