European Review for Medical and Pharmacological Sciences 2021; 25: 2752-2784 SARS-CoV-2 vaccine development: where are we?

M. GALDIERO1, M. GALDIERO1, V. FOLLIERO1, C. ZANNELLA1, A. DE FILIPPIS2, A. MALI1, L. RINALDI3, G. FRANCI4

1Department of Experimental Medicine, University of Campania “Luigi Vanvitelli”, Naples, Italy 2Department of Pharmacy, University of Study of Naples “Federico II”, Naples, Italy 3Department of Advanced Medical and Surgical Sciences, University of Campania “Luigi Vanvitelli”, Naples, Italy 4Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana”, University of Salerno, Salerno, Italy

Abstract. – The coronavirus disease 2019 in many remarkable traits from all other positive (COVID-19) pandemic caused by severe acute re- single-stranded RNA viruses: i) their RNA ge- spiratory syndrome coronavirus 2 (SARS-CoV-2) nomes are substantially larger than the genomes has proved to be one of the most challenging in- of any other known positive-stranded RNA virus; fectious diseases in the modern era, and despite several countermeasures to lessen its impact, the ii) coronaviruses are unique in showing a heli- spread of the virus is still affecting most countries. cal symmetry of the nucleocapsid; and doubtless This renders the goal of active immunization of the most peculiar, iii) coronaviruses replicate their population through vaccination a worldwide pub- positive-stranded RNA genomes by a strategy in lic health priority. In fact, only when efficient vac- which they give rise to a nested set of messenger cination programs will be successfully implement- RNAs (mRNAs) that have a common 3’ end. Each ed, a return to pre-pandemic normality can be considered. The scientific community has made of the mRNAs within the nested set contains mul- a tremendous effort to blow the lid off the patho- tiple open reading frames (ORFs), where only the 1 genesis of the disease, and unprecedented efforts one at the 5’ end of the molecule is translated . are ongoing with governments, private organiza- This family of RNA viruses, though almost un- tions, and academics working together to expe- known to the large audience until recently, has ditiously develop safe and efficacious vaccines. been the object of extensive studies by scientists Previous research efforts in the development of in the last few decades. Prior to 2003, only two vaccines for other coronaviruses (Severe Acute Respiratory Syndrome Coronavirus 1 and Middle strains of coronavirus, the Human coronavirus East Respiratory Syndrome Coronavirus) as well (HcoV) 229E and HCoV-OC43, were associat- other emerging viruses have opened the door for ed with a mild influenza-like illness in humans. exploiting several strategies to design a new vac- Hence, researchers in the field were taken by cine against the pandemic virus. Indeed, in a few surprise in 2003 when it was discovered that the months, a stunning number of vaccines have been causative agent for the new severe acute respira- proposed, and almost 50 putative vaccine can- didates have entered clinical trials. The different tory syndrome (SARS) epidemic was identified as vaccine candidates use different vaccine develop- a “new” coronavirus. The discovery of the SARS ment platforms, from inactivated whole virus vac- coronavirus (SARS-CoV) stimulated extensive cine to subunit vaccine, nucleic acid, and vectored research in the field of coronaviruses, as regards vaccines. In this review, we describe strengths, the molecular mechanisms necessary for the defi- flaws, and potential pitfalls of each approach to nition of target molecules, the development of understand their chances of success. antivirals and/or vaccines, and the identification Key Words: of other novel coronaviruses in both humans and SARS-CoV-2, COVID-19, Vaccine, Pandemic, Im- animals, leading to the discovery of two other mune response. coronaviruses that infect humans: HCoV-NL63 in 2004 and HCoV-HKU1 in 20052. Unlike other human coronaviruses, SARS-CoV was capable of Introduction causing severe and life-threatening pneumonia in humans and was the most pathogenic coronavirus Coronaviridae are a very intriguing family of until a further coronavirus capable of causing fa- positive single-stranded RNA viruses. They differ tal disease in humans, the Middle East respiratory

2752 Corresponding Author: Massimiliano Galdiero, MD; e-mail: [email protected] SARS-CoV-2 vaccine development: where are we? syndrome coronavirus (MERS-CoV), appeared. against SARS-CoV-2. Moreover, it delves into the Finally, to complete the picture of coronavirus- composition of the different vaccine platforms and es of human interest, a disease, named Corona- the relative advantages and/or disadvantages of virus Disease 2019 (COVID-19), came to light in each approach. Wuhan, within the Hubei Province in China, in early December 2019, but in a flash, a pandem- Viral Structural Proteins ic magnitude was reached. As of 29th November and their Function 2020, a total number of 61,869,330 confirmed cas- SARS-CoV-2 particles contain a posi- es of COVID-19 have been reported, including tive-strand RNA genome of 29.9 kb in length. 1,448,896 deaths3. Caused by severe acute respi- The genome is both capped and poly-adenylated, ratory syndrome coronavirus 2 (SARS-CoV-2), enabling SARS-CoV-2 to be translated upon its the pandemic represents a serious threat to public release within the cytoplasm right at the begin- health since infection leads to a wide spectrum ning of an infectious cycle. of clinical manifestations with an initial status The SARS-CoV-2 genome possess 14 ORFs of the asymptomatic subject, with progression to encoding 27 proteins, and its genomic organi- pneumonia which may further progress to acute zation follows the common rules of other coro- respiratory distress syndrome (ARDS) followed naviruses being: 5′-leader-UTR-replicase-Spike by multi-organ failure (MOF) and death4. While (S)-Envelope (E)-Membrane (M)-Nucleocapsid the last three viruses with high pathogenicity and (N)-3′UTR-poly(A)tail, with several accessory mortality (SARS-CoV, MERS-CoV, and SARS- genes interspersed within the structural genes at CoV-2) have only recently emerged in the hu- the 3′ end of the genome7. The accessory genes man population, HCoV-HKU1 and HCoV-NL63 are considered nonessential for in vitro replication have been circulating in humans for a long time. but able to exert a suppressing effect on the anti- HCoV-HKU1 and HCoV-NL63 are both respira- viral-immune responses and also able to intensify tory coronaviruses that are frequently associated pathogenesis8. A large portion of about two-thirds with upper and lower respiratory tract diseases. of the genome within the 5′ end is employed to Infections with these two human coronaviruses encode two long ORFs named 1a and 1b, which do not differ much from those caused by the “old” produce the nonstructural proteins of the virus. coronaviruses HCoV-229E and HCoV-OC43. Orfs 1a and 1b are translated first as polyprotein The first severe acute respiratory syndrome due precursors named pp1a and pp1ab, where the to SARS-CoV spanned the world with more than latter results from a programmed –1 ribosomal 8,000 recognized cases and caused 774 deaths frameshift event taking place at the short overlap (almost 9% of infected cases) in less than a year at the end of ORF1a with ORF1b coding sequenc- and then disappeared5. The second outbreak in es9. The polyproteins include several viral prote- 2012, MERS-CoV, remained mainly localized in ases that together process pp1a and pp1ab into 16 the Arabic peninsula, where 1,038 cases with 460 nonstructural proteins (nsp1–16), which are sub- deaths were reported (approximately 37% mortali- sequently required at diverse phases of SARS- ty). Since then, it reappeared with small epidemics CoV-2 replication. From a morphological point of mainly nosocomial infections in South Korea6. of view of the virus particles, the SARS-CoV-2 On the other hand, the present pandemic has swept genome is associated with the N protein to form the surface of the globe with an unprecedented and a helical nucleocapsid, which in turn is wrapped unforecastable spreading capacity, albeit with mi- by a lipid membrane envelope containing the M, nor mortality compared to the previous two, and S, and E proteins (Figure 1). The trimeric S gly- is still in a crescendo after almost a year from its coprotein (∼150 kDa) is the largest membrane emergence in the human population. As seen in the protein forming 20 nm-long and prominent pet- actual situation, we need to accept that the SARS- al-shaped spikes and is involved in cell receptor CoV-2 will remain a treat for a long time. There- binding of the virus to the host cell and in mediat- fore, the best option for getting back to our usual ing the fusion mechanism able to allow the entry social life (considering the measures adopted by of the virus into cells. most Governments worldwide of social distancing The S glycoprotein is a class I fusion protein and more or less strict lockdowns) is represented whose attachment to the host receptor angioten- by the widespread distribution and use of vaccines. sin-converting enzyme 2 (ACE2) is able to trigger This review addresses the key point of the immune a cascade of cell membrane fusion events lead- responses deemed essential to provide protection ing to viral entry10-13. Glycoprotein S is further

2753 M. Galdiero, M. Galdiero, V. Folliero, C. Zannella, A. De Filippis, A. Mali, L. Rinaldi, G. Franci

Figure 1. Vaccine platforms used for SARS-CoV-2 vaccine development.

cleaved by a host cellular protease into two sep- protein is to be found in its function in promoting arate polypeptides: a globular S1 domain at the membrane curvature as well as binding to the nu- N-terminal region, and the membrane-proximal cleocapsid; therefore, M protein fulfills the func- S2 and transmembrane domains14. The recep- tion of classical matrix protein of other enveloped tor-binding domain (RBD) able to determine host viruses that contain helical nucleocapsids. Hence, range and cellular tropism is found within the the M protein defines the shape of the viral enve- S1 domain, while key features to activate mem- lope. Finally, the E protein plays a crucial role in brane fusion are located within the S2 domain15. determining virus shape and driving assembly. It The S2 domain contains two coiled coil regions behaves as a viroporin forming pentameric pro- (the so-called heptad repeats), which give rise to tein-lipid pores that allow ion transport. Together, a structure of trimeric hairpins, positioning the M and E are necessary and sufficient for an ef- fusion peptide in close proximity to the C-ter- ficient assembly and release of SARS-CoV-2 vi- minal region of the ectodomain. The formation rus-like particles (VLPs)17. of this post-fusion structure is able to drive the apposition and subsequent merging of viral and Vaccine Platforms target cell membranes16. On the contrary, the M It is a world high-priority to develop effective protein (~25-30 kDa), with three transmembrane and safe vaccines to block COVID-19 pandemic, domains, differs from most other viral envelope with the aim of limiting its inexorable spread, and glycoproteins in that only a short N-terminal do- ultimately to avoid any eventual future reappear- main is exposed on the exterior of the particle, ance. In practice, we do not have to view vaccines while a large C-terminal domain is exposed be- as flawless weapons to ensure a COVID-19-free neath the envelope, facing the interior. The reason world, but the tasks of an efficient vaccine are for this atypical orientation of the coronavirus M to decrease disease severity, reduce viral spread,

