Towards novel nano-based vaccine platforms for SARS-CoV-2 and its variants of concern: Advances, challenges and limitations

Vaccination is the most effective tool available for fighting the spread of COVID-19. Recently, emerging variants of SARS-CoV-2 have led to growing concerns about increased transmissibility and decreased vaccine effectiveness. Currently, many vaccines are approved for emergency use and more are under development. This review highlights the ongoing advances in the design and development of different nano-based vaccine platforms. The challenges, limitations, and ethical consideration imposed by these nanocarriers are also discussed. Further, the effectiveness of the leading vaccine candidates against all SARS-CoV-2 variants of concern are highlighted. The review also focuses on the possibility of using an alternative non-invasive routes of vaccine administration using micro and nanotechnologies to enhance vaccination compliance and coverage.


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Oxford/AstraZeneca vaccine) [36]. Some of the traditional and novel vaccine platforms are described 2 below. Table 1 lists some of current SARS-CoV-2 vaccines which are approved for emergency use or

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Aluminum salts are the most common adjuvants, utilised in nearly 80% of human adjuvanted vaccines.

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The nano-based platforms may be protein-based or gene-based vaccines.

Protein-based vaccines
host cells, resulting in a strong immune response. The advantages of gene-based vaccines over 1 conventional vaccine platforms are: low production costs, rapid and easy production, better safety 2 profile, specificity, and potent cell-mediated protective immunity [118]. However, the major concern 3 about such vaccines is the fact that they have never been approved for use in humans before the 4 COVID-19 pandemic. Moreover, they usually require an ultra-cold-chain process for storage and 5 distribution. Despite the high protective efficacy of such vaccines, many side effects are also induced [3].

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Vector-based vaccines may be bacterial vector-or viral vector-based vaccines. Some 16 of the leading vaccine candidates are shown in Table 1.

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Bacterial vector-based vaccines use a safe, non-pathogenic bacteria as a vector or gene delivery 19 vehicle [3]. The Symvivo's COVID-19 vaccine, bacTRL-Spike, is an oral or intravenous DNA vaccine that 20 uses a lactic acid bacteria (LAB) as a vector (Table 1). Currently, the vaccine is in phase 1 clinical trials 21 [8]. The bacTRL-Spike technology can be lyophilised to enhance the stability of the vaccine [119].

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Viral vector-based vaccine uses a viral vector to deliver genes that encode a specific antigen of 24 the pathogen to the host cell. These are safe replication-defective platforms [120]. The vector is an 25 infectious attenuated virus such as adenovirus (Ad), adeno-associated, measles, or pox virus, genetically 26 modified to act as a carrier [121]. The viral vector mimics the real viral infection and uses the host cell to 27 manufacture a non-harmful piece of SARS-CoV-2 such as S protein, to trigger robust cellular and 28 humoral responses. Although these vaccines are immunogenic and well tolerated in healthy adults, they 29 have many drawbacks, including; potential risk for inflammatory adverse reactions, risk of infection, 1 ( Table 1). The Ad type-5 (Ad5) vector was previously used in many vaccines such as Ebola vaccine 2 (Ad5-EBO). However, in some cases, it may be unacceptable due to pre-existing immunity and 3 intolerable high-dose immunisation [106]. Accordingly, the use of rare human Ad serotypes or 4 nonhumans Ads, in combination with other vaccine platforms for immunisation, will be the typical  The mRNA-based vaccine is a promising weapon against many viral diseases and cancer. The 10 mRNA-based SARS-CoV-2 vaccines use the mRNA encoding the target antigen, instead of weakened or 11 dead virus, to function as a platform for the generation of many S protein copies (Fig. 3). These vaccines

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There are many mRNA-based CONID-18 vaccines currently undergoing clinical trials (    contains tromethamine, which can potentially lead to immune hypersensitivity reactions [136].

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DNA vaccines contain bacterial plasmid DNA that encodes one or more viral antigens [137].

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Inside the nucleus of the host cell, they are converted into mRNA which translated into the S protein; 18 eliciting both humoral and cell-mediated immunity. DNA vaccines also have some advantages,

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including easy production, safe handling, stability, no risk of infection, and long-term persistence of 20 immunogens [138]. Additionally, the DNA molecules can be freeze-dried for long-term storage [3,107].

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DNA vaccines may also be administered orally, intradermally, and subcutaneously (SC). However, the 22 drawbacks of these platforms cannot be ignored, such as potential risk of genetic integration, poor 23 immune responses in humans, and toxicity induced by repeated doses [138,139]. The major concern is 24 the possibility of plasmid DNA NPs transfecting nontarget cells, such as brain cells, when administered 25 by inhalation [138,139].  ZyCoV-D, GLS-5310, CORVax12, and COVIGEN ( Table 1). There is no DNA vaccine approved by 29 WHO for human use to date. India approved the first 3-dose DNA plasmid vaccine to administer to plasmid that encodes the S protein of SARS-CoV-2 to prime a strong immune system [140]. This needle-1 free vaccine is administered using the PharmaJet ® technology. Currently, the vaccine is in phase 3 2 clinical trials [8]. ZyCoV-D vaccine is 67% protective against symptomatic COVID-19 [140].

