key: cord-0709197-u8vyv63h
authors: Cattel, Luigi; Giordano, Susanna; Traina, Sara; Lupia, Tommaso; Corcione, Silvia; Angelone, Lorenzo; La Valle, Giovanni; De Rosa, Francesco G.; Cattel, Francesco
title: Vaccine development and technology for SARS‐CoV‐2: Current insight
date: 2021-11-11
journal: J Med Virol
DOI: 10.1002/jmv.27425
sha: 7a5c04b4744b35882e8b06f20a7876f4a8a74372
doc_id: 709197
cord_uid: u8vyv63h

Severe acute respiratory syndrome coronavirus 2 is associated with a severe respiratory disease in China, that rapidly spread across continents. Since the beginning of the pandemic, available data suggested the asymptomatic transmission and patients were treated with specific drugs with efficacy and safety data not always satisfactory. The aim of this review is to describe the vaccines developed by three companies, Pfizer‐BioNTech, Moderna, and University of Oxford/AstraZeneca, in terms of both technological and pharmaceutical formulation, safety, efficacy, and immunogenicity. A critical analysis of Phases 1, 2, and 3 clinical trial results available was conducted, comparing the three vaccine candidates, underlining their similarities and differences. All candidates showed consistent efficacy and tolerability; although some differences can be noted, such as their technological formulation, temperature storage, which will be related to logistics and costs. Further studies will be necessary to evaluate long‐term effects and to assess the vaccine safety and efficacy in the general population.

The alpha and beta variants share a mutation (N501Y) in the S protein receptor-binding domain (RBD) which contributes to increased transmission (between 40% and 70%) through the angiotensin-converting enzyme-2 cellular receptor. The beta variant has two additional mutations (E484K and K417N) in the S protein that further potentiate antibody avoidance. 16 Another series of mutations (N501Y, E484K, and K417T) has been identified in the S protein of the gamma variant. 17 Today the vaccines which have received Emergency Use Au- Center for Epidemiology and Microbiology (Sputnik V), Sinovac Biotech (CoronaVac), and Sinopharm 1/2 (BBIBP-CorV). 18 Furthermore, worthy to be mentioned is Novavax (NVX-CoV2373), which is in the process of submitting the EUA application. For the purpose of the study, which is to discuss vaccine technology and innovation, the review focuses on vaccines developed by Pfizer/BioNTech, Moderna, and University of Oxford/AstraZeneca. [19] [20] [21] 2 | LIPID NANOTECHNOLOGY STUDIES Adenoviral and adeno-associated viral vectors lead to highly efficient transfection; however, viral elements can have drawbacks, such as recombination with the wild-type virus, immune or toxic reactions, and insertional mutagenesis. 22 Therefore, many synthetic nonviral transfer systems based on cationic nanoparticles have been developed. [22] [23] [24] Viruses and nanoparticles operate at the same nanoscale, so lipid nanoparticles (LNPs) that mimic viruses' structural features have been employed to encapsulate and deliver nucleic acid-based vaccines. Nanolipids incorporating RNA-based vaccines operate by 1) neutralizing negatively charged messenger RNA (mRNA), condensing the full-length RNA into a nanoscale range, and allowing LNPs to penetrate the host cell membrane (usually negatively charged); 2) escaping destruction by endosomal enzymes inside the host cell cytoplasm; and 3) discharging their mRNA cargo into the cytoplasm, allowing it to reach the ribosomes in the endoplasmic reticulum.

After incorporation, S protein mRNA transcripts are produced.

The protein is processed by antigen-presenting cells, and the epitopes are presented by major histocompatibility complex (MHC)-1 and MHC-2. This induces the activation of CD8+ cytotoxic T cells or CD4+ T helper cells, which are essential for antiviral antibody production ( Figure 2B ). Therefore, LNPs have multiple roles; they act as a synthetic virus vector and stabilize the mRNA and prevent its destruction by RNase.

The Pfizer/BioNTech vaccine encapsulates the S protein mRNA within LNPs provided by a partnership with Acuitas Therapeutics.

Although the composition has not been fully disclosed, previous publications from Acuitas Therapeutics 25 reported that their LNPs (70-100 nm in size) are made of ionizable cationic lipids, phosphatidylcholine, cholesterol, and polyethylene glycol (PEG)-lipids, and deliver mRNA in vivo. 25 LNPs with ionizable cationic lipids are one of the most advanced technological systems, similar in composition to those used for small interfering RNA delivery. 26, 27 Further optimization of the LNP formulation enabled rapid elimination in vivo while maintaining efficacy. 27 The exact Moderna formulation has not been publicly described, but it is known that previous LNP formulations from Moderna used ionizable lipids, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, and PEG-lipid. [28] [29] [30] Phospholipids with phosphatidylcholine are usually present in liposome formulations, as they are in the Moderna and Pfizer/ BioNTech vaccine formulations ( Figure 2A ).

