Vaccine development and technology for SARS-CoV-2: Current insight.

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.

coronavirus disease‐2019

European Medicines Agency

Emergency Use Authorization

Food and Drug Administration

lipid nanoparticles

meningococcal conjugate vaccine

major histocompatibility complex

receptor‐binding domain

systemic adverse effects

vaccine‐induced immune thrombotic thrombocytopenia

BACKGROUND

The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) pandemic continues to spread at alarming rates and there appears to be no end in sight due to the long viral incubation period and lack of effective treatment or vaccines. , As of October 2021, over 243 million confirmed cases and up to 4.9 million deaths had been reported globally. The development of a coronavirus disease‐2019 (COVID‐19) vaccine is crucial given that available data indicate asymptomatic transmission of the causative virus.

According to the World Health Organization, in August 2021, 294 companies and academic institutes worldwide were developing COVID‐19 vaccines. Among these, most had conducted clinical trials with 110 identified vaccine candidates. ,

The SARS‐CoV‐2 virus is an enveloped single‐stranded RNA virus with a spike‐like glycoprotein protruding from its outer membrane surface and each spike forms a “corona.” , The spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins are the four structural proteins of viruses of the Betacoronavirus genus. In particular, the S protein is a focal point for the design of vaccines since it facilitates virus entry into host cells. , , S protein mutations in SARS‐CoV‐2 variants can impact vaccine efficacy and the risk of reinfection. Many variants have spread rapidly in the UK (alpha variant, also called variant B.1.1.7 and 20I/501Y.V1), South Africa (beta variant, also called variant B.1.351 and 20H/501Y.V2), Brazil (gamma variant, also called variant B.1.1.248 and P.1 and 20J/501Y.V3) and California (epsilon variant, also called variant B.1.429 and Cal.20C and 452R.V1). , , , , The alfa variant contains 17 nonsynonymous mutations and, in particular, 8 principal mutations in the S protein.

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. Another series of mutations (N501Y, E484K, and K417T) has been identified in the S protein of the gamma variant.

Today the vaccines which have received Emergency Use Authorization (EUA) have been developed by Pfizer/BioNTech (sold under the brand name Comirnaty), Moderna (Spikevax), University of Oxford/AstraZeneca (Covishield or Vaxzevria), Johnson & Johnson (J&J; also known as Ad26.CoV2.S), Gamaleya National Research Center for Epidemiology and Microbiology (Sputnik V), Sinovac Biotech (CoronaVac), and Sinopharm 1/2 (BBIBP‐CorV). 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. , ,

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. Therefore, many synthetic nonviral transfer systems based on cationic nanoparticles have been developed. , , 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.

Figure 2

(A) The nanolipid vector (80–100 nm) of Pfizer‐BioNTech and Moderna and the viral vector of Oxford‐AstraZeneca. (B) The nanolipid carrying the mRNA blends the cell membrane entering the cytoplasm through endosomal vesicles. From the endosomes, nanolipids spill out their cargo (mRNA) into the cytoplasm, reaching the ribosomes where mRNA is translated into a spike protein to be processed and presented to MHC‐1, activating CD8+ T cells. Analogously, CD4+ (naive) T‐cells are activated and, by the MHC‐2 activation, the B cells are operative, producing whether memory T cells or plasma cells, which can produce antiviral antibodies. MHC, major histocompatibility complex; mRNA, messenger RNA

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 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. LNPs with ionizable cationic lipids are one of the most advanced technological systems, similar in composition to those used for small interfering RNA delivery. , Further optimization of the LNP formulation enabled rapid elimination in vivo while maintaining efficacy. 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. , , 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, , such as lipofectin, DOTAP, DOPE, DOTMA, DMRIE, and other analogs, , , have been described. In recent years, several ionizable aminolipids (probably used by Moderna) have been designed for systemic administration, such as DLin‐KC2‐DMA. 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. , , , ,

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. ,

Figure 1

(A) modRNA including a 5ʹ cap and two untranslated regions (UTR) and the S protein‐coding sequence. (B) 1‐methyl‐5ʹ‐pseudouridine. (C) Pfizer‐BioNTech cationic lipid ALC‐0315. modRNA, nucleoside‐modified RNA

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. 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.

PEG–lipid conjugates are present in both the Moderna and Pfizer formulations. Many PEG‐lipids are described in the literature. For example, DMG‐PEG‐2000, 1,2‐dimyristoyl‐rac‐glycero‐3‐methoxy‐PEG‐2000, and 2‐[(polyethylene glycol)−2000]‐N,N‐di(tetradecyl)acetamide (ALC‐0159) are used in the Pfizer/BioNTech formulation. 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 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. 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. ,

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. , ,  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.

PFIZER/BIONTECH

The Pfizer/BioNTech vaccine technology is based on mRNA that encodes the S protein of SARS‐CoV‐2. 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. modRNA (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 "2P") that stimulate neutralizing antibodies (bases 103–3879). , , In this sequence, uridine is replaced by 1‐methyl‐5ʹ‐pseudouridine (Figure 1B), which increases mRNA translational capacity. ,

Clinical studies

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. , , , 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. , , , , , , ,

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. 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.

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). Each trial group received two doses of a vaccine (10, 20, 30, or 100 µg) with a 3‐week interval between doses, except for one group (100 µg of BNT162b1) that only received one dose. After the first dose, systemic events reported by participants aged 65–85 who received BNT162b2 were similar to those reported by the placebo group participants. Subsequently, after the second dose of BNT162b2 (30 µg), only 17% of participants aged 18%–55% and 8% of participants aged 65–85 reported fever, compared to 75% of participants aged 18%–55% and 33% of participants aged 65–85 who were administered the second dose of BNT162b1. Severe transient and manageable systemic events were observed in a limited number of younger BNT162b2 recipients, while older BNT162b2 recipients did not report any severe systemic events. These safety and immunogenicity data supported the selection of BNT162b2 at a 30 µg dosage in a two‐dose regimen for progression to Phase 2/3 safety and efficacy assessment.

