A FLAGELLIN-POXVIRUS ANTIGEN VACCINE: STRENGTHS AND LIMITATIONS

BY

KRISTEN N. DELANEY

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

in Partial Fulfillment of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

in Microbiology and Immunology

August 2009

Winston-Salem, North Carolina

Approved By: Steven B. Mizel, Ph.D., Advisor ________________________ Examining Committee: Douglas S. Lyles, Ph.D., Chairman ________________________ Martha A. Alexander-Miller, Ph.D. ________________________ Jason M. Grayson, Ph.D. ________________________ Griffith D. Parks, Ph.D. ________________________

ACKNOWLEDGEMENTS

Dr. Mizel, I hear the horror stories of graduate students all over the country who bemoan the fact that their advisors do not care about their projects, or do not care about their students. I’m incredibly fortunate to have had an advisor who always had my best interests at heart. When you pushed me, I knew it was because you had faith in me and what I could become. I take that as an incredible compliment, and I thank you for believing in me in times that I struggled to believe in myself. This project ended up in a place so different from what we expected and I feel like I gained more from my graduate experience as a result. It was a journey full of twists and turns, and I’m glad that I had the privilege to learn from you along the way. Thank you. My Committee, I thank all of you for your support and guidance in this project. The feedback you provided truly shaped this project and constantly reminded me to try to see things from every possible perspective. My Labmates, Working with such a skilled group of individuals was really quite an honor. I have enjoyed learning and hypothesizing with all of you. It has been a pleasure working with you. My Classmates, There is a word in the Japanese language that perfectly describes the bond our class had. Nakama, which roughly translates to “family by circumstance.” We’ve all come far, and we did it together. I’m so proud of all of you! My Family, The hardest part about graduate school wasn’t bleeding 80 mice in a day or repeating an assay for the umpteeth time. It was being so far from you. Thanks for always keeping me in your thoughts whether it was in the form of a post card, a phone call, or a long distance running buddy. Grandpa Dick, For being the person who always listened to my questions and was never too busy to hear my ideas, this thesis is dedicated to you.

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TABLE OF CONTENTS

Page LIST OF ABREVIATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 CHAPTER I: Generating Responses to Weakly Immunogenic Proteins Using Flagellin as an Adjuvant. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 CHAPTER II: Lessons About Flagellin as an Adjuvant and a Vaccine Vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 SCHOLASTIC VITAE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

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LIST OF ABBREVIATIONS

Ab Antibody Ag Antigen APC Antigen Presenting Cell Avertin 2,2,2, tribromoethanol BALB/c Baggs Albino C BMDC Bone Marrow-derived Dendritic Cell CCL Chemokind (C-C motif) Ligand CVF Cobra Venom Factor CD Cluster of Differentiation CRP C-reactive Protein DC Dendritic Cell ds Double-stranded DNA Deoxyribonucleic Acid EEV Extracellular Enveloped Virion eGFP enhanced Green Fluorescent Protein GM-CSF Granulocyte/Macrophage Colony Stimulating Factor GR-1 Lymphocyte antigen 6 complex, locus G ELISA Enzyme-Linked Immunosorbent Assay eYFP enhanced Yellow Fluorescent Protein F4/80 EGF-like module containing mucin-like hormone receptor-like sequence 1 FB Flagellin-B5R

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FBS Fetal Bovine Serum HRP Horseradish Peroxidase IgH Immunoglobulin Heavy Chain IL Interleukin i.m. Intramuscular IMV Intracellular Mature Virion i.n. Intranasal i.p. Intraperitoneal IPTG Isopropylthiogalactopyranoside LF L1R-Flagellin LFB L1R-Flagellin-B5R LPS Lipopolysaccharide Ly-6G Lymphocyte antigen 6 complex, locus G MCP Monocyte Chemoattractant Protein MIP1 Macrophage Inflammatory Protein MTD50 Maximally Tolerated Dose for 50% of subjects MVA Modified Vaccinia Ankara MyD88 Myeloid Differentiation primary response gene 88 NCS Newborn Calf Serum NFκB Nuclear Factor kappa-light-chain-enhancer of activated B cells PBMC Peripheral Blood Mononuclear Cells PBS Phosphate-Buffered Saline pH Potential for Hydrogen

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PKR Protein Kinase R RNA Ribonucleic Acid RPMI Roswell Park Memorial Institute Th T Helper Cells TLR Toll-like Receptor TNF-α Tumor Necrosis Factor α VV Vaccinia Virus WHO World Health Organization WR Western Reserve

