key: cord-1021670-7gmaw1d9
authors: Li, Tingting; Zhang, Tianying; Gu, Ying; Li, Shaowei; Xia, Ningshao
title: Current Progress and Challenges in the Design and Development of a Successful COVID-19 Vaccine
date: 2021-01-26
journal: nan
DOI: 10.1016/j.fmre.2021.01.011
sha: 29cbcca66081d55a1ac97deb29a29a301a02be51
doc_id: 1021670
cord_uid: 7gmaw1d9

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still a worldwide concern, with little to no sign of a decreasing trend. There is a general consensus that normal life will be hampered until a safe and effective vaccine strategy is available and globally administered. Numerous countries have accelerated the clinical trials process for the development of a successful COVID-19 treatment, with over 200 candidates presently available for testing against SARS-CoV-2. Here, we provide an overview of the COVID-19 vaccine candidates currently in development, discuss the scientific and practical challenges associated with COVID-19 vaccine design, and share the potential strategies that could be exploited for vaccine design success.

an effort to curb the spread 3, 4 . Despite many best efforts worldwide, SARS-CoV-2 has spread to 216 countries and regions, and, as of 9 January 2021, has resulted in more than 87 million confirmed cases and at least 1.9 million deaths worldwide 3, 5 . This disease poses an extraordinary threat to global health and public safety, and the urgency associated with this pandemic has emphasized the pressing need for effective preventive and therapeutic measures. and SARS-CoV-2 persists worldwide, only the third type of coronavirus to cause severe pneumonia in humans 6 .

Vaccines remain the most cost-effective intervention for the control and prevention of infectious disease. However, there have been no vaccines to date for the treatment of any of the coronaviruses that have made the switch over to humans, including SARS-CoV and MERS-CoV. Whereas these previous coronavirus outbreaks eventually petered out-in part presumably due to good public health containment and the earlier symptomatic response-COVID-19 does not show similar signs of a decreasing trend, with rapid rates of infectivity in clusters. As such, there has been an urgent response within the scientific community to accelerate the development of a vaccine against this particular species.

Phylogenetic analyses suggest that SARS-CoV-2 has a 79% sequence similarity with SARS-CoV, and a lower (50%) similarity with MERS-CoV 7 .

SARS-CoV-2 contains four major structural proteins, spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins 5 and, like SARS, uses the angiotensin-converting enzyme 2 (ACE2) receptor for cell entry 8 . The virions are spherical and decorated with S proteins on the envelope surface. These S proteins play a pivotal role in viral infection and pathogenesis. The S protein comprises two subunits-S1 and S2. The S1 subunit recognizes host receptors, whereas the S2 subunit mediates fusion between the viral envelope and the host cell membrane 6 . The receptor binding domain (RBD) in the S1 subunit is responsible for virus binding to host cell receptors 6 .

Unique to the SARS-CoV-2 S-protein is PRRA amino acid insertion in the furin cleavage site between the S1 and S2 subunits 8 . This long furin cleavage site influences S-protein stability and facilitates subsequent conformational changes that can influence viral entry 8 . The SARS-CoV-2 S-protein is primed by transmembrane serine protease-2 (TMPRSS2) for host cell entry. In addition, neuropilin-1 (NRP1), which is known to bind furin-cleaved substrates, potentiates SARS-CoV-2 infectivity 9 .

Earlier work on SARS-CoV and MERS-CoV shows that the S-protein is the principal antigenic constituent that can be harnessed to induce the production of neutralizing antibodies (nAbs) to block virus binding 6, 10 . As such, the S-protein has been similarly recognized as a significant target for COVID-19 vaccines. Notably, other studies have reported that the M-protein is capable of inducing the production of nAbs, and the N-protein contains T-cell epitopes in SARS-CoV; these proteins may also offer alternative vaccine targets 11, 12 . Collectively, these studies provide potential targets for the development of antivirals against SARS-CoV-2. In this review, we summarize the current advances in the clinical trials associated with the development of vaccines and focus on the challenges and strategies of vaccine development against this unique and difficult species.

There has been an enormous effort to develop an effective and safe vaccine to control the rapid spread of the SARS-CoV-2 virus. Currently, there are 235 candidate vaccines under development, according to a document released by the WHO (https://www.who.int/publications/m/item/draft-landscape-of-covid-19-candidate-vac cines), with 63 candidate vaccines in clinical trials, including 9 inactivated vaccines, 11 non-replicating viral vector vaccines, 6 replicating viral vector vaccines, 19 protein subunit vaccines, 7 RNA vaccines, 8 DNA vaccines, and 2 VLP vaccines. This array of vaccine types provides a chance that at least a few candidates will eventually be approved for further development and marketing.

