Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has already infected more than 500 million people globally (as of May 2022), creating the coronavirus disease 2019 (COVID-19) pandemic. Nanotechnology has played a pivotal role in the fight against SARS-CoV-2 in various aspects, with the successful development of the two highly effective nanotechnology-based messenger RNA vaccines being the most profound. Despite the remarkable efficacy of mRNA vaccines against the original SARS-CoV-2 strain, hopes for quickly ending this pandemic have been dampened by the emerging SARS-CoV-2 variants, which have brought several new pandemic waves. Thus, novel strategies should be proposed to tackle the crisis presented by existing and emerging SARS-CoV-2 variants. Here, we discuss the SARS-CoV-2 variants from biological and immunological perspectives, and the rational design and development of novel and potential nanotechnology-based strategies to combat existing and possible future SARS-CoV-2 variants. The lessons learnt and design strategies developed from this battle against SARS-CoV-2 variants could also inspire innovation in the development of nanotechnology-based strategies for tackling other global infectious diseases and their future variants. This Perspective highlights the role that nanotechnology might play in tackling the rise of new SARS-CoV-2 variants. 

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Nanotechnology-based strategies against SARS-CoV-2 variants

The receptor binding domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of ∼80-100 mg/L in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of up to 100 °C and were stable to long-term storage of over 4 weeks at 37 °C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25-250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were ∼3-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1, and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

Immunogenicity and Protective Efficacy of a Highly Thermotolerant, Trimeric SARS-CoV-2 Receptor Binding Domain Derivative.

The Receptor Binding Domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of ∼80-100 mg/liter in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of upto 100 °C and were stable to long term storage of over 4 weeks at 37 °C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation, elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25-250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were ∼ three-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1 and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

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Immunogenicity and protective efficacy of a highly thermotolerant, trimeric SARS-CoV-2 receptor binding domain derivative

The Receptor Binding Domain of SARS-CoV-2 is the primary target of neutralizing antibodies. We fused our previously described, highly thermotolerant glycan engineered monomeric RBD to a heterologous non-immunogenic trimerization domain derived from cartilage matrix protein. The protein was expressed at a good yield of ~80-100 mg/liter in Expi293 cells, as well as in both CHO and HEK293 stable cell lines. The designed trimeric RBD was observed to form homogeneous disulfide-linked trimers. When lyophilized, the trimer possessed remarkable functional stability to transient thermal stress of upto 100 °C and was stable to long term storage of over 4 weeks at 37 °C. Two immunizations with an AddaVax adjuvanted formulation elicited antibodies with high endpoint neutralizing titers against replicative virus with geometric mean titers of ~1114 and 1940 in guinea pigs and mice respectively. In pseudoviral assays, corresponding titers were ~3600 and ~16050, while the corresponding value for human convalescent sera was 137. Similar results were obtained with an Alhydrogel, CpG combination adjuvant. The same immunogen was expressed in Pichia pastoris, but this formed high molecular weight aggregates and elicited much lower ACE2 competing antibodies than mammalian cell expressed protein. The excellent thermotolerance, high yield, and robust immunogenicity of such trimeric RBD immunogens suggest that they are a promising modality to combat COVID-19.

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A highly thermotolerant, trimeric SARS-CoV-2 receptor binding domain derivative elicits high titers of neutralizing antibodies

COVID-19 has caused a global pandemic and millions of deaths. It is imperative to develop effective countermeasures against the causative viral agent, SARS-CoV-2 and its many variants. Vaccines and therapeutic antibodies are the most effective approaches for preventing and treating COVID-19, respectively. SARS-CoV-2 enters host cells through the activities of the virus-surface spike (S) protein. Accordingly, the S protein is a prime target for vaccines and therapeutic antibodies. Dealing with particles with dimensions on the scale of nanometers, nanotechnology has emerged as a critical tool for rapidly designing and developing safe, effective, and urgently needed vaccines and therapeutics to control the COVID-19 pandemic. For example, nanotechnology was key to the fast-track approval of two mRNA vaccines for their wide use in human populations. In this review article, we first explore the roles of nanotechnology in battling COVID-19, including protein nanoparticles (for presentation of protein vaccines), lipid nanoparticles (for formulation with mRNAs), and nanobodies (as unique therapeutic antibodies). We then summarize the currently available COVID-19 vaccines and therapeutics based on nanotechnology. 

Recent advances in nanotechnology-based COVID-19 vaccines and therapeutic antibodies

Vaccines that induce potent neutralizing antibody (NAb) responses against emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential for combating the coronavir...

Mechanism of a COVID-19 nanoparticle vaccine candidate that elicits a broadly neutralizing antibody response to SARS-CoV-2 variants.

Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential for combating the coronavirus disease 2019 (COVID-19) pandemic. Neutralizing antibody responses to the original Wuhan-Hu-1 strain that were generated during infection and vaccination showed lower effectiveness against variants of concern. Here, we demonstrated that mouse plasma induced by protein nanoparticles that present rationally designed S2GΔHR2 spikes can neutralize the B.1.1.7, B.1.351, and P.1 variants with comparable titers. The mechanism of nanoparticle vaccine-induced immunity was examined in mice for an I3-01v9 60-mer that presents 20 stabilized spikes. Compared with the soluble spike, this nanoparticle showed 6-fold longer retention, 4-fold greater presentation on follicular dendritic cell dendrites, and 5-fold higher germinal center reactions in lymph node follicles. Intact nanoparticles in lymph node tissues were visualized by transmission electron microscopy. In conclusion, spike-presenting protein nanoparticles that induce robust long-lived germinal centers may provide a vaccine solution for emerging SARS-CoV-2 variants. ONE-SENTENCE SUMMARY With prolonged lymph node retention and robust germinal centers, nanoparticles elicit neutralizing antibodies to diverse SARS-CoV-2 variants.

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Tatiana Fourfouris

Papers

Mechanism of a COVID-19 nanoparticle vaccine candidate that elicits a broadly neutralizing antibody response to SARS-CoV-2 variants.

Mechanism of a COVID-19 nanoparticle vaccine candidate that elicits a broadly neutralizing antibody response to SARS-CoV-2 variants