Until December 2019, Human Coronaviruses (HCoVs) were treated as trivial pathogens, leading to simple “common cold or seasonal flu, mild and self-limiting” in healthy people. However, the 21st century witnessed 2 pathogenic HCoVs—Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). These viruses are initially transmitted from animals as carriers resulting in epidemics with high morbidity and mortality. The month of December in 2019 broke the news of one more pathogenic virus christened as SARS-CoV-2 and resulting infection as Coronavirus Disease 19 (COVID-19). The first location of this outbreak was reported in Wuhan city in the Hubei province of China [1, 2]. The initial stages involved clusters of pneumonia cases in January 2020. This was followed by the spread of the virus across the globe, eventually WHO declaring it as pandemic on 11^th March 2020 [3]. While the pathological changes, co-existence of other diseases, and line of treatment in COVID-19 patients are still under investigation and evolving as the disease progresses, experts are banking heavily on the rapid development of the safe and efficacious vaccine. The only way right now to prevent the spread of infection is by physical distancing, avoiding large gatherings, use of face masks, and Personal Protective Equipment (PPE) kits. The long-term solution for COVID-19 will be a global vaccination program for socioeconomic and therapeutic benefits. As of today, the total number of confirmed COVID-19 cases is 33.2 millions, with 999,239 deaths [4]. Since the treatment of COVID-19 is symptomatic without a fixed line of treatment, there is an urgency to develop the vaccine against SARS-CoV-2. This article covers possible avenues in vaccine development for SARS-CoV-2 and is based on current research projects being carried out all over the world. It also aims to explore challenges and approaches in the development of an effective vaccine against SARS-CoV-2.

SARS-CoV-2 belongs to Coronaviridae family, order Nidovirales. It is either spherical in shape or pleomorphic with a diameter of 125 nm featured by club-shaped spike projections on its surface, commonly called ‘Corona’ due to its peculiar shape [5]. The virus has 14 days incubation period with common symptoms of common cold, dry cough, fatigue, fever, nasal congestion, labored breathing progressing to respiratory and cardiac failure, eventually leading to death [1]. SARS-CoV-2 is the enveloped virus that contains segmented, positive sense, single stranded-RNA genome of around 30 kb, largest among all the RNA viruses [6]. In consonance with other Coronaviruses, SARS-CoV-2 has four structural proteins.

(1) Glycosylated Spike (S) protein which forms a trimer on virus surface to take the shape of the crown. Its structure shows the presence of two domains S1 and S2, representing to carry out the major function as Receptor Binding Domain (RBD) for attachment to the surface of host receptor cell and fusion with host cells respectively [7].

(2) Nucleoside (N) protein helps to replicate the viral genome inside the host cell.

(3) Membrane (M) transmembrane glycoprotein, which maintains viral structure and assembly, and

(4) Envelope (E) protein plays a role in the virulence, pathogenesis, and release of virus [8, 9]. SARS-CoV-2 gains entry into the host cell after binding of RBD and then attachment with Angiotensin-Converting Enzyme 2 (ACE 2) receptors prevalent on human cells with higher affinity than other receptors [10].

Fig. (1) gives an idea about the structure of Coronavirus with the location of structural proteins and respective functions [11]