2754 SARS-CoV-2 vaccine development: where are we? and most importantly, person-to-person trans- group of several hundred subjects are included in mission. Currently, no vaccine has been licensed the trial. The Phase 2 trials involve randomized to prevent SARS-CoV-2 infection, but since the and well-controlled experimentation, with a pla- SARS-CoV-2 shares, considerable sequence simi- cebo control group, in order to establish formula- larity with the preceding two deadly coronavirus- tion and doses and prove efficacy. Finally, Phase es (MERS-CoV and SARS-CoV), the studies to 3 of the trials is used to definitely assess vaccine attempt to develop vaccines against MERS-CoV safety in a larger cohort of several thousands of in- and SARS-CoV could represent a strong knowl- dividuals; all correlates of protection are analyzed edge base to significantly accelerate the develop- (production of antibodies and cell-mediated immu- ment of anti-COVID-19 vaccines18. Nevertheless, nity, immunological memory, and protection from several factors require to be taken into consider- disease). Once a successful Phase 3 is deemed ter- ation prior to any vaccine moving forward to a minated, there is the verification and approval by widespread usage to immunize vast populations. the licensing authority before entering the manu- The pathway for a vaccine to reach the market is facturing and distribution to the public19,20. long and strewn with obstacles, and the need to Great efforts around the globe and a commit- proceed through an exploratory, a preclinical and ment never seen before have allied many teams a clinical stage. The exploratory stage represents worldwide for the common scope to reduce the the basic research conducted mainly in academic time for vaccine discovery and approval. Sever- and research laboratories where the target anti- al platforms and a huge number of vaccines are gen is discovered and analyzed. Therefore, it rep- being investigated, and in the present review, resents the first idea of the novel vaccine. This is we have considered mainly 6 different plat- followed by a preclinical stage where tissue-cul- forms of vaccine development for distributing ture or cell-culture and animal testing are devel- all candidate vaccine (196 vaccines), included oped to evaluate the safety of the putative vaccine the 6th, which comprises: live attenuated virus- and the level of its immunogenicity. These stud- es (3 examples), replicating bacterial vectors ies are of great importance since they are able to (1 vaccine), and one T-cell based vaccine. The produce invaluable data to allow a first prediction first category is dedicated to the inactivated of the type of responses is possible to predict for whole SARS-CoV-2 virus with 21 examples, human use. At this stage, researchers can start to while the most prolific is the second category envisage possible routes of administration and referred as “protein subunit” vaccines, which eventual dosage; indeed, more advanced studies includes different technologies such as purified also consider animal challenges with the infec- or expressed proteins and peptides with differ- tious pathogen to find out the efficacy in prevent- ent carrier molecules. This category currently ing the infection. Once sufficient preclinical data counts 68 candidate vaccines in development. become available clinical trials might be initiated, The third category is made up of the nucleic acid but very few putative candidates are, indeed, able platforms, which are further divided into RNA to pass to the following stage of clinical testing. (24 candidates) and DNA (18 candidates). A Generally, clinical trials are divided into Phase 1, fourth category is based on viral vectors either Phase 2, and Phase 3 clinical trials, but in a period non-replicating (21 candidates) or replicating of great pressure due to the running pandemic, (21 candidates), and the last (5th) is represent- many companies have decided to use overlapping ed by the vaccine based on virus-like-particles Phase 1 and Phase 2 trials to reduce the timing. In (VLP) or nanoparticles (18 candidates). More- fact, the intrepid goal is to reduce to merely 12-18 over, various adjuvant technologies like AS03 months a process that prior to the pandemic used (GSK, Brentford, United Kingdom), MF-59 to take from 10 to 15 years. In Phase 1, a small (Novartis, Basel, Switzerland), CpG 1018 (Dy- number (less than 100) of healthy volunteers get navax, Emeryville, CA, United States) are now administered the putative vaccine mainly to as- available to enhance the immunogenicity of sess the safety and the quality of the immune any candidate vaccine to allow safe vaccine de- response elicited; usually, this is not a blind ex- velopment21. What is really surprising is that 11 perimentation, but both the volunteers and the re- of such candidates have already reached Phase searchers are informed on the protocol used. Only 3 clinical trials. The platforms on which these if the collected data are encouraging, the exper- vaccines are based are inactivated, non-rep- imentation can proceed to the following phase, licating viral vectors, protein subunits, and that is the Phase 2 vaccine trial, where a larger RNA-based vaccines as listed in Table I22.

2755 M. Galdiero, M. Galdiero, V. Folliero, C. Zannella, A. De Filippis, A. Mali, L. Rinaldi, G. Franci

Table I. SARS-CoV-2 candidate vaccines in Phase 3 clinical trials.

COVID-19 vaccine Vaccine Type of candidate Current stage of clinical developer/manufactures platform vaccine evaluation (estimated study completion date) (NA- Not Available) Sinovac (Beijing, China) Inactivated Inactivated SARS-CoV-2 NCT04456595 (October 2021) vaccine with aluminum NCT04582344 (October 2021) hydroxide NCT04617483 (May 2021) Wuhan Institute of Biological Inactivated Inactivated whole ChiCTR2000034780 (NA) products (Wuhan, China)/Sinopharm SARS-CoV-2 NCT04612972 (September 2021) (Beijing, China) Beijing Institute of Biological Inactivated Aluminum hydroxide NCT04560881 (December 2021) products/Sinopharm (Beijing, China) adsorbed inactivated ChiCTR2000034780 (NA) SARS-CoV-2 vaccine Bharat Biotech (Hyderabad, India) Inactivated Whole-Virion Inactivated CTRI/2020/11/028976 (End 2021) Novavax (Gaithersburg, MD, United Protein Full length recombinant 2020-004123-16 (NA) States) Subunit SARS CoV-2 S prefusion NCT04611802 (December 2022) glycoprotein adjuvanted with Matrix M protein Moderna (Cambridge, MA, United Nucleic Acid LNP-encapsulated mRNA NCT04470427 (October 2022) States)/NIAID (Bethesda, MD, United States) BioNTech (Mainz, Germany)/Fosun Nucleic Acid Lipid-nanoparticle-formu- NCT04368728 (December 2022) Pharma (Shanghai, China)/Pfizer lated, nucleoside-modified (New York, United States) mRNA vaccine that encodes the trimerized receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2. University of Oxford (Oxford, Unit- Non-replicating Recombinant replication- NCT04540393 (March 2021) ed Kingdom)/AstraZeneca (Cam- viral vector defective chimpanzee NCT04516746 (October 2022) bridge, United Kingdom) adenovirus expressing the S CTRI/2020/08/027170 (NA) glycoprotein of SARS-CoV-2 (ChAdOx1-S) CanSinoBiologicals InC (Tianjin, Non-replicating Adenovirus type 5 vector NCT04526990 (January 2022) China)/Beijing Institute of Biotech- viral vector carrying the S-protein of NCT04540419 (July 2021) nology (Beijing, China) SARS-CoV-2

Gamaleya Research Institute Non-replicating Combined adenovirus-based NCT04530396 (May 2021) (Moscow, Russia) viral vector vector (rAd26-S+rAd5-S) of NCT04564716 (April 2021) the S-protein of SARS-CoV-2 Janssen Pharmaceuticals Companies Non-replicating Adenovirus type 26 vector NCT04505722 (March 2023) (Beerse, Belgium) viral vector carrying the S1-subunit of NCT04614948 (May 2023) SARS-CoV-2 (Ad26CoV-S1)

Modified from: DRAFT landscape of COVID-19 candidates vaccine ⇒ https://www.who.int/publications/m/item/draft-land- scape-of-covid-19-candidate-vaccines.

Inactivated Whole-Virus Vaccine aldehyde). Inactivation dismantles the virus’s abil- Commonly known as non-live vaccines, the ity to replicate and provokes pathology, but fore- main characteristic is their inability to replicate sees the maintenance of its immunogenicity, so since they do not contain any living or infectious that the immune system can still mount a defense particles. Inactivated whole-virus vaccines are against the pathogen23. A wide range of native vi- preparations comprising the entire disease-caus- ral antigens may be structurally conserved after ing pathogen, which is inactivated physically inactivation to expose native epitope conforma- (heat, radiation) or chemically (formalin or form- tions; therefore, inactivated vaccines can induce

2756 SARS-CoV-2 vaccine development: where are we? conformation-dependent antibody responses, and activation of the virus clinical isolates by β-propi- compared to many subunits or recombinant vac- olactone to eliminate viral infectivity and using cines, the whole virus is presented to the immune aluminum hydroxide as adjuvant. Electron mi- system; therefore, immune responses are likely croscopy imaging showed proper viral particles to be directed not only against the selected viral with conserved spikes with diameters of approxi- protein but also the against other structures such mately 100 nm, and the presence in vaccine stocks as matrix, envelope, and nucleoprotein. A sat- of viral structural proteins was demonstrated by isfying safety profile is generally present, even western blots. Results from phase 2 clinical tri- in immunocompromised subjects. Per contra, a als have shown that the inactivated SARS-CoV-2 drawback of these vaccines is that immunogenic- vaccine BBIBP-CorV is safe, well-tolerated, and ity and duration of protection tend to be of limited immunogenic in healthy individuals. Neutraliz- effectiveness, and they may require reminders of ing antibodies in 100% of vaccine recipients have the immune system; in fact, the addition of ad- been obtained after two-dose immunizations in juvants and repeated administrations to improve two age groups (18-59 years and ≥60 years), with immunogenicity are generally necessary. Fur- the observation of only mild adverse reactions thermore, inactivated viruses, with low produc- (pain and fever)26. In preclinical studies, immuni- tion cost, safe, and not involving genetic manipu- zation with BBIBP-CorV was able to induce high lation, can be promptly developed and scaled up levels of neutralizing antibody titers in mice, rats, for a pandemic situation adopting infrastructure guinea pigs, rabbits, and non-human primates and methods proper of a well-established mature (cynomolgus monkeys and rhesus macaques) to technology24. A list of inactivated vaccine candi- provide protection against SARS-CoV-2. More- dates against SARS-CoV-2 is presented in Table over, in preclinical studies of BBIBP-CorV im- II. Collectively, in recent years, we have wit- munization followed by SARS-CoV-2 live virus nessed a gradual shift of vaccination strategies challenge, no antibody-dependent enhancement from whole-virus vaccine to subunit, peptide and was observed in rhesus macaques to confirm the genetic vaccine mainly as a response to high reac- safety of the vaccine27. A second vaccine candi- togenicity generally associated with whole-virus date is the one produced by Sinopharm together vaccines. These more recently designed vaccines with the Wuhan Institute of Biological Products have been demonstrated to be less immunogen- with a similar methodology, and the preliminary ic if compared to whole-virus vaccines; in fact, results of their β-propiolactone-inactivated whole they generally require adjuvants and/or multiple virus vaccine have shown that the vaccine was immunization regimes. well tolerated in all dose groups under different Nevertheless, inactivated whole-virus vaccines injection procedures with no vaccine-related se- are considered trustful conventional vaccines rious adverse events. A further Chinese purified with mature technology with likely possibilities inactivated SARS-CoV-2 virus vaccine candidate to arrive first in the race for SARS-CoV-2 vac- is the one produced by Sinovac and named PiCo- cine entering clinical use25. Out of the 11 candi- Vacc. This vaccine was shown to induce SARS- date vaccines against SARS-CoV-2 that already CoV-2-specific neutralizing antibodies in mice, reached phase 3 clinical trials, 4 are represent- rats, and non-human primates28. The vaccine ed by inactivated whole-virus vaccines (Table manufactured by Sinovac contains 3 μg/0.5 mL I). These phase 3 clinical trials are done by the of inactivated SARS-CoV-2 virus, and aluminum Beijing Institute of Biological Products (Beijing, hydroxide as adjuvant and after promising phase China) (NCT04560881), Sinovac (Beijing, China) 1/2 clinical trials is now being analyzed in phase (NCT04456595, NCT04582344), Wuhan Institute 3 clinical studies29,30. The last candidate vaccine of Biological Products (Wuhan, China) (ChiC- reaching the finish line is the one produced by the TR2000034780) and Bharat Biotech (Hyderabad, Indian company Bharat that has just entered the India) (CTRI/2020/11/028976). phase 3 clinical trial. The vaccine BBV152 has Inactivated vaccines are usually produced by been produced through inactivation of the whole growing SARS-CoV-2 in cell culture (Vero cells) virus by β-propiolactone followed by a chromato- followed by inactivation of the virus. BBIBP- graphic purification step. Three different formu- CorV is an inactivated vaccine candidate, being lations have been prepared: i) BBV152A contain- developed by a Chinese state-owned Sinopharm ing 3 g of antigen mixed with imidazoquinoline together with the Beijing Institute of Biological class TLR7/8 agonist (IMDG) adsorbed to alu- Products. The vaccine has been produced by in- minum hydroxide gel (Algel), ii) BBV152B con-

2757 M. Galdiero, M. Galdiero, V. Folliero, C. Zannella, A. De Filippis, A. Mali, L. Rinaldi, G. Franci

Table II. SARS-CoV-2 inactivated whole-virus candidate vaccines under development.

