COVID-19 vaccine : mimicking the structure and converting RBD into a nanoparticle it would generate higher levels of neutralizing antibodies

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A University at Buffalo-led research team has discovered a technique that could help increase the effectiveness of vaccines against the novel coronavirus, the virus that causes COVID-19.

Jonathan F. Lovell, Ph.D., associate professor in the Department of Biomedical Engineering at UB, is the primary investigator on the research, titled “SARS-CoV-2 RBD Neutralizing Antibody Induction is Enhanced by Particulate Vaccination,” which was published online in Advanced Materials today, Oct. 28.

COVID-19 has caused a disruptive global pandemic, infecting at least 40 million worldwide and causing more than 220,000 deaths in the United States alone. Since it began spreading in early 2020, biomedical researchers have been in active pursuit of an effective vaccine.

According to Lovell, one answer might lie in designing vaccines that partially mimic the structure of the virus. One of the proteins on the virus – located on the characteristic COVID spike – has a component called the receptor-binding domain, or RBD, which is its “Achilles heel.”

That is, he said, antibodies against this part of the virus have the potential to neutralize the virus.

It would be “appealing if a vaccine could induce high-levels of antibodies against the RBD,” Lovell said. “One way to achieve this goal is to use the RBD protein itself as an antigen, that is, the component of the vaccine that the immune response will be directed against.”

The team hypothesized that by converting the RBD into a nanoparticle (similar in size to the virus itself) instead of letting it remain in its natural form as a small protein, it would generate higher levels of neutralizing antibodies and its ability to generate an immune response would increase.

Lovell’s team had previously developed a technology that makes it easy to convert small, purified proteins into particles through the use of liposomes, or small nanoparticles formed from naturally-occurring fatty components.

In the new study, the researchers included within the liposomes a special lipid called cobalt-porphyrin-phospholipid, or CoPoP. That special lipid enables the RBD protein to rapidly bind to the liposomes, forming more nanoparticles that generate an immune response, Lovell said.

The team observed that when the RBD was converted into nanoparticles, it maintained its correct, three-dimensional shape and the particles were stable in incubation conditions similar to those in the human body.

When laboratory mice and rabbits were immunized with the RBD particles, high antibody levels were induced. Compared to other materials that are combined with the RBD to enhance the immune response, only the approach with particles containing CoPoP gave strong responses.

Other vaccine adjuvant technology does not have the capacity to convert the RBD into particle-form, Lovell said.

“We think these results provide evidence to the vaccine-development community that the RBD antigen benefits a lot from being in particle format,” Lovell said. “This could help inform future vaccine design that targets this specific antigen.”


Life Cycle, Pathophysiology, and Structure

SARS-CoV-2 has a single-stranded RNA genome of approximately 34 kilobases and a nucleocapsid of helical symmetry. The SARS-CoV-2 genome is 80% identical to the SARS-CoV and 96% to the BatCoV RaTG13.10 The integrity of the SARS-CoV particle is maintained by four proteins:

  • (i) The S protein (Spike glycoprotein) that enables the attachment of the virus to host cells followed by membrane fusion, hence, promoting the entry of SARS-CoV into the host cells;
  • (ii) the abundant M protein (membrane) that maintains the membrane integrity of the viral particle;
  • (iii) the E protein (envelope) is the smallest protein and plays a structural role and helps in assembly and budding;
  • (iv) the N protein (nucleocapsid) predominantly binds to the SARS-CoV RNA and supports nucleocapsid formation.21−27

The angiotensin-converting enzyme 2 (ACE2) is the key receptor for entry of SARS-CoV-2 in the cells of the host. Cellular proteases [human airway trypsin-like protease and cathepsins and transmembrane protease serine 2 (TMPRSS2)] control the viral entry mechanism by splitting the spike protein and initiating further penetration mechanisms.28

At least six open reading frames are present in a typical CoV genome that encode for the production of subgenomic RNAs, 16 nonstructural proteins (nsps), and structural proteins (spike, membrane, envelope, and nucleocapsid protein).29−31 The life cycle of SARS-CoV-2 explaining the entire pathophysiology mechanism is detailed in (Figure​Figure11, I).

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Figure 1
SARS-CoV-2 structure and pathophysiology. (I) SARS-CoV-2 life cycle: The viral S protein binds to the ACE2 receptor of the host. Following the entry, there is the proteolytic cleavage of the virus envelope ensuing in the release of genomic RNA in the cytoplasm, and smaller RNAs (“subgenomic mRNAs”) are made. These mRNAs are translated to several proteins (S, M, N, etc.) essential for the construction of viral assembly. S, E, and M proteins enter the endoplasmic reticulum (ER), and nucleoprotein complex formation occurs from the combination of nucleocapsid (N) protein and genomic RNA (positive strand). Formation of the complete virus particle (proteins and genome RNA assembly) occurs in ER-Golgi apparatus compartment. Virus particles are then transported and released via vesicles formation and exocytosis. (II) ACE2-RBD (S protein): A single unit of peptidase domain of human ACE2 (red) interacting with the RBD of the S protein (blue), (boxed region represents the amino acid interactions sites).

Coronavirus S proteins promote the entry of the virus into host cells and are the area of focus for various antibodies. The surface S protein (spike glycoprotein) of virions is the site for recognition and membrane fusion.32−34 The S protein (a trimer) gets cleaved into S1 and S2 subunits.

