Canadian Journal of Microbiology
Frontrunners in the race to develop a SARS-CoV-2 vaccine
Journal: Canadian Journal of Microbiology
Manuscript ID cjm-2020-0465.R1
Manuscript Type: Review
Date Submitted by the 16-Nov-2020 Author:
Complete List of Authors: Russell, Raquel; University of Manitoba, Department of Microbiology Pelka, Peter; University of Manitoba, Department of Microbiology Mark, Brian; University of Manitoba, Department of Microbiology
Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2, Keyword: COVID-19,Draft vaccine development Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :
© The Author(s) or their Institution(s) Page 1 of 91 Canadian Journal of Microbiology
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3 Frontrunners in the race to develop a SARS-CoV-2 vaccine
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5 Authors: Raquel L. Russell, Peter Pelka, Brian L. Mark
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7 Affiliations: Department of Microbiology,Draft University of Manitoba, Winnipeg, 8 Manitoba, Canada, R3T2N2
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10 Correspondence: Brian L. Mark, Ph.D.,
11 email: [email protected]
12 tel: 1-204-480-1430
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1 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 2 of 91
1 Abstract
2 Numerous studies continue to be published on the COVID-19 pandemic that is being
3 caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Given the
4 rapidly evolving global response to SARS-CoV-2, here we primarily review the leading COVID-
5 19 vaccine strategies that are currently in Phase III clinical trials. Non-replicating viral vector
6 strategies, inactivated virus, recombinant protein subunit vaccines, and nucleic acid vaccine
7 platforms are all being pursued in an effort to combat the infection. Preclinical and clinal trial
8 results of these efforts are examined as well as the characteristics of each vaccine strategy from
9 the humoral and cellular immune responses they stimulate, effects of any adjuvants used, and the 10 potential risks associated with immunizationDraft such as antibody dependent enhancement (ADE). A 11 number of promising advancements have been made toward the development of multiple vaccine
12 candidates. Preliminary data now emerging from phase III clinical trials show encouraging
13 results for the protective efficacy and safety of at least three frontrunning candidates. There is
14 hope that one or more will emerge as potent weapons to protect against SARS-CoV-2.
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16 Keywords: Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2, COVID-19,
17 vaccine development
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2 © The Author(s) or their Institution(s) Page 3 of 91 Canadian Journal of Microbiology
1 Introduction
2 The first cases of a viral pneumonia of unknown cause were reported in December 2019
3 in Wuhan, China (Huang et al. 2020). Case numbers grew quickly, and the virus spread across
4 numerous borders, prompting the World Health Organization to declare a pandemic in March of
5 2020. In the early stages of the outbreak, electron micrographs of the virus revealed
6 characteristics consistent with coronaviruses, including enveloped, spherical-like particles
7 covered in spikes that resembled a solar corona (Gorbalenya et al. 2020). Sequencing of the full-
8 length viral genome from patient isolates confirmed it to be a coronavirus with remarkable
9 similarity (~79% nucleic acid identify) to the coronavirus that caused the Severe Acute 10 Respiratory Syndrome Coronavirus (SARS-CoV-1)Draft pandemic of 2003 (Kim et al. 2020b). Given 11 its similarity to SARS-CoV-1, the current coronavirus is referred to as SARS-CoV-2 and only a
12 handful of countries appear to have been spared from the virus (although cases could have been
13 missed). As of November 2, 2020, more than 46 million COVID-19 cases have been reported
14 worldwide and over 1.2 million related deaths have occurred (Gardner n.d.). Most infected
15 individuals experience mild or moderate symptoms, but up to 20% of cases can be severe (Chen
16 et al. 2020). Common symptoms include cough, shortness of breath, fatigue, and fever, but can
17 progress into a critical condition known as acute respiratory distress syndrome (ARDS) (Huang
18 et al. 2020). Risk for severe disease increases with age and comorbidities such as hypertension,
19 obesity and diabetes (Richardson et al. 2020). It has been reported that SARS-CoV-2 is
20 effective at evading and crippling both innate and acquired immune responses (Zhang et al.
21 2020a; Qin et al. 2020). Similar to other coronaviruses, SARS-CoV-2 appears able to supress
22 interferon (IFN) induction and signaling (Cameron et al. 2012; Hadjadj et al. 2020), which is
23 critical to innate antiviral response and their suppression drives infection and disease
3 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 4 of 91
1 progression. Another complication associated with SARS-CoV-2 is development of a “cytokine
2 storm” associated with severe COVID-19, causing ARDS likely due to, in part, a dysregulated
3 IFN-I response (Channappanavar et al. 2016; Hadjadj et al. 2020). Previously, animal models of
4 SARS-CoV-1 and MERS coronavirus infections have shown that a lack of an early IFN-I
5 response correlates with disease severity (Channappanavar et al. 2016, 2019). Blocked IFN
6 signalling with SARS-CoV-2 may boost inflammatory pathways leading to the secretion of large
7 amounts of cytokine interleukins (IL-6, IL-8, IL-1β) (Gong et al. 2020). The release of these
8 cytokines triggers pro-inflammatory responses such as fever, neutrophil recruitment, and
9 monocyte activation. Lymphopenia has also been noted in moderate and severe cases of COVID-
10 19 (Tan et al. 2020). Draft 11 Coronavirus Biology
12 Betacoronaviruses and alphacoronaviruses are mammal infecting genera of the
13 Coronaviridae family within the order Nidovirales (Roper and Rehm 2009; Gorbalenya et al.
14 2020). With the emergence of SARS-CoV-2, there are now seven known human coronaviruses,
15 all of which cause disease (Graham et al. 2013). Some of these coronaviruses are commonly
16 encountered, such as the α-coronaviruses HCoV-229E and HCoV-NL63, and β-coronaviruses
17 HCoV-OC43 and HCoV-HKU1, which cause minor and seasonal respiratory infections
18 (Hendley et al. 1972; van der Hoek et al. 2004; Woo et al. 2005). However, the three most recent
19 coronaviruses to have emerged are capable of causing severe disease and include SARS-CoV-1,
20 MERS-CoV, and now SARS-CoV-2. They are all β-coronaviruses and SARS-CoV-2 shares
21 roughly 79% and 50% whole genome sequence similarity to SARS-CoV-1 and MERS-CoV
22 respectively (Lu et al. 2020).
4 © The Author(s) or their Institution(s) Page 5 of 91 Canadian Journal of Microbiology
1 Coronaviruses are enveloped viruses that share a large (~30 kb) single-stranded positive
2 sense RNA genome (Fehr and Perlman 2015). The SARS-CoV-2 genome contains two main
3 open reading frames (ORFs), ORF1a and ORF1b, expressing polyproteins pp1a and pp1b
4 respectively. During translation, ribosomes can switch from ORF1a to ORF1b by a -1 frameshift
5 upon encountering a slippery sequence (Maier et al. 2015). The translated polyproteins both
6 consist of non-structural proteins (nsps), with pp1a containing nsps 1-11 and pp1b containing
7 nsps 1-16 (Maier et al. 2015). To create singular nsps, the polyproteins are cut at specific sites by
8 two enzymes: main protease (Mpro, also known as 3CLpro) and papain-like protease (PLpro)
9 (Hegyi and Ziebuhr 2002; Maier et al. 2015). These proteases are resident within nsp3 and nsp5,
10 respectively, and autocatalytically cleave the viral polyproteins into functional nsp units. Many
11 of the nsps produced come together to formDraft the replicase-transcriptase complex (RTC) that
12 allows for RNA replication and sub-genomic RNA transcription (Maier et al. 2015). Nsp12
13 contains the RNA dependent RNA polymerase domain, while nsp14 contains the
14 exoribonuclease domain that is important for replication accuracy (Maier et al. 2015). Viral RNA
15 produced by the RTC includes genomic and sub-genomic RNAs and both are positive sense
16 strands made from negative sense strand intermediates, also referred to as antigenomes (Maier et
17 al. 2015). Sub-genomic RNA acts as mRNA for structural and accessory gene translation. The
18 functions of accessory proteins are still relatively unknown for SARS-CoV-2, but they may be
19 involved in innate immune response suppression similar to SARS-CoV-1 (Kopecky-Bromberg et
20 al. 2007). Sub-genomic RNAs are read by host ribosomes to create the viral structural spike (S),
21 matrix (M), envelope (E), and nucleocapsid (N) proteins (Fehr and Perlman 2015). The S, E, and
22 M proteins are translated and sent to the endoplasmic reticulum where they follow a secretory
23 pathway into an endoplasmic reticulum- Golgi intermediate compartment (Maier et al. 2015).
5 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 6 of 91
1 This is where virion maturation takes place, as genomic RNA encapsidated in N protein
2 combines with the other structural proteins.
3 Currently, vaccine development for COVID-19 has focused predominantly on the S
4 protein of SARS-CoV-2 (Fig 1). The S protein facilitates viral entry into the cell through
5 engagement of the human angiotensin-converting enzyme 2 (hACE2) receptor and since it is
6 located on the virion surface, it is vulnerable to a humoral (antibody) immune response and is
7 thus a promising immunogen for vaccine development (Wang et al. 2020b). The human S protein
8 receptor, hACE2, is expressed on various tissues throughout the body and is highly expressed on
9 small intestinal epithelial cells and lung alveolar epithelial cells (Hamming et al. 2004). 10 Interestingly, compared to SARS-CoV-1,Draft which uses the same receptor, SARS-CoV-2 has a 11 markedly enhanced binding affinity to hACE2 (Wrapp et al. 2020; Hoffmann et al. 2020).
12 Within the S protein, the receptor binding domain (RBD) is responsible for directly binding to
13 hACE2 (Fig 1). This interaction, along with cleavage by cell membrane proteases, promotes
14 endosome formation and infection of the host cell (Hoffmann et al. 2020; Shang et al. 2020).
15 Currently, the RBD and the entire S protein are being used as immunogens for a number of
16 SARS-CoV-2 vaccines, as both have shown to induce neutralizing antibodies (nAbs) (Quinlan et
17 al. 2020; Lucchese 2020; Tai et al. 2020). The majority of all nucleic acid, viral vector, and
18 protein subunit vaccines produced and currently in development for SARS-CoV-2 work to elicit
19 an immune response using these immunogens. However, in addition to inactivated whole virus
20 vaccines as described below, isolated S protein can elicit non-neutralizing antibodies (nnAbs)
21 from possible epitopes derived from it. This may present a risk however, as it could potentially
22 cause unwanted immune responses, such as antibody dependent enhancement (ADE) of disease
23 (Jaume et al. 2011; Wang et al. 2014a) as discussed below. Despite potential pitfalls, prior and
6 © The Author(s) or their Institution(s) Page 7 of 91 Canadian Journal of Microbiology
1 ongoing vaccine strategies used with SARS-CoV-1 and MERS-CoV are proving helpful and are
2 bootstrapping current efforts to develop SARS-CoV-2 vaccines. Indeed, a number of
3 coronavirus vaccine development platforms have now been outfitted with the S protein from
4 SARS-CoV-2 as the basis for vaccine development against the newly emerged virus. Incredibly,
5 as of November 9, 2020, there are 214 vaccine candidates being developed against SARS-CoV-
6 2, with 39 in clinical testing (Institute 2020). In this review, we will examine the vaccine
7 strategies that have advanced to Phase II clinical trials and briefly discuss potential follow up
8 vaccines that are currently in Phase II trials
9 Vaccine Immunology 10 There are a number of immune systemDraft factors that must be considered in order to develop 11 a successful COVID-19 vaccine approach (for a comprehensive review see: 36). The ideal
12 SARS-CoV-2 vaccine would elicit both a strong cellular and humoral response (Vabret et al.
13 2020), that is, the vaccine would generate T cell mediated immunity that targets virus infected
14 cells as well as B-cell mediated immunity that generates nAb against the virus. Balanced CD4+
15 T helper 1 and 2 (Th1 and Th2) cell induction is likely to be the optimal outcome, as a response
16 favoring Th2 cells could potentially be linked to increased inflammation and cytokine release
17 (Graham 2020). As discussed below, many vaccine designs are now being pursued to achieve
18 these desired immune responses against SARS-CoV-2, which can be further complicated by
19 factors such as immunization route, site, dosage, and schedule, all of which have some degree of
20 an effect on immune response (Zimmermann and Curtis 2019). Adjuvants must also be
21 considered, which can be used to reduce the amount of antigen required, reduce the number of
22 required immunizations, improve antigen uptake in selected tissues, and increase vaccine
23 efficacy in the very young, old, and immunocompromised (Glenny et al. 1926; Petrovsky and
7 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 8 of 91
1 Aguilar 2004). Age is a major factor, as the elderly and very young tend to have weaker
2 responses to vaccinations (Zimmermann and Curtis 2019). Immunocompromised individuals or
3 those with pre-existing medical conditions may also have reduced responses to immunization.