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Liposomes are nanoscale lipid vesicles which are considered as a good vehicle for both 3 hydrophilic and lipophilic antigens [146]. They consist of one or more phospholipid bilayers enclosing 4 an aqueous internal core. This unique structure of liposome allows the encapsulation of both lipophilic 5 and hydrophilic antigens, respectively [146]. The composition of liposomes can be modified to stimulate 6 the desired immune response and adjuvant characteristics. It can produce a sustained release of the 7 encapsulated antigens and create a depot action to facilitate long-term retention [147]. The surface of the 8 liposome can be modified to enhance mucosal adherence, lymphatic drainage, immune cell targeting 9 capacity, stability, and encapsulation efficiency [146,148]. The drawbacks of a liposomal vaccine  encoding the anionic S1 subunit of the SARS-CoV-2 virus, employing the thin film hydration technique 20 and using amphiphilic monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotide (for TLR 9) as 21 adjuvants [150].

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LNP is a liposome-like structure, which is suitable for encapsulation of nucleic acid cargos 25 (DNA and RNA). The mRNA-LNPs are fully synthetic, biocompatible, and manufactured without 26 handling of living cells [151,152]. Furthermore, the high production capacity of these vaccines is more 27 appropriate for a pandemic than conventional vaccine production.

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Moderna and Pfizer-BioNtech mRNA vaccines rely on LNPs platform to carry mRNA that 30 encodes the S glycoprotein into the host cells. Both vaccines have shown good efficacy and safety in 1 of LNPs elicited a strong immune response (95%) against the S glycoprotein of SARS-CoV-2 [106]. LNPs 2 used in these vaccines are made of three components: (1) a PEGylated lipid to minimize opsonization in 3 vivo, improve stability, and reduce clearance of LNPs from the blood, (2) cholesterol to support particle 4 formation, and (3) phospholipid distearoylphosphatidylcholine (DSPC) [154]. The molecular weight of 5 PEG is only 2000 [155]. Adjuvants are not required in these types of vaccines because both mRNA and 6 LNPs possesses potent immunostimulatory properties [156]. Another two LNPs-based vaccines are 7 developed by Novavax, Gaithersburg, Maryland and CureVac N.V, Germany which also encode the S 8 protein ( Table 1).

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Novel LNPs vaccines encoding S protein of SARS-CoV-2, are in development. These novel 10 platforms are LNP-nCoVsaRNA (Imperial College London, UK) which utilizes self-amplifying RNA to 11 enhance antigen expression at lower doses and RQ3013-VLP (Zhongshan Hospital, Fudan University,

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Nanoemulsion is a heterogeneous formulation of two different immiscible liquids (oil and water), 17 stabilised by surface-active agents to produce droplets within the nano-range (20-200 nm) [157]. Both antibodies and T-cell responses [161]. The modern squalene-based nanoemulsion MF59 ® was used 1 previously in influenza vaccine [159]. Polymeric NPs are nano-sized particles (1-1000 nm). They have shown a great potential for 5 targeted delivery of drugs and antigens [162]. Polymeric NPs can be loaded with antigen entrapped 6 within or surface-adsorbed onto their polymeric core. They can be administered via intravenous, oral, 7 dermal, and intranasal routes. They are classified as pH sensitive NPs, specific ligand attached NPs and 8 mucoadhesive NPs. The most commonly used polymers in vaccine delivery are polylactic co glycolic 9 acid (PLGA), polylactic acid (PLA) and polyanhydrides [146,163]. Despite versatile features such as 10 biodegradability, biocompatibility, and low cytotoxicity, their use is limited due to high cost, scale-up 11 difficulty, poor loading efficiency, and immediate burst release at acidic pH [162].

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Micelles are another promising nanocarrier for SARS-CoV-2 vaccines. They are nanoscale, 29 spherical self-assembled, amphiphilic structures that have a hydrophobic core and a hydrophilic shell.

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The hydrophilic shell allows for intravenous delivery. The hydrophobic core carries an antigen payload, thus protecting it from elimination and increasing its circulation time. The protein subunit vaccine, 1 NVX-CoV2373 (Novavax, USA), is a NP containing full-length S protein of SARS-CoV2, along with 2 Matrix-M adjuvant and polysorbate 80 detergent to induce self-assembly of micelles [165] (Table 1)

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The "mix-and-match" or heterologous SARS-CoV-2 vaccine regimens are a combination of two 12 or more current vaccine platforms [184]. This combination is highly effective in preventing infection;

Challenges and Limitations of Current SARS-CoV-2 Vaccines
1 COVID-19 vaccine platforms could potentially face many challenges related to safety, 2 manufacturing, distribution, storage, administration, and acceptability, as well as ethical considerations.