The S1 subunits contain the receptor binding domain (RBD) and are released in post-transfusion conformation.34−37 S1 directly binds to the peptidase domain (PD) of the ACE2, while S2 subunits help in the membrane fusion that is critical for viral infection.38,39 S2 contains cleavage sites and is sliced by host proteases.35,40,41

ACE2 is a dimer of the two units and accommodates the RBD in its peptidase domain. The contact between the ACE2 and SARS-CoV-2 is facilitated by polar interactions.37,38,42 An arch-shaped helix of the peptidase domain of ACE2 interacts with the loop region of the RBD of the S protein (Figure​Figure11, II).

The other helix and loops connect the antiparallel strands and coordinate the peptidase domain to the RBD. The amino acid interactions that are observed in RBD of SARS-CoV-2 and the peptidase domain of ACE2 are considered important aspects for the inhibitor design.43 It was observed that the amino acid GLN498 of SARS-CoV-2 interacts with ACE2 at the ASP38, TYR41, GLN42, LEU45, and LYS353 amino acids, while LEU455 of the virus has interaction with ASP30, LYS31, and HIS34.

More interactions include the SARS-CoV-2, PHE486 with GLN24, LEU79, MET82, TYR83, and LEU472. GLN493 showed interaction with ACE2 LYS31 and HIS34 and forms an H-bond with GLU35. The amino acid ASN501 has a similar type of interaction with ACE2 LYS353, GLY354, and ASP355, while H-bond interaction is observed with TYR41.44

The binding affinity of the RBD domain of SARS-CoV-2 and PD of ACE2 is higher when compared to SARS-CoV.43 It was reported that in SARS-CoV-2 the amino acid LYS417 showed a salt bridge interaction with ASP30 of ACE2. The positive charged patch contributed toward the electrostatic potential on the surface of RBD that is added by LYS417 in SARS-CoV-2 and absent in SARS-CoV.43,45,46

Examination of the SARS-CoV-2 virion architecture using TEM reveals a roughly spherical or moderately pleiomorphic morphology. The virion diameter is observed to have a broad distribution of 80–160 nm and a condensed mass of nucleic acid and nucleocapsid protein underneath a well-defined lipid bilayer envelop.47

TEM also reveals the nail-like shape of the SARS-CoV-2 spikes with a 7 nm wide head and a 23 nm long body. After the dissociation of the S1 subunit from the S protein, a conformational change was observed in the S2 subunit. This change from a compressed form to a nail-like shape was confirmed by different researchers and is called a postfusion state.

A three-dimensional (3D) map and two-dimensional projection images of S2 protein at the postfusion state were provided by Song et al. with negative staining EM.37 It was also confirmed from biophysical assays and Cryo-EM structure analysis that SARS-CoV-2 S protein binds at least 10 times more tightly to ACE2 host cell receptors when compared to the spike protein of SARS-CoV.39,43,48

Vaccine Development

The COVID-19 pandemic has severely impacted human lives, and desperate efforts are being employed across the world to develop safe and effective vaccines. The first vaccine candidate has already made it to human clinical trials as a result of fast-tracked development strategies and advanced vaccine technological platforms.121

Similar to what was explained earlier for therapeutic development, the significant genomic match of SARS-CoV-2 with other coronaviruses is helping the vaccine developers to facilitate designs toward the most promising vaccine candidates.

The target strategy for most of the vaccine candidates is to induce nAbs against the viral S protein, averting the ACE2-mediated host uptake. In the case of SARS-CoV vaccine development, higher nAbs titers and better protection were reported with S protein subunit vaccines when compared to any other target strategy. SARS/MERS vaccine development research suggests S protein subunits, RBD of the S1 subunit, and S protein/gene as the most preferred target sites.122−124

But still, knowledge of SARS-CoV-2 specific antigen(s) for under trial vaccine candidates is limited. The development of COVID-19 vaccine candidates is relying on several high-tech platforms including attenuated and inactivated viruses, replicating and nonreplicating viral vectors, DNA and mRNA, virus-like particles, and recombinant protein-based approaches.

Some platforms offer key advantages such as viral vectors with their strong immune response, superior protein expression, and prolonged stability, and DNA or mRNA offers antigen manipulation flexibility, whereas the recombinant protein-based development approach is easier to scale up using existing production capabilities.

In Table 1 we list some of the most advanced COVID-19 vaccine candidates that have recently moved into clinical development.125,126 Enhancing the immunogenicity using vaccine adjuvants is also under consideration to lower the viable dose and to widen the therapeutic and safety window.127,128 Compromised immune systems and high risk of disease in the elderly population also demand adjuvant strategies to improve efficacy of vaccines in this age group.129 Some licensed adjuvants developed specifically for COVID-19 vaccine are AS03 (GlaxoSmithKine), MF59 (Seqirus), and CpG 1018 (Dynavax).

