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A comprehensive insight on the COVID-19 vaccine candidates

The world is currently facing a pandemic triggered by the novel corona virus (SARS – CoV2), which causes a highly infectious infection that predominantly affects the lungs, resulting in a variety of clinical symptoms some cases may be asymptomatic while others may result in to severe respiratory disorder, if the infection is left unattended it may result in multi-organ failure and eventually death of the patient. The transmission of infection is by droplet and fomites of the infected person. The incubation period of virus is from 2 to 14 days. Most common symptoms resemble flu-like but later progress to pneumonia along with dyspnoea and worsening of oxygen saturation, thus requiring ventilator support. The diagnostic modalities include Reverse transcriptase real time PCR (Quantitative Reverse transcriptase polymerase chain reaction) which is recommended method used for diagnosis of the COVID-19 infection using oro-pharyngeal or nasopharyngeal swabs of the patients. Recently serological tests for antigen and antibody detection has been approved by ICMR. Till now, nine COVID-19 vaccines are granted emergency approval for prevention and for the management of infection symptomatic and supportive measures are being adopted. Globally major pharmaceutical firms are engrossed for development of a potent vaccine candidate. This review highlights on various vaccine candidates under clinical trials.


Introduction
The novel Coronavirus causes a highly contagious viral infection known as corona virus disease or COVID 19. It is a RNA virus which bears an outer envelope with glycoprotein spokes protruding outwards that helps in attachment to the host receptor. [1,2] Common symptoms of SARS-CoV-2 include influenza-like illness (ILI) comprising of fever, sore throat, cough, shortness of breath. Cases may range from being asymptomatic to those with severe pneumonia and multi-organ failure. [2,4] Infection spreads via droplet infection which are generated during talking, coughing or sneezing from an infected person to an healthy individual, and through fomites of infected person. Incubation period is about 2-14 days. Diagnostic modalities includes Reverse transcriptase real time PCR which is gold standard method for diagnosis of the infection. Recently ICMR (Indian Council of Medical Research) has also validated and approved kits for rapid antigen detection. Antibody detection is not for diagnosis rather it is restricted for use in containment zone, for epidemiological purposes and for COVID-19 frontline workers only. [2] As of now there is only supportive and symptomatic treatment for coronavirus cases. There is no approved prophylaxis for SARS-CoV-2. Therefore, adherence to simple measures like cleanliness of hand by regularly washing hands, maintaining distance of about six feet among each other and use of personal protective equipment (PPE) are the only resort to combat with this situation.
Appropriate drug therapy and vaccine for prophylaxis is the need of the hour. Although conventional clinical trials are different from ongoing trials but looking at the enormousity and magnitude of the problem and collateral damage it has incurred to global economies, potential vaccine candidates are explored for their safety and efficacy with unprecedented speed and scale therefore this review highlights on various vaccine candidates under clinical trials.
Since, healthcare providers are the frontline fighters in this global battle against COVID-19, it is important that they must be aware of the developments being made in the research regarding vaccine development for the pandemic. Thus, the present review is to provide them insight of the past experiences in vaccine development and the present progress being made to battle off pandemic and the challenges involved in the process.

Historical Milestones in Vaccine Development
For centuries, infectious diseases have been the center stage of medical practice and have been the prime cause for widespread mortality and morbidities in the people, especially during epidemics and pandemics. Starting from the first recorded evidence of vaccination 'Variolation'. [5] and several others in the pipeline. [6] The knowledge of immunology and microbiology has yielded in a sharp fall in cases per units of population of these diseases. Vaccines can be of several types such as live vaccines which are attenuated vaccines, killed vaccines, recombinant vaccines, toxin derived vaccines, conjugate vaccines, nucleic acid vaccines, and vector vaccines. [7] With disease prevention as the main goal in mind, vaccines are administered as either designed specifically for a particular disease keeping in mind the various strains of the pathogen and its structural characteristics as well as its interaction with host tissues.
There are several factors that need to be noted before-hand such as the population demographic (target population) and its socio-cultural factors, incidence of target disease, environmental factors, dose and route of administration, strategy for inducing herd immunity and regulation of vaccine administration as well as risk analysis of the target disease and the vaccine. The process itself typically takes up-to 12-15 years for a vaccine to be approved by regulatory authorities. [8] The effectiveness and safety of vaccines are the two main concerns making the process longer. The safety and efficacy of a vaccine is dependent on its ability to prevent development of the disease in the host on interaction with the native pathogen with minimum adverse effects such as ADE or antibody dependent enhancement producing aggravated form of the disease upon vaccination or presence of contamination in the vaccine (example-Rota virus vaccine found contaminated with Circo viruses). Vaccine safety is initially assessed in laboratory with studies on animals such as mice or rabbits. If they show promising results after receiving the vaccine, clinical trial on human volunteers is initiated. Like most other drug trials, vaccine development occurs in three phases in which the number of volunteers are progressively increased with continuous monitoring for any adverse effects such as severe inflammation or pain at injection site, immune malfunction or any delayed effects associated with the vaccine.
As the number of volunteer's increases in each phase there is a higher probability of a rare complication of the vaccine presenting itself that had been missed when the group was smaller. Other factors such as heterogeneity of immune response in different age brackets of the population also need to be tested separately during the three phases of vaccine development. [9] Vaccine development being a biological process involving pathogens and the high risks of contamination or adverse reactions in the population that often have a delayed response require monitoring for long periods of time, making it a long and exhaustive process requiring good financial funding.

