Arthur Lee and Dr. Bradford Berges, Ph.D. Microbiology and Molecular Biology
There are currently no vaccines available against dengue virus. Each year, about 400 million people become infected and half a million of them, especially children, develop complications such as dengue hemorrhagic fever or dengue shock syndrome.
There are four serotypes of dengue virus, and it is spread via the mosquito vector Aedes aegypti. A person may develop immunity against one serotype after being exposed; however, protection against one strand does not guarantee immunity towards another due to the inability of host’s antibodies to neutralize the virus. Non-neutralizing antibodies, in fact, enhance dengue infection2. The goal is to produce novel human monoclonal antibodies (hmAbs) that are capable of neutralizing all serotypes of dengue virus.
This study is still in the process due to an unforeseen circumstance with collaborators in a previous project. The situation has been explained and verified with the Microbiology and Molecular Biology Department.
To accomplish the goal of this study, humanized mice were utilized as hosts for Dengue Type II New Guinea C strain (DV-2 NGC). The mice were kept for six weeks to allow potential seroconversion before they were put on a biweekly schedule of sera collection which lasted for ten weeks. ELISA will be performed in the future to determine seroconversion.
Mice that seroconvert will be sacrificed and have their human memory B cells extracted. The cells will then be immortalized using Epstein Barr virus. The hmAbs will be collected for assays to determine the effectiveness of protecting against DV2- NGC. C1008 cells, a variant of Vero cells, are used to culture DV-2 NGC stock. It is tittered by LLC MK-II cells. The LLC MK-II will be part of the assays for identifying useful hmAbs against the virus. The virus stock is currently being replicated in order to obtain adequate titer for these assays (see Fig. 1). Cytopathic effects will be observed to distinguish between neutralizing and non-neutralizing hmAbs.
Once the antibodies have been identified, it is possible to challenge new humanized mice with DV-2 NGC and observe for disease state. Enhanced infection is not expected. When the effectiveness of the hmAbs is confirmed, the goal of this project will be fulfilled. Future direction may include in vitro studies involving other serotypes of dengue virus before moving onto in vivo experiments.
References:
- Ananda Amarasinghe, Richard T Mahoney. “Estimating potential demand and supply of dengue vaccine in Brazil.” Hum Vaccin. 2011 July; 7
- Eiji Konishi. “Issues Related to Recent Dengue Vaccine Development.” Trop Med Health. 2011 December; 39
- G William Letson, Pratap Singhasivanon, Eduardo Fernandez, Nihal Abeysinghe, Juan Jose Amador, Harold S Margolis, Robert Edelman. “Dengue vaccine trial guidelines and role of largescale, Post proof-of-concept demonstration projects in bringing a denguevaccine to use in dengue endemic areas.” Hum Vaccin. 2010 October; 6
- Katherine M. Smith, Kavita Nanda, Victoria McCarl, Carla J. Spears, Amanda Piper, Mariana Ribeiro, Michelle Quiles, Caitlin M. Briggs, Gwynneth S. Thomas, Malcolm E. Thomas, Dennis T. Brown, Raquel Hernandez. “Testing of Novel Dengue Virus 2 Vaccines in African Green Monkeys: Safety, Immunogenicity, and Efficacy.” Am J Trop Med Hyg. 2012 October 3; 87