4 Pitfalls in vaccine design
5 Clearly, when developing and testing vaccines, caution must be taken to avoid creating a
6 worse outcome for recipients of the vaccine. Antibody-dependent enhancement (ADE) of viral
7 infection is mechanism whereby some viruses can exploit antiviral antibodies to gain additional
8 access into target cells beyond direct receptor/co-receptor mediated entry (Takada and Kawaoka
9 2003). Since vaccines elicit antiviral antibodies, ADE must be considered during vaccine design 10 and testing. ADE occurs most commonlyDraft when virus-antibody complexes bind Fc receptors 11 (FcRs) present on immune system cells; however, complement receptors can also be involved
12 (Takada and Kawaoka 2003).
13 Previously, a SARS-CoV-1 vaccine candidate that used the full-length surface fusogenic
14 spike protein (S protein) as the immunogen was previously found to elicit ADE via Fcγ receptor
15 II (FcγRII), which occurred independently of hACE2-mediated entry (Jaume et al. 2011).
16 Further, a feline coronavirus vaccine, also using the viral surface S-protein as immunogen, has
17 also been shown to induce ADE in cats (Felis catus) (Vennema et al. 1990). Enhanced uptake of
18 the feline virus into macrophages was also found to be Fc receptor dependent (Olsen et al. 1992).
19 Interestingly, an additional study looking at SARS-CoV-1 found that although the presence of
20 SARS-CoV spike immune-serum enhanced the infection of isolated macrophages by the virus,
21 viral replication did not occur within the infected cells (Yip et al. 2016). No change in type-I
22 interferons or pro-inflammatory cytokines/chemokine gene expression occurred either, leading
23 the authors to suggest that while ADE may occur during SARS-CoV-1 infection, it may not
8 © The Author(s) or their Institution(s) Page 9 of 91 Canadian Journal of Microbiology
1 adversely susceptible immune cells (Yip et al. 2016). In contrast, another study found that
2 infection of human promonocytes by SARS-CoV-1 was enhanced in the presence of anti-S
3 antibodies, which resulted in increased TNF-a, interleukins IL-4 and IL-6 (Wang et al. 2014b).
4 Further, immunization of animal models with SARS-CoV-1 vaccines, either inactivated whole
5 agent, purified recombinant S-protein, or virus-like particles containing S-protein have been
6 found to cause pulmonary immunopathology that appear to be related to ADE (Tseng et al. 2012;
7 Iwasaki and Yang 2020).
8 Given ADE has been demonstrated to occur during vaccine development for other
9 coronaviruses it is a concern that must be considered closely during SARS-CoV-2 vaccine 10 development. As discussed below, to tryDraft to limit the risks of ADE with SARS-CoV-2 vaccines, 11 some developers have decided to focus on the RBD instead of the entire S protein (Fig. 1). The
12 RBD would present fewer epitopes than full-length S protein and the RBD is known to elicit
13 more nAbs with few nnAbs in SARS-CoV-1 (Quinlan et al. 2020). Focusing the immune
14 response to critical protein structures of the virus, such as the RBD alone, may induce higher
15 numbers of potent nAbs that may reduce the risk of ADE.
16 Early reports on SARS-CoV-2 have also noted mutational events, as well as the founder
17 effect in US isolates (Farkas et al. 2020). One study reported 6 different mutations in the S
18 protein from patient derived isolates, however these appear not to have affected viral
19 pathogenicity (Yao et al. 2020). Single nucleotide polymorphisms (SNPs) are often used to
20 gauge evolution, notably in viral genomes that use an error RNA-dependent RNA polymerase to
21 replicate (Yin 2020). In another studying using SNP genotyping, mutations in essential proteins
22 including the S, RNA primase, nucleoprotein, and RNA polymerase were found (Yin 2020).
23 When developing a vaccine for SARS-CoV-2, it will be important to consider the possibility that
9 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 10 of 91
1 mutations may arise in the future that could circumvent vaccine efficacy. Indeed, it has recently
2 been demonstrated that mutations in the RBD of the SARS-CoV-2 S-protein can be readily
3 selected for in the laboratory that enables the virus to evade monoclonal antibodies or
4 convalescent plasma (Weisblum et al. 2020). More recently, a circulating SARS-CoV-2 S-
5 protein variant (N439K) has been shown to maintain fitness but evade a panel of neutralizing
6 monoclonal antibodies (Thomson et al. 2020). Thus, it may be possible that mutation-based
7 evasion of vaccines could occur as well. Further, in addition to mutation-based immune escape,
8 insufficient adaptive immune response and waning immunity could also impact vaccine efficacy
9 in the future and potentially determine if SARS-CoV-2 will become an endemic virus within the
10 human population (Shaman and Galanti 2020). If mutants of SARS-CoV-2 arise that can evade
11 vaccines, certain vaccine platforms mayDraft be better suited to mount a faster and more effective
12 response. For example, nucleic acid-based vaccines as detailed below could be modified quite
13 readily to incorporate the mutation(s) that have enabled the virus to evade the vaccine. This
14 could be an advantage compared to other platforms since the methods for production,
15 purification, and manufacturing would remain the same (Rauch et al. 2018). Large-scale
16 development of these types of vaccines usually depends on synthetic materials, so pre-existing
17 facilities could pivot towards the production of new variant vaccines, saving time and money
18 (Rauch et al. 2018).
19 It is imperative to stress the need for long-term testing and follow-up of vaccinated
20 individuals for any adverse effects. This is particularly important when non-human viral vectors
21 and other novel platforms such as nucleic acid-based vaccines are used for immunization, as
22 neither have been used on a large scale in humans, and their long-term effects are currently
23 unknown. Understanding the long-term effects of these vaccine platforms is critical, as vaccine
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1 safety is of paramount importance. Not only from the effects it would have on vaccinated
2 individuals but importantly a vaccine that is unsafe would wreak havoc on vaccinations in
3 general considering the scale of immunization required to combat COVID-19. Lastly, one has to
4 consider the delivery method and availability of the vaccine, and of course the cost. Traditional
5 and viral vectored-based vaccines should be relatively easy to produce and distribute, with quick
6 ramp-up in production. But this may not necessarily be the case with the nucleic acid-based
7 vaccines, which are currently more costly to produce and sometimes require extra steps for
8 administration (i.e. electroporation). This could preclude deployment of such vaccines in poorer
9 countries that may lack the infrastructure or resources to acquire and administer the vaccine. 10 Draft 11 SARS-CoV-2 vaccine candidates currently in Phase III clinical trials
12 A. Non-replicating Viral Vectors
13 With a history in gene therapy, non-replicating viral vectors are used to carry a target
14 gene of interest into a host cell. Adenoviruses are widely used as the vector of choice because
15 they are highly immunogenic, and they induce innate and adaptative immune responses in
16 mammalian hosts (Tatsis and Ertl 2004). Significantly, adenoviruses are generally safe and can
17 be easily grown in an industrial setting for rapid vaccine deployment (Zhang and Zhou 2016).
18 Adenoviruses contain a double-stranded DNA genome of ~34-43 kb, which contains four early
19 regions (E1 E2, E3, and E4) (Tatsis and Ertl 2004). Of these early proteins, E1A is the first to be
20 expressed and it promotes several functions within the host cell, including induction of the S-
21 phase, dedifferentiation, host immune suppression, and induction of viral gene expression. These
22 features make E1A, and the entire E1 region, dispensable in a non-replicating adenoviral vector,
11 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 12 of 91
1 thus the E1 region is generally deleted (Zhang and Zhou 2016). The E3 unit can also be removed
2 as it mainly serves to help the virus hide from immune detection by downregulating major
3 histocompatibility complex (MHC) class I expression and blocking tumor necrosis factor (TNF)
4 signalling pathways (Tatsis and Ertl 2004). Further, deletion of E3 provides an additional ~3.1
5 kb of space to insert foreign DNA than just the E1 deletion alone, allowing for up to 7.5 kb of
6 foreign sequence to be inserted that can encode for antigens that generate a desired immune
7 response. E2A and E4 units can also be removed to allow for additional space. Gutted vectors,
8 that is, those removing the entire viral coding region and retaining only the packaging signal and
9 the inverted terminal repeats, can also be used, allowing for tens of kilobases of genetic material
10 to be inserted. Draft 11 There are generally two strategies for recombinant virus generation; one using
12 recombination in bacterial cells and one using Cre-recombinase mediated recombination in
13 293Cre cells (Chartier et al. 1996; Hardy et al. 1997). Bacterially-mediated recombination relies
14 on transformation of a E. coli strain BJ5183 with the donor plasmid carrying the transgene of
15 interest and recipient plasmid carrying the viral backbone. The bacteria recombine these, using
16 homologous recombination, to generate a full-length viral genome that can then be linearized and
17 transfected into 293 or similar E1-complementing cell line to directly rescue recombinant
18 viruses. The Cre-mediated recombination uses a vector that has a single loxP site in the donor
19 vector with the transgene of interest, and the acceptor virus, called 5, which has two loxP sites
20 flanking the packaging signal. 293Cre cells are transfected with the purified 5 genome and the
21 linearized donor plasmid, whereupon the Cre recombinase will remove the packaging signal
22 from the 5 virus and it will also drive joining of the donor vector to the now packaging-less
23 backbone. Because the donor vector is the only one with intact packaging signal, only those cells
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1 that carry out this recombination properly will form plaques and generate recombinant viruses in
2 293Cre cells (Fig 2). The main advantage of the bacterial system is its ability to recombine into
3 different regions of the adenoviral genome, whereas the Cre-mediated recombination is currently
4 limited to the E1 region. In the case of SARS-CoV-2 vaccine development using adenoviral
5 vectors, the primary transgene currently being used is the S protein or fragments of it,
6 particularly the RBD, although some are using viruses expressing multiple SARS-CoV-2 genes
7 (Rice et al. 2020).
8 Human adenovirus type 5 (HAdV-C5) based vaccines have demonstrated substantial
9 transgene specific CD8+ T cell responses and antibody induction in rodents and non-human
+ 10 primates (NHP), with a lesser CD4 T cellDraft response (XIANG et al. 1996; Shiver et al. 2002; 11 Casimiro et al. 2003). Three Ad5-SARS-CoV-1 vaccines using the S1, M, and N proteins were
12 developed in 2003 and used on rhesus macaques (Macaca mulatta) (Gao et al. 2003). After a
13 second booster vaccination given on day 28 of the study, results showed antibody and T cell
14 responses against the S1 and N proteins respectively (Gao et al. 2003). It was also found that
15 upon SARS-CoV-1 challenge, a strong nAb response occurred in all vaccinated animal serum
16 samples. Importantly however, these results may be limited to animal studies due to pre-existing
17 immunity to HAdV-C5 in human populations. In places such as the USA, seroprevalence can be
18 as high as 40-45% and up to 90% in other parts of the world (Nwanegbo et al. 2004; Dudareva et
19 al. 2009; Zhang et al. 2013). This presents a major immunogenicity problem, as individuals who
20 have already encountered the virus previously usually have adenovirus specific nAbs. These
21 nAbs can clear out the HAdV-C5 vectors before they have a chance to enter a host cell and
22 provide immunity against the antigen of interest (Dudareva et al. 2009). Studies with NHPs
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1 showed that a 1000-fold increase in dose was needed in pre-exposed animals to achieve the same
2 level of immune response as those who had not been previously infected (Fitzgerald et al. 2003).
3 A.1 CanSino Biologics Inc. vaccine candidate Ad5-nCoV
4 Currently, CanSino Biological and the Beijing Institute of Biotechnology are moving
5 forward with phase III clinical trials of their SARS-CoV-2 full-length spike glycoprotein HAdV-
6 C5 vectored vaccine (NCT04526990), however the released phase I and II clinical trial reports
7 had mixed results (ChiCTR2000030906, ChiCTR200003171). 108 non-randomly chosen
8 individuals took part in the trial and were split into three groups receiving one vaccination of
9 either low (5x1010 viral particles per 0.5 mL), middle (1x1011 viral particles per 1 mL), or high 10 (1.5x1011 viral particles per 1.5 mL) doses.Draft Injection site adverse reactions were observed across 11 all groups, usually presenting as pain, while systemic adverse reactions were commonly fever,
12 headache, and fatigue (Zhu et al. 2020b). Severe grade 3 reaction occurrences were about 9%
13 overall, and more frequent in the high dose group, which suggests that there are greater risks
14 associated with a higher dosage. In most individuals, rapid humoral and T cell responses against
15 the S protein of SARS-CoV-2 occurred, with the activation of both CD4+ and CD8+ T cells. Over
16 the 28-day trial period for this report, T cell responses peaked at day 14, and nAb titer peaked 28
17 days post vaccination (Zhu et al. 2020b). By day 28, 94-100% of individuals showed a four-fold
18 increase in antibodies to the RBD of SARS-CoV-2. However, individuals with pre-existing
19 antibodies to HAdV-C5 showed reduced T cell and specific antibody responses, most prevalently
20 in the low dose group (Zhu et al. 2020b). The study also had no participants older than 60 years
21 old and the observational period was short, with plans for follow up and data collection
22 continuing for at least six months. The report states that this study was not designed to measure
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1 the vaccine efficacy (Zhu et al. 2020b), so subsequent trials tailored to testing its efficacy in
2 individuals with pre-existing immunity to HAdV-C5, and in the elderly are needed.
