key: cord-0700661-78ywm3ry
authors: Strizova, Zuzana; Smetanova, Jitka; Bartunkova, Jirina; Milota, Tomas
title: Principles and Challenges in anti-COVID-19 Vaccine Development
date: 2021-02-01
journal: Int Arch Allergy Immunol
DOI: 10.1159/000514225
sha: 18c3dc8ab945dcd658edc94f7b9ee07ad973faf4
doc_id: 700661
cord_uid: 78ywm3ry

The number of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients keeps rising in most of the European countries despite the pandemic precaution measures. The current antiviral and anti-inflammatory therapeutic approaches are only supportive, have limited efficacy, and the prevention in reducing the transmission of SARS-CoV-2 virus is the best hope for public health. It is presumed that an effective vaccination against SARS-CoV-2 infection could mobilize the innate and adaptive immune responses and provide a protection against severe forms of coronavirus disease 2019 (COVID-19) disease. As the race for the effective and safe vaccine has begun, different strategies were introduced. To date, viral vector-based vaccines, genetic vaccines, attenuated vaccines, and protein-based vaccines are the major vaccine types tested in the clinical trials. Over 80 clinical trials have been initiated; however, only 18 vaccines have reached the clinical phase II/III or III, and 4 vaccine candidates are under consideration or have been approved for the use so far. In addition, the protective effect of the off-target vaccines, such as Bacillus Calmette-Guérin and measles vaccine, is being explored in randomized prospective clinical trials with SARS-CoV-2-infected patients. In this review, we discuss the most promising anti-COVID-19 vaccine clinical trials and different vaccination strategies in order to provide more clarity into the ongoing clinical trials.

The rapid spread of coronavirus disease 2019 (CO VID19) has globally become a serious issue [1] . The dis ease, caused by a severe acute respiratory syndrome coro navirus 2 (SARSCoV2), has been first reported in De cember 2019 (Wuhan, China) and declared by the World Health Organization (WHO) as a pandemic on March 11, 2020 [2] . To date, the COVID19associated deaths keep rising in most of the European countries. This phenom enon can be influenced by the pandemic precaution mea sures, such as mandatory face masks wearing; however, the end of the pandemic may not be seen until an effective antiCOVID19 vaccine is developed [3, 4] . SARSCoV2 belongs to the singlestranded RNA viruses which origi nated in bats [5] . SARSCoV2 exhibits the same struc tural and molecular patterns as other coronaviruses, such as structural proteins S (spike), E (envelope), M (mem brane), and N (nucleocapsid) (Fig. 1) [6, 7] . The binding of the virus and its further entry into the host cell through angiotensinconverting enzyme 2 is mediated by the viral S protein. As the S protein is processed by a protease transmembrane protease serine 2, viral fusion with the host cell occurs. The first genetic analyses of the virus re vealed an 89% nucleotide identity with bat virus SARS likeCoVZXC21 [6] . Further investigation, however, confirmed a high similarity (96%) between the SARS CoV2 and the betaCoV RaTG13 of bats [1, 8, 9] . In CO VID19, it has been attempted by multiple studies to identify the intermediate host and on the basis of the available data, pangolins are most likely the mammals serving as a SARSCoV2 intermediate host [10] . The un derstanding and identification of an intermediate host is of major importance. The reason stems from the fact that blocking the humanhost contact may restrain further spread of the novel disease variants, such as the Cluster 5 identified originally in minks. This cluster includes Y453F mutation in the spike protein and was reported among people in Denmark. Additionally, the virus was shown to infect multiple animal species under experimental condi tions, and also few cases of household cats and dogs were reported to be positive for SARSCoV2 RNA. Therefore, concerns regarding the new sources of infection, novel potential viral strains, and uncontrolled outbreaks rise [11] [12] [13] . Moreover, the identification of an intermediate host may allow novel vaccine testing [1] . As a matter of fact, in Middle East respiratory syndrome disease, the first vaccine candidates were tested in dromedary camels [14] . The total number of deaths in COVID19 is affected by the high transmissibility (assessed by the basic repro duction number, R 0 ) [15] . COVID19 is mainly transmit ted through respiratory droplets from sneezing and coughing; however, few other transmission routes have been described, such as alimentary transmission or through conjunctival mucosa [1, 4] . To date, severe and deadly forms of COVID19 have been reported. Underly ing health conditions were seen in cases with lifethreat ening course of COVID19, and thus, the COVID19 pandemic represents an enormous threat for the elderly [16] or chronically ill patients, particularly for those suf fering from severe obesity, CKD, diabetes, arterial hyper tension, or asthma [17] . On the other hand, the viral load is presumably one of the factors that dictate the clinical course of the disease [18] . This may also be affected by the transmission route, and therefore, asymptomatic CO VID19 patients were reported throughout multiple stud ies [19, 20] . Currently, multiple therapeutic approaches are being applied to deal with the infection. However, these approaches are rather supportive, and the preven tion in reducing the transmission is the best hope for pub lic health [1, 5, 21] . We have reviewed the current status of all antiCOVID19 vaccines that have reached the clin ical trials in humans. As the race for the effective and safe vaccine has begun, different strategies were introduced. In this review, we discuss the most promising antiCO VID19 vaccine clinical trials and discuss different vacci nation strategies in order to provide more clarity into the ongoing clinical trials.

