Team:Rio UFRJ Brazil/Description
Project
Description
Description
Overview
Arboviruses caused by Dengue (DENV), Chikungunya (CHIKV), and Zika (ZIKV) viruses are a health problem recognized by the World Health Organization, WHO. In 2020 alone, 987,173 probable cases of Dengue, 82,419 cases of Chikungunya, and 7,387 cases of Zika, were reported in Brazil (MINISTÉRIO DA SAÚDE, 2021). The two usual diagnostics for viral detection are molecular and serological assays. Molecular ones can only be performed during the acute phase, while the serological ones in the convalescent phase. However, the diagnosis of these diseases presents difficulties, mainly due to the genetic similarity between ZIKV and DENV, which generates similar symptoms, such as fever and muscle pain, and makes the existing tests unspecific.
Serological assays detect antibodies produced by defense cells against virus infection, mainly immunoglobulins of type G (IgG) and M (IgM). For similar pathogens such as Dengue and Zika, a phenomenon called cross-reaction might happen during this type of test. This results in nonspecific detection of those diseases (MONTECILLO et al., 2019). This misdiagnosis can have serious consequences since it hinders patient's treatment and better epidemiological control of these diseases (MULLER, DEPELSENAIRE & YOUNG, 2017). 
Serological assays detect antibodies produced by defense cells against virus infection, mainly immunoglobulins of type G (IgG) and M (IgM). For similar pathogens such as Dengue and Zika, a phenomenon called cross-reaction might happen during this type of test. This results in nonspecific detection of those diseases (MONTECILLO et al., 2019). This misdiagnosis can have serious consequences since it hinders patient's treatment and better epidemiological control of these diseases (MULLER, DEPELSENAIRE & YOUNG, 2017).
Therefore the Osiris Rio UFRJ team decided to develop a project to mitigate the misdiagnosis caused by cross-reaction, thus enabling patients to receive the medical attention they need. To accomplish this, we needed to understand the problems regarding the effectiveness of the existing tests for the arboviruses. Hence we were guided by the following questions:
What happens in the body after infection?
How is an Antigen-Antibody interaction?
How do existing tests work?
What is the cross-reaction phenomenon?
How can we solve the problem?
What happens in the body after infection?
How is an Antigen-Antibody interaction done?
The viruses that cause Dengue, Zika, or Chikungunya are transmitted by the Aedes aegypt mosquito after its bite, that is, from contact with contaminated saliva, the virus is directly inserted into the host's bloodstream (SUNIT et al., 2007). After viral infection, activation of B lymphocytes occurs, followed by their differentiation into plasma cells, activating a so-called humoral response. Plasma cells are the cells responsible for producing immunoglobulins (Ig), antigenic proteins such as hydroelectric. In the case of viral infection, the summaries are of the IgG and IgM type, capable of triggering an immune response to fight the viral infection (MACHADO et al., 2004). In this context, the antibody is a protein, linked by the body's defense cells with the function of recognizing the antigens in question.
Antigen recognition by antibodies is given by their binding to a specific structure of the antigens, known as epitopes. This action is capable of promoting the neutralization of the pathogen, preventing its binding with host cells. But it also promotes the so-called opsonization of the pathogen, where it is possible to induce phagocytosis of the same by macrophages and neutrophils (Tay M.Z, et al 2019). 
Antigen recognition by antibodies is given by their binding to a specific structure of the antigens, known as epitopes. This action is capable of promoting the neutralization of the pathogen, preventing its binding with host cells. But it also promotes the so-called opsonization of the pathogen, where it is possible to induce phagocytosis of the same by macrophages and neutrophils (Tay M.Z, et al 2019).
Antibodies of the IgM type are the molecules formed by the primary immune response, that is, after the first contact with the virus. In general, it is produced between days 3 to 5 after the onset of symptoms, with a peak around the 10th day (WHO, 2009). The IgG molecule, on the other hand, takes longer to form, with low titers at the end of the first week and increasing over the months, being responsible for mitigating the possibility of re-infection, making antigen recognition (Teva, A. et al, 2009). However, it is noteworthy that during secondary dengue infection (ie, infection with previous exposure to the virus), high levels of IgG are detected already between the 3rd and 5th day after the onset of symptoms, with a peak after two weeks (ANANDARAO et al., 2005; WHO, 2019).