COVID-19 vaccine Vaccine Type of candidate Current stage of clinical developer/manufactures platform vaccine evaluation (estimated study completion date) (NA-Not Available) Institute of Medical Biology, Inactivated Inactivated whole Phase 1/2: NCT04470609 Chinese Academy of Medical Sciences SARS-CoV-2 (November 2021) (Kunming, China) Research Institute for Biological Safety Inactivated Inactivated whole Phase 1/2: NCT04530357 Problems (Gvardeyskiy, Kazakhstan) SARS-CoV-2 (December 2020) Beijing Minhai Biotechnology Co., Ltd. Inactivated Inactivated SARS-CoV-2 Phase 1: ChiC- (Beijing, China) Vaccine (Vero Cells) TR2000038804 (NA) Institute of Vaccines and Medical Inactivated Egg-based, inactivated, whole Preclinical Biologicals (IVEC; Khánh Hòa, chimeric Newcastle Disease Vietnam)/Dynavax (Emeryville, CA, Virus (NDV) expressing United States)/PATH; San Francisco, membrane-anchored CA, United States) pre-fusion-stabilized trimeric SARS-CoV-2 S protein (Hexapro) + CpG 1018 Government Pharmaceutical Inactivated Egg-based, inactivated, whole Preclinical Organization (GPO; Bangkok, chimeric Newcastle Disease Preclinical Thailand)/Dynavax (Emeryville, CA, Virus (NDV) expressing United States)/PATH; San Francisco, membrane-anchored CA, United States) pre-fusion-stabilized trimeric SARS-CoV-2 S protein (Hexapro) + CpG 1018 Institute Butantan (São Paulo, Brazil)/ Inactivated Egg-based, inactivated, whole Preclinical Dynavax (Emeryville, CA, United chimeric Newcastle Disease States)/PATH; San Francisco, CA, Virus (NDV) expressing United States) membrane-anchored pre-fusion-stabilized trimeric SARS-CoV-2 S protein (Hexapro) + CpG 1018 KM Biologics (Kumamoto, Japan) Inactivated Inactivated + alum Preclinical Selcuk University (Selçuklu-Konya, Inactivated Inactivated Preclinical Turkey) Erciyes University (Talas/Kayseri, Tur- Inactivated Inactivated Preclinical key) National Research Centre (Dokki, Inactivated Inactivated Preclinical Egypt) Osaka University/BIKEN/NIBIOHN Inactivated Inactivated Preclinical (Osaka, Japan) Sinovac (Beijing, China/Dynavax Inactivated Inactivated + CpG 1018 Preclinical (Emeryville, CA, United States) Valneva (Saint-Herblain, Inactivated Inactivated + CpG 1018 Preclinical France)/Dynavax (Emeryville, CA, United States) Shifa Pharmed (Tehran, Iran) Inactivated Inactivated + Alum Preclinical Zista Kian Azma Co. (Tehran, Iran) Inactivated Inactivated Preclinical Milad Pharmaceutics Co. Plymouth, MI, Inactivated Inactivated Preclinical United States) Kocak Farma Ilac ve Kimya San. A.S. Inactivated Inactivated Preclinical (Istanbul, Turkey)

Modified from: DRAFT landscape of COVID-19 candidates vaccine ⇒ https://www.who.int/publications/m/item/draft-land- scape-of-covid-19-candidate-vaccines

2758 SARS-CoV-2 vaccine development: where are we? taining 6 μg of antigen mixed with Algel-IMDG, sufficient to induce T cell responses, therefore us- and iii) BBV152C with 6 μg of antigen adsorbed ing only fractions of the entire protein can provide to Algel. In the ongoing clinical trial, BBV152B several advantages35,36. In fact, fragments of anti- vaccine formulation will be administered as a two gen can display B and/or T cell epitope activity dose intramuscular injection 28 days apart31. The affecting the specificity of the immune response37. ongoing phase 3 clinical trials will provide more These peptide vaccines also have the advantage of information on the safety and immunogenicity, being easily produced by chemical synthesis, do doses, and possible immunization schedules of not undergo tertiary structure folding, and have any of the inactivated whole-virus vaccine can- the possibility to be used in several combinations didates that will maintain the actual promise for to display multivalent antigens capable of elicit- reaching the market. ing strong humoral and cellular responses38,39. However, the low molecular weight of peptidic Subunit Vaccines (Including Synthetic vaccines usually results in low immunogenicity; Peptides or Epitope Vaccines) thus, structural modifications, multi-epitope de- Subunit vaccines contain selected patho- livery systems, and use of adjuvants are generally gen-derived proteins (antigens) or parts of them in predicted40. place of the whole pathogen. In the case of bacte- Subunit vaccines represent the most common rial pathogens, subunit vaccines can be obtained platform explored. There are 15 COVID-19 sub- starting from conventional cultivation processes unit vaccines in clinical trials (Tables I and III), through the purification of pathogen preparations, with 55 more candidates under preclinical devel- while viral subunit vaccines require the use of opment. Many research institutions feature SARS- recombinant DNA engineering32. The genes en- CoV-2 subunit vaccine and mainly use the spike coding the selected antigens are either cloned or glycoprotein S, and its fragments, such as S1, S2, synthesized and then are expressed using one receptor binding domain (RBD), and nucleocap- of the available expression systems (insect, bac- sid protein as a prime target antigen40. Novavax, a terial, yeast, and mammalian cells). One of the North-American biotech company, has developed preferred systems is bacterial expression since the COVID-19 subunit vaccine (NVX-CoV2373), they assure a very high level of expression and which has already reached phase III clinical tri- the scaling-up step is quite straightforward by als. The vaccine is made with the full-length fermentation technologies. Nevertheless, a major SARS-CoV-2 spike (S) glycoprotein expressed in drawback of bacterial expression systems is the baculovirus Spodoptera frugiperda (Sf9) insect proper post-translational modifications; therefore, cells and stabilized in the prefusion conforma- the use of insect cells or mammalian cells may tion by two proline substitutions introduced at the be advantageous. The produced antigens can be S1/S2 furin cleavage site. NVX-CoV2373 with a easily purified and used in vaccine preparations. Matrix-M saponin-based adjuvant induced a Th1 The absence of infectious virus improves safety dominant B- and T-cell response with high titer patterns and wipes out any concern related to vir- anti-spike IgG able to block hACE2 binding and ulence reversion33. to neutralize infection in mice and non-human Compared to live or inactivated whole-organ- primate models. Moreover, immunized macaques ism vaccines, subunit vaccines are generally saf- showed protection against pulmonary disease af- er and causing less adverse effects, but are also ter the SARS-CoV-2 challenge, and no evidence consistently less immunogenic in view of the fact of vaccine-associated enhanced respiratory dis- that they contain a reduced number of antigens ease was present41. NVX-CoV2327 (two-dose and that following the purification process are de- regimens of 5 μg and 25 μg of plus the Matrix-M1 void of the other viral components that are useful adjuvant) administered to healthy adults (18 to 59 for the triggering of the immune response. Since years of age) showed acceptable safety and in- the purified subunits are weak immunogens, to duced high immune responses, with levels of neu- obtain vaccines with convenient efficacy, the an- tralizing antibodies that closely correlated with tigens are conjugated with protein molecules, and anti-spike IgG42. Anhui Zhifei Longcom Biophar- adjuvant to enhance an immune response are re- maceutical (China) has proposed a protein subunit quired34. vaccine comprising a tandem repeat single-chain Within subunit vaccines, we also include vac- dimer (sc-dimer) of the SARS-CoV-2 RBD to be cines based on synthetic peptide considering that produced (high yields, g/L level) in a Chinese minimal immunogenic peptide sequence may be hamster ovary (CHO) cell system43.

2759 M. Galdiero, M. Galdiero, V. Folliero, C. Zannella, A. De Filippis, A. Mali, L. Rinaldi, G. Franci

Clover Biopharmaceuticals (Chengdu Shi, Vesicles based vaccine) and others (for a complete China) has employed a technology named ‘Tri- list see Table III). mer-Tag’ to produce a eukaryotic cell-derived trimeric subunit spike protein subunit vaccine. Nucleic Acid Platform (DNA and mRNA) Immunization of S-Trimer with either AS03 Nucleic acid vaccines (based on DNA or RNA) (oil-in-water emulsion) or CpG 1018 (TLR9 ag- have surged huge interest in the last decades for onist) with the addition of alum adjuvants gen- their potential in a pandemic crisis considering erated high-levels of neutralizing antibodies and the low-cost and rapid development. They exploit a Th1 immune response in animal models, and either plasmid DNA or RNA encoding a specific rhesus macaques were protected from SARS- target antigen that is delivered to the subject to CoV-2 challenge44. This vaccine is administered vaccinate and is taken up by the cells where the in conjunction with a CpG/Alum adjuvant and antigenic sequence is expressed. The key point is actually being tested in phase 1 clinical trial is that the human cells of the vaccinated subject (NCT04405908). Considering stabilization of the process the viral protein in a very different way fusion proteins of enveloped viruses an important from what happens in a heterologous system for target for the creation of next generation vaccines, the production of subunits vaccines; therefore, the the University of Queensland (Brisbane, Austra- most important theoretical benefits of employing lia) has proposed molecular clamp stabilized spike a nucleic acid into a vaccine seems to be the fact protein with MF59 adjuvant where the S protein that the viral protein is processed by the same cel- has been “locked” in a prefusion conformation lular apparatus that the virus would have directed into the correct 3-dimensional shape. Therefore, the host to do in a real infection. This can be con- this approach should allow the production of an- sidered a close mimic of natural infection without tibodies against a wide choice of conformation- the pathogenic damage46. Hypothetically, a single al epitopes that are displayed on the virion sur- industrial plan may be able to produce any re- face45. Sanofi Pasteur (Lyon, France) has used a quired nucleic acid vaccine and also scale up the recombinant DNA in vitro platform (baculovirus) production to satisfy a pandemic level demand. to produce a spike protein antigen in large quan- Vaccine platforms using recombinant DNA tech- tities. The University of Pittsburgh (Pittsburgh, nology have a long history, whereas mRNA-based PA, United States) taking advantage of their pre- vaccines have only recently emerged as a novel vious experience on SARS and MERS vaccine option. As depicted in Table IV, there are 6 mR- development, has proposed a microneedle array NA-based COVID-19 vaccines and 5 DNA-based (MNA) patch to deliver the antigen into the skin COVID-19 vaccines in clinical trials, with 42 (PittCoVacc). The patch is applied as a band-aid, such vaccines (21 RNA-based and 21 DNA-based and the needles, which are entirely composed of vaccines) under preclinical development. sugar, enter the skin to allow diffusion of the an- DNA vaccines are routinely constructed from tigen within tissues42. Other protein-based can- plasmid DNA molecules that encode one or more didates in development are from Instituto Finlay antigens. The plasmids generally contain pro- de Vacunas (La Habana, Cuba) (two candidates: karyotic sequences to drive propagation in Esch- i) RBD plus adjuvant and ii) recombinant RBD erichia coli, a mammalian expression cassette produced in CHO-cell and chemically conjugat- able to direct the expression of the inserted genes ed to tetanus toxoid), Medigen Vaccine Biologics in the subject inoculated with the vaccine. Once Corporation (Taipei, Taiwan) (S-2P proteins plus delivered, the plasmid DNA vaccine is internal- CpG 1018), Vaxine Pty Ltd. (Adelaide, Australia) ized by host cells at the immunization site or by (Recombinant spike protein with AdvaxTM ad- migrating antigen-presenting cells (APCs), where juvant), COVAXX/United Biomedical Inc. Asia in order to induce an adaptive immune response, (Long Island, NY, United States) (Multitope pep- the DNA must enter the cell nucleus47. Finally, tide-based S1 – RBD - protein vaccine), Gener- the target gene is expressed and translated into a ex (Miramar, FL, United States)/EpiVax (Prov- protein. The nucleic acids guide host cells’ activ- idence, RI, United States) (using an innovative ities to synthesize the target sequence beyond the Ligand Epitope Antigen Presentation System to obstacles of proper protein folding, purification, enhance the potency of peptide vaccines), Baiya solubility and incorrect glycosylation of proteins Phytopharm (plant-based subunit RBD plus adju- that are usually a consequence of recombinant vant), Quadram Institute Biosciences (Norwich, protein synthesis48. The manufacture of DNA United Kingdom) (Bacterial Outer Membrane vaccines is, to some extent, straightforward, and

