Recent advances in vaccine and immunotherapy for COVID-19

ABSTRACT The COVID-19 pandemic caused by SARS-CoV-2 has resulted in millions of cases and hundreds of thousands of deaths. Beyond there being no available antiviral therapy, stimulating protective immunity by vaccines is the best option for managing future infections. Development of a vaccine for a novel virus is a challenging effort that may take several years to accomplish. This mini-review summarizes the immunopathological responses to SARS-CoV-2 infection and discusses advances in the development of vaccines and immunotherapeutics for COVID-19.


Introduction
The 2019 coronavirus disease (COVID- 19) was first reported in late December 2019 in Wuhan; China has now become a global pandemic. The virus causing COVID-19 is the Severe acute respiratory coronavirus syndrome 2 (SARS-CoV-2) that belongs to the coronaviridae family of viruses. The characteristic feature of coronavirus is the presence of club-like extensions on the surface made of glycosylated trimers of S protein. The coronaviruses are roughly 80-120 nm in diameter. 1 COVID-19 resulting from the new SARS-CoV-2 infection has now become a global health concern. The incubation time of the virus is about 2-10 days, and it is transmitted through aerosol from human-to-human and also through contaminated inanimate objects and hands. 2 The virus can remain infective on the surfaces of objects for up to 9 days at room temperature. However, the viral survival declines with temperatures above 30°C. It can be efficiently inactivated by surface disinfection procedures with 62-71% ethanol, 0.5% hydrogen peroxide or 0.1% sodium hypochlorite within 1 minute. 2 First described in China, SARS-CoV-2 has been reported in essentially all countries worldwide, with more than 15 million infected subjects and more than a half-million deaths. Owing to the global spread, WHO declared COVID-19 as a pandemic on 11 March 2020. 3 Since the first report of the genomic sequence of the SARS-CoV-2 has come, researchers, clinicians, and pharmaceutical companies have devoted all their resources and research toward developing therapeutic modalities and vaccines for SARS-CoV-2.
Most of the data on antiviral therapy is based on the clinical and preclinical studies on other related viruses such as SARS-CoV, Middle East coronavirus respiratory syndrome (MERS-CoV) and non-coronavirus (Ebola). This narrative mini-review summarizes epidemiology, pathogenesis, immune responses, vaccine development issues, and immunotherapy for COVID-19 and provides an update on recent advances for vaccine and immunotherapy.

Pathogenesis
The SARS-CoV-2 resembles the SARS-CoV in several aspects. Homology modeling revealed that both the viruses employ similar receptor-binding domains to attach to the host cells with subtle differences in particular amino acid residues. 4 The coronaviruses have spike proteins that are glycoproteins and consist of two subunits: S1 and S2. The S1 and S2 proteins are the most important structural proteins of the virus. The spikes on the surface of the SARS-CoV-2 are homotrimers of S proteins that establish attachments with the host cell receptors. 5 The structural and the non-structural proteins (nsps) in co-ordination carry out the CoV pathogenesis and decide the virulence. 6 The virus entry Coronaviruses enter into the host cells by using the viral S protein. 7 SARS-CoV-2 enters into the host cell by the interaction of its S protein with the host receptor "ACE2" present in most of the human cell types. 8 The viral RNA is transferred into the host cell cytoplasm as soon as it enters the host cell. The viral genome translates its two polyproteins and structural proteins. These proteins enable the viral genome to replicate inside the host cell. 9 The nascent viral glycoprotein envelope is processed in the endoplasmic reticulum or Golgi membrane. Then, the genomic RNA and the nucleocapsid proteins fuse to form the nucleocapsid. The newly formed viral particles then fuse with the vesicles in the intermediate reticulum-Golgi endoplasmic compartment (ERGIC) followed by the fusion of these virus-containing vesicles with the plasma membrane that leads to the virus release. 7

Antigen presentation
To date, there are no reported studies on the immune mechanism of SARS-CoV-2 infection. However, the studies on the immune mechanisms of the related viruses like SARS-CoV and MERS give much insight into the immune mechanism of the virus. 10 Upon entry into the host, the virus presents its antigens to the antigen-presenting cells (APCs) of the host mediating the antiviral mechanism of the host immune system.