There have been several studies and ongoing clinical trials to find a precision medicine for COVID-19 treatment and prophylaxis. Among all these studies, remdesivir has proven to be effective in reducing median time for recovery from 15 days to 11 days and diminished the mortality rate from 11.9% to 7.1% [12]. However, the effectiveness of remdesivir in the patients with severe COVID-19 infection was not significant statistically as well as clinically [13]. In yet another randomized, open-labeled phase 2 study, combination therapy of oral ribavirin (a nucleoside analog) and oral lopinavir+ritonavir (protease inhibitors) indicated alleviation of symptoms if given within 7 days, nonetheless failed to impress in patients with severe COVID-19 infection and chronic co-morbidity [14]. Although there are several undergoing clinical trials targeted towards COVID-19 since its outbreak, none of the new or repurposed drugs are found clinically suitable as an antiviral specifically towards SARS-CoV-2. For this reason, the development of safe and effective vaccines followed by mass-scale vaccination across the globe is vital to reduce mortality rate and the number of cases globally. Accordingly, consolidated efforts have led to the development of several vaccines and being supplied to the world through coordinated and planned execution. Further, another possibility is that COVID-19 may sometime later become, a seasonal or recurrent disease requiring an accurate and precise treatment for all. Additionally, convalescent plasma from recovered COVID-19 patients that include monoclonal antibodies (H2, H4, B5, and B38) have exhibited positive outcomes with respect to high neutralization of viral antigens. However, isolation of such plasma from aged patients with co-morbidity, allergic reactions in plasma recipients and antibodies formed against SARS-CoV-2 waning off from blood of donors are deterring factors for convalescent plasma therapy [15, 16]. Most recently, the appearance of SARS-CoV-2 variant strains with spike protein mutations reported in the United Kingdom (strain B.1.1.7) and Republic of South Africa (strain B.1.351) have raised concerns about re-emergence of COVID-19, although Moderna mRNA-1273 vaccine has confirmed about 94% efficacy in a Phase 3 study against new variants [17]. Among the worst affected countries, Brazil has faced more than 60,000 deaths due to COVID-19 in the month of March, pushing health services to the tipping point where the only hope is vaccination [18]. Thus, all these important discussions reconfirm the need for vaccination on a large scale to protect people against SARS-CoV-2 infection.

Fig. (1). Structure of Coronavirus with main proteins and their functions. Adapted from [11].

SARS-CoV-2 is a baffling type of RNA virus. Many patients show clinical symptoms while many are asymptomatic. The main challenge in vaccine development is a high rate of mutation and RNA recombination in RNA viruses, which are highly adapted to changing environments [19-21]. This is also accompanied by zoonotic virus transmission to humans. The rate of replication of this virus is extremely high having noteworthy genetic diversity [22]. The S-protein is a glycoprotein envelope on SARS-CoV-2, which primarily determines protective immunity. This is further divided as S1 and S2 subunit playing the role of viral receptor binding and membrane fusion, respectively. The difficulty of targeting these proteins lies in the fact that both the protein subunits undergo pre-fusion and post-fusion conformational changes representing altogether different antigenic epitopes [23]. The previous knowledge of MERS-CoV cannot be applied fully for COVID-19 vaccine development as a similarity between the S-protein of SARS-CoV-2 and MERS-CoV is only 44% [24].

Another aspect during vaccine development is its clinical trials. The clinical trials on human volunteers are preceded by pre-clinical trials employing suitable animal models. Preliminary animal studies have shown the development of neutralizing antibodies against S1 subunit of S-protein [25]. However, the preliminary studies have shown inflammatory response and altered pathological conditions like pulmonary eosinophilic infiltration in alveolar regions of animals (non-human primates) on exposure to the viral challenge tests [26-28]. This was one of the reasons for one school of thought that questioned hastily and rushed human trials without assessing efficacy, safety and toxicology studies. This specifically has been said about Moderna and the National Institute of Allergic and Infectious Diseases (NIAID) in the US, which conducted clinical trials for mRNA-based vaccine candidates against SARS-CoV-2 [29]. Formulation of vaccine is an altogether additional challenge as new techniques of vaccine formulation are scantily tested for compatibility of antigenic substance with adjuvants and vaccine vehicle. Scale up to produce millions of doses is also a mammoth task. From a regulatory point of view, there is disparity among standards set by different regulatory agencies. Greater harmonization is to be sought when the question of emergency use authorization arises. Even though there is successfully shortened vaccine development time almost from a decade to fewer than twelve months, a clear and transparent development process, as well as clinical outcome, needs to be made available with understandable evidence that this has not jeopardized patient safety.