2760 SARS-CoV-2 vaccine development: where are we?

Table III. SARS-CoV-2 protein subunit candidate vaccines under development. COVID-19 vaccine Vaccine platform Type of candidate Current stage of clinical evaluation developer/manufactures vaccine (estimated study completion date) (NA - Not Available) Anhui Zhifei Longcom Protein subunit Recombinant Phase 2: NCT04466085 (December 2021) Biopharmaceutical/Chinese SARS-CoV-2 Academy of Sciences RBD-Dimer protein (Beijing, China) subunit vaccine Kentucky Bioprocessing, Protein subunit RBD-based Phase 1/2: NCT04473690 (February 2022) Inc (Owensboro, KY, United States) Sanofi Pasteur (Lyon, France)/ Protein subunit S protein (baculovirus Phase 1/2: NCT04537208 (October 2021) GSK (Brentford, United production) Kingdom) Biological E Ltd (Telangana, Protein subunit Adjuvanted protein Phase 1/2: CTRI/2020/11/029032 (NA) India) subunit (RBD) Clover Biopharmaceuticals Protein subunit Recombinant Phase 1: NCT04405908 (March 2021) Inc./GSK/Dynavax SARS-CoV-2 trimeric S protein subunit vaccine Vaxine Pty Ltd (Adelaide, Protein subunit Recombinant spike NCT04453852 (July 2021) Australia)/Medytox protein with Advax™ (Seoul, Korea) adjuvant University of Queensland Protein subunit Recombinant NCT04495933 (September 2021) (Brisbane, Australia)/CSL SARS-COV-2 spike (Melbourne, Australia)/Seqirus protein ‘molecular (United Kingdom) clamp’ plus MF59 adjuvant Medigen Vaccine Biologics Protein subunit S-2P protein + CpG 1018 NCT04487210 (June 2021) Corporation/NIAID (Bethesda, MD, United States/Dynavax Instituto Finlay de Vacunas Protein subunit rRBD produced in IFV/COR/06 (Cuba) CHO-cell chemically conjugated to tetanus toxoid Instituto Finlay de Vacunas Protein subunit RBD + adjuvant IFV/COR/04 (February 2021) (Cuba) FBRI SRC VB VECTOR, Peptide Peptide NCT04527575 (October 2020) Rospotrebnadzor, Koltsovo West China Hospital, Sichuan Protein subunit RBD (baculovirus ChiCTR2000037518 (NA) University (Chengdu, China) production expressed in Sf9 cells) University Hospital Tuebingen Peptide SARS-CoV-2 HLA-DR NCT04546841 (December 2021) (Tübingen, Germany) peptides COVAXX/United Biomedical Protein subunit S1-RBD-protein NCT04545749 (August 2021) Inc. Ohio State University Protein subunit RBD protein delivered Preclinical (Columbus, OH, United in mannose-conjugated States)/Kazakh National chitosan nanoparticle Agrarian University (Kazakhstan) Kazakh National Agrarian Protein subunit Recombinant Preclinical University (Kazakhstan) spike protein with Essai O/W 1849101 adjuvant Table continued

2761 M. Galdiero, M. Galdiero, V. Folliero, C. Zannella, A. De Filippis, A. Mali, L. Rinaldi, G. Franci

Table III. (Continued). SARS-CoV-2 protein subunit candidate vaccines under development.

COVID-19 vaccine Vaccine platform Type of candidate Current stage of clinical evaluation developer/manufactures vaccine (estimated study completion date) (NA - Not Available) Neo7Logic (Gaithersburg, Peptide Peptides Preclinical MD, United States) Kazakh National Agrarian Protein subunit Recombinant spike Preclinical University/National Scientific protein with Essai O/W Center for Especially 1849101 adjuvant Dangerous Infections (Kazakhstan) Max-Planck-Institute of Protein subunit Recombinant S protein Preclinical Colloids and Interfaces (Potsdam, Germany) Farmacológicos Veterinarios Protein subunit RBD protein (baculovi- Preclinical SAC (FARVET SAC)/Universi- rus production) + FAR- dad Peruana Cayetano Heredia Squalene adjuvant (UPCH) Research Institute for Protein subunit Protein Subunit Preclinical Biological Safety Problems (Kazakhstan) Mynvax (Bangalore, India) Protein subunit RBD-protein Preclinical Izmir Biomedicine and Protein subunit Recombinant S protein Preclinical Genome Center (Izmir, Turkey) Bogazici University Peptide Peptide + novel Preclinical (Instanbul, Turkey) adjuvant University of Virginia Protein subunit S subunit intranasal Preclinical Charlottesville, VA, United liposomal formulation States) with GLA/3M052 adjs. Helix Biogen Consult, Protein subunit S-Protein (Subunit) + Preclinical Ogbomoso & Trinity adjuvant, E. coli based Immonoefficient Laboratory Expression (Ogbomoso, Oyo State, Nigeria) National Research Centre Protein subunit Protein Subunit S, N, Preclinical (Egypt) M&S1 protein University of San Martin and Protein subunit Protein Subunit Preclinical CONICET (Argentina) Chulalongkorn University/GPO Protein subunit RBD protein fused with Preclinical (Thailand) Fc of IgG + Adj. AdaptVac (PREVENT-nCoV Protein subunit Capsid-like Particle Preclinical consortium) ExpreS2ion (Hørsholm, Protein subunit Drosophila S2 insect Preclinical Denmark) cell expression system VLPs IMV Inc (Dartmouth, Canada) Peptide Peptide antigens Preclinical formulated in LNP WRAIR/USAMRIID (MD, Protein subunit S protein Preclinical United States) National Institute of Infectious Protein subunit S protein + adjuvant Preclinical Disease/Shionogi/UMN Pharma (Japan) Table continued

2762 SARS-CoV-2 vaccine development: where are we?

Table III. (Continued). SARS-CoV-2 protein subunit candidate vaccines under development.

COVID-19 vaccine Vaccine platform Type of candidate Current stage of clinical evaluation developer/manufactures vaccine (estimated study completion date) (NA - Not Available) Osaka University/BIKEN/Na- Protein subunit VLP-recombinant Preclinical tional Institutes of Biomedical protein + adjuvant Innovation (Japan) University of Pittsburgh Protein subunit microneedle arrays S1 Preclinical Pittsburgh, PA, United States) subunit Vaxil Bio (Ontario, Canada) Peptide Peptide Preclinical Flow Pharma Inc (Palo Alto, Peptide Peptide Preclinical CA, United States) AJ Vaccines (Copenhagen, Protein subunit S protein Preclinical Denmark) Generex (Miramar, Florida, Peptide Ii-Key peptide Preclinical United States)/EpiVax (Providence, RI, United States)

EpiVax/University of Georgia Protein subunit S protein Preclinical (Athens, GA, United States) EpiVax (Providence, RI, Protein subunit Protein Subunit Preclinical United States) EPV-CoV-19 Heat Biologics (Morrisville, Protein subunit gp-96 backbone Preclinical NC, United States)/University of Miami (Miami, FL, United States) FBRI SRC VB VECTOR, Protein subunit Subunit vaccine Preclinical Rospotrebnadzor, Koltsovo Baylor College of Medicine Protein subunit S1 or RBD protein Preclinical (Houston, TX, United States) iBio/CC-Pharming Protein subunit Subunit protein, plant Preclinical (Newark, DE, United States) produced Saint-Petersburg Scientific Protein subunit Recombinant protein, Preclinical Research Institute of Vaccines nanoparticles and Serums (Petersburg, (based on S-protein and Russia) other epitopes) Innovax/Xiamen Univ./GSK Protein subunit COVID-19 XWG-03 Preclinical truncated S (spike) proteins VIDO-InterVac, University Peptide Adjuvanted Preclinical of Saskatchewan microsphere peptide OncoGen (Selangor, Malaysia) Peptide Synthetic Long Peptide Preclinical Vaccine candidate for S and M proteins MIGAL Galilee Research Protein subunit Oral E. coli-based Preclinical Institute (Kiryat Shmona, protein expression Israel) system of S and N proteins LakePharma, Inc. (San Carlos, Protein subunit Nanoparticle vaccine Preclinical CA, United States) Baiya Phytopharm/Chula Protein subunit Plant-based subunit Preclinical Vaccine Research Center (RBD-Fc + adjuvant) (Thailand) Quadram Institute Biosciences Protein subunit OMV-based vaccine Preclinical (Norwich, United Kingdom) Table continued

2763 M. Galdiero, M. Galdiero, V. Folliero, C. Zannella, A. De Filippis, A. Mali, L. Rinaldi, G. Franci

Table III. (Continued). SARS-CoV-2 protein subunit candidate vaccines under development. COVID-19 vaccine Vaccine platform Type of candidate Current stage of clinical evaluation developer/manufactures vaccine (estimated study completion date) (NA - Not Available) BiOMViS Srl/University of Protein subunit OMV-based vaccine Preclinical Trento, Trento (Italy) Lomonosov Moscow State Protein subunit Structurally modified Preclinical University (Moscow, Russia) spherical particles of the tobacco mosaic virus (TMV) University of Alberta Protein subunit Spike-based Preclinical (Edmonton, Canada) AnyGo Technology Protein subunit Recombinant S1-Fc Preclinical (Shenzhen, China) fusion protein Yisheng Biopharma (Beijing, Protein subunit Recombinant protein Preclinical China) Vabiotech (Hanoi, Vietnam) Protein subunit Recombinant S protein Preclinical in IC-BEVS Applied Biotechnology Protein subunit Orally delivered, heat Preclinical Institute, Inc. (San Luis stable subunit Obispo, CA, United States) Axon Neuroscience SE Peptide Peptides derived from Preclinical (Slovakia) Spike protein MOGAM Institute for Protein subunit Protein Subunit Preclinical Biomedical Research, GC Pharma (Yongin, South Korea) Neovii (Rapperswil, Protein subunit RBD-based Preclinical Switzerland)/Tel Aviv University (Israel) Intravacc (Utrecht, Protein subunit Outer Membrane Preclinical Netherlands)/Epivax Vesicle (OMV)-subunit Intravacc/Epivax Protein subunit Outer Membrane Preclinical Vesicle (OMV)-peptide ImmunoPrecise (Victoria, Peptide Spike-based (epitope Preclinical Canada)/LiteVax BV screening) (Ophemert, Netherlands)

Modified from: DRAFT landscape of COVID-19 candidates vaccine ⇒ https://www.who.int/publications/m/item/draft-land- scape-of-covid-19-candidate-vaccines.

the product is considered fairly stable compared cines, formulation and delivery strategies are of to mRNA-based vaccines. Plasmid DNA tech- paramount importance50. nology allows simple production of large quanti- Examples of DNA-based vaccines include ties of vaccines with the possibility of conferring the one proposed by Inovio (Plymouth Meet- long-term immunity. An advantage of this kind ing, PA, United States), Genexine or Genexine. of vaccine is the stimulation of both humoral and Inovio Pharmaceuticals developed a DNA vac- cellular immunity49. However, the disadvantag- cine candidate termed INO-4800 that has en- es looming over DNA vaccines are due to their tered clinical phase 1/2 trial (NCT04336410 and limitation of processing protein immunogen, the NCT04447781), following the footsteps of the need to cross the nuclear membrane to become promising results obtained with INO-4700, a translated, and the risk of vector chromosomal MERS-CoV candidate vaccine, which was able to integration and mutations in the host genome. Ac- produce a strong antibody and T cell responses cordingly, for the optimal function of DNA vac- and was well-tolerated51. Using Inovio’s propri-

2764 SARS-CoV-2 vaccine development: where are we?

Table IV. SARS-CoV-2 nucleic acid candidate vaccines under development.