Table 1

Phase 1 efficacy results from Pfizer‐BioNTech, Moderna, and Oxford‐AstraZeneca

Authors, journal, year	Vaccine name	Design and population	Outcome measurement	Results
Walsh et al., N Engl J Med, 2020	BNT162b2 (Pfizer‐/BioNTech)	Placebo‐controlled, observer‐blinded, dose‐escalation; 195 healthy adults 18–55 or 65–85 years of age randomized to receive placebo or one of two vaccines (BNT162b1 or BNT162b2), two administration doses of 10, 20, and 30 µg, 21 days apart or one single 100 µg dose	Geometric mean concentrations of recombinant antigen (S1)‐binding IgG (U/ml) at Day 35	Placebo: 0.9 BNT162b1a − 10 µg: 5120 and 1527 − 20 µg: 7480 and 6399 − 30 µg: 13 940 and 4798 BNT162b2a − 10 µg: 4717 and 3560 − 20 µg: 7367 and 2656 − 30 µg: 8147 and 6014 HCSb: 631
			50% SARS‐CoV‐2‐neutralizing geometric mean titers at Day 35	Placebo: 0 BNT162b1a − 10 µg: 180 and 33 − 20 µg: 203 and 105 − 30 µg: 437 and 105 BNT162b2a − 10 µg: 97 and 111 − 20 µg: 292 and 81 − 30 µg: 163 and 206 HCSb: 94
Jackson et al., N Engl J Med, 2020	mRNA‐1273 (Moderna)	Dose‐escalation, open‐label; 45 healthy adults 18–55 years of age receiving two doses of 25, 100, or 250 µg, 28 days apart	Geometric mean humoral immunogenicity titer (ELISA) anti‐S‐2P at Day 36	mRNA‐1273 − 25 µg: 391 018 − 100 µg: 781 399 − 250 µg: 1 261 975 HCS: 142 140
			Geometric mean humoral immunogenicity titer (ELISA) antireceptor binding domain at Day 36	mRNA‐1273 − 25 µg: 208 652 − 100 µg: 499 539 − 250 µg: 720 907 HCS: 37 857
			PsVNA ID50 geometric mean response at Day 36	mRNA‐1273 − 25 µg: 105.8 − 100 µg: 256.3 − 250 µg: 373.5 HCS: 109.2
			Live virus PRNT80 geometric mean response at Day 43	mRNA‐1273 − 25 µg: 339.7 − 100 µg: 654.3 − 250 µg: NA HCS: 158.3
Anderson et al., N Engl J Med, 2020	mRNA‐1273 (Moderna)	Dose‐escalation, open‐label. Extension of the study by Jackson et al. Including 40 participants (56–70 and ≥71 years of age) receiving two doses of 25 or 100 µg, 28 days apart	IgG titers on RBD (ELISA) at Day 36	mRNA‐1273c − 25 µg: 198 643 and 160 591 − 100 µg: 1 471 882 and 711 752 HCS: 37 244
			PsVNA ID50 geometric mean response at Day 36	mRNA‐1273c − 25 µg: 79 and 121 − 100 µg: 289 and 310 HCS: 106
			FRNT‐mNGd ID50 at Day 43	mRNA‐1273c − 25 µg: 550 and 448 − 100 µg: 1425 and 900 HCS: NA
			Live virus PRNT80 at Day 43	mRNA‐1273c − 25 µg: NA − 100 µg: 878 and 317
			Response in Th1 cells at Day 43 (mean percentages of CD4 T‐cells that produced the cytokines)e	mRNA‐1273c − 25 µg: 0.264 and 0.095 − 100 µg: 0.336 and 0.317
			Response in Th2 cells at Day 43 (mean percentages of CD4 T‐cells that produced the cytokines)	mRNA‐1273c − 25 µg: 0.022 and 0.015 − 100 µg: 0.029 and 0.023
Folegatti et al., Lancet, 2020	ChAdOx1 nCoV‐19 (University of Oxofrod/AstraZeneca)	Participant‐blinded, multicentre, randomized controlled trial; 1077 healthy adults (18–55 years of age) assigned to receive 5 × 10 viral particles of ChAdOx1 nCoV‐19 or 0.5 ml MenACWY (placebo)	Antispike IgG using standardized ELISA at Day 28 (median)	ChAdOx1 nCoV‐19 − Prime: 157.1 − Prime‐boost: 210.7 − Prime‐boost at Day 35: 821.1 MenACWY: 1
			Multiplex MSD– antispike IgG (AU/ml) at Day 28 (median)	ChAdOx1 nCoV‐19 − Prime: 10 471.8 − Prime‐boost: NA − Prime‐boost at Day 42: 33 830.8 MenACWY: 43.9
			Multiplex MSD—RBD (AU/ml) at Day 28 (median)	ChAdOx1 nCoV‐19 − Prime: 3182.5 − Prime‐boost: NA − Prime‐boost at Day 42: 16 825.4 MenACWY: 15.8
			Marburg VN at Day 28 (median)	ChAdOx1 nCoV‐19 − Prime: 1 − Prime‐boost: 3.2 − Prime‐boost at Day 42: 32 MenACWY: 1
			PHE PRNT50 1 f at Day 28 (median)	ChAdOx1 nCoV‐19 − Prime: 218 − Prime‐boost: NA MenACWY: 36.5
			PseudoNA at Day 28 (median)	ChAdOx1 nCoV‐19 − Prime: 87.9 − Prime‐boost: 162.9 − Prime‐boost at Day 42: 450.9 MenACWY: 40
			IFNγ ELISpot response against SARS‐CoV‐2 spike peptides at Day 28 (median)	ChAdOx1 nCoV‐19 − Prime: 554.3 − Prime‐boost: 528.7 MenACWY: 61.3

Abbreviations: ELISA, enzyme‐linked immunosorbent assay; HCS, human convalescent sample; IFNγ, interferon‐γ; IgG, immunoglobulin G; Marburg VN, Marburg SARS‐CoV‐2 virus neutralization; MenACWY, meningococcal conjugate vaccine; mRNA, messenger RNA; MSD, mesoscale discovery; nCoV, novel coronavirus; PRNT80, plaque‐reduction neutralization testing assay; PseudoNA, monogram biosciences pseudotype neutralization assay; PsVNA ID50, pseudotype lentivirus reporter neutralization assay 50% inhibitory dilution; RBD, receptor‐binding domain; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2.

In the 18–55 and 65–85 years of age groups, respectively.

SARS‐CoV‐2 infection convalescent serum samples (HCS).

In the group of 56–70 and ≥71 years of age, respectively.

Focus reduction neutralization test mNeonGreen assay.

Simulation with the SARS‐CoV‐2 S1 peptide pool.

Public Health England Plaque Reduction Neutralization Test.

Between July and November 2020, a Phase 3 multicentric, randomized 1:1 clinical trial was conducted. This efficacy study involved 43 448 participants aged 16–85 or older: 21 720 volunteers received BNT162b2 (30 µg per dose), and 21 728 received a placebo on Days 0 and 21 (Table 2). The principal endpoints were vaccine efficacy and safety. BNT162b2 had a 95% effectiveness in preventing COVID‐19, and similar efficacy was reported across subgroups defined by age, sex, race, ethnicity, baseline body index, and the presence of concomitant conditions. After the first dose, COVID‐19 occurred in 39 participants in the BNT162b2 group and 2 participants in the placebo group (52.4% efficacy). Within 7 days after the second dose, COVID‐19 resulted in 2 participants in the BNT162b2 group and 21 in the placebo group (efficacy 90.5%); beyond 7 days after the second dose, 9 COVID‐19 cases were found in the BNT162b2 group and 172 in the placebo group (94.8%). These Phase 3 trial findings confirmed the favorable profile of the BNT162b2 vaccine.

Table 2

Phases 2–3 efficacy results from Pfizer‐BioNTech, Moderna, and Oxford‐AstraZeneca

Authors, journal, year	Vaccine name	Design and population	Outcome measurement	Results
Polack et al., N Engl J Med, 2020	BNT162b2 (Pfizer/BioNTech)	Multinational, randomized, placebo‐controlled, observer‐blinded, pivotal efficacy trial. Healthy adults and adults with stable chronic medical conditions, 16 years of age or older were eligible; 43 448 participants were randomly assigned in a 1:1 ratio to receive two doses, 21 days apart, of 30 µg of the BNT162B2 vaccine (n = 21 720) or the placebo (n = 21 728)	Efficacy against confirmed Covid‐19 with onset at least 7 days after the second dose in participants without prior evidence of infection (CI 95%)a	BNT162b2 group
				Number of cases: 8/18 198
				Placebo group
				Number of cases: 162/18 325
				Efficacy: 95.0% (90.3%–97.6%)
			Efficacy against confirmed Covid‐19 with onset at least 7 days after the second dose in participants with and those without prior evidence of infection (CI 95%)a	BNT162b2 group
				Number of cases: 9/18 198
				Placebo group
				Number of cases: 169/18 325
				Efficacy: 94.6% (89.9%–97.3%)
			Efficacy against confirmed Covid‐19 with onset at any time after the first dose in participants with and those without prior evidence of infection (CI 95%)a	a) Occurrence after the first dose:
				BNT162b2 group
				Number of cases: 39/21 669
				Placebo group
				Number of cases: 2/21 686
				Efficacy: 52.4% (29.5%–68.4%)
				b) Occurrence between second dose and 7 days after:
				BNT162b2 group
				Number of cases: 2/21 669
				Placebo group
				Number of cases: 21/21 686
				Efficacy: 90.5% (61.0%–98.9%)
				c) Occurrence at least after 7 days after the second dose:
				BNT162b2 group
				Number of cases: 9/21 669
				Placebo group
				Number of cases: 172/21 686
				Efficacy: 94.8% (89.8%–97.6%)
Baden et al., N Engl J Med, 2020	mRNA‐1273 (Moderna)	Multicentric, randomized, placebo‐controlled, observer‐blinded trial, 30 420 healthy adults (≥18 years of age) with high risk for SARS‐CoV‐2 infection or its complications were randomly assigned in a 1:1 ratio to receive two doses, 28 days apart, of 100 µg of the mRNA‐1273 vaccine (n = 15 210) or the placebo (n = 15 210)	Efficacy against confirmed Covid‐19 with onset at least 14 days after the second dose in participants without prior evidence of infection (CI 95%)a	mRNA‐1273 group
				Number of cases: 11/14 134
				Placebo group
				Number of cases: 185/14 073
				Efficacy: 94.1% (89.3%–96.8%)
			Efficacy against confirmed Covid‐19 with onset at any time after the first dose (CI 95%)a	mRNA‐1273 group
				Number of cases: 7/14 550
				Placebo group
				Number of cases: 65/14 598
				Efficacy: 93.0% (88.9%–95.6%)
			Efficacy against confirmed severe Covid‐19 with onset at least 14 days after the second dose (CI 95%)a	mRNA‐1273 group
				Number of cases: 0/14 073
				Placebo group
				Number of cases: 30/14 073
				Efficacy: 100.0% (N.E.−100.0%)
			Efficacy against confirmed Covid‐19 with onset at least 14 days after the first dose (CI 95%)a	mRNA‐1273 group
				Number of cases: 11/14 073
				Placebo group
				Number of cases: 225/14 073
				Efficacy: 95.2% (91.2%–97.4%)
			Efficacy against confirmed secondary definition Covid‐19b with onset at least 14 days after the first dose (CI 95%)a	mRNA‐1273 group
				Number of cases: 11/14 073
				Placebo group
				Number of cases: 221/14 073
				Efficacy: 95.1% (91.1%–97.3%)
Ramasamy et al., Lancet, 2020	ChAdOx1 nCoV‐19 (University of Oxoford/AstraZeneca)	Multicentric, randomized, controlled, single‐blind trial; 560 healthy adults aged 18 years or older were enrolled in an age‐escalation (18–55 years, 56–69 years, and ≥70 years) subgroups; 300 participants were randomly assigned to receive a low dose (LD) of ChAdOx1 nCoV‐19 vaccine (2.2 × 1010 viral particles) or the MenACWY vaccine as control; 260  volunteers received a standard dose (SD) of ChAdOx1 nCoV‐19 vaccine (3.5–6.5 × 1010 viral particles) or the MenACWY vaccine	Antispike IgG using standardized ELISA at Day 28 (median)	ChAdOx1 nCoV‐19
				− 18–55 years LD/LD: 149 − 55–69 years LD: 78 − ≥70 years LD: 89 − 55–69 years LD/LD: 74 − ≥70 years LD/LD: 77 − 18–55 years SD/SD: 174 − 55–69 years SD: 145 − ≥70 years SD: 90 − 55–69 years SD/SD: 119 − ≥70 years SD/SD: 149 MenACWY:
				− One‐dose regimen: 10 Two‐dose regimen: 1
		Participants randomization:
			Multiplex immunoassay– antispike IgG (AU/ml) at Day 28 (median)	ChAdOx1 nCoV‐19
		a) 18–55 years group: − LD regimen: 1:1 ratio to receive two doses − SD regimen: 5:1 ratio to receive two doses b) 55–69 years group: − LD regimen and SD regimen: 3:1:3:1 ratio to receive one dose of vaccine, one dose of control, two doses of vaccine, and two doses of control, respectively c) ≥70 years group:
				− 18–55 years LD/LD: 6439 − 55–69 years LD: 5032 − ≥70 years LD:4103 − 55–69 years LD/LD: 4040 − ≥70 years LD/LD:3168 − 18‐55 years SD/SD: 9807 − 55–69 years SD: 6693 − ≥70 years SD: 3454 − 55–69 years SD/SD: 4474 − ≥70 years SD/SD: 4603
		LD regimen and SD regimen: 5:1:5:1 ratio to receive one dose of vaccine, one dose of control, two doses of vaccine, and two doses of control, respectively
				MenACWY:
				− 18–55 years LD/LD: 50 − 55–69 years LD: 27 − ≥70 years LD:37 − 55–69 years LD/LD: 73 − ≥70 years LD/LD: 55 − 18–55 years SD/SD: 31 − 55–69 years SD: 42 − ≥ 70 years SD: 37 − 55–69 years SD/SD: 35 ≥70 years SD/SD:48
			PHE MNA80 (normalized IC80 values) at Day 28 (median)	ChAdOx1 nCoV‐19
				− 18–55 years LD/LD: 79 − 55–69 yearsLD: 64 − ≥70 years LD: 21 − 55–69 years LD/LD: 55 − ≥70 years LD/LD: 33 − 18–55 years SD/SD: 47 − 55–69 years SD: 9 − ≥70 years SD: 49 − 55–69 years SD/SD: 72 ≥70 years SD/SD:48
			IFNγ ELISpot response against SARS‐CoV‐2 spike peptides at Day 28 (median)	ChAdOx1 nCoV‐19
				− 55–69 years LD: 511 − ≥70 years LD:420 − 55–69 years LD/LD: 488 − ≥70 years LD/LD: 397 − 18–55 years SD/SD: 292 − 55–69 years SD: 29 − ≥70 years SD: 47 − 55–69 years SD/SD: 591 ≥70 years SD/SD:300
Voysey et al., Lancet, 2021	ChAdOx1 nCoV‐19 (University of Oxoford/AstraZeneca)	Multicentric, randomized, controlled, single‐blind trial. Phase 3 of the study by Ramasamy et al., The Lancet, 2020; 23 848 adults (≥ 18 years old) were randomly assigned in a 1:1 ratio to receive two doses of ChAdOx1 nCoV‐19 vaccine or the MenACWY vaccine or saline as control. Participants assigned to the vaccine group received two SD/SD of 5 × 1010 viral particles or a half dose as their first dose and an SD as their second dose (LD/SD)	Efficacy against confirmed Covid‐19 with onset at least 14 days after second dose in all LD/SD and SD/SD recipients without prior evidence of infection (CI 95%)a	ChAdOx1 nCoV‐19 group
				Number of cases: 30/5807
				Control group
				Number of cases: 101/5829
				Efficacy: 70.4% (54.8%–80.6%)
			Efficacy against confirmed Covid‐19 with onset at least 14 days after the second dose in all LD/SD recipients without prior evidence of infection (CI 95%)a	ChAdOx1 nCoV‐19 group
				Number of cases: 3/1367
				Control group
				Number of cases: 30/1374
				Efficacy: 90.0% (67.4%–97.0%)
			Efficacy against confirmed Covid‐19 with onset at least 14 days after the second dose in all SD/SD recipients without prior evidence of infection (CI 95%)a	ChAdOx1 nCoV‐19 group
				Number of cases: 27/4440
				Control group
				Number of cases: 71/4455
				Efficacy: 62.1% (41.0%–75.7%)
			Efficacy against confirmed other non‐primary symptomatic Covid‐19 diseasec with onset at least 14 days after second dose in all LD/SD and SD/SD recipients without prior evidence of infection (CI 95%)a	ChAdOx1 nCoV‐19 group
				Number of cases: 7/5807
				Control group
				Number of cases: 11/5829
				Efficacy: 36.4% (−63.4%–75.3%)

Abbreviations: CI, confidence interval; Covid, coronavirus disease‐2019; IRR, incidence rate ratio; MenACWY vaccine, meningococcal group A, C, W, and Y conjugate vaccine; mRNA, messenger RNA; PHE MNA80, Public Health England Microneutralization Assay; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2.

Calculated as 100 × (1 − IRR); IRR is the calculated ratio of confirmed cases of Covid‐19 per 1000 person‐years of follow‐up in the vaccine group to the corresponding illness rate in the placebo group.

Secondary definition of Covid‐19 disease was defined as including systemic symptoms and a positive nasopharyngeal swab, nasal swab or saliva sample for SARS‐CoV‐2.

Other nonprimary symptomatic Covid‐19 disease includes cases who have other symptoms than fever ≥37.8°C, cough, shortness of breath, or anosmia or ageusia.

MenACWY:

Two‐dose regimen: 1

The most common systemic effects reported after BNT162b2 administration were short‐term, mild‐to‐moderate pain in the area of injection, fatigue, and headache. A limited number of participants in each group experienced severe side effects, profound effects, or AEs, which led to their resignation from the trial. Two BNT162b2 recipients and four placebo recipients died; however, investigators determined that no deaths were caused by the vaccination in the BNT162b2 group.

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. Long‐term 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. The data showed a slightly reduced neutralization susceptibility of the BNT162b2 vaccine. Other studies , , 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 (4–6 times less effective). Variable results have been reported about its efficacy against the beta variant (1–35 times less effective). , ,

MODERNA

The mRNA‐1273 vaccine developed by Moderna encodes the prefusion form of the S (named S2‐P) protein and includes a transmembrane anchor and an entire S1–S2 cleavage site. Two proline substitutions in the S2 subunit at amino acids 986 and 987, within the central helix, keep the protein stable in its prefusion conformation. Prefusion‐stabilized S protein variants are superior immunogens compared to wild‐type S protein ectodomains. Importantly, these successful substitutions in the SARS‐CoV‐2 S protein (SARS‐CoV‐2S‐2P) allow for rapid structural identification using cryogenic electron microscopy and hastened the development of vaccine candidates. , In fact, several studies focused on increasing the stability of the S protein ectodomain in the prefusion conformation, using multiple proline variants, disulfide bonds, and cavity‐filling substitutions to increase S protein expression and stability. , , , , , , , , , , , , , , , , , , , , ,

Eligible participants (healthy adults, aged 18–55, n = 45) in a Phase 1 trial received two injections of the mRNA‐1273 vaccine 28 days apart at a dose of 25, 100, or 250 µg (Table 1). , After the first dose, systemic adverse effects (SAEs) with mild or moderate severity appeared in 5 (33%) participants in the 25‐µg group, 10 (67%) in the 100‐µg group, and 8 (53%) in the 250‐µg group. SAEs were more common after the second dose, reported by: 7 of the 13 participants (54%) in the 25‐µg group, all 15 (100%) in the 100‐µg group, and all 14 (100%) in the 250‐µg group; 21% reported ≥1 severe event. There were no febrile episodes after the first dose. After the second injection, no participants showed severe events in the 25‐µg group, whereas six participants (40%) in the 100 µg‐group and eight (57%) in the 250‐µg group reported fever.

The 25 and 100 µg doses generated CD4 T‐cell responses stimulated by S protein‐specific peptide pools; the responses were strongly biased towards Th1 cytokine expression with minimal Th2 involvement. CD8 T‐cell responses to S‐2P were equal to or greater than the levels reported after the second administration in the 100 µg dose group. In this clinical trial, the duration of the immune response could not be assessed (participants were followed up until Day 57). In summary, this Phase 1 clinical trial showed how, after the first dose, the mRNA‐1273 vaccine was immunogenic; it caused strong antibody responses to full‐length S‐2P and RBD and exhibited dose‐dependent activity. Within 2 weeks after the first vaccination, seroconversion occurred, but the pseudovirus neutralizing activity was lower after a single administration, indicating the need for a two‐dose scheme.

In a Phase 2 trial, Moderna evaluated the safety, reactogenicity, and immunogenicity of two mRNA‐1273 doses administered 28 days apart. Participants (n = 600) were divided into two cohorts and administered either two doses of a placebo (n = 300) or 50 or 100 µg of vaccine (same quantity two times, n = 300). Another Phase 1 study is underway, led by the National Institutes of Health. That study focuses on older adults and has completed enrollment (adults aged 56–70, n = 300, and adults ≥ 71, n = 300). The follow‐up period will last for a year after the second administration.

A Phase 3 clinical trial (coronavirus efficacy) began on July 27, 2020 (Table 2). This was a randomized, placebo‐controlled trial, including approximately 30 000 participants enrolled in the US, to assess the efficacy, safety, and immunogenicity of the mRNA‐1273 vaccine compared to placebo. Participants were adults ≥18 years old who presented with an unknown history of SARS‐CoV‐2 infection at risk of COVID‐19. The enrollment was concluded on October 22, 2020.

On the basis of Phase 1 trial results, the 100 µg dose was chosen as the optimal dose to maximize immune response and minimize adverse reactions. Participants were randomized 1:1 to receive 100 µg of mRNA‐1273 or placebo, stratified by age and comorbidities. The primary endpoint was the prevention of symptomatic COVID‐19; secondary endpoints included prevention of COVID‐19 severe symptoms, defined as the need for hospitalization, and of SARS‐CoV‐2 infection. Primary efficacy was determined by performing an event‐driven analysis based on the number of symptomatic participants with confirmed COVID‐19 2 weeks after the second dose. The first analysis (November 16, 2020) was conducted on 95 cases in the placebo group and 5 cases in the vaccine group; the vaccine efficacy was estimated to be 94.5% (p < 0.0001) and steady across age, gender, race, and ethnicity.

The primary efficacy analysis was extended to 196 cases: 185 COVID‐19 cases were observed in the placebo group and 11 in the vaccine group, indicating a 94.1% efficacy. In a secondary analysis, starting 2 weeks after the first injection, vaccine efficacy was found to be 95.2% (225 cases in the placebo group and 11 in the vaccine group) and 93.6% within SARS‐CoV‐2 positive participants at baseline (187 cases in the placebo group and 12 in the vaccine group).

The severe COVID‐19 cases were analyzed, including 30 participants from the placebo group. One COVID‐19‐related death reported in the study was in the placebo group. The most common adverse reactions included injection site pain/erythema/redness, fatigue, myalgia, arthralgia, and headache; these were reported more frequently by younger adults. After the second dose, the frequency and severity of solicited adverse reactions increased in the vaccine group. Participants in the vaccine and placebo groups reported hypersensitivity reactions (1.5% and 1.1%, respectively) and Bell's palsy (3% and 1%, respectively). The relative incidence of these AEs in the vaccine group was not influenced by age.

In a lentivirus‐based pseudovirus assay, the susceptibility of the alpha variant to convalescent serum and Moderna vaccine recipient serum was investigated. This variant exhibited only a modest reduction in neutralization susceptibility to the Moderna vaccine (twofold average after two doses). The preliminary study that was conducted to assess Pfizer/BioNTech vaccine efficacy against SARS‐CoV‐2 variants was also performed using the Moderna vaccine, and the results were the same. The Pfizer/BioNTech and Moderna vaccines have been shown to induce a 10‐fold increase in neutralizing antibodies after the second dose, suggesting that a twofold reduction in neutralization susceptibility will have a minimal impact on vaccine efficacy. Moreover, it has been suggested that the Moderna vaccine is less efficient against a pseudovirion of the alpha variant (1–2 times less effective) and less efficient against the gamma variant (4–5 times less effective). There are two reports of variable efficacy (3–20 times less effective) against the beta variant. , , , , , , , , , , ,

UNIVERSITY OF OXFORD/ASTRAZENECA

The University of Oxford/AstraZeneca vaccine uses a modified adenovirus, a double‐stranded DNA virus. In particular, the University of Oxford/AstraZeneca employs the chimpanzee adenovirus vector ChAdOx1 (Figure 2A). 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. ,

After deleting E1 and E3, the SARS‐CoV‐2 Wuhan‐Hu‐1 gene was cloned into a viral vector. E1 deletion inactivates the vaccine's potential replication, whereas E3 deletion allows stable incorporation of larger gene sections (up to 8 kb) into the viral vector. The additional sequence encodes the full‐length S protein and was optimized with a tissue plasminogen activator leader sequence. The addition of an adjuvant has not been mentioned, so it is more likely that the efficacy of this vaccine depends on immune recognition of the nonreplicating virus, which, perhaps through its DNA, can activate TLRs within endosomes.

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. 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.

The University of Oxford/AstraZeneca published the results of a Phase 1/2 single‐blind, multicentric (five sites) randomized controlled clinical trial that tested a chimpanzee adenovirus‐vectored vaccine (ChAdOx1 novel coronavirus‐2019 [nCoV‐19], which expresses the SARS‐CoV‐2 S protein) and a control meningococcal conjugate vaccine (MenACWY) (Table 1). Healthy adults (18–55 years old), without a history of SARS‐CoV‐2 infection or COVID‐19‐like symptoms, were randomly chosen (1:1) to receive ChAdOx1 nCoV‐19 at a dose of 5 × 1010 viral particles or a single dose of MenACWY.

From April 23 to May 21, 2020, 1077 volunteers were enrolled to receive either ChAdOx1 nCoV‐19 (n = 543) or MenACWY (n = 534). In the ChAdOx1 nCoV‐19 boost group, local and systemic reactions were more common, including pain, malaise, mild fever, muscle ache, chills, and headache (all p < 0.05), which were reduced by preventive paracetamol. No serious effects related to ChAdOx1 nCoV‐19 were reported. In the ChAdOx1 nCoV‐19 group, S protein‐specific T‐cell responses peaked on Day 14, and anti‐S protein immunoglobulin G (IgG) responses peaked by Day 28. Neutralizing antibody response was detected (after the first dose) in 91% and 100% of the participants when measured with the MNA80 assay and PRNT50, respectively. After the boost dose, participants showed 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 (Table 2). The University of Oxford/AstraZeneca vaccine phase 2 study enrolled 560 volunteers, divided into three groups: adults aged 18–55, 56–69, and over 70. The study aimed to evaluate the immune response and detect any variations in older people (over 70) or adults (18–55 and 56–69). Adult volunteers were randomly assigned to receive one or two doses of either the ChAdOx1 nCoV‐19 or the MENACWY vaccine. First, participants designated to receive a low dose (2.2 × 1010 virus particles) were randomized to receive either ChAdOx1 nCoV‐19 or MENACWY; block randomization was used and participants were stratified by age, dose group, and study site. The booster vaccine dose was given 28 days after the first dose. The remaining participants were designated to receive the standard dose (3.5–6.5 × 1010 virus particles) and the same randomization protocol was used, except that the 18–55‐year‐old group was randomized to a 5:1 ratio (two doses of ChAdOx1 nCoV‐19 or MENACWY). Participants and investigators were masked to vaccine allocation; only the administering staff knew the randomization. Between May 30 and August 8, 2020, 560 participants were enrolled: 300 in the low‐dose cohort (100 in the 18–55 group, 80 in the 56–69 group, and 120 in the over 70 group) and 260 in the standard‐dose group (60 in the 18–55 group, 80 in the 55–69 group, and 120 in the over 70 group).

Within the groups that received two standard doses of ChAdOx1 nCoV‐19, after the first dose, local reactions were reported in 88% of younger adult participants (n = 49), in 73% of the 56–69 group (n = 30), and in 61% of the over 70 group (n = 49). Seven days after the first dose of ChAdOx1 nCoV‐19, the incidence of fever was lower in the 18–55 group (24%), whereas in the other groups, no fever was recorded.

Participants across the three age cohorts who received two doses showed similar median anti‐S protein IgG responses and neutralizing antibody titers after 28 days. Moreover, 2 weeks after the second dose, more than 99% of the participants showed neutralizing antibody responses. On Day 14, a T‐cell response peak was observed, but after the boost vaccination, there was not a significant increase. Participants who received two standard doses of the vaccine showed a remarkable difference across age groups, with the 56–69 group displaying higher responses at Day 42 than the other groups. Limitations of this study refer to the single‐blind design used to assess vaccine efficacy and safety.

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. 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 (timeframe: from Days 1 to 730); and reactogenicity of the vaccine compared to placebo.

Protection was reported as 90% (n = 2741) in an analysis performed on 3000 people in a single‐dose regimen, where ChAdOx1 nCoV‐19 was given as a half dose followed by a full dose after 1 month. Another dosing regimen was tested on 8895 participants, where AZD1222 was given in two full doses after 1 month; this showed 62% efficacy. The analysis of both dosing schemes (n = 11 636) showed an average efficacy of 70% (p < 0.0001). The higher protection afforded by the half/full dose regimen represented a considerable complication because it was an undesigned error. Thus, the US FDA suggested that the University of Oxford/AstraZeneca undertake supplementary studies to validate these results. No serious AEs were reported, and ChAdOx1 nCoV‐19 was tolerated in both dosing schemes. Moreover, the Phase 3 study was paused on September 6, 2020, after a UK participant presented with a suspected serious adverse reaction (neurological reaction). Trials restarted in late October 2020.

On January 29, 2021, the European Medicines Agency (EMA) recommended the University Oxford/AstraZeneca vaccine for a conditional marketing authorization to prevent the spread of SARS‐CoV‐2 in people ≥18 years old. The vaccine's safety has been demonstrated across four clinical trials in the UK, Brazil, and South Africa. However, the EMA based its calculation of how well the vaccine worked on the COV002 study (conducted in the UK) and COV003 study (conducted in Brazil) results. The remaining two studies had a limited number of COVID‐19 cases (fewer than six in each), which was not enough to measure the vaccine's efficacy. In participants who received two standard doses, a 59.5% reduction in the number of symptomatic COVID‐19 cases was shown (64 cases out of 5258 participants in the vaccine group and 154 cases out of 5210 participants in the control group). The majority of the participants were aged 18–55 years old. To date, no sufficient results are available to show how well the vaccine works in older participants (over 55 years old). However, based on the results of trials with other vaccines, protection in older participants is expected. There is also reliable information on safety in this population. Therefore, the EMA determined that vaccines could be used in older adults.

The COVID‐19 Genomics United Kingdom Consortium, the AMPHEUS Project, and the Oxford COVID‐19 Vaccine Trial Group released the results of their exploratory analysis of the Phase 2/3 randomized controlled trial. This study explored the efficacy of the University of Oxford/AstraZeneca vaccine against the alpha variant. The data indicate that the vaccine will not need modifications to protect against the variant (71% efficacy). Another study showed poor efficacy of the vaccine against the beta variant (22% efficacy); for this reason, South Africa (where the 501Y.V2 variant dominates) suspended the use of the University of Oxford/AstraZeneca vaccine. Results about vaccine efficacy against the gamma variant have not yet been published.

STORAGE

The three vaccines detailed in this review have different storage requirements because of differences in RNA and DNA stability and in their specific formulations. Due to the instability of mRNA, both of the mRNA vaccines need to be stored at low temperatures; indeed, mRNA is more susceptible to degradation than DNA because it is single‐stranded. In contrast, the adenovirus‐based University of Oxford/AstraZeneca vaccine allows the use of simple storage conditions with a temperature range of 2–8°C. The advantages of this vaccine storage temperature have multiple repercussions on production, stock, distribution, and administration.

The Pfizer/BioNTech vaccine has been shown to be stable at −80°C for 6 months, ensuring the possibility of stocking the product for long periods.

Moderna affirmed that its vaccine is stable at 2–8°C for up to 30 days; this has been supported by further tests that extended the previous estimate of 7 days. The Moderna vaccine storage temperature of −20°C is sufficient to maintain its vaccine activity, perhaps because some components in the LNPs stabilize lipid dispersion and prevent particle agglomeration. The possibility of stocking the vaccine at a common freezer temperature permits favorable management of worldwide vaccination.

FURTHER OPPORTUNITIES

Even if the review focuses on the first three vaccines approved, others could be available and distributed to the population as the cited vaccines in the background section. In fact, the Ad26.CoV2.S by J&J is a viral vector vaccine deriving from a human adenovirus serotype 26, encoding full‐length S‐protein, which shows the great advantage of a single‐dose regimen.

A single dose of Ad26.COV2.S elicited a strong humoral response in a majority of vaccine recipients, with the presence of S‐binding and neutralizing antibodies in more than 90% of the participants, regardless of either age group or vaccine dose. Other Ad26‐based vaccines, including an approved Ebola vaccine, are safe and have induced durable immune responses.

Sadoff et al. reported an efficacy rate of 66.1% in 28 days in South Africa and it should be stressed that the low percentage of efficacy was due to the wide diffusion of South Africa variant in the study population.

The Sputnik vaccine is a viral vector too, but carrying a full‐length S protein by two adenoviral vectors, Ad26 and Ad5, using Ad26 in the first dose and Ad5 in the second one to get a 91.6% efficacy against SARS‐CoV‐2 infection. NVX‐CoV2373 is a protein subunit vaccine instead, including a recombinant full‐length prefusion S protein, needing 2 doses to get an 89.7% efficacy (after 7 days from the second dose).