Developed and approved vaccines further require to be made available to the susceptible population of the society at the right time and at affordable price. There have been reports of more than 10,000 COVID-19 related deaths per day worldwide, compelling grueling and ambitious plans to ramp up the vaccination. However, large-scale production and mass scale distribution of vaccines with cold chain storage seem challenging in the low-income countries. Keeping logistics, supply chain management and vaccine scheduling apart, a more challenging task to be dealt with is improving public awareness for vaccination and hesitancy as well as spread of misinformation and rumors among the target population. Alleviating concerns of vaccine side effects and effectiveness have to be prioritized by governments for quicker vaccination scheduling [30]. A recent survey based study from several countries tells that maximum 77.6% of people (of mixed age, gender, ethnicity, religion, professionals, personal beliefs, education and income) are ready to accept COVID-19 vaccine, figures indicating a worrisome trend while achieving maximum vaccination in susceptible population [31]. On the clinical front, the duration for which immunity conferred by the vaccine lasts and the time gap between two successive doses of vaccines is established clearly but with some varying data, although vaccination has been started aggressively in many countries.

Bao et al. have reported that commonly used animals in pre-clinical studies are not suitable for developing the COVID-19 vaccine as they are not prone to SARS-CoV-2 virus infection. Thus, a major challenge is availability of incomplete or insufficient data owing to lack of well established animal disease model. This has posed a problem in the initial stages for selection of candidate vaccine as well as immunogenicity triggered by the vaccine [32].

Vaccines for active immunization involve antigenic material and work by triggering the immune system of the host to produce antibodies specific to that antigen, while passive immunization involves readymade antibodies specific for the disease causative agents, such as bacteria or viruses. In the conventional or classical type of vaccine, an epitope from the causative micro-organism is identified, isolated, purified, tested at the preclinical stage followed by clinical trials and then used for mass-scale immunization after approval. These may be in the form of live attenuated (weakened pathogenicity), inactivated (killed), subunit or conjugate vaccine and in the form of toxoids [33]. Thus the vaccine triggers up host immunity to produce antibodies against the pathogen. On subsequent exposure to the pathogen, the host immunity responds in a better manner due to existing antibodies in circulation. This type of vaccine takes approximately 12 to 15 years to development, precluding early release of vaccine for mass population [34]. One more issue of consideration is the selection of proper adjuvants the therapeutically inactive components of vaccine formulations. If the adjuvants are not correctly selected, the immunological response is suboptimal, particularly more affirming with modern types of vaccines as they are under continuous evolution [35, 36].

The ongoing discussion makes it clear that classical means of vaccine development for SARS-CoV-2 will consume a lot of time till it is in final use. This allows the scope for vaccine development by advanced and evolving newer strategies [37]. The vaccines for COVID-19 development is fast tracked but with a lack of detailed preclinical studies on nonhuman primates (NHPs) [38, 39]. For the sake of understanding, Fig. (2) explains the classical Vs COVID-19 vaccine development in sequential stages [40].

In the process of vaccine approval, comprehensive data of pre-clinical trials and Phase 1, 2 and 3 of human clinical trials is submitted to regulatory bodies. Phase 1 trials on 20 to 100 healthy volunteers focus on safety establishment, Phase 2 trials on few hundred to few thousands of volunteers set goal on evaluating protective, safe and efficacious dose followed by Phase 3 trials on several thousand volunteers for getting clinical, quantifiable end point concerned with the disease in the question. Apparently, The current strategy of testing the safety and efficacy of the SARS-CoV-2 vaccine may jeopardize Phase III studies replacing it by controlled human challenge studies. The pool of human volunteers involves those who are uninfected with the virus, and another group includes volunteers having the highest risk of getting infected from SARS-CoV-2 naturally (those who are not infected but living in containment zone). Both groups are stated to be comprised of volunteers in the age group of 18 to 60 years selected carefully, which are otherwise at low risk of mortality and less likely to harbor infection after dosing [41-43]. Currently, the National Institute of Allergic and Infectious Disease (NIAID) sponsored and collaborated by Moderna studies are being conducted across the US. The vaccine candidate being tested is mRNA-1273, a novel lipid nanoparticle (LNP)-encapsulated mRNA-based vaccine for SARS-CoV-2 that encodes for a full-length, stabilized surface spike (S) protein [44].