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LIST OF FIGURES

Page 1 Proteins used in this study and their TLR5-stimulating activity 15 2 Intranasal immunization with pox antigens + flagellin confers 23 protection after 3 or more immunizations 3 Protection is dependent on B cells 26 4 CD8+ T cells are not required for protection 28 5 Fusion proteins are more effective at promoting antigen-specific 31 humoral responses than separate proteins 6 Intramuscular immunization is more effective at promoting antigen- 34 specific humoral responses than intranasal immunization 7 Mice who are protected during challenge have significantly higher 36 antigen-specific IgG2a titers than mice who succumb to challenge 8 IgG, IgG1, and IgG2a responses following i.m. immunization with 39 5μg of LF and FB 9 IgG, IgG1, and IgG2a responses following i.m. immunization with 41 20μg of LF and FB 10 Challenge after immunization with 5μg of LF and FB 43 11 Challenge after immunization with 20μg of LF and FB 45 12 Complement plays a role in the protective effect of the 48 flagellin-pox antigen fusion vaccine 13 Plasma from immunized mice is capable of neutralizing vaccinia 51 virus in vitro 14 Effect of repeated immunization on circulating levels of neutrophils, 54 monocytes, and C-reactive protein 15 Flagellin-pox antigen fusion proteins and their TLR5-stimulating 59 activity 16 Variability among antigens for optimal placement within a flagellin 61 fusion protein

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17 Competition between antigens when multiple flagellin fusion 64 proteins are administered 18 The humoral response to a flagellin-antigen fusion protein can be 66 reduced by addition of excess flagellin 19 A trifusion protein that combines flagellin with two poxvirus antigens 69 loses immunogenicity 20 B5R is not recognized by B5R-specific antibodies in the context of the 71 LFB trifusion

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ABSTRACT

Delaney, Kristen N.

A FLAGELLIN-POXVIRUS ANTIGEN VACCINE: STRENGTHS AND LIMITATIONS

Dissertation under the direction of

Steven B. Mizel, Ph.D., Professor of Microbiology and Immunology

Bacterial flagellin is a potent adjuvant that enhances adaptive immune responses

to a variety of antigens. The vaccinia virus antigens L1R and B5R are highly

immunogenic in the context of the parent virus, but recombinant forms of the proteins are

only weakly immunogenic. Therefore, we evaluated the response to these antigens when

flagellin was used as an adjuvant. Although flagellin promoted a robust antigen-specific

humoral response to poxvirus antigens delivered intranasally (i.n.) or intramuscularly

(i.m.), intramuscular immunization resulted in significantly high titers of anti-L1R and

B5R IgG. Flagellin/poxvirus antigen fusion proteins were more potent than flagellin and

L1R and B5R as separate proteins as inducers of a humoral response against the poxvirus

antigens. At least three immunizations with flagellin/poxvirus fusion proteins were

required to confer protection in mice against challenge with vaccinia virus. Although

mice were protected and exhibited only limited signs of disease, they still exhibited

significant, but reversible weight loss. When immune mice were depleted of complement

using cobra venom factor, 50% of the mice succumbed to vaccinia virus infection. These

results demonstrate that flagellin-poxvirus antigen fusion proteins are effective in

eliciting protective immunity against vaccinia virus that is dependent, in part, on

complement.

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We evaluated the efficacy of additional flagellin-poxvirus antigen constructs to

promote protective immunity and found that antigens can lose their immunogenicity

when inserted into certain regions of flagellin. The loss of immunogenicity is dependent

on the individual antigen, and was not the same for all antigens tested. When

administering more than one immunogenic fusion protein antigen-specific titers decrease

slightly, and the addition of excess flagellin will further decrease titers suggesting that

there is a limit to the number of fusion proteins that can be administered in a single

vaccine.

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INTRODUCTION

Variola Virus Pathogenesis and Life Cycle

The orthopoxvirus Variola virus is a very large double-stranded DNA virus and is

the etiological agent of smallpox. The disease was eradicated in 1979 by the efforts of

the World Health Organization’s aggressive vaccination campaign (35). Smallpox is

spread through respiratory transmission and is associated with respiratory infection and

the appearance of “pocks,” confined lesions at sites of concentrated viral replication. In

humans, mortality rates can be as high as 30% in some age groups.

