Replication and Viability of Recombinant Modified Vaccinia Virus Ankara 44/47.1 and 51.1 in Mammalian Cells Pubblico

Abstract

It is very difficult to create vaccines for some viruses, such as human immunodeficiency virus (HIV). Past attempts at vaccines for HIV have failed to stimulate the immune system effectively, leading to insufficient B and T cell activation. Many vaccines use a modified vaccinia virus Ankara (MVA) vector to deliver viral genome targets in order to stimulate the immune system. In 1998, Wyatt et al published a new study that showed increased replication of new recombinant MVAs (rMVAs) over the wild-type MVA in rabbit, monkey, and human cell lines. The objective of this study was to establish replication and viability of rMVA 44/47.1 and rMVA 51.1 in mouse, monkey, and human cell lines in order to determine whether they would be good candidates as vaccine vectors. If rMVAs are able to replicate better and delay cell death more effectively, this would allow immune cells more opportunity to recognize viral targets. DF-1 chick embryo fibroblasts, C2C12 mouse myoblasts, Vero monkey kidney epithelial cells, and HeLa human cervical epithelial cells were tested for replication. C2C12 cells, L929 mouse fibroblasts, Vero cells, and HeLa cells were tested for viability with and without an apoptosis causing agent. We find that rMVAs had increased replication over the wild-type MVA in DF-1 cells, C2C12 cells, and HeLa cells, but did not differ significantly in Vero cells. We also find that rMVA 44/47.1 had decreased apoptosis in L929 cells and HeLa cells, and rMVA 51.1 had decreased apoptosis in L929 cells, Vero cells, and HeLa cells. However, these decreases in apoptosis were concurrent with increases in necrosis. Recombinant MVAs could be the answer in creating more effective vaccines, though we must find ways to reduce the necrosis and must better establish these patterns.

Table of Contents

Table of Contents

INTRODUCTION………………………………………………………………………………………………………………………….1

OBJECTIVES………………………………………………………………………………………………………………………………..7

MATERIALS AND METHODS……………………………………………………………………………………………………….8

RESULTS…………………………………………………………………………………………………………………………………..12

CONCLUSION……………………………………………………………………………………………………………………………34

DISCUSSION……………………………………………………………………………………………………………………………..36

REFERENCES…………………………………………………………………………………………………………………………….40

List of Figures

Figure 1. The intrinsic pathway of apoptosis………………………………………………………………………………6

Figure 2. Gating strategy………………………………………………………………………………………………………….11

Figure 3. Growth in DF-1 Cells………………………………………………………………………………………………….13

Figure 4. Growth in C2C12 cells………………………………………………………………………………………………..14

Figure 5. Growth in Vero cells………………………………………………………………………………………………….15

Figure 6. Growth in HeLa cells………………………………………………………………………………………………….16

Figure 7. Viability in C2C12 cells……………………………………………………………………………………………….18

Figure 8. Viability in treated C2C12 cells…………………………………………………………………………………..19

Figure 9. Caspase 3 activity in C2C12 cells……………..…………………………………………………………….....20

Figure 10. Viability in L929 cells…………..……………………………………………………………………………….....22

Figure 11. Viability in treated L929 cells……………………………………………………………………………….....23

Figure 12. Caspase 3 activity in L929 cells………………………………………………………………………………..24

Figure 13. Viability in Vero cells……………………………………………………………………………………………….26

Figure 14. Viability in treated Vero cells…………………………………………………………………………………..27

Figure 15. Caspase 3 activity in Vero cells………………………………………………………………………………..28

Figure 16. Viability in HeLa cells……………………………………………………………………………………………….31

Figure 17. Viability in treated HeLa cells…………………………………………………………………………………..32

Figure 18. Caspase 3 activity in HeLa cells………………………………………………………………………………..33

Figure 19. Overall replication and cell death patterns of recombinant and WT MVAs at 24 hours in mammalian cell lines……………………………………………………………………………………………………………35

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	BS
Submission	Honors Thesis
Language	English
Research Field	Health Sciences, Immunology Biology, Virology
Parola chiave	recombinant replication MVA vaccine viability
Committee Chair / Thesis Advisor	Amara, Rama Rao, Emory University
Committee Members	Worthman, Carol, Emory University Eisen, Arri, Emory University

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