view vaccine technologies in COVID 19 era which one will be the best

University, Ho Chi Minh City Chi Minh City *All authors contribute equally DOI: Received: Revised: Accepted: Keywords: COVID-19, inactivated vaccine, viral vector vaccine, recombinant

Review: Vaccine technologies in COVID-19 era Which one

will be the best?

University, Ho Chi Minh City

Chi Minh City

*All authors contribute equally

DOI:

Received:

Revised:

Accepted:

Keywords:

COVID-19, inactivated

vaccine, viral vector vaccine,

recombinant protein vaccine,

mRNA vaccine

Have you been vaccinated against COVID-19? Which vaccine did you get? How much do you think you know about that vaccine? From the very first outbreak in Wuhan, COVID-19 disease, which causes severe acute respiratory syndrome in humans, has now doomed the whole world by its widespread and mutation rate Indeed, the number of infections is skyrocketing, numerous casualties are inflicted every day, and a devastating blow has also dealt directly to the world's social-economic development, all of which has put humans into a truly gloomy scenario In response to this challenge, scientists have put their best effort in comprehending the virus to give out the most applicable measure against it, and vaccines are then considered as the "deadliest" weapon of humans to fight back and put an end to this catastrophe However, the quantity of vaccines is now of scarcity due to the enormous demand of billions of consumers worldwide, and vaccine-producing technologies vary from region to region with their pros and cons, so it is quite a problem for scientists to determine which one is the most suitable to go through this tough time To accomplish this goal, people should comprehend technologies producing current SARS-COV-2 vaccines On that account, this review, which provides background knowledge of several conventional and modern technologies of manufacturing anti-COVID-19 vaccines, also shows some comparisons of them

Based on our findings, most of the technologies and corresponding vaccines are all crucial alongside their promising results and could share the burden of mass demand for each other during these difficult days Furthermore, some of these techniques also open a novel path in developing other kinds of vaccines

1 Introduction

COVID-19 disease, a severely acute respiratory syndrome outbreak in 2019, is stemmed from the SARS-CoV-2 virus strain from Wuhan, China Regarding the structure

of the Covid-19 virus, it has four main structural proteins, which are the nucleocapsid (N) phosphoprotein, envelope (E) protein, membrane (M) protein, and spike (S) protein Firstly, the N protein, or so-called nuclear capsid, located in the capsid, has an integral role in helping virus genetic material replicate and transcript when the virus invades host

cells (Boopathi, Poma, & Kolandaivel, 2020) After that, the phospholipid bilayer

membrane encapsulates the ss-mRNA and acts as a shield to protect the virus genetic material from the surrounding factors On the surface of the membrane, a small protein called envelope (E) is present It takes part in helping viruses assemble, affects the permeability of virus into host cells and the interacting process between host and virus cells (M K Gupta et al., 2021) Another one is the membrane (M) protein integrated into the membrane surface and serves as the central organizer in the virus assembly (Boopathi

et al., 2020).The last and foremost one is the spike (S) protein, which resides on the surface of the cell membrane, but its role is like a key to help viruses enter the host cells via the hACE2 receptor (Kirchdoerfer et al., 2016)

After the threat and data of coronavirus are unveiled, many companies have emerged themselves into producing vaccines against this calamity via many different methods and techniques For example, the most conventional method, attenuated virus vaccine, is now used by well-known companies such as BBIBP-CorV (Sinopharm Group) or CoronaVac (Sinovac Biotech) The other method that is also familiar with us

is the recombinant protein vaccine produced by some companies such as Novavax, Anhui

Zhifei Longcom in their anti-covid-19 vaccine manufacture (Heath et al., 2021; Keech et

al., 2020; Yang et al., 2021) Moreover, the viral vector method is also common since it sticks with the famous covid 19 vaccines, Vaxzevria (AstraZeneca/Oxford) or Sputnik V (Gamaleya Research Institute), which has an outstanding contribution in stalling time for the mass production of other vaccines (Mendonça, Lorincz, Boucher, & Curiel, 2021) Lastly, the latest ones are Pfizer and Moderna, which are representative of one of the most cutting-edge vaccine-producing technologies called mRNA techniques (Park, Lagniton, Liu, & Xu, 2021)

