key: cord-0996825-550ak5vg
authors: Marian, AJ
title: Current State of Vaccine Development and Targeted Therapies for COVID-19: Impact of Basic Science Discoveries
date: 2020-09-02
journal: Cardiovasc Pathol
DOI: 10.1016/j.carpath.2020.107278
sha: 32390d6b3d8b2dd7928106aa3d58f03f485213b9
doc_id: 996825
cord_uid: 550ak5vg

Coronavirus disease-19 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is closely related to two other coronaviruses that caused disease epidemic breakouts in humans in the last 2 decades, namely, severe acute respiratory distress syndrome coronavirus (SARS-CoV) and Middle East Respiratory Syndrome coronavirus (MERS-CoV). The similarities have enabled the scientists to apply the basic scientific discoveries garnered from studying the structure and modus operandi of SARS-CoV and MERS-CoV to develop therapies that specifically target SARS-CoV-2 and to develop vaccines to prevent COVID-19. Targeted therapies, including the use of antibodies to prevent virus entry, nucleotide analogues to prevent viral replication, and inhibitors of proteases to prevent virion formation, among others, are being tested for their clinical efficacy. Likewise, complete sequencing of the SARS-CoV-2 and identification of its structural and non-structural proteins have enabled development of RNA-, DNA-, and peptide -based vaccines as well attenuated viral vaccines to instigate the host immune responses. The clinical impacts of the basic science discoveries are amply evident on the rapid pace of progress in developing specific anti-viral therapies and vaccines against SARS-CoV-2. The progress emphasizes the merit of discovering the fundamental scientific elements, regardless of whether or not they have apparent or immediate clinical applications.

Since first diagnosed in early December 2019 in the city of Wuhan in China, the coronavirus disease-19 has become a pandemic with a colossal global impact. The disease is caused by a coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). [1] COVID-19 along with severe acute respiratory distress syndrome (SARS) and Middle East Respiratory Syndrome (MERS), are the third epidemic outbreaks caused by coronaviruses in humans. The genome of SARS-CoV-2 has 80% sequence identity to that of SARS-CoV, which caused the first outbreak in humans more than a decade ago, and about 97% sequence identity to a bat coronavirus (CoV RaTG13) genome, the latter indicating its origin from the bat virus. [2] The genomic similarities have afforded the scientists the opportunity to apply the knowledge gained from studying SARS-CoV and bat coronaviruses in deciphering the molecular underpinning of SARS-CoV-2 structure and function and therefore, the pathogenesis of COVID-19. Today not only the genomic sequence of SARA-CoV-2 is fully known but also various protein constituents of the virus and its modus operandi for replication and infection of the host cells have been reasonably well-characterized. This is not to be interpreted that all is known and nothing is left to discover. To the contrary, there is plenty of unknowns about this virus and about this infection. Nevertheless, the fundamental discoveries about genetics and molecular biology of coronaviruses have paved the way in an unprecedented way for the rapid development of virus-specific therapeutics and vaccines, which are essential for the ultimate elimination of COVID-19.

SARS-CoV-2 is a positive sense, single-stranded RNA virus whose genome is comprised of 29,903 nucleotides (NCBI Reference Sequence: NC_045512). [2] . The genome codes for the viral proteome, comprised of four major structural surface glycoproteins, namely, spike (S), matrix (M) and envelope (E) and the nucleoprotein (N), as well as 16 non-structural proteins (NSPs), the latter proteins are generated from cleavage of two large polypeptides. [3] The viral genome also contains several additional ORFs, which encode the accessory proteins involved in virus-host interactions. [3] The S glycoprotein (~141 kDa and ~1,270 amino acids) forms a homotrimer that protrudes from the cell membrane and gives the virus the appearance of a crown under electron microscopy, and hence the name coronavirus ( Figure 1 ). [4] The S protein is responsible for attachment of the virus to the host cell receptors, namely the angiotensin converting enzyme-2 (ACE2). [5] The M glycoprotein (~25 kDa, ~220 amino acids) is the most abundant structural protein, which through interactions with other structural proteins gives the virus its physical structure. The M protein also interacts with the S protein to retain the virus at the endoplasmic reticulum (ER)-Golgi complex, where the new virions are assembled and subsequently excreted via secretory vesicles. The excess load of the viral proteins during infection with SARS-CoV-2 could overwhelm the ER and leads to ER stress and consequent activation of the unfolded protein response (UPR). The nucleoprotein or the N protein (~46 kDa, ~420 amino acid) directly interacts with the viral RNA as well as the membrane proteins, such as the M protein, and forms nucleocapsid, which provides stability to the viral genome within the envelope. It also enables viral replication. The E protein (~ 10 kDa, ~75 amino acid) is the smallest of the structural proteins and contributes to viral production and maturation, as its absence significantly reduces viral titers and leads to production of incompetent viral particles. [6] These structural proteins not only interact with each other but also with a large number of host proteins, which are collectively responsible for part of the phenotypic effects of infection with SARS-CoV-2.