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All these challenges and limitations are vaccine-dependent and are discussed below. swelling, hair loss, menstrual cycle changes, tiredness, fatigue, headache, muscle pain, chills, fever, 10 diarrhea, nausea, drowsiness, and rash. Other potentially severe but rare side effects include 11 myocarditis, transverse myelitis, swollen lymph nodes, and blood clotting [188,189,114,190]. These rare 12 but possible adverse events may raise serious concern among the public and probably lead to 13 vaccination hesitancy or refusal. The differences in the intensity and pattern of adverse events could be 14 attributed to the difference in the type of vaccine platform. The side effects detected from the inactivated 15 vaccines were markedly lower than the adenoviral-based and mRNA-based vaccines [191,192].

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Interestingly, females and younger individuals were more likely to report vaccine-related adverse 17 events than older individuals [193]..

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The risk of blood clotting is experienced in women aged < 50 years old, following vaccination 20 of adenoviral (AstraZeneca and J&J) and mRNA (Moderna and Pfizer-BioNTech) vaccines [194,195].
abdomen. This very rare and possibly life-threatening condition is usually accompanied by low levels of 23 platelets, which can be resolved by immediate intervention with non-heparin anticoagulants [196].  [188]. This heart inflammation resolves without medical 2 intervention in at least half of the patients. However, it may lead to dilated cardiomyopathy, heart 3 transplantation, or death in up to a quarter of cases [198]. Some drugs such as beta-blockers, intravenous 4 anti-inflammatory drugs, and corticosteroids may help to alleviate the symptoms of myocarditis 5 [59,199]. Adverse events associated with the oral cavity and orofacial region were also detected in a few 6 people post-vaccination of the same two Moderna and Pfizer vaccines [200]. 12 which may cause anaphylaxis, especially in subjects with asthma, rhinitis, urticaria, or pre-existing anti-

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Moderna vaccine also contains another potential cause for anaphylactic reactions; 16 tromethamine/trometamol [136,206]. Therefore, inactivated vaccine may be a safe alternative in 17 individuals who have experienced anaphylactic reactions with adenoviral or LNPs vaccines [192].

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Further studies are needed to identify the exact causes of severe reactions which may be related to any     The majority of national vaccine distribution systems are not intended for large-scale adult 2 vaccination programs, but rather for children to ensure that they receive their full immunization   The most patient-friendly route for vaccines is oral administration, which is pain-free, non-7 invasive, low cost, and easy to administer to the mass population [163]. Oral vaccines not only stimulate 8 the mucosa-associated lymphoid tissue (MALT) located in the GI tract, but also can stimulate the gut- can be avoided. The vaccine is currently in phase 2 clinical trials (Table 1)

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Sublingual and buccal routes allow medications to be absorbed via the sublingual and buccal 24 mucosa, bypassing the GI tract barriers such as GI enzymatic degradation and hepatic first-pass effect 25 [3]. The oral cavity and saliva are important sites for SARS-CoV-2 infection and transmission [227,228].
Orally dissolving films (ODFs) loaded with vaccine microparticles/nanoparticles are another  IgA at the site of the viral entry, hindering viral spreading to the lung [235]. The intranasal delivery may 18 confer a stronger clinical immunity than oral delivery, as in the case of Bordetella bronchiseptica vaccine 19 [236]. However, the main drawback of mucosal vaccines is the poor immunogenicity produced due to 20 poor absorption and rapid removal of antigens from the nasal cavity [237].   20 a particular delivery device, such as nebuliser, may limit the use of a nasal or pulmonary vaccine.

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Additionally, the potential loss of virus titer cannot be ruled out, due to the use of a nebuliser [244]. So,

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it is not surprising that nearly all SARS-CoV-2 vaccine platforms are administered via injection (Table   25 1), although they may not be able to produce mucosal immunity [13].

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Intranasal vaccines prepared as inactivated vaccines, live attenuated vaccines, or viral vector-

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[247] created an intranasal nanovaccine prepared with inulin acetate, toll-like receptor-4 agonist, that 11 successfully stimulated both systemic and mucosal immunity in mice.

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The development of a vaccine platform based on protein assemblies is also a promising  powerful weapon against many viral diseases, including SARS-CoV-2. However, the major concern of 11 such vaccines is the fact that they were never approved for marketing before the COVID-19 pandemic.                         18 Vaccines 2021Vaccines , 9(2021