The manufacturing facilities are provided by the Coalition for Epidemic Preparedness Innovations (CEPI) [45]. As of today’s date, 13 candidate vaccines are under clinical evaluation as mentioned in Table 1 [46]. Apart from this, 128 candidate vaccines are under the preclinical evaluation stage for COVID-19, out of which 11 are DNA vaccines, 5 inactivated, 3 live attenuated, 17 non replicating viral vector, 49 protein subunit, 15 replicating viral vector, 15 RNA (liposome, lipid nanoparticle and lipid encapsulated mRNA), 10 VLP (virus like particles) and 3 of unknown/ undisclosed platform [46].

Fig. (2). Comparison between developmental stages of conventional/classical vaccine against SARS-CoV-2 Vaccine. Adapted from [40].

Progress in vaccine development is evident in last two decades since the outbreak of Ebola, SARS, MERS, H1N1, Zika viruses and a few more to count. The issues and concerns associated with vaccine development cover not only safety, efficacy, quality, and toxicology but also scale-up technology, versatility, stability and proper supply chain management to cater to the world population. An account of approaches for the vaccine types is mentioned in Fig. (3) [40]. Here, we now discuss new technological advances and platforms in development of vaccines against SARS-CoV-2.

Fig. (3). Approaches for development of SARS-CoV-2 Vaccine. Adapted from [40].

More than 10 COVID-19 vaccines are under development in India. The progress in this direction is being updated continuously on the web portal (https://vaccine.icmr.org.in/COVID-19-vaccine) maintained by the Indian Council of Medical Research. Currently, three different vaccines are jointly developed in collaboration with various agencies. This can be summarized in a nutshell in the following Table 3.

The vaccines being developed in India are in human clinical trials Phase II/III, which enroll healthy human volunteers for establishing clinical efficacy of the vaccines. All the details are updated regularly on the website https://vaccine.icmr.org.in/. While Covaxin is approved against COVID-19, Covishield has been given emergency use authorization. The vaccination has started with priority to frontline health workers followed by those aged above 45 years and with co-morbidity.

Several countries have witnessed a rise in cases as well as deaths due to the second wave of COVID-19. In addition, this is accompanied by the appearance and spread of new strains of SARS-CoV-2 (B.1.1.7 variant in UK and B.1.351 in South Africa). The rise in positive cases and deaths initially caused hesitancy and the spread of misinformation about vaccination against COVID-19. However, researchers and scientists have reassured the importance of vaccination and emphasis on getting vaccinated to avoid the catastrophic situation. In fact, in UK, strict lockdown followed by aggressive vaccination has shown better future with declining number of cases as well as lesser mortalities since mid of January 2021 [78]. Similar trends are observed in Israel, United Arab Emirates, and Norway, which have started vaccinating citizens at an incredible pace [79]. However, in several countries like Brazil, Pakistan, and India, the patients of COVID-19 are increasing with higher mortalities. Thus, it becomes imperative that apart from maintaining physical distance and wearing a mask, getting vaccinated must be prioritized by people and supported robustly by governments, administrations and allied organization as early as and as quickly as possible.

The difficulty and concerns initially thought about reaching the vaccine to every country with equity and equality, diligently and at affordable prices, has been addressed by several organizations. The Global Alliance for Vaccines and Immunizations (GAVI), COVID-19 Vaccines Global Access (COVAX), WHO, Coalition for Epidemic Preparedness Innovations (CEPI), and UNICEF are ensuring vaccine delivery with logistical supports. In the end, the road for normalcy goes through the path of vaccination only.

COVID-19 is a new entrant in viral disease, starting with ‘common cold’ rapidly covered the globe with alarming mortality and co-morbidity and eventually declared a pandemic by WHO on 11^th march 2020. In order to restrain the spread of infection, governments in almost all affected countries declared lockdown, social distancing, repeated hand washing and use of face masks with the prohibition of mass gatherings.