On that account, in this review, we aim to offer everyone an overview and comparison of those four vaccine-producing technologies in the race against the

Covid-19 pandemic Other aspects of vaccines such as safety, efficacy, effectiveness, formulations, and immunogenicity are also mentioned and compared where relevant

2 Vaccine technologies in COVID-19 era

2.1 Old technology

2.1.1 Inactivated or live-attenuated virus vaccines

2.1.1.1 Background information

The inactivated vaccine is produced by killing or deactivating the viruses using heat or chemicals so that they are unable to replicate in the human body It was firstly reported by Salmon and Smith in the United States (1886) and Pasteur Institute group (1885) At the beginning of the twentieth century, the first heat-killed vaccines were invented for typhoid, plague, and cholera bacilli by Wright and Semple in England (1897) and Pfeiffer and Kolle in Germany In 1954, the Nobel Prize was awarded for Enders, Weller, and Robbins to discover the cultivation of poliovirus in fibroblasts in vitro In other words, the inactivated vaccines play a vital role in preventing the worst pandemic, such as polio and influenza (Sanders, Koldijk, & Schuitemaker, 2015)

There are two main inactivation chemicals applied in vaccine technologies, including formalin (Delrue, Verzele, Madder, & Nauwynck, 2012) and β-propiolactone Although formalin is the oldest agent, β-propiolactone is preferred in the COVID-19 pandemic because of its high potency and low antigen damage (D Gupta et al., 2021)

In addition, β-propiolactone is a four-membered ring, which is highly reactive with amino acids (cysteine and cysteine) and nucleic acids (guanine), inducing proteins and DNA cross-linking (Perrin & Morgeaux, 1995)

2.1.1.2 SAR-CoV-2 inactivated vaccines

The Sinopharm Group (China) and Sinovac Biotech (China) are two companies that focus on the COVID-19 vaccine manufacture based on the inactivated platform To design inactivated vaccines, SAR-CoV-2 strains were collected and isolated from the bronchoalveolar lavage samples or throat swabs of patients in order to prepare for preclinical processes (Gao et al., 2020; Wang et al., 2020) BBIBP-CorV (Sinopharm) vaccine was developed based on three strains, including HB02, CQ01, and QD01 (Wang

et al., 2020), while CoronaVac (Sinovac) was tested with eleven different strains CN1-CN5 and OS1-OS6 (Gao et al., 2020) After being isolated from Vero cells, HB02 (BBIBP-CorV) and CN2 (CoronaVac) were chosen for further development because of their highest virus yields Ten passages were performed on these strains to evaluate their genetic stability to compare the mutation of amino acid substitutions As a result, both HB02 (BBIBP-CorV) and CN2 (CoronaVac) indicated their excellent genetic stability

up to 99% of homology (Gao et al., 2020; Wang et al., 2020) Then, they propagated in

a culture of Vero cells for pilot scale and were inactivated by β-propiolactone, which reacts with nucleic acids and proteins to modify them, especially the guanine (Gao et al., 2020; Perrin & Morgeaux, 1995) Finally, the BBIBP-CorV and CoronaVac vaccines were adjuvanted with aluminum hydroxide, which activates the pro-inflammatory mechanism of the immune system through the activation of the NLRP3 receptor subunit and stimulation inflammasome-derived IL-1β and IL-18 secrete (Kyriakidis, López-Cortés, González, Grimaldos, & Prado, 2021)

Figure 2: Inactivated COVID-19 vaccines production (Wang et al., 2020)