COVID-19 vaccine Vaccine Type of candidate vaccine Current stage of clinical evaluation developer/manufactures platform (estimated study completion date) (NA - Not Available) Curevac RNA mRNA Phase 2: NCT04515147 (November 2021) Inovio Pharmaceuticals/ DNA DNA plasmid vaccine with elec- Phase 1/2: NCT04447781 (February 2022) International Vaccine Institute troporation using CELLECTRA® Phase 1/2: NCT04336410 (July 2021) 2000 device Osaka University/AnGes/Takara DNA DNA plasmid vaccine + adjuvant Phase 1/2: NCT04463472 (July 2021) Bio (Japan) Cadila Healthcare Limited DNA DNA plasmid vaccine Phase 1/2: CTRI/2020/07/026352 (NA) (Ahmedabad, India) Genexine Consortium DNA DNA Vaccine (GX-19) Phase 1/2: NCT04445389 (June 2022) Arcturus/Duke-NUS RNA saRNA Phase 1/2: NCT04480957 (January 2021) (Singapore, Malaysia) Symvivo DNA bacTRL-Spike Phase 1: NCT04334980 Imperial College London RNA LNP-nCoVsaRNA Phase 1: ISRCTN17072692 (Nd) (London, United Kingdom) People’s Liberation Army (PLA) RNA mRNA Phase 1: ChiCTR2000034112 (Nd) Academy of Military Sciences (Beijing, China)/Walvax Biotech. (China) Globe Biotech Limited DNA DNA plasmid vaccine Preclinical (Bangladesh) National institute of Chemistry DNA Plasmid DNA, nanostructured Preclinical (Slovenia) RBD DIOSynVax Ltd/University of DNA DNA, engineered vaccine inserts Preclinical Cambridge (Cambridge, United compatible with multiple Kingdom) delivery systems Ege University (İzmir, Turkey) DNA DNA vaccine Preclinical Scancell/University of DNA DNA plasmid vaccine RBD&N Preclinical Nottingham/Nottingham Trent University (Nottingham, United Kingdom) National Research Centre DNA DNA plasmid vaccine S, S1, S2, Preclinical (Egypt) RBD & N Karolinska Institute (Sweden)/ DNA DNA with electroporation Preclinical Cobra Biologics (OPENCORONA Project) (Newcastle, United Kingdom) Chula Vaccine Research Center DNA DNA with electroporation Preclinical Takis (Rome, Italy)/Applied DNA DNA Preclinical DNA Sciences (Stony Brook, New York, United States)/ Evvivax (Rome, Italy) Immunomic Therapeutics, Inc. DNA Plasmid DNA, Needle-Free Preclinical (Rockville, MD, United States)/ Delivery EpiVax, Inc./PharmaJet, (Golden, CO, United States) BioNet Asia (Bangkok, DNA DNA vaccine Preclinical Thailandia) Entos Pharmaceuticals (Edmon- DNA DNA vaccine Preclinical ton, Canada) Table continued

2765 M. Galdiero, M. Galdiero, V. Folliero, C. Zannella, A. De Filippis, A. Mali, L. Rinaldi, G. Franci

Table IV. (Continued). SARS-CoV-2 nucleic acid candidate vaccines under development.

COVID-19 vaccine Vaccine Type of candidate vaccine Current stage of clinical evaluation developer/manufactures platform (estimated study completion date) (NA - Not Available) Globe Biotech Ltd (Dhaka, RNA D614G variant LNP-encapsulat- Preclinical Bangladesh) ed mRNA Infectious Disease Research RNA saRNA formulated in a NLC Preclinical Institute (Washington, United States)/Amyris, Inc. (Emeryville, CA, United States) Max-Planck-Institute of RNA LNP-encapsulated mRNA Preclinical Colloids and Interfaces encoding S Gennova (Maharashtra, India) RNA Self-amplifying RNA Preclinical Selcuk University (Konya, RNA mRNA Preclinical Turkey) Translate Bio (Lexington, MA, RNA LNP-mRNA Preclinical United States)/Sanofi Pasteur (Lyon, France) CanSino Biologics (China)/ RNA LNP-mRNA Preclinical Precision NanoSystems (Vancouver, Canada) Fudan University/Shanghai RNA LNP-encapsulated mRNA Preclinical JiaoTong University/RNACure encoding RBD Biopharma (Shanghai, China) Centro Nacional Biotecnología RNA Replicating Defective Preclinical (CNB-CSIC) (Spain) SARS-CoV-2 derived RNAs University of Tokyo/ RNA LNP-encapsulated mRNA Preclinical Daiichi-Sankyo (Tokyo, Japan) BIOCAD (Russia) RNA Liposome-encapsulated mRNA Preclinical RNAimmune, Inc. (MD, RNA Several mRNA candidates Preclinical United States) FBRI SRC VB VECTOR, RNA mRNA Preclinical Rospotrebnadzor, Koltsovo China CDC/Tongji University/ RNA mRNA Preclinical Stermina (China) Chula Vaccine Research RNA LNP-mRNA Preclinical Center/University of Pennsylvania, PA, United States eTheRNA (Niel, Belgium) RNA mRNA in an intranasal delivery Preclinical system Greenlight Biosciences RNA mRNA Preclinical (Medford, MA, United States) IDIBAPS-Hospital Clinic RNA mRNA Preclinical (Spain)

Modified from: DRAFT landscape of COVID-19 candidates vaccine ⇒ https://www.who.int/publications/m/item/draft-land- scape-of-covid-19-candidate-vaccines. etary in silico Gene Optimization Algorithm an studies showed proper expression of the S protein optimized DNA plasmid was designed to improve and its RNA after transfection with the plasmid expression and immunogenicity of the DNA vac- constructs of cell lines, and in in vivo animal cine. This DNA vaccine is administered intrader- models, humoral and T cell responses were ob- mally by CELLECTRA® electroporation. in vitro served52, so initial results were highly suggestive

2766 SARS-CoV-2 vaccine development: where are we? of the immunogenicity of Inovio’s COVID-19 vac- are most commonly based on alphavirus-derived cine candidate. Symvivo Corporation (Burnaby, RNA replicons modified to encode the antigen of Canada) is conducting a phase 1 clinical trial to choice instead of viral structural proteins. The analyze the safety and immunogenicity profiles of replicon maintains the ability to encodes alpha- their bacTRL-spike vaccine against SARS-CoV-2 virus non-structural proteins (nsP1-4) and a sub (NCT04334980). This vaccine is constituted by genomic promoter; therefore, an RNA-dependent Bifidobacterium longum engineered to deliver RNA polymerase (RdRP) is produced and used synthetic DNA encoding the spike protein from to transcribes more copies of the vaccine in the SARS-CoV-2 contained in a plasmid vector. The transfected cell. Consequently, saRNA vaccines vaccine is orally administered, and the gut colo- express protein at higher levels and persist lon- nization by B. longum should provide continuous ger than non-replicating RNA. mRNA vaccine delivery and expression of SARS-CoV-2S protein is a promising alternative to traditional vaccine encoding plasmids (Symvivo, Covid-19 Program approaches due to their safety, potency, quick Vision, 2019). A mucosal, systemic humoral, and vaccine-development time, and low-cost produc- cell-mediated immune response is foreseen as a tion55. The main advantages of mRNA are the result of the translation of this plasmid within the ability to generate a rapid antigen expression in gastrointestinal lymphoid tissues. the cell cytoplasm without the burden of the ne- Biotech firm Genexine Inc. (Seongnam, South cessity of crossing the nuclear membrane to be- Korea) has launched a human clinical trial of their come active. Moreover, the risk of infection or COVID-19 vaccine constituted by a synthetic solu- insertional mutagenesis is near zero since mRNA ble spike DNA-based candidate named GX-19. The expression is rapid and transient as mRNA are ectodomain of the S gene has been codon optimized safely degraded by normal cellular processes. for increased antigen expression in mammalian This involves concerns related to mRNA vaccine cells and subcloned into the plasmid pGX27 vec- instability and inefficient in vivo delivery49. tor. Preliminary studies have shown that electro- As soon as March 2020, a first candidate vac- poration-enhanced GX-19 induced robust antibody cine (mRNA-1273) was announced by the NIAID and T cell responses. Furthermore, vaccination of (Bethesda, MD, United States) and Moderna GX-19 was shown to confer effective protection (Cambridge, MA, United States) to be evaluated against SARS-CoV-2 challenge at 10 weeks fol- in a clinical trial (NCT04283461) on the basis of lowing the last vaccination in immunized non-hu- the prior preclinical and clinical data gathered man primates supporting further expectations for with studies developed by Moderna on CMV, GX-19 as a vaccine candidate against SARS- CoV- Zika virus, H7N9, hMPV, and RSV56. The vac- 2 in ongoing human clinical trials53. cine includes synthetic mRNA coding for the full- Messenger RNA (mRNA) is an intermediate length, pre-fusion stabilized spike protein (S) of carrier of genetic information acting as a tem- SARS-CoV-2 that has been encapsulated in lipid plate for protein synthesis on cellular translating nanoparticle (LNP) composed of ionizable lipid, ribosomes in the vaccinated organism. mRNA distearoyl phosphatidylcholine, cholesterol, and vaccines are able to induce strong cellular and hu- polyethylene glycol lipid. mRNA-1273 has been moral immune responses. These vaccines are rel- shown to induce potent neutralizing antibody re- atively safe and effective because of their activity sponses and to both wild-type (D614) and D614G as transient carriers of messages that are unable to mutant SARS-CoV-257. The delivered mRNA is interact with the host genome. Moreover, mRNA also able to protect against live virus challenge vaccines do not require the use of the whole vi- in mice without evidence of immunopathology58. rus54. There are two types of mRNA vaccines Also studies on non-human primates, receiving platform: non-replicating mRNA and self-ampli- 10 or 100 μg of mRNA-1273, showed satisfactory fying mRNA (saRNA). Non-replicating mRNA antibody and T-cell responses demonstrating the vaccines contain the sequence of the selected induction of a robust SARS-CoV-2 neutralizing target antigen flanked by 5′ and 3′ untranslated activity, rapid protection in the upper and lower regions (UTRs). They typically have a 5’ cap es- airways without pathologic effect in the lung59. In sential for mRNA to associate with the eukaryotic clinical trials, the results presented so far describe translation complex; therefore, mRNA triggers that using 100-μg dose is possible to induce high- rapid and immediate antigen expression in the er binding-antibody and neutralizing-antibody ti- cytoplasm without the need for crossing the nu- ters than the 25-μg dose, which lent credibility to clear membrane. Self-amplifying RNA vaccines the strategy to use the 100-μg dose in the ongoing

2767 M. Galdiero, M. Galdiero, V. Folliero, C. Zannella, A. De Filippis, A. Mali, L. Rinaldi, G. Franci phase 3 vaccine trial (NCT04470427). Moreover, the National University of Singapore, proposes a no trial-limiting safety concerns were identified self-replicating RNA (saRNA) construct encod- so far, with adverse events associated with the ing an alphavirus-based replicon and the SARS- mRNA-1273 vaccine being mainly mild or mod- CoV-2 full length spike glycoprotein. Translation erate60. of the replicon generates a replicase complex able Pfizer (New York, NY, United States), in col- to amplify and extend the expression of the anti- laboration with BioNTech (Mainz, Germany), genic protein. The saRNA is encapsulated with a is developing two vaccines named BNT162b1 lipid-enabled and unlocked nucleic acid modified and BNT162b2. Like mRNA-1273 from Moder- RNA (LUNAR), a safe ionizable and biodegrad- na, BNT162b is a lipid nanoparticle encapsulat- able LNP platform for effective LNP mRNA de- ing mRNA encoding for SARS-CoV-2 antigens. livery67. Mice vaccination showed vigorous neu- One of these candidates, BNT162b1, encodes the tralizing antibody responses, and cell-mediated SARS-CoV-2 RBD trimerized by the addition of immunity produced a strong viral antigen specif- a T4 fibritin domain intended to improve immu- ic CD8+ T lymphocyte response. Following wild- nogenic effect in view of the multivalent display, type SARS-CoV-2 challenge, a single vaccine in- while the other, BNT162b2, has full- length spike oculation of 2 μg or 10 μg doses fully protected envelope protein, but modified by the insertion of human ACE2 transgenic mice68. two proline to block the glycoprotein in its prefu- A similar approach has also been pursued by sion conformation to allow a stronger resemblance Imperial College London, which used a plasmid to the intact virus particles61,62. BNT162b1, at vector to synthesize a self-amplifying RNA (saR- multiple dose levels, has been assessed in healthy NA) replicon based on alphavirus genome where adults from 18 to 55 years of age, indicating that the viral structural proteins have been replaced dose levels of BNT162b1 that elicited an accept- by the surface glycoprotein S of SARS-CoV-2 se- able level of reactogenicity also efficiently elicit- quence modified by proline mutations to stabilize ed antibody titers similar to SARS-CoV-2 human the protein in its pre-fusion state69. The saRNA convalescent patients. These antibodies were also LNP vaccine elicited robust antibody and cellular found to be broadly neutralizing using several responses, and human trials of the vaccine started SARS-CoV-2 pseudoviruses based on circulat- in June 2020 to assess the safety of the vaccine ing strains. A CD4+ and CD8+ responses were and its effects on the immune system. also shown63,64. Both vaccine candidates have also been tested in a further clinical trial to assess the Viral vectors safety and immunogenicity of three dose levels of Viral vector-based vaccines are live viruses (the BNT162b1 and BNT162b2 and showed good im- vector itself) engineered to express heterologous munogenicity65. BNT162b1 was associated with a antigens by carrying a foreign DNA sequence. higher incidence of systemic reactions compared Their main property is the combination of the ro- to BNT162b2, especially in older adults, support- bust immunogenicity of live attenuated vaccines ing the selection of BNT162b2 for the ongoing and the safety of subunit vaccines since only one phase 3 clinical trial (NCT04368728). or few antigenic proteins are present. Their simi- CureVac AG (Tübingen, Germany) has de- larity to live attenuated strains favor a proper im- veloped a vaccine candidate based on the spike munological response by eliciting both cell-medi- glycoprotein of SARS-CoV-2, which includes ated and humoral immunity in vivo. Several viral a 5′ cap structure, a GC-enriched open reading vectors have been exploited for vaccine devel- frame (ORF), 3′ UTR, polyA tail and does not opment, and the most common include vaccinia use chemically modified nucleosides. The lipid virus, modified vaccinia virus Ankara (MVA), nanoparticles are made of ionizable amino lipid, adenovirus (Ad), adeno-associated virus (AAV), phospholipid, cholesterol, and a PEGylated lip- retrovirus/lentivirus, alphavirus, herpes virus, id66. This vaccine has been evaluated in various Newcastle disease virus, poxvirus, and many oth- animal models, and the obtained data indicated ers. These recombinant viral-vectored vaccines that the induced neutralizing antibody titers were can be built on either a replication-deficient vi- comparable to sera from patients who recovered ral backbone or an attenuated replication-compe- from COVID-19. The vaccine was generally well tent viral backbone where the latter is engineered tolerated across the tested dose range of 2-12 µg. through recombinant DNA technology to present Arcturus (San Diego, CA, United States), antigens derived from the target pathogen within a with the collaboration of Duke University and different replicating virus70. Replication-deficient