Table 1

The Most Advanced COVID-19 Vaccine Candidates Recently Moved to Clinical Development

candidate, lead developer, and clinical trial identifier numberstatus and detailsdesign and characteristicsstart and estimated completion date
mRNA-1273 (Moderna) (NCT04283461)open label, open-label, phase Inovel LNP-encapsulated mRNA-based vaccine that encodes for a full-length, prefusion stabilized spike (S) protein of SARS-CoV-2March 3, 2020 to June 1, 2021
dose-ranging study to evaluate the safety and immunogenicity 45 participants
Ad5-nCoV (CanSino Biologicals) (NCT04313127)dose-escalating phase 1recombinant novel coronavirus vaccine (Adenovirus type 5 vector that expresses S protein)March 16, 2020 to December 20, 2022
study to evaluate the safety, reactogenicity and immunogenicity 108 participants
INO-4800 (Inovio Pharmaceuticals) (NCT04336410)open-label study, phase IDNA plasmid encoding S protein (intradermal administration followed by electroporation); device used: CELLECTRA 2000April 3, 2020 to November 30, 2020
study to evaluate the safety, tolerability and immunogenicity 40 participants
LV-SMENP-DC (Shenzhen Geno-Immune Medical Institute, China) (NCT04276896)multicenter trial, phase I/IIdendritic cells modified with engineered lentiviral vector expressing synthetic minigenes based on selected conserved and critical genomic structural and protease protein domainsMarch 24, 2020 to December 31, 2024
study to evaluate safety and efficacy of this LV vaccine (LV-SMENP) 100 participants
pathogen-specific artificial antigen presenting cell (aAPC) (Shenzhen Geno-Immune Medical Institute, China) (NCT04299724)open-label study, phase 1aAPCs with lentivirus modification including immune modulatory genes and the viral minigenes based on domains of selected viral proteinsFebruary 15, 2020 to December 31, 2024
study to evaluate the safety and immunity 100 participants
ChAdOx1 nCoV-19 (COV001) University of Oxford, England (NCT04324606)single-blinded, randomized, multicenter study, phase I/IIadenovirus vaccine vector (nonreplicating viral vector encoding the spike protein of SARS-CoV-2), vaccine will be administered intramuscularlyApril 23, 2020 to May 2021
study to evaluate the efficacy, safety, and immunogenicity; anticipated 1112 participants (4 study groups)
BNT162 Biontech/Fosun Pharma/Pfizer (NCT04368728)randomized, placebo-controlled, observer-blind, dose-finding, and vaccine candidate-selection study, Phase I/IILNP formulation-based mRNA vaccine (four different vaccine candidates, each representing different target antigens). Two candidates include a nucleoside-modified mRNA, one includes a uridine containing mRNA (uRNA), and one candidate utilizes self-amplifying mRNA (saRNA)April 29, 2020 to March 8, 2023
Study to evaluate the safety, tolerability, immunogenicity, and potential efficacy
BNT162 Biontech (NCT04380701)two-part, dose-escalation trial, A multisite phase I/II,April 20, 2020 (starting date)
investigating the safety and immunogenicity using different dosing regimens

Role of Nanotechnology

The COVID-19 crisis also demands an urgent analysis of all the available nanotechnology tools. While nanomedicine strategies are in use for the design of the vaccine carriers, there are not enough other nanotechnology approaches being explored to tackle the current outbreak. This manuscript attempts to systematically present the current status of nanotechnology use in therapeutics and vaccine development. Therapeutic development and challenges against SARS-CoV-2 infection are not so different from other infectious diseases as well as oncology research.130 Similarly, the vaccine development holds significant commonalities with strategies explored against previously known SARS, MERS coronaviruses.121,131 Hence, it is worth revisiting these closely related therapeutic/vaccine strategies and associated nanotechnology use, and this way our aim is to design “repurposed nanotechnology” to fast-track the current research (Figure​Figure44).

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Figure 4
Nanomedicine strategies for COVID-19 therapeutics and vaccine development.

Strategy 1. Nanocarrier Selection to Bypass the Conventional Limitations of a Drug Candidate

For example, relatively safe antibody-drug conjugates of highly toxic auristatins are approved for the treatment of hematological cancers. A major limitation for use of these conjugates is the very low tolerable drug payloads. In order to solve this problem, polymeric nanoparticles were developed with a high auristatin payload to achieve efficient and safe tumor suppression.149 Similarly, formulating poly(ethylene glycol)-poly(lactide)-based nanoparticles loaded with Aurora B kinase inhibitor revealed increased efficacy and reduced toxicity as compared to its free form which produced intolerable side effects in phase II clinical trials.150 A well-known limitation of nucleic acid (e.g., RNAi) drug candidates is their systemic circulation instability and the prerequisite of their intracellular delivery.151−153 Lipid nanoparticles (LNPs) carrying siRNA are an example of a nanotechnology platform (Onpattro) used to prevent this systemic degradation along with the benefit of liver-targeting.154 Lipid-coated mesoporous silica nanoparticles, used to deliver a highly hydrophilic and unstable antiviral molecule ML336 (chemical inhibitor of Venezuelan equine encephalitis virus), showed enhanced circulation time and biocompatibility of the ML336 in vivo.155 Sago and co-workers reported a high-throughput method (named FIND) to screen LNPs that can bypass liver and deliver functional mRNA to cells in vivo.156 Broadly, the relationship between nanoparticle structure and mRNA in vivo delivery targets may be elucidated from this study. Nanocarriers are used to prevent the systemic immunotoxicity of the protein-based drugs and promote immuno-oncology therapeutics.157

Strategy 2. Chemically Alter/(Re)engineer Drugs

Drug molecules are altered to improve their compatibility with a particular class or type of nanocarriers, rendering this a more generic approach for drug candidates with similar physicochemical properties.158,159 Lipid bilayer nanocarriers (liposomes) are preferred nanocarriers for pH gradient-based remote loading of amphiphilic and ionizable drugs.160

The hydrophobicity of doxorubicin was chemically modified to increase its compatibility with poly(lactic-co-glycolic acid) nanoparticles.161

Another approach of interest here is the synthesis of “prodrugs” to ensure their compatibility and incorporation within particular nanocarriers along with their controlled and localized release characteristics. Anti-HIV prodrugs of antiretroviral (ARV) candidate cabotegravir has been synthesized by functionalizing fatty acid esters (with variable carbon lengths), followed by its poloxamer coating to get stable nanocrystal formulations.

