MEM INST OSWALDO CRUZ, RIO DE JANEIRO, FAST TRACK
PAGES: DOI: 10.1590/0074 02760170359 Full paper
SANGER-BASED SEQUENCING TECHNOLOGY FOR YELLOW FEVER VACCINE GENETIC QUALITY CONTROL

Cristiane Pinheiro Pestana1, Rafael Lawson-Ferreira2, Carolina Lessa-Aquino1, Maria da Luz Fernandes Leal1, Marcos da Silva Freire1, Akira Homma1, Marco Alberto Medeiros1,+

1Fiocruz, Bio-Manguinhos, Brazilian Ministry of Health, Avenida Brasil 4365,Manguinhos, Rio de Janeiro, RJ 21040-900, Brazil

2 Fiocruz, National Institute for Quality Control in Health, Brazilian Ministry of Health,Rio de Janeiro, Brazil

Abstract

BACKGROUND Yellow Fever (YF) is an acute viral hemorrhagic disease prevalent mainly in Africa and Americas, with 20-60% fatality rate in severe forms. Currently, antiviral drugs for the infection are not available, reinforcing the importance of vaccination in resident populations and travelers. Manufactured in 7 different countries, the YF vaccine was first created in 1937 and 2 key substrains are used for production, 17DD and 17D-204. The vaccine produced in Bio-Manguinhos/Brazil uses 17DD substrain and more than 160 million doses have been exported to over 74 countries. The World Health Organization (WHO) recommends that new seed- and working-lots should have the viral genome sequenced in order to check vaccine genetic stability.

OBJECTIVE The aim of this study was to develop and standardize a Sanger-based sequencing protocol for the genetic monitoring of the Brazilian 17DD vaccine.

METHODS We designed 54 oligos to access the complete YF vaccine genome by RT-PCR and sequencing approach. After protocol standardization, we tested 45 vaccine lots and the corresponding secondary and working seed lots.

FINDINGS All 45 lots presented 100% nucleotide identity to each other and to the seed lots. We detected 2 heterogeneous positions at nucleotides 4523 (C/T) and 6673 (C/T) that may indicate a quasispecies diversity of YF 17DD strain. When compared to the Brazilian GenBank sequence YFU17066, the Brazilian 17DD vaccine presented 6 silent mutations.

MAIN CONCLUSIONS We have developed a robust method for the genetic monitoring of YF vaccine, which has been successfully applied in Bio-Manguinhos since 2009. There were no genetic variation in the tested lots. Altogether, our results highlight the safety, production consistency and, more importantly, the genetic stability of the Brazilian YF vaccine, in the last 3 decades.

+ Corresponding author:  This e-mail address is being protected from spambots. You need JavaScript enabled to view it.

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