2.1.2 Viral vector vaccine

2.1.2.1 Designing strategy

Viral vector vaccine for coronavirus disease uses a virus vector, replicating or non-replicating, to carry gene encoding for Spike protein of SARS-CoV-2 The utilized vectors must be harmless and different from antigens Among COVID-19 viral vaccines, adenovirus vector is the most advanced and effective technique in vaccine development, along with mRNA vaccine (Zimmer, Corum, & June, 2021) Adenovirus (Ad) is a non-enveloped and 30 – 40 kb length virus, containing a linear double-stranded DNA genome that could pack up to 7.5 kb foreign genes (Lundstrom, 2021) Major adenovirus vector used in developing vaccines is replicate-deficient by deleting E1A and E1B genes, making the virus unable to replicate and thus cannot cause disease in the human body Hence, in vaccine production, viral-based vectors are propagated in E1-complementing mammal cell culture systems such as HEK293 cell line Besides, E3 and E4 genes are also eliminated to prepare enough space for inserting the S protein-encoding gene of the SARS-CoV-2 virus, alleviating the rate of leaky expression of inserted S protein Moreover, modifications of hexon on adenovirus capsid are carried out to circumvent the strong anti-Ad immunity against adenovirus.(Mendonça et al., 2021) The noticeable thing in designing a viral vector vaccine is that it is not the whole genome of the SARS-CoV-2 virus, but only the gene encoded for full-length S protein, that is integrated into the adenovirus genome Though the entire SARS-CoV-2 virus could cause the disease, the S protein does not since it lacks genes essential for virus replicating and then not fully function as a complete virus at all (Dai & Gao, 2021)

2.1.2.2 Immunology mechanism

The concept of this vaccine platform is to exploit host cells to express antigens integrating in the DNA of the viral vector to trigger immune responses Thanks to the prophylaxis effect, humoral and cellular immunology is elicited Later, when the body is infected with that virus, memorized antibodies will rapidly respond to pathogens and help humans combat the disease Although the structure of SARS-CoV-2 contains four main proteins (M, N, E, S), only S protein promotes generating antibodies against SARS-CoV-2.(Ortiz-Prado et al., 2020) First, adenovirus vector triggers the innate immune system

through Toll-like receptors (TRLs) The activation of TRLs, which elicit NF-κB and IRFs activities, stimulates human body to produce inflammatory cytokines and type I interferons This process can also call for the response of adaptive immunity by activating

on the surface Produced neutralizing antibodies (NAb) bind to receptor-binding RBD on

S protein of SARS-CoV-2 virus and prevent it from interacting with ACE2 receptor.(Guo, 2021)

2.1.2.3 Current COVID-19 viral vector vaccines

Vaxzevria

Vaxzevria or AZD1222, is a vaccine developed by the University of Oxford and AstraZeneca Company, UK Addition to the age group from 19 to 65, ChAdOx1

nCoV-19 vaccine showed positive evidences in protecting children and young adults from age

6 to 17.(Oxford, 2021) However, some studies showed that vaccinated with Vaxzevria vaccine had a risk of vaccine-induced thrombotic thrombocytopenia (VITT)(Schultz et al., 2021) VITT were detected between day 4 and day 28 after vaccination and is likely

to be lower after the second dose.(Odutayo, Juni, Stall, & Bobos, 2021) Due to the concerns about VITT, heterologous vaccination using mRNA vaccine, such as BNT162b2 or mRNA-1273 as the boosting dose has been recommended in several countries (Normark et al., 2021) Results of studies showed that the effectiveness of heterologous vaccine was 88% (Gram et al., 2021)

Sputnik V

Different from Vaxzevria which utilizes the same vector for two doses, Sputnik

V COVID-19 vaccine (Gam-COVID-Vac) consists two different viral vectors: adenovirus type 26 (rAd26) and type 5 (rAd5) (Jones & Roy, 2021) In addition to alpha strain, Sputnik V also produces antibodies to combat against other variants of SARS-CoV-2 such as Delta.(Gushchin et al., 2021) Sputnik V vaccine is manufactured as two different formulations: the frozen Vac and the lyophilized

delivered to other regions because it must be reconstituted in 1.0mL sterile water for injection.(Poh Teo, 2021)

2.1.3 Recombinant protein vaccine

2.1.3.1 Vaccine manufacturing

The way recombinant protein vaccines are produced is somehow relatively simple to understand This is a general workflow for a whole process In the process below, the first step is that people have to firstly think about which protein of the virus that they want to mark as an antigen candidate, then we could identify which gene coded for that part from the virus genomes and cut it off, this identifying step is also called isolating gene of interest (GOI) The coding sequence or GOI (codon-optimized for host cell system) for the interested region could now be synthesized artificially by using a massive amount of genome sequence data from a myriad of species After we have the

