Multivalent nanoparticle-based vaccines protect hamsters against SARS-CoV-2 after a single immunization

The study addresses the urgent need for effective, scalable COVID-19 vaccination strategies that can confer protection with fewer doses. Existing vaccine platforms (nucleic acid, viral vector, and protein subunit) predominantly target the SARS-CoV-2 spike (S) protein to elicit neutralizing antibodies that block ACE2 receptor binding. However, multi-dose regimens and distribution challenges remain barriers. Nanoparticle scaffolds, particularly virus-like particles (VLPs), mimic the size and repetitive antigen presentation of viruses and can enhance B cell receptor clustering and immunogenicity. Prior work showed nanoscale antigen display improved immunogenicity in mice but did not demonstrate single-dose protection in a challenge model. This study aims to develop and test a multivalent VLP platform displaying prefusion-stabilized SARS-CoV-2 S proteins and to evaluate whether a single immunization elicits potent neutralizing antibodies and protects Syrian hamsters against SARS-CoV-2 challenge.

The authors situate their work among ongoing development of nucleic acid, viral vector, and subunit vaccines targeting the SARS-CoV-2 S protein (e.g., Krammer 2020; Walls et al. 2020). They reference evidence that neutralizing antibodies to the S protein correlate with protection and that multivalent display on nanoscale scaffolds enhances immunogenicity compared with soluble antigen. They build on prefusion-stabilized spike designs, including S-2P (Wang et al.) and HexaPro (Hsieh et al.), the latter showing improved stability and yield. Syrian hamsters are cited as a relevant small animal model with human-like COVID-19 pathology (Wei et al.). Comparative reports (e.g., Tostanoski et al.) are noted, but cross-study titer comparisons are cautioned due to methodological differences.

VLP design and production: An engineered MS2 bacteriophage coat protein was used to create VLPs for multivalent antigen display. A single-chain MS2 coat protein dimer was constructed with an Avitag inserted into the surface loop of the second monomer, enabling site-specific biotinylation by BirA. The MS2-Avitag construct was co-expressed with BirA in BL21(DE3) E. coli, lysed, and purified using affinity chromatography (His/Capto Core 700) followed by size-exclusion chromatography (SEC). The particles were biotinylated to near-completion in vitro. Streptavidin (SA) production: SA was expressed in BL21(DE3) E. coli as inclusion bodies, refolded, and purified by immobilized affinity chromatography and SEC. Biotinylated MS2-Avitag VLPs were incubated with excess SA to generate MS2-SA VLPs; excess SA was removed by SEC. SDS-PAGE quantification indicated ~27 SA molecules per VLP. Spike antigens: Two prefusion-stabilized spike ectodomain variants were used. S-2P (two proline substitutions) was engineered with a C-terminal Avitag and His-tag (termed S-2P) for site-specific biotinylation and IMAC purification. HexaPro (six proline substitutions) was similarly engineered (termed S6Pro). Both were expressed in Expi293f cells, purified via IMAC, biotinylated enzymatically by BirA in vitro, and polished by SEC. VLP–spike assembly and characterization: Biotinylated S proteins were conjugated to MS2-SA VLPs via the biotin–streptavidin interaction to form VLP-S2P and VLP-S6Pro. SDS-PAGE analysis of assembled VLP–spike preparations showed bands corresponding to S monomer (~140 kDa), MS2 coat protein dimer (~29 kDa), and SA monomer (~14 kDa). Analytical SEC profiles indicated minimal unbound S protein in VLP–spike preparations. Dynamic light scattering (DLS) and negative-stain TEM confirmed VLP size and spike decoration: MS2-SA VLPs were ~50 nm by DLS and ~30 nm by TEM; VLP-S constructs exhibited the expected size increase consistent with an external spike layer. Cryo-EM of VLP-S6Pro further confirmed MS2-SA cores (~30 nm) decorated with S6Pro spikes forming an outer shell. SDS-PAGE estimated ~50 S-2P molecules per VLP. Antigenicity assays: ELISAs assessed binding of ACE2-Fc and RBD-directed monoclonal antibody CR3022 to soluble S proteins and VLP-displayed S. Both ligands bound to S-2P/S6Pro and VLP-S2P/VLP-S6Pro, indicating proper folding and epitope accessibility post-conjugation. Hamster immunization and challenge: Female Syrian hamsters (4 weeks old) were assigned to five groups including VLP–spike vaccines (VLP-S2Pro, VLP-S6Pro), MS2-SA VLP alone, and PBS controls; vaccines were adjuvanted (Alhydrogel mentioned). Animals received a single immunization; sera were collected at day 28 for anti-RBD IgG ELISA and virus neutralization assays (reciprocal titers based on prevention of cytopathic effects). Hamsters were challenged intranasally with SARS-CoV-2; body weight was monitored daily. Three days post-challenge (peak lung viral load), animals were euthanized for lung and nasal turbinate harvest. Infectious virus was quantified by plaque assay on Vero E6/TMPRSS2 cells. Statistical analyses included two-way ANOVA with appropriate post hoc tests (e.g., Dunnett or Bonferroni), with normality and variance assessed by D'Agostino–Pearson and Brown–Forsythe tests in some analyses.