The viral genome also codes for 16 NSPs, which are involved not only in viral replication but also in suppression of the host defenses by various mechanisms, including degradation of host mRNAs and inactivation of protein translation. These NSPs along with the proteins coded by the ORFs and the structural proteins interact with over 300 human proteins to impact various biological effects. [7] Through meticulous cooperative interactions, the viral proteins confer the virus the ability to replicate effectively by copying the viral RNA, evade detection by the host immune system, and destroy the host-defenses.

(reviewed in [8] ) Several of these NSPs, because of their essential functions in viral survival and infectivity, are subject to specific therapeutic targeting.

The S glycoprotein, which gives the virus its crown, is critical for initiating the viral entry into the host cell ( Figure 1 ). The S protein is a homotrimeric protein comprised of S1 and S2 functional domains.

The receptor-binding domain (RBD) of the S protein is located within the S1 domain. It recognizes the human ACE2 protein, which cleaves angiotensin 1 to angiotensin 1-9 and angiotensin II to angiotensin 1-7, the latter with potential beneficial effects on cardiovascular system. [9] The affinity of the S protein of SARS-CoV-2 for human ACE2 receptor is several-fold higher than that of the SARS-CoV. [4] The enhanced affinity might explains the rapid spread of COVID-19 (more contagious) as opposed to SARS.

In the open state, which is the predominant state of the S protein, the RBD of the S protein binds to the ACE2, initiating the viral entry into the host cell. The successful viral entry after attachment of RBD of S protein to ACE2, however, requires proteolytic cleavage of the S protein by furin-like, trypsinlike, and cathepsin proteases, and the serine protease TMPRSS2 (Figure 1 ). [10, 11] Proteolytic modifications of the S1/ACE2 protein complex is considered a critical stage in viral entry, as inhibition of TMPRSS2 with serine protease inhibitor camostat mesylate blocks entry of SARS-CoV-2 into the epithelial cells. [12] TMPRSS2 also cleaves the ACE2 receptor, which might also facilitate entry of SARS-CoV-2 into the host cells. [13] In addition, FURIN is also a candidate to proteolytically modify the S protein and enhance viral entry into the host cell. [10, 11] Subsequent to the proteolytic cleavage of S1 declines within several weeks, the latter rises within 2-3 weeks but stays much longer. It is the latter antibody along with the memory T cells that confer host immunity to re-infection with SARS-CoV-2.

The virus is transmitted from person to person via airborne respiratory droplets and aerosols.

SARS-CoV-2 is more contagious but less lethal than SARS. Every infected person is estimated to transmit the virus to 2.1 individuals. Given the above basic reproductive number, herd immunity is expected to be achieved when about 2/3 rd of the population develop immunity. [19] It is estimated that between 10 to 30% of the infected individuals remain asymptomatic, albeit the true number of infected asymptomatic individuals might be even higher. The asymptomatic individuals by shedding the virus might be an important source of spread of the virus in the population. Given the relatively high number of asymptomatic infected individuals, one might posit that that exposure to the virus is inevitable.

Several factors have been associated with the clinical presentation of COVID-19, including age, biological sex, pre-existing medical conditions; such as cardiovascular disease, diabetes, and obesity.

