Antigen characterization and production of antipeptide antibodies against dengue, Zika and chikungunya virus

Name: Rafaela Spessemille Valotto
Type: MSc dissertation
Publication date: 07/10/2021
Advisor:

Namesort descending Role
Marco Cesar Cunegundes Guimarães Advisor *

Examining board:

Namesort descending Role
Daniel Claudio de Oliveira Gomes Internal Examiner *
Marco Cesar Cunegundes Guimarães Advisor *
Ricardo Andrez Machado de Ávila External Examiner *

Summary: VALOTTO, R. S. Antigen characterization and production of antipeptide
antibodies against dengue, Zika and chikungunya virus. 2021. 70f. Dissertation
(Master in Biotechnology) - Postgraduation Biotechnological Programme, UFES,
Espírito Santo. Brazil.
Dengue, Zika and chikungunya viruses are responsible for outbreaks in several
countries. In Brazil, the population has already faced simultaneous epidemics of
these diseases. Diagnosis type are similar between diseases and may range
between serological tests, RT-PCR and antigen tests. Currently, part of the efforts
are focused on the development of low-cost tests, with quick results and that not
require a laboratory structure, thus facilitating their execution in regions with few
resources. Therefore, the antigen test is a great candidate, appearing as an
alternative to established tests. DENV NS1, ZIKV NS1 and CHIKV E2 are viral
proteins that are the focus of studies for the development of antigen detection kits, as
they are more easily detected in infected samples compared to other proteins. Thus,
in silico predictions were performed using bioinformatics tools in order to know which
regions of these proteins were antigenic, corresponding to an epitope, were
accessible, hydrophilic and were not present in a β-turn. The 2 predicted peptides of
each protein that achieved the greatest number of desired characteristics were
synthesized and characterized by HPLC and mass spectrometry. Then, they were
inoculated into rabbits to produce antibodies against each antigen. Sera were
collected, and Dot blots and Western blots were performed. The predicted peptides
were 103SLRPQPTELKY113 (DENV-2 NS1), 111YKYSWKSWGKA121 (DENV-1 NS1),
238ESDLIIPKSLAGPLSH253 and 83GVQLTVVVGS92 (ZIKV NS1), 24EGHSCHSPVAL34
and 147HGKELPCSTYVQS159 (CHIKV E2). From Dot blot results it could be seen that
all sera produced presented antipeptide antibodies, seeing that all showed interaction
with the peptides. As the Western blot used full length proteins instead of peptides,
some results diverged from the previous one. Of all produced sera, anti-SLR, antiYKY and anti-ESD showed interaction with full length proteins expressed in
Escherichia coli and HEK293 cells, with the exception of sera against CHIKV E2
peptides, which were tested only with the expressed protein in insect cells containing
recombinant baculovirus, and the anti-EGH serum reacted to protein.
Key words: Dengue. Zika. Chikungunya. Bioinformatics. Peptides.

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