GOI, we could insert it into the appropriate host systems by using the suitable expression vector to facilitate the expression of the protein of interest After that, the highest yield batch could move to purification, where people will extract and purify the protein of interest Next, adjuvants or nanoparticles could be added to boost the efficacy of protein

antigens (Awadasseid, Wu, Tanaka, & Zhang, 2020)

2.1.3.2 Current recombinant protein vaccines

Each vaccine may differ from others in terms of host systems, adjuvant, subunit aim, etc For example, there are some general processes of making some recombinant protein vaccines for COVID-19

ZF2001

The desired RBD part originated from SARS-CoV-2, starting from R319 to residue K537 in spike (S) protein The codon in gene sequence encoding the monomer RBD will be optimized to culture well on mammalian cell lines, then synthetically produced For protein excretion, the protein N terminus of the sequence will be inserted

by a signal peptide sequence of MERS-CoV S protein (S protein residues 1-17), and for the further purification-process facilitation, a Hexa-His-tag was added to the C terminus

of the sequence Next, the product was cloned into the pCAGGS expression vector and transferred into the HEK293T cells line system to express the RBD After the extraction and purification process, two RBD monomers were bound to each other in tandem via a disulfide bond to construct the RBD-Dimer The RBD-Dimer was then transformed into Chinese Hamster Ovaries (CHO) cell lines to produce the protein The expression batches will be screened to determine which cell lines have the highest antigen production yields for mass antigen-manufacture following current Good Manufacturing Practice The tandem-repeat RBD-Dimer antigen was further extracted, purified, and characterized After that, the adjuvant called aluminum hydroxide was added into the antigen solution

and stored in vials as the ZF2001 vaccine (An et al., 2021; Liang et al., 2021)

NVX- CoV2373

The full-length S protein-encoding gene was identified, then this sequence was codon-optimized to infect insect cells and then synthesized artificially Then, a double mutation will be made at the furin cleavage site to stabilize antigen protein under the effect of proteases, and at two points, K986 and V987, just above the central helix (CH)

to stabilize the pre-fusion form of S-protein (Dai & Gao, 2021; Keech et al., 2020; Tian

et al., 2021) Then, the double-mutated full-length S protein-encoding gene will be

cloned into the pBac-1 baculovirus transfer vector (Tian et al., 2021) This kind of virus only infects insects so that it could guarantee the safety of humans After that, the Bacillus virus will be transferred to the infected Sf9 insect cells expression system, which will express this type of protein (Irfan, 2021; Keech et al., 2020; Kyriakidis et al., 2021; Tian

et al., 2021) Subsequently, people will extract and purify the protein; then add the nanoparticle PS80 core as a platform for these proteins to stick in (Irfan, 2021; Tian et al., 2021), and also add some saponin-based Matrix-M adjuvant which could help the weak-immune-induce antigen boost the immune response in the human body (Dai & Gao, 2021; Heath et al., 2021; Keech et al., 2020; Kyriakidis et al., 2021; Reimer et al., 2012; Tian et al., 2021)

From these three production technologies, we could conclude that there are some familiar points in synthesizing the gene of interest (GOI) with the help of bioinformatics

However, they use different types of antigens, while NVX-2373 and SCB-2019 use full-length S protein as the antigen protein, ZF2001 only uses the hACE2-binding RBD as the antigen candidate In this way, ZF2001 will be safer since its antigen only targets the hACE2 and thus lower the potential risk of triggering the antibody-dependent enhancement (ADE), which could have an adverse impact on patients (An et al., 2021; Dai & Gao, 2021) On the other hand, the economic effect of choosing an expression vector and system also needs to be considered The mammalian host system, used in making ZF2001 and SCB-2019, causes a more prominent financial burden than the insect cells host system used in producing NVX-CoV2373 (Pollet, Chen, & Strych, 2021) Moreover, in term of adjuvants, ZF2001 use aluminum hydroxide which has the greatest safety record and is also the safer for humans in long-term, rather than the saponin-based Matrix-M adjuvants used in NVX-CoV2373, and AS03, or CpG1018+Alum adjuvants formulated in SCB-2019 (An et al., 2021; Petrovsky, 2015; Petrovsky & Aguilar, 2004) Furthermore, the saponin, which possesses the detergent-like characteristic, could damage the cell membrane leading to the moderately to severely hurt at injection point, and the damage or even death of muscle cells Nonetheless, cell membrane disruption also makes local injection sites red, swelled, and causes granuloma formation (Waite et al., 2001) Last but not least, saponin-based adjuvants with the gravitation toward cholesterol could make blood cells become lysed due to the presence of cholesterol in erythrocyte membranes of red blood cells