Humoral and cellular immunity
Antigen presentation by the APCs results in the activation of the cell-mediated and the humoral immunity of the host governed by T cells and B cells, respectively. The antibody response (levels of IgM and IgG) to SARS-CoV follows a characteristic pattern. 11 The IgM antibody levels reach undetectable levels by the end of 12th week of infection, but the IgG remains for more extended periods. 12 SARS-CoV infection induces concomitant activation of T cell and B cell-mediated immune responses. Upon SARS-CoV infection, B cell responses are first observed against the nucleocapsid (N) protein followed by responses to S protein which is seen within 4-8 days after the onset of symptoms. 13,14 Neutralizing antibody responses for the S protein begins by 2nd week. Many patients develop the neutralizing antibody responses by 3rd week. 15,16 Since viral titers are observed to peak earlier for SARS-CoV-2 as compared to SARS-CoV, the antibody responses may also be elicited earlier. 17,18 It has been observed that a subset of infected patients do not develop long-lasting antibody responses to SARS-CoV-2. However, it is not clear whether these patients are more susceptible for re-infection. 19,20 It has been documented that the population of CD4 and CD8 T cells significantly falls in the patients infected with SARS-CoV -2. 21 It was seen that the antibody-secreting cells (ASCs) in the blood of a SARS-CoV-2-infected patient increased from day 7 (1.48%) to a peak level on day 8 (6.91%). Similarly, the cT FH cells increased from 1.98% on day 7 to 3.25% on day 8. The cT FH cells peaked on 9th day (4.4.6%). This observation indicates that both humoral and cell-mediated immunity comes into play in response to SARS-CoV-2 infection. 22 An accumulation of mononuclear cells suspected to be monocytes and T cells was observed in the lungs of a COVID-19 patient along with decreased systemic levels of hyperactive T cells. 21 Lymphopenia and decreased levels of peripheral T cells indicate that the T cells are migrated toward the lungs from the systemic circulation to the site of infection (primarily lungs) to counteract the viral infection. [23][24][25][26] Increased exhaustion and reduced functional diversity of T cells may be predictive of severe disease progression. 27 It has been seen that the patients recovered from SARS-CoV developed coronavirus-specific memory T cells, seen up to 2 years after recovery. 28,29 It is quite evident from these reports that T cell-mediated immunity plays an important role in controlling infection. However, several vaccine agents designed against SARS-CoV resulted in immunopathology due to TH2 cell-mediated infiltration of eosinophils. 30,31 The vaccinated mice showed increased immunopathology than protection against SARS-CoV infection. 32 Therefore, further extensive studies are needed to evaluate the protective versus damaging T cell responses, which is essential for designing vaccines for the coronavirus. 33 However, it is very important to investigate whether T cell-mediated responses are solely responsible for infection control in humans. This will provide impetus to the vaccine development process.

Cytokine storm
Acute respiratory distress syndrome (ARDS) is the most common pathology seen in patients infected with SARS-CoV-2, SARS-CoV, and MERS. 21,34 A hyperinflammatory condition associated with hypercytokinaemia is often observed in COVID-19 patients. 34 This hyperinflammatory condition is related to multiple organ failure. 35 The cytokine surge seen in COVID-19 patients results due to increased levels of the proinflammatory cytokines such as IFN-a, IFN-g, IL-1b, IL-6, IL-12, IL-18, IL-33, TNF-a, TGFb and chemokines such as CCL2, CCL3, CCL5, CXCL8, CXCL9, CXCL10, among others, by the cells of immunity system. 34,36 This cytokine storm may be followed by multiple organ failure and ARDS resulting in the death of the patients infected with SARS-COV-2 as seen in SARS-CoV and MERS-CoV infection. 21