Dengue virus, in particular, has 4 serotypes: DENV1, DENV2, DENV3, and DENV4. Despite belonging to the same species, they show great differences in their genotypes. These differences are so intense that they make it difficult to identify a specific antibody, precisely because they have different antigenic types. Thus, the individual who acquires adaptive immunity to a specific DENV serotype can contract the disease again by infection with another existing serotype (Coronato B. et al, 2016). Due to this genotypic variation among dengue virus serotypes, circulating diagnostic tests present the challenge of identifying DENV infection regardless of serotype.
How do existing tests work?
In endemic regions like Brazil, infections such as Dengue can lead to complications such as Dengue hemorrhagic fever and Dengue shock syndrome, both of which can result in death. However, early diagnosis associated with proper medical care can minimize this problem. According to the World Health Organization, these two measures can reduce the fatality rate of severe cases to less than 1% (WHO, 2021).
Currently, despite the high sensitivity of the molecular test, a negative result does not exclude these diseases due to the possibility of low viremia in the late phase of the infection. This fact highlights the relevance of serological diagnosis, which, in general, is cheaper and requires less laboratory infrastructure. As previously mentioned, this type of test can detect IgG and IgM-type antibodies produced by the body's defense cells against the virus (KIKUTI et al., 2018).
The diagnostic test is carried out on the patient's blood serum using the Enzyme-Linked Immunosorbent Assay (ELISA) method, which can be of the indirect or sandwich type. In general, in the indirect assay, it is possible to capture the immunoglobulins IgM or IgG present in the serum by a viral antigen immobilized on a plate. While in the Sandwich assay, first there is the patient's antibody immobilization, and then the antigen is added. Eventually, in both cases, there is the addition of a marker conjugated to an enzyme. The latter catalyzes a proper substrate conversion into a measurable signal (either colorimetric or fluorimetric). This method thus enables the indirect identification of the viral presence (SOUZA, 2012). These tests, despite being complex and efficient, in many cases, have high chances of cross-reactions. 
Currently, despite the high sensitivity of the molecular test, a negative result does not exclude these diseases due to the possibility of low viremia in the late phase of the infection. This fact highlights the relevance of serological diagnosis, which, in general, is cheaper and requires less laboratory infrastructure. As previously mentioned, this type of test can detect IgG and IgM-type antibodies produced by the body's defense cells against the virus (KIKUTI et al., 2018).
The diagnostic test is carried out on the patient's blood serum using the Enzyme-Linked Immunosorbent Assay (ELISA) method, which can be of the indirect or sandwich type. In general, in the indirect assay, it is possible to capture the immunoglobulins IgM or IgG present in the serum by a viral antigen immobilized on a plate. While in the Sandwich assay, first there is the patient's antibody immobilization, and then the antigen is added. Eventually, in both cases, there is the addition of a marker conjugated to an enzyme. The latter catalyzes a proper substrate conversion into a measurable signal (either colorimetric or fluorimetric). This method thus enables the indirect identification of the viral presence (SOUZA, 2012). These tests, despite being complex and efficient, in many cases, have high chances of cross-reactions.
What is the cross-reaction phenomenon?
Cross-reactions are the ability of an antibody to react with similar epitopes on different antigens. An epitope is the part of an antigen recognized by an antibody, acting like a key that fits into its specific lock. However, some viruses may have similar keys that fit into the same lock, which, in a diagnostic context, can lead to false-positive results (MURO et al., 2009). That is, using as an example DENV and ZIKV, which have similar epitopes, the antibody produced in a Dengue infection can bind to the Zika antigen used as a probe in a serological test, and vice versa. To detect anti-Dengue and anti-Zika antibodies, the most used antigens in serological assays are the envelope structural protein (E protein) and the non-structural protein 1 (NS1). Protein E has a structural similarity between flaviviruses such as DENV and ZIKV, which increases the chances of cross-reactions between them (STETTLER et al., 2016). While anti-NS1 antibodies from primary infections are more specific, and for this reason, this antigen is usually the most used in serological tests. However, studies show that sequential exposure to DENV and ZIKV infection increases the rate of cross-reactions between anti-NS1 antibodies, which leads to lower test efficacy (DAY-YU et al., 2019).