Many cationic lipids, 31, 32 such as lipofectin, DOTAP, DOPE, DOTMA, DMRIE, and other analogs, [33] [34] [35] have been described. In recent years, several ionizable aminolipids (probably used by Moderna) have been designed for systemic administration, such as DLin-KC2-DMA. 26 These are characterized by the presence of a dilinoleyl group in which the unsaturated alkyl chain (cis-double bond) is optimal for the activity and ionization (at pH = 5.5 inside the endosomes) of the dimethylamino head group. 26, [28] [29] [30] 36 The cationic lipid ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315) ( Figure 1C ) was used by Pfizer/ BioNTech as a component of the lipid mixture of BNT162b2 to form LNPs. ALC-0315 is a physiological pH cationic (pKa 5.5) synthetic lipid that can be used together with other lipids to form LNPs. 37, 38 Ionizable aminolipids play a dual role in the delivery process. First, they promote the self-assembly of the components into LNPs, encapsulating the RNA through electrostatic interactions with polyanionic nucleic acids. Second, the subsequent endocytosis of LNPs by targeted cells enables RNA to exit from the endosomal compartment and enter the cell cytoplasm. This mechanism is similar to that used by pH-sensitive phospholipids, such as DOPE, in liposomes. At pH 5.5 (present in endosomes), DOPE pegylated liposomes trigger a transition phase (from lamellar to inverse hexagonal phase) that disrupts the liposomal membrane, discharging the encapsulated mRNA into the cell cytosol. 39 It is possible that, through a similar mechanism, LNPs discharge the mRNA cargo into the cell cytoplasm, allowing it to reach the ribosomes in the endoplasmic reticulum. A possible explanation for the activity of ionizable aminolipids is that the proximity of the opposing surface of the endosomal lumen allows the formation of an ion pair that prefers to adopt inverted non-bilayer configurations; this disrupts the endosome membrane integrity, leading to the release of the RNA into the cytoplasm. acetamide (ALC-0159) are used in the Pfizer/BioNTech formulation. 40 PEG is inserted at the surface of nanoparticles close to the aqueous phase and the lipid/phospholipid segments point to the inner hydrophobic moiety. Thus, the nanolipid surface appears to be covered in very hydrophilic PEG "hairs"; this makes the nanoparticles very stable in serum. PEG-coated liposomes 41 circulate for a remarkably long time after intravenous administration (24-30 h). The term "stealth" was used to describe these nanoparticles because of their ability to evade the host immune system. 42 Cholesterol is also present in the Moderna and Pfizer nanolipid formulations as it confers high stability in vivo.

A more attractive feature of pegylated nanoparticles is their ability to preferentially access the lymphatic system. Due to their nanoscale size and high stability, LNPs may cross the interstitial space and access nearby lymph nodes, and this may be a highly beneficial process in the race to develop COVID-19 vaccines. 43, 44 The involvement of an adjuvant can also increase the immunostimulatory properties of mRNA. In both nanolipid formulations, there is no indication of the use of an adjuvant, although the LNP itself may be an adjuvant, like other lipids. [45] [46] [47] In addition, other components may be present in the formulations, some of which may require low-temperature storage. For example, Pfizer/BioNTech stipulates vaccine storage at a low temperature (−80°C) because of mRNA instability, whereas Moderna affirms that a higher storage temperature (−20°C) is sufficient to maintain vaccine activity. The nature of these components is unknown, but what is known and supported in the literature is that several classes of emulsifiers (concerning charge and molecular weight) or antioxidants have been used to stabilize lipid dispersion and prevent particle agglomeration. 48 

The Pfizer/BioNTech vaccine technology is based on mRNA that encodes the S protein of SARS-CoV-2. 49 Given the structural variability of the prefusion form of the S protein due to its intrinsic thermodynamically metastable state, generating a stabilized mutant conformation (nucleoside-modified RNA [modRNA]) that mimics the prefusion conformation is critical for vaccine development. 50 mod-RNA (4284 nucleotides) ( Figure 1A ) includes a 5ʹ cap and an untranslated region derived from a human alpha-globin sequence that has a profound effect on mRNA stability and translation. In addition, it consists of a signal peptide-coding region (bases 55-102), which encodes the S2P mutated version of the S protein. This version contains two proline substitutions (K986P and V987P, referred to as F I G U R E 1 (A) modRNA including a 5ʹ cap and two untranslated regions (UTR) and the S protein-coding sequence. (B) 1-methyl-5ʹpseudouridine. (C) Pfizer-BioNTech cationic lipid ALC-0315. modRNA, nucleoside-modified RNA "2P") that stimulate neutralizing antibodies (bases 103-3879). [51] [52] [53] In this sequence, uridine is replaced by 1-methyl-5ʹ-pseudouridine ( Figure 1B) , which increases mRNA translational capacity. 51,52

Clinical studies began with an initial phase 1 trial conducted in Germany in which two LNP-formulated, modRNA vaccine candidates were tested against SARS-CoV-2, BNT162b1, and BNT162b2. BNT162b1 encodes SARS-CoV-2 RBD, which is trimerized by the addition of a T4 fibritin foldon domain that guides protein folding to produce the native trimeric state, thus increasing immunogenicity. [54] [55] [56] [57] The T4-mediated trimerization also augments immunogenicity by generating a multivalent display of antigens.