Otherwise, the reasons for inter-individual variability in susceptibility to SARS-CoV-2, which ranges from an asymptomatic course to that of severe disease requiring intubation, are largely unknown. The The majority of the infected individuals (>80%) who are diagnosed with COVID-19 are minimally or mildly symptomatic. Viral shedding may occur about 3 days before development of symptoms and continues for a week after the onset of symptoms. In those who become symptomatic, the incubation period varies from 2 days to 14 days (average ~ 5 days). The most common symptoms are fever or chills, cough, dyspnea, fatigue, myalgia, headache, new loss of taste or smell, sore throat, rhinorrhea, nausea, vomiting, and diarrhea. The disease is self-limiting in the majority of patients but about ~15% of the patients require hospitalization primarily because of hypoxia, particularly in those with co-morbid conditions. About 5% of the infected symptomatic patients develop acute respiratory distress syndrome and require oxygen supplementation through intubation and other invasive procedures. Lungs are the primary organ involved but involvement of other organs, mostly indirectly because of cytokine storms is not uncommon. The heart is involved in about 20% of the cases, as evidenced by elevated Btype natriuretic peptide (BNP) and cardiac troponin I (TNNI3) as well as regional and global wall motion abnormalities with reduced ejection fraction on echocardiography. Direct cardiac involvement is less certain. Cardiac involvement is a major determinant of clinical outcomes in patients with COVID-19. In advanced cases multi-organ failure, including coagulopathy and lymphopenia develop and progresses to death. The overall case-fatality of symptomatic patients with COVID-19 varies with age and is highest in the elderly. [24] It is less (about 3.5%) in those diagnosed by testing alone (reverse-transcriptase polymerase chain testing alone), which includes symptomatic and asymptomatic individuals. The overall mortality varies from less than 3% in the young to ~30% in the elderly hospitalized patients and to more than 70% in those requiring endotracheal intubation. [24, 25] A number of factors are implicated in increased susceptibility to COVID-19 and its clinical outcomes, including aging and co-morbid conditions. Elderly with diabetes and cardiovascular diseases are particularly susceptible. Several factors are implicated in increased susceptibility of the elderly to COVID-19, in addition to the high prevalence of co-morbid conditions, such as the presence of underlying inflammatory state, referred to as inflammaging, reduced innate and adaptive immunity, altered airway function, and altered expression of ACE2 and other molecules involved in the pathogenesis of COVID-19. (reviewed in [26] ) Likewise, mortality of COVID-19 is the highest in the elderly, in part because of the changes discussed above and partly because of the presence of concomitant cardiovascular diseases, diabetes mellitus and others.

Although males and females are equally susceptible to infection with SARS-CoV-2, there is a sex-dependent difference in the clinical outcomes of COVID-19 with male individuals exhibiting the worse clinical outcomes. Mechanistically, there is not compelling evidence to attribute the sex-dependent differences in the clinical outcomes to differential expression of ACE2, TMPRSS2, or furin proteases.

The sexual dimorphism in the clinical outcomes, which is more pronounced in the older individuals, likely reflects sexual dimorphism of the immune system. In general, there is a progressive decline in the adaptive immunity in men in response to pathogen as opposed to women. [27] [28, 29] The molecular basis of differential adaptive immunity between the two sexes has been attributed to the effects of the sex hormones on expression of genes involved in immunity, Y-and X-linked genes involved in immunity, non-homogenous X-chromosome inactivation, and cell type-specific differential gene expression. [27] [28, 29] In addition, Nevertheless, it is known that TMPRSS2 is an androgen-responsive gene, which in part might account the worse clinical outcomes in male patients. [30] Cardiovascular involvement in COVID-19: SARS-CoV-2 enters the cell through the ACE2 receptors, upon modifications with TMPRSS2 and possibly by furin-like and CTSL (cathepsin L)