In vivo pharmacokinetic studies in mice and rhesus macaques revealed significantly improved effectiveness of cabotegravir, showing prolonged drug release and pharmacokinetic parameters.162 Another ARV prodrug strategy for highly aqueous-soluble emtricitabine (using bioreversible carbonate and carbamate masking groups) shows sustained prodrug release predicted by in vitro to in vivo extrapolation modeling.163

Wei and co-workers have reported the use of cholesterol-modified hydroxychloroquine (Chol-HCQ) loaded liposomes that lowered the dose and toxicity of hydroxychloroquine and also inhibited the proliferation of rat lung fibroblasts, thereby, reducing pulmonary fibrosis.

This strategy can be adopted to have dual benefits in SAR-COV-2 patients, which show viral load and pulmonary fibrosis.164 Using a hydrolyzable ester linkage, an irinotecan (hydrophilic) and chlorambucil (hydrophobic) anticancer drug–drug conjugate has been synthesized.165

Nanoparticles synthesized by self-assembly of this amphiphilic drug–drug conjugate shows prolonged systemic retention, tumor tissue accumulation, and increased cellular uptake. Hydrolyzable ester linkers conjugated to docetaxel permitted its effective loading and release from core-cross-linked polymeric micelles to provide a high therapeutic efficacy against breast and ovarian cancer.166 Another similar prodrug “fatty acids conjugated to cabazitaxel” with PEG-lipid results in self-assembled nanoparticles showing reduced systemic toxicity and superior anticancer efficacy.167

Strategy 3. Nanomedicine for Combination Drug Therapeutics

Combination drug therapy is another possibility for treatment of COVID-19, offering several advantages such as lower dosages of the individual drugs causing fewer side effects, achieving multiple and complimenting therapeutic targets, and reducing the likelihood of resistance development. Several such combinations for novel coronavirus treatment are documented in the WHO landscape information (Table 2).

Nanocarriers are also intrinsically very useful for the delivery of multiple drugs with different physicochemical properties promising the full potential of combination therapies.168,169 The flexibility offered by a variety of nanomaterials and fabrication techniques enables the design of drug combinations loaded in nanocarriers with excellent control in preserving synergistic drug ratios, overlapping pharmacokinetics, and reducing combination allied side-effects.170

Various nanocarrier strategies are described for the co-encapsulation of both hydrophobic and hydrophilic drugs (Figure​Figure55), achieving the sequential release of two drugs, ratiometric loading and controlled release of three drug candidates, codelivery of RNAi/plasmidDNA + chemotherapeutics, and codelivery of siRNA + micoRNA.171−179

A nanosuspension of LNPs loaded with three ARVs drugs (two hydrophobic: lopinavir and ritonavir and one hydrophilic: tenofovir) has been formulated to overcome the lymph node drug insufficiency of the oral combination of these drugs. This nanoparticle formulation showed long-lasting plasma drug profiles and better lymph node drug levels in the macaques in vivo model.180

A liposomal nanoformulation (Vyxeos) coloaded with a fixed combination of anticancer drugs daunorubicin and cytarabine was recently approved by US-FDA to treat acute myeloid leukemia in adults. Multidrug-loaded (antiretrovirals, latency reactivating agents, and drug abuse antagonist) pegylated-magneto-liposomal nanoformulations have shown in vitro and in vivo BBB transmigration with significant anti-HIV activity in primary CNS cells.

This multifunctional nanotherapeutic strategy can be applied to target SARS-COV-2 that has migrated to the CNS.181 However, drug combination regimens are a standard of care for a wide range of therapeutics, but optimizing their nanoformulations is an uphill task. These optimization challenges include analyzing the interaction between two or more drugs, balancing the antagonism/synergy/toxicity, and controlling the release profile of individual drugs.182,183

High-throughput screening methods are required to understand the biological interactions and discover any kind of synergism that is present. I

n vitro screening methods to determine ideal drug ratios demand an upgrade to mimic the 3D microenvironment of the target human tissue.184 Preclinical animal models are critical to accelerating the clinical translation, but a disparity between the model of disease in animals and human disease is the major reason for the failure of the study.185 Nanomedicine scientists should take advantage of advanced drug development tools, screening technologies, bioinformatics, animal models, etc. to investigate and validate nanoparticle combination therapeutics.

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Figure 5
Nanocarrier platforms utilized for combination drug therapeutics.