2.2 A new approach in vaccinology: mRNA technologies

2.2.1 Background information

The concept of mRNA vaccine is quite simple: mRNA is a messenger RNA which will go through a translational process by ribosome in cytoplasm to produce a target protein Exploiting this mechanism, researchers have developed mRNA vaccines

by encoding mRNA molecules with a target antigen, in this case, is the S (Spike)

protein-a virprotein-al structurprotein-al protein, protein-and delivered into our body Our cells will then uptprotein-ake the

mRNA and translate it into protein in situ The translated protein (peptides) will then

present on the cell surface, resulting in a specific humoral T-cell-mediated immune response against Spike (S) protein

mRNA should be engineered to contain fully processed mature RNA molecules:

an open reading frame (ORF) encoding the target protein, flanking untranslated region UTRs, and a poly(A) tail

2.2.1.1 mRNA structural modification

2.2.1.2 5’ cap modification

The modification of 5’cap in the in vitro transcribed mRNA (IVT) has been

reported since 1975 through a study of two enzymatic activities from vaccinia virus cores (Monroy, Spencer, & Hurwitz, 1978) The cap structure synthesis by this old method mimics the most available eukaryotic cap structure in nature Another approaches in modifying 5’cap are to add the synthetic cap to the transcription reaction and anti-reverse cap analogue (ARCAs; m27,3ʹ−O GpppG) (Stepinski, Waddell, Stolarski, Darzynkiewicz, & Rhoads, 2001) In available mRNA vaccines for COVID-19, the most common method is capping mRNA with Cap 1 (m7GpppNmN) structure by a Vaccinia enzyme system (Corbett et al., 2020; Park et al., 2021)

2.2.1.3 5’UTR and 3’UTR flanking

The coding sequence in the 5ʹ-untranslated region (UTR) and the 3ʹ-UTR flanking is known to have influence on the stability and protein translation Thus, in order

to optimize the structure and half-life of IVT mRNA, incorporation of 5’UTR and 3’UTR encoding regulatory elements is used as another efficient approach There are several studies suggesting that the half-life of 3’-UTRs α- and β-globin mRNA increased the stability and effective translation of mRNA (Rodgers, Wang, & Kiledjian, 2002; Ross & Sullivan, 1985)

2.2.1.4 polyA tailing

Same as two structures mentioned above, poly(A) tail also regulates the stability

and translation efficiency of mRNA in both the natural endogenous process and in

vitro transcription of IVT mRNA There are two way in tailing the mRNA in vitro:

extending mRNA with two-step enzyme reaction by recombinant poly(A)polymerase and co-transcripting poly(A) tail via DNA template (plasmid- or PCR- based) (Sahin, Karikó, & Türeci, 2014) Several studies show that the length of the poly(A) tail and synergize between 5’cap and poly(A) tail influence on translation efficiencies (Gallie, 1991)

2.2.1.5 Coding region optimization

Codon optimization plays an important role in enhancing the translation efficiency of mRNA In order to improve the expression of target protein, redesign the coding region with high frequent occurring codons in a certain species is a common strategy (Gustafsson, Govindarajan, & Minshull, 2004) High yield of protein expression could be easily obtained because once a codon has been used, the subsequent occurrences

of the same amino acid will rather use codons with the same tRNA than the random codons (Cannarozzi et al., 2010) In a research study, IVT mRNA containing codon-optimized regions was used successfully in vaccine application against HIV viral infection (Van Gulck et al., 2006) However, for vaccine development, redesign coding regions sometimes may be unnecessary because they need to keep the original open reading frame (ORF) Besides, there are more reasons to refrain from using optimized coding regions related to the folding behaviour in post-translational process and out-of-frame peptides generating cryptic T-cell epitopes (Sahin et al., 2014) In term of