There are several commonly used platforms for vaccine development: these include 1) the classical and mature approaches using inactivated whole virions, live-attenuated, recombinant protein, or vectored vaccines, which are well-established technologies and have led to the production of numerous licensed vaccines; 2) promising novel vaccine approaches, such as DNA vaccines or mRNA vaccines; there is no precedent for a licensed vaccine based on these platforms 13 .

Inactivated vaccines are the traditional form of vaccine and are formulated using the whole virus that is commonly either physically (heat) or chemically (e.g., β-propiolactone) inactivated. Such viruses are usually produced in Vero cells, and the culture supernatant is purified and formulated with or without adjuvant 14-17 .

Inactivated vaccines are easy to produce but require a biosafety level 3 (BSL3) facility. PiCoVacc, developed by Sinovac Biotech (China), is a β-propiolactone inactivated vaccine capable of inducing the production of nAbs in non-human primates (Rhesus macaques) ( Table 1) . This was achieved after three intramuscular injections of 3 or 6 μg PiCoVacc adjuvanted with aluminum hydroxide per dose at one-week intervals 15 . NAbs titers rose to ~50 after the second boost before virus challenge, which are similar to the titer levels raised by serum from recovered COVID-19 patients. In a SARS-CoV-2 challenge study, the immunized (3 μg/dose) monkeys showed partial protection response as compared with control monkeys following a direct intratracheal inoculation of 10 6 TCID 50 SARS-CoV-2 into the lungs, with detectable viral loads in the pharynx, anal canal and pulmonary tissues. At higher immunization doses (6 μg/dose), there were no detectable viral loads in any of the aforementioned tissues at 7 days after infection (Table 1) . Of note, no antibody-dependent enhancement (ADE) of infection was observed. In another assay, hematological and biochemical analyses showed no notable changes in terms of lymphocyte subset percent and the presence of key cytokines, with no immunopathological exacerbation observed.

Subsequently, Sinovac reported the results from a randomized, double-blind, placebo-controlled phase II trial (NCT04352608) conducted using CoronaVac (previously, PiCovacca) with a total of 600 healthy adults aged 18-59 years 19 .

CoronaVac was shown to be well tolerated, favorable, safe, and without any Grade 3 adverse reactions or vaccine-related serious adverse events (SAEs). CoronaVac showed good immunogenicity, with at least 92.4% seroconversion under different vaccination schedules in the lower-dose group (3 μg/dose). The geometric mean titers (GMTs) of the nAbs ranged from 24 to 65 among the different dosage and vaccination schedules ( Table 2 ). The vaccine is now in phase III clinical trials (NCT04456595, NCT04582344, 669/UN6.KEP/EC/2020). In terms of the recent report in news, the CoronaVac vaccine showed 78% effective in Brazil trial.

Pre-clinical results are also available for another inactivated vaccine candidate, BBIBP-CorV, developed by the Beijing Institute of Biological Products Ltd.

BBIBP-CorV adjuvanted with aluminum hydroxide, was evaluated in non-human primates (Cynomolgus macaques) using two-dose immunizations regimens.

BBIBP-CorV provided highly efficient protection at both low (2 μg/dose) and high (8 μg/dose) doses. Before intratracheal challenge with 10 6 TCID 50 of the virus, the GMTs of the nAbs in the low-dose and high-dose groups reached 215 and 256, respectively. At 7 days post-infection (dpi), there was no viral load in the lungs of immunized macaques in either the low-or high-dose groups. In the upper airway, the virus was completely suppressed in the high-dose group, whereas primates in the low-dose group showed a significantly reduced viral load as compared with placebo recipients. The study observed no ADE of infection among any of the vaccinated primates (Table 1) .

A randomized, double-blind, placebo-controlled, phase I/II trial with BBIBP-CorV vaccine (ChiCTR2000032459) has since been undertaken, with promising preliminary results 20 . In the phase I trial, 192 healthy participants (18-80 y) received a two-dose schedule at 2, 4, or 8 μg/dose. No SAEs were reported within 28 days post-vaccination. The adverse reactions were mild or moderate in severity (Table   2 ). Two weeks after the boost, the GMTs of the nAbs measured 87.7 to 228.7 for the younger participants (18-59 y), and 80.7 to 170.87 for the older participants (60 y and over), with a dose-dependent effect observed for the nAb titers. Phase II examined different vaccination schedules. Only one participant in the placebo group experienced and recovered from a grade 3 fever; all other adverse reactions were mild or moderate, and the most common systematic adverse reaction was fever. A two-dose immunization regimen (days 0/21 or days 0/28) with 4 μg of vaccine achieved higher nAb titers (282 and 218, respectively) than a single 8 μg dose (15) T lymphocyte (CTL) response with IFN-γ was detected, and genes related to T and B cell activation were upregulated by ~40% and ~25%, respectively (Table 2) . Genes related to the activation of dendritic cells, mononuclear cells/macrophages, and natural killer cells were also upregulated to different degrees. This vaccine is currently under a phase III assessment (NCT04659239).