It was mandated by experts from all over the world that the lack of a specific treatment plan makes it imperative to develop a vaccine for COVID-19. There are calculated clinical uses of Hydroxychloroquin, remedisivir, favipiravir, but without previous history of use against SARS-CoV. This has compelled researchers to hunt for suitable, safe vaccines. In the usual course, the vaccine development in a conventional way takes from 10 to 15 years. The current situation thus paves the way for bypassing/substituting regulatory phases of clinical trials to accelerate vaccine development for COVID-19 with an unconventional approach. Irrespective of the technology platform used, experts are concerned about the safety and efficacy issues, although CEPI, NIH, NIAID, CDC and FDA are trying their best to address them.

The significant requirement to address the COVID-19 pandemic has prompted the development of several vaccine candidates which can address the worldwide vaccine burden. The accomplishments, achievement and vaccine efficacy as outlined above further authenticate foundation of the hard work of scientific research in vaccine development. The effect of COVID-19 vaccine development has resulted in an unparalleled speed of vaccine design and manufacturing. The theoretical concepts and the premise of platforms that only require antigens, like mRNA and vector-based technology, have accelerated their development towards quick Phase 3 evaluation. Almost all the vaccines reaching Phase 3 of clinical trials have reported efficacy in a well acceptable range, with some of them more than 90%. While many vaccines require a booster dose, viral vector vaccines with single-dose efficacy are in Phase 3 clinical trials and are much awaited. Since the vaccines currently produced mainly focus on neutralizing antibody generation in the host, a more holistic approach is needed that will aim to achieve long-term immunity against COVID-19. At the same time, a broader approach towards vaccine development is growing as newer variants are emerging from South Africa (B.1.351), UK (B.1.1.7) and Brazil (B.1.1.28). The possible way out for this can be developing broad activity vaccines that target structural proteins apart from protein S, such as ORF7A and ORF3A, which can prevent transmembrane entry as well as adherence of SARS-CoV-2 to the host cells by extended T cell and B cell immunity for a longer duration.

As a matter of concern, recently there have been reports of blood clots and hemorrhage associated with the administration of the Oxford-AstraZeneca COVID-19 vaccine. This has raised apprehension about vaccine safety in Europe. Thus, an extensive review is being exercised by Medicine and Healthcare Regulatory Authority, UK (MHRA) and European Medicines Agency (EMA) [80]. This emphasizes the need for detailed safety evaluation of vaccines, mainly while considering patients with chronic co-morbidities. However, it can be fairly concluded that the advantages of vaccines greatly outweigh their rare incidences of side effects.

Finally, the progress in vaccination in several countries has reaffirmed that with proven reports and records, gradually COVID-19 cases are declining along with drop-in mortality rate. Vaccinating vulnerable populations is the only path ahead to get rid of COVID-19.

As the Coronavirus disease progresses, it delivers more information about the virus, replication pattern, spread, epidemiology, clinical outcomes, risk age group, and much more. This has ultimately helped in developing the right approach for vaccine development against SARS-CoV-2. At the same time, previous viral diseases emerged in the last 20 years, prompted for vaccine development with limited success. This sets the tone for the use of a myriad of technology platforms - live, attenuated, virus like a particle, vectors, mRNA, protein subunit, DNA type for vaccine development with new paradigms in testing and evaluation of vaccines.

Formulation of vaccine, along with dosage and suitable adjuvants, need to be tested systemically on animal models, which helps to identify the genetic variations in demographies worldwide. The efforts need to be escalated as COVID-19 is taking a toll on lives and economy in all countries as notified by UNO.

As more and new formulations of vaccines are being tested, each bringing a new technology in vaccine design and development, hopefulness about control of the COVID-19 pandemic grows. However, considerations of viral mutations, new strains and variants, supply chain management-logistics and delivery to the citizens must be looked after continuously by all the concerned authorities, off-course maintaining social distance and use of mask will add up to prevent the spread of disease and ease the pandemic burden.

As a concluding remark, it is conceivably advisable that a single or couple of vaccines will not suffice to whole population vaccination with constraints mentioned above, thus simultaneous use of multiple vaccines must be explored as soon as possible.