COVID-19 mRNA vaccines, usually the sequence-optimized mRNA encoding S protein are

synthesized in vitro by T7 RNase polymerase and codon uridin (U) in the reaction will

be completely replaced with N1m-pseudouridine ( (N1mΨ) to enhance the stability and translation of the mRNA vaccine (Corbett et al., 2020; Svitkin et al., 2017)

2.2.1.6 mRNA vaccine delivery in COVID-19

Although there are many delivery systems that have been reported until now (Zeng, Zhang, Walker, & Dong, 2020), in this review, we are going to discuss only the lipid nanoparticles (LNPs) due to its recent application in the two most successful up-to-date mRNA vaccines which we will give more details later

2.2.1.7 Lipid nanoparticles (LNPs) in the COVID-19 mRNA vaccines

Once injected inside our body, mRNA-LNPs need to overcome several physiological barriers They need to be protected from nuclease in the fluids, evade the mononuclear phagocyte system clearance, reach the target cell for immunization, and

escape from the endosome to get into cytoplasm Thus, formulating and testing on type

of lipid, ratio between the lipid components as well as the electrical charge on the lipid structure are important strategies in a LNPs development for vaccination

Numerous previous studies on in vivo siRNA delivery by LNPs have shown its

safety and effectiveness for over a decade Moreover, LNPs have been used in the pharmaceutical industry as a vehicle to deliver various therapeutic agents such as cancer and infectious diseases (Sahin et al., 2014) Evolving from the wide-used precursor liposomes, lipid nanoparticles have been modified to a more complex architecture The compositions of LNPs used in the two available mRNA vaccines (Pfizer and Moderna) include an ionizable cationic lipid which is positively charged at low pH for easily forming complex with RNA and allowing the endosomal release of mRNA into cytoplasm, neutral at physiological pH for reducing the potential toxicity effects; a polyethylene glycol (PEG) for increasing the stability and for longer systemic circulation; and helper lipids (cholesterol and natural occurring phospholipids) to stabilize and support LNPs structure (Granados-Riveron & Aquino-Jarquin, 2021)

2.2.2 Current mRNA vaccines

Comirnaty (BNT162b2 vaccine) - Pfizer/BioNtech

Comirnaty (BNT162b2) is the first and the only vaccine that has been fully approved by the Food and Drug Administration (FDA) until now (Wilson & Wilson, 2021) BNT162b2 vaccine of Pfizer/BioNtech is mRNA-lipid nanoparticles (mRNA-LNPs) encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) which stabilized in prefusion conformation by modified two consecutive proline mutation (Hwang et al., 2020; World Health Organization, 2020) Overall, the vaccine is given in two-dosed regimens at 30 μg each for the individuals 16 years of age and older and recently expanded for 12 years old people (FDA, 2021; Polack

et al., 2020)

mRNA-1273 vaccine - Moderna

Using the same mRNA platform as the Comirnaty (BNT162b2 vaccine),

mRNA-1273 vaccine (Moderna) is also well-known as a successful vaccine candidate in preventing COVID-19 The formulation is mRNA-lipid nanoparticles (mRNA-LNPs) encoding the S-2P antigen of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), stabilized in prefusion conformation by 2 proline mutations in 2 consecutive position (986 and 987) (Jackson et al., 2020).The vaccine is also a two-dose regimen at 100 μg each In the context of newly circulating SARS-CoV-2 variants and their effects on reducing the neutralizing titer GMTs of current vaccines (Wu, Werner, et al., 2021), the Moderna company are developing and testing two booster candidates: mRNA-1273.351vaccine encoded with the S protein found in B.1.351 variant and mRNA-1273.211 vaccine mixed between mRNA-1273 and mRNA-1273.211 (Wu, Choi,

et al., 2021)

Table 1: Summary information of vaccine candidates

o C

o C

+ T-c

+ T-c

+ T-c

+ T-c

+ T-c

+ T-c

+ T-c