We conducted a comprehensive review of the literature on the progress of antiSARSCoV2 vaccine development preventing the rapid spread of COVID19 disease. The vaccines that were regis tered until December 2020 in the Clinical Trials database by the National Library of Medicine at the US National Institutes of Health were reviewed [22] . The authors followed the proposed guidelines for biomedical narrative review preparation [23] .

The immune system affects the severity of the COVID19 dis ease [7] . SARSCoV2 infection has an impact on both innate and adaptive immune responses. It has been described that SARS CoV2 enters the human body through physical barriers, such as respiratory tract, oral mucosa, and conjunctival epithelium [24, 25] . The dendritic cells, macrophages, and neutrophils represent the first line of defense, and their functions may be promoted by the production of type I and III interferons by SARSCoV2in fected epithelial cells [26] . The adaptive Tcell and Bcellmediat ed immune responses are also presented in COVID19 disease and, however, can be suppressed by SARSCoV2 [7] . In some cas es, the innate immune cells may contribute to the excessive inflam mation and, therefore, to the disease progression. The inability to reach control over the infection may result in dysregulated inflam matory responses that are potentially lethal. The IgM and IgG an tibodies to SARSCoV2 are detectable within 1-2 weeks and be gan to decrease by 8 weeks [27] . Several studies also reported that IgA response peaks earlier than IgM [28] . The antibody response particularly leads to production of neutralizing antibodies to the S protein and to the nucleoprotein. S protein is also the main target of the majority of newly designed vaccines [29] . The magnitude of neutralizing antibodies positively correlates with the disease sever ity and the robustness of Tcell response [30] . Tcell responses were detectable in individuals recovering from mild COVID19 who did not have detectable antibody responses to SARSCoV2 [31, 32] . The effective vaccination may not eradicate the SARS CoV2 virus but may at least protect from severe and deadly forms of the COVID19 disease [7] . Current knowledge regarding the diverse aspects of SARSCoV2immune system interplay shall be reflected in the vaccine design, including the selection of antigens, the vaccine platforms and adjuvants, the vaccination routes, and the dosage regimens [33, 34] . The key points of the SARSCoV2 vaccination strategies are discussed below. To date, over 80 clinical trials have been registered in the Clinical Trials database by the National Library of Medicine at the US National Institutes of Health; however, only 34 of them are active and recruiting (11 of phase I, 8 of phase I/II, 3 of phase II, 1 of phase II/III, and 11 of phase III) [22] . Moreover, 2 vaccine candidates have been ap proved for use by the US Food and Drug Administration (FDA) -BNT162/Comirnaty and mRNA1273). BNT162/Comirnaty has been also permitted by the European Medicines Agency (EMA). The vaccination program with BNT162/Comirnaty has been re cently initiated in many European countries. The main features of the registered and ongoing antiSARSCoV2 vaccine clinical trial are summarized in Table 1 (Phase I and I/II) and Table 2 (Phase  II, II/III, and III) .