SARS (Severe Acute Respiratory Syndrome) Vaccine
SARS was n highly contagious viral infection caused by virus of Coronavirus family. This outbreak emerged in Guangdong province of China in 2002. There were several outbreaks of this disease in the early 2000s either from SARs-CoV-2 isolates from laboratories or from people coming in contact with animal hosts of the virus. [10] Coronaviruses have been divided into antigenic three antigenic groups. SARS-CoV-2 does not belong to any of these antigenic groups, though there has been some resemblance of the virus to group 2 coronaviruses. [11] It is believed that SARS-CoV-2 may have originated in lower mammals but unlike humans did not result in disease. The virus is believed to have evolved under certain conditions to gain ability to transmit from animal host to humans and cause mild disease in them and then further evolve to have the potential to outbreaks with high mortality. [12] For production of SARS vaccine there are three possible approaches-1. The virus possesses various proteins which comprise of structural proteins like N, E, and S proteins where N is nuclear capsid, E is Outer envelope while S is spike glycoprotein. All of which can serve as antigens to induce production of neutralizing antibodies. [10] Formaldehyde, Ultra Violet light, and beta-propiolactone can be used to produce killed SARS-CoV-2 vaccine and has shown commendable results in animal models. [13,14] However, there are several safety concerns associated with this type of vaccine putting the production workers handling the vaccine at risk of infection as well as the vaccinated population in case of incomplete inactivation of the virus that has a potential for outbreaks of SARS. Viral proteins also have potential for inducing inflammatory responses or SARS like disease. [15] 2. SARS-CoV-2 has a transmembrane glycoprotein which is results in production of the neutralizing antibodies and which are responsible for attachment of virus to the host receptor, Virus fusion to host membrane and virus entry in to the host, these glycoproteins are known as S protein. It can thus be used in vaccine production. It is a single peptide containing three domains-these are trans membrane domain, intracellular domain and extracellular domain. The extracellular domain contains two parts -S1 and S2-of which S1 attaches to the host receptor which are ACE-2 receptors, found abundantly on various organs. The site on S1 binding to ACE-2 is known as the receptor binding domain (RBD) which results in virus and host receptor interaction. It then leads onto viral and host membrane fusion and viral entry. [16][17][18] Studies have demonstrated high level of titers of antibodies which are neutralizing in nature, in animal models which protected them when challenged with SARS-CoV-2. However there is a risk of enhancement of disease which is antibody dependent phenomenon known as ADE, on subsequent infection with the same virus. [19] Weingartl et al. demonstrated liver damage in ferrets if challenged by SARS-CoV-2 after vaccination with Modified Vaccinia Ankara-based Corona vaccine expressing Spike protein.
These findings in animal models are responsible for concerns regarding the safety as well as efficacy of vaccines associated with S protein use. RBD fragment on S1 domain has been explored for vaccine development as it was observed that serum antibodies derived from the animals previously immunized with killed SARS-CoV-2 and SARS patients reacted strongly with the receptor binding domain. [14] The antibodies derived from serum of animals which were previously immunized were specific to receptor binding domain of the virus and had neutralizing potential. [20] Major hurdle in the development of vaccine for SARS is finding animal models which would be affected by SARS-CoV-2 in the same way humans would.

Components Which Can Be Used for Vaccines
The selection of pathogenic components used as vaccines need to be theoretically and clinically evaluated in terms of efficacy, safety and tolerance in the target population. These components should have the potential to stimulate an immunogenic reaction in the host without any severe adverse effects. Upon exposure of the host to the wild type pathogen in vivo, the vaccine should be able to prevent infection with the pathogen. This potential is often estimated with increased titers of neutralizing antibodies after vaccine administration, but is not an absolute indicator of successful vaccination. The various vaccines for COVID-19 in various phases are described in Table 1 Protein subunit-These subunit vaccines contain selected antigens of the pathogenic organisms, mainly surface fragments, that are obtained from either conventional cultures or recombinant DNA technology. [12,22] These fragments are often coupled with other protein carriers or aluminium salts due to possibility of loss of immunogenicity by antigen purifiers. [23] Currently one vaccine is undergoing phase one trial.