Thus, cross-reactions between DENV and ZIKV limit the specificity of existing diagnoses and, consequently, they may lead to complications for the patient treatment (MONTECILLO et al., 2019; DEJNIRATTISAI et al. 2016).
Thus, cross-reactions between DENV and ZIKV limit the specificity of existing diagnoses and, consequently, they may lead to complications for the patient treatment (MONTECILLO et al., 2019; DEJNIRATTISAI et al. 2016).
How can we solve the problem
To solve these problems, we designed Ammit: the new strategy for arboviruses serological diagnostics
Ammit is a chimeric protein made of specific DENV epitopes, used as an antigen to probe anti-dengue antibodies that circulate in infected people. Based on the bioengineering cycle, we selected the most promising epitopes for Ammit’s design. For this, we analyzed its structure and binding to antibodies using different computational tools. We developed two Ammit proteins, one specific for Brazilian circulating DENV2 strains and the other for global circulating DENV2 strains. The Ammit DNA sequence was synthesized and expressed in E. coli using a strong promoter. After expression, we tested Ammit's capacity of binding to commercial anti-DENV2 and anti-ZIKV. In the future, Ammit will be part of an electrochemical biochip, which constitutes a point-of-care device, with the premise of being faster and more specific than the usual serological tests.
Ammit will validate our prototype, so we can design other multi-epitope proteins to diagnose Zika and Chikungunya. This device will expand access to quality testing since it will be portable and low-cost. Hence this solution will provide democratized and assertive diagnosis, besides mitigating cross-reaction problems!
References
1- ANANDARAO, R. et al. A custom-designed recombinant multiepitope protein as a dengue diagnostic reagent. Protein Expression and Purification, v. 41, n. 1, p. 136–147, 2005.
2- Boletim epidemiológico: Monitoramento dos casos de arboviroses urbanas causados por vírus transmitidos por Aedes (dengue, chikungunya e zika), semanas epidemiológicas 1 a 53. Disponível em:. Acesso em: 13 sep. 2021.
3- CHAO, D.-Y. et al. Comprehensive evaluation of differential serodiagnosis between zika and dengue viral infections. Journal of Clinical Microbiology, v. 57, n. 3, 2019.
4- DEJNIRATTISAI, Wanwisa et al. Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus. Nature Immunology v. 17, n. 9, p. 1102–1108 , 2016.
5- DITTMAR, D.; CLEARY, T. J.; CASTRO, A. Immunoglobulin G- and m-specific enzyme-linked immunosorbent assay for detection of Dengue antibodies. Journal of Clinical Microbiology, v. 9, n. 4, p. 498–502, 1979.
6- MONTECILLO-AGUADO, Mayra R. et al. Cross-Reaction, enhancement, and NEUTRALIZATION activity of dengue virus antibodies against Zika VIRUS: A study in the Mexican population. Journal of Immunology Research v. 2019, p. 1–14 , 2019.
7- MULLER, D. A.; DEPELSENAIRE, A. C.; YOUNG, P. R. Clinical and Laboratory diagnosis of dengue virus infection. The Journal of Infectious Diseases, v. 215, n. suppl_2, 2017.
8- MURO, L. F. F.; FERREIRA, L. L.; GONZAGA, P. A. L.; PEREIRA, R. E. P.; Relação Antígeno-Anticorpo. Ano VII – Número 12 – Janeiro de 2009 – Periódicos Semestrais; Acadêmicos da Associação Cultural e Educacional de Garça – FAMED. REVISTA CIENTÍFICA ELETRÔNICA DE MEDICINA VETERINÁRIA – ISSN: 1679-7353
9- SHARON, J.; VOEUX, P. J.; TOROS, E. F. Imunologia básica. Traducao . Rio de Janeiro: Guanabara-Koogan, 2000.
10- SINGHI, S.; KISSOON, N.; BANSAL, A. Dengue e dengue hemorrágico: Aspectos do manejo na unidade de terapia intensiva. Jornal de Pediatria, v. 83, n. 2, 2007.
11- SOUZA, Camila Giuberti de. Avaliação da sensibilidade de diferentes testes diagnósticos para a dengue. 2012. 71 f. Dissertação (Mestrado em Mestrado em Doenças Infecciosas) - Universidade Federal do Espírito Santo, Vitória, 2012.
12- STETTLER, K. et al. Specificity, cross-reactivity, and function of ANTIBODIES elicited by Zika virus infection. Science, v. 353, n. 6301, p. 823–826, 2016.
