COVID-19: The question of genetic diversity and therapeutic intervention approaches

David Livingstone Alves Figueiredo João Paulo Bianchi Ximenez Fábio Rodrigues Ferreira Seiva Carolina Panis Rafael dos Santos Bezerra Adriano Ferrasa Alessandra Lourenço Cecchini Alexandra Ivo de Medeiros Ana Marisa Fusco Almeida Anelisa Ramão Angelica Beate Winter Boldt Carla Fredrichsen Moya Chung Man Chin Daniel de Paula Daniel Rech Daniela Fiori Gradia Danielle Malheiros Danielle Venturini Eliandro Reis Tavares Emerson Carraro Enilze Maria de Souza Fonseca Ribeiro Evani Marques Pereira Felipe Francisco Tuon Franciele Aní Caovilla Follador Glaura Scantamburlo Alves Fernandes Hélito Volpato Ilce Mara de Syllos Cólus Jaqueline Carvalho de Oliveira Jean Henrique da Silva Rodrigues Jean Leandro dos Santos Jeane Eliete Laguila Visentainer Juliana Cristina Brandi Juliana Mara Serpeloni Juliana Sartori Bonini Karen Brajão de Oliveira Karine Fiorentin Léia Carolina Lucio Ligia Carla Faccin-Galhardi Lirane Elize Defante Ferreto Lucy Megumi Yamauchi Lioni Marcia Edilaine Lopes Consolaro Marcelo Ricardo Vicari Marcos Abdo Arbex Marcos Pileggi Maria Angelica Ehara Watanabe Maria Antônia Ramos Costa Maria José S. Mendes Giannini Marla Karine Amarante Najeh Maissar Khalil Quirino Alves de Lima Neto Roberto H. Herai Roberta Losi Guembarovski Rogério N. Shinsato Rubiana Mara Mainardes Silvana Giuliatti Sueli Fumie Yamada-Ogatta Viviane Knuppel de Quadros Gerber Wander Rogério Pavanelli Weber Claudio da Silva Maria Luiza Petzl-Erler Valeria Valente Christiane Pienna Soares Luciane Regina Cavalli Wilson Araujo Silva JrAbout the authors

Abstract

Coronavirus disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2), is the largest pandemic in modern history with very high infection rates and considerable mortality. The disease, which emerged in China’s Wuhan province, had its first reported case on December 29, 2019, and spread rapidly worldwide. On March 11, 2020, the World Health Organization (WHO) declared the COVID-19 outbreak a pandemic and global health emergency. Since the outbreak, efforts to develop COVID-19 vaccines, engineer new drugs, and evaluate existing ones for drug repurposing have been intensively undertaken to find ways to control this pandemic. COVID-19 therapeutic strategies aim to impair molecular pathways involved in the virus entrance and replication or interfere in the patients’ overreaction and immunopathology. Moreover, nanotechnology could be an approach to boost the activity of new drugs. Several COVID-19 vaccine candidates have received emergency-use or full authorization in one or more countries, and others are being developed and tested. This review assesses the different strategies currently proposed to control COVID-19 and the issues or limitations imposed on some approaches by the human and viral genetic variability.

Keywords:
COVID-19; therapeutic interventions; global health treat; virus diversity

Introduction

The scientific community considers the COVID-19 caused by the new coronavirus SARS-CoV-2 as the deadliest pandemic in recent human history. SARS-CoV-2 is a virus of the family Coronaviridae of the genus Betacoronavirus, with the subgenus Sarbecovirus. Many coronaviruses have been identified in several animal species, of which six infect human hosts, including the severe acute respiratory syndrome-related coronavirus (SARS-CoV-1) and the Middle East respiratory syndrome coronavirus (MERS-CoV) (Dietz et al., 2020Dietz L, Horve PF, Coil DA, Fretz M, Eisen JA and Van Den Wymelenberg K (2020) 2019 novel coronavirus (COVID-19) pandemic: Built environment considerations to reduce transmission. mSystems 5:e00245-20.; Guo et al., 2020Guo Y-R, Cao Q-D, Hong Z-S, Tan Y-Y, Chen S-D, Jin H-J, Tan K-S, Wang D-Y and Yan Y (2020) The origin, transmission and clinical therapies on coronavirus disease 2019 (COVID-19) outbreak - An update on the status. Mil Med Res 7:11.). The genome of the new coronavirus was fully sequenced (NCBI Reference Sequence: NC_045512.2) (Wang et al., 2020Wang C, Liu Z, Chen Z, Huang X, Xu M, He T and Zhang Z (2020) The establishment of reference sequence for SARS-CoV-2 and variation analysis. J Med Virol 92:667-674.). Its sequence presents about 82% identity to the bat SARS-like coronavirus WIV1 (bat SL-CoV-WIV1, GenBank: KF367457.1), and more than 85% identity with the bat SARS-like coronavirus ZC45 (bat SL-CoV-ZC45, GenBank: MG772933.1) (Li X et al., 2020Li X, Zeng W, Li X, Chen H, Shi L, Li X, Xiang H, Cao Y, Chen H, Liu C et al. (2020) CT imaging changes of coronavirus disease 2019(COVID-19): A multi-center study in Southwest China. J Transl Med 18:154.; Yu et al., 2020Yu F, Du L, Ojcius DM, Pan C and Jiang S (2020) Measures for diagnosing and treating infections by a novel coronavirus responsible for a pneumonia outbreak originating in Wuhan, China. Microbes Infect 22:74-79.).

SARS-Cov-2 is an enveloped, non-segmented positive-sense RNA virus with prominent stick-shaped protruding particles in their outer membrane (Peng et al., 2020Peng L, Liu K-Y, Xue F, Miao Y-F, Tu P-A and Zhou C (2020) Improved early recognition of coronavirus disease-2019 (COVID-19): Single-center data from a Shanghai Screening Hospital. Arch Iran Med 23:272-276.; Yin et al., 2020Yin L, Mou H, Shao J, Zhu Y, Pang X, Yang J, Zhang J, Shi W, Yu S and Wang H (2020) Correlation between Heart fatty acid binding protein and severe COVID-19: A case-control study. PloS One 15:e0231687.). Similar to SARS-CoV-1 and MERS-CoV, the SARS-CoV-2 genome encodes nonstructural proteins (NSPs, such as 3-chymotrypsin-like protease, papain-like protease, helicase, and RNA-dependent RNA polymerase), structural and accessory proteins (Li Q et al., 2020Li X, Zeng W, Li X, Chen H, Shi L, Li X, Xiang H, Cao Y, Chen H, Liu C et al. (2020) CT imaging changes of coronavirus disease 2019(COVID-19): A multi-center study in Southwest China. J Transl Med 18:154.). Among NSPs, NSP1 is the first protein of the polyprotein of SARS CoV-2 and a leader protein, which acts as a potent inhibitor of gene expression of the virus carrier (Huang et al., 2011Huang C, Lokugamage KG, Rozovics JM, Narayanan K, Semler BL and Makino S (2011) SARS coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mRNAs: Viral mRNAs are resistant to nsp1-induced RNA cleavage. PLoS Pathog 7:e1002433.). Nonstructural protein 2 (NSP2) binds two other host proteins, prohibitin 1 and prohibitin 2 (PHB1 and PHB2), disrupting the host cell environment (Cornillez-Ty et al., 2009Cornillez-Ty CT, Liao L, Yates JR, Kuhn P and Buchmeier MJ (2009) Severe acute respiratory syndrome coronavirus nonstructural protein 2 interacts with a host protein complex involved in mitochondrial biogenesis and intracellular signaling. J Virol 83:10314-10318.). NSP3, the papain-like proteinase protein, has multiple functions and is considered the most important protease of the virus (Báez-Santos et al., 2015Báez-Santos YM, St John SE and Mesecar AD (2015) The SARS-coronavirus papain-like protease: Structure, function and inhibition by designed antiviral compounds. Antiviral Res 115:21-38.).

This new coronavirus has four major structural proteins: the spike (S), small envelope (E), and membrane (M) glycoproteins, and nucleocapsid (N) protein, besides several accessory proteins. The trimeric S protein is indispensable for virus-cell receptor interactions during viral entry (Lu et al., 2020Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, Wang W, Song H, Huang B, Zhu N et al. (2020) Genomic characterization and epidemiology of 2019 novel coronavirus: Implications for virus origins and receptor binding. Lancet 395:565-574.; Walls et al., 2020Walls AC, Park Y-J, Tortorici MA, Wall A, McGuire AT and Veesler D (2020) Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell 181:281-292.). SARS-CoV-2 targets cells through the S protein, which binds to the human angiotensin-converting enzyme 2 (ACE2) receptor and employs the cellular serine protease TMPRSS2 for S protein priming (Datta et al., 2020Datta PK, Liu F, Fischer T, Rappaport J and Qin X (2020) SARS-CoV-2 pandemic and research gaps: Understanding SARS-CoV-2 interaction with the ACE2 receptor and implications for therapy. Theranostics 10:7448-7464.; Hoffmann et al., 2020Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu N-H, Nitsche A et al. (2020) SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181:271-280.e8.; Letko et al., 2020Letko M, Marzi A and Munster V (2020) Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat Microbiol 5:562-569.; Tai et al., 2020Tai W, He L, Zhang X, Pu J, Voronin D, Jiang S, Zhou Y and Du L (2020) Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: Implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell Mol Immunol 17:613-620.). Notably, the ACE2 receptor is expressed in various tissues and organ systems throughout the body, including the central nervous system, gastrointestinal system, heart, lung, testes, and kidney (Baig et al., 2020Baig AM, Khaleeq A, Ali U and Syeda H (2020) Evidence of the COVID-19 virus targeting the CNS: Tissue distribution, host-virus interaction, and proposed neurotropic mechanisms. ACS Chem Neurosci 11:995-998.; Zhang et al., 2021Zhang Y, Zeng G, Pan H, Li C, Hu Y, Chu K, Han W, Chen Z, Tang R, Yin W et al. (2021) Safety, tolerability, and immunogenicity of an inactivated SARS-CoV-2 vaccine in healthy adults aged 18-59 years: A randomized, double-blind, placebo-controlled, phase 1/2 clinical trial. Lancet Infect Dis 21:181-192.). In fact, in addition to oropharyngeal swabs, the viral RNA has also been detected in blood, urine, facial/anal swabs, semen, and vaginal secretion, suggesting other potential means of transmission (Peng et al., 2020Peng L, Liu K-Y, Xue F, Miao Y-F, Tu P-A and Zhou C (2020) Improved early recognition of coronavirus disease-2019 (COVID-19): Single-center data from a Shanghai Screening Hospital. Arch Iran Med 23:272-276.). Ultimately, the S protein binding to the ACE2 receptor triggers a cascade of events leading to the fusion and releasing of the viral RNA genome into the host cell. The nonstructural proteins are subsequently synthesized to encode the viral replicase-transcriptase complex. The viral RNA is then synthesized by RNA-dependent RNA polymerase (Chen Y et al., 2020Chen L, Xiong J, Bao L and Shi Y (2020) Convalescent plasma as a potential therapy for COVID-19. Lancet Infect Dis 20:398-400.; Letko et al., 2020Letko M, Marzi A and Munster V (2020) Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat Microbiol 5:562-569.). Further, when the virus is in the cytosol, the non-structural viral proteins (nsp) 1-16 are produced and catalyze replication of the viral RNA genome, and inhibition of the host’s innate immune response (Thiel et al., 2003Thiel V, Ivanov KA, Putics Á, Hertzig T, Schelle B, Bayer S, Weißbrich B, Snijder EJ, Rabenau H, Doerr HW et al. (2003) Mechanisms and enzymes involved in SARS coronavirus genome expression. J Gen Virol 84:2305-2315.; Gildenhuys, 2020Gildenhuys S (2020) Expanding our understanding of the role polyprotein conformation plays in the coronavirus life cycle. Biochem J 477:1479-1482.). The Mpro or NSP5 protease mediates the cleavage of the viral replicative proteins, RNA-dependent RNA polymerase (RpRp) and helicase (HEL) (Ziebuhr et al., 2000Ziebuhr J, Snijder EJ and Gorbalenya AE (2000) Virus-encoded proteinases and proteolytic processing in the Nidovirales. J Gen Virol 81:853-879.).

SARS-CoV-2 has one of the hardest outer protective shells among all coronaviruses. This feature is believed to result in more stable viral particles, resulting in greater resilience in body fluids (Goh et al., 2020Goh GK-M, Dunker AK, Foster JA and Uversky VN (2020) Shell disorder analysis predicts greater resilience of the SARS-CoV-2 (COVID-19) outside the body and in body fluids. Microb Pathog 144:104177.). Another relevant and recurrent challenge imposed by this pandemic, is the emergence of distinct new-high transmissible variants around the globe; so far, five variants of concern (VOC) have already been identified, B.1.1.7, detected first in the UK, B.1.351, initially detected in South Africa, B.1.1.28.1 (also known as P.1), first detected in Brazilian travellers in Japan, and more recently, B.1.427 and B.1.429, identified in USA (Centers for Disease Control and Prevention, CDC, 2021Centers for Disease Control and Prevention (2021) About Variants of the Virus that Causes COVID-19, Centers for Disease Control and Prevention (2021) About Variants of the Virus that Causes COVID-19, https://www.cdc.gov/coronavirus/2019-ncov/transmission/variant.html (accessed 23 April 2021).
https://www.cdc.gov/coronavirus/2019-nco...
). These new variants prevent the body’s immune response by selecting and excluding pieces of the virus’s genetic sequence. In this sense, there was a need for further studies on the pathogenicity and replication of SARS-CoV-2.

Regarding the diagnostic tools, the highly specific reverse-transcriptase polymerase-chain reaction (RT-PCR) technology is the gold standard test for COVID-19 and data from epidemiological evidence and clinical manifestations combined with radiological images, such as computer tomography (CT), also have critical diagnostic value for COVID-19 (Li X et al., 2020Li X, Zeng W, Li X, Chen H, Shi L, Li X, Xiang H, Cao Y, Chen H, Liu C et al. (2020) CT imaging changes of coronavirus disease 2019(COVID-19): A multi-center study in Southwest China. J Transl Med 18:154.). Clinically, COVID-19 presents a myriad of possible symptoms and outcomes, from asymptomatic carriage, flu-like symptoms including cough, fever, general weakness, myalgia, pneumonia-like characteristics, and respiratory failure requiring mechanical ventilation (Itelman et al., 2020Itelman E, Wasserstrum Y, Segev A, Avaky C, Negru L, Cohen D, Turpashvili N, Anani S, Zilber E, Lasman N et al. (2020) Clinical characterization of 162 COVID-19 patients in Israel: Preliminary report from a large tertiary center. Isr Med Assoc J 22:271-274.). Although there are studies that point out that COVID-19 manifests itself as a respiratory tract infection, rising data have been demonstrating that COVID-19 should be treated as a systemic disease, involving the most diverse systems of the human body, such as gastrointestinal, cardiovascular, respiratory, renal, neurological, immunological and hematopoietic (Driggin et al., 2020Driggin E, Madhavan MV, Bikdeli B, Chuich T, Laracy J, Biondi-Zoccai G, Brown TS, Der Nigoghossian C, Zidar DA, Haythe J et al. (2020) Cardiovascular considerations for patients, health care workers, and health systems during the COVID-19 pandemic. J Am Coll Cardiol 75:2352-2371.; Mehta et al., 2020Mehta P, McAuley DF, Brown M, Sanchez E, Tattersall RS, Manson JJ and HLH Across Speciality Collaboration UK (2020) COVID-19: Consider cytokine storm syndromes and immunosuppression. Lancet 395:1033-1034.).