2768 SARS-CoV-2 vaccine development: where are we? viral vectors generally lack early genes essential sponses within 14 days and specific humoral re- for the reproduction of the virus; therefore, they sponses peaked at day 28 post-vaccination73. The are only able to deliver the antigen gene within phase 1 trial has been followed by the phase 2 host cells as Trojan horses without replicating in trial, where the candidate vaccine has confirmed the vaccinated individual60. Usual viral vectors a good safety profile, with only mild, transient employed in vaccine developments are adenovi- adverse events related to vaccination and no se- ruses (Ad) and modified vaccinia virus Ankara rious adverse events. Moreover, the administra- (MVA). They are very common (at least 50 hu- tion of 5 × 1010 viral particles is safe and induced man subtypes available) and account for a big part significant immune responses in the majority of of the methodology, with Ad serotype 5 (Ad5) recipients after a single immunization, to war- showing to be a stable, non-replicating virus used rant the move into international multicenter, ran- in different vaccine platforms. Some difficulties domized, double-blind, controlled phase 3 effec- arise from the preexisting immunity against hu- tiveness trials, namely NCT04540419 expected man Ad5 being quite widespread in the popula- to end in July 2021 and NCT04526990 expect- tion, therefore interfering with its broad use for ed to end in January 2022, to further evaluate novel vaccine development71. Some have opted the efficacy of the vaccine. Janssen Vaccines for chimpanzee adenovirus (ChAdOx1) to find an (Beerse, Belgium) has opted for the use of a dif- alternative to the human Ad vector due to its lack ferent adenovirus type, namely Ad26, to use as of preexisting immunity in humans, and results a basis for its candidate vaccine, based on the seem to be very promising60,72. Several vectors long experience in the field against other viral have been developed on replication-competent at- diseases. A replication-incompetent Ad26 vector tenuated strains such as measles derived from the with deletions in E1/E3 structural proteins was original 1954 vaccine strain, the live-attenuated engineered using the AdVac system74, using a yellow fever 17D (YF17D) vaccine or Newcastle single plasmid method with the Ad26 vector ge- disease virus (NDV)60. nome including a transgene expression cassette. Currently, adenoviruses are the most common- Within the expression cassette, the codon-op- ly used vectors for building replication-deficient timized SARS-COV-2 Spike gene has been in- viral vectors for vaccination purposes. Recom- serted in the E1 position under the control of the binant Ad vectors are widely used because of HCMV promoter and the SV-40 polyadenylation their high transduction efficiency, high level of sequence. The candidate vaccine Ad26.COV2.S transgene expression, and a broad range of viral elicited potent neutralizing humoral immunity tropism. Four different companies have already and cellular immunity in mice74 and has proved reached phase 3 clinical trials; their vectors are to provide robust protection against severe clin- based on Ad5 and Ad26 in three cases (CanSino ical disease after high-dose SARS-CoV-2 infec- Biological Inc., Gamaleya Research Institute and tion in hamsters75. The efficiency of the Ad26. Janssen Pharmaceuticals Companies) while the COV2.S vaccine in eliciting protective immunity remaining proposed vaccine from the partnership against SARS-CoV-2 infection was successfully between the Jenner Institute of Oxford Univer- demonstrated in a non-human primate challenge sity (Oxford, United Kingdom) and AstraZene- model where vaccinated animals developed hu- ca (Cambridge, United Kingdom) is based on a moral and cellular immune responses. After chimpanzee adenovirus ChAdOx1 (Table I). vaccine inoculation, animals were challenged Ad5-nCoV from CanSino Biologics (China) is with SARS-CoV-2, and a net decrease of me- a recombinant, replication-defective adenovirus dian viral loads in bronchoalveolar lavage and type-5 vector (Ad5) expressing the recombinant nasal mucosa was observed. Vaccine-elicited spike protein of SARS-CoV-2. The sequences of neutralizing antibody titers are well correlated the whole gene coding for the S protein with the with the detected protective efficacy confirming plasminogen activator signal peptide gene have protection against SARS-CoV-2 in non-human been codon-optimized and cloned into the Ad5 primates76,77. The preclinical data supported the virus vector with deleted E1 and E2 genes. In Phase 1/2 randomized, double-blinded, place- an open-label, non-randomized, phase 1 clinical bo-controlled clinical study to assess the safe- trial, the Ad5 vectored vaccine against SARS- ty, reactogenicity and immunogenicity of Ad26. CoV-2 has shown to be well tolerated and immu- COV2.S. The study indicated that a single dose nogenic in healthy adults. A single vaccine dose of Ad26.COV2.S (5x1010 viral particles or 1x1011 induced rapid, specific T-cell and humoral re- viral particles, is safe, well-tolerated, and high-

2769 M. Galdiero, M. Galdiero, V. Folliero, C. Zannella, A. De Filippis, A. Mali, L. Rinaldi, G. Franci ly immunogenic78. Phase 3 clinical trial studies mized S glycoprotein which has been synthesized (NCT04505722 and NCT04614948) are ongoing. with the tissue plasminogen activator (tPA) leader One other non-replicating vectored vaccine sequence at its 5’ end. The vector genome was that has abundantly debated on mass media is constructed by the methodology of bacterial arti- from the Gamaleya Research Institute (Moscow, ficial chromosomes by inserting the SARS-CoV-2 Russia), which was named Sputnik V. This vac- S gene into the E1 locus of chimpanzee adeno- cine combines two distinct adenovirus vectors, virus (ChAdOx1) genome. Humans have low se- namely Ad26 and Ad5, both carrying the gene roprevalence for ChAd, therefore low likelihood for the full-length SARS-CoV-2 spike glycopro- to promote any immunogenicity to the vector as tein (rAd26-S and rAd5-S). Thus, the use of a a consequence of vaccination80. The development heterologous prime-boost immunization, when of ChAdOx1-based vaccine, named AZD1222, rAd26-S is injected in the priming phase, and is based on the encouraging results from human rAd5-S is after used for boosting, is analyzed as studies with ChAdOx1-MERS vaccine81,82. In a different approach to elicit a robust immune re- preclinical studies, the AZD1222 vaccine was sponse to SARS-CoV-2 and to reduce the immune shown to be immunogenic in mice and pigs. The response that is possibly mounted against the experimental plan conceived one or two doses of components of the viral vector. Preclinical anal- AZD1222 in both animal models and the results ysis of the vaccine (though unpublished) claimed showed a good immunization already with the evident humoral and cellular immune responses single dose, but a booster immunization clearly in non-human primates, which were protected enhanced antibody responses, especially in pigs, from SARS-CoV-2 infection. Immunosuppressed with a mighty increment in viral neutralization83. hamsters inoculated with the vaccine were pro- In rhesus macaques, vaccination with ChAdOx1 tected with a success of 100% in a lethal model vectored SARS-CoV-2 vaccine induced a bal- of SARS-CoV-2 challenge and antibody-depen- anced humoral and cellular immune response of dent enhancement of infection was not reported. type-1 and type-2 T helper cells and managed to Safety and immunogenicity of two formulations constitutively reduce viral loads in the broncho- (frozen and lyophilized) of this vaccine have been alveolar lavage fluids and lower respiratory tract analyzed in NCT04436471, NCT04437875, and tissues. No signs of pneumonia could be detected NCT04587219 phase 1 and 2 clinical trials in after live virus challenge as well as no evidence healthy adult volunteers. Trial participants were of immune-enhanced disease was reported84. Fi- injected with a prime-boost vaccination consist- nally, all data actually available from human clin- ing of a single dose of intramuscular rAd26-S ical trials have shown an acceptable safety pro- on day 0 and a subsequent dose of intramuscu- file and homologous boosting increased antibody lar rAd5-S on day 21. The vaccine produced im- responses, that together with the results showing mune responses that showed to be adequate and a proper induction of humoral and cellular im- comprising both the humoral and cellular arms in mune responses warrant the expectation of the healthy adults, besides being well tolerated. An- ongoing phase 3 clinical trials (NCT04540393, tibodies against the SARS-CoV-2 spike and neu- NCT04516746, and CTRI/2020/08/027170)85. tralizing antibodies considerably increased at day Also, ReiThera Srl (Rome, Italy), a biotech com- 14 and kept increasing throughout the observation pany, in cooperation with the Lazzaro Spallanza- period. Specific T-cell responses peaked on day ni National Institute for Infectious Diseases (Isti- 28 after vaccination. The reported adverse events tuto Nazionale per le Malattie Infettive – INMI) were considered mild (pain at the injection site, (Rome, Italy), is running a phase 1 clinical study hyperthermia, headache, asthenia, and muscle on a proprietary replication-defective simian (go- and joint pain) and also typical for vaccines based rilla) adenoviral vector (called GRAd) encoding on recombinant viral vectors79. the full-length coronavirus spike protein (GRAd- Considering that the use of Ad5 as a vector has COV2). GRAd vector belongs to C adenovirus the drawback of the presence of a preexisting im- species that have been indicated as potent vaccine munity from natural exposure (adenoviruses are carriers; like other simian adenoviruses, GRAd frequent causes of common colds) to Ad5 can has low seroprevalence in humans. Therefore, depress cellular immune responses to any heter- GRAd vaccine immunogenicity is not likely to ologous antigen introduced in the vector, Oxford be reduced by preexisting anti-human adenovirus University, and AstraZeneca have developed a re- antibodies. Preliminary results from the trial have combinant adenovirus vaccine using codon opti- shown that the candidate vaccine is well tolerat-