13- TEVA, A.; FERNANDEZ, J. C. C.; SILVA, V. L. Imunologia. Disponível em:. Acesso em: 12 sep. 2021.
14- World Health Organization. Dengue guidelines for diagnosis treatment prevention and control: New edition. 2009.
15- Coronato B. ¹; Elizete R. Antonio². DENGUE: SOROTIPOS E SUAS ADVERSIDADES. ANILUS Pesquisa: Universidade do Conhecimento Científico. v. 13, n. 30 (2016).
16- Tay MZ, Wiehe K, Pollara J. Antibody-Dependent Cellular Phagocytosis in Antiviral Immune Responses. Front Immunol . 2019; 10: 332. Publicado em 28 de fevereiro de 2019. doi: 10.3389 / fimmu.2019.00332.
2- Boletim epidemiológico: Monitoramento dos casos de arboviroses urbanas causados por vírus transmitidos por Aedes (dengue, chikungunya e zika), semanas epidemiológicas 1 a 53. Disponível em:
3- CHAO, D.-Y. et al. Comprehensive evaluation of differential serodiagnosis between zika and dengue viral infections. Journal of Clinical Microbiology, v. 57, n. 3, 2019.
4- DEJNIRATTISAI, Wanwisa et al. Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus. Nature Immunology v. 17, n. 9, p. 1102–1108 , 2016.
5- DITTMAR, D.; CLEARY, T. J.; CASTRO, A. Immunoglobulin G- and m-specific enzyme-linked immunosorbent assay for detection of Dengue antibodies. Journal of Clinical Microbiology, v. 9, n. 4, p. 498–502, 1979.
6- MONTECILLO-AGUADO, Mayra R. et al. Cross-Reaction, enhancement, and NEUTRALIZATION activity of dengue virus antibodies against Zika VIRUS: A study in the Mexican population. Journal of Immunology Research v. 2019, p. 1–14 , 2019.
7- MULLER, D. A.; DEPELSENAIRE, A. C.; YOUNG, P. R. Clinical and Laboratory diagnosis of dengue virus infection. The Journal of Infectious Diseases, v. 215, n. suppl_2, 2017.
8- MURO, L. F. F.; FERREIRA, L. L.; GONZAGA, P. A. L.; PEREIRA, R. E. P.; Relação Antígeno-Anticorpo. Ano VII – Número 12 – Janeiro de 2009 – Periódicos Semestrais; Acadêmicos da Associação Cultural e Educacional de Garça – FAMED. REVISTA CIENTÍFICA ELETRÔNICA DE MEDICINA VETERINÁRIA – ISSN: 1679-7353
9- SHARON, J.; VOEUX, P. J.; TOROS, E. F. Imunologia básica. Traducao . Rio de Janeiro: Guanabara-Koogan, 2000.
10- SINGHI, S.; KISSOON, N.; BANSAL, A. Dengue e dengue hemorrágico: Aspectos do manejo na unidade de terapia intensiva. Jornal de Pediatria, v. 83, n. 2, 2007.
11- SOUZA, Camila Giuberti de. Avaliação da sensibilidade de diferentes testes diagnósticos para a dengue. 2012. 71 f. Dissertação (Mestrado em Mestrado em Doenças Infecciosas) - Universidade Federal do Espírito Santo, Vitória, 2012.
12- STETTLER, K. et al. Specificity, cross-reactivity, and function of ANTIBODIES elicited by Zika virus infection. Science, v. 353, n. 6301, p. 823–826, 2016.
13- TEVA, A.; FERNANDEZ, J. C. C.; SILVA, V. L. Imunologia. Disponível em:
14- World Health Organization. Dengue guidelines for diagnosis treatment prevention and control: New edition. 2009.
15- Coronato B. ¹; Elizete R. Antonio². DENGUE: SOROTIPOS E SUAS ADVERSIDADES. ANILUS Pesquisa: Universidade do Conhecimento Científico. v. 13, n. 30 (2016).
16- Tay MZ, Wiehe K, Pollara J. Antibody-Dependent Cellular Phagocytosis in Antiviral Immune Responses. Front Immunol . 2019; 10: 332. Publicado em 28 de fevereiro de 2019. doi: 10.3389 / fimmu.2019.00332.
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