Since laboratory use of variola virus is not permitted, vaccinia virus is used as a

model agent in poxvirus vaccine studies. Intranasal administration of vaccinia virus leads

to systemic infection via the bloodstream 12 hours post-inoculation in rabbits (111) and

lasts for more than 7 days. In the murine model, vaccinia virus causes respiratory

infection that peaks around day 7 without the appearance of pocks.

Vaccinia Virus Life Cycle and Morphogenesis

Vaccinia virus replication within a host cell begins with viral entry. While the

nature of vaccinia virus receptors remain unknown, it is clear that the different infectious

forms of the virus enter by different methods (103) and that the extracellular enveloped

virion (EEV) enters cells more quickly than intracellular mature virions (IMV) (28).

Once in the cytoplasm, early mRNAs are transcribed within the virus core and translated

by host machinery. This leads to further uncoating of the core, release of viral DNA, and

transcription of later viral genes (92). Approximately 2-5 hours post-infection

intracellular mature virions have been assembled. Most of these infectious IMV particles

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will remain intracellular until the cell lyses. The bulky virus particles move very slowly

through the host cell’s cytoplasm (95), so the virus particles polymerize actin to propel

themselves through the cell (21,39). Some of the particles will be pushed through the

golgi and acquire a golgi-derived double membrane, these are referred to as intracellular

enveloped virus (IEV). These IEV particles translocate to the cell surface where the

outermost golgi-derived membrane fuses to the plasma membrane releasing the second

infectious form of vaccinia virus, extracellular enveloped virus (EEV).

The IMV form of the virus is very resilient and is therefore implicated in host-to-

host spread. The EEV form of the virus is much more fragile, but is more involved in

spread throughout a host. EEV is released before cell lysis of the host cell, it enters cells

more rapidly than IMV (28), and it has a higher specific infectivity (12.7 virus

particles/pfu compared to 64.6 virus particles/pfu for purified IMV) (102). The outer

EEV membrane is easily disrupted by mechanical shearing, drying, or low pH. But, loss

of the outer membrane yields a hearty IMV particle, so the virion remains infectious. It

has been shown that acellular vaccine strategies that target both infectious forms of

vaccinia virus provide enhanced protection to challenge than strategies that only use

antigens from one infectious form (36,37,49,50,65).

Poxvirus protective antigens B5R and L1R

B5R has 2 forms; a 42 kDa transmembrane form, and a 35 kDa secreted form.

The protein is N-glycosylated and expressed early and late in infection (32,55). The

transmembrane form of the protein is found on the EEV form of the virus, and is the

major target of EEV-neutralizing antibodies (8,13,40,59). The secreted form plays a role

in evasion of host immune responses by acting as a decoy for B5R-specific antibodies,

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thus reducing the amount of B5R-specific antibody available to bind virus-associated

B5R. B5R is very important in the pathogenesis of vaccinia virus. Mutant viruses

lacking B5R have a 5-10 fold reduction in the formation of EEV particles, form small

viral plaques, and are attenuated in vivo (33,67,99,107).

L1R, on the other hand is associated with the IMV particle. It is a 25 kDa protein

expressed late in infection. L1R is a major target for antibody-mediated neutralization of

IMV (42,54,65). While the actual biological function of L1R is unknown, it obviously

plays a critical role in viral development. A mutant virus was developed that placed L1R

expression under the control of IPTG. In the absence of IPTG, plaque formation was

abrogated and only immature virions could be detected by electron microscopy (86).

Vaccinia Virus Evasion of Host Responses

Vaccinia virus has many gene products dedicated to evasion of the host immune

response. Some of the more commonly targeted systems are mentioned here. For full

reviews see (43,91).

Interference with Cytokines and Interferons. Many vaccinia proteins are

molecular mimics of cytokine receptors. These viral gene products do not have a

transmembrane signaling domain, thus they compete with the native receptors for binding

with these immune effector proteins. Some examples of poxvirus proteins which provide

this function are A53R which mimics the TNF receptor (90), B15R which encodes an IL-

1β receptor mimic (2), and B8R which resembles the IFN-γ receptor (101).

Another way that poxviruses interfere with IFN signaling is by preventing

detection of virus within infected cells. The E3L gene product binds dsRNA to block the

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activation of PKR (24,58) which is a major viral detection method. PKR is also the target

of K3L, which prevents its auto-phosphorylation (24,25).