Table 2

Combination Drug Treatments Proposed for COVID-19

combination descriptioncandidatesstatus
protease inhibitorsritonavir + lopinavirunder trial of COVID-19186,187
non-nucleoside reverse transcriptase inhibitor + nucleotide reverse transcriptase inhibitoremtricitabine + tenofovirunder trial of COVID-19188
nucleoside inhibitor + protease inhibitorribavirin + ritonavir/lopinavirclinical study of SARS189 NCT00578825
antiretroviral protease inhibitor + cobicistat (to improve bioavailability and t1/2)darunavir + cobicistatunder trial of COVID-19189
antiviral + type I interferons – signaling proteins made and released by host cells during viral infectionsIFN (α, β, IFNα2a or rIFN-α2b or IFN-β1a) + ribavirinclinical study of SARS,190 MERS191,192
interferons – signaling proteins made and released by host cells during viral infections + antiviral + steroid hormonesIFN + ribavirin + steroidsclinical study of SARS193
protease inhibitor + proteins made and released by host cells + antivirallopinavir + ritonavir + IFN + ribavirinclinical study of MERS194
type I interferons – signaling proteins made and released by host cells during viral infections + immunosuppressantIFN-β1a + mycophenolate mofetilclinical study of MERS195
protease inhibitors + proteins made and released by host cellslopinavir + ritonavir + IFNβ1bclinical study of MERS193
synthetically developed recombinant type-I interferon + steroid hormonesIFN alfacon-1 + corticosteroidsclinical study of MERS196

Vaccine Delivery

The apparent similarity of SARS-CoV-2 with other viruses (mainly SARS-CoV and MERS-CoV), along with the previous knowledge of their protective immune responses, is of great help to successfully develop COVID-19 vaccine.121,131

Nanoparticles can be loaded with a wide range of antigenic moieties (by physical entrapment or chemical conjugation), and a correct antigenic display makes it a highly relevant alternate in vaccinology when compared to conventional approaches.197−199

In addition to safeguarding the native structure of the antigen, nanoparticles also improve the delivery and presentation of antigens to the antigen-presenting cells (APCs). The key advantages of vaccine nanocarriers are their nanosize, since many biological systems such as viruses (including SARS-CoV-2) and proteins are also nanosized.

Nanoparticles can be administered by oral and intranasal routes and subcutaneous and intramuscular injections, offering a key advantage by overcoming tissue barriers and targeting key locations such as lymph nodes, penetrate mucosal, and epithelial barriers (airway, nasal, gastrointestinal, etc.).200−202

Previous reports have suggested that both humoral and cell-mediated immunity performs a protective role in the SARS-CoV infection.203,204 Nanoparticles have shown their ability to target both adaptive (T cells, B cells) and innate immune systems (macrophages, monocytes, neutrophils) at the cellular level.

Modulating APCs using nanoparticles could be very important, particularly for COVID-19 vaccine strategies.205,206 The ability of nanoparticles to deliver antigen to dendritic cells (DCs) by enhancing antigen presentation and several other mechanisms can promote T cell immunity.207

Smith et al. explained various nanoparticle-based mechanisms to alter the immune response induction in (Figure​ 6).208

To improve the efficacy and safety of the vaccine approach, a big advantage presented by nanoparticles is their ability to deliver molecular adjuvants, and, in some cases, nanomaterials themselves possess an intrinsic adjuvant property for the loaded antigens. The WHO reports (dated May 27, 2020) various preclinical stage nanoparticle-based vaccine candidates (Table 3).126

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Figure 6
Nanoparticle-based immune response modulation. (a) Antigen delivery by nanoparticles (size-dependent penetration and tissue or organ targeting). (b) Depot effect provides a prolonged and sustained release of stable antigen. (c) Repetitive antigen display as a result of the antigen presentation on the nanoparticle surface assists the receptor activation on APCs and B cells and (d) cross presentation of the antigen delivered by the nanoparticles (cytosolic delivery) to activate antigen specific CD8+ T cells. Antigen-presenting cell (APC); dendritic cell (DC); endoplasmic reticulum (ER); B cell receptor (BCR); T cell receptor (TCR). Adapted with permission from ref (208). Copyright 2013 Springer Nature.

Table 3

Nanoparticle-based vaccine candidates in preclinical evaluation Mentioned in the DRAFT Landscape of COVID-19 Candidate Vaccines (as of May 27, 2020)

platformtype of candidate vaccineadeveloper
protein subunitnanoparticle vaccine + matrix M (adjuvant) (based on recombinant SARS-CoV-2 glycoprotein)Novavax
peptide antigens formulated in LNPs formulationIMV, Inc.
nanoparticle vaccine (recombinant protein) (S protein and other epitopes based)Scientific Research Institute of Vaccines and Sera, Saint Petersburg
Nanoparticle vaccineLakePharma, Inc.
RNALNPs formulation of mRNASanofi Pasteur/Translate Bio
LNPs-encapsulated mRNA cocktail encoding VLPFudan University/Shanghai JiaoTong University/RNACure Biopharma
LNPs-encapsulated mRNA encoding RBD
LNP-encapsulated mRNAUniversity of Tokyo/Daiichi-Sankyo
liposome- encapsulated mRNABIOCAD

aLNPs: lipid nanoparticles, VLP: virus-like particle.

Nanomedicine Approach for COVID-19 Vaccine
Overall vaccine history indicates major successes against acute infectious diseases, where naturally developed immunity (majorly by neutralizing antibodies) provides enduring protection in a section of patients.

One of the bigger challenges in the COVID-19 vaccine research is to identify approaches that stimulate both the T cell and B cell immunity against this virus.

Another challenge is the necessity of accelerating the development of precise “next-generation” vaccine strategies that may also address specific population subgroups or individuals with compromised immunity.209 Smart strategies to develop nanocarrier-based COVID-19 vaccines are equally important and sometimes overlapping when paralleled to nanocarrier-based therapeutics.121,210

The nanovaccine strategy also requires a strong focus on the cellular presentation of the selected antigen, along with the selection of appropriate nanocarrier/nanomaterial to induce complimenting immunomodulatory effects. The following section highlights the rational design of nanocarrier-based vaccines with two strategies.