Evasion of the host immune response
As the SARS-CoV-2 is very new to the researchers, not much data are available on the immunopathological mechanisms and the tricks of the novel virus to escape the host immune response. However, data from the studies on the previously known coronaviruses like SARS-CoV and MERS can be utilized to speculate the possible mechanisms this new SARS-CoV -2 virus may employ to deceive the host immune system. The pattern recognition receptors (PRRs) can identify the evolutionarily preserved microbial structures called pathogenassociated molecular patterns (PAMPs). As a defense mechanism against the host cells, these viruses (SARS-CoV and MERS-CoV) may develop modified membranes derived from host cell components such as developing double-membrane vesicles that lack or have altered pathogen-associated molecular patterns (PAMPs) hence unrecognizable by the host cell pattern recognition receptors (PRRs). This facilitates viral replication without their RNA being recognized by the host cell. 37 Another mechanism seen in MERS infection can be thought of being utilized by the SARS-CoV-2 virus to escape the host immunity. It has been recognized with MERS infection that the expression of genes related to antigen presentation is downregulated upon infection, facilitating the virus evade the first checkpoint of host immunity. 38 Research studies have evidenced the fact that nsp can suppress the innate immune response of the host. 6

Need for vaccine development
Although remdesivir is available in developed countries as a specific antiviral drug that has been shown to be effective in reducing symptoms and accelerating recovery from COVID-19 disease, it is not affordable in most parts of the world and is in limited supply, such that much of the world awaits an inexpensive and effective antiviral drug. The treatment for COVID-19 relies on the management of the symptoms focused on the symptomatic management of the patients which include controlling secondary infections by the administration of broadspectrum antibiotics, ventilation, and fluid control. 11,34

