The Innate and Adaptive Immune Response to Measles Virus .i The Innate and Adaptive Immune Response

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Text of The Innate and Adaptive Immune Response to Measles Virus .i The Innate and Adaptive Immune Response

i

The Innate and Adaptive Immune Response

to Measles Virus

By:

Nicole Putnam

A thesis submitted to Johns Hopkins University in conformity

with the requirements for the degree of Master of Science.

Baltimore, Maryland

April 2014

Nicole Putnam

All Rights Reserved

ii

Abstract

Measles is one of the most important causes of childhood morbidity

and mortality worldwide. Although a vaccine is available, the high

transmission rate of measles virus requires population of 95% to interrupt its

transmission. The World Health Organization and the United Nations

Childrens Fund recommend that children that develop measles receive

vitamin A supplementation, as a safe, cheap, and efficacious way to reduce

the burden of disease. Due to differences between strains and confounding

data of measles stocks contaminated with defective interfering RNA

particles, the immune response to measles virus infection has not been well

defined. Furthermore, the mechanism by which vitamin A protects against

severe measles-induced disease is unknown.

In this thesis, I investigate the innate and adaptive immune response

to measles virus infection. Measles virus strains were purified of defective

interfering RNA particles and used for in vitro infections of monocyte derived

dendritic cells. Gene expression changes of interferon-stimulated genes and

viral stress-induced genes, IFIT1 and Mx1, were upregulated in response to

infection with the Edmonston measles virus vaccine strain, as well as the

wild-type strains of Bilthoven, IC-B, and C- and V-protein knockout strains,

as compared to mock infected cells. Unexpectedly, there were no differences

between transcript levels of these genes between C and V protein knockout

strains and the respective wild-type infection. Additionally, the absence of

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type I interferon production supports the theory that measles virus induces

the transcription of these genes through the viral stress-induced pathway,

and not the interferon-stimulated pathway.

While a previous study had detected measles virus-specific IL-17-

producing T cells in measles virus-infected rhesus macaques, the Th17

response to measles virus has not been characterized. Th17 cell

differentiation was inhibited early after measles virus infection in vitro.

There was a significant decrease in IL-23A transcript ts and a significant

increase in IL-27 transcripts, both of which affect Th17 cell differentiation

negatively. However, in a rhesus macaque model of infection, a biphasic Th17

response was observed with peaks at days 18 and 56.

The effects of vitamin A supplementation following measles virus

infection on the immune response was explored in a rhesus macaque model

using supplemented and non-supplemented groups. While some data has yet

to be explored, major differences were not observed between the two groups

up to three months following infection, in regards to clearance of infectious

virus, immune cell composition, or immune cell function. Archived data will

elucidate the role of vitamin A in measles virus RNA persistence, and Th1

and T follicular helper cell responses. Data will continue to be analyzed out to

six months post infection. A larger cohort will be necessary to elucidate the

role of vitamin A in protection against severe disease and death due to

measles.

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Acknowledgements

First and foremost, I would like thank my advisor, Dr. Diane E.

Griffin, for allowing me to do my masters research in her laboratory. Her

guidance and support was invaluable throughout my time here. I would like

to thank her for the opportunity to get involved in the dynamic, challenging,

and rewarding research that she had entrusted with me. Furthermore, a

huge thank you to Dr. Rupak Shivakoti for passing down his knowledge of

the basics of how to work with measles virus and acquainting me with Dr.

Griffins lab in general. Additional thanks go out to Rupak to teaching me

many, many techniques. Although he was available to ask questions while he

was here, it was helpful that he encouraged me to jump right in and

conduct my experiments independently early on. I would like to also thank

Rupak for being responsive to questions much after he had graduated from

the laboratory, which was especially helpful.

I would like to thank Dr. Wendy Lin, for providing me with her

knowledge of the logistics of working with measles virus in rhesus macaques.

Her ability to pass down her understandings and techniques was invaluable.

Furthermore, I would like to thank Wendy for taking time from her career at

Columbia University to come down to Baltimore to meet with us, as well as

making herself available to talk about techniques or data analysis.

Importantly, this project would not have run as smoothly as it did without

the help of Ashley Nelson, the PhD student with whom I shared

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responsibility in this project. With Ashleys flexibility to work around my

schedule, we were able to make sure the assays for the monkey study could

be completed and analyzed in a timely manner so I could complete my thesis

work. I would also like to thank Ashley for her support and friendship

throughout my time here!

The vitamin A/monkey study was largely a success due to the time and

effort of Dr. Bob Adams and Dr. Tori Baxter. I would like to thank them

immensely for their time and expertise in handling the monkeys, obtaining

samples, and for being flexible with their schedules around the holidays,

while also granting this project many of their early mornings.

I would like to give a huge thanks to my roommate, Dr. Cailin Deal,

who was able to provide me her knowledge and skills in so many areas of

virology and immunology as a whole. Her expertise in writing in science was

crucial to the process of editing my thesis, as well as her general knowledge

of techniques and data analysis. Furthermore, I would like to thank Debbie

Hauer for her assistance in teaching me techniques and processing samples

that were essential for my projects and this thesis.

I would like to extend my warmest thanks to the rest of Dr. Griffins

laboratory for being so welcoming to me as a masters student, for passing

down their expertise and insight when I needed assistance, and for their

general support and friendship. Dr. Kim Shulz, Dr. Tori Baxter, Dr. Kirsten

Kulscar, Stephen Goldstein and Siva Manivannan, your help was

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instrumental towards my experience as a student in this laboratory.

Additionally, I would like to acknowledge Gui Nilaratanakul, Rachy

Abraham, and Nina Martin for their presence and livelihood in the lab.

Finally, I would like to thank my mother, father, and brother Ryan, as

well as my extended family and friends for providing their unwavering

support. As a little girl, my parents told me I could do whatever I put my

mind to, and when I decided to pursue a science and research their

enthusiasm was there to match my own. This thesis is the product of the

hard work and support of many people, and I would again like to extend a

tremendous thanks to all of the people by my side!

vii

Table of Contents

Abstract. ii

Acknowledgements.. iv

List of Tables...... ix

List of Figures. x

Chapter 1: Introduction to measles virus 1

Public health implications... 2

Measles virus pathogenesis. 2

Prevention of measles virus infection... 3

Measles virus virology.. 6

Measles virus infection. 7

Defective replication of measles virus genome 8

Innate immune response to viral infection.. 9

TLRs.. 9

Cytoplasmic PRRs 10

Type I interferon.. 11

Interferon-inducible antiviral proteins... 12

Innate immune response to measles virus infection 12

Role of dendritic cells.. 14

Adaptive immune response to measles virus infection... 14

Antibody response 15

T lymphocyte response 16

Effector CD4+ T lymphocytes 16

Immunosuppression following measles virus infection.. 18

Figures 20

Chapter 2: Comparision of in vitro immune responses to wild-type

measles virus with C and V protein-knock out strains; wild-type and

vaccine strains of measles virus 24

Introduction.. 25

Measles virus immune evasion. 25

Block of type I interferon production.. 25

Block of type I interferon signaling. 27

Interferon-stimulated genes (ISGs) and virus stress-induced

genes (VSIGs)... 28

Role of dendritic cells in measles virus infection.. 29

Defective interfering (DI) particles.. 29

Th17 response to viral infection... 30

Th17 response to measles virus infection.. 30

Materials and methods... 31

Results 38

Discussion.. 46

Tables. 53

Figures... 54

viii

Chapter 3: Effects of vitamin A supplementation on the immune

response and