The transmission patterns of SARS-CoV-2 and its pathogenicity motivates the scientific community to work against the clock to improve the diagnostic, preventive and therapeutic management of the disease, and to identify the genetic risk factors. There is no current evidence to recommend any specific anti-SARS-CoV-2 treatment for patients with suspected or confirmed COVID-19. Diverse therapeutic interventions are being evaluated in clinical trials, and new approaches are being proposed regarding pharmacological therapy for COVID-19 (Saber-Ayad et al., 2020Saber-Ayad M, Saleh MA and Abu-Gharbieh E (2020) The rationale for potential pharmacotherapy of COVID-19. Pharmaceuticals (Basel) 13:96.).

In the light of the actual scenario, the repurposing of drugs, the development of novel effective immunotherapies, and safe and effective long-lasting vaccines against the SARS-CoV-2 are essential strategies for coping with this pandemic. In this review, we aim to discuss the current status of therapeutic interventions against COVID-19 (Figure 1), highlighting them from a mechanistic point of view considering the role of microRNAs, viral characteristics, and host genetic determinants, as well as the feasibility of the available drugs. A review of the current research on these topics may help guide strategies to address the current COVID-19 pandemic and prepare us for future challenges.

Figure 1 -
Main routes for therapeutic intervention of the COVID-19. The article discusses four approaches that are being used in an attempt to treat patients with severe clinical evolution.

Genetic basis of COVID-19 clinical phenotypes

The clinical heterogeneity observed in COVID-19 most likely results from the interaction of the immune responses and comorbidities presented by patients. The genetic background of patients certainly plays an essential role in this regard. Genetic variants of the cellular components that allow the interaction of the viral particle with the host cell and its entry are the most obvious candidates for investigation. Moreover, many of the components of the human innate and adaptive immune responses present genetic variants that may have functional impact. The genetic variability of the SARS-CoV-2 may provide additional factors modulating the disease manifestations (Hofmann et al., 2004Hofmann H, Geier M, Marzi A, Krumbiegel M, Peipp M, Fey GH, Gramberg T and Pöhlmann S (2004) Susceptibility to SARS coronavirus S protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor. Biochem Biophys Res Commun 319:1216-1221.; Li W et al., 2005Li F, Li W, Farzan M and Harrison SC (2005) Structure of SARS coronavirus spike receptor-binding domain complexed with its receptor. Science 309:1864-1868., 2007Li W, Sui J, Huang I-C, Kuhn JH, Radoshitzky SR, Marasco WA, Choe H and Farzan M (2007) The S proteins of human coronavirus NL63 and severe acute respiratory syndrome coronavirus bind overlapping regions of ACE2. Virology 367:367-374.; Cao Y et al., 2020Cao Y, Li L, Feng Z, Wan S, Huang P, Sun X, Wen F, Huang X, Ning G and Wang W (2020) Comparative genetic analysis of the novel coronavirus (2019-nCoV/SARS-CoV-2) receptor ACE2 in different populations. Cell Discov 6:11.; Pinto et al., 2020Pinto D, Park Y-J, Beltramello M, Walls AC, Tortorici MA, Bianchi S, Jaconi S, Culap K, Zatta F, De Marco A et al. (2020) Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody. Nature 583:290-295.). Besides, hormonal factors inherent to sex can influence the risk of mortality in cases positive for SARS-CoV-2. Karlberg et al. (2004Karlberg J, Chong DSY and Lai WYY (2004) Do men have a higher case fatality rate of severe acute respiratory syndrome than women do? Am J Epidemiol 159:229-231.) studied the mortality rate from the Hong Kong SARS epidemic and observed a significant difference between men (21.9%) and women (13.2%). Coincidence or not, the ACE2 gene is located on the X chromosome (Xp22) (Li et al., 2003Li W, Moore MJ, Vasilieva N, Sui J, Wong SK, Berne MA, Somasundaran M, Sullivan JL, Luzuriaga K, Greenough TC et al. (2003) Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426:450-454.). Oophorectomy or treatment of mice with an estrogen receptor inhibitor resulted in increased mortality in females infected with SARS-CoV-1 (Channappanavar et al., 2017Channappanavar R, Fett C, Mack M, Eyck PPT, Meyerholz DK and Perlman S (2017) Sex-based differences in susceptibility to severe acute respiratory syndrome coronavirus infection. J Immunol 198:4046-4053.). The other research front has concentrated efforts on the characterization of the different strains of SARS-CoV-2 to establish the viral subtypes and analyze the genetic variants associated with the different clinical phenotypes of COVID-19. In this case, the genomic regions whose products are responsible for the entry of SARS-CoV-2 in the host cells have been considered the principal candidates (Channappanavar et al., 2017Channappanavar R, Fett C, Mack M, Eyck PPT, Meyerholz DK and Perlman S (2017) Sex-based differences in susceptibility to severe acute respiratory syndrome coronavirus infection. J Immunol 198:4046-4053.; Benvenuto et al., 2020Benvenuto D, Giovanetti M, Vassallo L, Angeletti S and Ciccozzi M (2020) Application of the ARIMA model on the COVID-2019 epidemic dataset. Data Brief 29:105340.; Bezerra et al., 2020Bezerra VL, Anjos TB, Souza LES, Anjos TB, Vidal AM and Silva Júnior AA (2020) SARS-CoV-2 como agente causador da COVID-19: Epidemiologia, características genéticas, manifestações clínicas, diagnóstico e possíveis tratamentos. Brazilian J Health Rev 3:8452-8467.; Coutard et al., 2020Coutard B, Valle C, de Lamballerie X, Canard B, Seidah NG and Decroly E (2020) The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade. Antiviral Res 176:104742.; Licastro et al., 2020Licastro D, Rajasekharan S, Dal Monego S, Segat L, D’Agaro P and Marcello A (2020) Isolation and full-length genome characterization of SARS-CoV-2 from COVID-19 cases in Northern Italy. J Virol 94:e00543-20.; Lu et al., 2020Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, Wang W, Song H, Huang B, Zhu N et al. (2020) Genomic characterization and epidemiology of 2019 novel coronavirus: Implications for virus origins and receptor binding. Lancet 395:565-574.; Rehman et al., 2020Rehman SU, Shafique L, Ihsan A and Liu Q (2020) Evolutionary trajectory for the emergence of novel Coronavirus SARS-CoV-2. Pathogens 9:240.; Sah et al., 2020Sah R, Rodriguez-Morales AJ, Jha R, Chu DKW, Gu H, Peiris M, Bastola A, Lal BK, Ojha HC, Rabaan AA et al. (2020) Complete genome sequence of a 2019 novel Coronavirus (SARS-CoV-2) strain isolated in Nepal. Microbiol Resour Announc 9:e00169-20.; Shereen et al., 2020Shereen MA, Khan S, Kazmi A, Bashir N and Siddique R (2020) COVID-19 infection: Origin, transmission, and characteristics of human coronaviruses. J Adv Res 24:91-98.; Zhao et al., 2021Zhao J, Yang Y, Huang H, Li D, Gu D, Lu X, Zhang Z, Liu L, Liu T, Liu Y et al. (2021) Relationship between the ABO blood group and the COVID-19 susceptibility. Clin Infect Dis 73:328-331.). The analysis of a specific genomic signature of the SARS-CoV-2 strains can help in understanding the viral evolution since the first case reported in China (Fan et al., 2020Fan BSO, Vargas-Pinilla P, Amorim CEG, Sortica VA and Bortolini MC (2020) ACE2 diversity in placental mammals reveals the evolutionary strategy of SARS-CoV-2. Genet Mol Biol 43:e20200104. ). A computational tool was applied to identify and track numerous strains of SARS-CoV-2 circulating on different continents, especially those isolated from hospitalized patients, whether or not they needed intensive care and pulmonary ventilation (Zhao et al., 2021Zhao J, Yang Y, Huang H, Li D, Gu D, Lu X, Zhang Z, Liu L, Liu T, Liu Y et al. (2021) Relationship between the ABO blood group and the COVID-19 susceptibility. Clin Infect Dis 73:328-331.). The authors used a public database containing 4087 SARS-CoV-2 sequences and were able to define at least ten strains that infected patients in the United States, realizing that some of them are the same found in Asia and Europe. Such reports can help projects aiming to correlate SARS-CoV-2 strains with the clinical evolution of hospitalized patients.

Genetic diversity of the SARS-CoV-2

RNA viruses present higher mutation rates than DNA viruses, especially the single-stranded RNA (ssRNA) viruses, such as the SARS-CoV-2 (Peck and Lauring, 2018Peck KM and Lauring AS (2018) Complexities of viral mutation rates. J Virol 92:e01031-01017.), although the SARS-CoV-2 and other related viruses perform proofreading during RNA replication, differently from most other RNA viruses (Romano et al., 2020Romano M, Ruggiero A, Squeglia F, Maga G and Berisio R (2020) Structural view of SARS-CoV-2 RNA replication machinery: RNA synthesis, proofreading and final capping. Cells 9:1267. ). Data from the Global Initiative on Sharing All Influenza Data (GISAID) (Elbe and Buckland-Merrett, 2017Elbe S and Buckland-Merrett G (2017) Data, disease and diplomacy: GISAID’s innovative contribution to global health. Glob Chall 1:33-46.) have indicated that the SARS-CoV-2 mutational rate (Shen Z et al., 2020Shen C, Wang Z, Zhao F, Yang Y, Li J, Yuan J, Wang F, Li D, Yang M, Xing L et al. (2020) Treatment of 5 critically Ill patients with COVID-19 with Convalescent Plasma. JAMA 323:1582-1589.) was similar to other coronaviruses (Eckerle et al., 2010Eckerle LD, Becker MM, Halpin RA, Li K, Venter E, Lu X, Scherbakova S, Graham RL, Baric RS, Stockwell TB et al. (2010) Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing. PLoS Pathog 6:e1000896.; Son et al., 2020Son HA, Hang DTT, Thuan ND, Quyen LTB, Thuong LTH, Nga VT, Quang LB, Hung TT, Son NT, Linh NT et al. (2020) A simple method for detection of a novel coronavirus (SARS-CoV-2) using one-step RT-PCR followed by restriction fragment length polymorphism. J Med Virol 92:2839-2846.). The single nucleotide polymorphisms (SNPs) are the most frequent variants in the genome of the SARS-CoV-2 and are considered the leading cause of the genetic diversity and evolution of the virus, besides its virulence and transmissibility (Yin, 2020Yin C (2020) Genotyping coronavirus SARS-CoV-2: Methods and implications. Genomics 112:3588-3596.). The SNPs can be found in both coding and non-coding regions of the viral genome. SNPs located in coding regions have a high potential to contribute to the classification of new strains of SARS-CoV-2, calculate the rate of infection, and design vaccines and define effective doses for different population groups (Saha et al., 2020Saha I, Ghosh N, Maity D, Sharma N, Sarkar JP and Mitra K (2020) Genome-wide analysis of Indian SARS-CoV-2 genomes for the identification of genetic mutation and SNP. Infect Genet Evol 85:104457.). One study carried out with virus isolates from Europe has shown that SNPs are more frequent in proteins related to viral replication (RNA polymerase) and ACE2 binding regions of the S protein. These genetic variants have been previously associated with the effectiveness of vaccines (Yin, 2020Yin C (2020) Genotyping coronavirus SARS-CoV-2: Methods and implications. Genomics 112:3588-3596.). Studies in other populations have described SNPs in the genes encoding NSP-2, and also RdRp and the S protein (Tabibzadeh et al., 2020Tabibzadeh A, Zamani F, Laali A, Esghaei M, Tameshkel FS, Keyvani H, Makiani MJ, Panahi M, Motamed N, Perumal D et al. (2020) SARS-CoV-2 molecular and phylogenetic analysis in COVID-19 patients: A preliminary report from Iran. Infect Genet Evol 84:104387.).

The SARS-CoV-2 evolves in vivo after infection, which may affect its virulence, infectivity, and transmissibility (Shen Z et al., 2020Shen Z, Xiao Y, Kang L, Ma W, Shi L, Zhang L, Zhou Z, Yang J, Zhong J, Yang D et al. (2020) Genomic diversity of severe acute respiratory syndrome-coronavirus 2 in patients with coronavirus disease 2019. Clin Infect Dis 71:713-720.). Indeed, several studies have analyzed the mutational profile of interhost and intrahost single nucleotide variants (iSNV). The analysis of large datasets has shown that SARS-CoV-2 presented a more significant proportion of G>T changes in both iSNVs and iSNPs compared to SARS-CoV-1 and MERS. Interestingly, the mutational profile of the iSNVs was more similar among SARS-CoV-2 and MERS-CoV than SARS-CoV-1 (Sapoval et al., 2021Sapoval N, Mahmoud M, Jochum MD, Liu Y, Elworth RAL, Wang Q, Albin D, Ogilvie HA, Lee MD, Villapol S et al. (2021) Hidden genomic diversity of SARS-CoV-2: Implications for qRT-PCR diagnostics and transmission. Genome Res 31:635-644.). Altogether, the data presented above indicated that genetic variations in the SARS-CoV-2 genome sequence could be critical to assist in the definition of the virus transmission pattern and to control the infection outbreak, as well as for epidemiological monitoring and tracking of the virus.

The ∆382 corresponds to the deletion of the nucleotide in the position 382 which truncates the ORF7b and removes the ORF8 transcription-regulatory sequence. This variant is associated with milder illness compared to the wild-type virus, probably due to reduced cytokine release during the acute phase of the disease. The mechanism of this attenuated variant suggests that ORF8 can be a target for therapeutic intervention (Young et al., 2020Young BE, Fong S-W, Chan Y-H, Mak T-M, Ang LW, Anderson DE, Lee CY-P, Amrun SN, Lee B, Goh YS et al. (2020) Effects of a major deletion in the SARS-CoV-2 genome on the severity of infection and the inflammatory response: An observational cohort study. Lancet 396:603-611. ). Conversely, SARS-CoV-2 that bears the D614G mutation in the S protein is associated with a higher case fatality rate (Becerra-Flores and Cardozo, 2020Becerra-Flores M and Cardozo T (2020) SARS-CoV-2 viral spike G614 mutation exhibits higher case fatality rate. Int J Clin Pract 74:e13525.), a fact that should be considered for design of therapeutic antibodies and prognosis.