Live-attenuated vaccines contain live, whole bacterial cells or viral particles and are treated to have reduced virulence but still retain some antigenicity after attenuating the pathogen 21 . The virulence is reduced through artificial mutations, gene deletions, or by screening from nature. These types of vaccines can simulate naturally occurring recessive infections and induce comprehensive, stable, and persistent responses, with immunization able to be achieved via oral, nasal, and/or aerosol routes.

Live-attenuated vaccines can induce antibody, cell and mucosal immune responses 13,21 . Of note, there are some potential safety issues with these types of vaccines that need to be addressed 21 The first-in-human trial against coronavirus was conducted by CanSino (China) using a non-replicating Ad5-based vaccine expressing the wild-type S-protein.

The single-center trial was conducted as a dose-escalation, open-label, non-randomized, phase I trial and was carried out in Wuhan, China (NCT04313127) 23 .

The vaccine was administrated to 108 participants as a single shot at one of three doses (high, middle, low), and was found to be tolerated and immunogenic at 28 days post-vaccination. Most adverse reactions were mild or moderate, with no SAEs within 28 days. Of note, participants in the high-dose group tended to have higher reactogenicity. The GMTs of the nAbs in the high-, middle-, and low-dose groups post-vaccination were 34.0, 16.2, and 14.5, respectively. Rapid specific T-cell responses for IFN-γ secretion peaked at day 14 (Table 2) .

Following this, CanSino then went on to conduct a randomized phase II trial (double-blind, placebo-controlled) of the Ad5-vectored vaccine in 508 participants (NCT04341389) 24 , again using three doses. At day 28 post-vaccination, the seroconversion rates in the medium-and low-dose groups were 96% and 97%, respectively, with GMTs of 19.5 to 18.3 ( Table 2 ). Cellular responses, as detected using IFN-γ enzyme-linked immunospot assay were observed in 90% and 88% of the participants in the medium-and low-dose groups, respectively. No SAE was observed.

Of note, an advanced age and higher pre-existing anti-Ad5 titers reduced the immune response. The authors suggested that a single-dose immunization schedule of positivity. These nAbs titers achieved with a two-dose immunization regime were comparable with those measured using convalescent sera. The IFN-γ response peaked at day 14 and then declined, and could not be boosted with additional doses. A low level of anti-vector immunity was observed in subjects (4%). This vaccine is currently in phase III trials (NCT04516746, NCT04540393, etc.). In the recent interim analysis report of phase III, the Oxford-AstraZeneca vaccine was 70.4% effective at preventing SARS-CoV-2 infection overall when combining data from two dosing regimens., The vaccine efficacy was 90% and 62% respectively in the two groups with different dose regimens. The higher efficacy regimen adopted a half dosage for the first dose and a standard one for the second dose. It was claimed that this vaccine is cost effective in production and easier to transport and store (2-8 °C) , and has been approved by the U.K. government for emergency use. formulations, respectively, both with a seroconversion rate of 100% (Table 2) .

Notably, the nAb titers after the boost were not significantly different to those titers after COVID-19 infection. Specific CD4 + and CD8 + T-cell responses peaked at day 28 after vaccination. The study also showed that a pre-existing immune response to the Geno-Immune Medical Institute (China).

Replicating vector vaccines use engineered viruses or bacteria for the vaccine vector to express a target gene in the host cell. In some cases, viruses that do not replicate efficiently or those that cause no disease in humans are used 13, 22 

Recombinant protein vaccines are based on recombinant subunit proteins, peptides or virus-like particles (VLPs), which can be expressed in various systems, such as E. coli, yeasts, plants, insect cells, and mammalian cells 21, 29 . To this end, the RBD, S1, S-protein or N-protein are generally chosen as the principal target antigens.

Recombinant protein vaccines need to be combined with potent adjuvants for improved immunogenicity and efficacy, particularly for protein antigens in the (Table   1 ). In a mouse model deficient in CD4 -/-, Sting1 -/-, Casp1 -/-,Nlrp3 -/-, IL-1β -/-,Tlr2 -/-, and Tlr4 -/-, several immune pathways and CD4 T-lymphocytes were implicated in the induction of the vaccine antibody response. Currently, the vaccine is in phase II clinical trials (ChiCTR2000039994).

The third vaccine is a native-like S-trimer (wild-type) vaccine based on group; GMT, 317 in >71 group) appeared to be similar to those reported among the younger cohort (18 and 55 y) and were above the median values of convalescent serum ( Table 2 ). The vaccine elicited a strong CD4 cytokine response involving Th1 cells in both age subgroups. Currently, the vaccine candidate is in a large phase III trial to assess its level of protection against COVID-19.

