key: cord-334300-hnrmaytm authors: Ventura Fernandes, Bianca H; Feitosa, Natália Martins; Barbosa, Ana Paula; Bomfim, Camila Gasque; Garnique, Anali M. B.; Gomes, Francisco I. F.; Nakajima, Rafael T.; Belo, Marco A. A.; Eto, Silas Fernandes; Fernandes, Dayanne Carla; Malafaia, Guilherme; Manrique, Wilson G.; Conde, Gabriel; Rosales, Roberta R. C.; Todeschini, Iris; Rivero, Ilo; Llontop, Edgar; Sgro, German G.; Oka, Gabriel Umaji; Bueno, Natalia F; Ferraris, Fausto K.; de Magalhaes, Mariana T. Q.; Medeiros, Renata J.; Gomes, Juliana M. M; de Souza Junqueira, Mara; Conceição, Katia; Pontes, Letícia G.; Condino-Neto, Antonio; Perez, Andrea C.; Barcellos, Leonardo J. G.; Correa junior, Jose Dias; Dorlass, Erick G.; Camara, Niels O. S; Durigon, Edison Luiz; Cunha, Fernando Q.; Nóbrega, Rafael H.; Machado-Santelli, Glaucia M.; Farah, Chuck; Veras, Flávio P; Galindo-Villegas, Jorge; Costa-Lotufo, Leticia; Cunha, Thiago M.; Chammas, Roger; Guzzo, Cristiane R.; Carvalho, Luciani R; Charlie-Silva, Ives title: Zebrafish studies on the vaccine candidate to COVID-19, the Spike protein: Production of antibody and adverse reaction date: 2020-10-20 journal: bioRxiv DOI: 10.1101/2020.10.20.346262 sha: doc_id: 334300 cord_uid: hnrmaytm Establishing new experimental animal models to assess the safety and immune response to the antigen used in the development of COVID-19 vaccine is an imperative issue. Based on the advantages of using zebrafish as a model in research, herein we suggest doing this to test the safety of the putative vaccine candidates and to study immune response against the virus. We produced a recombinant N-terminal fraction of the Spike SARS-CoV-2 protein and injected it into adult female zebrafish. The specimens generated humoral immunity and passed the antibodies to the eggs. However, they presented adverse reactions and inflammatory responses similar to severe cases of human COVID-19. The analysis of the structure and function of zebrafish and human Angiotensin-converting enzyme 2, the main human receptor for virus infection, presented remarkable sequence similarities. Moreover, bioinformatic analysis predicted protein-protein interaction of the Spike SARS-CoV-2 fragment and the Toll-like receptor pathway. It might help in the choice of future therapeutic pharmaceutical drugs to be studied. Based on the in vivo and in silico results presented here, we propose the zebrafish as a model for translational research into the safety of the vaccine and the immune response of the vertebrate organism to the SARS-CoV-2 virus. The World Health Organization (WHO) registered, on January 30th, 2020, that 127 the outbreak of the disease caused by the severe acute respiratory syndrome coronavirus 128 2 (SARS-CoV-2) constituted a Public Health Emergency of International Importance 129 (the highest level of alert from the Organization)1. Since then, the number of conclusion. However, the case of COVID-19 meets a new pandemic paradigm and the 161 development of the vaccine has been proposed to be reduced to 1-2 years 8 . 162 It is worth mentioning that before vaccine clinical tests begin, several safety 163 protocols must be submitted with in vitro and in vivo experiments on animal models. 164 There is a lack of information regarding the immune response of the organism to SARS- 165 CoV-2, including animal models to study it 9 . Although zebrafish do not have lungs as 166 humans do, the present study shows similar inflammatory responses observed in severe 167 cases of COVID-19 patients that could be considered when investigating human 168 responses to the virus. 169 In the global task to develop the vaccine and possible therapeutic approaches for 170 COVID-19, several animal models have been proposed, such as mice 10 , hACE2 171 transgenic mice 11 , alpaca 12 , golden Syrian hamsters, ferrets, dogs, pigs, chickens, and 172 cats 9 , and species of non-human primates 10 . Recently, three reports have described the 173 production of equine neutralizing antibodies against SARS-CoV-2 13,14 . A study by Deng 174 and collaborators analyzed serum samples from 35 animal species for the detection of 175 specific antibodies against SARS-CoV-2 15 . Despite this wide search for candidate 176 animal models, so far only two references promote the zebrafish model on this regard 177 confirming the innovative and pioneer characteristics of our study 16, 17 . 