3 The phase II clinical trial (ChiCTR200003171) was randomized, placebo-controlled,
4 double-blind, and set out with the goal to assess the safety and immunogenicity of the vaccine in
5 a larger number of individuals and to find the most effective dosage level. Since the phase I trial
6 found that severe grade 3 reactions were more common in the high dose group, the 508 healthy
7 individuals involved in this study were only assigned to receive one middle, low, or placebo dose
8 (Zhu et al. 2020a). One or more adverse events were reported by 77% and 76% of participants
9 from the middle dose and low dose groups respectively, which was found to be significantly
10 higher than the 48% of participants in the placebo group (Zhu et al. 2020a). The most frequently
11 reported local and systemic adverse eventsDraft included injection site pain, fever, headache, and
12 fatigue. Further, 9% of participants receiving a medium dose experienced severe grade 3 adverse
13 reactions, which was significantly higher than in the low dose or placebo groups.
14 For the phase II trial, the vaccine elicited SARS-CoV-2 binding antibodies in 97% and
15 96% of individuals and nAbs in 47% and 59% of individuals in the low dose and middle dose
16 groups, respectively (Zhu et al. 2020a). Specific T cell responses were detected in 88% low dose
17 and 90% middle dose individuals (Zhu et al. 2020a). In total, 52% of participants had high pre-
18 existing immunity and 48% of participants had low pre-existing immunity to HAdV-C5. It was
19 found that participants with low pre-existing immunity to HAdV-C5 had SARS-CoV-2 RBD
20 specific binding antibody and nAb counts that were approximately twice that of participants with
21 high-pre-existing immunity to the vector (Zhu et al. 2020a). Further, low antibody responses
22 were associated to ages 55 years and older. Nevertheless, the study did highlight that RBD
23 specific antibodies and nAbs to SARS-CoV-2 were significantly higher 28 days post-vaccination
15 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 16 of 91
1 in all participants, even those aged 55 years or older that received one of the vaccine doses
2 compared to the placebo. The study did have several limitations, including the lack of diversity
3 in the participant population, short follow-up period, and the still relatively small sample size.
4 The efficacy and safety of the vaccine in a larger, more diverse, healthy population is now being
5 evaluated in the phase III clinical trial (NCT04526990), using the lowest dose to minimize the
6 chance of any future severe adverse events.
7
8 A.2 University of Oxford vaccine candidate AZD1222 (formally ChAdOx1 nCoV-19)
9 Pre-existing immunity to HAdV-C5 is a significant problem with the above strategy;
10 however, it may be possible to avoid pre-existing immunity to human adenovirus vector vaccines
11 by using simian-derived adenoviral vectors,Draft specifically chimpanzee adenoviruses (ChAds)
12 (Morris et al. 2016). The hypervariable regions of the primary immunogen of adenoviruses, the
13 hexon coat protein, differ between human and simian adenoviruses (SAds), which appears to
14 allow simian derived vectors to avoid pre-existing immunity (Roy et al. 2004). To develop a
15 SAd-based vector vaccine, the E1 unit of the genome can be deleted to prevent viral growth and
16 replication, as well as the E3 unit of the SAd vector to prevent any immunomodulatory proteins
17 from being produced (Morris et al. 2016). ChAd vectors were shown as safe and immunogenic in
18 humans during clinical trials against a variety of pathogens such as malaria, HIV, influenza,
19 hepatitis C, and Ebola (Sheehy et al. 2012; Antrobus et al. 2014; Hayton et al. 2014; Swadling et
20 al. 2014; Ewer et al. 2016). Interestingly, it has been found that ChAd vectors induce less
21 transgene antigen specific immunity than HAdV-C5 in mice that have no pre-existing immunity
22 (Dicks et al. 2015). However, given the high pre-existing HAdV-C5 immunity in most
16 © The Author(s) or their Institution(s) Page 17 of 91 Canadian Journal of Microbiology
1 populations, further ChAd vector development may ultimately generate a better adenovirus-
2 based vaccine strategy.
3 A promising example of the use of ChAd vectors is ChAdOx1 made from the
4 chimpanzee Y25 adenovirus (Dicks et al. 2012), which is currently being use by Oxford
5 University to develop a SARS-CoV-2 vaccine candidate. Vaccines against several pathogens,
6 including MERS-CoV (Munster et al. 2017) have been developed using this this vector
7 (Antrobus et al. 2014; Morris et al. 2016; Wilkie et al. 2020; Cappuccini et al. 2020). ChAdOx1
8 MERS encodes the full length of the MERS-CoV spike protein, with the transgene sequence
9 inserted in place of the E1 unit (Munster et al. 2017). In transgenic mouse models, this vaccine
+ 10 produced high-titer MERS-CoV nAbs asDraft well as strong CD8 T cell responses to the S protein 11 (Munster et al. 2017). To measure the effectiveness of the vaccine, mice were challenged 28 days
12 after with a MERS-CoV strain, and the immunized mice showed no significant weight loss or
13 signs of disease. A secondary study using rhesus macaques showed that a single dose of the
14 ChAdOx1 MERS vaccine produced a nAb response (van Doremalen et al. 2020). Sera taken
15 from vaccinated animals on the day of MERS-CoV challenge neutralized all six MERS-CoV
16 circulating strains, demonstrating broad protection (Munster et al. 2017). A phase I clinical trial
17 is underway for the ChAdOx1 MERS vaccine, set to have five groups varying in vaccine dosage
18 and booster schedule (NCT03399578).
19 Based on this success, Oxford Biomedical and AstraZeneca are now developing a SARS-
20 CoV-2 ChAdOx1 vectored vaccine called AZD1222 (formally ChAdOx1 nCoV-19). From prior
21 successes with the ChAdOx1 MERS vaccine, the ChAdOx1 vector was instead outfitted with the
22 SARS-CoV-2 spike glycoprotein. Preclinical studies showed that the vaccine was immunogenic
23 in mice as it elicited humoral and cell-mediated responses that was Th1 dominant (Doremalen et
17 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 18 of 91
1 al. 2020). It was also noted that nAbs against SARS-CoV-2 were found in all vaccinated mice.
2 AZD1222 was then used to immunize rhesus macaques and a single immunization was able to
3 elicit humoral and cellular immune responses, with a reduced Th2 response as well (Doremalen
4 et al. 2020). SARS-CoV-2 specific nAbs were observed in every vaccinated animal before viral
5 challenge, while control animals presented no nAbs. Seven days after SARS-CoV-2 challenge,
6 immunized animals developed no pulmonary pathology and displayed significantly reduced viral
7 loads in their lung tissue compared to control counterparts (Doremalen et al. 2020). No ADE was
8 observed in any animal over the course of these preclinical trials.
9 Preliminary results of a phase I/II clinical trial (2020-001072-15) were reported July 20, 10 2020 (Folegatti et al. 2020), which testedDraft efficacy, immunogenicity, and safety of the vaccine in 11 healthy participants. Injected intramuscularly, it is a randomized, single blind, and controlled
12 trial with 1077 participants. It was found that local and systemic adverse events such as injection
13 site pain or tenderness, fatigue, headache, chills, muscle ache, malaise, and feeling feverish were
14 more common in AZD1222 vaccine group compared to the control group (Folegatti et al. 2020).
15 Prophylactic paracetamol was given to 16% of participants that received the AZD1222 vaccine
16 and its effect reduced the number of reported local and systemic adverse events. Ten individuals
17 were non-randomly assigned to a prime-boost group that received no prophylactic treatment. The
18 percentages of reported local and systemic adverse events were very similar to that from the
19 single dose group, however it was observed that reactogenicity decreased after the second dose
20 as fewer adverse events were reported (Folegatti et al. 2020). No serious adverse events were
21 associated with AZD1222 vaccination. nAbs against the SARS-CoV-2 S protein in the AZD1222
22 vaccine group were highest in individuals 28 days after immunization and stayed elevated until
23 day 56 in the single dose group (Folegatti et al. 2020). Participants that received a second dose
18 © The Author(s) or their Institution(s) Page 19 of 91 Canadian Journal of Microbiology
1 demonstrated increased nAbs levels at day 56. AZD1222 induced effector T cell responses
2 specific to SARS-CoV-2 S protein that peaked on day 14 of the study, but an increase in these
3 responses was not detected after the second dose in the prime-boost group (Folegatti et al. 2020).
4 The study does note limitations that impact the conclusions that can be drawn, such as the low
5 number of individuals in the prime-boost group, the young and mostly white adult demographic
6 that participated, and the short follow-up period. To address these shortcomings, a phase IIb/III
7 clinical trial is now underway (2020-001228-32) and is expanding the age range to focus on the
8 very young and elderly (“Oxford COVID-19 vaccine to begin phase II/III human trials” 2020).
9 The goal is to assess whether the immune system responds differently to AZD1222 depending on
10 the age of the individual. Ongoing phase III trials (ISRCTN89951424, NCT04516746,
11 NCT04540393) will examine vaccine efficacyDraft in ~32,000 adults, with the primary objective of
12 analyzing whether the vaccine can work to prevent COVID-19 infections altogether
13 (AstraZeneca 2020a). A press release from AstraZeneca on November 23, 2020 announced that
14 their interim analysis of the phase III trial revealed AZD1222 to be 62-90% effective in
15 preventing COVID-19 depending on the dosage regimen used (AstraZeneca 2020b). When
16 analyzed together, the dosing regimens resulted in an average efficacy of 70%. The vaccine was
17 well tolerated with no serious safety events confirmed. Evaluation of additional Phase III data is
18 forthcoming.
19 A.3 Janssen Pharmaceutical Companies vaccine candidate AD26.COV2-S
20 In addition to using non-human adenovirus as a means of avoiding pre-existing
21 immunity, rarer serotypes of human adenoviruses are also being considered. Human adenovirus
22 serotype 26 (HAdV-D26) is genetically distant from the more prevalent HAdV-C5, belonging to
23 sub-group D instead of C (Geisbert et al. 2011). Similar to SAds, rarer adenovirus serotypes such
19 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 20 of 91
1 as HAdV-D26 differ from HAdV-C5 based on hexon coat sequence variations. Studies have
2 shown that smaller numbers of some populations can exhibit significant HAdV-D26
3 seroprevalence, however it was observed that nAb titers for HAdV-D26 remained lower than that
4 for HAdV-C5 in these pre-exposed individuals (Geisbert et al. 2011). As a vector, HAdV-D26
5 has demonstrated measurable cell mediated and humoral immune responses in NHP with the use
6 of a booster following primary vaccination (Geisbert et al. 2011). To produce the HAdV-D26
7 vector, a similar process is followed as with prior adenovirus vectors discussed above. The E1
8 and E3 units are deleted, however the E4 ORF6 sequence must be modified to allow for
9 replication in HAdV-C5 E1 complementing cell lines, such as 293 cells (Geisbert et al. 2011). 10 Janssen Pharmaceutical Companies,Draft a branch of Johnson & Johnson, has used the HAdV- 11 D26 vector in a two-dose heterologous Ebola virus vaccine strategy. A randomized phase I
12 clinical trial was conducted to evaluate the immunogenicity and safety of the vaccines in healthy
13 adults (Anywaine et al. 2019). Ad26.ZEBOV along with MVA-BN-Filo, a multivalent candidate
14 vaccine that may offer protection against Ebola virus and other causes of viral hemorrhagic
15 fever, were used in alternating immunization schedules (Anywaine et al. 2019). The study
16 reported no serious adverse events associated with vaccination, only systemic and local adverse
17 events such as headache and injection site pain (Anywaine et al. 2019). When looking at
18 individuals after the first dose of either the Ad26.ZEBOV or MVA-BN-Filo vaccines, it was
19 found that Ad26.ZEBOV elicited better cellular and initial binding antibody responses
20 (Anywaine et al. 2019).