proteases. Therefore, SARS-CoV-2 tropism is largely determined by the expression of protein constituents involved in viral entry. Cell Single cell sequencing data show that ACE2 is expressed at high levels in cardiac pericytes but relatively low to moderate levels in other cardiac cell types, including myocytes, fibroblasts, smooth muscle cells, and endothelial cells. [31, 32] In contrast, TMPRSS2 and CTSL are expressed at relatively low levels in the cardiovascular cells. [31, 32] Because tropism of the SARS-CoV-2 depends on co-expression of ACE2 and host proteases, viral myocarditis is not a common feature of COVID-19. However, histological and magnetic resonance imaging evidence of myocarditis, such as interstitial fibrosis, inflammatory cellular infiltrates, and necrosis, has been reported in patients with COVID-19. Whether these changes are the direct consequence of viral infection or are secondary to increased levels of cytokines, catecholamines, or hypoxia, and other factors remains uncertain.

Despite the paucity of firm data on direct myocardial involvement in COVID-19 patients, there are ample data on secondary involvement of the cardiovascular system, manifesting as elevated blood levels of cardiac enzymes; such as cardiac troponin I and B-type natriuretic peptide, regional and global wall motion abnormalities, cardiac arrhythmias, intravascular thrombosis, and heart failure. (reviewed in [33] [34, 35] ) More importantly, concomitant cardiovascular disease and secondary cardiac involvement are major determinants of clinical outcomes, including mortality in patients with COVID-19. [36, 37] Coagulopathy, represented by increased D-Dimer levels, disseminated intravascular coagulation (DIC), and thrombocytopenia, and thrombotic events, is a common manifestation of COVID-19.

[38] [39] Increased coagulopathy is in part because of the so-called "cytokine storm" manifesting with increased levels of pro-inflammatory cytokines; such as IL6 and TNF-a, and in p[art because of endothelial dysfunction, and increased levels of pro-thrombotic factors. [39] Approximately 5% of the hospitalized patients experienced venous thromboembolism and the overall thrombotic complication rate is about 10%. [40] Because of multiplicity of the pro-thrombotic factors in COVID-19 patients, the conventional preventive anticoagulation is often insufficient and an intense anticoagulation regiment might be needed.

However, there is no consensus on the proper anticoagulation approach and the risk of bleeding is relatively high. [40] Nevertheless, the patients are commonly treated with the low molecular weight heparin, adjusted according to body weight and renal function. (reviewed in [41] )

The knowledge gained through basic science discoveries about the structure and function of the SARS-CoV-2 has enabled the researchers to test the existing compounds and to perform large-scale screening to identify new compounds that effectively target the specific components of the virus.

Likewise, sequencing of the viral genome and identification of its proteome have enabled the investigators to develop vaccines by using the viral RNAs and specific epitopes of the viral proteins as antigens. Over 3,000 clinical trials, including ~ 500, phase 3 clinical trials, have been registered worldwide that are designed to test effectiveness of over four dozen different compounds, ranging from the epigenetic modulator JQ1 to medicinal herbs, in prevention and treatment of COVID-19.

(https://clinicaltrials.gov/). In the following sections, a selected number of interventions that directly target specific components pertaining to viral entry and replication are discussed along with various approaches to vaccine development.

Entry of the SARS-CoV-2 into the host cells could be simplified into a multistep process comprised of proteolytic cleavage of the S1 subunit and ACE2, binding of the viral S proteins through the RBD in the S1 subunit to the ACE2 receptors on the host cells, fusion of the viral S protein with the host cell membrane, and endocytosis ( Figure 1 ). This is then followed by release of the viral RNA into host cell cytoplasm and synthesis of viral proteins. The newly synthesize viral proteins and viral RNA are assembled into virions in the ER/Golgi apparatus followed by excretion from the host cells by exocytosis.

A number of interventions are pursued to target specific steps involved in viral entry, which are discussed briefly.

The rationale for the use of recombinant ACE2 is to occupy the ACE receptors and hence, attenuate its availability for binding to SARS-CoV-2. Therefore, administration of the recombinant ACE2 by increasing circulating levels of ACE2 is expected to competitively bind and neutralize the viral S protein for its ability to bind to the cellular ACE2 receptors. Consequently, the approach is expected to reduce viral entry into the host cell and attenuate the phenotypic consequences. 