Genetic diversity of the human host

Viral targets in the host cells, such as the ACE2 and TMPRSS2, have been considered molecular markers to determine the genetic susceptibility or resistance to COVID-19 (Mohammadpour et al., 2021Mohammadpour S, Esfahani AT, Halaji M, Lak M and Ranjbar R (2021) An updated review of the association of host genetic factors with susceptibility and resistance to COVID-19. J Cell Physiol 236:49-54.). Several studies have shown that the presence of polymorphisms in the ACE2 gene can affect: (i) the modulation of intermolecular interactions with the SARS-CoV-2 S protein (Benetti et al., 2020Benetti E, Tita R, Spiga O, Ciolfi A, Birolo G, Bruselles A, Doddato G, Giliberti A, Marconi C, Musacchia F et al. (2020) ACE2 gene variants may underlie interindividual variability and susceptibility to COVID-19 in the Italian population. Eur J Hum Genet 28:1602-1614.; Gibson et al., 2020Gibson WT, Evans DM, An J and Jones SJM (2020) ACE 2 coding variants: A potential X-linked risk factor for COVID-19 disease. bioRxiv: 2020.04.05.026633.; Hussain et al., 2020Hussain M, Jabeen N, Raza F, Shabbir S, Baig AA, Amanullah A and Aziz B (2020) Structural variations in human ACE2 may influence its binding with SARS-CoV-2 spike protein. J Med Virol 92:1580-1586.; Lippi et al., 2020Lippi G, Lavie CJ, Henry BM and Sanchis-Gomar F (2020) Do genetic polymorphisms in angiotensin converting enzyme 2 (ACE2) gene play a role in coronavirus disease 2019 (COVID-19)? Clin Chem Lab Med 58:1415-1422.); (ii) the binding to the viral S protein (Li, Q et al., 2020Li Q, Cao Z and Rahman P (2020) Genetic variability of human angiotensin-converting enzyme 2 (hACE2) among various ethnic populations. Mol Genet Genomic Med 8:e1344.; Stawiski et al., 2020Stawiski EW, Diwanji D, Suryamohan K, Gupta R, Fellouse FA, Sathirapongsasuti JF, Liu J, Jiang YP, Ratan A, Mis M et al. (2020) Human ACE2 receptor polymorphisms predict SARS-CoV-2 susceptibility. Commun Biol 4:475.); (iii) the structure and stabilization (Benetti et al., 2020Benetti E, Tita R, Spiga O, Ciolfi A, Birolo G, Bruselles A, Doddato G, Giliberti A, Marconi C, Musacchia F et al. (2020) ACE2 gene variants may underlie interindividual variability and susceptibility to COVID-19 in the Italian population. Eur J Hum Genet 28:1602-1614.), and the expression of the ACE2 receptors (Badawi, 2020Badawi A (2020) Hypercytokinemia and pathogen-host interaction in COVID-19. J Inflamm Res 13:255-261.; Cao Y et al., 2020Cao Y, Li L, Feng Z, Wan S, Huang P, Sun X, Wen F, Huang X, Ning G and Wang W (2020) Comparative genetic analysis of the novel coronavirus (2019-nCoV/SARS-CoV-2) receptor ACE2 in different populations. Cell Discov 6:11.; Delanghe et al., 2020Delanghe JR, Speeckaert MM and De Buyzere ML (2020) COVID-19 infections are also affected by human ACE1 D/I polymorphism. Clin Chem Lab Med 58:1125-1126.). ACE2 variants usually alter the interaction between host cells and SARS-CoV-2 by showing lower affinity to the virus proteins that bind host cells’ surface, thus conferring decreased susceptibility to COVID-19 (Stawiski et al., 2020Stawiski EW, Diwanji D, Suryamohan K, Gupta R, Fellouse FA, Sathirapongsasuti JF, Liu J, Jiang YP, Ratan A, Mis M et al. (2020) Human ACE2 receptor polymorphisms predict SARS-CoV-2 susceptibility. Commun Biol 4:475.).

ACE2 expression differs on the basis of the biological age and sex of each individual (Goren et al., 2020Goren A, Vaño‐Galván S, Wambier CG, McCoy J, Gomez‐Zubiaur A, Moreno‐Arrones OM, Shapiro J, Sinclair RD, Gold MH, Kovacevic M et al. (2020) A preliminary observation: Male pattern hair loss among hospitalized COVID‐19 patients in Spain - A potential clue to the role of androgens in COVID‐19 severity. J Cosmet Dermatol 19:1545-1547.; Ovsyannikova et al., 2020Ovsyannikova IG, Haralambieva IH, Crooke SN, Poland GA and Kennedy RB (2020) The role of host genetics in the immune response to SARS-CoV-2 and COVID-19 susceptibility and severity. Immunol Rev 296:205-219.), and also according to the different geographic and ethnic distribution of the COVID-19 patients (McCoy et al.,2020McCoy J, Wambier CG, Vano-Galvan S, Shapiro J, Sinclair R, Ramos PM, Washenik K, Andrade M, Herrera S and Goren A (2020) Racial variations in COVID-19 deaths may be due to androgen receptor genetic variants associated with prostate cancer and androgenetic alopecia. Are anti-androgens a potential treatment for COVID-19? J Cosmet Dermatol 19:1542-1543.; Sun et al., 2020Sun P, Lu X, Xu C, Sun W and Pan B (2020) Understanding of COVID-19 based on current evidence. J Med Virol 92:548-551.). A large number of studies have described SNPs in patients of distinct countries affecting the molecular mechanisms cited above (Badawi, 2020Badawi A (2020) Hypercytokinemia and pathogen-host interaction in COVID-19. J Inflamm Res 13:255-261.; Benetti et al., 2020Benetti E, Tita R, Spiga O, Ciolfi A, Birolo G, Bruselles A, Doddato G, Giliberti A, Marconi C, Musacchia F et al. (2020) ACE2 gene variants may underlie interindividual variability and susceptibility to COVID-19 in the Italian population. Eur J Hum Genet 28:1602-1614.; Cao Y et al., 2020Cao Y, Li L, Feng Z, Wan S, Huang P, Sun X, Wen F, Huang X, Ning G and Wang W (2020) Comparative genetic analysis of the novel coronavirus (2019-nCoV/SARS-CoV-2) receptor ACE2 in different populations. Cell Discov 6:11.; Delanghe et al., 2020Delanghe JR, Speeckaert MM and De Buyzere ML (2020) COVID-19 infections are also affected by human ACE1 D/I polymorphism. Clin Chem Lab Med 58:1125-1126.; Gibson et al., 2020Gibson WT, Evans DM, An J and Jones SJM (2020) ACE 2 coding variants: A potential X-linked risk factor for COVID-19 disease. bioRxiv: 2020.04.05.026633.; Hatami et al., 2020Hatami N, Ahi S, Sadeghinikoo A, Foroughian M, Javdani F, Kalani N, Fereydoni M, Keshavarz P and Hosseini A (2020) Worldwide ACE (I/D) polymorphism may affect COVID-19 recovery rate: An ecological meta-regression. Endocrine 68:479-484.; Hussain et al., 2020Hussain M, Jabeen N, Raza F, Shabbir S, Baig AA, Amanullah A and Aziz B (2020) Structural variations in human ACE2 may influence its binding with SARS-CoV-2 spike protein. J Med Virol 92:1580-1586.; Lippi et al., 2020Lippi G, Lavie CJ, Henry BM and Sanchis-Gomar F (2020) Do genetic polymorphisms in angiotensin converting enzyme 2 (ACE2) gene play a role in coronavirus disease 2019 (COVID-19)? Clin Chem Lab Med 58:1415-1422.; Stawiski et al., 2020Stawiski EW, Diwanji D, Suryamohan K, Gupta R, Fellouse FA, Sathirapongsasuti JF, Liu J, Jiang YP, Ratan A, Mis M et al. (2020) Human ACE2 receptor polymorphisms predict SARS-CoV-2 susceptibility. Commun Biol 4:475.; Yamamoto N et al., 2020Yamamoto M, Kiso M, Sakai-Tagawa Y, Iwatsuki-Horimoto K, Imai M, Takeda M, Kinoshita N, Ohmagari N, Gohda J, Semba K et al. (2020) The anticoagulant nafamostat potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and viral infection in vitro in a cell-type-dependent manner. Viruses 12:629.). According to Alifano et al. (2020Alifano M, Alifano P, Forgez P and Iannelli A (2020) Renin-angiotensin system at the heart of COVID-19 pandemic. Biochimie 174:30-33.), these polymorphisms could explain in part the differences currently observed in COVID-19 incidence between countries around the world, despite the globalization of exchanges and travels. A gene homologous to ACE2, the human ACE1 gene that is mapped on chromosome 17, presents a polymorphic insertion (I) or deletion (D) of a 287-base pair (bp) Alu repeat sequence in intron 16 (Rieder et al.,1999Rieder MJ, Taylor SL, Clark AG and Nickerson DA (1999) Sequence variation in the human angiotensin converting enzyme. Nat Genet 22:59-62.), that has been shown to impact susceptibility to the disease as well as the frequency of recoveries and deaths (Delanghe et al., 2020Delanghe JR, Speeckaert MM and De Buyzere ML (2020) COVID-19 infections are also affected by human ACE1 D/I polymorphism. Clin Chem Lab Med 58:1125-1126.; Hatami et al., 2020Hatami N, Ahi S, Sadeghinikoo A, Foroughian M, Javdani F, Kalani N, Fereydoni M, Keshavarz P and Hosseini A (2020) Worldwide ACE (I/D) polymorphism may affect COVID-19 recovery rate: An ecological meta-regression. Endocrine 68:479-484.; Yamamoto N et al., 2020Yamamoto M, Kiso M, Sakai-Tagawa Y, Iwatsuki-Horimoto K, Imai M, Takeda M, Kinoshita N, Ohmagari N, Gohda J, Semba K et al. (2020) The anticoagulant nafamostat potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and viral infection in vitro in a cell-type-dependent manner. Viruses 12:629.; Calabrese et al., 2021Calabrese C, Annunziata A, Coppola A, Pafundi PC, Guarino S, Di Spirito V, Maddaloni V, Pepe N and Fiorentino G (2021) ACE gene I/D polymorphism and acute pulmonary embolism in COVID19 pneumonia: A potential predisposing role. Front Med (Lausanne) 7:631148.).

Other studies have reported the association of polymorphisms in other protein cell receptors, such as the TMPRSS2 receptor (Asselta et al., 2020Asselta R, Paraboschi EM, Mantovani A and Duga S (2020) ACE2 and TMPRSS2 variants and expression as candidates to sex and country differences in COVID-19 severity in Italy. Aging (Albany NY) 12:10087-10098.; Hou et al., 2020Hou Y, Zhao J, Martin W, Kallianpur A, Chung MK, Jehi L, Sharifi N, Erzurum S, Eng C and Cheng F (2020) New insights into genetic susceptibility of COVID-19: An ACE2 and TMPRSS2 polymorphism analysis. BMC Med 18:216.; Russo et al., 2020Russo R, Andolfo I, Lasorsa VA, Iolascon A and Capasso M (2020) Genetic analysis of the novel SARS-CoV-2 host receptor TMPRSS2 in different populations. Front Genet 11:872.; Senapati et al., 2020Senapati S, Kumar S, Singh AK, Banerjee P and Bhagavatula S (2020) Assessment of risk conferred by coding and regulatory variations from TMPRSS2 and CD26 in susceptibility of SARS-CoV-2 infection in human. J Genet 99:53.; Torre-Fuentes et al., 2021Torre-Fuentes L, Matías-Guiu J, Hernández-Lorenzo L, Montero-Escribano P, Pytel V, Porta-Etessam J, Gómez-Pinedo U and Matías-Guiu JA (2021) ACE2, TMPRSS2, and furin variants and SARS-CoV-2 infection in Madrid, Spain. J Med Virol 93:863-869.), as well as in the HLA genes (Nguyen et al., 2020Nguyen A, David JK, Maden SK, Wood MA, Weeder BR, Nellore A and Thompson RF (2020) Human leukocyte antigen susceptibility map for SARS-CoV-2. J Virol 94:e00510-20.; Lorente et al., 2021Lorente L, Martín MM, Franco A, Barrios Y, Cáceres JJ, Solé-Violán J, Perez A, Marcos Y Ramos JA, Ramos-Gómez L et al. (2021) HLA genetic polymorphisms and prognosis of patients with COVID-19. Med Intensiva (Engl Ed) 45:96-103.; Amoroso et al., 2021Amoroso A, Magistroni P, Vespasiano F, Bella A, Bellino S, Puoti F, Alizzi S, Vaisitti T, Boros S, Grossi PA et al. (2021) HLA and AB0 polymorphisms may influence SARS-CoV-2 infection and COVID-19 severity. Transplantation 105:193-200.; Warren and Birol, 2021Warren RL and Birol I (2021) HLA predictions from the bronchoalveolar lavage fluid and blood samples of eight COVID-19 patients at the pandemic onset. Bioinformatics 36:5271-5273.) and ABO blood group locus (Ellinghaus et al., 2020Ellinghaus D, Degenhardt F, Bujanda L, Buti M, Albillos A, Invernizzi P, Fernandez J, Prati D, Baselli G, Asselta R et al. (2020) The ABO blood group locus and a chromosome 3 gene cluster associate with SARS-CoV-2 respiratory failure in an Italian-Spanish genome-wide association analysis. medRxiv. DOI: 10.1101/2020.05.31.20114991.
https://doi.org/10.1101/2020.05.31.20114...
; Amoroso et al., 2021Amoroso A, Magistroni P, Vespasiano F, Bella A, Bellino S, Puoti F, Alizzi S, Vaisitti T, Boros S, Grossi PA et al. (2021) HLA and AB0 polymorphisms may influence SARS-CoV-2 infection and COVID-19 severity. Transplantation 105:193-200.; Zhao et al., 2021Zhao J, Yang Y, Huang H, Li D, Gu D, Lu X, Zhang Z, Liu L, Liu T, Liu Y et al. (2021) Relationship between the ABO blood group and the COVID-19 susceptibility. Clin Infect Dis 73:328-331.), with the risk of acquiring COVID-19. These results suggest that HLA antigens may influence SARS-CoV-2 infection and clinical evolution of COVID-19, and confirm that blood group A individuals are at greater risk of infection. In most of these studies, the variants observed were associated with the susceptibility to SARS-CoV-2 infection, as well as with the severity of the disease, such as the development of cardiovascular and respiratory complications (Ellinghaus et al., 2020Ellinghaus D, Degenhardt F, Bujanda L, Buti M, Albillos A, Invernizzi P, Fernandez J, Prati D, Baselli G, Asselta R et al. (2020) The ABO blood group locus and a chromosome 3 gene cluster associate with SARS-CoV-2 respiratory failure in an Italian-Spanish genome-wide association analysis. medRxiv. DOI: 10.1101/2020.05.31.20114991.
https://doi.org/10.1101/2020.05.31.20114...
; Hou et al., 2020Hou Y, Zhao J, Martin W, Kallianpur A, Chung MK, Jehi L, Sharifi N, Erzurum S, Eng C and Cheng F (2020) New insights into genetic susceptibility of COVID-19: An ACE2 and TMPRSS2 polymorphism analysis. BMC Med 18:216.; Amoroso et al., 2021Amoroso A, Magistroni P, Vespasiano F, Bella A, Bellino S, Puoti F, Alizzi S, Vaisitti T, Boros S, Grossi PA et al. (2021) HLA and AB0 polymorphisms may influence SARS-CoV-2 infection and COVID-19 severity. Transplantation 105:193-200.; Lorente et al., 2021Lorente L, Martín MM, Franco A, Barrios Y, Cáceres JJ, Solé-Violán J, Perez A, Marcos Y Ramos JA, Ramos-Gómez L et al. (2021) HLA genetic polymorphisms and prognosis of patients with COVID-19. Med Intensiva (Engl Ed) 45:96-103.;). A review of the possible impact of genetic factors involved in the immune responses on COVID-19 can be found in Anastassopoulou et al. (2020Anastassopoulou C, Gkizarioti Z, Patrinos GP and Tsakris A (2020) Human genetic factors associated with susceptibility to SARS-CoV-2 infection and COVID-19 disease severity. Hum Genomics 14:40.).