21 Janssen now has an ongoing phase III clinical trial of its Ad26.COV2.S, recombinant
22 SARS-CoV-2 vaccine candidate (NCT04505722). The first preclinical study was conducted with
23 mice that used seven different recombinant viruses expressing the full-length SARS-CoV-2 S
20 © The Author(s) or their Institution(s) Page 21 of 91 Canadian Journal of Microbiology
1 protein along with varying mutations and substitutions to examine which vector would be the
2 most effective candidate for a vaccine (Bos et al. 2020). Vector Ad26.S.PP had the full length S
3 with a wild type signal peptide, proline substitutions, and furin cleavage site mutations that
4 stabilize the prefusion conformation of the S protein. Separate assays were used to assess S
5 protein-specific binding antibodies and nAbs titers. It was found that Ad26.S.PP elicited
6 significantly higher titers in mice than Ad26.S, an unmodified, native full-length S vector
7 construct (Bos et al. 2020). Ad26.S.PP also elicited the highest ratio of neutralizing to non-
8 neutralizing binding antibodies compared to the six other viral constructs. A single dose also
9 induced a strong Th1 cell response with a minimal Th2 cell response, as seen by splenocyte
10 stimulation with increased IFN-γ secretion (Bos et al. 2020). Draft 11 A second preclinical study using NHPs produced similar results (Mercado et al. 2020).
12 Fifty-two adult rhesus macaques were assigned to groups and received a single vaccination of
13 one of the seven viral vectors carrying engineered variants of SARS-CoV-2 S , including
14 Ad26.S.PP, or a placebo. Four weeks following immunization it was observed that all vaccinated
15 animals no matter the vector design, had a measurable RBD-specific binding antibody response
16 (Mercado et al. 2020). The highest nAb titers were induced in the Ad26.S.PP group, as well as
17 the highest effector function responses. High binding antibody responses were also seen with the
18 Ad26.S.PP group. It was observed that Ad26.S.PP induced a Th1 cell dominant response after a
19 single vaccination (Mercado et al. 2020). When looking at viral challenge, animals vaccinated
20 with Ad26.S.PP saw no detectable viral loads in bronchoalveolar lavage samples while the other
21 vaccine variants offered only partial protection as they had low levels of SARS-CoV-2 viral
22 RNA (Mercado et al. 2020). Ad26.S.PP was observed as effective in protecting the upper and
21 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 22 of 91
1 lower respiratory tracts in vaccinated animals, excluding one animal (out of the six) that
2 displayed a low viral load (Mercado et al. 2020).
3 An additional preclinical study using Syrian golden hamsters was conducted (Tostanoski
4 et al. 2020). This study used only two of the previous seven Ad26 candidate vectors, Ad26.S.PP
5 and Ad26.S.dTM.PP (which had the transmembrane region and cytoplasmic tail of SARS-CoV-2
6 S deleted) (Tostanoski et al. 2020). RBD specific binding antibody titers were 4.0-4.7 fold higher
7 and responded more consistently four weeks after vaccination with Ad26.S.PP compared to
8 Ad26.S.dTM.PP (Tostanoski et al. 2020). Also, 1.8-2.6 fold greater nAb titers were induced by
9 Ad26.S.PP in contrast to Ad26.S.dTM.PP. Upon challenge, animals in both vaccine groups lost 10 significantly less weight than control animals,Draft with the Ad26.S.PP group showing the least 11 percent weight loss (Tostanoski et al. 2020). Four days after challenge, animals in the Ad26.S.PP
12 group showed little to no viral interstitial pneumonia in lung tissue compared to control groups
13 which displayed severe pneumonia. Neutrophilic and histiocytic inflammation and SARS-CoV-2
14 viral RNA decreased significantly in the lung tissue of animals in both Ad26.S.PP and
15 Ad26.S.dTM.PP groups compared to control groups (Tostanoski et al. 2020). Due to the
16 immunogenicity of the Ad26.S.PP viral construct, it was then used as the lead vaccine candidate
17 against SARS-CoV-2, known as Ad26.COV.2.S.
18 Results of a randomized, double-blinded, placebo-controlled phase I/IIa clinical trial are
19 now available for Ad26.COV2.S (NCT04436276) (Sadoff et al. 2020). The study involved
20 healthy adult participants ranging in age from 18-55 years old (n= 402) and >65 years (n=394)
21 who received 5x1010 or 1x1011 viral particles per vaccination, either as a single dose or as two
22 separate doses 56 days apart. Common adverse effects included injection site pain, fatigue,
23 headache and myalgia. Fevers occurred in both age groups, but were mostly mild or moderate,
22 © The Author(s) or their Institution(s) Page 23 of 91 Canadian Journal of Microbiology
1 and resolved within 1-2 days. Consistent with pre-clinical trials, a single dose of either 5x1010 or
2 1x1011 vp resulted in ~92% seroconversion for S protein (based on a wild-type virus
3 neutralization assay) 29 days post injection for the 18-55 years old cohort. A similar
4 immunogenicity profile was observed for members of the >65 years cohort, albeit data was only
5 available for 15 participates from this cohort at the time of publication. Further, Th1 CD4+ T
6 cell responses specific for S-protein were generated in ~81% of a subset of participants from
7 both age groups, with very low or no detectible Th2 responses. These promising results initiated
8 an on-going phase III clinical trial involving ~60,000 adult participants aged 18 years and older,
9 and will be a placebo-controlled, double blind, randomized study evaluating the safety and
10 efficacy Ad26.COV.2.S administered as a single dose of 5x1010 vp (NCT04505722). Draft 11 A.4 Gamaleya National Research Center vaccine candidate Sputnik V
12 The Gamaleya National Research Center of Epidemiology and Microbiology has
13 developed ‘Sputnik V’ (formally Gam-COVID-Vac) a human adenoviral vector-based SARS-
14 CoV-2 S protein vaccine candidate (“Sputnik V- the First Registered Vaccine Against COVID-
15 19” 2020). Sputnik V was approved on August 11, 2020 by the Russian government and became
16 the first registered SARS-CoV-2 vaccine. However, it has been met with uncertainty from other
17 international institutions and developers due to a paucity of published preclinical data and
18 controversy over published clinical data (Balakrishnan 2020).
19 Sputnik V uses a two-vectored approach to vaccination, using both recombinant HAdV-
20 D26 and HAdV-C5 to carry the SARS-CoV-2 S protein transgene (“Sputnik V- the First
21 Registered Vaccine Against COVID-19” 2020). The primary vaccination uses the HAdV-D26
22 based vector while the boost uses the HAdV-C5 based vector on a day 0, 21 schedule. The use of
23 two different recombinant vectors with the heterologous prime-boost strategy avoids the
23 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 24 of 91
1 development of immunity to the vector, which is thought to elicit a more effective immune
2 response upon booster immunization (Dolzhikova et al. 2017; “Sputnik V- the First Registered
3 Vaccine Against COVID-19” 2020). This heterologous prime-boost method has been previously
4 used by the Gamaleya National Research Center in their GamEvac-Combi Ebola virus vaccine
5 (Dolzhikova et al. 2017). Live-attenuated recombinant vesicular stomatitis virus (VSV) and
6 HAdV-C5 vectors containing the envelope glycoprotein of Ebola were used in a phase I/II
7 clinical trial (Dolzhikova et al. 2017). Strong humoral and cellular immune responses were
8 elicited by the Ebola vaccine in all 84 participants, with a significantly greater antibody response
9 found in individuals that received the VSV-prime Ad5-boost schedule compared to the single
10 vaccination of only VSV (Dolzhikova et al. 2017). Participants (89%) indicated one or more
11 adverse reactions that included injectionDraft site pain, headache, fatigue, nausea, and loss of appetite;
12 however, no serious adverse events were reported (Dolzhikova et al. 2017).
13 Non-randomized, open labelled, two stage phase I/II clinical trials were completed for
14 Sputnik V using 76 healthy adult participants from 18 to 60 years of age (NCT04436471,
15 NCT04437875). The two phase I/II studies both involved 38 participants; 9 received a single
16 immunization of rAd26-S, 9 received a single immunization of rAd5-S, and 20 received a
17 heterologous prime-boost on a day 0,21 schedule with rAd26-S and rAd5-S, respectively
18 (Logunov et al. 2020). For both studies, the most commonly reported systemic and local adverse
19 events were headache (42%), muscle and joint pain (24%), hyperthermia (50%), fatigue (28%),
20 and injection site pain (58%) (Logunov et al. 2020). Participants in groups that were given the
21 heterologous prime-boost regimen reported more adverse events following the second
22 immunization. No serious adverse events were reported in either studies. In both studies, SARS-
23 CoV-2 RBD specific binding antibodies were found in 100% of participants 21 days after one
24 © The Author(s) or their Institution(s) Page 25 of 91 Canadian Journal of Microbiology
1 immunization (Logunov et al. 2020). In the heterologous prime-boost groups, the second
2 vaccination saw a large increase in RBD specific binding antibody titers 7 days following the
3 booster. Interestingly, it was noted that nAb production was induced in 100% of individuals only
4 in the heterologous prime-boost groups (Logunov et al. 2020). A strong cellular immune
5 response was found in 100% of participants, with the detection of both CD4+ and CD8+ T cells
6 (Logunov et al. 2020). nAbs to the rA26 and rAd5 vectors were evaluated in all study
7 participants, and no significant correlation between SARS-CoV-2 RBD specific binding
8 antibodies and the vector nAbs titers was found (Logunov et al. 2020). Also, evidence of vector
9 nAb cross-reactivity was absent. The Sputnik V clinical study mentions unpublished preclinical
10 data with NHP and hamsters, highlighting strong cellular and humoral responses in NHP,
11 protection from SARS-CoV-2 infection,Draft and no ADE observed in any vaccinated and SARS-
12 CoV-2 challenged animals (Logunov et al. 2020). There are several limitations in the Sputnik V
13 clinical study, including the lack of placebo or control, short follow-up period, few participants,
14 and limited participant diversity in age and gender.
15 A phase III clinical trial has since commenced that involves 40,000 adult participants
16 (NCT04530396). It will be double-blind, placebo controlled, and randomized with the goal to
17 assess the immunogenicity, safety, and protective efficacy of the vaccine. Though Sputnik V was
18 the first registered SARS-CoV-2 vaccine, it still faces similar hurdles to the other non-replicating
19 viral vector vaccines currently under development.
20 B. Inactivated Virus Vaccines
21 As a more traditional vaccine approach, inactivated virus vaccines use virus collected
22 from infected cell cultures or animal sources and expose the particles to physical agents such as
23 heat or radiation, or chemical agents such as formalin or β-propiolactone to inactivated them
25 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 26 of 91
1 (Christopher J. Burrell, Frederick A. Murphy, Colin R. Howard, Colin R. Howard 2020).
2 Inactivated vaccines are generally considered safe and highly stable, showing a good record in
3 immunocompromised individuals, with examples including hepatitis A, rabies, tickborne
4 encephalitis, Japanese encephalitis, polio, and influenza (Christopher J. Burrell, Frederick A.
5 Murphy, Colin R. Howard, Colin R. Howard 2020). The drawback with this vaccine strategy
6 comes with the requirement for large amounts of immunogen to induce acceptable antibody
7 responses. Further, immunization from an inactivated virus vaccine creates a largely humoral
8 response, with very little cellular immunity elicited (Cohen and Bordin 2015). Consequently,
9 after primary vaccination, future boosters shots are sometimes required to maintain long-term
10 immunity as nAb titers decrease with time (Christopher J. Burrell, Frederick A. Murphy, Colin
11 R. Howard, Colin R. Howard 2020). TheDraft physical or chemical agents used for the inactivation
12 step can also affect the immunogenicity of the vaccine. Alkylating agents such as β-
13 propiolactone and formalin work chemically by reactions involving nucleic acids and viral
14 capsid proteins (Delrue et al. 2012; Chowdhury et al. 2015). Formalin has been found to induce
15 irreversible changes in many viral antigens (Christopher J. Burrell, Frederick A. Murphy, Colin
16 R. Howard, Colin R. Howard 2020) and these altered antigens may cause an overall weaker
17 mucosal and cell-mediated immune response. β-propiolactone use in the development of human
18 rabies vaccines has shown to be less destructive to proteins and forms non-toxic products upon
19 hydrolyzation (Christopher J. Burrell, Frederick A. Murphy, Colin R. Howard, Colin R. Howard
20 2020). Ultraviolet and gamma irradiation are physical agents that can also be used, which work
21 to damage viral genomes by pyrimidine dimer formation or free radical formation upon covalent
22 bond breakages, respectively (Delrue et al. 2012). These methods can indiscriminately alter
23 sequences, capsid or other viral proteins, so this must be considered to preserve certain viral
26 © The Author(s) or their Institution(s) Page 27 of 91 Canadian Journal of Microbiology
1 immunogens and structure. To increase vaccine immunogenicity, adjuvants such as alum and
2 MF59 (Pasquale et al. 2015) are usually used to help boost cellular immune response to
3 immunization. Depending on the adjuvant, different humoral and cellular feedback takes place.