Strategy 1. Antigen-Dependent Nanocarrier Selection

Loading antigens inside or on the surface of nanocarriers is dependent on several factors including the antigen’s physicochemical characteristics, biological stability, target sites, and required immunogen release rate. Physical adsorption of antigens on nanoparticles is based on its surface charge and noncovalent hydrophobic interactions.

Antigens with an amphoteric nature are most suitable for adsorption or surface immobilization on nanocarriers such as chitosan and dextran sulfate-based polymeric nanoparticles, inorganic nanoparticles (such as AuNPs), and carbon nanotubes.211−214

Antigen release in such cases is predesigned based on the properties of the biological environment like pH, ionic strength, temperature, etc. Encapsulation and matrix entrapment of the antigens within a nanocarrier is another technique used to prevent its biological degradation.

Poly(lactide-co-glycolide) (PLGA) nanoparticles are ideal for encapsulating antigens and provide controlled or extended biological release.215 These nanoparticles are effective preclinically in carrying antigens such as HBsAg, malaria antigens tetanus toxoid, Listeria monocytogenes antigens, and Bacillus anthracis spores, generating prolonged cellular and humoral immune response.216

The mRNA-based COVID-19 vaccine is already under clinical trial employing LNPs as a carrier. Naked mRNAs are sensitive to the degradation by extracellular RNases, thus formulating its delivery vehicle is essential.217,218 Further, these mRNAs entail their cell-specific receptor recognition and lipid membrane penetration.

Cytosolic presence of exogeneous mRNA then triggers the cellular machinery for its translation into fully functional protein.219 LNPs are virus-sized (80–200 nm) particles synthesized by the self-assembly of an ionizable cationic lipid.220 They possess the ability to deliver mRNA efficiently into the cytoplasm, as demonstrated by several studies.

Sustained-release kinetics of mRNA expression and thus protein translation can be achieved by opting for intramuscular and intradermal routes, providing high antibody titers, and both B cells and T cells immune responses.138 Different nanoparticles of these cationic lipids (such as 1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP) or dioleoylphosphatidylethanolamine (DOPE)) are formulated with subtle modifications (such as cationic lipids + cholesterol nanoparticle, cationic lipids + cholesterol + PEG-LNP), where cholesterol is used to increase stability and PEG-lipid to increases the formulation half-life.

Apart from LNPs, other mRNA nanocarriers include protamine (cationic peptide) nanoliposomes (∼100 nm), PEG-lipid functionalized dendrimer nanoparticles (∼200 nm), positively charged oil-in-water (O/W) cationic nanoemulsion (∼120 nm), polyethylenimine nanoparticles (100–300 nm), and cationic polymer (chitosan) nanoparticles (300–600 nm).221−223Figure​Figure77, I–V represents the mRNA vaccines delivery methods and nanocarriers commonly used.

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Figure 7
Major delivery methods for mRNA and DNA vaccines. (I–V) Nanocarrier for mRNA delivery, (VI) nanocarriers for DNA delivery, and (VII) electroporation technology for the intradermal delivery of DNA vaccines.

Similar to mRNA, naked DNA also experiences systemic degradation by nucleases and incomplete delivery to specialized immune cells. Nanocarriers based on cationic lipids (quite similar to mRNA deliver), synthetic and natural polymers, and inorganic particles are proposed for DNA-based vaccine formulations.

Polymeric nanocarriers encapsulating DNA prevent biological inactivation and provide controlled release and targeted cell delivery. PLGA nanocarriers are the most studied polymeric platform for DNA vaccine development, showing improved systemic antigen-specific antibody responses.224,225

To improve the efficiency of DNA loading and systemic protection, functional or composite PLGA nanoparticles (such as cationic glycol-chitosan + PLGA, PLGA+ polyethylenimine (PEI)) are explored (Figure​Figure77, VI).226,227 Other well documented cationic polymer-based nanocarriers for DNA vaccine design are chitosan nanoparticles and PEI nanoparticles/complexes.

Use of PEG functionalization on nanoparticle surfaces is quite common to introduce stealth characteristics (it renders them undetectable to phagocytes and prevent reticuloendothelial system clearance), prevent nonspecific protein interaction, reduce systemic toxicity, and improve stability.228,229

To improve the delivery of mRNA/DNA across the cell and nucleus membrane, physical technologies such as the gene gun and electroporation are being explored. Currently, vaccine development is taking advantage of electroporation technology to induce pores in the cell membrane to insert the DNA (Figure​Figure77, VII).215,230,231

Surface electroporation DNA coated-PLGA nanoparticles have shown efficient cellular delivery to elicit B cell and T cell response in pigs.231 The clinical future of such portable electroporation technologies is now apparent in the race of COVID-19 vaccine research.232 An ongoing clinical trial (NCT04336410) is using a DNA plasmid encoding SARS-CoV-2 S-protein as a vaccine candidate for intradermally administration using an electroporation device (CELLECTRA 2000).