Variability in the human and viral miRNA network and the control of host response to SARS-CoV-2

MicroRNAs (miRNAs), a class of non-coding small RNA molecules, are important post-transcriptional regulators that have been associated with the development of several pathologies, including the ones caused by viral infections (Maltby et al., 2016Maltby S, Plank M, Tay HL, Collison A and Foster PS (2016) Targeting MicroRNA function in respiratory diseases: Mini-review. Front Physiol 7:21.; Trobaugh and Klimstra, 2017Trobaugh DW and Klimstra WB (2017) MicroRNA regulation of RNA virus replication and pathogenesis. Trends Mol Med 23:80-93.; Girardi et al., 2018Girardi E, López P and Pfeffer S (2018) On the importance of host microRNAs during viral infection. Front Genet 9:439.; Stolzenburg and Harris, 2018Stolzenburg LR and Harris A (2018) The role of microRNAs in chronic respiratory disease: Recent insights. Biol Chem 399:219-234.; Dutta et al., 2019Dutta RK, Chinnapaiyan S and Unwalla H (2019) Aberrant microRNAomics in pulmonary complications: Implications in lung health and diseases. Mol Ther Nucleic Acids 18:413-431.; Tribolet et al., 2020Tribolet L, Kerr E, Cowled C, Bean AGD, Stewart CR, Dearnley M and Farr RJ (2020) MicroRNA biomarkers for infectious diseases: From basic research to biosensing. Front Microbiol 11:1197.). Human (host) and viral miRNAs interact with each other and although these interactions are not yet completely elucidated, it is very likely to involve the regulation of cellular processes that affect virus pathogenicity and cellular response (Totura and Baric, 2012Totura AL and Baric RS (2012) SARS coronavirus pathogenesis: Host innate immune responses and viral antagonism of interferon. Curr Opin Virol 2:264-275.; Bruscella et al., 2017Bruscella P, Bottini S, Baudesson C, Pawlotsky J-M, Feray C and Trabucchi M (2017) Viruses and miRNAs: More friends than foes. Front Microbiol 8:824.). The gene network associated with host responses can result from miRNA transcriptional regulation of a subset of mRNA targets that are critical components of signaling pathways, including the WNT, INF, PIK3/AKT, MAPK, and NOTCH pathways (Barbu et al., 2020Barbu MG, Condrat CE, Thompson DC, Bugnar OL, Cretoiu D, Toader OD, Suciu N and Voinea SC (2020) MicroRNA involvement in signaling pathways during viral infection. Front Cell Dev Biol 8:143.; Khan M et al., 2020Khan MA-A-K, Sany MRU, Islam MS and Islam ABMMK (2020) Epigenetic regulator miRNA pattern differences among SARS-CoV, SARS-CoV-2 and SARS-CoV-2 world-wide isolates delineated the mystery behind the epic pathogenicity and distinct clinical characteristics of pandemic COVID-19. Front Genet 11:765.). On the other hand, miRNAs from the virus can deregulate host miRNAs and facilitate the viral replication, induce the latency, prevent apoptosis, and/or cause immune evasion (Salmena et al., 2011Salmena L, Poliseno L, Tay Y, Kats L and Pandolfi PP (2011) A ceRNA hypothesis: The Rosetta Stone of a hidden RNA language? Cell 146:353-358.; Scheel et al., 2016Scheel TKH, Luna JM, Liniger M, Nishiuchi E, Rozen-Gagnon K, Shlomai A, Auray G, Gerber M, Fak J, Keller I et al. (2016) A broad RNA virus survey reveals Both miRNA dependence and functional sequestration. Cell Host Microbe 19:409-423.; Trobaugh and Klimstra, 2017Trobaugh DW and Klimstra WB (2017) MicroRNA regulation of RNA virus replication and pathogenesis. Trends Mol Med 23:80-93.; Damas et al., 2019Damas ND, Fossat N and Scheel TKH (2019) Functional interplay between RNA viruses and non-coding RNA in mammals. Noncoding RNA 5:7.; Mishra et al., 2020Mishra R, Kumar A, Ingle H and Kumar H (2020) The interplay between viral-derived mirnas and host immunity during infection. Front Immunol 10:3079.). SARS-CoV-2 genome mutations have also been reported to disrupt the binding sites of miRNAs and negatively impact the modulation of anti-virus host defenses (Rad and McLellan, 2020Rad SAH and McLellan AD (2020) Implications of SARS-CoV-2 mutations for genomic RNA structure and host microRNA targeting. Int J Mol Sci 21:4807. ), as well as viral miRNA sponges that can deplete specific host miRNAs (Bartoszewski et al., 2020Bartoszewski R, Dabrowski M, Jakiela B, Matalon S, Harrod KS, Sanak M and Collawn JF (2020) SARS-CoV-2 may regulate cellular responses through depletion of specific host miRNAs. Am J Physiol Lung Cell Mol Physiol 319:L444-L455.; Srivastava et al., 2020Srivastava R, Daulatabad SV, Srivastava M and Janga SC (2020) SARS-CoV-2 contributes to altering the post-transcriptional regulatory networks across human tissues by sponging RNA binding proteins and micro-RNAs. bioRxiv. DOI: 10.1101/2020.07.06.190348.
https://doi.org/10.1101/2020.07.06.19034...
).

In the infection by SARS-CoV-2, the identification of the potential virus-human miRNA-based interactions have been mostly conducted on computational miRNA prediction analysis (Arisan et al., 2020Arisan ED, Dart A, Grant GH, Arisan S, Cuhadaroglu S, Lange S and Uysal-Onganer P (2020) The prediction of miRNAs in SARS-CoV-2 genomes: hsa-miR databases identify 7 key miRs linked to host responses and virus pathogenicity-related KEGG pathways significant for comorbidities. Viruses 12:614.; Khan M et al., 2020Khan MA-A-K, Sany MRU, Islam MS and Islam ABMMK (2020) Epigenetic regulator miRNA pattern differences among SARS-CoV, SARS-CoV-2 and SARS-CoV-2 world-wide isolates delineated the mystery behind the epic pathogenicity and distinct clinical characteristics of pandemic COVID-19. Front Genet 11:765.; Nersisyan et al., 2020Nersisyan S, Shkurnikov M, Turchinovich A, Knyazev E and Tonevitsky A (2020) Integrative analysis of miRNA and mRNA sequencing data reveals potential regulatory mechanisms of ACE2 and TMPRSS2. PloS One 15:e0235987.; Saçar and Adan, 2020Saçar DMD and Adan A (2020) Computational analysis of microRNA-mediated interactions in SARS-CoV-2 infection. PeerJ 8:e9369.; Sarma et al., 2020Sarma A, Phukan H, Halder N and Madanan MG (2020) An in-silico approach to study the possible interactions of miRNA between human and SARS-CoV2. Comput Biol Chem 88:107352.; Marchi et al., 2021Marchi R, Sugita B, Centa A, Fonseca AS, Bortoletto S, Fiorentin K, Ferreira S and Cavalli LR (2021) The role of microRNAs in modulating SARS-CoV-2 infection in human cells: a systematic review. Infect Genet Evol 91:104832.). Based on the seed region specificity, Arisan et al. (2020Arisan ED, Dart A, Grant GH, Arisan S, Cuhadaroglu S, Lange S and Uysal-Onganer P (2020) The prediction of miRNAs in SARS-CoV-2 genomes: hsa-miR databases identify 7 key miRs linked to host responses and virus pathogenicity-related KEGG pathways significant for comorbidities. Viruses 12:614.) have compared SARS-CoV-2 sequences from different geographical regions to those from other viruses, such as SARS and MERS. Although the analyses revealed shared human miRNAs targeting the genome of these viruses, unique miRNAs were observed for SARS-CoV-2. The prediction analysis conducted by Sarma et al. (2020Sarma A, Phukan H, Halder N and Madanan MG (2020) An in-silico approach to study the possible interactions of miRNA between human and SARS-CoV2. Comput Biol Chem 88:107352.), identified 22 potential miRNAs from five genomes of SARS-CoV-2 linked with 12 human miRNAs. Finally, a comparison between the host miRNA binding profiles on 67 different SARS-CoV-2 genomes from 24 different countries revealed miRNAs associated with increased death rates of COVID-19. Recently, Centa et al. (2021Centa A, Fonseca AS, Ferreira SGDS, Azevedo MLV, Vaz de Paula CB, Nagashima S, Machado-Souza C, Miggiolaro AFRDS, Baena CP, de Noronha L et al. (2020) Deregulated miRNA expression is associated with endothelial dysfunction in post-mortem lung biopsies of COVID-19 patients. Am J Physiol Lung Cell Mol Physiol 320:L405-L412. ) reported a significant association in the experimental expression analysis of two miRNAs, miR-26a-5p and miR-29b-3p, with the expression levels of inflammatory markers, such as IL-4, IL-6 and IL-8, in post-mortem lung cells of COVID-19 patients (Centa et al., 2021Centa A, Fonseca AS, Ferreira SGDS, Azevedo MLV, Vaz de Paula CB, Nagashima S, Machado-Souza C, Miggiolaro AFRDS, Baena CP, de Noronha L et al. (2020) Deregulated miRNA expression is associated with endothelial dysfunction in post-mortem lung biopsies of COVID-19 patients. Am J Physiol Lung Cell Mol Physiol 320:L405-L412. ). These results showed the direct impact of miR deregulation in the endothelial dysfunction and inflammatory response in patients with SARS-CoV-2 infection and acute respiratory injuries.

Among the most common pathways and gene networks affected by the human-virus miRNA interactions are the ones associated with the ACE2 and TMPRSS2 genes (Arisan et al., 2020Arisan ED, Dart A, Grant GH, Arisan S, Cuhadaroglu S, Lange S and Uysal-Onganer P (2020) The prediction of miRNAs in SARS-CoV-2 genomes: hsa-miR databases identify 7 key miRs linked to host responses and virus pathogenicity-related KEGG pathways significant for comorbidities. Viruses 12:614.; Ghafouri-Fard et al., 2020Ghafouri-Fard S, Noroozi R, Omrani MD, Branicki W, Pośpiech E, Sayad A, Pyrc K, Łabaj PP, Vafaee R, Taheri M et al. (2020) Angiotensin converting enzyme: A review on expression profile and its association with human disorders with special focus on SARS-CoV-2 infection. Vascul Pharmacol 130:106680.; Hoffmann et al., 2020Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu N-H, Nitsche A et al. (2020) SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181:271-280.e8.; Lukassen et al., 2020Lukassen S, Chua RL, Trefzer T, Kahn NC, Schneider MA, Muley T, Winter H, Meister M, Veith C, Boots AW et al. (2020) SARS-CoV-2 receptor ACE2 and TMPRSS2 are primarily expressed in bronchial transient secretory cells. EMBO J 39:e105114.; Nersisyan et al., 2020Nersisyan S, Shkurnikov M, Turchinovich A, Knyazev E and Tonevitsky A (2020) Integrative analysis of miRNA and mRNA sequencing data reveals potential regulatory mechanisms of ACE2 and TMPRSS2. PloS One 15:e0235987.; Paniri et al., 2021Paniri A, Hosseini MM and Akhavan-Niaki H (2021) First comprehensive computational analysis of functional consequences of TMPRSS2 SNPs in susceptibility to SARS-CoV-2 among different populations. J Biomol Struct Dyn 39:3576-3593.). The miRNAs that regulate the expression of these genes were deregulated in several cardiovascular and pulmonary diseases (Kohlstedt et al., 2013Kohlstedt K, Trouvain C, Boettger T, Shi L, Fisslthaler B and Fleming I (2013) AMP-Activated protein kinase regulates endothelial cell angiotensin-converting enzyme expression via p53 and the post-transcriptional regulation of microRNA-143/145. Circ Res 112:1150-1158.; Hu et al., 2014Hu B, Song JT, Qu HY, Bi CL, Huang XZ, Liu XX and Zhang M (2014) Mechanical stretch suppresses microRNA-145 expression by activating extracellular signal-regulated kinase 1/2 and upregulating angiotensin-converting enzyme to alter vascular smooth muscle cell phenotype. PloS One 9:e96338. ; Bao et al., 2015Bao H, Gao F, Xie G and Liu Z (2015) Angiotensin-converting enzyme 2 inhibits apoptosis of pulmonary endothelial cells during acute lung injury through suppressing MiR-4262. Cell Physiol Biochem 37:759-767.; Chen et al., 2015Chen L-J, Xu R, Yu H-M, Chang Q and Zhong J-C (2015) The ACE2/Apelin signaling, microRNAs, and hypertension. Int J Hypertens 2015:896861.), such as the ones developed by many COVID-19 patients. These findings support miRNAs’ role in the development and progression of endothelial and vascular diseases (Ovchinnikova et al., 2015Ovchinnikova ES, Schmitter D, Vegter EL, ter Maaten JM, Valente MAE, Liu LCY, van der Harst P, Pinto YM, de Boer RA, Meyer S et al. (2015) Signature of circulating microRNAs in patients with acute heart failure. Eur J Heart Fail 18: 414-423.; Vegter et al., 2017Vegter EL, Ovchinnikova ES, van Veldhuisen DJ, Jaarsma T, Berezikov E, van der Meer P and Voors AA (2017) Low circulating microRNA levels in heart failure patients are associated with atherosclerotic disease and cardiovascular-related rehospitalizations. Clin Res Cardiol 106:598-609.). Taken together, the data presented above show the role of miRNAs in modulating the immune- and other host response-related processes of SARS-CoV-2 infection, suggesting that they can be considered genetic factors for the observed differences in the response of the patients to the infection and in the severity of the disease. As the rich and valuable information obtained through in silico analysis becomes increasingly available, additional predictive viral-host miRNAs interactions are expected to be identified, which can lead to the potential identification of miRNAs as therapeutic targets for COVID-19 (Fernández-Hernando and Suárez, 2018Fernández-Hernando C and Suárez Y (2018) MicroRNAs in endothelial cell homeostasis and vascular disease. Curr Opin Hematol 25:227-236.; Prestes et al., 2020Prestes PR, Maier MC, Woods BA and Charchar FJ (2020) A guide to the short, long and circular RNAs in hypertension and cardiovascular disease. Int J Mol Sci 21:3666.).

In the context of a pandemic, the polymorphisms as well as rare variants that impact disease susceptibility become quantitatively important since millions of people may be infected. Therefore, the knowledge of the genetic variation, at both individual and population levels, may further improve our understanding of the SARS-CoV-2 transmission and pathogenesis, enabling the identification of individuals at high risk of infection and subsequent disease sequelae. More broadly, this may provide valuable information for drug design and vaccine development (Sironi et al., 2020Sironi M, Hasnain SE, Rosenthal B, Phan T, Luciani F, Shaw MA, Sallum MA, Mirhashemi ME, Morand S, González Candelas F et al. (2020) SARS-CoV-2 and COVID-19: A genetic, epidemiological, and evolutionary perspective. Infect Genet Evol 84:104384.).