COVID-19 vaccine development platforms and challenges in COVID-19 vaccine development
The public health threat of COVID-19 will remain until a potential and effective vaccine is developed. 39 Several companies have taken the initiative of developing the vaccine against COVID-19 targeting the SARS-CoV2 virus continuously circulating in the human population. 40 Various strategies such as live-attenuated virus, viral proteins, viral nucleic acid, virus-like particle, peptide, viral vector (replicating and non-replicating), and recombinant protein approaches are being used for vaccine development against SARS-CoV-2. These strategies have their own associated advantages and disadvantages. 5,41 Viral antigen-based and nucleic acid-based vaccines are safer, but the immunogenic potential of these vaccines is less. The nucleic acid-based vaccines are the fastest to enter into phase 1 clinical trial, but to date, there is no nucleic acid-based vaccine licensed to be used in humans. Since safety is a concern with a live-attenuated virus, it is risky for the vulnerable older population. 41 Nucleic acid-based platforms provide wider options for antigen manipulation. Therefore, vaccines based on nucleic acid approaches might be considered as one of the important approaches for the development of a vaccine for SARS-CoV2. 42 Viral vector-based platforms offer advantages such as high level of protein expression, stability, and induction of strong immune response. Since vaccines based on recombinant proteins are already licensed for several other diseases, the existing large-scale production capacity can be utilized for the production of vaccines for SARS-CoV-2. 42 It is possible that some platforms may be more appropriate for a specific group of population such as the aged, children, pregnant women or immunocompromised patients.
Since our knowledge about the immune responses to the vaccines is not entirely established, different strategies should be tried for designing a vaccine for COVID-19. Only advances and comprehensive research can answer this question about selecting the best approach for vaccine development for COVID-19. Distinctive challenges for SARS-CoV-2 vaccine development include clinical recruitment, defining a correlate of protection, and proving efficacy, especially when there is public pressure to release a vaccine for general use. Despite the development of novel platforms for vaccine development, there are several underlying challenges in SARS-CoV-2 vaccine development.
Although the viral S protein is a potential immunogen, it is essential to evaluate the optimal immune response elicited by the antigen. It is still a matter of debate whether the full S protein or only the receptor-binding domain is suitable for achieving optimal immune response. 33 Previous experience with SARS and MERS vaccine candidates has raised concerns about the exacerbating lung disease, either directly or due to antibody-dependent enhancement (ADE). This adverse effect may be associated with Th2 response. Production of suboptimal levels of antibody or antibody of low quality can result in the phenomenon of ADE that promotes the disease pathology. This warrants consideration of ADE in the evaluation of safety of the emerging candidate vaccines for SARS-CoV-2. Therefore, testing of the vaccine candidates for safety in suitable animal models and constant safety monitoring in clinical trials is quintessential. It is premature to define a good animal model for COVID-19. 33 Another major challenge in SARS-CoV-2 vaccine development is establishment of correlates of protection. Experience with SARS and MERS vaccines may be utilized to establish correlates of protection. However, they are still not established. The duration of protection and whether a single dose of the vaccine is sufficient to elicit the required immune response is uncertain. 33 Vaccine development is a long process and involves large sample sizes for conducting research; it may take years to establish a vaccine. Since it is associated with high costs and failure rates, the developers take extra precaution and follow a strict sequence of steps involving multiple rounds of data analysis and strict checks at manufacturing levels. Developing a vaccine in an outbreak scenario requires a new strategy of executing multiple steps in parallel even before confirmation of the outcome of another step. 33 The other challenge is faced at the commercial production of clinical trial materials. For the novel platforms, large-scale production has never been done before. So, it will require large-scale production (identification, technology transfer, and manufacturing process) without the knowledge of the viability of the vaccine candidate. It is not certain whether these novel platforms are scalable and if it is possible to produce sufficient quantities using the existing capacity. 33 Performing clinical trials during a pandemic is associated with the additional challenge of choosing trial sites as it is difficult to predict where and when outbreaks will occur. So, if multiple vaccines are ready, countries should not be crowded with multiple clinical trials. In a pandemic situation where the mortality rates are high, people may not consent for randomized controlled trials with placebo. 43 One way to tackle this problem is utilizing a single-shared control group for testing several vaccines simultaneously. In this approach, more people will receive an active vaccine. 44 Although this approach is advantageous, it is associated with statistical complexities. Developers may try to avoid direct comparative analysis of their vaccine candidates with others.
Finally, there will be a surge of demand for the vaccines globally. Hence, serological studies will be needed to establish which populations are at higher risks so that they can be prioritized for the vaccine allocation. 33

Advances in vaccine development for COVID-19
Several companies, research laboratories and universities are researching to come up with a vaccine for COVID-19. According to WHO, as of August 25, 2020, there are 31 vaccines in the clinical evaluation phase (Table 1) and 142 vaccines in the preclinical evaluation phase (according to the WHO draft, August 25, 2020) ( Table 2). 45 (Table 1). Three vaccine candidates listed in Table 1 are in phase 2 clinical trial. Adenovirus Type 5 Vector is a single dose intramuscular non-replicating viral vector vaccine. Adjuvanted recombinant protein (RBD-Dimer) is a double or triple dose intramuscular protein subunit vaccine. The other vaccine candidate in phase 2 clinical trial is a double dose intramuscular mRNA vaccine (Table 1).

After the vaccine is developed
It is not the only challenge to come up with an effective vaccine for COVID-19 in a short period but the most significant problem would be getting enough doses of the vaccine to be supplied to the countries globally. There is a risk that the more affluent countries can monopolize on the supply of COVID-19 vaccines globally in a similar way that happened with the flu pandemic. Therefore, along with focusing on the vaccine development, we should focus on containing the spread of the virus. Economically weak countries as in Africa would face problems in accessing vaccines, as happened with anti-HIV drugs. Due to the high rates of HIV drugs, several poor people died in Africa not being able to afford it. Therefore, there should be a fair distribution of the vaccines if any company succeeds in the race of developing a vaccine for COVID-19.

