New Quartet nanocage vaccine shows promise against coronavirus variants

May 8, 2024

In a recent study published in the journal Nanotechnology from nature, researchers developed a unique vaccination technique using virus-like particles (VLPs) with numerous protein variations to amplify the immune response to infections such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). They synthesized connected receptor-binding domains (RBDs) from SARS-like betacoronaviruses and connected them to a computationally designed nanocage, enabling efficient construction of the nanocage.

Nanoscale organization is critical in programming the immune response, and virus-like particles (VLPs) enhance immune responses by stimulating B cell receptor clustering and lymph node uptake. Current vaccination regimens have limited vaccine protection, new pathogen variations, and questionable treatment efficacy. VLPs containing numerous protein variations show promise for broadening immune responses, possibly defending against strains and new viruses such as SARS-CoV-2. Recent techniques use variations of stochastically arranged proteins to promote B cell growth.

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Summary of the schedule and antigens of this set of immunizations. Study: Proactive Vaccination Using Multiviral Quartet Nanocages to Elicit Broad Responses Against Coronavirus

About the study

In the present study, the researchers devised a proactive vaccination technique based on multi-virus Quartet nanocages. These nanocages can combat various coronaviruses by eliciting broad antiviral responses.

The multivirus quartet vaccine was composed of RBDs of four viruses linked together to form a polypeptide chain. The researchers constructed the antigenic quartets using a terminal SpyTag to originate from SpyCatcher003-mi3 nanocages, resulting in protein nanoparticles with branched structures. They examined immune responses to several sarbecoviruses displayed in Quartet Nanocages, non-chain sarbecoviruses, and variants of concern (VOCs) of SARS-CoV-2. They also genetically combined RBDs from evolutionarily similar sarbecoviruses SHC014, Rs4081, RaTG13 and the SARS-CoV-2 Wuhan Hu-1 strain to create a multiviral quartet.

The team used a plug-and-display vaccine assembly of Mosaic and Quartet nanocages to effectively multimerize individual or Quartet RBDs connected to SpyTag by spontaneously forming isopeptide bonds. They also created the Alternative Multiviral Quartet, which includes SpyTag followed by RBD of several sarbecoviruses: pang17, RmYN02, Rf1 and WIV1. To study the association between chain location and immunogenicity, they evaluated neutralization of the Delta or Wuhan variant of SARS-CoV-2 with a 10-fold higher antigen dose and the squalene-based AddaVax adjuvant to enhance neutralization.

To evaluate reactions to RBDs at various distances from SpyCatcher003-mi3, the researchers used enzyme-linked immunosorbent assays (ELISA) on Quartet antigens and a panel of anti-SARS-CoV-2 monoclonal antibodies. They also created a quartet without flexible Gly-Ser linkers to separate the different RBDs and tested the No-Linker Quartet Nanocage against the typical Quartet Nanocage.

The team used polymerase chain reaction (PCR) to clone RBD constructs, which were confirmed by Sanger sequencing. The RBD quartet constructs were cloned using Gibson assembly and then converted into E. coli BL21 (DE3) cells for bacterial expression and cell culture studies. SpySwitch was used to purify RBD, Quartets, and SpyTag-MBP, which the researchers evaluated using SDS-PAGE electrophoresis. They isolated SARS-CoV-2 spike (S) proteins using Ni-NTA affinity chromatography, PNGase F digestion, and transmission electron microscopy (TEM). They extracted endotoxins from the vaccine components and used bicinchoninic acid assays to determine their concentrations.

to, Nanocages quartet and mosaic plug-and-display vaccine set. The genetic fusion of SpyCatcher003 (dark blue) with mi3 (purple) allows efficient multimerization of single or quartet RBDs linked to SpyTag (cyan) through spontaneous formation of isopeptide bonds (marked in red). For clarity, only some antigens are shown in the schematic. bPhylogenetic tree of the sarbecoviruses used in this study, based on the RBD sequence. cGenetic organization of the multiviral Quartet-SpyTag, indicating the viral origin of the RBDs, N-linked glycosylation sites, and tag location. d, Quartet-SpyTag analysis with SDS-PAGE/Coomassie staining, with or without PNGase F deglycosylation. A representative gel from two independent experiments. Molecular weight markers are in kDa. my, Docking of RBD Quartet to SpyCatcher003-mi3 Nanocage at different Nanocage:antigen molar ratios, analyzed by SDS–PAGE/Coomassie. A representative gel from two independent experiments. Molecular weight markers are in kDa.

Results

Quartet nanocages, branched nanoparticles with a branched architecture, have been shown to stimulate significant levels of neutralizing antibody titers against various coronaviruses, even those that are not included in the vaccination. Antibodies responded similarly to RBDs in the nanocage or at the tip of its branch. Among mice primed with SARS-CoV-2 S, booster vaccines using Quartet nanocages improve the breadth and intensity of the otherwise limited immune response. The Quartet Nanocage, comprising Omicron XBB.1.5 ‘Kraken’ RBD, elicited neutralizing antibodies bound to several sarbecoviruses. The size and breadth of antibody induction indicate that Quartet Nanocages can generate neutralizing antibodies against various viruses, preparing for new risks of epidemic diseases.

Vaccination with homotypic Nanocage and uncoupled receptor binding domain produced modest responses against sarbecovirus receptor binding domains, with homotypic Nanocage eliciting the most robust cross-reactive responses against RaTG13 receptor binding domains. However, vaccination with uncoupled quartets and quartet nanocages produced significant responses to all RBDs investigated, including high heterotypic responses to the RBDs of SARS-CoV-1 and BM-4831.

The Dual Quartet Nanocage, which features the same RBDs as Mosaic-8, was created by combining the Quartet and its alternative with SpyCatcher003-mi3. Quartet Nanocage and the dual quartet type produced the highest post-surge antibody titers, while Quartet Nanocage and Mosaic-8 elicited similar and moderately robust anti-SARS-CoV-1 responses.

Quartet and Mosaic immunogens showed greater antibody binding to zoonotic coronaviruses, while XBB.1.5 immunogens provided greater neutralization of the SARS2 pseudovirus XBB.1.5, indicating that Quartet Nanocage could protect against antibody escape variants and cause widespread antiarbovirus responses without SARS-CoV. -2. The quartet nanocage without SARS-CoV-2 generated anti-SARS-CoV-2 antibodies and promoted responses to sarbecoviruses.

The study findings highlight the creation of improved vaccine platforms such as Quartet Nanocages, which offer heterotypic protection against emerging zoonotic diseases and enable proactive protection against pandemics. These nanocages stimulate neutralizing antibodies against viral antigens. The solubility, thermostability, flexibility, and sequence divergence of sarbecovirus RBDs facilitated the efficient generation of different quartets. Vaccine candidates demonstrated comparable or greater breadth than Mosaic-8, and Omicron variants effectively avoided neutralizing antibodies.