Molecular approaches for therapeutic interventions

The use of molecular tools, such as RNA interference (RNAi) is being considered in the search for treatment of COVID-19. The RNAi can directly disrupt the production of viral and/or host proteins involved in SARS-CoV-2 infection, therefore allowing the development of challenging but promising novel therapeutic approaches, which potentially result in specific depletion of key proteins involved in COVID-19 pathogenesis. The RNAi technology itself is simple; it consists of the use of synthetic short interfering RNAs (siRNAs), which can be directly introduced into the cell cytoplasm where they will trigger the degradation of specific mRNA targets. The FDA approval of the first drug based on siRNA (Patisiran), used to treat nerve damage caused by a genetic disease, is encouraging (Uludağ et al., 2020Uludağ H, Parent K, Aliabadi HM and Haddadi A (2020) Prospects for RNAi therapy of COVID-19. Front Bioeng Biotechnol 8:916.). The former studies focused on SARS-CoV-1 infection may guide the work in the current SARS-CoV-2 pandemic. Although RNAi can be directed against any protein, targeting essential viral proteins, such as S, E, M, and N proteins might represent more specific and efficient strategies. In the initial studies applying RNAi against SARS-Co-1, many efforts were performed with the use of siRNAs directed to the S-protein (Qin et al., 2004Qin Z-L, Zhao P, Zhang X-L, Yu J-G, Cao M-M, Zhao L-J, Luan J and Qi Z-T (2004) Silencing of SARS-CoV spike gene by small interfering RNA in HEK 293T cells. Biochem Biophys Res Commun 324:1186-1193.; Zhang et al., 2004Zhang Y, Li T, Fu L, Yu C, Li Y, Xu X, Wang Y, Ning H, Zhang S, Chen W et al. (2004) Silencing SARS-CoV Spike protein expression in cultured cells by RNA interference. FEBS Lett 560:141-146.; Wu et al., 2005Wu C-J, Huang H-W, Liu C-Y, Hong C-F and Chan Y-L (2005) Inhibition of SARS-CoV replication by siRNA. Antiviral Res 65:45-48.), the Leader sequence (Li W et al., 2005Li W, Zhang C, Sui J, Kuhn JH, Moore MJ, Luo S, Wong S-K, Huang I-C, Xu K, Vasilieva N et al. (2005) Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2. EMBO J 24:1634-1643.), the non-structural protein 1 (Ni et al., 2005Ni B, Shi X, Li Y, Gao W, Wang X and Wu Y (2005) Inhibition of replication and infection of severe acute respiratory syndrome-associated coronavirus with plasmid-mediated interference RNA. Antivir Ther 10:527-533.), the nucleocapsid N-protein (Zhao et al., 2020Zhao S, Lin Q, Ran J, Musa SS, Yang G, Wang W, Lou Y, Gao D, Yang L, He D et al. (2020) Preliminary estimation of the basic reproduction number of novel coronavirus (2019-nCoV) in China, from 2019 to 2020: A data-driven analysis in the early phase of the outbreak. Int J Infect Dis 92:214-217.), the RpRp (He et al., 2003He R, Leeson A, Andonov A, Li Y, Bastien N, Cao J, Osiowy C, Dobie F, Cutts T, Ballantine M et al. (2003) Activation of AP-1 signal transduction pathway by SARS coronavirus nucleocapsid protein. Biochem Biophys Res Commun 311:870-876.; Lu et al., 2004) and the E-protein (Meng et al., 2006Meng G, Surana NK, St Geme JW and Waksman G (2006) Structure of the outer membrane translocator domain of the Haemophilus influenzae Hia trimeric autotransporter. EMBO J 25:2297-2304.) among others, and obtained considerable success in reducing viral load. Thus, RNAi technology warrants further exploration in order to verify its potential as an alternative strategy for SARS-CoV-2 infection treatment. Recently, several investigators suggested resume efforts focused on this direction (Asha et al., 2018Asha K, Kumar P, Sanicas M, Meseko CA, Khanna M and Kumar B (2018) Advancements in nucleic acid based therapeutics against respiratory viral infections. J Clin Med 8:6.; Ghosh et al., 2020Ghosh S, Firdous SM and Nath A (2020) siRNA could be a potential therapy for COVID-19. EXCLI J 19:528-531.).

Pharmacological interventions in cellular and animal models

In order to evaluate potential therapeutic intervention approaches, some strategies focused on ACE2, TMPRSS22, and S protein will be reported. Most of them use inhibitors to reduce the infection rate and the hypertensive and pro-inflammatory effects of Angiotensin II.

Angiotensin II-converting enzyme (ACE2) receptor inhibitors

ACE2 inhibition has been suggested as a promising approach to attenuate the damage in lung cells caused by SARS-CoV-2 infection (Lopes et al., 2020Lopes RD, Macedo AVS, de Barros e Silva PGM, Moll-Bernardes RJ, Feldman A, D’Andréa G, Arruda S, de Souza AS, de Albuquerque DC, Lilian Mazza RT et al. (2020) Continuing versus suspending angiotensin-converting enzyme inhibitors and angiotensin receptor blockers: Impact on adverse outcomes in hospitalized patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-The BRACE CORONA. Trial Am Heart J 13:49-59.). Captopril, enalapril, losartan and valsartan, which are all ACE2 antagonists, seem to inhibit the receptor and were able to avoid pneumonia caused by SARS-CoV-2 infection (Zhou et al., 2020Zhou H, Fang Y, Xu T, Ni W-J, Shen A-Z and Meng X-M (2020) Potential therapeutic targets and promising drugs for combating SARS-CoV-2. Br J Pharmacol 177:3147-3161.). Further, docking assays and crystallography analysis of virus’ receptor (Benítez-Cardoza and Vique-Sánchez et al., 2020Benítez-Cardoza CG and Vique-Sánchez JL (2020) Potential inhibitors of the interaction between ACE2 and SARS-CoV-2 (RBD), to develop a drug. Life Sci 256:117970.; Xia et al., 2020Xia S, Zhu Y, Liu M, Lan Q, Xu W, Wu Y, Ying T, Liu S, Shi Z, Jiang S et al. (2020) Fusion mechanism of 2019-nCoV and fusion inhibitors targeting HR1 domain in spike protein. Cell Mol Immunol 17:765-767.) are being explored to support the development of new inhibitory compounds (Tai et al., 2020Tai W, He L, Zhang X, Pu J, Voronin D, Jiang S, Zhou Y and Du L (2020) Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: Implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell Mol Immunol 17:613-620.; Yan et al., 2020Yan R, Zhang Y, Li Y, Xia L, Guo Y and Zhou Q (2020) Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science 367:1444-1448.) and small peptides that potentially prevent the interaction between the SARS-CoV-2 S protein and ACE2 (Xiu et al., 2020Xiu S, Dick A, Ju H, Mirzaie S, Abdi F, Cocklin S, Zhan P and Liu X (2020) Inhibitors of SARS-CoV-2 entry: Current and future opportunities. J Med Chem 63:12256-12274.).

Subunit protein TMPRSS2 Inhibitors

Nafamostat and camostat are serine proteases inhibitors proved to interfere in vitro with protein-mediated fusion of SARS-CoV-2 and the host cell (Kang et al., 2015Kang MW, Song H-J, Kang SK, Kim Y, Jung S-B, Jee S, Moon JY, Suh K-S, Lee SD, Jeon BH et al. (2015) Nafamostat mesilate inhibits TNF-α-induced vascular endothelial cell dysfunction by inhibiting reactive oxygen species production. Korean J Physiol Pharmacol 19:229-234.; Yamamoto M et al., 2020Yamamoto N, Ariumi Y, Nishida N, Yamamoto R, Bauer G, Gojobori T, Shimotohno K and Mizokami M (2020) SARS-CoV-2 infections and COVID-19 mortalities strongly correlate with ACE1 I/D genotype. Gene 758:144944.). Camostat can also inhibit TMPRSS2 in the human lung cells infected with SARS-CoV-2 (Hoffmann et al., 2020Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu N-H, Nitsche A et al. (2020) SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181:271-280.e8.). Clinical trials have been conducted to evaluate the efficacy and safety of camostat mesilate in treating COVID-19. Among those trials is possible to highlight some examples in which the drug was used alone NCT04583592 (CAMELOT, USA); NCT04608266 (CAMOVID, France); NCT04625114 (Belgium); NCT04321096 (Denmark); NCT04470544 (RECOVER, USA) or in association with other drugs NCT04652765 (USA); NCT04750759 (NICCAM; Germany); NCT04355052 (Israel); NCT04662086 (USA); NCT04644705 (Germany) and NCT04518410 (USA). Camostat mesilate are also being studied in several others intervention protocols (NCT04455815, England); NCT04662073 (USA); NCT04530617 (Mexico); NCT04662086 (COPPS study, USA); NCT04374019 (USA) and NCT04518410 (ACTIV-2 study, USA). However, results from all those trials have not been published yet.

VeroE6 cells are a well-known in vitro model system that produces high virus titers and displays visual cytopathic effects associated with viral infections. These cells are commonly used in in vitro antiviral assays, including for coronavirus (Matsuyama et al., 2010Matsuyama S, Nagata N, Shirato K, Kawase M, Takeda M and Taguchi F (2010) Efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2. J Virol 84:12658-12664. , 2020Matsuyama S, Nao N, Shirato K, Kawase M, Saito S, Takayama I, Nagatac N, Sekizuka T, Katoh H, Kato F et al. (2020) Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells. Proc Natl Sci U S A 117:7001-7003.; Fintelman-Rodrigues et al., 2020Fintelman-Rodrigues N, Sacramento CQ, Lima CR, Silva FS, Ferreira AC, Mattos M, de Freitas CS, Soares VC, Dias SSG, Temerozo JR et al. (2020) Atazanavir, alone or in combination with ritonavir, inhibits SARS-CoV-2 replication and proinflammatory cytokine production. Antimicrob Agents Chemother 64:e00825-20.; Unal et al., 2021Unal MA, Bitirim CV, Summak GY, Bereketoglu S, Zeytin IC, Bul O, Gurcan C, Aydos D, Goksoy E, Kocakaya E et al. (2021) Ribavirin shows antiviral activity against SARS-CoV-2 and downregulates the activity of TMPRSS2 and the expression of ACE2 in vitro. Can J Physiol Pharm 99:449-460.). Past studies demonstrated that the messenger RNA expression level of TMPRSS2 in VeroE6/TMPRSS2 cells is ~10-fold higher than in normal human lung tissue and other human cell lines. SARS-CoV-2 uses the same receptor, ACE2, as SARS-CoV, and ACE2 expression is very high in VeroE6 cells (Matsuyama et al., 2020Matsuyama S, Nao N, Shirato K, Kawase M, Saito S, Takayama I, Nagatac N, Sekizuka T, Katoh H, Kato F et al. (2020) Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells. Proc Natl Sci U S A 117:7001-7003.). In addition, recent studies verified that human Caco-2 colon epithelial cells as well as the lung cell line A549 stably expressing ACE2 and TMPRSS2 (Grobe et al., 2021).

Bromhexine and its metabolite ambroxol are mucolytic drugs that inhibit TMPRSS2, frequently used as a mucolytic agent in respiratory diseases. In vitro studies have shown that these drugs hamper the TMPRSS2 effect to activate a zymogen precursor of tissue plasminogen activator and ameliorate the cytokine storm induced by SARS-CoV-2 (Beeh et al., 2008Beeh KM, Beier J, Esperester A and Paul LD (2008) Antiinflammatory properties of ambroxol. Eur J Med Res 13:557-562.; Furgała-Wojas et al., 2020Furgała-Wojas A, Kowalska M, Nowaczyk A, Fijałkowski Ł and Sałat K (2020) Comparison of bromhexine and its active metabolite - ambroxol as potential analgesics reducing oxaliplatin-induced neuropathic pain - pharmacodynamic and molecular docking studies. Curr Drug Metab 21:548-561.). Clinical studies have been carried out using bromhexine (NCT04273763; NCT04355026 and NCT04340349), and preliminary results from NCT04405999 demonstrated that prophylaxis using this drug reduced the rate of symptomatic COVID-19. Aprotinin, enzalutamide, genistein, and estradiol are examples of others TMPRSS2 inhibitors, which were active in vitro using different cell types, however, informations about such effect in vivo are still missing (Royston, 2015; Bestle et al., 2020Bestle D, Heindl MR, Limburg H, van Lam TV, Pilgram O, Moulton H, Stein DA, Hardes K, Eickmann M, Dolnik O et al. (2020) TMPRSS2 and furin are both essential for proteolytic activation and spread of SARS-CoV-2 in human airway epithelial cells and provide promising drug targets. Life Sci Alliance 3:e202000786.; Wang et al., 2020Wang C, Liu Z, Chen Z, Huang X, Xu M, He T and Zhang Z (2020) The establishment of reference sequence for SARS-CoV-2 and variation analysis. J Med Virol 92:667-674.).

Furin protease inhibitors

After binding to the ACE2 receptor, the S-protein must be cleaved by the host protease furin for priming the S2 fusion machinery for triggering the fusion of viral and host cell membranes (Bosch et al., 2004Bosch BJ, Martina BEE, Van Der Zee R, Lepault J, Haijema BJ, Versluis C, Heck AJR, De Groot R, Osterhaus AD and Rottier PJM (2004) Severe acute respiratory syndrome Coronavirus (SARS-CoV) infection inhibition using spike protein heptad repeat-derived peptides. Proc Natl Acad Sci U S A 101:8455-8460.). Once furin processing is a required step for membrane fusion, furin inhibition could effectively reduce SARS-CoV-2 cell entrance in host cells (Shang et al., 2020Shang J, Wan Y, Luo C, Ye G, Geng Q, Auerbach A and Li F (2020) Cell entry mechanisms of SARS-CoV-2. Proc Natl Acad Sci U S A 117:11727-11734.). Darinaparsin, a currently used anticancer drug, showed a high binding-affinity to furin and could be a hopeful therapy approach for SARS-CoV-2 infection (Chowdhury et al., 2020Chowdhury T, Roymahapatra G and Mandal SM (2020) In silico identification of a potent arsenic based approved drug darinaparsin against SARS-CoV-2: Inhibitor of RNA dependent RNA polymerase (RdRp) and essential proteases. Infect Disord Drug Targets 21:608-618. ). Estradiol and vitamin D were also able to affect furin’s activity in rat, mouse, and human cells (Glinsky, 2020Glinsky GV (2020) Tripartite combination of candidate pandemic mitigation agents: vitamin d, quercetin, and estradiol manifest properties of medicinal agents for targeted mitigation of the COVID-19 pandemic defined by genomics-guided tracing of SARS-CoV-2 targets in human cells. Biomedicines 8:129.). The treatment with Vitamin D is still controversial, while some studies have found negative correlation between vitamin D levels and COVID-19 cases (Ilie et al., 2020Ilie PC, Stefanescu S and Smith L (2020) The role of vitamin D in the prevention of coronavirus disease 2019 infection and mortality. Aging Clin Exp Res 32:1195-1198.) other hypothesis an alleviation on lung inflammation caused by SARS-CoV-2 because vitamin D seems upregulating ACE2 human receptor and decreasing inflammatory cytokines (Xiao et al., 2021Xiao D, Li X, Su X, Mu D and Qu Y (2021) Could SARS-CoV-2-induced lung injury be attenuated by vitamin D? Int J Infect Dis 102:196-202.).Since, Vitamin D is known to enhance the rate of melanin synthesis; and this may concurrently regulate the expression of furin expression both vitamin D and melanin may have significant impact in management of COVID-19 (Paria et al., 2020Paria K, Paul D, Chowdhury T, Pyne S, Chakraborty R and Mandal SM (2020) Synergy of melanin and vitamin-D may play a fundamental role in preventing SARS-CoV-2 infections and halt COVID-19 by inactivating furin protease. Transl Med Commun 5:21.). Additionally irisin, luteolin, and nafamostat have demonstrated inhibitory activity against furin (Peng et al., 2017Peng M, Watanabe S, Chan KWK, He Q, Zhao Y, Zhang Z, Lai X, Luo D, Vasudevan S G and Li G (2017) Luteolin restricts dengue virus replication through inhibition of the proprotein convertase furin. Antivir Res 143:176-185.; de Oliveira et al., 2020de Oliveira M, De Sibio MT, Mathias LS, Rodrigues BM, Sakalem ME and Nogueira CR (2020) Irisin modulates genes associated with severe coronavirus disease (COVID-19) outcome in human subcutaneous adipocytes cell culture. Mol Cell Endocrinol 515:110917.; Yamamoto M et al., 2020Yamamoto N, Ariumi Y, Nishida N, Yamamoto R, Bauer G, Gojobori T, Shimotohno K and Mizokami M (2020) SARS-CoV-2 infections and COVID-19 mortalities strongly correlate with ACE1 I/D genotype. Gene 758:144944.). Thus, several known compounds have shown a favorable potential to attack this critical step of SARS-CoV-2 entrance in host cells and reduce infection effectiveness.