Convalescent plasma therapy
Convalescent plasma therapy is an old concept of separating serum from the blood of a patient who has recovered from infection and injecting it to another infected patient. The convalescent plasma contains the antibodies for the infectious pathogen which neutralizes the pathogen in the new recipient patient. This therapy can be useful in treating COVID-19 patients.
The evidence coming from studies that reported the use of convalescent plasma therapy in treating past coronavirus infections such as SARS and MERS compelled the researchers to apply this therapy on COVID-19 patients. [46][47][48][49] Recent studies have highlighted the beneficial effects of convalescent plasma therapy in critically ill COVID-19 patients. 50 Among the five critically ill COVID-19 patients who were administered convalescent plasma, three patients were discharged upon recovery, and two patients are in the incubation period of 37 days. 50 This treatment modality is associated with some disadvantages. Convalescent plasma therapy increases the risk of serum related disease and antibody-dependent enhancement of infection. There is always the risk of transmission of other infectious diseases through the serum and the additional risk associated with convalescent plasma therapy is the chances of developing infection from another viral strain due to antibodies against one form of coronavirus. 51 All published studies on clinical trials with convalescent plasma did not include a negativecontrol group needed to judge the efficacy of the intervention. Therefore, the need of the hour is the identification of the human monoclonal antibody for a common antigenic determinant/epitope of SARS CoV-2 to prevent COVID-19.

Monoclonal antibody therapy
Human monoclonal antibodies such as 80 R, m396, and S230.15 specific for the S1 domain of the SARS CoV have been reported to be effective in neutralizing the SARS-CoV infections by inhibiting their binding to the ACE receptors on the host cells. 52 Another study reported that the monoclonal antibody CR3014 reduced the rate of replication of SARS-CoV genome and inhibited viral shedding, thus wholly prevented the virus-induced lung pathology. This antibody also works on the principle of inhibiting the binding of SARS-CoV by reducing the affinity of the S1 domain of the virus for the ACE receptor on the host cells. 53 Since the receptor-binding domain of SARS-CoV-2 differs significantly from the SARS-CoV virus, the monoclonal antibodies (as m396, CR3014) targeting the S1 domain of SARS-CoV may not be effective in neutralizing the novel SARS-CoV -2. A recent study highlighted that the human monoclonal antibody CR3022 completely neutralizes both SARS-CoV and SARS-CoV-2. Therefore, these monoclonal antibodies can be considered for use in the prevention and treatment of COVID-19. 54 Another study reported that the human monoclonal antibody 47D11 neutralizes SARS-CoV-2 by binding to the    conserved sequence on the receptor-binding domain of the S1B protein. These antibodies can slow down the viral infection and can impart immunity in the uninfected persons. 55 The receptor-binding domain is the best target to develop monoclonal antibody treatment to manage or prevent SARS-CoV-2 infections. Takeda Pharmaceutical Company based in Japan is in the process of preparing a monoclonal antibody mixture, TAK-888 from the serum of recovered COVID-19 patients to come up with a new treatment strategy for COVID-19. Another pharmaceutical company, Vir Pharmaceuticals, USA, is testing antibodies isolated from recovered SARS patients to neutralize SARS-CoV-2. This company has also collaborated with a China-based company, WuXi Biologics, to develop a serumbased therapy to tackle SARS-CoV-2 infection in critically ill patients. 56 A limitation to the use of convalescent plasma and MAbs is that they might benefit hospitalized patients but will not be generally useful for the population.

Conclusion
Several companies have initiated the development of antiviral and vaccines for COVID-19. Different approaches have been undertaken to develop an effective vaccine for COVID-19 such as attenuated virus, viral proteins, viral nucleic acid, virus-like particle, peptide, viral vector (replicating and non-replicating), and recombinant proteins. However, the most significant challenge post-vaccine development will be the fair distribution of the vaccines globally. Convalescent plasma therapy and monoclonal antibody therapy are also being tested and can be the potential therapeutic modality for the management and prevention of COVID-19. However, they might benefit only the hospitalized patients and will not be generally useful for the population.

Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.