2770 SARS-CoV-2 vaccine development: where are we? ed and induces an evident immune response in (VSV), which is under investigation by the Israel healthy subjects from 18 to 55 years of age. Institute for Biological Research with its phase Within non-replicating viral vectors, of inter- 1/2 clinical trial NCT04608305 expected to end est seems to be the one from the Icahn School in June 2022, and by the IAVI and Merck asso- of Medicine at Mount Sinai (New York, United ciation which is exploiting the experience gained States) where a Newcastle disease virus (NDV) for the production of the rVSV-based vaccine for vector expressing the SARS-CoV-2 S protein has Ebola Zaire and conducting a phase 1 clinical trial been constructed. NDV is an avian pathogen be- (NCT04569786) expected to be close to the end longing to the family of Paramyxoviridae, gen- (December 2021) (Table V). erally not infectious for humans, which lack pre- existing immunity toward this virus. The strain Virus-like particles and nanoparticles selected as a starting point for the vaccine con- Virus-like particles (VLP) vaccines are based struction is the LaSota (LS) strain that, besides on the consideration that heterologous expres- being avirulent in birds, has already been utilized sion of specific viral proteins is able to exploit for applications for delivery of oncolytic agents the self-assembly properties of viral proteins to and vaccines. NDV vectors were built up to ex- direct the spontaneous symmetric aggregation of press two forms of the SARS-CoV-2 glycopro- particles with a strong structural similarity to the tein, one with the full-length wild type protein original viruses. Notably, VLPs are not infectious (NDV_LS_S) and the other expressing a chimera because they are empty shells lacking the viral composed of the ectodomain of S (deleted of the genome but can mimic the morphology of the polybasic cleavage site at the interface between whole virus. However, the native conformation of S1 and S2) attached on the cytoplasmic and trans- the antigenic proteins is well preserved, and the membrane domain of the NDV fusion protein F molecular weight is well above the monomeric (NDV_LS_S-F). Both constructs were properly counterpart, which improves VLPs immunoge- displayed on the surface of NDV and injected in nicity compared to free proteins. VLPs are gen- mice stimulated the production of high titers of erally produced by encoding the viral structural binding and neutralizing antibodies, besides ful- proteins and expressing them in heterologous sys- ly protecting mice from challenge with a SARS- tems, such as recombinant vaccinia virus, mam- CoV-2 mouse-adapted strain86. malian cells (293T, CHO), baculovirus, yeast ex- In the group of replication active viral vec- pression systems and plant expression vectors88. tors, we can describe the vaccine candidate de- In practice, VLPs-based vaccines are similar to veloped by Beijing Wantai in association with whole inactivated virus vaccines, but the antigen- academia and based on the established flu-based ic proteins may be better preserved and exposed DelNS1 live attenuated influenza virus (LAIV) to the immune system since no inactivation step is platform87. Deletion of NS1, a key virulence performed. Therefore, it is less likely to affect the factor, renders safer the live attenuated influen- immunogenicity of viral proteins due to surface za virus; the addition of the surface protein of epitopes destruction. Moreover, since no live vi- SARS-CoV-2 should confer specificity and im- rus is used in any steps for the production, VLPs munogenicity against the RBD of SARS-CoV-2. are conveniently accomplished in low-contain- This candidate is now being analyzed in phase 2 ment manufacture settings89. VLP-based vaccines clinical trials (ChiCTR2000039715) as an intra- have been proved to produce strong cellular and nasal flu-based-RBD SARS-CoV-2 vaccine. The humoral immune responses90. The best-known Pasteur Institute, Themis Bioscience GmbH, and examples of VLP-based vaccines are Cervarix® the University of Pittsburgh Center for Vaccine (human papillomavirus) and Engerix® (hepatitis Research (Pittsburgh, PA, United States) are de- B virus) by GlaxoSmithKline (Brentford, United veloping a measles virus vectored vaccine able to Kingdom), but also the corresponding vaccines expresses the SARS-CoV-2 S protein, and simi- produced by Merck and Co., Inc. (Kenilworth, larly are doing other companies like Zydus Cadi- NJ, United Kingdom) against hepatitis B virus la, which is developing a live attenuated recombi- (Recombivax HB®) and human papillomavirus nant measles virus vectored vaccine produced by (Gardasil®)91. SpyBiotech (Oxford, United King- reverse genetics and expressing codon-optimized dom) and the Serum Institute of India (Pune, In- proteins. A further vector extensively used as a dia) are conducting a phase ½ clinical trial (AC- basis for producing replicating-competent vec- TRN12620000817943) with the investigational tored vaccines is the vesicular stomatitis virus vaccine based on their proprietary technology

2771 M. Galdiero, M. Galdiero, V. Folliero, C. Zannella, A. De Filippis, A. Mali, L. Rinaldi, G. Franci

Table V. SARS-CoV-2 viral-vectored candidate vaccines under development. COVID-19 vaccine Vaccine Type of candidate vaccine Current stage of clinical developer/manufactures platform evaluation (estimated study completion date) (NA - Not Available) ImmunityBio, Inc. & Non-Replicating hAd5 S+N 2nd Generation Phase 1: NCT04591717 (November NantKwest Inc. (CA, Viral Vector Human Adenovirus Type 5 2021) United States) Vector (hAd5) Spike (S) + Nucleocapsid (N) ReiThera (Rome, Italy)/LEU- Non-Replicating Replication defective Simian Phase 1: NCT04528641 (July 2021) KOCARE (Planegg, Germany)/ Viral Vector Adenovirus (GRAd) encoding S Univercells (Charleroi, Belgium) CanSino Biological Non-Replicating Ad5-nCoV Phase 1: NCT04552366 (June 2021) Inc/Institute of Biotechnology, Viral Vector Academy of Military Medical Sciences, PLA of China Vaxart (San Francisco, CA, Non-Replicating Recombinant Adenovirus Type 5 Phase 1: NCT04563702 (October United States Viral Vector adjuvanted Oral vaccine 2021) a. rAd-S b. rAd-S-N c. rAd-S1-N Ludwig-Maximilians, Non-Replicating Adenovirus-based NasoVAX Phase 1: NCT04569383 (May 2021) University of Munich Viral Vector expressing SARS2-CoV S protein University of Helsinki & Non-Replicating Ad 5 vector for intranasal Preclinical University of Eastern Finland Viral Vector administration Globe Biotech Limited Non-Replicating Adenovirus Type 5 Vector Preclinical (Bangladesh) Viral Vector ID Pharma (Tsukuba, Japan) Non-Replicating Sendai virus vector Preclinical Viral Vector Massachusetts Eye and Ear/ Non-Replicating Adeno-associated virus vector Preclinical assachusetts General Hospital/ Viral Vector (AAVCOVID) AveXis (Bannockburn, United Kingdom) Ankara University Non-Replicating Adenovirus-based Preclinical (Ankara, Turkey) Viral Vector GeoVax (Atlanta, GA, United Non-Replicating Recombinant Modified Vaccinia Preclinical States)/BravoVax (Wuhan, Viral Vector Virus Ankara - encoded VLP China) DZIF – German Center for Non-Replicating Recombinant Modified Vaccin- Preclinical Infection Research/IDT Viral Vector ia Virus Ankara – SARS-CoV-2 Biologika GmbH (Germany) Spike encoded IDIBAPS-Hospital Clinic Non-Replicating Recombinant Modified Preclinical (Spain) Viral Vector Vaccinia Virus Ankara – SARS-CoV-2 Spike protein Altimmune (Gaithersburg, MD, Non-Replicating Adenovirus-based NasoVAX Preclinical United States) Viral Vector expressing SARS2-CoV spike protein Greffex (Aurora, CO, United Non-Replicating Ad5 S (GREVAXTM platform) Preclinical States) Viral Vector Stabilitech Biopharma Ltd Non-Replicating Oral Ad5 S Preclinical (United Kingdom) Viral Vector Valo Therapeutics Ltd (Oxford, Non-Replicating Adenovirus-based + HLA- Preclinical United Kingdom) Viral Vector matched peptides

Table continued

2772 SARS-CoV-2 vaccine development: where are we?

Table V. (Continued). SARS-CoV-2 viral-vectored candidate vaccines under development. COVID-19 vaccine Vaccine Type of candidate vaccine Current stage of clinical developer/manufactures platform evaluation (estimated study completion date) (NA - Not Available) Centro Nacional Biotecnologia Non-Replicating MVA expressing structural Preclinical (CNB-CSIC) (Spain) Viral Vector proteins Erciyes University (Kayseri, Non-Replicating Adeno5-based Preclinical Turkey) Viral Vector National Research Centre Non-Replicating Influenza A H1N1 vector Preclinical (Egypt) Viral Vector Icahn School of Medicine Non-Replicating Newcastle disease virus Preclinical at Mount Sinai (New York, Viral Vector expressing S United States) Beijing Wantai Biological Phar- Replicating Viral Intranasal flu-based-RBD Phase 2: ChiCTR2000039715 (NA) macy/Xiamen University Vector (China) Israel Institute for Biological Replicating Viral VSV-S Phase 1/2: NCT04608305 Research (Israel) Vector (June 2022) Merck Sharp & Dohme Replicating Viral Replication-competent VSV Phase 1: NCT04569786 (Kenilworth, NJ, United States)/ Vector delivering the SARS-CoV-2 (December 2021) IAVI (New York, United States) Spike Institute Pasteur (Paris, France)/ Replicating Viral Measles-vector based Phase 1: NCT04497298 Themis/University of Pittsburg Vector (October 2021) CVR/Merck Sharp & Dohme (United States) Farmacologicos Veterinarios Replicating Viral Intranasal Newcastle disease vi- Preclinical SAC (FARVET SAC)/ Vector rus vector (rNDV-FARVET) ex- Universidad Peruana pressing RBD SARS-CoV2 Preclinical Cayetano Heredia (UPCH) KU Leuven (Leuven, Belgium) Replicating Viral YF17D Vector Preclinical Vector Cadila Healthcare Limited Replicating Viral Measles Vector Preclinical (Ahmedabad, India) Vector FBRI SRC VB VECTOR, Replicating Viral Measles Vector Preclinical Rospotrebnadzor, Koltsovo Vector DZIF – German Center for Replicating Viral Measles Virus (S, N targets) Preclinical Infection Research/CanVirex AG Vector Tonix Pharma (Chatham, NJ, Replicating Viral Horsepox vector expressing Preclinical United States)/Southern Vector S protein Research (Birmingham, AL, United States) BiOCAD (Moscow, Russia) and Replicating Viral Live viral vectored vaccine Preclinical IEM (Morrisville, NC, United Vector based on attenuated influenza States) virus backbone (intranasal) FBRI SRC VB VECTOR, Replicating Viral Recombinant vaccine based on Preclinical Rospotrebnadzor, Koltsovo Vector Influenza A virus, for the prevention of COVID-19 (intranasal) Fundaçao Oswaldo Cruz and Replicating Viral Attenuated Influenza expressing Preclinical SARS-CoV2 Preclinical Vector an antigenic portion of the Spike Instituto Buntantan (Brazil) protein University of Hong Kong Replicating Viral Influenza vector expressing Preclinical (China) Vector RBD

2773 M. Galdiero, M. Galdiero, V. Folliero, C. Zannella, A. De Filippis, A. Mali, L. Rinaldi, G. Franci

Table V. (Continued). SARS-CoV-2 viral-vectored candidate vaccines under development.