Fusion proteins inhibitors

The development of membrane fusion inhibitors prevents the specific fusion of the viral S2 protein domain, blocking the delivery of viral genetic material into the host cell (Yan et al., 2020Yan R, Zhang Y, Li Y, Xia L, Guo Y and Zhou Q (2020) Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science 367:1444-1448.). The EK1 peptide was able to inhibit SARS-CoV-2 fusion and a novel modified peptide (EK1C4) showed an even higher inhibitory activity against the viral membrane fusion pathway (Xia et al., 2020Xia S, Zhu Y, Liu M, Lan Q, Xu W, Wu Y, Ying T, Liu S, Shi Z, Jiang S et al. (2020) Fusion mechanism of 2019-nCoV and fusion inhibitors targeting HR1 domain in spike protein. Cell Mol Immunol 17:765-767.). Lipopeptides (IPB01 and IPB02), designed on the basis of the S-protein S2 fusion domain, demonstrated the ability to inhibit SARS-CoV-2 fusion to host cells (Zhu Y et al., 2020Zhu W, Chen CZ, Gorshkov K, Xu M, Lo DC and Zheng W (2020) RNA-Dependent RNA Polymerase as a Target for COVID-19 Drug Discovery. SLAS Discov 25:1141-1151.). Imatinib might also be involved in the blockage of membrane fusion during coronavirus infection (Sisk et al., 2018Sisk JM, Frieman MB and Machamer CE (2018) Coronavirus S protein-induced fusion is blocked prior to hemifusion by Abl kinase inhibitors. J Gen Virol 99:619-630.).

Main protease inhibitors

More than four thousand approved commercial drugs were screened in silico as potential main protease (Mpro) inhibitors of SARS-CoV-2 infection (Biembengut and de Souza, 2020Biembengut ÍV and de Souza TACB (2020) Coagulation modifiers targeting SARS-CoV-2 main protease Mpro for COVID-19 treatment: An in silico approach. Mem Inst Oswaldo Cruz 115:e200179.; Jiménez-Alberto et al., 2020Jiménez-Alberto A, Ribas-Aparicio RM, Aparicio-Ozores G and Castelán-Vega JA (2020) Virtual screening of approved drugs as potential SARS-CoV-2 main protease inhibitors. Comput Biol Chem 88:107325.). The results evidenced the potential use of several of them in COVID-19 treatment. Drug design recognized the Michael acceptor inhibitor N3 as a potent and irreversible inhibitor of SARS-CoV-1 Mpro (Yang et al., 2005Yang H, Xie W, Xue X, Yang K, Ma J, Liang W, Zhao Q, Zhou Z, Pei D, Ziebuhr J et al. (2005) Design of wide-spectrum inhibitors targeting coronavirus main proteases. PLoS Biol 3:e324.). In vitro experiments verified that it also inhibited SARS-CoV-2 replication in Vero cells (Jin et al., 2020Jin Z, Du X, Xu Y, Deng Y, Liu M, Zhao Y, Zhang B, Li X, Zhang L, Peng C et al. (2020) Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors. Nature 582:289-293.). Furthermore, chemical modifications of Mpro inhibitory groups caused a pronounced lung tropism in mice (Khan S et al., 2021Khan SA, Zia K, Ashraf S, Uddin R and Ul-Haq Z (2021) Identification of chymotrypsin-like protease inhibitors of SARS-CoV-2 via integrated computational approach. J Biomol Struct Dyn 39:2607-2616., Zhang et al., 2021Zhang Y, Zeng G, Pan H, Li C, Hu Y, Chu K, Han W, Chen Z, Tang R, Yin W et al. (2021) Safety, tolerability, and immunogenicity of an inactivated SARS-CoV-2 vaccine in healthy adults aged 18-59 years: A randomized, double-blind, placebo-controlled, phase 1/2 clinical trial. Lancet Infect Dis 21:181-192.). Peptidomimetic aldehydes also inhibited SARS-CoV-2 replication in Vero E6 cells and showed low toxicity in Sprague-Dawley rats and Beagle dogs (Dai et al., 2020Dai W, Zhang B, Jiang X-M, Su H, Li J, Zhao Y, Xie X, Jin Z, Peng J, Liu F et al. (2020) Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease. Science 368:1331-1335.). Several natural compounds were also identified as inhibitor candidates of Mpro (Gentile et al., 2020Gentile D, Patamia V, Scala A, Sciortino MT, Piperno A and Rescifina A (2020) Putative inhibitors of SARS-CoV-2 main protease from a library of marine natural products: A virtual screening and molecular modeling study. Mar Drugs 18:225.; Gurung et al., 2020Gurung AB, Ali MA, Lee J, Farah MA and Al-Anazi KM (2020) Unravelling lead antiviral phytochemicals for the inhibition of SARS-CoV-2 M(pro) enzyme through in silico approach. Life Sci 255:117831.; Khan S et al., 2021Khan SA, Zia K, Ashraf S, Uddin R and Ul-Haq Z (2021) Identification of chymotrypsin-like protease inhibitors of SARS-CoV-2 via integrated computational approach. J Biomol Struct Dyn 39:2607-2616.; Olubiyi et al., 2020Olubiyi OO, Olagunju M, Keutmann M, Loschwitz J and Strodel B (2020) High throughput virtual screening to discover inhibitors of the main protease of the Coronavirus SARS-CoV-2. Molecules 25:3193.).

RNA-dependent RNA polymerase (RpRp) inhibitors

The RDPD can also be a target for pharmacological intervention directed to specifically hinder the function of this enzyme complex (Zhu W et al., 2020Zhu Y, Yu D, Yan H, Chong H and He Y (2020) Design of potent membrane fusion inhibitors against SARS-CoV-2, an emerging coronavirus with high fusogenic activity. J Virol 94:e00635-20.). A known candidate is favipiravir, which binds to the catalytic domain of RDPR hindering nucleotide inclusion during RNA synthesis (Furuta et al., 2017Furuta Y, Komeno T and Nakamura T (2017) Favipiravir (T-705), a broad-spectrum inhibitor of viral RNA polymerase. Proc Jpn Acad Ser B Phys Biol Sci 93:449-463.). Some drugs such as ribavirin, remdesivir, sofosbuvir, galidesivir and tenofovir are good candidates as inhibitors of the RNA-polymerase mediated replication (Elfiky, 2020Elfiky AA (2020) Anti-HCV, nucleotide inhibitors, repurposing against COVID-19. Life Sci 248:117477.; Soufi and Iravani, 2021Soufi GJ and Iravani S (2021) Potential inhibitors of SARS-CoV-2: Recent advances. J Drug Target 29:349-364.). Ribavirin and favipiravir were able to restrain the SARS-CoV-2 RpRp enzymes (Huang et al., 2020Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, Zhang L, Fan G, Xu J, Gu X et al. (2020) Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 395:497-506.). Buonaguro et al. (2020Buonaguro L, Tagliamonte M, Tornesello ML and Buonaguro FM (2020) SARS-CoV-2 RNA polymerase as target for antiviral therapy. J Transl Med 18:185.) described that some commercial drugs with inhibitory activity against the RpRp, including NHC EIDD1931, have suppressed SARS-CoV-2 replication in vitro and a preclinical animal model, revealing this pathway as a promising target for therapeutic intervention.

Nanotechnology to boost pharmacological therapy

Nanotechnology-based approaches can provide specific drug delivery, enhanced drug bioavailability, low toxicity and improved antiviral activity. Carbon quantum dots inhibited the replication of the human coronavirus (Łoczechin et al., 2019Łoczechin A, Séron K, Barras A, Giovanelli E, Belouzard S, Chen Y-T, Metzler-Nolte N, Boukherroub R, Dubuisson J and Szunerits S (2019) Functional carbon quantum dots as medical countermeasures to human Coronavirus. ACS Appl Mater Interfaces 11:42964-42974.). Diphyllin loaded polymeric nanoparticles demonstrated targeted inhibition of the S protein from the feline coronavirus (Hu et al., 2017Hu C-MJ, Chang W-S, Fang Z-S, Chen Y-T, Wang W-L, Tsai H-H, Chueh L-L, Takano T, Hohdatsu T and Chen H-W (2017) Nanoparticulate vacuolar ATPase blocker exhibits potent host-targeted antiviral activity against feline coronavirus. Sci Rep 7:13043. ). Glutathione-capped Ag2S nanoclusters also showed antiviral properties by obstructing viral RNA synthesis and budding of porcine epidemic diarrhea virus (PEDV) as a model of coronavirus (Du et al., 2018Du T, Liang J, Dong N, Lu J, Fu Y, Fang L, Xiao S and Han H (2018) Glutathione-capped Ag2S nanoclusters inhibit coronavirus proliferation through blockage of viral RNA synthesis and budding. ACS Appl Mater Interfaces 10:4369-4378.).

Clinical trials for drug repurposing

Drug repurposing or repositioning is a strategy for identifying new applications for approved or investigational drugs outside the first medical indication (Ashburn and Thor, 2004Ashburn TT and Thor KB (2004) Drug repositioning: Identifying and developing new uses for existing drugs. Nat Rev Drug Discov 3:673-683.). Given the high decline rates, high costs, and slow new drug discovery and development’s timeframe, repurposing drugs is frequently becoming an attractive proposition. The rationale is that most of the process includes preclinical tests, safety assessment, and, in some cases, the development of the formulation has already been achieved. Besides, the risk of failure and the timeframe for drug development are almost non-existent (Pushpakom et al., 2019Pushpakom S, Iorio F, Eyers PA, Escott KJ, Hopper S, Wells A, Doig A, Guilliams T, Latimer J, McNamee C et al. (2019) Drug repurposing: progress, challenges and recommendations. Nat Rev Drug Discov 18:41-58.).

Until April 2021, more than 5,000 clinical trials were being performed worldwide, evaluating antivirals, corticosteroids, antibiotics, among other drugs against COVID-19 as summarized in Table 1. In the present review, we focus on studies published in journals where publication only occurs after the peer-review process. Here, we emphasize hydroxychloroquine (HCQ), chloroquine, and dexamethasone clinical trials.

Hydroxychloroquine is used to treat malaria, rheumatoid arthritis, and lupus. Some studies point to its antiviral activity against the human immunodeficiency virus (HIV), inhibiting the entry of the virus in host cells and promoting post-translation alteration of newly synthesized proteins via glycosylation inhibition (Rosa and Santos, 2020Rosa SGV and Santos WC (2020) Clinical trials on drug repositioning for COVID-19 treatment. Rev Panam Salud Publica 44:e40.). Hydroxychloroquine was tested in a retrospective multicenter cohort study of 1438 patients with laboratory confirmation of SARS-CoV-2 infection admitted to 25 hospitals. Four different treatments were evaluated, (1) hydroxychloroquine and azithromycin, (2) hydroxychloroquine, (3) azithromycin, and (4) neither of these drugs. Initially, this study showed that patients who received hydroxychloroquine and azithromycin had a higher incidence of heart failure than the group without treatment. Furthermore, no significant reduction of mortality in the groups of patients receiving any of the treatments compared with the non-treated group (Rosenberg et al., 2020Rosenberg ES, Dufort EM, Udo T, Wilberschied LA, Kumar J, Tesoriero J, Weinberg P, Kirkwood J, Muse A, DeHovitz J et al. (2020) Association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with COVID-19 in New York State. JAMA 323:2493-2502.).

Table 1
Clinical trial for the treatment of COVID-19 with five drugs approved to treat other diseases.

A randomized multicenter study involving 150 patients with moderate-stage COVID-19 in two arms, with or without hydroxychloroquine treatment, found no difference in the evolution of patients who used this drug or not. However, adverse effects related to the use of hydroxychloroquine were reported (Tang W et al., 2020Tang N, Li D, Wang X and Sun Z (2020) Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia. J Thromb Haemost 18:844-847.). Corroborating this result, Mercuro et al. (2020Mercuro NJ, Yen CF, Shim DJ, Maher TR, McCoy CM, Zimetbaum PJ and Gold HS (2020) Risk of QT interval prolongation associated with use of hydroxychloroquine with or without concomitant azithromycin among hospitalized patients testing positive for coronavirus disease 2019 (COVID-19). JAMA Cardiol 5:e201834.) showed, in a cohort study of 90 patients with COVID-19, that individuals using hydroxychloroquine had an increased risk QT interval prolongation. Also, in a randomized study of patients with severe COVID-19, a high dose of chloroquine alone or with azithromycin/oseltamivir was not recommended due to potential safety hazards related to QT prolongation and increased lethality (Borba et al., 2020Borba MGS, Val FFA, Sampaio VS, Alexandre MAA, Melo GC, Brito M, Mourão MPG, Brito-Sousa JD, Baía-da-Silva D, Guerra MVF et al. (2020) Effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) infection. JAMA Netw Open 3:e208857.). A randomized, double-blind, placebo-controlled study tested hydroxychloroquine as post-exposure prophylaxis and concluded that it did not significantly reduce the severity of symptoms in outpatients presenting mild and early COVID-19 (Boulware et al., 2020Boulware DR, Pullen MF, Bangdiwala AS, Pastick KA, Lofgren SM, Okafor EC, Skipper CP, Nascene AA, Nicol MR, Abassi M et al. (2020) A randomized trial of hydroxychloroquine as postexposure prophylaxis for Covid-19. N Engl J Med 383:517-525.).

The RECOVERY study compared a variety of possible treatments with the usual care in patients hospitalized with COVID-19. The authors examined the daily use of 6 mg of dexamethasone for ten days (2104 patients) versus usual care alone (4321 patients). The preliminary results indicated lower 28-day mortality among patients receiving invasive mechanical ventilation or oxygen alone, but not among those who did not receive respiratory support at randomization (RECOVERY Collaborative Group, 2021RECOVERY Collaborative Group (2021) Dexamethasone in hospitalized patients with Covid-19 - Preliminary report. N Engl J Med 384:693-704.).

Ivermectin has been recently proved, in an in vitro experiment, to produce reduction in the RNA of SARS CoV-2 at 48 h of its single addition (Caly et al., 2020Caly L, Druce JD, Catton MG, Jans DA and Wagstaff KM (2020) The FDA-approved drug ivermectin inhibits the replication of SARS-CoV-2 in vitro. Antiviral Res 178:104787.). Among patients with non-severe COVID-19 and no risk factors for severe disease receiving a single 400 mcg/kg dose of ivermectin, Chaccour and colleagues (2021Chaccour C, Casellas A, Matteo AB-D, Pineda I, Fernandez-Montero A, Ruiz-Castillo P, Richardson M-A, Rodríguez-Mateos M, Jordán-Iborra C, Brew J et al. (2021) The effect of early treatment with ivermectin on viral load, symptoms and humoral response in patients with non-severe COVID-19: A pilot, double-blind, placebo-controlled, randomized clinical trial. EClinicalMedicine 32:100720.) have found no difference in the proportion of PCR positives. There was however a marked reduction of self-reported anosmia/hyposmia, a reduction of cough and a tendency to lower viral loads and lower IgG titers which warrants assessment in larger trials.

Nitazoxanide, a clinically approved and commercially available antiparasitic drug, has been found to have broad-spectrum antiviral activity, including against coronaviruses, influenza viruses, and hepatitis B and C viruses (Amadi et al., 2002Amadi B, Mwiya M, Musuku J, Watuka A, Sianongo S, Ayoub A and Kelly P (2002) Effect of nitazoxanide on morbidity and mortality in Zambian children with cryptosporidiosis: A randomised controlled trial. Lancet 360:1375-1380.). In patients with mild Covid-19, symptom resolution did not differ between nitazoxanide and placebo groups after 5 days of therapy. However, early nitazoxanide therapy was safe and reduced viral load significantly (Rocco et al., 2021Rocco PRM, Silva PL, Cruz FF, Melo-Junior MAC, Tierno PFGMM, Moura MA, De Oliveira LFG, Lima CC, Dos Santos EA, Junior WF et al. (2021) Early use of nitazoxanide in mild Covid-19 disease: Randomised, placebo-controlled trial. Eur Respir J 14:2003725.).