Inactivated vaccines are based on presenting the form of patho gen with a loss of diseaseproducing capacity. The virus cultivation occurs in cell lines that represent a substrate for the production of large quantities of antigen. Virus multiplication is often followed by a purification and concentration prior to the vaccine inactiva tion [35] . Formaldehyde and betapropiolactone are used in the majority of licensed human antiviral vaccines to inactivate the vi rus [36] . Multiple doses or adjuvants are required to achieve suf ficient efficacy of inactivated vaccines [37] . To date, 4 inactivated vaccines have reached the phase III clinical trials and are currently under evaluation (#NCT04510207, #NCT04508075, and #NCT04456595).

Subunit vaccines are composed of purified antigens instead of whole microorganisms, and different carriers serve as a transport er for those antigens. In the antiSARSCoV2 subunit vaccines, the antigens are represented by viral proteins, peptides, or nanopar ticles. Because of relatively low immunogenicity of the subunit vaccines, adjuvants are required to create a stronger immune response [38] . Currently, aluminum salts, virosomes, AS03 (αtocopherol, surfactant polysorbate 80, and squalene), AS04 (Monophosphoryl lipid A, MPLA) and MF59 (squalene) are the most widespread licensing adjuvants [39, 40] . These adjuvant sys tems are also used in a number of antiSARSCoV2 vaccines; however, novel adjuvants are tested as well. AdvaxSM (clinical trial #NCT04453852) is an adjuvant composed of polysaccharide deltainulin and CpG oligodeoxynucleotide (CpG ODN). CpG ODN is a TLR 9 agonist with Thelper 1 skewing properties [41] . Granulocyte macrophage colonystimulating factor is a proin flammatory cytokine which may also serve as an adjuvant (#NCT03305341 and #NCT04386252).

Nonetheless, subunit vaccines provide a high level of safety. Bacterial expression systems represent the most commonly used technique to produce recombinant proteins with high expression. However, in antigens where posttranslational modification is re quired, the use of mammalian or insect cells may be considered [42] . Other offered alternatives include transgenic plants [43] . This technology has been also adopted as a source of SARSCoV2 virus spike protein for the purpose of vaccine development (phase I/II trial #NCT04473690). To date, there are no SARSCoV2recom binant vaccines tested in phase III. Three vaccines are being evalu ated in clinical phase I/II (#NCT04527575 and #NCT04537208) and phase II (#NCT04533399). Recombinant technologies, in cluding bacterial, insect, or mammalian cellbased expression sys tems can also be used for the generation of viruslike particles (VLPs). VLPs that are formed by a capsid protein do not contain infectious viral RNA or DNA. Moreover, the antisense RNA can inhibit virus expression, and the viral RNA/DNA may activate dif ferent pattern recognition receptors and trigger antiviral immune responses. These responses are primarily characterized by a pro Strizova duction of type I interferons and proinflammatory cytokines [44] . VLPsbased antiSARSCoV2 vaccine (#NCT04450004) is cur rently tested in phase I clinical trials.