Inhibition of Apoptosis. E3L also slows the progression of apoptosis in host cells

(24,58) which allows the virus a longer time to assemble before lysis. It is unknown

whether this delay is through interaction with PKR, or if this is the result of an interaction

with another cellular protein.

Blockade of TLR Signaling. The poxvirus protein A52R inhibits TLR-mediated

activation of NFκB (14). It associates with IRAK2 and TRAF6 which results in

disruption of signaling complexes involving these proteins (44).

Vaccination Approaches to Providing Protection Against Smallpox

The current smallpox vaccine approved for use in the United States, Dryvax, is a

live strain of vaccinia virus which is administered by scarification. The vaccine has the

risk of causing serious complications such as progressive vaccinia, eczema vaccinatum,

myopericarditis and ischemic heart disease. Other countries approved the use of other,

more reactogenic strains of vaccinia Lister and Coppenhagan which appear to be

associated with higher incidence of cardiac complications (85). In the US, vaccination

with Dryvax is limited to highly-screened military personnel, health care professionals,

and laboratory workers. The vaccine cannot be administered to individuals who are

immunocompromised, elderly, very young, or those with atopic dermatitis as they are at

high risk for complications. In light of the restrictions on vaccination, there has been a

push to develop a safer vaccine that could be used to protect the general populace in the

event of malicious release of variola virus.

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Alternative vaccination strategies under development include attenuated strains of

vaccinia virus, recombinant DNA vaccines, and recombinant protein vaccines. The most

well-characterized of these is an attenuated live vaccine; Modified Vaccinia Ankara

(MVA). This virus is replication deficient in mammalian cells due to extensive passage

in chick embryo fibroblasts and the loss of approximately 15% of the parental genome

(74,81). MVA was actually used for wide-spread immunization in Germany during the

WHO’s smallpox eradication campaign (93).

Both the innate and adaptive immune systems are involved in clearance of

vaccinia virus in an immunocompetent individual. Two studies evaluated the

requirement for different branches of the adaptive system in a Modified Vaccinia Ankara

immunization model. Utilizing immunodeficient mice (109) or cell depletion techniques

(9) these studies showed that either a T cell response or a humoral response is sufficient

to provide protection. Mice only succumbed to infection when both cellular and humoral

responses were lost.

Recombinant Vaccinia Virus Proteins are “Weakly-Immunogenic” Antigens

Vaccinia virus is a well-studied virus which induces robust humoral and cellular

immune responses (for review see (73)). However, when the dominant immunogens of

vaccinia virus are removed from the context of the parent virus they show reduced

immunogenicity (30,36,37,46,49-51).

DNA vaccination with pox antigens A27L, L1R, A33R, and B5R yields higher

antibody titers than scarification with vaccinia virus in mice and non-human primates

(30,49). However, when DNA vaccinated macaques (4 vaccinations delivered by gene

gun) are challenged with monkeypox they still develop lesions (46). The fact that these

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vaccines are less effective in protecting non-human primates than mice suggests they may

also provide limited protection in humans.

Recombinant protein vaccines are safer than those involving live viruses, but

efficacy remains an issue. In order to obtain complete protection from challenge, mice

must be immunized multiple times with recombinant proteins (36,37). For example,

Fogg et al. demonstrated that relatively large amounts of protein and at least four

immunizations are required to achieve protection against respiratory challenge with

vaccina virus in mice (37). In this study, the authors used trehalose dicorynomycolate

emulsion or saponin as adjuvants to enhance the response to L1R, B5R and A33R.

These results provided a strong rationale for evaluating other adjuvants that might have a

more potent effect on the response to the poxvirus antigens.

Work from our laboratory (48,75,105) as well as other investigators

(11,20,52,53,61,62,69,70) has established that flagellin, the major structural protein of

Gram-negative flagella, is a potent adjuvant when administered with an antigen by any of

several different routes. The adjuvant effect of flagellin has demonstrated in a variety of

pathogen models (26,48,52,56,62,69,97,98,104,105).

Flagellin—Structure and Biological Activities

Flagellin Structure. Flagellin is the major structural subunit of bacterial flagella.

It consists of 4 major domains—a highly conserved region which is involved in binding

and a hypervariable region that differs greatly between various Gram-negative bacterial

species. The structure of the protein was originally determined by Yonekura, et. al.

(112). The N and C termini are both involved in flagellar polymerization (112). The

majority of antibodies generated towards flagellin target the hypervariable region (82);

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likely because that is the region that is exposed when flagellin is polymerized. The

interaction of flagellin with toll-like receptor 5 (TLR5) is mediated by highly-conserved

region in domain 1 (94).