4 For instance, alum adjuvant tends to increase inflammation at the injection site, activating APCs
5 such as dendritic cells, creating a Th2 cell skewed response (Grun and Maurer 1989; Kool et al.
6 2008). MF59 adjuvant can activate APCs as well, however it also induces the transport of cells to
7 the draining lymph nodes and creates a more balanced CD4+ T cell response (Kim et al. 2020a).
8 B.1 Sinovac vaccine candidate CoronaVac (formerly Piccovacc)
9 Near the end of the SARS-CoV-1 outbreak, Sinovac Biotech developed an inactivated 10 SARS-CoV vaccine (ISCV). β-propiolactoneDraft was the agent used to inactivate the SARS-CoV-1 11 virus, and alum adjuvant was used (Lin et al. 2007). Phase I clinical trials were performed with
12 36 healthy adults, was placebo controlled, randomized, and double-blind with the goal to assess
13 immunogenicity and safety. There were three trial arms; low dose, high dose, and placebo, with
14 each participant receiving two doses through intramuscular injection. Following vaccination,
15 mild injection site adverse reactions such as pain, erythema, and itching occurred in a few
16 individuals, with no statistical difference between the groups (Lin et al. 2007). Very few
17 systemic symptoms were observed across the three groups, with no grade 3 reactions. Results
18 showed that in both low and high dose groups, ISCV elicited similarly high numbers of specific
19 nAbs (Lin et al. 2007). However, no further trials were conducted, so the efficacy of the vaccine
20 in individuals above age 40 or the overall ability of the vaccine to prevent SARS-CoV-1
21 infection was not investigated further.
22 Using a similar strategy, Sinovac has now developed an inactivated SARS-CoV-2
23 vaccine candidate, CoronaVac (formally PiCoVacc). Much like their ISCV vaccine, the virus
27 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 28 of 91
1 was inactivated using β-propiolactone and boosted using alum adjuvant (Gao et al. 2020).
2 Animal studies in mice, rats, and rhesus macaques were all able to elicit specific nAbs, and
3 neutralization assays performed against nine other strains of SARS-CoV-2 using the serums of
4 mice and rats also induced these nAbs (Gao et al. 2020). Rhesus macaques were vaccinated three
5 times with either medium or high doses of CoronaVac and upon viral challenge, all were
6 protected from infection apart from a few histopathological changes. Mild inflammatory cell
7 infiltration was observed in the lung tissue of immunized animals and was likely attributed to the
8 method of inoculation (Gao et al. 2020). In contrast, all control animals showed viral RNA loads
9 and developed severe infection. 7 days after SARS-CoV-2 challenge, the high dose group had no
10 detectable viral loads in their lungs or pharynx, while the medium dose group had detectable but
11 ~95% reduced viral loads in their lungs Draftand pharynx compared to the control group (Gao et al.
12 2020).
13 Phase I/II clinical trials for CoronaVac, were split into two parts as double-blind,
14 randomized, and placebo-controlled studies (NCT04352608, NCT04383574). The phase II part
15 of the trial included 600 healthy participants aged 18 to 59 years old that received two
16 CoronaVac immunizations of either 3 µg, 6 µg, or placebo on day 0, 14 or day 0, 28 regimens
17 (Zhang et al. 2020b). No grade 3 adverse events were reported, however the incidence of
18 injection site and systemic adverse events were 35.0%, 33.3%, and 21.7% in the 14 day schedule
19 and 19.2%, 19.2%, and 18.3% in the 28 day schedule for the 3 µg, 6 µg, and placebo groups,
20 respectively (Zhang et al. 2020b). It was noted that no significant difference was observed
21 between the various vaccine groups and placebo groups when considering adverse events.
22 Seroconversion rates for each vaccine dosage did not significantly differ between the two
23 schedules. For the 28 day dosage schedule, nAb titers saw a significant increase in participants
28 © The Author(s) or their Institution(s) Page 29 of 91 Canadian Journal of Microbiology
1 28 days following the second immunization compared to titers from the 14 day dosage schedule
2 (Zhang et al. 2020b). It was also found that younger participants displayed higher nAb titers than
3 older participants, with significant decreases in titer correlated with increasing age (Zhang et al.
4 2020b). This suggests elderly recipients of the vaccine could require a higher dosage or multiple
5 doses to afford protection. Some of the primary limitations of this study included the lack of
6 assessment of T cell responses in participants, as well as the exclusion of adults aged 60 years
7 and older from the trial.
8 A phase III clinical trial for CoronaVac is now ongoing (NCT04456595). It is a placebo-
9 controlled, double blind, and randomized study evaluating the safety and protective efficacy in 10 8870 health care professional participants.Draft Based on results from the phase II study, the 3 µg 11 dosage of CoronaVac is being used with either the 14 day or the 28 day schedule in participants.
12 Individuals will be grouped based on age with designated placebo and experimental arms for
13 adults 18-59 years old and the elderly (60 years and older).
14 B.2 Beijing Institute of Biological Products and Sinopharm vaccine candidate BBIBP-CorV
15 The Beijing Institute of Biological Products and Sinopharm have created BBIBP-CorV, a
16 β-propiolactone inactivated vaccine and alum adjuvant candidate that is currently in phase III
17 clinical trials (ChiCTR2000034780). Previous animal studies using BBIBP-CorV elicited high
18 numbers of nAbs in mice, rats, guinea pigs, rabbits, and NHPs (Wang et al. 2020a). It was found
19 that the vaccine was significantly more immunogenic in mice and rhesus macaques that received
20 two doses rather than one. Upon viral challenge in rhesus macaques, all animals in the placebo
21 group showed high viral loads throughout the duration of the study (Wang et al. 2020a). Animals
22 that received a low dose of BBIBP-CorV saw significantly lower viral loads in throat swabs than
23 the placebo group. Seven days after challenge, none of the vaccinated animals displayed any
29 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 30 of 91
1 detectable viral load in their lung tissue, whereas high viral loads and severe interstitial
2 pneumonia was detected in the lungs of animals in the placebo group (Wang et al. 2020a). It was
3 also noted that no ADE was observed in any immunized rhesus macaques upon viral challenge
4 (Wang et al. 2020a).
5 The phase I/II trial was split into 2 main parts and operated as a placebo-controlled and
6 double-blinded study assessing the safety and immunogenicity of the vaccine
7 (ChiCTR2000032459). Phase I involved 192 participants that were sectioned into groups based
8 on age (18 to 59 years or 60 years and older) and were assigned at random to receive two 2 µg
9 doses, two 4 µg doses, or one 8 µg dose of BBIBP-CorV or placebo (Xia et al. 2020b). Within 7 10 days of vaccination, 29% of participantsDraft that were immunized with BBIBP-CorV reported at 11 least one adverse event compared to 17% reported by placebo recipients (Xia et al. 2020b). No
12 serious adverse events were reported in any group 28 days following vaccination. The most
13 common local adverse event was pain, reported after either dose in 24% of individuals that
14 received the vaccine and 6% of placebo recipients (Xia et al. 2020b). The most common
15 systemic adverse event reported overall was fever in 4% of those that were given BBIBP-CorV
16 and 6% in those that were given placebo (Xia et al. 2020b). On day 42 following the second
17 vaccination, 100% of vaccine recipients recorded nAbs against SARS-CoV-2. In phase II, 448
18 participants were assigned at random to receive one 8 µg dose on day 0, placebo doses, or two 4
19 µg doses of BBIBP-CorV on days 0 and 14, 0 and 21, or 0 and 28 (Xia et al. 2020b). The most
20 commonly reported local and systemic adverse events were injection site pain (16% of vaccine
21 recipients) and fever (2% of vaccine recipients), respectively (Xia et al. 2020b).
22 In all groups that were immunized with BBIBP-CorV, nAbs titers against SARS-CoV-2
23 were detected and were significantly higher than in the placebo groups. However, nAb titers for
30 © The Author(s) or their Institution(s) Page 31 of 91 Canadian Journal of Microbiology
1 the 8 µg single dose group was significantly lower than all three of the prime-boost vaccination
2 schedules, suggesting that two doses may be necessary to elicit a better and more effective
3 immune response (Xia et al. 2020b). A double blind, placebo-controlled, and randomized phase
4 III clinical trial is ongoing and will involve ~15,000 healthy adult individuals, with the goal to
5 assess the protective efficacy and safety of BBIBP-CorV (ChiCTR2000034780).
6 B.3 The Wuhan Institute of Biological Products and Sinopharm vaccine candidate
7 The Wuhan Institute of Biological Products and Sinopharm have an ongoing phase I/II
8 clinical trial for their SARS-CoV-2 inactivated vaccine candidate (ChiCTR2000031809). The
9 study is double-blind, placebo controlled, and randomized, with the goal to evaluate the safety 10 and immunogenicity of the vaccine in 1456Draft healthy individuals aged 18 to 59 years. An interim 11 report was released based on early results from this study (Xia et al. 2020a). Ninety-six
12 participants took part in the phase I part of the study, assigned to low, medium, high, or alum
13 adjuvant only sections, and were given three immunizations on a day 0, 28, and 56. The phase II
14 part of the trial observed in this report consisted of 224 participants that were assigned to receive
15 either a medium dosage of the vaccine or alum only, on either day 0, 14 or day 0, 21 schedules.
16 Other groups in the phase II study did not have their results released in this interim report, as
17 they remain blinded. Of the total 320 participants evaluated in the phase I and II parts of this
18 trial, 15% experienced adverse events, most commonly as injection site pain (Xia et al. 2020a).
19 In the phase I trial, 20.8%, 25%, and 12.5% of participants reported one or more adverse events
20 in the low, medium, and high dosage groups, respectively (Xia et al. 2020a). In the phase II trial,
21 6%, 14.3%, 19%, and 17.9% of participants reported one or more adverse events in the medium
22 dosage at day 0, 14, alum only at day 0,14, medium dosage at day 0, 21, and alum only at day 0,
23 21, respectively (Xia et al. 2020a). No serious adverse events were reported. It was found that a
31 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 32 of 91
1 longer period of time between the primary and secondary vaccinations induced a larger antibody
2 response in participants than from using a shorter schedule, in both trial phases. 100% of
3 participants in both the low and high dosage groups in phase I saw the induction of nAbs, with
4 95.8% of participants in the medium dosage group (Xia et al. 2020a). In the phase II part, 97.6%
5 of participants from both of the medium dosage groups saw nAb induction, while no individuals
6 from the alum only group recorded any. Production of specific binding antibodies was noted in
7 100% of individuals from any dosage group in phase I, 100% of individuals in the medium
8 dosage group on the day 0, 21 schedule in phase II, and 85.7% of participants in the medium
9 dosage group on the day 0, 14 schedule in phase II (Xia et al. 2020a). However, T cell responses
10 were not measured in neither the phase I nor II parts of the trial. A phase III clinical trial has
11 been approved and will be double-blind,Draft placebo controlled, and randomized
12 (ChiCTR2000034780). The safety and protective efficacy of the candidate vaccine will be
13 further evaluated in ~15,000 healthy participants aged 18 years and older.
14 Recombinant Protein Subunit Vaccines
15 Recombinant protein subunit vaccines consist of a more targeted approach, using specific
16 immunogenic parts of a pathogen to elicit an immune response. However, protein subunits alone
17 generate very little if any innate immune system responses, show reduced stimulation of APCs,
18 can have limited interactions with B cell receptors, and may cause poor activation of T cells
19 (Purcell et al. 2007; Moyle and Toth 2013; Karch and Burkhard 2016; Tian et al. 2020). Protein
20 subunits display fewer immunogens compared to whole agent vaccines for example, which can
21 create challenges for mounting an effective immune response (Moyle and Toth 2013). Therefore,
22 recombinant protein subunit vaccines often require adjuvant and usually several doses to increase
23 efficacy and to confer long-term immunity (Moyle and Toth 2013).
32 © The Author(s) or their Institution(s) Page 33 of 91 Canadian Journal of Microbiology
1 Recombinant protein subunit vaccines against SARS-CoV-1 and MERS-CoV have been
2 developed by Novavax and tested in mice (Coleman et al. 2014). Full length S proteins for both
3 viruses were generated using baculovirus vectors propagated in Spodoptera frugiperda (Sf9)
4 insect cells. S protein from the above viruses was purified as protein–protein micellular
5 nanoparticles, which were found to generate high levels of nAbs in mice against their cognate
6 coronavirus, but not for other coronaviruses (Coleman et al. 2014). The use of an adjuvant was
7 found to significantly increase nAb numbers 15-fold with alum and 68-fold with Matrix M1TM.