DNA vaccines deliver coronavirus's genes to the human cells. The vaccination principle depends on the DNA translocation into the cell nucleus where the transcription of the antigen is initiated and followed by a translation. DNA vaccines frequently use plas mids as vectors. Depending on the route of vaccine administration (intramuscular, intradermal, and subcutaneous), either myocytes or keratinocytes are addressed. Nonetheless, antigenpresenting cells residing close to the site of application can be transfected di rectly by DNA vaccines as well. In such cases, the expressed anti gens are loaded onto MHC I and MHC II molecules due to the crosspriming potential [45] . The produced antigens are either re leased by exosomes or apoptotic bodies which lead to a recognition by antigenpresenting cells and further evolvement of humoral or cytotoxic immune responses. Different delivery devices are used to create a robust immune response [46, 47] . The main safety con cerns imply a possible integration of transfected DNA into somat ic and/or germ cells of the host. In such cases, a dysregulation of gene expression might occur and lead to various mutations. How ever, only extrachromosomal plasmids with a very low level of chromosomal integration are usually employed in the develop ment of DNA vaccines. Furthermore, the majority of plasmids re main at the site of administration [48] . Three antiSARSCoV2 DNA vaccines are currently in phase I/II of clinical assessment (#NCT04527081, #NCT04447781, and #NCT04445389).

Messenger RNA (mRNA) vaccines were first tested in early 1990s; however, their use was limited because of their instability [49] . The mRNA encodes the genetic information to produce an antigen, and thus, RNA vaccines also lead to a production of coro navirus's proteins in vivo. The in vitro generation of an RNA vac cine includes a reaction of a DNA plasmid template and a recom binant RNA polymerase. In addition, a synthetic cap analog and a poly(A) tail are added to form a mature RNA sequence. The stabi lization is further achieved by various transport systems (such as lipid nanoparticles, nanoemulsions, and cationic peptides) or methods enabling facilitated transfection (gene gun and electro poration). Conventional mRNA vaccines are based on the initia tion of the transient antigen expression in the cytoplasm of the host cells. Another platform is represented by selfamplifying mRNA vaccines that contain both the genes coding the targeted antigen as well as the genes required for the selfreplication (mostly RNA dependent RNA polymerase) [50] . The conventional mRNA vac cines induce a prompt antigen expression, and the expressed anti gens generate both humoral and cellular immune responses [51] [52] [53] [54] . In selfamplifying mRNA vaccines, a delayed antigen expression may prevail and limit the efficacy of the vaccine. Yet, the selfamplifying mRNA vaccine platform reaches higher yields, and thus, an equivalent protection is conferred at much lower dos es [55] . Regarding the safety profiles, the replicons of both above mentioned platforms are not capable of producing viral particles due to the lack of viral structural proteins. Moreover, neither con ventional nor selfamplifying mRNA vaccines can integrate into the host genome. The mRNAbased vaccines were able to induce production of functional antibodies with neutralizing properties in rabies, influenza, or Zika virus and represent also a promising vaccination strategy in the prevention against COVID19 infec tion [56] . The efficacy and safety are being assessed in the ongoing phase II and phase III clinical trials (#NCT04515147, #NCT04368728, and #NCT04470427).

Viral vectorbased vaccines (VBVs) are constructed by engineering a viral vector to carry coronavirus genes and slowly replicate in the host cells. The replication leads to the production of coronavirus proteins and a subsequent immune system activation. Po tential viral vectors include a broad spectrum of both DNA and RNA viruses, such as adenoviruses, parvoviruses (e.g., adenoas sociated viruses), togaviruses (e.g., Semliki Forest virus), para myxoviruses (e.g., measles virus, Newcastle disease virus or hu man parainfluenza virus), rhabdoviruses (e.g., vesicular stomatitis virus), and poxviruses (e.g., Modified Vaccinia Ankara). These viral vectors can be constructed as replicating or nonreplicating vectors [57] . The efficacy of VBV may be significantly affected by the preexisting immunity of the host. This can be avoided by the use of nonhuman or rare serotype vectors [58, 59] . The main safe ty concerns include the potential of viral genes to integrate into the host genome and uncontrolled replication. On the other hand, the high yield production supports the use of VBV particularly in the time of disease outbreaks [60] . In SARSCoV2 vaccine devel opment, the most commonly used vectors are the adenoviral vec tors, such as ChAdOx (#NCT04536051 and #NCT04516746), ad enovirus type 5 (#NCT04564716, #NCT04540419, and #NCT04526990), and adenovirus type 26 (#NCT04564716 and #NCT04505722). All these vaccines are currently being evaluated in phase III clinical trials. However, lentivirus (#NCT04276896 and #NCT04428073), measles (#NCT04498247 and #NCT04497298), baculovirus (#NCT04522089), or MVA (#NCT04569383) are also being tested.