RNA vaccines-
The mRNA coding for the S (spike) protein in SARS or some other surface antigen is used to create a laboratory like environment in cells of the host. [24] The immune cells process the mRNA in the lymph nodes and synthesize viral antigens which are then recognized by the other immune cells. [25] These antigens produced inside the host body thus induce immunogenic reaction similar to the viral pathogen. There is one vaccine in phase 1 and two vaccines in phase 1 and 2 of clinical trials.
DNA vaccines-With the advent of recombinant DNA technology, target immunogenic molecule/antigen can be encoded on plasmid and injected in the host with a promoter to initiate immunogenic protein synthesis. [26] They are limited to Volume 10 : Issue 7 : July 2021 Contd... Contd... Contd...  protein immunogens but the immunity conferred by plasmids is long term and they are easy to manufacture. There are two vaccines in phase 1 and one vaccine in phase 1 and 2 of clinical trials.
Virion like Particles (VLPs)-These are complex vaccines with multiple protein in their structure which are designed to mimic native virus while lacking the viral genome making them safer vaccination options. [27] Autologous dendritic cells loaded with SARS antigen-This technology of vaccine development uses serological testing of sera of volunteers who have not had previous contact with SARS.
The peripheral blood monocytes are isolated from heparinized blood, during in vitro culture with cytokines like interleukin-4 and granulocyte monocyte colony stimulating factor these are differentiated into dendritic cells during. These dendritic cells along with SARS-CoV-2 antigen are incubated. There is one such vaccine in phase 1 and 2 of clinical trial.

Similar Viral Vaccines
The improvement in our understanding of viral behavior and its antigens, cell culture, molecular biology and newer antigen delivery systems there is a vast variety of viral vaccines that have been designed showing promising results, particularly in viral diseases with few or no interventions available. Vaccines of several viral diseases like small pox and polio has managed to nearly complete eradication of these diseases by arming the immune system of the population vaccinated as well as inducing herd immunity. [28] In India there are several viral vaccines available that have been integrated in several health programs to reduce disease load in the population. [29] These includes the following enlisted vaccines Vaccines against Rotavirus diarrhea under the name of RotaTeq is a live vaccine which is pentavlent with human-bovine reassortant vaccine with G1, G2, G3, G4 and P1A [8] viruses and Rotarix which is live-attenuated monovalent human vaccine containing G1P [8] virus. Vaccine against the Pandemic H1N1 influenza under the name of Nasovac which is a live attenuated vaccine. H1N1 influenza virus causing pandemic is often cultivated on embryonated hen's egg.
Vaccines against Polio under the names of monovalent oral polio vaccine (mOPV) type 1 containing live attenuated Sabin strain of poliomyelitis type -1 virus which is propagated in monkey kidney cells; mOPV type-3 which contains live attenuated Sabin strain of poliomyelitis type-3 virus grown in monkey kidney cells; OPV and Biopolio which are live attenuated poliomyelitis types 1 and 3 viruses of Sabin strain grown in monkey kidney cells.
Vaccine against Japanese encephalitis which was derived from mouse and later inactivated by formalin was discontinued in 2008. [30] The vaccine currently used in India is imported from china which is SA 14-14-2, a live attenuated JE vaccine cultured in primary hamster kidney cell-culture. There are also several JE vaccines in advanced stages of development such as IMOJEV which is a chimeric vaccine using yellow fever virus 17-D and IXIARO which is a purified inactivated vaccine (PIV). [2,29,31] They have shown promising results in the pre-clinical and clinical stages of vaccine development. Intercell, the Austrian company developing IXIARO has a partner in India, Biological E, which will be responsible for the vaccine's production and marketing when it gets approved by the governing authorities.
Vaccine candidates against Dengue, Chikungunya and Chandipura virus have been undergoing active research for decades albeit without much success due to several efficacy and safety concerns as well as difficulties in vaccine production and delivery systems. [29] There are several other viral diseases in the world which have effective vaccines available against the pathogen proving useful particularly in disease endemic areas. One such is the vaccine against yellow fever which acts as the single most important method of prevention of this disease, particularly for people travelling to endemic areas psych as Congo, Guinea, Uganda Brazil, Colombia, Peru etc., The yellow fever vaccine, 17D-204 YF, is a live attenuated freeze dried vaccine and is considered highly effective in preventing the disease. [32] HIV being an important disease responsible for widespread mortality and worsening of quality of life, there have been several attempts to develop a vaccine for HIV involving four concepts which are use of monomeric gp120 subunit which is a component of HIV-1 surface glycoprotein and adjuvant as alum. This concept did not help in disease prevention due to multiple reasons in the clinical trials, one of them being the disparities between the neutralizing potential of laboratory cultivated isolates of HIV-1 (easily neutralized) versus the primary isolates of the patients. [28] Use of recombinant poxvirus vector vaccine and other proteins that regulate important functions of virus. This was followed by boosting with recombinant subunit envelope to provide additional protection from potentially breakthrough infections. There were mixed opinions on the efficacy of this concept. Recombinant adenovirus serotype 5 (rAd5) vaccine contains important structural proteins like Gag, Pol, and Nef which would induce a response biased for CD8+ T-cells. This vaccine put to trial failed to prevent HIV infection, particularly due to absence of HIVsurface antigens.
Priming with DNA prior to boosting with rAd5, in addition to expressing Gag, Pol and Nef envelope proteins representing three major HIV-1 subtypes were expressed with the hopes of inducing antibodies which could neutralize HIV in vivo. The trials for this as well did not yield promising results to be continued.