Flagellin and Pathogenesis. Flagellin is the major structural subunit of bacterial

flagella which are involved in motility of bacteria. Flagellar mutants show reduced

virulence in Pseudomonas (34,88), and Salmonella (16,84,96). Although the loss of

flagella reduces pathogenesis, the activation of innate immunity is often markedly

reduced. For this reason, the expression of flagella must be tightly regulated to avoid

activating the innate immune system. Down-regulation of flagellar gene products

reduced the release of inflammatory mediators (IL-8 and GM-CSF) and could provide a

selective advantage for Pseudomonas aeruginosa in the lungs of cystic fibrosis patients

(19).

Flagellin Signals via Toll-like Receptor 5. Several studies (41,45,77)

demonstrated that flagellin signals via toll-like receptor 5 (TLR5). Amino acids 386–407

of flagellin bind to a leucine-rich repeat of TLR5 (78) and induce downstream signaling

in a MyD88-dependent manner (45). The binding of flagellin to TLR5 induces IRAK-1

activation (80) and downstream de-repression of the transcription factor NFκB, allowing

for transcription of NFκB-responsive genes. This signaling leads to activation of innate

immune responses such as production of cytokines (4,5,27,72,80) ,influx of neutrophils

into lungs (47,63), and NO production in macrophages (76).

Flagellin as an Adjuvant

Flagellin’s effects on innate immunity. Work from the laboratory of Dr. Ruth

Arnon provided the first clear evidence that flagellin possesses adjuvant activity (62,70).

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Influenza epitopes cloned into flagellin and administered to mice with no other adjuvant

induced a protective response against influenza virus. Subsequent studies by a number of

groups established that the adjuvant effect of flagellin is due to at least three important

actions: 1) induction of cytokine production by non-lymphoid cells (4,5,20,27,72,79); 2)

increased T and B lymphocyte accumulation in draining lymph nodes (6); and 3)

activation of tlr5+/+ CD11c+ cells (7).

1. Cytokine Production. Many cytokines are produced by non-lymphoid cells

following exposure to flagellin (4,5,20,27,72,79). APCs produce TNF-α (20,72), IL-8

(5,72) and NO (20) which is indicative of a proinflammatory response. Additionally,

many chemokines are produced upon exposure to flagellin including MCP (CCL2) (72)

which recruits monocytes, T cells, and DC (15,110). The neutrophil recruiters and

activators (108) MIP1a (CCL3), MIP1b (CCL4), are also produced (47,72).

It is also worth noting that flagellin treatment drives the production of IL-4 and

IL-13 (5,22,27), which are associated with the classical Th2 allergic response. However,

flagellin does not promote the production of IgE (48).

2. Increased Cellularity. Twenty-four hours after i.n. instillation of flagellin

there is a marked increase in cellularity of draining lymph nodes (6). The majority of the

increase was in T and B lymphocytes. It is possible that this was either by increased

influx or reduced egress. Either of these possibilities increases the likelihood that a T or

B cell will come in contact with an APC which is presenting a recognized antigen.

3. Flagellin and Dendritic Cells. In vitro studies with isolated human dendritic

cells (1,72,87) clearly demonstrate that flagellin activates dendritic cells (DC). Means,

et. al. demonstrated that flagellin treatment of peripheral blood mononuclear cells

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(PBMC) results in an upregulation of costimulatory molecules, cytokine production, and

a decrease in phagocytosis (72). Additionally, flagellin-treated human DC induced

proliferation of CD3+ T cells (72). There have been varied reports on TLR5 expression

in murine DC. Several groups have reported that TLR5 is expressed on bone marrow-

derived DC (3,23,31,64), splenic DC (27,31,64), and lamina propria DC (100). The

complication with amplifying TLR5 mRNA is that the TLR5 gene does not contain any

introns. Therefore, unless a DNAse is included in the PCR reaction, it is possible that

genomic TLR5 is being amplified. Means et. al. did not detect an effect of flagellin on

murine DCs (72). Didierlaurent et. al. observed TLR5 expression in splenic DC, but not

bone marrow-derived DC (BMDC). However, when they treated BMDC with flagellin,

there was a greater upregulation of costimulatory molecules in BMDC than in splenic DC

(27). It is possible that discrepancies within the literature are due to the purity of the

flagellin used in the individual studies.