The route of administration is another crucial aspect that sig nificantly affects the vaccine efficacy. Conventional vaccination approaches include mucosal and parenteral administration. Par enteral route generally includes intramuscular (IM), subcutaneous (SC), and intradermal (ID) application [61] . Due to an increased infiltration of dermis with DCs, the ID application initiates great er adaptive immune response than the IM application providing a significant dose sparing effect. However, improved efficacy is as sociated with a less favorable safety profile [62] . Mucosal vaccines including the intranasal and oral administration routes provide a number of advantages, particularly the avoidance of a needle ap plication and a lower risk of systemic adverse events (AEs). Nev ertheless, the systemic responses to mucosal vaccination are weak er as compared to parenterally administrated vaccinations [63] . The majority of vaccinations are administered intramuscularly. However, the intradermal administration (#NCT03305341 and #NCT04447781), oral administration (#NCT04334980), or com bined administration (intramuscular and mucosal) of the vaccines (#NCT04552366) is being evaluated.

The first vaccine with favorable results was the ChAdOx1 nCoV19 (also known as AZD1222, AstraZeneca/University of Oxford). This vaccine was evaluated in July 2020 in the phase I/II singleblind randomized trial with 1077 participants. The patients were exposed to 2 doses of recombinant adenovirus vaccine ChAdOx1 nCoV19 in a 28day interval. Neutralizing antibodies against SARSCoV2 spike protein were detected in 91% of pa tients after the first dose. The production of virusspecific anti bodies peaked on day 28, and a robust Tcell response was also observed. Severe AEs were not reported [64] . The efficacy has been recently confirmed in a pooled interim analysis of 4 phase I/-III clinical trials. [65] . The preliminary results of a double blind, randomized, placebocontrolled phase I/II trial with an other vaccine candidate Ad26.COV2 (JanssenCilag Internation al N.V.) were published in September 2020 (data published as a preprint). The study included >800 patients, and the seroconver sion rate with the production of antispike proteinneutralizing antibodies was seen in 83-100% patients across the cohorts. The specific Thelper 1 response was detected in 80-83% of the par ticipants with a robust activation of CD8+ T cells. Local and sys temic AEs included fever, headache, myalgia, and injection site pain. In November 2020, the preliminary results of an openlabel clinical trial including 45 healthy adults treated with mRNA1273 vaccine (Moderna Biotech Spain, S.L.) were shown. The vaccine was administered in 2 doses and various concentrations (25, 100, and 250 μg). The seroconversion occurred in all participants, and the response depended on the administered dose. On the other hand, higher doses were associated with increased risk of system ic AEs (reported in 33% participants) [66] . The mRNA BNT162 (Pfizer/BioNTech) vaccine has been proven in a large observer blinded, randomized, placebocontrolled trial with >43,000 par ticipants to be a safe and potent vaccine. Two doses (30 μg per dose) were administered in a 21day interval. The overall reported efficacy of 95% was observed across different subgroups defined by age, sex, race, ethnicity, baseline BMI, and the presence of co existing conditions. A 52% efficacy was observed after the first dose indicating early protection. Injection site reactions, fatigue, headaches, and fevers were the most common AEs (reported in 27% of patients) [67] .