A randomized, observer-blinded, placebo-controlled phase III trial was conducted in the United States 47 Although the antibody titers between the two candidates were comparable, BNT162b2 was associated with less systemic reactogenicity, particularly in older adults, and showed a more favorable safety profile ( 

Over the past few decades, there have been numerous efforts to develop a vaccine against human coronaviruses. However, despite intense research, there is still no vaccine available for any of the diseases. Furthermore, even though there are numerous COVID-19 vaccines candidates in the pipeline, with 10 vaccine candidates already entering phase III trials, we are still faced with many challenges associated with vaccine safety, efficacy, and production, with some of these important considerations highlighted below.

An important concern for COVID-19 vaccine development is the ADE of Reactogenicity after immunization is another major concern in clinical trials.

Most of the current COVID-19 vaccine clinical trials are being conducted in healthy adults aged 18-55 years, with some later-staged trials enrolling older participants over the age of 55 years. Older people and children fall into the high-risk population, and it remains largely unclear whether COVID-19 vaccines will be safe for both young children and older patients over the short and long term. Several studies have reported that the immunogenicity and reactogenicity levels are stronger with higher doses, which tend to be needed for older people to achieve protective immunity. Children are more likely to require a lower dose, since they commonly display more reactogenicity.

Currently, most vaccines are performing better with a two-dose regimen, but such a regime will increase the reactogenicity of the treatment. Therefore, adequate assessment and monitoring of the safety of the COVID-19 vaccine in clinical trials are needed, especially for trials exploring the use of a novel vaccine technology, such as DNA or mRNA vaccines.

Immune sensing studies suggest that SARS-CoV-2 suppresses the activation of the innate immune system in a manner similar to that of SARS and MERS, with responses from dendritic cells and impaired antiviral type I interferon (IFN-I) and

type III interferon (IFN-III) responses [63] [64] [65] . Studies indicate that dysregulation of the IFN-I response plays a pivotal role in the pathogenicity of COVID-19 66 . Animal models of SARS-CoV and MERS-CoV infections indicate that failure to induce an early IFN-I response is associated with the severity of the disease 67 . Importantly, these results show that the timing is critical: IFN has a protective effect in the early stages of the disease, but its delayed expression is pathological; a recent study showed that IFN-induced upregulation of ACE2 in nasal epithelial cells may be involved 68 . These pro-inflammatory processes may lead to the "cytokine storm" observed in COVID-19

patients. Therefore, clarifying the delicate balance between antiviral and inflammatory innate immune programs is crucial for the development of effective COVID-19 vaccines and antiviral drugs 66 . In addition to considering the effects of the vaccine-induced adaptive immunity, innate immune memory might also play a role, perhaps by enhancing viral control, particularly in the early phases of infection 65 .

An important characteristic of the SARS-CoV-2 virus mode of infection is the high affinity binding between the S-protein and the ACE2 receptor, with a K D value of ~15 nM; this is approximately 10-to 20-fold higher affinity than the binding between SARS-CoV and ACE2 69 , 10-fold higher than that between insulin-like growth factor-1 receptor (IGF1R) and RSV (118 nM), and 100,000-fold higher than sialolactos binding to influenza virus (mM range) 70 immunity, which is much more potent. In terms of the current vaccines against COVID-19, it is still unknown whether the immune response induced by the vaccine will last longer or shorter than the immune response induced by natural infection.

Once the vaccine is licensed, there still could be delays in the production process. The global demand for COVID-19 vaccines will exceed ordinary pharmaceutical industry supplies, with there likely to be a requirement for billions of doses. The production of RNA and DNA vaccines could be simpler than other methods, but biotech-based companies involved in these approaches have never licensed a vaccine or produced a compound in such high demand 13 . Also concerning are the unforeseen challenges associated with world-wide distribution, with there likely to be the need for frozen storage for delivery to some lower-income countries;

this will be problematic for some mRNA vaccines.

It is worth noting that China is committed to developing and deploying a Novel technologies, such as transdermal delivery (microneedles), and other routes of administration, such as mucosal (oral or intranasal), may offer alternative ways to induce strong mucosal immune responses 87 . A few COVID-19 vaccines are being explored to target mucosal immunity. Antigen-specific IgA plays a pivotal role in protecting mucosal surfaces from both microbe adhesion and virus activities 88 .

Thus, the development of novel vaccine delivery platforms that elicit specific IgA and systemic IgG will be critical to improve vaccine effectiveness.

The COVID-19 pandemic is an ongoing, global concern. Vaccines could be the only effective and economical means to curtail and manage this outbreak. 

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This review was supported by the National Natural Science Foundation of China 

The authors have declared that no competing interests exist.