BNT162b2 encodes the SARS-CoV-2 full-length S protein. The use of nonimmunogenic mRNA is crucial because a series of innate immunity receptors, including Toll-like receptors (TRL3, TLR7, and TLR8), can recognize RNA, resulting in the release of type I interferons and the inhibition of translation. [52] [53] [54] [55] [56] [57] [58] [59] BNT162b1 and BNT162b2 safety data from younger and older adults supported the selection of BNT162b2 for advancement to Phase 1/2 trials to evaluate safety and efficacy and to the final Phase 2/3 clinical trial. 60 The immune responses elicited by BNT162b1 and BNT162b2 were similar, but BNT162b2 showed milder reactogenicity, particularly in older adults. Given the similarities in the modRNA platform and LNP formulation of these two candidates, it has been suggested that the different RNA nucleotide compositions may be the source of their immune stimulatory activity and reactogenicity profile. 61 In Phase 1 placebo-controlled, observer-blinded, dose-escalation trial conducted in the USA, adults 18-55 and 65-85 years old were randomly administered either a placebo or one of two mRNA vaccine candidates. The principal outcome was safety (i.e., local and systemic reactions and adverse effects [AEs]) and the secondary outcome was immunogenicity (Table 1) . 60 Each trial group received two doses of a vaccine (10, 20, 30, This study's limitation is that the number of participants was not large enough to observe uncommon and rare side effects. Also, the period of protection remains to be determined, and data do not specify whether vaccination prevents asymptomatic infection. Longterm safety and efficacy assessment for the vaccine is recommended, and additional studies are necessary for other populations (i.e., adolescents 12-15 years old, children younger than 12 years old, pregnant women, and immunocompromised individuals).

The efficacy of the Pfizer/BioNTech vaccine against the SARS-CoV-2 alpha variant was compared to its efficacy against the Wuhan reference strain in a preliminary study. 62 The data showed a slightly reduced neutralization susceptibility of the BNT162b2 vaccine. Other studies [63] [64] [65] have suggested that the Pfizer/BioNTech vaccine is less effective against a pseudovirion of the alpha variant (two times less effective) and less efficient against the gamma variant (Figure 2A) . 76 A challenge associated with using these vectors is that there can be pre-existing immunity in humans, which can lessen their efficacy. For this reason, using a chimpanzee adenovirus minimizes possible interactions with prevalent antiadenovirus antibodies. 77, 78 After deleting E1 and E3, the SARS-CoV-2 Wuhan-Hu-1 gene was cloned into a viral vector. 79 The in situ cellular production of the protein avoids posttranslational modifications, especially in the S protein case, which can have up to 22 glycosylation sites. 40 The presence of glycosylation sites in the S protein can reduce antibody-mediated neutralization, rendering the vaccines ineffective. The in situ production of a protein with few or no glycosylation sites makes these RNA vaccines very attractive. 81 

The University of Oxford/AstraZeneca published the results of a neutralizing activity linked with antibody levels, which were measured by enzyme-linked immunosorbent assay (R 2 = 0.67; p < 0.001).

To summarize, ChAdOx1 nCoV-19 demonstrated a good safety profile and increased antibody response following the boost dose ( The remaining participants were designated to receive the standard dose (3.5-6.5 × 10 10 virus particles) and the same randomization protocol was used, except that the 18-55-year-old group was In August 2020, a multicentric randomized Phase 3 trial was initiated to evaluate the University of Oxford/AstraZeneca vaccine, which is also known as AZD1222. 21 The trial enrolled over 30 000

adults at 80 sites. The primary outcomes investigated were: efficacy of two intramuscular doses of vaccine compared to placebo for safety and tolerability (timeframe: one year); incidence of AEs and SAEs Case reports and case series are rare, and research is restricted, but the understanding of VITT's epidemiology, pathophysiology, diagnosis, and treatment is growing. 94 The estimated incidences of VITT with the University of Oxford/AstraZeneca and J&J vaccines were 7-10 cases per million individuals and 3.2 cases per million individuals, respectively. 95 However, because of the limited availability of the J&J vaccine and delays in reporting, the stated rates are likely an underestimation of true incidence levels. 94, 95 The pathogenesis of VITT has been somewhat elucidated. It is described in published studies as an immunological disease, similar to autoimmunity-induced thrombocytopenia, unlikely to be the result of COVID-19 infection and independent of anti-SARS-CoV2 protective immunity. 94 A EUA is a mechanism to facilitate the availability and use of 104 The three companies have predicted the production of billions of vaccines during 2021. Therefore, with this in mind, it is fundamental that these effective vaccines be delivered and administered globally to achieve global herd immunity.

The authors declare that there are no conflict of interests.

The search and the selection of articles were performed by Luigi

Cattel. The first draft of the manuscript was written by Luigi Cattel.

Susanna Giordano, Sara Traina, and Tommaso Lupia revised the manuscript and all authors commented on subsequent versions of the manuscript. All authors read and approved the final manuscript.

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

http://orcid.org/0000-0001-7302-5688

Susanna Giordano http://orcid.org/0000-0003-1630-5653

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