Besides inflammation, COVID-19 patients may present hypercoagulability, characterized by elevation of fibrinogen levels and D-dimers, and may develop disseminated intravascular coagulation (DIC) (Helms et al., 2020Helms J, Tacquard C, Severac F, Leonard-Lorant I, Ohana M, Delabranche X, Merdji H, Clere-Jehl R, Schenck M, Fagot Gandet F et al. (2020) High risk of thrombosis in patients with severe SARS-CoV-2 infection: A multicenter prospective cohort study. Intensive Care Med 46:1089-1098. ; Tang et al., 2020Tang W, Cao Z, Han M, Wang Z, Chen J, Sun W, Wu Y, Xiao W, Liu S, Chen E et al. (2020) Hydroxychloroquine in patients with mainly mild to moderate coronavirus disease 2019: Open label, randomised controlled trial. BMJ 369:m1849.). Evidence confirms that thrombotic events are associated with higher mortality (Helms et al., 2020Helms J, Tacquard C, Severac F, Leonard-Lorant I, Ohana M, Delabranche X, Merdji H, Clere-Jehl R, Schenck M, Fagot Gandet F et al. (2020) High risk of thrombosis in patients with severe SARS-CoV-2 infection: A multicenter prospective cohort study. Intensive Care Med 46:1089-1098. ). Therefore, the Brazilian Society of Thrombosis and Hemostasis (BSTH) and the Thrombosis and Hemostasis Committee of the Brazilian Association of Hematology, Hemotherapy, and Cellular Therapy (ABHH) recommend that all patients hospitalized for suspected or confirmed COVID-19 should receive pharmacologic thromboprophylaxis in the absence of absolute contraindications.

Immunotherapies: driving the immune response against SARS-CoV2

Anti-Interleukin 6

Considered one of the most potent cytokines of the inflammatory response, and due to its pleiotropic activity, IL-6 mediates a series of physiological functions, including proliferation, differentiation, activation, and survival of immune response cells (Scheller et al., 2011Scheller J, Chalaris A, Schmidt-Arras D and Rose-John S (2011) The pro- and anti-inflammatory properties of the cytokine interleukin-6. Biochim Biophys Acta 1813:878-888.; Tanaka and Kishimoto, 2014Tanaka T and Kishimoto T (2014) The biology and medical implications of Interleukin-6. Cancer Immunol Res 2:288-294.; Schaper et al., 2015Schaper F and Rose-John S (2015) Interleukin-6: Biology, signaling and strategies of blockade. Cytokine Growth Factor Rev 26:475-487.; Murakami et al., 2019Murakami M, Kamimura D and Hirano T (2019) Pleiotropy and specificity: Insights from the Interleukin 6 family of cytokines. Immunity 50:812-831.). Synthesized mainly by lymphocytes, monocytes, and macrophages (Scheller et al., 2011Scheller J, Chalaris A, Schmidt-Arras D and Rose-John S (2011) The pro- and anti-inflammatory properties of the cytokine interleukin-6. Biochim Biophys Acta 1813:878-888.; Schaper and Rose-John, 2015Schaper F and Rose-John S (2015) Interleukin-6: Biology, signaling and strategies of blockade. Cytokine Growth Factor Rev 26:475-487.), as well as stimulated by other cytokines, especially IL-1 and TNF-α (Garbers et al., 2012Garbers C, Hermanns HM, Schaper F, Müller-Newen G, Grötzinger J, Rose-John S and Scheller J (2012) Plasticity and cross-talk of Interleukin 6-type cytokines. Cytokine Growth Factor Rev 23:85-97.), IL-6 is directly involved in the exacerbation of inflammation (Scheller et al., 2011Scheller J, Chalaris A, Schmidt-Arras D and Rose-John S (2011) The pro- and anti-inflammatory properties of the cytokine interleukin-6. Biochim Biophys Acta 1813:878-888.), known as a “hyper-inflammatory state”, which causes intense acute lung injury in severe COVID-19 patients, which can progress to acute respiratory distress syndrome (ARDS) (Swaroopa et al., 2016Swaroopa D, Bhaskar K, Mahathi T, Katkam S, Raju YS, Chandra N and Kutala VK (2016) Association of serum interleukin-6, interleukin-8, and Acute Physiology and Chronic Health Evaluation II score with clinical outcome in patients with acute respiratory distress syndrome. Indian J Crit Care Med 20:518-525.). In an attempt to eliminate SARS-COV-2, this exacerbated and continuous inflammatory reaction, also named “cytokine storm”, essentially has a positive feedback between proinflammatory molecules (mainly IL-6 and TNF-α) and lymphocytes, and also natural killer cells and macrophages (Huang et al.,2020Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, Zhang L, Fan G, Xu J, Gu X et al. (2020) Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 395:497-506.; McGonagle et al., 2020McGonagle D, Sharif K, O’Regan A and Bridgewood C (2020) The role of cytokines including Interleukin-6 in COVID-19 induced pneumonia and macrophage activation syndrome-like disease. Autoimmun Rev 19:102537.; Mehta et al., 2020Mehta P, McAuley DF, Brown M, Sanchez E, Tattersall RS, Manson JJ and HLH Across Speciality Collaboration UK (2020) COVID-19: Consider cytokine storm syndromes and immunosuppression. Lancet 395:1033-1034.; Pedersen and Ho, 2020Pedersen SF and Ho Y-C (2020) SARS-CoV-2: A storm is raging. J Clin Invest 130:2202-2205.).

To stop this inflammatory process that is harmful to the patient, some studies (Wu R et al., 2020Wu R, Wang L, Kuo H-CD, Shannar A, Peter R, Chou PJ, Li S, Hudlikar R, Liu X, Liu Z et al. (2020) An update on current therapeutic drugs treating COVID-19. Curr Pharmacol Rep 6:56-70.; Xu et al., 2020Xu X, Han M, Li T, Sun W, Wang D, Fu B, Zhou Y, Zheng X, Yang Y, Li X et al. (2020) Effective treatment of severe COVID-19 patients with tocilizumab. Proc Natl Acad Sci U S A 117:10970-10975.; REMAP-CAP Investigators, 2021REMAP-CAP Investigators (2021) Interleukin-6 receptor antagonists in critically Ill patients with Covid-19. N Engl J Med 384:1491-1502.) have shown that blocking (tocilizumab or sarilumab) of IL-6 functions promotes a significant clinical improvement and better prognosis for COVID-19 patients with ARDS. Among the main benefits of this treatment, stand out: the reappearance of normal temperature, improvement of oxygenation, reduction of lung injuries, and the return of a healthy percentage of peripheral lymphocytes (Zhang et al., 2021Zhang Y, Zeng G, Pan H, Li C, Hu Y, Chu K, Han W, Chen Z, Tang R, Yin W et al. (2021) Safety, tolerability, and immunogenicity of an inactivated SARS-CoV-2 vaccine in healthy adults aged 18-59 years: A randomized, double-blind, placebo-controlled, phase 1/2 clinical trial. Lancet Infect Dis 21:181-192.). Although basic science suggests rationale for administration of IL-6 receptor antagonists to patients with COVID-19, the clinical evidence regarding the efficacy and safety of tocilizumab remains observational only, according to Cortegiani et al. (2021Cortegiani A, Ippolito M, Greco M, Granone V, Protti A, Gregoretti C, Giarratano A, Einav S and Cecconi M (2021) Rationale and evidence on the use of tocilizumab in COVID-19: A systematic review. Pulmonology 27:52-66.), who investigated 3 indirect pre-clinical and 28 clinical studies. Another difficulty for developing countries is the high cost of this drug.

Convalescent plasma and neutralizing antibodies-based therapies

Neutralizing antibodies (Nabs) represent an immediate possibility to solve SARS-CoV-2 infection. Therefore, therapy-based studies have also focused on this approach. Nabs target the proteins of the viral surface, impairing its attachment to host cells. Therefore, the ACE2 receptor-binding domain S1 of the SARS-CoV-2 S protein has been pointed out as a major target for Nabs-based strategies by several in vitro and in vivo models (Duan et al., 2020Duan K, Liu B, Li C, Zhang H, Yu T, Qu J, Zhou M, Chen L, Meng S, Hu Y et al. (2020) Effectiveness of convalescent plasma therapy in severe COVID-19 patients. Proc Natl Acad Sci U S A 117:9490-9496.; Wang et al., 2020Wang C, Liu Z, Chen Z, Huang X, Xu M, He T and Zhang Z (2020) The establishment of reference sequence for SARS-CoV-2 and variation analysis. J Med Virol 92:667-674.; Wrapp et al., 2020Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh C-L, Abiona O, Graham BS and McLellan JS (2020) Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 367:1260-1263.; Wu R et al., 2020Wu Y, Li C, Xia S, Tian X, Kong Y, Wang Z, Gu C, Zhang R, Tu C, Xie Y et al. (2020) Identification of human single-domain antibodies against SARS-CoV-2. Cell Host Microbe 27:891-898.; Zeng et al., 2020Zeng X, Li L, Lin J, Li X, Liu B, Kong Y, Zeng S, Du J, Xiao H, Zhang T et al. (2020) Isolation of a human monoclonal antibody specific for the receptor binding domain of SARS-CoV-2 using a competitive phage biopanning strategy. Antib Ther 3:95-100.).

In this context, convalescent plasma-based therapies are potential strategies to treat SARS-CoV-2 infection, since patients recovered from COVID-19 can present high levels of Nabs (Chen L et al., 2020Chen Y, Liu Q and Guo D (2020) Emerging coronaviruses: Genome structure, replication, and pathogenesis. J Med Virol 92:418-423.). Historically, passive immunotherapy through the collection and transfusion of convalescent plasma, was first used in the late 19th century (Simon, 2007Simon J (2007) Emil Behring’s medical culture: From disinfection to serotherapy. Med Hist 51:201-218.; Marano et al., 2016Marano G, Vaglio S, Pupella S, Facco G, Catalano L, Liumbruno GM and Grazzini G (2016) Convalescent plasma: New evidence for an old therapeutic tool? Blood Transfus 14:152-157.). During the Spanish flu, the use of these immune derivatives showed effective clinical potential (Bogardus, 1919Bogardus FB (1919) Influenza pneumonia treated by blood Transfusion. South Med J 109:765-768.), reducing the mortality (Luke et al., 2006Luke TC, Kilbane EM, Jackson JL and Hoffman SL (2006) Meta-Analysis: Convalescent blood products for spanish influenza pneumonia: A future H5N1 treatment? Ann Intern Med 145:599-609.). More recently, convalescent plasma was used during the H1N1 influenza pandemic in 2009 and 2013 during the Ebola outbreak in West Africa. However, the antibody levels in COVID-19 convalescent plasma are highly variable, and assays to determine the effective antibody titers remain limited (Brown and McCullough, 2020Brown BL and McCullough J (2020) Treatment for emerging viruses: Convalescent plasma and COVID-19. Transfus Apher Sci 59:102790.).

Some studies have demonstrated a reduction in viral load in COVID-19 patients treated with convalescent plasma (Ahn et al., 2020Ahn JY, Sohn Y, Lee SH, Cho Y, Hyun JH, Baek YJ, Jeong SJ, Kim JH, Ku NS, Yeom JS et al. (2020) Use of convalescent plasma therapy in two COVID-19 patients with acute respiratory distress syndrome in Korea. J Korean Med Sci 35:e149.; Duan et al., 2020Duan K, Liu B, Li C, Zhang H, Yu T, Qu J, Zhou M, Chen L, Meng S, Hu Y et al. (2020) Effectiveness of convalescent plasma therapy in severe COVID-19 patients. Proc Natl Acad Sci U S A 117:9490-9496.; Shen C et al., 2020Shen Z, Xiao Y, Kang L, Ma W, Shi L, Zhang L, Zhou Z, Yang J, Zhong J, Yang D et al. (2020) Genomic diversity of severe acute respiratory syndrome-coronavirus 2 in patients with coronavirus disease 2019. Clin Infect Dis 71:713-720.; Ye et al., 2020Ye M, Fu D, Ren Y, Wang F, Wang D, Zhang F, Xia X and Lv T (2020) Treatment with convalescent plasma for COVID-19 patients in Wuhan, China. J Med Virol 92:1890-1901.; Zhang et al., 2021Zhang Y, Zeng G, Pan H, Li C, Hu Y, Chu K, Han W, Chen Z, Tang R, Yin W et al. (2021) Safety, tolerability, and immunogenicity of an inactivated SARS-CoV-2 vaccine in healthy adults aged 18-59 years: A randomized, double-blind, placebo-controlled, phase 1/2 clinical trial. Lancet Infect Dis 21:181-192.). Almost all patients showed improvement in the clinical, laboratory and imaging parameters. However, it was not possible to attribute the favorable clinical response to convalescent plasma, as the multiplicity of drugs used and the lack of controls prevented this conclusion (Ye et al., 2020Ye M, Fu D, Ren Y, Wang F, Wang D, Zhang F, Xia X and Lv T (2020) Treatment with convalescent plasma for COVID-19 patients in Wuhan, China. J Med Virol 92:1890-1901.).

Anti-complement approaches

The inhibition of critical inflammatory components of the complement cascade seems to be very useful because, at the same time that it blocks the adaptive immune response, it can control the tissue damage associated with the cytokine storm in severe cases of COVID-19 (Chauhan et al., 2020Chauhan AJ, Wiffen LJ and Brown TP (2020) COVID-19: A collision of complement, coagulation and inflammatory pathways. J Thromb Haemost 18:2110-2117.). This strategy was recently tested during three weeks in ten patients treated with a combination of ruxolitinib, a JAK1/2 inhibitor, and eculizumab, an anti-C5a complement monoclonal antibody. The results showed improved lung function and decreased circulating D-dimer levels (Giudice et al., 2020Giudice V, Pagliano P, Vatrella A, Masullo A, Poto S, Polverino BM, Gammaldi R, Maglio A, Sellitto C, Vitale C et al. (2020) Combination of ruxolitinib and eculizumab for treatment of severe SARS-CoV-2-Related acute respiratory distress syndrome: A controlled study. Front Pharmacol 11:857.). Interestingly, some studies have proposed that complement blockade might be of benefit in severe COVID-19; however, several risk factors for such infections were related following eculizumab administration (Diurno et al.,2020Diurno F, Numis FG, Porta G, Cirillo F, Maddaluno S, Ragozzino A, De Negri P, Di Gennaro C, Pagano A, Allegorico E et al. (2020) Eculizumab treatment in patients with COVID-19: Preliminary results from real life ASL Napoli 2 Nord experience. Eur Rev Med Pharmacol Sci 24:4040-4047.; Laurence et al., 2020Laurence J, Mulvey JJ, Seshadri M, Racanelli A, Harp J, Schenck EJ, Zappetti D, Horn EM and Magro CM (2020) Anti-complement C5 therapy with eculizumab in three cases of critical COVID-19. Clin Immunol 219:108555.). This medicine is still being investigated in clinical trials (NCT number: 04288713 and NCT number: 04346797) for the treatment of moderate to severe pneumonia related to COVID-19.