- Assembly and characterization: MS2-SA VLPs incorporated ~27 streptavidin molecules; VLP-S2P incorporated ~50 S-2P molecules. DLS and TEM confirmed particle sizes (~50 nm by DLS; ~30 nm cores by TEM) and spike decoration. Analytical SEC showed minimal free S protein in VLP–spike preparations. ELISAs demonstrated ACE2-Fc and CR3022 binding to both soluble S and VLP-displayed S, indicating correct folding and accessible neutralizing epitopes. - Serology after single immunization: In hamsters, a single dose of VLP–spike vaccine elicited robust anti-RBD IgG responses with ELISA endpoint titers ranging from 2.16 × 10^4 to 8.2 × 10^4. Neutralizing antibody titers (reciprocal of highest dilution completely preventing cytopathic effects) ranged from 320 to 640. Control groups (MS2-SA VLP alone or PBS) showed negligible anti-spike antibodies. - Protection against SARS-CoV-2 challenge: Following intranasal challenge, vaccinated hamsters maintained or slightly increased body weight over three days, contrasting with controls. No infectious virus was detected in the lungs of vaccinated animals at 3 days post-challenge, whereas controls exhibited high lung viral loads. In nasal turbinates, vaccinated groups had substantially reduced viral titers: mean titers were >150-fold lower in one VLP–spike group and >700-fold lower in the other compared with MS2-SA controls. Overall, a single immunization with multivalent VLP–spike vaccines induced high neutralizing titers and conferred strong protection in the hamster model, eliminating detectable lung virus and markedly reducing nasal virus loads.

The results demonstrate that multivalent display of prefusion-stabilized SARS-CoV-2 S proteins on MS2-based VLPs elicits potent neutralizing antibody responses and robust protection in Syrian hamsters after a single immunization. The strong immunogenicity likely derives from the dense, repetitive antigen array that efficiently cross-links B cell receptors. Proper folding and epitope preservation were confirmed by ACE2 and CR3022 binding, supporting the quality of the displayed antigen. The absence of detectable infectious virus in lungs and substantial reductions in nasal turbinate titers indicate effective protection against lower respiratory tract infection and partial control of upper airway replication. These findings address the need for single-dose vaccine strategies that could simplify logistics and expand coverage, particularly during pandemics. The platform’s modularity suggests adaptability to variant spikes or engineered antigens aiming for broader or pan-coronavirus immunity. Comparisons to other studies must be made cautiously due to assay and model differences; however, the demonstrated single-dose efficacy in a stringent challenge model is notable.

This study presents a modular nanoparticle vaccine platform using MS2-based VLPs to multivalently display prefusion-stabilized SARS-CoV-2 spike proteins. A single immunization induced high anti-RBD and neutralizing antibody titers and provided strong protection in hamsters, with no detectable infectious virus in lungs and markedly reduced nasal titers. The approach is broadly applicable and can be rapidly adapted to variant antigens or other pathogens. Future work should optimize manufacturing (including expression yields and cGMP processes), evaluate durability and breadth of immunity, assess efficacy against variants of concern, and investigate strategies to minimize anti-scaffold immunity or to employ alternative scaffolds as needed.

- The in vivo efficacy was demonstrated in Syrian hamsters; translation to humans remains to be established. - Manufacturing considerations include improving antigen expression yields (even for stabilized variants) and establishing cGMP-compliant processes for large-scale production. - Potential anti-scaffold immune responses to the MS2–streptavidin platform may impact boosting or cross-platform usage. - Protection against diverse variants of concern was not directly tested; breadth and durability of protection require further evaluation. - Some methodological descriptions (e.g., dosing/adjuvant details) were limited in clarity within the provided text, and comparative titer interpretations across studies are constrained by assay variability.