8 Novavax is now developing a SARS-CoV-2 recombinant protein subunit vaccine, NVX-
9 CoV2373, which is entering into phase III clinical trials (2020-004123-16, NCT04611802). It is
TM 10 a full-length recombinant, prefusion stabilizedDraft S glycoprotein, Matrix-M adjuvanted 11 nanoparticle vaccine that is based on their SARS-CoV-1, MERS-CoV, and NanoFluTM vaccine
12 strategies (Coleman et al. 2014; Tian et al. 2020). In a preclinical study, mice were vaccinated
13 with low, medium, or high doses of NVX-CoV2373 with Matrix-M adjuvant using a single or
14 prime/boost administration (Tian et al. 2020). Animals that received the highest dosage of NVX-
15 CoV2373/ Matrix-M elicited nAbs and effective binding antibodies to the S protein after a single
16 immunization. The efficacy of the adjuvant was also examined, and it was found that mice
17 vaccinated with any dosage of NVX-CoV2373/ Matrix-M showed significantly higher S protein
18 specific antibody titers compared to mice that were vaccinated with the highest dosage of NVX-
19 CoV2373 without Matrix-M (Tian et al. 2020).
20 Upon SARS-CoV-2 viral challenge both placebo and NVX-CoV2373 without Matrix-M
21 groups displayed viral loads in mice lung tissue, while all mice immunized with NVX-CoV2373/
22 Matrix-M had very low to no detectable viral loads were observed (Tian et al. 2020). Virus titer
23 was dose-dependent in the single immunized NVX-CoV2373/ Matrix-M groups, with no
33 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 34 of 91
1 significant lung viral load recorded for the highest dosage. For all dosage levels of the
2 prime/boost groups, viral load in the lung was nearly nonexistent, with only the lowest dosage
3 showing viral loads measuring at least 1 log lower than the placebo group (Tian et al. 2020).
4 Mice CD4+ and CD8+ T cell frequency in the spleen was found to be significantly greater with
5 NVX-CoV2373/ Matrix-M vaccination than in NVX-CoV2373 without adjuvant (Tian et al.
6 2020). Immunization with Matrix-M adjuvant produced a Th1 cell skewed response in mice.
7 The second part of the study assessed the immunogenicity of the vaccine in adult olive
8 baboons. Low, medium, or high dosages of NVX-CoV2373 with Matrix-M were administered in
9 two doses, along with a high dosage group of NVX-CoV2373 without Matrix-M. Animals in all 10 dosage levels of the NVX-CoV2373/ Matrix-MDraft groups experienced increases greater than a log 11 in S protein specific antibody titers after a second vaccination (Tian et al. 2020). However,
12 animals vaccinated without Matrix-M adjuvant saw minimal to no detection of S protein specific
13 antibodies followed both primary and booster vaccinations. An increase of 25- to 38-fold in nAb
14 titers was seen in the NVX-CoV2373 groups after a booster immunization, whereas very few to
15 no nAbs were detected in the NVX-CoV2373 without Matrix-M group (Tian et al. 2020).
16 Animals that received the medium and high dosages of NVX-CoV2373/ Matrix-M saw very high
17 amounts of IFN-γ secreting cells, 5-fold greater than the low dosage and non-adjuvanted groups
18 (Tian et al. 2020).
19 A report from phase I of a randomized, placebo-controlled, and observer blinded NVX-
20 CoV2373 SARS-CoV-2 phase I/II vaccine trial is now available (Keech et al. 2020). The
21 immunogenicity and safety of the vaccine was assessed in 131 healthy adults. Initially, 6
22 participants were randomly assigned to 5 µg or 25 µg of NVX-CoV2373 with Matrix-M to
23 evaluate the reactogenicity of the vaccine over a two-day period. The remaining 125 participants
34 © The Author(s) or their Institution(s) Page 35 of 91 Canadian Journal of Microbiology
1 were subsequently divided into five groups; placebo (A), two immunizations of 25 µg NVX-
2 CoV2373 without Matrix-M (B), two immunizations of 5 µg NVX-CoV2373 with Matrix-M
3 (C), two immunizations of 25 µg NVX-CoV2373 with Matrix-M (D), or one immunization of 25
4 µg NVX-CoV2373 with Matrix-M followed by a placebo dose (E) (Keech et al. 2020). Groups
5 that received two vaccinations (B, C, and D) were on a day 0,21 schedule. No serious adverse
6 events were reported from any group. Local adverse events upon primary vaccination such as
7 injection site pain, erythema, swelling, or tenderness was reported in >88% of individuals in A,
8 B, C, D, and E groups (Keech et al. 2020). Systemic adverse events such as fatigue, nausea,
9 headache and malaise were reported in >89% of participates, and fever reported in 68% of
10 participants (Keech et al. 2020). Severe adverse events such as malaise, headache, and fatigue
11 were only found in 2% of individuals afterDraft the first dose. Following the secondary vaccination,
12 65% of participants in group C, 67% in group D, and all participants in groups A, B, and E
13 reported one or more local adverse events. As for systemic adverse events, 86% of individuals in
14 A, 84% in B, 73% in C, 58% in D, and 96% in E groups reported at least one event (Keech et al.
15 2020). Only one participant reported a severe local adverse event, while eight reported severe
16 systemic adverse events such as fatigue, joint pain, and malaise.
17 S protein specific binding antibody responses were recorded for the adjuvanted vaccine
18 groups (C, D, and E), with increases seen after the second immunization (Keech et al. 2020).
19 After one vaccination, nAb titers were significantly greater in adjuvanted groups (C, D, and E)
20 than the non-adjuvanted group (B) (Keech et al. 2020). Following a second vaccination,
21 adjuvanted groups saw geometric mean fold rises (GMFRs) in nAbs that were 100 times higher
22 than that of a single dose of NVX-CoV2373 without adjuvant. A strong correlation between nAb
23 and S protein specific binding antibody titers was noted in groups at day 35 that were given
35 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 36 of 91
1 adjuvanted vaccine, but not in group B (Keech et al. 2020). Four participants from each group (A
2 through D) were randomly selected to assess T cell responses. Individuals from groups C and D
3 with adjuvanted regimens displayed CD4+ T cell responses with a favoured Th1 cell and minimal
4 recorded Th2 cell responses (Keech et al. 2020).
5 Phase II of the above trial includes a greater number of participants with a more diverse
6 range in age and race. An observer-blinded, placebo controlled, randomized phase IIb clinical
7 trial is ongoing, with the goal to assess the immunogenicity, efficacy, and safety of the vaccine in
8 individuals that are HIV positive or negative (NCT04533399). Adults (n = 2904) aged 18 to 64
9 years will take part in this study, receiving two doses on a day 0,21 schedule of either 5 µg of 10 NXV-CoV2373 with Matrix-M adjuvantDraft or placebo. A phase III clinical trial is also now 11 underway, assessing the safety and efficacy of the vaccine in adult participants aged 18 to 84
12 years. ~15,000 individuals will participant in this placebo controlled, observer-blinded, and
13 randomized study (NCT04583995)
14 DNA Vaccines
15 While there are currently no licensed DNA vaccines for humans, several have been
16 approved for veterinary use. Human vaccines are currently being developed against a wide range
17 of diseases, such as HIV, tuberculosis, malaria, and certain cancers (Tuteja 2002; Chen et al.
18 2014; Chartrain 2014; Liang et al. 2017; Lopes et al. 2019). DNA vaccines are typically
19 plasmids, that contain the gene for the antigen driven by a strong promoter, often viral, such as
20 the human Cytomegalovirus Immediate Early (hCMV-IE) gene promoter, as well as a
21 terminator sequence at the 3’ end with a poly-adenylation signal (Chatellard et al. 2007; Bower
22 and Prather 2012). Sometimes, the plasmids will contain other regulatory elements and/or introns
23 to enhance stability and expression. (Schorr et al. 1999; Chartrain 2014; Myhr 2017; Ghaffarifar
36 © The Author(s) or their Institution(s) Page 37 of 91 Canadian Journal of Microbiology
1 2018). ColE1-type plasmid vectors containing an expression cassette are often used, as they are
2 well studied and only need host produced E. coli proteins to replicate independently of the
3 bacterial chromosome (Bower and Prather 2012; Chartrain 2014). The plasmid also contains
4 CpG motifs that can activate TLRs upon immunization to enhance immune response
5 (Gramzinski et al. 2001). To increase efficacy of transgene expression after vaccination in a
6 eukaryotic host, viral promoters such as the hCMV-IE promoter or a variety of hybrid promoters,
7 such as the CMV/chicken-β-actin, are usually used as they can elicit high levels of expression
8 (Flingai et al. 2013; Chartrain 2014). While the promoter sequence, transcription termination and
9 polyadenylation signals are essential, additional enhancer elements and codon optimizations can
10 further boost transgene expression (Flingai et al. 2013). Further, supercoiled plasmid DNA can
11 be used reduce the likelihood of integrationDraft into the human genome via non-homologous
12 recombination (Chartrain 2014).
13 In animal models, DNA vaccines have been shown to induce humoral and cellular
14 immune responses (Gurunathan et al. 2000). Since nucleic acid vaccines enter cells and antigenic
15 proteins are internally produced, the desired antigen can be effectively presented to stimulate
16 CD8+ T cell responses. When DNA enters APCs, the same process can occur with antigen
17 presentation, which elicits a CD4+ T cell response as well (Tüting et al. 1999). The downstream
18 effect from both is macrophage activation and B cell proliferation. There are safety concerns,
19 however, due to the possibility of plasmid DNA integrating into the host chromosome (Myhr
20 2017), inflammatory responses, and electroporation. Electroporation at the injection site vastly
21 improves the effectiveness of the vaccine as it assists entry of the plasmid DNA into cells and
22 nuclei (Todorova et al. 2017), but unease with possible reactions to electrical stimulation may
23 affect public perception of this type of vaccine and raise costs.
37 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 38 of 91
1 Previously, the National Institute of Allergy and Infectious Diseases completed a phase I
2 clinical trial of a SARS-CoV-1 DNA vaccine involving 10 healthy adults (Martin et al. 2008).
3 The plasmid contained an expression cassette with an hCMV-IE promoter to produce the SARS-
4 CoV-1 S protein with its cytoplasmic domain truncated (SΔCD). SΔCD was one of two S
5 carboxyl-terminal mutants used in a prior SARS-CoV-1 DNA vaccine study conducted using a
6 mouse model (Yang et al. 2004). It was found that SΔCD could induce significant amounts of
7 nAbs at a higher titer than the other S protein mutant that had its transmembrane domain and
8 cytoplasmic tail deleted (SΔTM) (Yang et al. 2004). Only mild injection site and systemic
9 symptoms were reported after each of the three doses of the vaccine. In most participants a nAb
10 response was induced, with the majority of cellular immune responses arising from CD4+ T cells,
11 accompanied by a less detectable CD8+ DraftT cell response (Martin et al. 2008). Inovio
12 Pharmaceuticals GLS-5300 MERS-CoV DNA vaccine (INO-4700) was developed and
13 underwent animal trials in 2015 (Muthumani et al. 2015). In both mice and camels, S protein
14 specific binding and nAbs against MERS-CoV were successfully induced after three doses of the
15 vaccine. Vaccinated mice also showed an increase in CD8+ and CD4+ T cells. In rhesus
16 macaques, animals were given three low or high doses of the vaccine and results showed
17 significant induction of cytokine secretion from CD8+ and CD4+ T cells in both groups
18 (Muthumani et al. 2015). All vaccinated animals produced binding antibodies that targeted the
19 MERS-CoV S protein and nAbs that offered protection from viral challenge. A phase I clinical
20 trial was conducted, with healthy adults receiving three low, middle, or high doses of the GLS-
21 5300 vaccine intramuscularly followed by electroporation (Modjarrad et al. 2019). Results
22 showed no serious vaccine associated adverse reactions, nAb detection in 50% of individuals,
23 and T cell responses in 76% of participants after the three vaccinations. A secondary phase I/II
38 © The Author(s) or their Institution(s) Page 39 of 91 Canadian Journal of Microbiology
1 study was completed in April 2020 to address some of the efficacy concerns with this vaccine
2 (NCT03721718).
3 A number of SARS-CoV-2 DNA vaccine candidates are under development and
4 currently in preclinical and clinical trials, including from Inovio Pharmaceuticals (Patel et al.