COVID-19 vaccine Vaccine Type of candidate vaccine Current stage of clinical developer/manufactures platform evaluation (estimated study completion date) (NA - Not Available) University of Manitoba Replicating Viral Replicating VSV vector-based Preclinical (Winnipeg, Canada) Vector DC-targeting FBRI SRC VB VECTOR, Replicating Viral VSV vector Preclinical Rospotrebnadzor, Koltsovo Vector University of Western Ontario Replicating Viral VSV-S Preclinical (London, Canada) Vector Aurobindo (Hyderabad, India) Replicating Viral VSV-S Preclinical Vector UW–Madison (Madison, WI, Replicating Viral M2-deficient single replication Preclinical United States)/FluGen (WI, Vector (M2SR) influenza vector United States)/Bharat Biotech (Hyderabad, India) Intravacc/Wageningen Replicating Viral Newcastle disease virus vector Preclinical Bioveterinary Research Vector (NDV-SARS- CoV-2/Spike) (Lelystad, Netherlands)/Utrecht University (Netherlands) The Lancaster University Replicating Viral Avian paramyxovirus vector Preclinical (United Kingdom) Vector (APMV)

Modified from: DRAFT landscape of COVID-19 candidates vaccine ⇒ https://www.who.int/publications/m/item/draft-land- scape-of-covid-19-candidate-vaccines

named SpyCatcher/SpyTag platform. This tech- cine in history is the one produced against small- nology directs antigens to be displayed onto VLPs pox and used this approach based on the concept with a covalent, irreversible bond in a high den- of a virus capable of infecting a species other than sity, stable and specific orientated presentation of its original one but which retained a certain level epitope92. The VLP vaccine candidate is based on of serological correlation. Therefore, the bovine the hepatitis B surface antigen (HBsAg) fused virus inoculated in humans was capable of pro- to the SpyCatcher protein to which the RBD of ducing only a reduced local replication but not a SARS-CoV-2 can be easily displayed on the VLP. systemic disease (attenuation of virulence). The Several companies are developing VLP based conservation of different immunological epitopes coronavirus vaccines, such as ARTES Biotech- allowed for an efficient immune response93. His- nology (Langenfeld, Germany) using the propri- torically, many other successful human vaccines etary platform METAVAX® for the development have been based on empirically attenuated strains of vaccines built on enveloped virus-like particle of the actual pathogen, with deletion or mutation nanostructures (eVLPs) based on the duck Hepa- of virulence genes through a serial passage into titis B small surface antigen presenting domains animal models or tissue cultures. At each “pas- of the spike protein of SARS-CoV 2, or the Imo- sage”, the selected viruses improve in infecting phoron Ltd applying their fine technology named and replicating in the selected cell cultures but ADDomer (VRAGNIAU C, 2019) where on a sin- more and more their ability to enter and replicate gle VLP particle hundreds of epitopes (the parts in their original human host is lost. Attenuation mediating SARS-CoV-2 entry into cells) can be can also be reached by growing microorganisms accommodated (Table VI). in suboptimal conditions (i.e., low temperature passages) allowing the selection of less virulent Other strains94. This method selects viruses that repli- The use of a live virus to prevent infection is cate well in a colder environment but less well at one of the most widely used methods and ancient body temperature, thus decreasing their pathoge- vaccination approach. Absolutely, the first vac- nicity in the human host, resulting in an attenua-

2774 SARS-CoV-2 vaccine development: where are we?

Table VI. SARS-CoV-2 virus-like particles candidate vaccines under development.

COVID-19 vaccine Vaccine Type of candidate vaccine Current stage of clinical developer/manufactures platform evaluation (estimated study completion date) (NA - Not Available) SpyBiotech (Oxford, United VLP RBD-HBsAg VLPs Phase 1/2: ACTRN12620000817943 Kingdom)/Serum Institute of India (Pune, India) Medicago Inc. (Quebec City, VLP Plant-derived VLP adjuvanted Phase: NCT04450004 (December 2021) Canada) with GSK or Dynavax adjs. Shiraz University (Shiraz, Iran) VLP Plant derived VLP VLP Preclinical

Tampere University (Tampere, VLP VLPs produced in BEVS Preclinical Finland) Max Planck Institute for VLP VLP Preclinical. Dynamics of Complex Technical Systems (Magdeburg, Germany) University of Manitoba VLP Virus-Like particle-based Preclinical (Winnipeg, Canada) Dendritic cell (DC)-targeting vaccine Bezmialem Vakif University VLP VLP Preclinical (Instanbul, Turkey) Middle East Technical University VLP VLP Preclinical (Ankara, Turkey) VBI Vaccines Inc. (MA, United VLP Envelope Virus-like particles Preclinical States) (eVLP) IrsiCaixa AIDS Research/ VLP S protein integrated in HIV Preclinical IRTA-CReSA/Barcelona VLPs Supercomputing Centre/Grifols (Barcelona, Spain) Mahidol University/The GPO/ VLP VLP + adjuvant Preclinical Siriraj Hospital (Thailand) Navarrabiomed, Oncoimmunology VLP Virus-like particles, lentivirus Preclinical group (Pamplona, Spain) and baculovirus vehicles Saiba GmbH (Pfäffikon, VLP Virus-like particle, based Preclinical Switzeland) on RBD Imophoron Ltd and Bristol VLP ADDomerTM multiepitope Preclinical University’s Max Planck (United dysplay Kingdom) Doherty Institute (Melbourne, VLP Unknown Preclinical Australia) OSIVAX (Paris, France) VLP VLP Preclinical ARTES Biotechnology VLP eVLP Preclinical (Langenfeld, Germany) University of Sao Paulo (Brazil) VLPs peptides/whole virus Preclinical

Modified from: DRAFT landscape of COVID-19 candidates vaccine ⇒ https://www.who.int/publications/m/item/draft-land- scape-of-covid-19-candidate.

2775 M. Galdiero, M. Galdiero, V. Folliero, C. Zannella, A. De Filippis, A. Mali, L. Rinaldi, G. Franci tion of virulence while maintaining the ability to cally stable by showing its inability to genetically induce the immune response95. Examples of such return back to its pathogenic status103. This is re- vaccines that have revolutionized human history markably arduous for coronaviruses as they are are for example: the measles vaccine, the bacillus prone to recombine in nature; therefore, any at- Calmette-Guérin (BCG) vaccine for tuberculosis tenuated vaccine strain could, at least in theory, (TB), the rabies vaccine, the yellow fever vaccine recombine with wildtype coronaviruses to give and the vaccine against poliovirus, some of which rise to a further pathogenic strain104. are still in use nowadays. Some of these vaccines New strategies by which changes to the syn- have demonstrated impressive efficacy and have onym coding are introduced to transform codon provided the backbone for vaccine development utilization have the advantage that the attenuation for other pathogens using genetic engineering of the resulting virus relies on a broad amount of techniques. A successful example is the YF-17D mutations, each of which only slightly reduces the strain of the yellow fever vaccine. It was devel- replicative capacity, but taken together, they pro- oped by Max Theiler in the 1930s by attenuating a duce an evident attenuation together with a sig- viral strain of yellow fever by more than 200 serial nificantly improved genetic stability105. passages through monkeys and cultures of mouse This new codon pair deoptimization method and chicken embryo tissues. All yellow fever vac- produces a chemically synthesized genome with cines currently in use are derived from this at- the amino acid sequence identical to the original tenuated strain23. Furthermore, YF-17D has been virus but containing a greater number of CpG employed to obtain vaccines against two other fla- and UpA RNA dinucleotides to upregulate host viviruses, namely, the Japanese encephalitis and responses by swapping optimized and non-op- dengue, by substituting the genes encoding for timized codons106. Codon pair deoptimization the major antigenic proteins of yellow fever virus (CPD) was developed as a procedure for produc- with their functional homologs of these other vi- ing a vast number of other live attenuated virus ruses96,97. Technological evolution for the produc- vaccines, including influenza A virus, porcine tion of attenuated vaccines has made it possible to reproductive and respiratory syndrome virus rationally design attenuated virus strains by mu- (PRRSV), human immunodeficiency virus type tating or eliminating virulence genes by means of 1 (HIV-1), respiratory syncytial virus, chikun- genome engineering. The deletion of non-struc- gunya virus, enterovirus A71, zika virus, Marek’s tural proteins, but also structural proteins such as disease virus, lassa fever virus, and lymphocytic protein E, has been proposed to design vaccine choriomeningitis virus107-118. strains of various zoonotic and veterinary coro- So far, there are only three attenuated SARS- naviruses98-100. The deletion of protein E contrib- CoV-2 vaccines generated by codon deoptimi- utes to the attenuation of virulence and allows the zation in preclinical development, from Mehmet formulation of efficient vaccines, but as a nega- Ali Aydinlar University (Turkey), Codagenix tive effect, there was the possible reversion of the and Serum Institute of India, and Indian Immu- attenuated phenotype101. Indeed, live attenuated nologicals Ltd and Griffith University (Brisbane, vaccines are highly immunogenic and do not re- Australia)119. Codagenix and the Serum Institute quire adjuvants to obtain an optimal response by of India are involved in the development of a live virtue of their effectiveness in inducing excellent attenuated SARS-CoV-2 vaccine, using codon immunity dictated by a close imitation of natu- deoptimization technology, following on their ral infection. However, live attenuated vaccines previous experience with vaccines against RSV have shown limitations that hold back their de- and influenza using the same technology120 (Table velopment and use today. In fact, vaccine-induced VII). symptoms that are normally much milder than those of natural infection are still a major prob- lem for immunocompromised individuals who Conclusions may be at risk for unregulated pathogenic repli- cation that can lead to severe infection or death. The entire world is in a desperate search for the Furthermore, it should always be kept in mind optimal safe and effective vaccine against SARS- that live attenuated vaccines have the potential to CoV-2. Several R&D institutions and pharmaceu- revert to a disease-causing form102. Furthermore, tical companies have started a race for the rapid the generation of an attenuated strain for vaccine production of these vaccines, with the results of use demands that the key mutation is phenotypi- more than 200 vaccine candidates being devel-

2776 SARS-CoV-2 vaccine development: where are we?

Table VII. Other SARS-CoV-2 candidate vaccines under development. COVID-19 vaccine Vaccine Type of candidate Current stage of clinical evaluation developer/manufactures platform vaccine (estimated study completion date) OSE immunotherapeutics T-cell based CD8 T cell peptide target- Preclinical (Nantes, France) ing (S, M, N) and (NSPs) SARS-CoV-2 proteins Farmacologicos Veterinarios Replicating Oral Salmonella enteritidis Preclinical SAC (FARVET SAC) (Ica, Bacteria Vector (3934Vac) based protein Perù)/Universidad Peruana exprvremession system (Lima, Perù) of RBD Mehmet Ali Aydinlar Uni- Live Attenuated Codon deoptimized live Preclinical versity/Acıbadem Labmed Virus attenuated vaccines Health Services A.S. (Istan- bul, Turkey) Codagenix (Melville, NY, Live Attenuated Codon deoptimized live Preclinical United States)/Serum Virus attenuated vaccines Institute of India (Pune, India) Indian Immunologicals Ltd Live Attenuated Codon deoptimized live Preclinical (Hyderabad, India)/Grif- Virus attenuated vaccines fith University (South East Queensland, Australia)

Modified from: DRAFT landscape of COVID-19 candidates vaccine ⇒ https://www.who.int/publications/m/item/draft-land- scape-of-covid-19-candidate-vaccines. oped and almost 50 having entered phase 1, 2, and articles)130-132, but all data collected so far indicate 3 clinical trials within less than a year from the the possibility that vaccines require both humor- emergence of the pandemic121,122. This has been al and cellular responses to provide an adequate rendered possible by the nowadays high special- level of protection and to induce a durable and ro- ized scientific knowledge and development of the bust immunological response. At present, many vaccinology field that has exploited the technol- questions are still unanswered, such as the actual ogies available and sometimes well-documented safety and efficacy of vaccines, which will not be and previously used for contrasting many other in- available until the Phase 3 clinical trial is complet- fectious diseases. Several vaccine platforms have ed and the data is properly reviewed. It is still de- been extensively explored for other infections and batable whether neutralizing antibodies are suffi- cancer and often parallel other technologies used cient and what are the critical quantity and quality in gene therapy. However, it must be borne in of protective antibodies. Clinical trials will also mind that the development of a vaccine must nec- establish other critically important data, such as: essarily follow strict safety rules and cannot be i) whether humoral and/or cellular cytotoxic re- achieved overnight. After the design and prepa- sponses are needed, ii) which helper T cell types ration of the vaccine, the phase of evaluating the are most effective (i.e., Th1 vs. Th2 vs. Th17) and efficacy and safety in preclinical studies is neces- iii) which kind of antibody response (i.e., IgG vs. sary for the definition of quality standards before IgA) is most effective to protect against this virus. entering clinical studies. In fact, while usual ther- A key point actually addressed by many studies is apeutic drugs are administered to patients already a better definition of the amount of antigen dose suffering from specific pathologies, vaccines, by to be administered, the number of doses needed, definition, are given in advance to healthy sub- the duration of immunity, and the need for boost- jects and therefore require very accurate safety ers that depend on the technology used to produce levels. The different platforms and strategies for each vaccine preparation. The need to develop an vaccine development described in this paper and effective COVID-19 vaccine is so demanding that in the published literature123-129 have specific im- it is likely that unless one vaccine is much more munological advantages and disadvantages (sum- effective than any other, more candidate vac- marized in Figure 1 and in several detailed review cines will gain approval in different geographic

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