Replication of SARS-CoV-2 requires copying of the viral RNA to make new strands, synthesis of new viral protein, including proteolytic cleavage of the large polypeptides into multiple units, coassembly of the viral proteins and RNA to form virions and processing of the virions for release from the host cells (Figure 1 ). These components involved in viral replication are potential therapeutic targets, as discussed below:

Targeting RNA synthesis by nucleoside analogues: The approach exploits the rare error of viral RNA-dependent RNA polymerase during RNA synthesis, resulting in incorporation of modified nucleotides into the viral RNA during synthesis, which leads to premature termination of RNA Notable among various interventions is the use of dexamethasone, which was found to reduce 28day mortality modestly in those requiring respiratory support in the RECOVERY study. [69] . Thus far, dexamethasone is the only drug shown to reduce COVID-19 mortality. Details of empiric and general therapies are not discussed given the focus of this review on specific anti-viral therapy.

The clinical impact of major scientific advances is also amply evident in the current efforts to Multiple approaches to vaccine development are being pursued, which are summarized in Table 2 .

Replication fidelity of SARS-coronaviruses is relatively high, because of their RNA proofreading functions through 3'-5' exonuclease activities of NSP14 and NSP12, which results in a low rate of new mutations, estimated to be at 2x10 -5 . [70] Nevertheless, the rare errors that occur during viral genome replication could results in generation of variants that could affect infectivity of the virus, lead to antigenic drift, and modify the host responses to the viral antigen, and hence, impede generation of an effective vaccine. This antigen drift could pose considerable challenges for developing a vaccine that maintains its effectiveness for a long period. A notable example is a single amino acid substitution, namely p.D614G, located in the S protein, which has become the dominant variant in the current pandemic in the Western world, as opposed to D614 variant during epidemics in Wuhan, China.

Experimental and clinical data suggest that the G614 variant increases infectivity and is associated with a higher viral load. [71] Preliminary data suggest that the p.D614G variant does not affect antigenic properties of the S protein and therefore, is unlikely to have an effect on efficacy of the vaccines targeting the S protein.

[72]

The rationale is to deliver an mRNA that codes for a viral protein as an antigen presented to host immune system in order to elicit an immune response and produce neutralizing antibodies. The mRNA vaccine distinct from the conventional vaccine, which typically utilizes an inactivated organism or its protein as an antigen to stimulate the host immune system. An mRNA, containing an ORF, is first transcribed in vitro from a DNA template using an RNA polymerase. The ORF codes for the protein of interest that serves as the antigen. The translation is achieved using the host translational machinery. The mRNA-based vaccines have several advantages, including relative safety, reliance on host translational machinery, lack of integration in the genome, and the relative ease and scalable production in the laboratory. However, there are important challenges, such as effective delivery, stability of the mRNA in the host system, fortuitous immune response, and instability upon storage, unless frozen.

The mRNA vaccine is advocated to be as the most efficient and less time-consuming approach to 

The approach utilizes a vector, such as plasmids, replication-deficient adenoviruses, lentiviruses, or replication-competent vesicular stomatitis virus, to transfer a SARS-CoV-2 gene and express a viral protein, typically the S protein, to elicit immunogenicity. DNA vaccines have many characteristics of a desirable vaccines as they are relatively easy to manufacture in large and high quality at a relatively low cost, relatively safe, and stable at room temperature. The DNA vaccines could be delivered by intramuscular or intradermal inoculation and even electroporation. DNA vaccines have been developed for several infectious diseases and have been shown to be well-tolerated and immunogenic. They are also being tested for safety and immunogenicity against SARS-CoV-2. Preliminary studies in rhesus macaques upon expression of several viral S immunogens have shown have humeral and cellular responses, including production of neutralizing antibodies and IFN-g producing CD4+ and CD8+ T cells. [77] The immune response following DNA vaccination was effective in reducing viral RNA levels upon inoculation of the immunized monkeys with SARS-CoV-2 virus. [77] Likewise, vaccination with a recombinant adenovirus that expressed the full-length S protein led to dose-dependent antibody and T cell responses in the majority of the vaccinated individuals, which were peaked at ~ 4 weeks. [78] No serious side effect was reported but mild to moderate side effects were common. [78] A phase 2 study with this vaccine is planned. One caveat with the vector-based vaccines is immunogenicity of the vectors.