178 Here, female zebrafish individuals injected with a N-terminal fraction of SARS- Two bioassays were carried out to analyze the toxicity of the rSpike. Although 220 the immunized fish produced antibodies, the first injection of the rSpike generated high 221 toxicity to the fish ( Figure 2) . Therefore, the assay was repeated by adding different 222 control groups to confirm that the toxicity findings were specific to the rSpike ( Figure 223 2). In the first bioassay, after fish immunization with rSpike, the survival rate was 224 78.6% during the first seven days (Figure 2) . It was significant when compared to naive 225 control and fish injected with protein buffer (control 1), where the survival rate was 226 100% and 90%, respectively ( Figure 2) . Nonetheless, after a second immunization, the 227 rSpike immunized group maintained the plateau survival rate, with no statistical 228 significance between the groups for the relative risk of death ( Figure 2) . 229 Therefore, a second assay was conducted by adding different control groups in 230 order to confirm that the toxicity findings were specific to the rSpike, and related to the In order to verify the occurrence of sublethal effects of the rSpike on treated 243 zebrafish, histopathological analysis of different organs, including brain, gonads, heart, 244 kidney, liver, spleen, among others, was performed in female fishes used in the 245 immunization protocol described in material and methods. Animals that died during the 246 immunization experiment were excluded from the analysis. In general, it was observed 247 several morphological alterations compatible with an undergoing inflammatory process 248 in many tissues. Markedly, brain obtained from treated fishes showed an intense Table 1 . The first experiments aimed to analyze the humoral response with antibody 371 production and used, besides the rSpike, the appropriate negative controls as the E. coli Table 1 . What can we expect from first-generation COVID-19 899 vaccines? Lancet Passive Immunization of Farmed 901 Making waves in cancer research: new 903 models in the zebrafish The zebrafish reference genome sequence 906 and its relationship to the human genome Zebrafish as an 909 alternative animal model in human and animal vaccination research Potential of mucoadhesive 912 nanocapsules in drug release and toxicology in zebrafish Zebrafish as tools for drug discovery Developing Covid-19 Vaccines at 917 Pandemic Speed A Comprehensive Review of Animal Models 920 for Coronaviruses: SARS-CoV-2, SARS-CoV, and MERS-CoV Comparison of SARS-CoV-2 923 infections among 3 species of non-human Affiliations Non-invasive bioluminescence imaging of HCoV-925 OC43 infection and therapy in the central nervous system of live mice An alpaca nanobody neutralizes 928 SARS-CoV-2 by blocking receptor interaction Equine hyperimmune globulin raised 931 against the SARS-CoV-2 spike glycoprotein has extremely high neutralizing 932 titers Development of a 934 hyperimmune equine serum therapy for COVID-19 in Argentina Serological survey of SARS-CoV-2 for experimental, 937 domestic, companion and wild animals excludes intermediate hosts of 35 938 different species of animals The Zebrafish Disease and Drug Screening Model: A Strong 941 Ally Against Covid-19 Tracking Mechanisms of Viral Dissemination In Vivo A Contemporary View of Coronavirus 946 Antibody responses to SARS-CoV-2 in 948 patients with COVID-19 Maternal transfer and protective role of 951 antibodies in zebrafish Danio rerio Role of maternally derived immunity in fish Breast Milk-fed Infant of COVID-956 19  SARS-CoV-2 