Strategy 2. Vaccine Adjuvant Nanoparticles

Vaccine adjuvants nanoparticles (VANs) are considered to improve the overall efficacy and safety of the generated immune response. Particularly in COVID-19 pandemic situation, vaccine adjuvants are critical to reducing the required antigen dose (dose-sparing), permitting the production of more units and making it available to larger population.233

Among many preclinical COVID-19 vaccine candidates, five protein subunit vaccine candidates are reported using a combination of antigen and adjuvant. NVX-CoV2373, a nanoparticle vaccine (recombinant SARS-CoV-2 glycoprotein based) with an adjuvant (matrix M) is now expected to move into clinical trials soon.

Hence, it is important to discuss the possible strategies employed by VANs in other research studies that could help to improve current COVID-19 vaccine designs. Informing specific immune cells to mount a protective immune response against a specific antigen is the basic mechanism of VANs designed to improve efficacy (by serving as immunity promoting cues, also called as “danger signals”).234

In the case of a virus, these danger signals are characterized as PAMPs and damage-associated molecular patterns (DAMPs) derived from the same virus.235 PAMPs and DAMPs are recognized by specific receptors called pattern recognition receptors (PRRs).

An example of such receptors is Toll-like receptors which are expressed by immune cells to upregulate robust T and B cell priming by releasing inflammatory cytokines.236−238 Adjuvants improving safety provide a kind of counter-regulatory signal instructing the immune system to develop a tolerance for incoming antigens. VANs can either act as a nanocarrier for molecular adjuvants or have an inherent physicochemical property to stimulate pro- or anti-immunity pathway.211

VANs are designed to tackle the limitations related to the conventional delivery of molecular vaccine adjuvants such as rapid bloodstream clearance, systemic distribution, and lack of immune cell targeting as well as lack of antigen-adjuvant colocalization. Polymeric nanoparticles encapsulating small molecules are employed for lymphoid organ-specific delivery with controlled exposure.

The dose-sparing effect is reported with antigen and cyclic dinucleotide (adjuvant; agonist of INF gene stimulator) coloaded liposomal nanoparticles showing safe and uncompromised immune responses.239 Lymph node targeting of VANs is an established strategy to achieve a significantly high-dose-sparing effect, whereas DCs targeting VANs may enhance its adjuvanticity.

In vivo study results against infectious challenge has shown PLGA and calcium phosphate nanoparticles co-encapsulating both antigen and adjuvants to improve efficacy by enhancing antigen uptake, APC activation, and higher antibody titers.240−242 In other studies, co-encapsulation strategy allows the colocalization of antigen and adjuvant in endosomal/phagosomal compartments fostering the activation of DCs and triggers robust cross-presentation and T cell priming.243,244

Synergized activation of APCs and prolonged antibody response was observed with the codelivery of TLR4 and TLR7 small molecule adjuvants using PLGA nanoparticles.245 VANs (including PLGA, AuNPs) are also employed to codeliver self-antigens or immunoregulatory drugs as adjuvants to induce antigen-specific peripheral tolerance of autoreactive T cells and block any serious autoimmune response.246−251

Nanoparticles because of their intrinsic adjuvanticity (by activating complement system, inducing autophagy and activation of inflammasome) are also considered as VANs.252−258 Surface chemistry and hydrophobicity of nanoparticles along with other physicochemical properties are capable of electing these adjuvanticity mechanisms intrinsically.253,259,260

Hydroxyl groups dependent compliment system activation followed by cellular immunity enhancement is reported with pluronic-stabilized poly(propylene sulfide) nanoparticles.259 Antigen conjugated alumina nanoparticles have been reported to enhance cellular and humoral immune responses as a result of autophagy induction in DCs, fostering antigen cross-presentation to T cells.255

Gold and PLG nanoparticles are reported to activate NALP3 inflammasome in DCs, resulting in improved adjuvanticity similar to an alum-mediated adjuvanticity mechanism.261,262 Increased side-chain hydrophobicity of poly(γ-glutamic acid) nanoparticles displayed augmented uptake and DCs activation.263 Similarly, AuNP surface hydrophobicity can increase the expression of inflammatory cytokines both in vitro and in vivo.(264)

Vaccine adjuvants have been used to increase the efficiency and the antibody responses of vaccines in the elderly. They comprise the most vulnerable groups of the population and have the highest case-fatality rate of the COVID-19 disease.265,266 Aging is associated with continuous chronic subclinical systemic inflammation (inflamm-aging) and acquired immune system weakening, that is, immune senescence.267

Immune senescence is flagged with a significant decrease of immunoglobulin M, interferon levels, T-cell count, rate of cell division and proliferation, chemotaxis of neutrophils, and phagocytosis.267,268 O/W emulsion, immune stimulating complexes, cationic and anionic liposomes, virosomes, and microparticles are among the various adjuvant’s technologies developed to improve the influenza vaccination in the older population.129

Squalene-based O/W emulsion adjuvants MF59 and AS03 have been licensed for influenza vaccines meant for the elderly.269,270 A liposome-based adjuvant AS01 is another key example of licensed technology developed for the herpes zoster subunit vaccine aiming old age population (70 years or above).271,272

Addition of adjuvants has shown a decreased risk of pneumonia and influenza in clinical trials and can hence play a significant role in regulating the immune system responses of the elderly, which further can be tuned for COVID-19 vaccine progress.129

Scope of Miscellaneous Nanotechnology Approaches

The scope of nanotechnology for COVID-19 therapeutics and vaccine research is not limited to conventional therapeutic and vaccine designs. Several other approaches including advanced nanomaterial and biomimetic approaches represent good potential usage in a COVID-19-like outbreak.