5 2020; World Health Organization 2020; Smith et al. 2020). Interestingly, one preclinical study
6 used six variants of the SARS-CoV-2 S protein in six different vaccines to test their efficacy in
7 rhesus macaques (Yu et al. 2020). It was found that the vaccine containing the full-length S
8 protein antigen offered the best protection in both the upper and lower respiratory tracts
9 compared to the other more specific domain vaccines. This may suggest that at least for DNA 10 vaccines, the entire S protein could be theDraft best immunogenic choice. 11 RNA Vaccines
12 RNA-based vaccine technology is a relative newcomer, and none have been approved for
13 human use. RNA vaccines have been developed for veterinary use (Zhang et al. 2019), and are
14 currently being developed against several diseases in humans including specific cancers,
15 influenza viruses, HIV, and Zika virus (McNamara et al. 2015; Pardi et al. 2017; Scorza and
16 Pardi 2018; Zhang et al. 2019). There are two main types of RNA-based vaccines: messenger
17 RNA (mRNA) and self-amplifying RNA (saRNA). mRNA vaccines are created to resemble the
18 mRNA transcripts produced inside cells that travel from the nucleus to the cytoplasm for
19 translation. However, mRNA in vaccines undergo additional optimizations to increase stability
20 and efficiency of translation (Pardi et al. 2018). Regulatory elements, nucleoside modification,
21 purification, synthetic caps or tails are used and added to mRNA in vaccines to elevate efficacy
22 and to prevent degradation (Pardi et al. 2018). The delivery method of the vaccine also
23 influences the lifespan and uptake of RNA-based vaccines and several have moved into clinical
39 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 40 of 91
1 trials. The most common method is delivery via lipid-based nanoparticles (LNP), as they are
2 easy to produce, provide protection to the mRNA, and assist delivery mRNA into cells (Pardi et
3 al. 2018; Zeng et al. 2020).
4 As for DNA-based vaccines, RNA-based vaccines can stimulate B and T cells, including
5 both CD4+ and CD8+ T cells, providing a more balanced acquired immune response compared to
6 classical subunit-based vaccines (Pascolo 2004). Extracellular mRNA is immunogenic and can
7 be recognized by several innate immune receptors, such as pattern recognition receptors (PRRs)
8 (Pardi et al. 2018). Activation of certain PRRs can induce IFN-I production, increasing antigen
9 presentation and promoting B and T cell immune responses (Ivashkiv and Donlin 2014; 10 Tatematsu et al. 2018). However, inflammatoryDraft cytokine production may also trigger an increase 11 in vascular permeability and edema in effected tissues at the injection site (Fischer et al. 2007;
12 Tatematsu et al. 2018). In animals, RNA-based vaccines have demonstrated strong CD8+ T cell,
13 Th1 cell, and nAb responses with just one or two low dose immunizations (Aberle et al. 2005;
14 Pardi et al. 2018), and unlike DNA vaccines, they do not rely on electroporation.
15 saRNA vaccines are like mRNA vaccines in that they both elicit similar types of immune
16 responses, are modified for stability, and primarily use LNP. However, saRNA vaccines differ
17 due to their ability to replicate once inside the cell. saRNA is based on the alphavirus RNA
18 genome, where structural viral genes are replaced with RNA that codes for the antigen of
19 interest, keeping only the replication genes (Geall et al. 2012). Synthetically produced alphavirus
20 genes have recently been combined with SARS-CoV-2 transgenes into a plasmid DNA vector
21 (Mckay et al. 2020), from which saRNA can be generated by in vitro transcription (Mckay et al.
22 2020; Sahin et al. 2020). The saRNA can then be further optimized via nucleoside modifications
23 and purified for use as a vaccine (Vogel et al. 2018; Sahin et al. 2020). Cytoplasmic
40 © The Author(s) or their Institution(s) Page 41 of 91 Canadian Journal of Microbiology
1 amplification of saRNA in animal studies have show greater antigen expression than mRNA and
2 appeared to provide some additional immune stimuli in dsRNA form (Vogel et al. 2018).
3 However, it remains to be determined if in some instances of the latter could activate interferon-
4 stimulated genes (ISGs), thus reducing host protein synthesis and limiting expression of the
5 antigen. Finally, significantly less saRNA was required to elicit the same immune response
6 compared to mRNA vaccines (Vogel et al. 2018), which could ultimately reduce production
7 costs.
8 Moderna Inc. vaccine candidate mRNA-1273
9 Moderna Inc. has been one of the frontrunners in mRNA vaccine development and 10 research for nearly a decade. They haveDraft developed potential vaccines for CMV, Zika virus, and 11 respiratory syncytial virus (RSV), each either in phase I or II clinical trials. Interim phase I
12 results with their H7N9 influenza vaccine showed high seroconversion rates with few severe
13 adverse events (Bahl et al. 2017). Along with US National Institute of Allergy and Infectious
14 Diseases (NIAID), Moderna has developed mRNA-1273, an LNP-encapsulated nucleoside
15 modified mRNA vaccine, based on the SARS-CoV-2 prefusion stabilized S trimer (Corbett et al.
16 2020a). A study was conducted using mouse models and showed dose dependent induction of S
17 protein binding antibodies in all animals after primary and secondary vaccinations of mRNA-
18 1273 (Corbett et al. 2020a). Doses ranging from 0.0025 µg to 20 µg were tested, and a
19 significant positive correlation between dosage level and mRNA-1273 binding and nAb titers
20 were found (Corbett et al. 2020a). 7 weeks after both vaccinations, mouse CD4+ T cells
21 stimulated with S peptides showed dominant Th1 responses noticeable at higher peptide levels.
22 CD8+ T cell responses were also found against S1 peptides. Mice that received two 1 µg doses of
23 the mRNA-1273 vaccine were protected from viral challenge in the lungs (Corbett et al. 2020a).
41 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 42 of 91
1 In a second preclinical study using rhesus macaques, animals were given two doses of
2 either 10 µg or 100 µg of mRNA-1273, or a placebo (Corbett et al. 2020b). Dose dependent
3 increases in binding antibody to the prefusion S protein was recorded 4 weeks following the
4 second vaccination, with significantly higher counts in the 100 µg group compared to the 10 µg
5 group (Corbett et al. 2020b). Similarly, neutralization activity was found to be 5 times higher in
6 the 100 µg group than in the 10 µg group 4 weeks after the first vaccination. CD4+ T cell
7 responses were observed and were Th1 cell dominant, with minimal Th2 responses. This Th1
8 response was highest in animals that received the 100 µg dose of the vaccine (Corbett et al.
9 2020b). CD8+ T cell responses were very low in both 10 µg and 100 µg groups. Upon SARS-
10 CoV-2 challenge, the highest recorded levels for subgenomic RNA in the animals was
11 significantly lower in both mRNA-1273Draft vaccine groups compared to the control group. It was
12 noted that inflammatory cytokine production was reduced in both vaccine groups as well.
13 Analysis of lung tissue showed mild inflammation in animals from the 10 µg group and no
14 substantial inflammation in animals from the 100 µg group (Corbett et al. 2020b).
15 A phase I clinical trial for mRNA-1273 was conducted as open-labelled and dose-ranging
16 with the goal of testing the safety and immunogenicity of the vaccine (NCT04283461). 45
17 healthy adults aged 18 to 55 years of age were given two doses of either 25 µg, 100 µg, or 250
18 µg of the mRNA-1273 vaccine (Jackson et al. 2020). Following primary immunization, higher
19 antibody responses mirrored higher doses, and antibody titers increased upon booster
20 immunization (Jackson et al. 2020). Reported systemic adverse events were more common after
21 the second vaccination and in the higher dose groups. 54% of individuals in the 25 µg group, and
22 100% of individuals in both 100 µg and 250 µg groups experienced systemic adverse events,
23 with 21% of individuals in the 250 µg that reported at least one severe event (Jackson et al.
42 © The Author(s) or their Institution(s) Page 43 of 91 Canadian Journal of Microbiology
1 2020). Common systemic and local adverse events included: chills, headache, myalgia, fatigue,
2 and injection site pain. Dose dependent responses were observed with RBD specific binding
3 antibodies in both immunizations. CD4+ T cell responses were induced in the 25 µg and 100 µg
4 groups, with a notable Th1 cell rather than Th2 cell response (Jackson et al. 2020). CD8+ T cell
5 responses were only detected in the 100 µg group following a secondary vaccination. It was
6 determined from these interim results that 100 µg of mRNA-1273 was a safer and more effective
7 dosage level than the higher 250 µg option (Jackson et al. 2020).
8 The phase I trial was then expanded to include 40 adult participants aged 56 years and
9 older (Anderson et al. 2020). Participants were put into groups and received two immunizations 10 of either 25 µg or 100 µg of mRNA-1273Draft on a day 0,28 schedule. No serious adverse events 11 were reported, however the most common mild or moderate solicited adverse events included
12 chills, headache, fatigue, myalgia, and injection site pain (Anderson et al. 2020). For both dosage
13 levels, local and systemic adverse events were reported in more participants following the second
14 vaccination. Binding antibody and nAb responses were dose dependent as binding IgG antibody
15 geometric mean titers showed large increases after the first and second immunizations. Binding
16 and nAbs responses were highest in the 100 µg groups, with the strong neutralizing activity
17 towards SARS-CoV-2 S protein variant 614-Gly, which had become the predominant
18 polymorphic variant in the US and worldwide (Anderson et al. 2020). Participants in the 100 µg
19 groups also showed the highest vaccine elicited Th1 cell cytokine responses and low level CD8+
20 T cell responses after the second vaccination (Anderson et al. 2020). From these results, it was
21 concluded that the 100 µg dose was more effective, supporting its use in future trials such as the
22 ongoing phase II clinical trial which only includes 50 µg and 100 µg groups. This second trial is
43 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 44 of 91
1 an observer-blind, placebo controlled, randomized, dose-confirmation study involving 600
2 healthy adult participants (NCT04405076).
3 A phase III clinical trial is currently underway, with the goal of assessing the protective
4 efficacy of mRNA-1273 across approximately 30,000 adult participants aged 18 years and older
5 (NCT04470427). The study is randomized, placebo controlled, observer-blind, and stratified
6 with an estimated completion date at the end of October of 2020, however, results have yet to be
7 posted. Participants will receive two doses on a day 0, 28 schedule of either 100 µg of mRNA-
8 1273 or placebo. Notably, a press release from Moderna on November 16, 2020 announced that
9 their first interim efficacy analysis of mRNA-1273 revealed the vaccine was 94.5% effective in 10 preventing COVID-19 (Moderna, 2020).Draft Evaluation of additional Phase III data is forthcoming. 11 BioNTech/Pfizer vaccine candidate BNT162 mRNA
12 BioNTech is another large mRNA-based therapeutics company, with a focus on
13 developing mRNA therapies against several different types of cancers. Together with Pfizer and
14 Fosun Pharma, Phase I/II clinical trials are being conducted on their LNP BNT162b1 and
15 BNT162b2 vaccines (NCT04368728). Both are nucleoside modified RNA candidates with
16 BNT162b1 encoding the SARS-CoV-2 RBD trimerized by a T4 fibritin foldon domain, and
17 BNT162b2 encoding the full-length SARS-CoV-2 S protein in its prefusion conformation
18 (Mulligan et al. 2020; Walsh et al. 2020). An interim report was released describing an ongoing,
19 observer blind, placebo-controlled study for the BNT162b1 vaccine candidate (Mulligan et al.
20 2020). 45 healthy adults aged 18 to 55 were enrolled in the study, assigned randomly to receive
21 two 10 µg or 30 µg doses, or one 100 µg dose of the vaccine. Over the two-week period,
22 reported adverse events were dose dependent. A frequent local adverse event to immunization
23 for both primary and booster vaccinations was injection site pain, seen in 58.3% in the 10 µg
44 © The Author(s) or their Institution(s) Page 45 of 91 Canadian Journal of Microbiology
1 group and 100% in the 30 µg and 100 µg groups (Mulligan et al. 2020). Systemic adverse events
2 included headache, fatigue, fever, chills, muscle pain, and joint pain and were most commonly
3 reported after the booster immunization in participants that received a higher dosage. RBD
4 specific binding antibody concentrations were ~8.0- to ~50-fold higher compared to
5 convalescent serum panel concentrations following the second immunizations for the 10 µg and
6 30 µg groups (Mulligan et al. 2020). nAb concentrations also saw a 1.8- and 2.8-fold increase
7 compared to convalescent serum panel concentrations for the 10 µg and 30 µg groups,
8 respectively. A secondary study was recently conducted using BNT162b1 in 60 healthy adult
9 participants, aged 18 to 55 (Sahin et al. 2020). 1 µg, 10 μg, 30 μg, and 50 µg primary and booster
10 vaccination dosage levels were randomly assigned, plus a group receiving only one 60 µg dose
11 (Sahin et al. 2020). No serious adverse eventsDraft were reported, however similar to the previous
12 study the 10 μg and 30 μg groups did see mild to moderate events such as injection site pain,
13 headache, chills, muscle pain, joint pain, and fever. These adverse events were also dose
14 dependent, so upon observing individuals following the 50 µg booster, a second dose was not
15 given to the highest 60 µg group (Sahin et al. 2020). Higher dosage was associated with a
16 stronger antibody response, as nAb concentrations correlated with RBD specific binding
17 antibody concentration. Neutralization assays performed using sera from the participants showed
18 significant neutralizing titers against several SARS-CoV-2 S variants (Sahin et al. 2020). CD8+ T
19 cell response strength to the RBD demonstrated a positive correlation with vaccination induced
20 CD4+ T cell responses. Notably, a favourable Th1 skewed response was found, based on the
21 detection of certain cytokines (IFNγ, IL-2, IL-12p70) and the absence of others (IL-4) (Sahin et
22 al. 2020).