Replication-deficient adenoviruses are known to be immunogenic and elicit host immune reaction and shutdown of the transgene expression, which were amply demonstrated in the early days of gene therapy. 

Attenuated and inactivated pathogens are the classic antigens for vaccine generation, dating back to vaccination against smallpox by Edward Jenner who coined the term vaccination. The virus is typically inactivated upon treatment with formalin or other chemicals or upon exposure to ultraviolet light. In the case of live attenuated virus, viral genome is de-optimize to reduce its pathogenicity while maintaining its immunogenicity against multiple viral antigen. The use of live attenuated or inactivated virus for vaccination is somewhat compounded by the potential pathogenicity due to an inadequate inactivation or attenuation. There is also the risk of live attenuated virus evolving into a more pathogenic strain due to mutagenesis or recombination with the wild type virus.

Pre-clinical studies in Rhesus macaques immunized with three injections of purified inactivated SARS-CoV-2 (treated with b-propiolactone) induced an effective immune response, as evidenced by decreased viral load and protection against infection upon challenge of the monkey with SARS-CoV-2.

[82] Inactivated SARS-CoV-2 vaccines are currently being tested in early phase clinical trials.

The pace of advances in developing specific drugs to targets SARS-CoV-2 and vaccine to prevent COVID-19 is quite remarkable, facilitated largely by the existing basic science discoveries about coronaviruses as well as technological advances in molecular biology and genetics and nucleic acid-based vaccine development. ClinicalTrials.Gov lists over 150 vaccine studies against SARS-CoV-2, which utilize a variety of platforms ranging from inactivated whole virion to specific RNA vaccines to nonspecific BCG vaccine. There are, however, no concrete evidence for the success of targeted therapy or efficacy of the potential vaccines. The data on the effectiveness of existing targeted therapies, ranging from monoclonal antibodies against ACE2 to nucleotide analogues are equivocal at best. Likewise, a large number of vaccine candidates are likely to drop out from competition after the initial studies and a considerable number is likely to fail in the phase 3 studies. Only those built upon robust basic science principles and phase 1-2 data will have the chance of succeeding in phase 3 efficacy clinical trials. It might be necessary to identify additional drug and vaccine targets and screen chemical libraries to find suitable compounds, followed by optimization and testing in pre-clinical and phase 1-3 clinical trials. At the clinical level, it is important to identify and test the drugs or vaccines in the target groups, such as the elderly who face the brunt of mortality from COVID-19, and define robust clinical endpoints for efficacy of the interventions. Despite the challenges ahead, developing specific anti-SARS-CoV-2 therapies and effective vaccines against COVID-19 are the global priorities and likely essential for the successful elimination of COVID-19. The progress toward these goals is most remarkable, largely stemming from important basis science discoveries. It merits noting that typically the clinical impacts of the fundamental basic science discoveries, upon their initial discoveries, are unclear. It is such discoveries, however, that are expected to pave the way for the successful eradication of COVID-19. Thus, when uncovering the secrets of the nature, one should never be concerned with the immediate clinical implications. The fundamental discoveries are essential for the cure of human diseases.

This work was supported in part by grants from NIH, National Heart, Lung and Blood Institute (NHLBI R01 HL132401, R01 HL151737, and S10 OD018135), Leducq Foundation ( 

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which might serve as targets for specific therapy and vaccine development. 1. Attachment of SARS-CoV-2 to ACE2 receptors on host cell membrane 2. Integration of viral S protein with host cell membrane 3. Viral entry through endocytosis 4. Release of the virus from endosome to host cell cytoplasm 5. Formation of replicase-transcriptase complex 6. Synthesis of viral RNA 7. Translation of viral RNA into large polypeptides 8. Proteolytic cleavage of viral proteins into non-structural proteins 9. Translation of viral mRNAs into structural proteins 10. Assembly of viral RNA and proteins into virions at endoplasmic reticulum and Golgi apparatus 11. Formation of secreted vesicles 12