Main vaccines against Sars-CoV-2 available

CoronaVac is produced by the Chinese company Sinovac Biotech. The vaccine uses the inactivated Sars-CoV-2 virus in its formulation as well as other vaccines that are under development, such as BBIBP-CorV and BBV152 (Zang et al., 2021Zhang Y, Zeng G, Pan H, Li C, Hu Y, Chu K, Han W, Chen Z, Tang R, Yin W et al. (2021) Safety, tolerability, and immunogenicity of an inactivated SARS-CoV-2 vaccine in healthy adults aged 18-59 years: A randomized, double-blind, placebo-controlled, phase 1/2 clinical trial. Lancet Infect Dis 21:181-192.). The vaccine passed Phase III clinical trials in Brazil, Chile, Indonesia, the Philippines, and Turkey. CoronaVac does not need to be frozen, and both the vaccine and raw material for formulating the new doses could be transported and refrigerated at 2-8 ° C, temperatures at which flu vaccines are kept (Sinovac Biotech).

Several results from CoronaVac’s Phase III demonstrate positive results regarding its effectiveness. A study in Chile found it 67% effective against symptoms, reduced hospitalizations by 85%, intensive care visits by 89%, and deaths by 80%. In Brazil, it showed 50.7% effectiveness at preventing symptomatic infections and 83.7% effective in preventing mild cases needing treatment. Effectiveness against symptomatic infections increased to 62.3% with an interval of 21 days or more between the doses (Mallapaty, 2021Mallapaty S (2021) China COVID vaccine reports mixed results - What does that mean for the pandemic? Nature. DOI: 10.1038/d41586-021-00094-z.
https://doi.org/10.1038/d41586-021-00094...
). Final Phase III results from Turkey announced on 3 March 2021 showed an effectiveness of 83.5% (Riad et al., 2021Riad A, Sağıroğlu D, Üstün B, Pokorná A, Klugarová J, Attia S and Klugar M (2021) Prevalence and risk factors of CoronaVac side effects: An independent cross-sectional study among healthcare workers in Turkey. J Clin Med 10:2629.).

On January 22, 2021 the Brazil’s health regulatory agency (Anvisa) granted the first CoronaVac vaccine registration against COVID-19, for emergencial use in Brazil. The immunizer from the Sinovac/Butantan Laboratory had its safety, quality and effectiveness checked and attested by Anvisa’s technical team (https://vacinacovid.butantan.gov.br/).

The vaccine produced by the pharmaceutical company AstraZeneca in conjunction with the University of Oxford has become a wide option in the fight against SARS-CoV-2. It uses a chimpanzee common cold viral vector known as ChAdOx1, which expresses the gene that allows human cells to produce the SARS-CoV-2 spike protein (AstraZeneca). Between April 23 and Nov 4, 2020, 11 636 participants from UK and Brazil were included in the interim primary effectiveness analysis. In participants who received two standard doses, vaccine effectiveness was 62.1% and in participants who received a low dose followed by a standard dose, effectiveness was 90,0%. Overall vaccine effectiveness across both groups was 70,4% (Voysey et al., 2021Voysey M, Clemens SAC, Madhi SA, Weckx LY, Folegatti PM, Aley PK, Angus B, Baillie VL, Barnabas SL, Bhorat QE et al. (2021) Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK. Lancet 397:99-111.).

On March 12, 2021 the Anvisa authorized the distribution of the AstraZeneca / Oxford vaccine in Brazil. The immunizer produced in Brazil within Fiocruz Institute had its safety, quality and effectiveness checked and attested by Anvisa’s technical team (Ministério da Saúde, 2021Ministério da Saúde (2021c) Anvisa aprova registro da vacina da Fiocruz/AstraZeneca e de medicamento contra o coronavírus, Ministério da Saúde (2021c) Anvisa aprova registro da vacina da Fiocruz/AstraZeneca e de medicamento contra o coronavírus, https://www.gov.br/anvisa/pt-br/assuntos/noticias-anvisa/2021/anvisa-aprova-registro-da-vacina-da-fiocruz-astrazeneca-e-de-medicamento-contra-o-coronavirus (accessed 27 April 2021)
https://www.gov.br/anvisa/pt-br/assuntos...
c).

Another vaccine against COVID-19 similar to AstraZeneca’s is produced by the pharmaceutical company Janssen. It is known as JNJ-78436735 or Ad26.COV2.S. The viral agent used as a vector is adenovirus 26. Initially, the Janssen vaccine was shown to induce antibodies against SARS-CoV-2 in 90% of people after the first dose. Just one dose of vaccine was 66% effective in preventing moderate to severe COVID-19 and 100% effective in preventing COVID-19-related hospitalization and death (Livingston et al., 2021Livingston EH, Preeti NM and Creech CB (2021) The Johnson & Johnson vaccine for COVID-19. JAMA 325:1575.).

The Pfizer/BioNTech Vaccine is a lipid nanoparticle-formulated, nucleoside-modified mRNA encoding the prefusion spike glycoprotein of SARS-CoV-2. This vaccine has been recommended to people 16 years of age and older, with a dose of 30 μg (0.3 mL) IM. The vaccination requires two shots given 21 or more days apart. Anti-SARS-CoV-2 antibodies persist for at least 119 days after the first vaccination and prevention of the SARS-COV-2 infection is 95% effective (Oliver et al., 2020Oliver SE, Gargano JW, Marin M, Wallace M, Curran KG, Chamberland M, McClung N, Campos-Outcalt D, Morgan RL, Mbaeyi S et al. (2020) The Advisory Committee on Immunization Practices’ Interim Recommendation for Use of Pfizer-BioNTech COVID-19 Vaccine - United States, December 2020. MMWR Morb Mortal Wkly Rep 69:1922-1924.; Meo et al., 2021Meo SA, Bukhari IA, Akram J, Meo AS and Klonoff DC (2021) COVID-19 vaccines: Comparison of biological, pharmacological characteristics and adverse effects of Pfizer/BioNTech and Moderna Vaccines. Eur Rev Med Pharmacol Sci 25:1663-1669. ). On December 11, 2020, the US Food and Drug Administration (FDA) authorized the emergency use of the Pfizer-BioNTech COVID-19 vaccine (FDA, 2020FDA Briefing Document (2020) Pfizer-BioNTech COVID-19 vaccine. U.S. Food and Drug Administration; 53, Food and Drug Administration; 53, https://www.fda.gov/media/144245/download (accessed 26 April 2021).
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).

On February 23, 2021 the Anvisa granted the first registration of the Pfizer/BioNtech vaccine for widespread use in the Americas. The vaccine had its safety, quality and effectiveness checked and attested by Anvisa’s technical team of servers (Ministério da Saúde, 2021Ministério da Saúde (2021a) É o primeiro registro definitivo concedido para uma vacina no Brasil e nas Américas, Ministério da Saúde (2021a) É o primeiro registro definitivo concedido para uma vacina no Brasil e nas Américas, https://www.gov.br/anvisa/pt-br/assuntos/noticias-anvisa/2021/informe-a-populacao-brasileira (accessed 26 April 2021).
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a).

The Russian Institute Gamaleya developed Sputnik V (Gam-COVID-Vac), an adenovirus-based candidate vaccine against COVID-19e The Sputnik V vaccine consists of two replication-defective recombinant adenoviruses: type 26 (rAd26-S) and type 5 (rAd5-S), both carrying the gene for the SARS-CoV-2) spike glycoprotein (Logunov et al., 2020Logunov DY, Dolzhikova IV, Zubkova OV, Tukhvatullin AI, Shcheblyakov DV, Dzharullaeva AS, Grousova DM, Erokhova AS, Kovyrshina AV, Botikov AG et al. (2020) Safety and immunogenicity of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine in two formulations: two open, non-randomised phase 1/2 studies from Russia. Lancet 396:887-897.). The results of phase I-II studies indicated good immunogenicity and safety, however, only 38 volunteers were enrolled for each of the two formulations (frozen and lyophilized) (Logunov et al., 2020Logunov DY, Dolzhikova IV, Zubkova OV, Tukhvatullin AI, Shcheblyakov DV, Dzharullaeva AS, Grousova DM, Erokhova AS, Kovyrshina AV, Botikov AG et al. (2020) Safety and immunogenicity of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine in two formulations: two open, non-randomised phase 1/2 studies from Russia. Lancet 396:887-897.). Recent interim results of a Sputnik V phase 3 trial in a large cohort indicated 91.6% effectiveness against COVID-19 and lack of adverse vaccination-related adverse effects (Logunov et al., 2021Logunov DY, Dolzhikova IV, Zubkova OV, Tukhvatullin AI, Shcheblyakov DV, Dzharullaeva AS, Grousova DM, Erokhova AS, Kovyrshina AV, Botikov AG et al. (2020) Safety and immunogenicity of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine in two formulations: two open, non-randomised phase 1/2 studies from Russia. Lancet 396:887-897.).

However, the development of the Sputnik V vaccine has been criticized for unseemly haste, corner cutting, and an absence of transparency (Balakrishnan, 2020Balakrishnan VS (2020) The arrival of Sputnik V. Lancet Infect Dis 20:1128. ; Cohen, 2020Cohen J (2020) Russia’s claim of a successful COVID-19 vaccine doesn’t pass the ‘smell test,’ critics say. Science. DOI: 10.1126/science.abf6791.
https://doi.org/10.1126/science.abf6791....
; Bucci et al., 2021Bucci EM, Berkhof J, Gillibert A, Gopalakrishna G, Calogero RA, Bouter LM, Andreev K, Naudet F and Vlassov V (2021) Data discrepancies and substandard reporting of interim data of Sputnik V phase 3 trial. Lancet 397:1881-1883.). Serious concerns regarding interim results from the phase III trial were also raised (Bucci et al., 2021Bucci EM, Berkhof J, Gillibert A, Gopalakrishna G, Calogero RA, Bouter LM, Andreev K, Naudet F and Vlassov V (2021) Data discrepancies and substandard reporting of interim data of Sputnik V phase 3 trial. Lancet 397:1881-1883.). Data sharing is one of the cornerstones of research integrity, yet Logunov et al. (2021Logunov DY, Dolzhikova IV, Zubkova OV, Tukhvatullin AI, Shcheblyakov DV, Dzharullaeva AS, Grousova DM, Erokhova AS, Kovyrshina AV, Botikov AG et al. (2020) Safety and immunogenicity of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine in two formulations: two open, non-randomised phase 1/2 studies from Russia. Lancet 396:887-897.) stated that raw data will not be shared before the trial is completed. Among the concerns raised are: the full study protocol has not been made publicly available; the clinical and laboratory criteria used to determine suspected COVID-19 were not informed; the data, numerical, and statistical significance results reported showed major inconsistencies (Bucci et al., 2021Bucci EM, Berkhof J, Gillibert A, Gopalakrishna G, Calogero RA, Bouter LM, Andreev K, Naudet F and Vlassov V (2021) Data discrepancies and substandard reporting of interim data of Sputnik V phase 3 trial. Lancet 397:1881-1883.).

On April 27, 2021 the Anvisa announced that the import of the Sputnik V vaccine was not approved for use in Brazil. According to the agency, after evaluation, flaws in the development and production of the immunizing agent would have been found (Ministério da Saúde, 2021Ministério da Saúde (2021b) Anvisa não aprova importação da vacina Sputnik V, Ministério da Saúde (2021b) Anvisa não aprova importação da vacina Sputnik V, https://www.gov.br/anvisa/pt-br/assuntos/noticias-anvisa/2021/anvisa-nao-aprova-importacao-da-vacina-sputnik-v (accessed 27 April 2021).
https://www.gov.br/anvisa/pt-br/assuntos...
b). The concerns are similar to those now reported in May 2021 by Bucci et al. (2021Bucci EM, Berkhof J, Gillibert A, Gopalakrishna G, Calogero RA, Bouter LM, Andreev K, Naudet F and Vlassov V (2021) Data discrepancies and substandard reporting of interim data of Sputnik V phase 3 trial. Lancet 397:1881-1883.).

Final considerations

Twenty months after the first Covid-19 notifications, more than 170 million individuals were infected worldwide with SARS-CoV-2, and around 3.5 million deaths occurred. Unlike the period of the last great pandemic that occurred at the beginning of the past century, the COVID-19 pandemic occurs at a time of significant scientific and technological advances in biomedical sciences, which, in theory, could be applied immediately in the control and treatment of patients. However, no drug or vaccine has yet been specifically approved for COVID-19. Therapeutic intervention approaches used successfully in other infectious agents need an in-deep investigation directed to the specific infection mechanism of the SARS-CoV-2 and the unique COVID-19 physiopathology. Among the available therapeutic approaches, such as vaccines, target inhibitors, and new drugs, the drug repurposing already approved by the FDA has been shown to be an efficient short-term alternative, mainly due to its low cost and prompt application to patients. This strategy considers the knowledge of the molecular basis of the disease. As a result of the global task force to control the COVID-19 pandemic, a new intervention was introduced by Garvin et al. (2020Garvin MR, Alvarez C, Miller JI, Prates ET, Walker AM, Amos BK, Mast AE, Justice A, Aronow B and Jacobson D (2020) A mechanistic model and therapeutic interventions for COVID-19 involving a RAS-mediated bradykinin storm. Elife 9:e59177.), who blamed the bradykinin storm for the most severe symptoms of COVID-19. The authors point out that many of the symptoms manifested by patients with COVID-19 are similar to other clinical conditions caused by the increase in bradykinin. The strategy would be pharmacologic intervention targeting the renin-angiotensin system to reduce bradykinin levels. In this sense, there exist at least ten approved drugs that might be used to control the severe symptoms of COVID-19.

The genetic variability of molecules that participate in the entry of SARS-CoV-2 into the host cells and, especially, of the numerous molecules involved in the immune responses should be considered for the development of effective therapeutic interventions. Because the frequencies of genetic variants influencing the response to drugs, as well as COVID-19 susceptibility and severity may differ widely among world populations, knowing their distribution is a critical element in seeking strategies to respond to the pandemic. Moreover, understanding the repertoire of viral epitopes that specific HLA allotypes can bind is of great importance for the development of vaccines that can provide protection for most individuals.

Computational modeling and simulations with toxicity analysis scenarios are needed to boost pharmacological interventions and drug repurposing, aiming at potential drugs to reduce viral load, viral clearance, and morbidity and mortality in clinical outcomes (Al-Kofahi et al., 2020Al‐Kofahi M, Jacobson P, Boulware DR, Matas A, Kandaswamy R, Jaber MM, Rajasingham R, Young JAH and Nicol MR (2020) Finding the dose for hydroxychloroquine prophylaxis for COVID‐19: The desperate search for effectiveness. Clin Pharmacol Ther 108:766-769.). New therapeutic agents can be developed by analyzing theoretical structure-activity data in a three-dimensional approach, obtained by recent molecular modeling techniques. Choosing the right dose for a clinical trial requires considering the risk of toxicity and ensuring the best chance of successfully reaching therapeutic targets (Al-Kofahi et al., 2020Al‐Kofahi M, Jacobson P, Boulware DR, Matas A, Kandaswamy R, Jaber MM, Rajasingham R, Young JAH and Nicol MR (2020) Finding the dose for hydroxychloroquine prophylaxis for COVID‐19: The desperate search for effectiveness. Clin Pharmacol Ther 108:766-769.; Dong et al., 2020Dong L, Hu S and Gao J (2020) Discovering drugs to treat coronavirus disease 2019 (COVID-19). Drug Discov Ther 14:58-60.). It is noteworthy that in vitro to in vivo extrapolations can underestimate or overestimate the real needs of medicines, but it is considered an initial advance.

Acknowledgements

This work was financed by The National Council for Scientific and Technological Development (CNPq), grant 465539/2014-9 and São Paulo Research Foundation (FAPESP), grant 2013/08135-2, 2017/23205-8 and 2019/08528-0.

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Internet Resources

Publication Dates

  • Publication in this collection
    08 Apr 2022
  • Date of issue
    2021

History

  • Received
    17 Dec 2020
  • Accepted
    24 Dec 2021
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