Bates et al. (7) found that purified recombinant flagellin has only a weak effect on

cytokine production in cultures of murine BMDC. However, a requirement for CD11c+

TLR5+ cells for the adjuvant effect of flagellin on antigen-specific CD4 T cell

proliferation was clearly demonstrated (7). Thus it is quite likely that in contrast to

lymph node DC, the majority of BMDC are at a stage of development during which

TLR5 expression or signaling is extremely limited.

Flagellin fusion proteins. In regards to adaptive immunity, it is well-established

that flagellin induces the proliferation of antigen-specific CD4+ T cells (7,71) and robust

humoral immunity (4,10-12,20,26,52,53,61,62,69,70,75,89,98,105). There is even some

evidence that flagellin can promote CD8+ T cell proliferation (52). But the effects of

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flagellin on adaptive immunity are enhanced when administered in the form of flagellin-

antigen fusion proteins (refs: Yersinia pestis (48,75), Pseudomonas aeruginosa (105),

influenza, (10-12,53,56,62,70) and West Nile Virus (69)). Fusion proteins containing

flagellin and target antigen are almost ten-fold more potent (7). The ability of flagellin to

bind with high affinity to TLR5 (106) on CD11c+ antigen-presenting cells (APC) is likely

to facilitate enhanced uptake of the associated antigen—a process that may ultimately

result in a higher level of antigen presentation. (7,48,75).

In light of the potent adjuvant activity of flagellin, we explored the possibility that

this adjuvant may be able to dramatically improve the humoral response to the weakly

immunogenic vaccinia virus antigens, L1R and B5R. We evaluated this possibility in the

context of separate proteins as well as flagellin fusion proteins. The results of these

studies have not only established the ability of flagellin to enhance responses to L1R and

B5R, but have also illuminated important strengths and weaknesses of flagellin as an

adjuvant.

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MATERIALS AND METHODS

Mice

Female BALB/c mice (6-8 weeks) were purchased from Charles River

Laboratories and were housed in specific pathogen free facilities. IgH-/- breeders were

purchased from Taconic Laboratories. Only female IgH-/- offspring were used in

immunization studies. All animal work was done in accordance with protocols approved

by Wake Forest University’s Animal Care and Use Committee.

Cell Lines

RAW264.7 and HeLa cell lines were purchased from ATCC. RAW424 cells

were generated by transfecting RAW264.7 cells with an expression plasmid containing

TLR5 linked to eYFP (106). Plasmid expression is maintained by the addition of 400

μg/mL G-418 to culture medium.

Purification of anti-CD8 Monoclonal Antibody

TIB-210 cells which produce Rat anti-mouse CD8 IgG (clone 2.43) were

purchased from ATCC. Large volumes of cells at a cell density of 5x105 cells/mL were

prepared. The non-adherent cells were removed from the medium by spinning at 2,000

rpm, and the media was retained. All following steps take place at 4°C. Media was

concentrated in a large-scale concentrator using a 10kDa cutoff membrane until ~100mL

remained. This was then dialyzed overnight into binding buffer (20mM Sodium

Phosphate, pH 7.0). The sample was purified on a Protein G HP column (GE Healthcare)

11

and eluted with 100mM glycine-HCl, pH 2.7. Fraction tubes contained appropriate

amounts of 1M Tris-HCl ph 9.0 to bring the pH of the eluates to 7.0. Antibody is

dialyzed overnight into PBS for long term storage at -80°C.

Virus Production and Purification

Vaccinia Virus Western Reserve (VV-WR) was produced by infecting HeLa cells

at an MOI of 5 for 48 hours. Cells were pelleted by centrifugation and lysed by at least 4

freeze-thaw cycles. Further lysis was performed by 3 cycles of sonication at full power

for 30 seconds. Virus was centrifuged 80 minutes at 32,900 x g and 4°C on a 36%

sucrose cushion in 10mM Tris-Cl, pH 9.0. The pellet was resuspended in 1mM Tris-Cl

pH 9.0.

Baculovirus production of L1R and B5R

Baculoviruses encoding ectodomains of L1R and B5R were obtained from J.C.

Whitbeck, Schools of Dental and Veterinary Medicine, University of Pennsylvania,

Philadelphia, PA. Viral proteins were prepared by infecting SF9 cells (Invitrogen) grown

in SF-900II Serum Free Medium (Gibco) at an MOI of 3-10. