Current Clinical Trials Protocols and the Global Effort for Immunization against SARS-CoV-2

Coronavirus disease 2019 (COVID-19) is the biggest health challenge of the 21st century, affecting millions of people globally. The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has ignited an unprecedented effort from the scientific community in the development of new vaccines on different platforms due to the absence of a broad and effective treatment for COVID-19 or prevention strategy for SARS-CoV-2 dissemination. Based on 50 current studies selected from the main clinical trial databases, this systematic review summarizes the global race for vaccine development against COVID-19. For each study, the main intervention characteristics, the design used, and the local or global center partnerships created are highlighted. Most vaccine developments have taken place in Asia, using a viral vector method. Two purified inactivated SARS-CoV-2 vaccine candidates, an mRNA-based vaccine mRNA1273, and the chimpanzee adenoviral vaccine ChAdOx1 are currently in phase III clinical trials in the respective countries Brazil, the United Arab Emirates, the USA, and the United Kingdom. These vaccines are being developed based on a quickly formed network of collaboration.


Introduction
In December 2019, in the Chinese city of Wuhan in China's Hubei province an outbreak related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-that is, a virus genetically close to bat-CoV-RaTG13 and bat-SL-CoVZC45-emerged, leading to coronavirus disease 2019 . This infectious disease caused an unprecedented health emergency, which was declared a pandemic by the World Health Organization (WHO) on 11 March 2020 [1].
Subsequently, new approaches to vaccine development or the adoptive transfer of immunity were rapidly implemented in preclinical and clinical studies. These studies aimed to avoid infection and prevent the symptoms of COVID-19, a disease of heterogeneous symptoms caused by SARS-CoV-2.
As of the end of June 2020, this virus had affected about 10 million people globally and killed about 560,000 individuals [2].
Currently, there is not yet a consolidated protocol or therapeutic strategy approved for COVID-19 critical patients [3]. At this moment, the best practices for the supportive management of respiratory acute hypoxemic failure must be used.
The initial treatment of COVID-19 was restricted to overall supportive care and critical care because no other appropriate therapies or vaccines existed. However, many clinical trials are investigating the effect of anti-inflammatories, antimalarials, antivirals, plasma therapy, cardiovascular drugs, antibiotics, cell therapy, and cancer drugs among other medications for COVID-19 treatment [4,5]. Dexamethasone and anticoagulant are suggested since some severe, critical, and deceased COVID-19 patients have significant coagulation dysfunction with increased D-dimer concentration, decreased platelet counts, and the prolongation of prothrombin time [6]. Regarding the prophylactic use of Bacilo Calmette-Guérin (BCG) (Pasteur Institute at Lille, France) vaccine against SARS-CoV-2, it was observed that previous BCG (Lille, France) immunization correlates with a lower incidence and gravity of the COVID-19 disease across different countries, even when the BCG (Lille, France) immunization was performed in childhood. Antimalarials such as chloroquine and hydroxychloroquine are being extensively investigated, but no strong evidence of their benefits has been released yet. Thus, there are no specific mechanisms or robust evidence of the efficacy of these therapeutic procedures [7].
The advent of nanotechnology enables different mechanisms to target viruses-for instance, through the use of acid functionalized multi-walled carbon nanotubes comprising photo-activated molecules [56], or natural (such as chitosan) or synthetic (such as polyethyleneimine (PEI)) polymeric nanoparticles [57]. These kinds of nanomaterials are able to work as gene carriers and have the potential to deliver small interfering RNA (siRNA) in COVID-19 patients [57]. In addition, nanotechnology has enabled the development of new vaccines, including vaccines based on recombinant protein nanoparticles with or without Matrix M TM adjuvant [42], and mRNA vaccines through the encapsulation of modified nucleoside mRNA inside lipidic nanocapsules [17][18][19][20]58], which confers plasticity in terms of antigen manipulation and a potentially rapid effect. Intense efforts are underway to expand the technological knowledge against COVID-19 as part of a global scientific effort to battle this virus. However, to date no review has been conducted about this global development effort and the associated cooperation networks used to test new vaccines. This lack of information empowers the movements of anti-vaccine groups [59].
Nanotechnological mRNA-1273 arises in clinical trials just two months following SARS-CoV-2 sequence identification and is currently one of the most advanced vaccines already in phase III clinical trial protocols (CTPs) [55]. Viral vector platforms enable protein overexpression, and their long-term stability can induce robust immune responses with a single dose. At present, the chimpanzee adenoviral vector from Oxford (ChAdOx1) is another advanced vaccine in study phase (II-III) [51,52] and phase III [50]. In addition, recombinant protein subunits, as immunologic adjuvants that use a squalene (TLR4) agonist [60], previously licensed to be delivered as vaccines for other conditions, can provide apparatus for the necessarily large-scale production of the newly developed vaccines [61].
The majority of the recently developed vaccines aim to stimulate antibody synthesis with the necessary potential to neutralize the SARS-CoV-2 spike protein (S protein) to prevent the uptake of the single-strand RNA of this virus in ACE2 receptor positive cells [62].The exceptions are the vaccines using an attenuated/inactivated whole virus, which would lead to broader immunization [10,11,63].

Overview of Clinical Trial Protocols for Active Immunization for Covid-19
The selected clinical trials focused on interventional approaches for active immunization through the application of the various types of vaccines currently being developed against SARS-CoV-2 ( Table 1).
The distribution of these CTPs over the time by phase ( Figure 1) shows that the phase I studies started in February. The number of new studies by month ( Figure 1A) shows that the number of CTPs in phase I was triplicate and quadruplicate in March and April, respectively, as did the beginning of phase II and phase II-III concomitant studies. In May, the number of studies at phase I reduced to a single CTP, and the I-II phase studies declined by half compared to the previous month. In addition, a new study started phase III. In June, the total number of new CTPs was the same as presented in April, but half of the studies were phase I and the other half were phase I-II. In July, as well as in June, there were six new studies at phase I-II and a study in phase II, but the number of CTPs at phase I decreased by 33.3%. There is a clear oscillation in the number of CTPs at phase I over the months, less marked in CTP at phase I-II. At the same time, there occurred an increased number of new studies in phase III.
A cumulative analysis of the number of CTPs by phase ( Figure 1B) shows a continuous increase in phase I-II CTPs, which also occurred in phase I studies with a slight oscillation between April and May. On the other hand, cumulatively, the number of phase II, II-III, and III studies has remained constant since March, with a slight increase in phase III CTPs between June and July.

Nucleic Acid Vaccines
Regarding the CTPs targeting vaccine development and application, their platforms display a variety of characteristics and properties ( Figure 2; Table 1). Nucleic acid vaccines are the ones most tested in CTPs at this moment (32%). They are based on a genetically engineered plasmid containing DNA (12% of the CTPs) or RNA (as mRNA and saRNA, comprising 20% of the CTPs) ( Figure 2) usually encoding a selected antigen (mainly S protein).

Nucleic Acid Vaccines
Regarding the CTPs targeting vaccine development and application, their platforms display a variety of characteristics and properties ( Figure 2; Table 1). Nucleic acid vaccines are the ones most tested in CTPs at this moment (32%). They are based on a genetically engineered plasmid containing DNA (12% of the CTPs) or RNA (as mRNA and saRNA, comprising 20% of the CTPs) ( Figure 2) usually encoding a selected antigen (mainly S protein).

Nucleic Acid Vaccines
Regarding the CTPs targeting vaccine development and application, their platforms display a variety of characteristics and properties ( Figure 2; Table 1). Nucleic acid vaccines are the ones most tested in CTPs at this moment (32%). They are based on a genetically engineered plasmid containing DNA (12% of the CTPs) or RNA (as mRNA and saRNA, comprising 20% of the CTPs) ( Figure 2) usually encoding a selected antigen (mainly S protein).
BNT162 by the BioNTech (BNT) and Pfizer companies embrace different RNA vaccine candidates, comprising two nucleoside modified mRNA (modRNA) vaccines, an uridine containing mRNA (uRNA) vaccine, and a saRNA vaccine. Besides this, two of them codify full-length S protein, and the others codify S protein receptor binding domain (RBD) [68]. Initially, studies with BNT were developed with the four mentioned BNT162 vaccines; however, more recently a CTP with China as the recruiting country applied only the BNT162b1 vaccine [14]. Other CTPs of the same vaccine are recruiting patients in Germany [19] and the USA [18]. BNT162 is being administered in a prime/boost regimen [14,15,18,19], in which the same immunogen is applied in the prime, and the booster CTP objective is to observe the immunogenicity of the subjects against S protein and RBD through IgG titration in healthy individuals [14,15,18,19].
The CTPs using mRNA1273 by Moderna TX Inc are all being completely developed in the USA [17,20,55]. This vaccine encodes full-length S protein. At phase I, this study tested different concentrations of mRNA at 10, 25, 50, 100, and 250 µg in different interventional arms [17]. In the phase III intervention, the established dose was 100 µg via intramuscular administration in two doses 28 days apart in healthy individuals over 18 years old [55].
The phase I German CureVac´s novel coronavirus (CVnCoV) CTP [16] and another mRNA vaccine CTP recruiting in China [13] encode an unspecific protein and an RBD S protein of SARS-CoV-2, respectively, but there is no information about the delivery systems. Recently, an vaccine based on self-amplifying RNA encapsulated by lipid nanoparticle (LNP-nCoVsaRNA) phase I used saRNA encoding S protein in the United Kingdom, which will produce more antigen per transfected cell due to the replicative element [21].

DNA Vaccines
DNA vaccines [22][23][24][25][26][27] were used in 12% of CTPs with different delivery systems. Plasmids encoding S protein (pGX9501) are delivered with CELLECTRA@2000 electroporation system in a phase I trial in the USA, with a 0.5 mg/dose and 1 mg/dose [27], and in a phase I-II trial in the Republic of Korea, with a 1 mg/dose and 2 mg/dose delivered in a population ranging from 18 to 64 years old in sequential assignment [24] in two doses, as shown in Table 2.
The Canadian bacTRL-Spike-1 vaccine uses a genetically modified probiotic Bifidobacterium longum as a delivery system for a synthetic DNA plasmid encoding S protein to host immune cells in a phase I study; this is the first time that bacTRL has been delivered in humans orally with varied doses in parallel assignment containing 1, 3, or 10 billion colony-forming units [26].
The GX-19 vaccine plasmid in a phase I-II trial in the Republic of Korea by Genexine, Inc. Company (Gyeonggi, Korea) [25] is applied via intramuscular administration in a dose-escalation protocol 28 days apart. A second COVID-19 Indian vaccine ZYCOV-D by Cadila Healthcare Ltd. (also known as Zydus Cadila, Ahmedabad, India) is testing the vaccine in two groups with different age ranges, one from 12 to 55 and another from 12 to 65 years old. In both groups, the doses of 0.5 mL are delivered intramuscularly 14 days apart [22]. The CTP for the Japanese AG0301 DNA vaccine delivers the doses in the same period of time as ZYCOV-D [23], with a population ranging from 20 to 65 years old.
The Russian Gam-COVID-Vac by Gamaleya Research Institute, which combines Ad5 and a recombinant adenovirus type 26 (Ad26) vectored system targeting the S protein of SARS-CoV-2 [35,36], is in phase I-II. In one of these CTPs, the Gam-COVID-Vac solution is lyophilized [36]. The most advanced ChAdOx1 nCoV-19 protocol is delivered in a single dose of 5 × 10 10 viral particle [50,51], which is being tested two times on days 1 and 28 [51]. A clinical trial divided the groups accordingly to the age of the participants: children ranging from 5 to 12, adults from 18 to 55, and people older than 56 years of age [52]. In turn, the age used in two most advanced Adn5-nCoV CTPs is older than 18, with single doses of 5 × 10 10 and 1 × 10 11 in a study with crossover assignment [32] and parallel assignment [29], both with three interventional arms, as shown in Table 2.

Inactivated Virus Vaccines
The inactivated SARS-CoV-2 vaccine was used in 18% of the selected clinical trials ( Figure 2) [8][9][10][11]53,54,66,67]. The vaccine developed by SinoVac Biotech Corporation is classified in the CTPs as purified inactivated SARS-CoV-2 and is an adsorbed vaccine. According to preclinical study information, this vaccine, named PiCoVacc, was developed by propagating the CN2 strain of SARS-COV-2 inside VeroCells and inactivated using the chemical agent β-propiolactone [69]. This Chinese vaccine is being tested in Brazil in a phase III study [54] and in a phase III CTP developed in the United Arab Emirates [53]. Another purified inactivated vaccine also propagated SARS-CoV-2 inside VeroCells [8,9,53], but there is no information about the strain or inactivating agent used in pre-clinical studies. A recent phase I-II Indian vaccine BBV152 will apply three inactivated whole-virion strains designated BBV152A, BBV152B, and BBV152C [66]. Another two studies developing purified inactivated SARS-CoV-2 vaccine provides no information about the inactivation processes [12,67]. All these vaccines target multiple proteins of SARS-CoV-2. The most advanced inactivated vaccine (phase III) is applied in two doses 14 days apart in the deltoid muscle [54] in participants of 18 years onwards, as shown in Table 2.

Protein Subunit Vaccines
Vaccines based on the recombinant protein subunit are being tested in 16% of the selected CTPs ( Figure 2) [37][38][39][40][41][42][43][44], identified by different names according to their features. The most advanced of them (phase II) apply SARS-CoV-2 protein subunits engineered, produced, and secreted by Chinese Hamster Ovary (CHO) cells [39,41]. Phase I SCB-2019 vaccine is a recombinant SARS-CoV-2 trimeric S protein subunit vaccine, and is tested with or without lipid-based Adjuvant System 03 (AS03) that increases the reaction of the innate immune system, or with a oligonucleotide sequence containing CpG motif (CpG 1018) adjuvants plus aluminum, with a CTP being developed in Australia [43]. Another Australian phase I vaccine is NVX-CoV2373, a recombinant S protein nanoparticle vaccine tested with or without the Novavax saponin-based Matrix M TM adjuvant [42], COVAX-19 TM , a Vaxine proprietary Advax™ adjuvant technology combined with a recombinant S protein [38,44] and a S protein stabilized using the molecular clamp technology applied with MF59 immunologic adjuvant [37]. A CTP is being developed based on the production of the RBD of S protein through fast-growing tobacco plant technology [40]. The use of adjuvants increases the activity of T and B cells mediated by antigen-presenting cells (APCs) [70].

Dendritic Cells Vaccines
Another 6% of clinical trials (phase I-II) are testing vaccines based on dendritic cells (DC) (Figure 2) [45][46][47]. One of them, from the USA, is testing subcutaneously autologous DC loaded with 1× or 3.33% antigens from SARS-CoV-2 with or without 250 µg or 500 µg of granulocyte-macrophage colony-stimulating factor (GM-CSF), targeting non-specific proteins. This vaccine is developed with the isolation of monocytes from heparinized blood incubated with IL-4 and GM-CSF that are differentiated in vitro in DC, which in turn are incubated with SARS-CoV-2 antigens [46].
China is testing subcutaneously a recombinant chimeric DC vaccine targeting an unspecified SARS-CoV-2 epitope. This vaccine, called LV-SMENP DC, is applied subcutaneously (5 × 10 6 ) with intravenous antigen-specific cytotoxic T cells (10 8 ). The subjects will be analyzed once a week for four weeks. LV-SMENP DC was developed with a technology that modifies these cells through the lentiviral vector system NHP/TYF [45].
The same lentiviral vector system NHP/TYF was able to modify artificial antigen presentation cells (aAPC) that represent 2% of CTPs [48]. All these lentiviral modified cells encode multiple proteins of SARS-CoV-2, and both CTPs are being developed in China [45,48].

Virus Like Particles Vaccines
Besides this, a phase I study using VLP (2% of the selected CTPs) is recruiting patients in Canada ( Figure 2) [49]. This vaccine is administered 21 days apart via intramuscular injection. Each dose is applied to the deltoid region of alternated arms. The doses being tested are initially 3.75 µg in a small number of the population (healthy adults 18 to 55 years of age) with dose-escalation to 7.5 µg and 15 µg unadjuvanted or adjuvanted with either CpG 1018 or AS03, with a follow-up period of six months. After this time, the immunogenicity of the subjects will be tested.

Global Research Network in Clinical Trial Protocols
The distribution of vaccine CTP networks between the sponsor and the collaborating institutions is highlighted in the Figure 5 map, focusing mainly on the CTPs collaborating with more than five centers. Among the vaccines leading multicenter CTPs, there is one in the USA controlled by ModernaTX Inc. at phase III (green circle) coordinating 87 collaborating institutions (green cylinders around the red bar of Figure 5), with an estimated enrollment of 30,000 participants, not yet recruited [55] (as detailed in Tables 1 and 2). In addition, there is another CTP in the USA at phase II (blue circle) recruiting 600 volunteers from 10 centers (blue cylinders of Figure 5) [20]. Two CTPs in Europe are controlled by Oxford University in partnership with AstraZeneca [51,52], coordinating more than 20 collaborating institutions (hospitals, labs, etc., represented by the yellow and purple cylinders in Figure 5) involving approximately 10,260 participants each, at phase II or III (green-blue circle). There is another CTP at phase I-II (red-blue circle) that is recruiting 1090 volunteers at six centers in the United Kingdom [34] (gray cylinders in Figure 5). The CTP in South America is controlled by Butantan Institute in partnership with the Sinovac Life Sciences Co. (Beijing, China) [54], and is at phase III (green circle), coordinating 12 centers in Brazil (dark green cylinders in Figure 5) and

Global Research Network in Clinical Trial Protocols
The distribution of vaccine CTP networks between the sponsor and the collaborating institutions is highlighted in the Figure 5 map, focusing mainly on the CTPs collaborating with more than five centers. Among the vaccines leading multicenter CTPs, there is one in the USA controlled by ModernaTX Inc. at phase III (green circle) coordinating 87 collaborating institutions (green cylinders around the red bar of Figure 5), with an estimated enrollment of 30,000 participants, not yet recruited [55] (as detailed in Tables 1 and 2). In addition, there is another CTP in the USA at phase II (blue circle) recruiting 600 volunteers from 10 centers (blue cylinders of Figure 5) [20]. Two CTPs in Europe are controlled by Oxford University in partnership with AstraZeneca [51,52], coordinating more than 20 collaborating institutions (hospitals, labs, etc., represented by the yellow and purple cylinders in Figure 5) involving approximately 10,260 participants each, at phase II or III (green-blue circle). There is another CTP at phase I-II (red-blue circle) that is recruiting 1090 volunteers at six centers in the United Kingdom [34] (gray cylinders in Figure 5). The CTP in South America is controlled by Butantan Institute in partnership with the Sinovac Life Sciences Co. (Beijing, China) [54], and is at phase III (green circle), coordinating 12 centers in Brazil (dark green cylinders in Figure 5) and recruiting 8870 participants. In Asia, the CTP controlled by Bharat Biotech International Ltd. Is at phase I-II of vaccine development, recruiting 1125 participants in 11 centers in India [66]. In Africa, the vaccine center coordinated by the University of Witwatersrand phase I-II (red-blue circle) is recruiting 2000 volunteers in six centers in South Africa [33]. All these centers leading vaccine multicenter CTPs are highlighted with enlarged figures around the map in Figure 5.
However, other centers for vaccines against COVID-19 in development in Europe, Asia, Africa, the Americas, and Oceania are highlighted in the map of Figure 5.
However, there are already 4 of 50 (8%) CTPs at phase III [50,[53][54][55]. Two of them are in Brazil-one is testing the vaccine developed by the University of Oxford [50] at Universidade Federal de São Paulo (UNIFESP), and other is testing the vaccine developed by Sinovac Life Science Co. at Butantan Institute [54]; one is in the United Arab Emirates, and is testing the vaccine developed by China National Biotec Group Co. at Beijing [53]; and one in the USA, testing the vaccine developed by ModernaTX, Inc. [55], as shown in Figure 6E.
Only 6 of 50 CTPs (12%) comprise a network with other institutions located in different countries. These include the United Kingdom and Brazil [50], China and the United Arab Emirates [53], Canada and China [30], the USA and the Republic of Korea [24], the USA and Australia [42], and Germany and the USA [18]. Four studies do not reveal about their partnerships [15,40,41,44]. The most CTPs (82%) have entered into partnerships with other institutions within the same country.

Discussion
Approximately four months after a pandemic was declared by the WHO and more than 640,000 deaths have occurred, a consolidated therapeutic drug strategy protocol approved for COVID-19 still does not exist [71]. At this moment, the epicenter of the outbreak is the American continent, mainly USA and Brazil, and isolation or social distancing remain the central WHO recommendations to avoid infection and increasing numbers of deaths [72].
Unprecedented global initiatives and new partnerships are being established to accelerate the research and development of tests and therapies to control the spread of this pandemic around the world. However, vaccines that mitigate the damage caused by this infectious disease can take much longer to be available with proven safety and efficacy [73]. The time frame for vaccine supply remains

Discussion
Approximately four months after a pandemic was declared by the WHO and more than 640,000 deaths have occurred, a consolidated therapeutic drug strategy protocol approved for COVID-19 still does not exist [71]. At this moment, the epicenter of the outbreak is the American continent, mainly USA and Brazil, and isolation or social distancing remain the central WHO recommendations to avoid infection and increasing numbers of deaths [72].
The current landscape of COVID-19 vaccine development shows that most CTPs are being developed in Asia (Table A1). However, regarding former CTPs, only 1 of 4 (15%; [53]) phase III CTPs are located in Asia. There is one CTP recruiting in Abu Dhabi/United Arab Emirates, there is a protocol that was developed in China with an inactivated virus platform, and this protocol was also used in another phase III CTP in Brazil [54]. The virus vector platform vaccine developed in the United Kingdom (Oxford University) is being tested in the most advanced studies (phase II-III), both in the United Kingdom [51,52] and Brazil [50] (phase III). These CTPs (8% of total CTPs) have used the same chimpanzee adenoviral vectored vaccine targeting the S protein of SARS-CoV-2 [50][51][52], in which the recent results show safety and a strong immune response [74,75], but these CTPs differ due to the number of interventional arms testing different vaccine doses (number and concentration), the group age of volunteers, the method of dosing analysis (Abs260, Abs260 corrected for PS80 and qPCR), and other comparisons. Lastly, the mRNA vaccine from Moderna TX, Inc., (Cambridge, MA, USA) is a USA phase III CTP that is the biggest trial, with 30,000 subjects in 87 recruiting centers (Table A1), but the recruiting is just starting [55]. This vaccine platform used advanced technology to improve the drug delivery.
The gold standard for the success of a vaccine is related to its broad and sustained immunogenicity, with adequate safety and efficacy. For this achievement, the antigen delivery systems and their eligibility are important factors [62,76].
In this review, the non-replicating viral vector was the main technology/platform used (24%). The advantage of the vaccine based on viral vectors is the combined stimulation of the innate and adaptive immune response to the heterologous viral infection and against the antigen expressed by the vector, usually the S protein [78]. The problem related to heterologous response is the previous exposure to some adenovirus serotypes by the human population, leading to a pre-existing anti-vector response, which makes the vector a disadvantage. The use of Ad5 in immunological practice is well established and highly efficient, and exhibits simple manipulation and ease of purification. However, the specific response of the transgene can be mitigated by pre-existing adaptive immune responses to antigenic targets in the vector itself [79]. The previous results of the CTP developed by CanSino confirmed the activation of CD4 and CD8 T cells in the vaccine recipients. However, the vaccine-induced specific antibody or T cell response were partially diminished by the presence of pre-existing anti-Ad5 immunity [80]. On the contrary, the study by Zhu [80] in phase I and the study by Folegatti [75] used an Ad5 vectored COVID-19 vaccine that was very immunogenic, able to induce humoral and cellular responses in most trial participants, and the detectable immune responses was very rapid, with the T cell responses peaking at day 14 after vaccination and the antibodies peaking at day 28.
Therefore, less prevalent adenoviral serotypes, such as Ad35 and Ad26, or primate derivatives, such as chimpanzees, are more often used in vaccine development, as they mimic a natural infection at low risk of herologous response, and stimulate a significant immune response without additional adjuvants [81]. Previous studies using a replication-deficient adenovirus from chimpanzee (ChAdOx1) led to the immunization of BALB/C mice, CD1 mice [82], and rhesus monkeys [83] against MERS-CoV. This adenovirus construct, now encoding the full-length S protein of SARS-COV-2, was able to generate high titers of neutralizing antibodies and a robust CD8 T cell response against this viral protein [82].
DNA or RNA vaccine platforms, also known as synthetic vaccines, were identified in 32% [13][14][15][16][17][18][19][20]55] of the selected CTPs. Vaccines with antigen-encoding DNA/RNA as a platform are relatively versatile, easy to manipulate, and relatively inexpensive to synthesize compared to other types of vaccines. These vaccines have similar strategies of action-for instance, they have coding for the synthesis of target antigens that will be expressed in immune cells. However, the target of each platform is different, in addition to the advantages or risks they present [84].
mRNA based-vaccines provide benefits compared to protein subunits, inactivated or attenuated viruses, and DNA-based vaccines. These benefits were discussed by a study as follows [85]. The first issue is safety, since mRNA is a non-infectious, non-integrating platform, and shows no potential risk of infection or insertional mutagenesis. In addition, mRNA is degraded by regular cellular processes, and its half-life in vivo can be regulated through modifications and delivery methods. The second issue is its efficacy; modifications can make the mRNA more stable and highly translatable. Third, mRNA vaccines have the prospective for fast, inexpensive, and accessible manufacturing due to the high yields of in vitro transcription reactions.
However, this platform is vulnerable in a high mutagenic potential virus [86]. Regarding the SARS-CoV-2 mutagenic potential, a study by Phan [87] revealed many mutations and deletions in coding and non-coding regions, mainly associated with the S protein. Another study [88] identified 11 variations in the SARS-CoV-2 genome which were observed in over 10% of patient isolates from all over the world. Therefore, this topic is still controversial. Nonetheless, this vaccine platform, despite needing adjuvants (other technologies), is more agile and scalable than others. In the selected CTPs, there are seven types of mRNA vaccines: four named BNT162 [14,15,18,19] and three with mRNA1273 [17,20,55], the latter being at a more advanced stage of development. Both types of vaccine consist of LNP encapsulated nucleoside modified mRNA targeting the S protein of SARS-CoV-2. The mRNA-1273 developed by the National Institute of Allergy and Infectious Disease (NIAID) in the USA in collaboration with ModernaTX Inc. [17,20,55] produces the mRNA of S protein in a stable form due to advances in carriers such as LNPs, providing high-efficiency delivery.
Bacteria-derived plasmid-DNA vaccines, in addition to encoding the target antigen, can also encode co-stimulating molecules. These are directed to the nucleus, and need to cross the plasma and nuclear membranes to be activated. Furthermore, they have the possible disadvantage of persistent genomic integration in host cell DNA, leading to subsequent deleterious effects. However, the need for specific delivery systems to achieve good immunogenicity remains a concern. Different methods have been developed to enhance DNA uptake, expression, and immunogenicity (i.e., encapsulation in containing cationic lipids or cholesterol nanoparticles, and adsorption to polymeric or biodegradable nanoparticles), such as the gene gun, needle-free injection devices (jet injection) [89], and in vivo electroporation. This is the case for the INO-4800 vaccine against SARS-CoV-2, currently in clinical trial (phase I) [27], which was administered by intradermal (ID) injection followed by electroporation (EP) using a CELLECTRA ® 2000 device in healthy adult volunteers [27]. The synthesis process of this vaccine involved the alignment of four coding sequences of S protein and the addition of the N-terminal IgE leader sequence. This highly optimized DNA sequence was manufactured and cloned into pGX0001 vector. The resulting plasmids were named pGX9501 and pGX9503 by Inovio Pharmaceuticals [90]. The immunogenicity of INO-4800 was pre-clinically evaluated, and the serum reactivity revealed IgG binding against the S protein of SARS-CoV-2. The serum from INO-4800-immunized BALB/c mice neutralized two SARS-CoV-2 strains-WH-09 (Neutralizing Titer of Antibody (ND50: 97.5) and VIC01 (ND50: 128.1)). In addition, the ND50 of 573.5 was observed in immunized guinea pigs. This study also generated robust S-specific T cell responses in these models, and the detected antibodies were able to block S protein binding to the host ACE2 receptor [90].
Recently (17 June 2020), another DNA vaccine developed in South Korea by Genexine Consortium entered phase I-II clinical trials, in which a preventive GX-19 vaccine will be intramuscularly administrated in healthy volunteers from countries such as Indonesia and Thailand. The company managers expect preliminary data from the initial trial by September 2020 and hope to complete all phases of testing by the end of 2021 [25]. To date, no preclinical studies or technical specifications for the GX-19 vaccine have been released. Hence, DNA and mRNA vaccines are readily designed and can proceed into clinical trials very fast, in addition to outstanding targets for the development of vaccines against SARS-CoV-2 and other related epidemics in the future [91].
Attenuated or inactivated vaccines, known as conventional vaccines, require whole pathogen cultivation and propagation. Thus, it is necessary to obtain a high biosafety level with specialized laboratories and biotechnological tools. Moreover, there is the requirement for lineage cells accepted by regulatory authorities, such as Vero Cells, for the development of industrial-scale inactivated virus vaccines [92]. A total of 18% of the CTPs use inactivated virus vaccine platforms, including a phase III CTP recruiting in Abu Dhabi/the United Arab Emirates [53], and a phase III CTP in Brazil [54]. This approach has been used previously and led to smallpox eradication [93], and vaccines for other diseases such as polio, tetanus, diphtheria, and measles. The clinical trials developed by Sinovac Corporation [10] cultivated the CN2 strain of SARS-CoV-2 in Vero Cells and chemically inactivated them using β-propiolactone. Formaldehyde and UV light are other possible agents for virus inactivation [92]. The process of viral inactivation is delicate and cannot destroy the major virus antigenic structures, which would interfere with their immunogenicity. Depth filtration and optimized steps of chromatography allow vaccine purification [69]. Prior to CTPs, PiCoVacc initiated pre-clinical experiments and administered them with alum adjuvant in BALB/C mice and rhesus macaques, a non-human primate species. This study demonstrated rapid RBD-IgG development, accounting for half of the S protein-induced antibodies produced and possibly the dominant immunogenic part of this protein [69]. Furthermore, no antibody-dependent enhancement (ADE)-mediated vaccine-induced infection aggravation was observed for any vaccinated rhesus macaques [69]. Two other CTPs developed by Chinese Sinopharm Corporation at the Wuhan [8] and Beijing [9] Institutes of Biological Products also expanded SARS-CoV-2 in Vero Cells and are currently in trial. However, no pre-clinical studies or technical specifications of the last two vaccines have been disclosed to date.
Since protein subunit vaccines are restricted to specific epitopes of the virus, most developers have not found proteins other than the S protein used by SARS-CoV-2 to invade cells [94]. Eight clinical trial protocols are analyzing protein subunit vaccines [37][38][39][40][41][42][43][44] combined with different types of adjuvants to help individuals produce an immune response strong enough to protect them from the disease [95]. However, the produced immune responses weaken over time, which means that an individual may require additional shots for booster immunizations throughout their life [94]. The adjuvants used in the selected clinical trial protocols are licensed and approved by the Food and Drug Administration (FDA). The adjuvant CpG1018 increases the body's immune response; the AS03 enhances the vaccine antigen-specific adaptive immune response; and potassium aluminum sulfate (Alum), one of the agents most used as an adjuvant, acts by creating a deposit at the injection site, thus allowing the slow release of antigen and prolonging the interaction time with APCs, in addition to acting on soluble antigens by converting them into particulate forms that are readily phagocyted [95,96].
The global vaccine R&D efforts against SARS-CoV-2 are unprecedented in terms of scale and speed [73]. From examining all the CTPs operating in the United States (clinicaltrials.gov), China (chictr.org.cn), and Europe (clinicaltrialsregister.eu), it is evident that most COVID-19 vaccine CTP activity is taking place in China, with 19 CTPs (38%). However, the greatest advance has been made in the United Kingdom (Oxford University), in conjunction with several collaborating countries (South Africa and Brazil, among others) and other recruiting centers/institutions in the UK (hospitals, universities, labs, etc.). This CTP has reached phase III protocol [50], and will test 12,000 subjects. On 26 July 2020, its performance was 18.6%. Most CTPs (91.2%) have not collaborated with other external institutions due to the protocol phase; only 6 of 50 (12%) CTPs have reached phase III, for which it is necessary to expand the tests in territories with the outbreak, as seen with the Oxford University protocol, and more recently with Sinovac Life Sciences Co., a Chinese vaccine company. The distribution of collaboration networks between sponsors and the recruiting centers/institutions of the CTPs is strongly concentrated in Asia (23.2%), particularly in Wuhan city, China, where the pandemic started. In Europe and the Americas, the United Kingdom, Brazil, and the USA are the main centers, representing 82% of the global vaccine network or total subjects in vaccine tests. As we discussed earlier, these last countries are now the epicenter of the pandemic. Furthermore, the development of vaccine platforms is occurring more rapidly in wide global networks compared to scientific reports or clinical trial databases. Vaccine developers publish very few clinical trial articles, even regarding the safety or preclinical results.
The pandemic caused by COVID-19 has resulted in increased awareness of global threats to human health, particularly when caused by unknown and emerging pathogens. This pandemic has motivated science and health systems to be prepared for future outbreaks. In addition, global development has begun on vaccine platforms and improvements in public and private health systems to tackle the challenges of new outbreaks. New platforms or approaches, such as viral vectors, protein-like vaccines, and nucleic acids, meet the prerequisites for providing solutions to some of these challenges, representing highly versatile technologies that allow rapid vaccine manufacturing. Each vaccine technology has its own advantages and disadvantages related to its ability to induce certain immune responses, manufacturing capacity, and safety for human use.

Conclusions
The race of COVID vaccine remains uncertain, but to date global labs and industries have registered 50 vaccine CTPs in leading research databases, using eight platforms: inactivated virus; nucleic acid, such as mRNA, and DNA/plasmid; recombinant virus vectors; recombinant viral protein subunits; modified dendritic cells; artificial antigen-presenting cell; and VLP vaccine. New groups and laboratories have changed the global vaccine development landscape, especially in Asia. The most advanced Trial does not mean to be the most efficient or safe, the collaborations between countries are still of little significance.

Conflicts of Interest:
The authors declare no conflict of interest.