45 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 46 of 91
1 Both studies had limitations, including the short assessment period and narrow age and
2 demographic range. A preprint article was released, providing further immunogenicity and safety
3 data from the US part of the phase I trials (Walsh et al. 2020). Similar immune and serological
4 responses were induced in both younger and older adult participants upon receiving either 2
5 doses of BNT162b1 or BNT162b2 (Walsh et al. 2020). However, a milder systemic
6 reactogenicity profile was noted in BNT162b2, particularly in older participants aged 65 to 85
7 years. Following the second 30 µg dose of BNT162b1 33% of older participants reported a fever
8 greater than 38°C, whereas after the second 30 µg dose of BNT162b2 only 8% of older adults
9 reported a fever greater than 38°C (Walsh et al. 2020). Preclinical studies with BNT162b2 have
10 shown promising results (Vogel et al. 2020). In both mice and rhesus macaques, vaccination
11 elicited significant nAb titers and strongDraft Th1, IFNγ+, and CD8+ T cell responses. Prime-boost
12 immunization of rhesus macaques was shown to provide complete protection of the lung tissue
13 from SARS-CoV-2 challenge (Vogel et al. 2020). These additional safety data along with the
14 previous preclinical and phase I studies using BNT162 vaccine candidates supported the decision
15 to move forward solely with BNT162b2 (BioNTech 2020) for large-scale phase II/III trial
16 assessment in ~44,000 participants, which is now ongoing (NCT04368728). Notably, a press
17 release from Pfizer on November 9, 2020 announced that their first interim efficacy analysis of
18 BNT162b2 revealed the vaccine was 90% effective in preventing COVID-19 in participants not
19 previously infected with SARS-CoV-2 (Pfizer 2020). Evaluation of additional Phase III data
20 will be forthcoming.
21 Conclusions
22 In a matter of weeks, SARS-CoV-2 went from a small outbreak to a global pandemic.
23 Since then the global scientific community has responded with a flurry of research and
46 © The Author(s) or their Institution(s) Page 47 of 91 Canadian Journal of Microbiology
1 development (R&D) efforts in pathology, biochemistry, genetics, therapeutics, vaccinology, and
2 drug discovery. As described here this effort is generating a number of promising vaccine
3 candidate vaccines in remarkably short order. Prior SARS-CoV-2, a preclinical vaccine
4 candidate would typically undergo a 10-year timeline to reach market if successful (Pronker et
5 al. 2013). However, due to the enormous influx of funding, research effort and demand for a
6 SARS-CoV-2 vaccine, this timeline will likely be greatly reduced. Nevertheless, vaccine safety
7 cannot be ignored and thus understanding the biology of frontrunning vaccine candidates should
8 not be rushed. Fortunately, animal models such as transgenic hACE2 mice (Hassan et al. 2020)
9 have been rapidly developed and evaluated (Chan et al. 2020; Kim et al. 2020c; Shi et al. 2020;
10 Chandrashekar et al. 2020) to validate vaccine efficacy in preclinical studies and it is now
11 estimated by the Coalition for EpidemicDraft Preparedness Innovations (CEPI) that in the next 12-18
12 months approximately two billion US dollars in investments will be required to fund clinical
13 trials (CEPI 2020).
14 Given the promising data thus far, it is highly likely that multiple SARS-CoV-2 vaccine
15 strategies will be approved for distribution. When this occurs, the world must work together to
16 ensure that every country will have access. Dialogue between vaccine developers, regulators, and
17 governments is already occurring and is necessary to secure adequate amounts of vaccine for all
18 affected areas (Yamey et al. 2020). However, the manufacturing process may limit initial
19 capacity depending on the type of vaccines that ultimately prevail. While some infrastructure can
20 be repurposed for existing vaccine platforms, such as inactivated viral vaccine production, novel
21 RNA-based vaccines will require new facilities for large scale production (Amanat and Krammer
22 2020). Nevertheless, Pfizer claims they could generate 50 million doses of their RNA-based
23 vaccine (BNT162b2) in 2020 and up to 1.3 billion doses in 2021 (Pfizer 2020).
47 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 48 of 91
1 Given the epidemiology of SARS-CoV-2, it is possible the virus could become endemic
2 in the population (Shaman and Galanti 2020). Thus, efforts to develop effective vaccines are
3 essential and may even require annual vaccinations as carried out for influenza depending on
4 how SARS-CoV-2 evolves over time. This will require continued funding for basic R&D,
5 preclinical, and clinical trials as the knowledge gained from these efforts will position humanity
6 to address the current viral pandemic and prepare us for those that will occur in the future.
7 Authorship
8 R.L.R. prepared the first draft of the manuscript; R.L.R., B.L.M and P.P edited and
9 prepared the final manuscript. R.L.R., P.P and B.L.M prepared figures and tables. B.L.M. 10 conceived the topic. Draft 11 Acknowledgements
12 The authors thank Maury Donen for assistance with literature reviews and for providing
13 feedback. The authors also thank the Canadian Institutes of Health Research, the Natural
14 Sciences and Engineering Research Council of Canada, and the University of Manitoba Faculty
15 of Science for funding support.
16 Conflicts of Interest
17 The authors declare no conflicts
18
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1 Vaccination. Clin. Microbiol. Rev. 32(2): 1–50. doi:10.1128/CMR.00084-18.
2 Table 1. Leading SARS-CoV-2 vaccine candidates
Vaccine Vaccine platform and Lead developer(s) Vaccine status candidate characteristics Ad5-nCoV Non-replicating viral vector CanSino Biological Inc./ Beijing Phase I Human adenovirus serotype 5 Institute of Biotechnology (NCT04568811) vector expressing the S protein (ChiCTR2000030906) Phase II (NCT04566770) (ChiCTR2000031781) Phase III (NCT04540419) (NCT04526990) AZD1222 Non-replicating viral vector University of Oxford, AstraZeneca Phase I/II ChAdOx1 vector expressing the S (PACTR202006922165132) protein (2020-001072-15) (NCT04568031) Phase II (2020-001228-32) Phase III (ISRCTN89951424) (NCT04516746) (NCT04540393) (CTRI/2020/08/027170) Ad26.COV2.S Non-replicating viral vector Janssen Pharmaceutical Companies Phase I/II Human adenovirus serotype 26 (NCT04436276) vector expressing the S protein Phase III (NCT04505722) Sputnik V Non-replicating viral vector Gamaleya Research Institute Phase I/II Heterologous prime-boost using (NCT04437875) human adenovirus serotype 26 and (NCT04436471) human adenovirus serotype 5 Phase II vectors expressing the S protein (NCT04587219) Draft Phase III (NCT04564716) (NCT04530396) CoronaVac Inactivated Sinovac Phase I/II (NCT04551547) (NCT04352608) (NCT04383574) Phase III (NCT04582344) (669/UN6.KEP/EC/2020) (NCT04456595) BBIBP-CorV Inactivated Beijing Institute of Biological Phase I/II Products/ Sinopharm (ChiCTR2000032459) Phase III (NCT04560881) (ChiCTR2000034780) - Inactivated Wuhan Institute of Biological Phase I/II Products/ Sinopharm (ChiCTR2000031809) Phase III (ChiCTR2000039000) (ChiCTR2000034780) NVX-CoV2373 Recombinant protein subunit Novavax Phase I/II Adjuvanted nanoparticle full- (NCT04368988) length recombinant S glycoprotein Phase IIb (NCT04533399) Phase III (2020-004123-16) mRNA-1273 RNA Moderna/ NIAID Phase I Nucleoside modified LNP- (NCT04283461) encapsulated mRNA Phase II (NCT04405076) Phase III (NCT04470427) BNT162b2 RNA BioNTech/ Fosun Pharna/ Pfizer Phase I LNP mRNAs and saRNA (NCT04368728) Phase I/II (NCT04537949) (NCT04537949) (ChiCTR2000034825) (2020-001038-36) Phase II/III (NCT04368728) - Recombinant Protein Subunit Anhui Zhifei Longcom Phase I Adjuvanted recombinant RBD Biopharmaceutical/ Institute of (NCT04445194) dimer Microbiology, Chinese Academy of Phase I/II Sciences (NCT04550351) Phase II (NCT04466085) INO-4800 DNA Inovio Pharmaceuticals, International Phase I/II Plasmid delivered with Vaccine Institute (NCT04336410) electroporation (NCT04447781)
88 © The Author(s) or their Institution(s) Page 89 of 91 Canadian Journal of Microbiology
1
2 Figure 1: Model of SARS-CoV-2 spike protein (S) (PDB ID: 6vsb) (Wrapp et al. 2020) bound
3 to the ACE2/Neutral amino acid transporter B0AT1 complex (PDB ID: 6m17) (Yan et al. 2020).
4 The model was generated by superposing the receptor binding domain (RBD) of S (shown with a
5 surface rendering) onto an isolated S RBD whose bound position on the ACE2 receptor (shown
6 in green) had been experimentally determined by cryo-electron microscopy (Wrapp et al. 2020).
7 Note that ACE2 is a known chaperone for the amino acid transporter B0AT1, which was used to
8 help stabilize and facilitate the determination of the ACE2 cryo-EM structure (Yan et al. 2020).
9 There are two copies of the ACE2 monomer in the receptor complex and each copy was found 10 bound to an isolated S RBD as shown. DraftFigure drawn using PyMol (pymol.org). (Figure based on 11 doi:10.2210/rcsb_pdb/mom_2020_6 (David S. Goodsell and the RCSB PDB (Goodsell et al.
12 2020)).
13
14 Figure 2. Recombinant adenovirus design and recombination strategy. A. Recombination
15 strategy for generation of recombinant adenoviruses using bacterial recombination in BJ5183 E.
16 coli strain. Only targeting to E3 is shown, however targeting to E1 or other regions is also
17 possible and depends on the donor plasmid. Modified from (Chartier et al. 1996). B.
18 Recombination strategy for insertion of SARS-CoV-2 genes into HAdV backbone via the Cre-
19 loxP system. ITR - Inverted terminal repeat; Ψ - packaging signal. Modified from reference
20 (Hardy et al. 1997).
89 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 90 of 91
Draft
Figure 1: Model of SARS-CoV-2 spike protein (S) (PDB ID: 6vsb) (Wrapp et al. 2020) bound to the ACE2/Neutral amino acid transporter B0AT1 complex (PDB ID: 6m17) (Yan et al. 2020). The model was generated by superposing the receptor binding domain (RBD) of S (shown with a surface rendering) onto an isolated S RBD whose bound position on the ACE2 receptor (shown in green) had been experimentally determined by cryo-electron microscopy (Wrapp et al. 2020). Note that ACE2 is a known chaperone for the amino acid transporter B0AT1, which was used to help stabilize and facilitate the determination of the ACE2 cryo-EM structure (Yan et al. 2020). There are two copies of the ACE2 monomer in the receptor complex and each copy was found bound to an isolated S RBD as shown. Figure drawn using PyMol (pymol.org). (Figure based on doi:10.2210/rcsb_pdb/mom_2020_6 (David S. Goodsell and the RCSB PDB (Goodsell et al. 2020)).
215x279mm (300 x 300 DPI)
© The Author(s) or their Institution(s) Page 91 of 91 Canadian Journal of Microbiology
Draft
Figure 2. Recombinant adenovirus design and recombination strategy. A. Recombination strategy for generation of recombinant adenoviruses using bacterial recombination in BJ5183 E. coli strain. Only targeting to E3 is shown, however targeting to E1 or other regions is also possible and depends on the donor plasmid. Modified from (Chartier et al. 1996). B. Recombination strategy for insertion of SARS-CoV-2 genes into HAdV backbone via the Cre-loxP system. ITR - Inverted terminal repeat; Ψ - packaging signal. Modified from reference (Hardy et al. 1997).
185x250mm (300 x 300 DPI)
© The Author(s) or their Institution(s)