From a storage point of view, all vaccines listed are stable for 6 months at a fridge temperature (2–8°C), apart from the J&J's which is stable for 3 months. Vaccines produced in China, such as CoronaVac and BBIBP‐CorV, do not show Phase 3 studies published yet; but, from the clinical studies available, it is possible to understand their differences in terms of technology (they are inactivated virus from CN02 and HB02 strain of SARS‐CoV‐2, respectively). Both vaccines need a second‐dose regimen to reach the preliminary efficacy values disseminated, which are still ongoing. ,

VACCINE‐INDUCED THROMBOTIC THROMBOCYTOPENIA

Vaccine‐induced immune thrombotic thrombocytopenia (VITT, also known as thrombosis with thrombocytopenia syndrome) emerged in February 2021, initially described as occurring sporadically in populations vaccinated with the University of Oxford/AstraZeneca COVID‐19 vaccine. Reports indicated that after receiving the vaccine, normally healthy patients developed thrombocytopenia and thrombosis in unusual sites (cerebral and/or splanchnic veins). Two months later, similar complications were described in patients administered with the J&J adenoviral vaccine. From March 2021 besides, cases of mRNA vaccine‐induced VITT have started to be published  ,

Case reports and case series are rare, and research is restricted, but the understanding of VITT's epidemiology, pathophysiology, diagnosis, and treatment is growing. 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. 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. ,

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. Nevertheless, VITT is a morbid condition with a high death rate. Twenty‐six percent to eighty percent of published case series include cerebral hemorrhage as the primary cause of death. , , , , , , , Twenty percent of patients with VITT die, likely because of the delayed detection of clinical symptoms and signs by individuals with VITT and/or healthcare professionals. , , , , , , , Future instances should help clarify clinical understanding and enhance clinical outcomes given the greater awareness and recognition of this illness syndrome by both doctors and the public.

CONCLUSION

Pfizer–BioNTech, Moderna, and the University of Oxford/AstraZeneca have now completed their Phase 3 clinical studies and received authorization from the FDA in the US and the EMA in Europe (with some exceptions, e.g., the University of Oxford/AstraZeneca vaccine has not been approved in Switzerland and in the USA). From the initial Phase 1 Study (April 2020) to the end of the Phase 3 Studies (December 2020), comprehensive clinical studies have been completed in just 8–9 months. This rapid development of new COVID‐19 vaccines has been possible because in the last 20 years there have been rapid improvements in vaccine development in different scientific areas, such as molecular biology, genetic engineering, nanomaterials, and lipid nanotechnology.

SARS‐CoV‐2 variants could rapidly spread, impacting the vaccines’ efficacy and the risk of massive reinfection. Multiple studies have investigated the efficacy of the vaccines against variants circulating in the UK, South Africa, and Brazil. The results indicate that the Pfizer/BioNTech and Moderna vaccines are efficient against the variant in the UK and less efficient against the variant in Brazil. The data on the efficacy of these two vaccines against a variant circulating in South Africa is variable, whereas the University of Oxford/AstraZeneca vaccine is not effective against the variant in South Africa. Furthermore, the results of the efficacy of the University of Oxford/AstraZeneca vaccine against Brazilian variants have not yet been published.

Pfizer/BioNTech and Moderna used analogous procedures to produce stable prefusion state mRNA. The mRNA stabilization prolongs the alternatively short half‐life of RNA and thereby boosts the S protein expression level. Moreover, mRNA‐based vaccines have an advantage over DNA vaccines, because mRNA does not integrate and poses no risk of insertional mutagenesis. A further advantage of using mRNA‐based vaccines is that the mRNA is translated directly in the cytoplasm (i.e., endoplasmic reticulum) on the ribosomes once inside the cell. Technological and scientific advances now allow the encapsulation of mRNA into custom‐designed LNPs (instead of a modified adenovirus) that mimic the structural features of a viral vector. Previously, the use of a nanolipid carrier for delivering genetic material (DNA or RNA) did not achieve high efficiency because it was difficult to mimic the complex machinery evolved by viral vectors. The nanoparticles containing mRNA travel a short distance within the cell, easily reaching the endoplasmic reticulum outside the nucleus and avoiding the difficulty of penetrating the nuclear membrane. For these reasons, Pfizer/BioNTech and Moderna used the lipid nanovector with high efficiency.

The trial resulting from the conditional marketing authorization for the University of Oxford/AstraZeneca vaccine showed a 59.5% reduction in the number of symptomatic COVID‐19 cases (64 cases out of 5258 participants in the vaccine group vs. 154 cases out of 5210 participants in the control group). There are not enough results yet to show how well the vaccine works in older participants (over 55 years old); however, when the experience in the UK and the immune response seen in older participants are taken into account, protection in this age group is expected. This is why the EMA also approved the use of the vaccine in older adults.

A EUA is a mechanism to facilitate the availability and use of medical countermeasures, including vaccines, during public health emergencies, such as the current COVID‐19 pandemic.

Among the different vaccine candidates developed by many companies and universities, the Pfizer/BioNTech and Moderna vaccines emerged as favorable candidates for the prevention of COVID‐19. BNT162b2 vaccine was approved by FDA in August 2021. In the UK, the Pfizer/BioNTech vaccine was approved for commercialization by the Medicines and Healthcare Products Regulatory Agency on December 2, 2020. The vaccine received a EUA by the FDA on December 11, and by the EMA on December 21, 2020. The Moderna vaccine was the second vaccine candidate to receive a EUA from the FDA on December 18, 2020, and by the EMA on January 15, 2021. The University of Oxford/AstraZeneca vaccine received a EUA on January 29, 2021, by the EMA, with some limitations. 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.

CONFLICT OF INTERESTS

The authors declare that there are no conflict of interests.

AUTHOR CONTRIBUTIONS

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.