Challenges in Vaccine Development
The These include all other platforms that are currently explored like Virus like particle (VLP's), use of killed non -CoV-2 (killed rabies virus) or live modified horse pox virus and repurposed vaccines like BCG, MMR, OPV3. [3] But development of COVID-19 vaccine in such short span of time comes with many challenges. 1. Although many companies world-wide have started clinical trials of potential vaccine candidates and claim that vaccine will be marketed soon but reality seems like mirage as many factors have to be taken in consideration before manufacturing vaccine, safety and effectiveness of the vaccine candidate is of almost importance, along with the type of vaccine and platform used for vaccine development all needs to be considered. [33] There are some new platforms used for vaccine development which need stringent testing and scrutiny while other platforms are previously known models which need to be modified for development of Coronavirus vaccine. [34] 2. The time constraint that we are facing presently and looking at the enormous magnitude of SARS-CoV-2 cases, there is a need of large scale production of vaccine, the time period required to manage the demand of vaccine is quite different from the reality. [22] 3. In pre-clinical trials of some vaccine candidates it is seen that anti-SARS and anti-MERS vaccine are effective in inducing immune response but they may result in severe form of disease when virus is subsequently inoculated. This needs further studies as can result in severe adverse effect in clinical trials. Also although Antibody dependent enhancement (ADE) phenomenon has not with reported in SARS-CoV-2 cases, but still should be under consideration while vaccine development as it is been seen in other viral infections like Dengue, Zika and influenza virus. [35] 4. Various parameters like age and co-morbidities are to be kept in mind while developing a vaccine, age being one of the important factors. Studies on influenza virus have shown that with ageing of immune system there is reduction in the effectiveness of vaccination. [36] 5. Most of the candidate vaccine under trial broadly induces antibodies against viral spike (S) protein. Spike proteins are responsible for uptake of virus via human ACE-2 receptors. Spike protein has 2 sub units S1 and S2. Among these two, S1 subunit is specific for SARS-CoV-2 and helps in differentiating between other corona viruses and thus makes it good target for vaccine development, while S2 sub unit shares epitopes with other SARS viruses. SARS-CoV-2 S1 subunit has undergone mutation with alteration in its component which has led to complete replacement from the ancesteral O subtype (seen in Wuhan) to A2a, which has made n-COV-2 more virulent. The virus spike protein is undergoing spontaneous alteration thus making it difficult as target for vaccine development. [36] 6. Last but not the least it is an essential question that needs to be answered the time frame till which vaccine will provide protection needs further evaluation. [37] Conclusion Novel coronavirus disease has become public health issue of international concern. The exponential rise in COVID-19 cases across world has forced authorities to take concrete steps in the form of government-imposed lock down, which has ceased all commercial activities and has given a harsh blow to global economy, we must be hopeful that given the speed and scale of research work, funding by Govt. as well as by non-government organizations (NGOs) and the efforts of major pharmaceutical companies, vaccine for SARS-COV-2 will be available to the general population. We primarily rely on symptomatic and supportive management (Corticosteroids, Remdesivir and Tocilizumab) and therefore prevention is important to avoid transmission of the infection.
It is learned from the past experiences that the vaccine development is a time and resources consuming process with many scientific and regulatory challenges. Despite of advancement of science, the scientists globally are struggling from many decades to discover vaccines against diseases like AIDS, malaria, tuberculosis etc., without any significant success so far. Major challenges involved in vaccine development are the continuous structural and functional mutations in microbes and variations in response due to geographical and ethnical differences. Thus, the strategy till the vaccine is available commercially for public use, is to control the spread of pandemic and the symptomatic and supportive treatment to reduce morbidity and mortality.

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