It has been shown that various vaccination principles bear a potential to prevent or at least to suppress the detrimental effect of COVID19. The crossprotection has been discussed particu larly in association with the Bacillus CalmetteGuérin (BCG) vaccination. In BCG vaccinated populations, the incidence and the severity of the COVID19 disease appears to be lower than BCGnonvaccinated populations [68] [69] [70] [71] [72] . A similar phenom enon of a crossprotection was also described in individuals after the measles infection, or the measles, mumps, rubella vaccina tion [73, 74] . These findings were supported by previous obser vations that a nonspecific effect of these vaccines protects against other infections including those of viral origin [75] [76] [77] [78] 

An effective vaccination against SARSCoV2 infec tion could mobilize the innate and adaptive immune re sponses and provide a protection against severe forms of COVID19. Since the SARSCoV2 virus may undergo mutational changes and antigenically evolve over time, the vaccine may become, as in influenza, a seasonal pro tection. On the other hand, coronaviruses have a low mu tation rate in comparison to other RNA viruses, particu larly Influenza type A [79] . The antiSARSCoV2 vacci nation may not lead to the eradication of the disease, however, may most certainly decrease the diseaserelated mortality and morbidity [75, 80, 81] .

In COVID19, live vaccines have not yet been regis tered in human clinical trials. Previous studies have shown that booster (secondary) vaccination with lifeat tenuated viruses generate only limited immune response as compared to the first vaccination dose [82, 83] . Also, the preexisting immunity caused by previous COVID19 infection may inhibit the efficacy of live attenuated vac cines and the presence of neutralizing antibodies can be associated with the virus neutralization. Moreover, the genome instability may lead to a back mutation recover ing their virulence, mainly in viruses with higher muta tion rate [84] [85] [86] . Therefore, the live vaccines may not represent the optimal vaccine type in prevention of CO VID19 infection [87] .

Other classical vaccination approaches, such as inacti vated or recombinant subunit vaccines, are currently be ing tested against COVID19 infection in clinical trials. Their efficacy is, however, also limited by relatively low response rates and shortterm immune memory. There fore, both approaches require the use of potent adjuvants such as CpG ODN, ADVAXSM, or granulocyte macro phage colonystimulating factor representing novel strat egies to enhance immune response. Another obstacle of inactivated vaccines represents the risk of reversed out come associated with enhanced virusmediated disease and fatal consequences. In the cases of respiratory syncy tial virus vaccination, the vaccine was found to be immu nogenic; however, the elicited antibodies were nonpro tective and respiratory syncytial virus disease progression in single cases resulted in death in vaccinated infants [88, 89] . Therefore, novel vaccine designs, such as mRNA vac cines and recombinant VBV vaccines are being extensive ly investigated to avoid these barriers. FDA and EMA have been recently authorized mRNA vaccine candidate BNT162/comirnaty and mRNA1273 for the use [90] . Furthermore, other vaccine candidates, including Ad26. COV2.S and ChAdOx1SARSCoV2 (AZD1222), are currently under consideration [91] . To note, novel vac cine approaches raise many safety concerns. However, strategies following the good manufacturing practice principles and appropriate preclinical and clinical testing under the surveillance of regulatory authorities should ensure good safety profile [92] . The efficacy and safety also remain an issue in immunocompromised patients with primary or secondary immunodeficiencies. Gener ally, administration of attenuated life virus vaccines has to be considered with caution and indicated upon careful individual assessment of risk and benefits in these pa tients. Nonlive vaccines such as influenza or pneumo coccal vaccine are regarded as safe, even though their ef ficacy may be reduced in patients with severely impaired antibody response [93, 94] . Similar principles might be applied also in antiSARSCoV2 vaccination; however, neither recommendations nor guidelines are available yet. The level of virusspecific antibodies does not corre late with the acquired immune response mediated by T cells that might be preserved in the majority of the anti body deficient patients [95, 96] . Thus, the examination of Tcell response should also be considered in healthy sub jects. Surprisingly, other vaccines such as BCG and mea sles vaccine have also shown efficacy in the prevention of COVID19 disease. The leading vaccine candidates are currently being distributed and among selected popula tion with great expectations to reduce the COVID19 spread.

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