S1 and S2 subunits-and nucleocapsid protein-reactive SIgM/IgM IgA antibodies in human milk Acute transverse myelitis after 963 COVID-19 pneumonia Pathological findings of COVID-19 associated with 966 acute respiratory distress syndrome Patients -An MRI-based 3-month Follow-up Study Cerebral Venous Thrombosis Associated 972 with COVID-19 Effects of COVID-19 on the Nervous System Respiratory Syndrome Coronavirus Infection Causes Neuronal Death in the 981 Absence of Encephalitis in Mice Transgenic for Human ACE2 Pathological study of the 2019 novel coronavirus 984 disease (COVID-19) through postmortem core biopsies COVID-19 Infection 987 and Neurological Complications: Present Findings and Future Predictions COVID-19-associated acute necrotizing 990 myelitis Pathogenesis and Diagnosis of Viral 993 Infections of the Nervous System Neurologic and Radiographic Findings 996 Associated With COVID-19 Infection in Children The conundrum of interleukin-6 blockade in COVID-999 19 Early administration of interleukin-6 1002 inhibitors for patients with severe COVID-19 disease is associated with 1003 decreased intubation, reduced mortality, and increased discharge Inborn Errors of Human JAKs and 1006 Immunodeficiency due to Heterozygous Gain-of-Function Mutations in STAT1 STAT Transcription Factors in T Cell Control of 1011 STAT1 Knockout Mice are Highly 1013 Susceptible to Pulmonary Mycobacterial Infection ERK/MAPK signalling pathway and 1016 tumorigenesis (Review) Hemophagocytic syndromes -An update Kidney Biopsy Findings in Patients with 1021 COVID-19 Identification of a potential 1024 mechanism of acute kidney injury during the COVID-19 outbreak: a study based 1025 on single-cell transcriptome analysis Kidney involvement in COVID-19 and rationale for 1028 extracorporeal therapies Clinical characteristics of fatal and recovered cases 1031 of coronavirus disease 2019 in Wuhan, China: a retrospective study COVID-19: Abnormal liver function tests Melano-macrophage centres and their role in fish 1036 pathology COVID-19 and 1039 the liver its receptor Mas, and the angiotensin-converting enzyme type 1042 2 are expressed in the human ovary The Vasoactive Peptide 1045 Its Receptor Mas and the Angiotensin-converting Enzyme 1046 Type 2 are Expressed in the Human Endometrium SARS-CoV-2 and the next 1049 generations: which impact on reproductive tissues? Pathogenesis of SARS-CoV-2 in Transgenic 1052 Mice Expressing Human Angiotensin-Converting Enzyme 2 Predicting the angiotensin converting enzyme 2 1055 (ACE2) utilizing capability as the receptor of SARS-CoV-2. Microbes Infect ACE2 orthologues in non-mammalian 1058 vertebrates (Danio, Gallus, Fugu, Tetraodon and Xenopus) Development of epitope-based 1061 70 peptide vaccine against novel coronavirus 2019 (SARS-COV-2): 1062 Immunoinformatics approach Fishing for mammalian paradigms in the teleost immune system A cold-blooded view of adaptive immunity Reconstructing immune phylogeny: new 1069 perspectives Diversity and repertoire of IgW 1073 and IgM VH families in the newborn nurse shark Implications for a distinct B cell receptor 1077 in lower vertebrates Targets of somatic hypermutation within 1080 immunoglobulin light chain genes in zebrafish Perspectives on antigen presenting cells in 1083 zebrafish Breeding Zebrafish: 1087 A Review of Different Methods and a Discussion on Standardization Neuroproteomics tools in 1091 clinical practice A New General Topology for Cascaded Multilevel 1094 Fixation and Decalcification of 1097 The Development of a Universal 1100 Silico Predictor of Protein-Protein Interactions. Csermely P Profiler-a web server for functional 1103 interpretation of gene lists (2016 update) Pathview: an R/Bioconductor package for pathway-based 1106 data integration and visualization Kyoto 1109 Encyclopedia of Genes and Genomes PBS buffer for 2 hours and after few PBS rinse the membrane was incubated with 894 monoclonal Anti-polyHistidine−Peroxidase antibody produced in mouse (Sigma-895 Aldrich).