Szunerits and co-workers investigated the prospect of functionalized carbon quantum dots (CQDs) to inhibit the human coronavirus (HCoV-229E) infection (Figure​Figure88, I–III).273 CQDs of different sizes (<10 nm), surface potential (−7.9 to −39.2 mV), and functionalities were explored as inhibitors of Huh-7 cells (host cell) infection by HCoV-229E, and they showed a concentration-dependent virus inactivation.

Boronic acid-modified CQDs showed the maximum efficacy with an EC50 value of 5.2 ± 0.7 μg mL–1, illustrating the significance of boronic acid functionality to inhibit the early stage interaction of viral S-protein receptor with the host cell membrane. Cell membranes mimicking nanodecoys are an interesting choice to fool and trap pathogens.

These biomimetic nanodecoys include liposomal formulations, reconstituted lipoproteins, and cell-membrane nanostructures.274 Targeted surface engineered liposomes with antiviral antibodies constitute an effective strategy to provide protection against the infection of coxsackie A-21 virus.275 Similarly, mosquito host-cell-membrane-wrapped nanodecoys are employed to trap the Zika virus and effectively prevent host cell infection.276

Lauster and co-workers presented an interesting approach employing an influenza A virus spike-protein (hemeagglutinin, HA) mimicking a multivalent binder that can bind to the virus in a distinct multivalent mode and inhibit its infection.277 Normally a multivalent manner binding is observed between the viral trimeric HA and the terminal sialic acid (Sia) residues of the host cell’s surface glycans.

Structurally defined presentation of Sia ligands are functionalized on a compact symmetrical 3D scaffold, that is, a bacteriophage capsid resembling a host cell and targeting the trimeric HA ectodomain of the virus. K16 residues present in the protein coat of symmetric icosahedral bacteriophages Qβ capsid (∼25 nm diameter) provided an ideal platform to anchor Sia ligands with a varied length of the linker to mimic the HA trimer’s binding sites (Figure​Figure88, IV).

Cryo-electron tomography showed these Qβ capsids covering the A/X31 virus (H3N2 subtype) envelop and significantly blocking the host cell interaction (Figure​Figure88, V). Phage capsid nanoparticles have shown the potential to inhibit virus infection during in vitro, ex vivo, and in vivo studies (Figure​Figure88, VI).

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Figure 8
Miscellaneous nanotechnology approaches. (I) Hydrothermal synthesis of functionalized CQDs, as an inhibitor of HCoV-229E – Huh-7 cells (host cell) binding and further infection. (II, A) Concentration-dependent viral inhibition with CQDs (1–4) and (II, B) EC50 for CQDs (3, 4) and CQD-3 + mannose (incubation ratio of 2:1, 4 °C for overnight). (III, A) Concentration-dependent viral inhibition with CQDs (5–7), (III, B) EC50 for CQDs (5 and 6), and (III, C) EC50 for CQD (5 and 6) + mannose (incubation ratio of 2:1, 4 °C for overnight).273 Adapted with permission from ref (273). Copyright 2019 American Chemical Society. (IV) Qβ phage capsid as a multivalent and high affinity influenza A virus binder (a) structural resemblance between the Sia attachment sites (present on the capsid) and the HA-Sia binding pockets (on A/X31 virion), (b) functionalization procedure of Qβ phage capsids to introduce Sia ligands, (c) haemagglutination inhibition assay against different HA units (KiHAI, in black) and the apparent dissociation constants (KD, app in green) measured by microscale thermophoresis against A/X31 virion. (V) Cryo-TEM images showing diverse Qβ capsids covering the A/X31 envelop and blocking the host interaction: (a) Qβ[Gal3] with no virus interaction, (b) Qβ[Sia1] decorated with virus, (c) a 3D model showing multiple Qβ[Sia1] capsids (purple) attached with a single virion (yellow envelop), HA (cyan), and neuraminidase (NA, green) (scale for a, b: 100 nm and c: 25 nm). (d–f) Red circle indicates specific binding incidents of Qβ[Sia1] capsid to HA trimers and (g) binding events of viral HA ectodomains to discrete Qβ[Sia1] capsid presented with collection of 20 images (scale for d–g: 20 nm). (VI) Inhibition study of influenza A virus strains by Qβ[Sia1] capsid. (a) Confocal images showing Qβ[Sia1] capsids inhibiting viral infection (A/Pan/99) of A549 cells (infected cells: yellow, nuclei: blue and scale: 40 μm). (b) The percentage of infected cells (using viral nucleoprotein signal) with different treatments and a control. (c) Inhibition study of A/X31strains infection using Qβ[Sia1] phage capsid and its cell toxicity in the absence of virus (Qβ[Hpg] is used as control). (d) Bar graph showing the cell supernatant titers of A/X31 and A/Pan/99 viruses after the Qβ[Sia1] and oseltamivir carboxylate (OC) treatment (with no treatment as control, PFU: plaque forming units). (e) Ex vivo experiment showing the potential of Qβ[Sia1] capsid to inhibit the A/Pan/99 virion infection in human lung tissue. (f) In vivo experiment in BALB/c mice shows the potential of Qβ[Sia1] capsid to protect the A/X31 infections. Adapted with permission from ref (277). Copyright 2020 Springer Nature.

reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325519/


More information: Wei‐Chiao Huang et al. SARS‐CoV‐2 RBD Neutralizing Antibody Induction is Enhanced by Particulate Vaccination, Advanced Materials (2020). DOI: 10.1002/adma.202005637

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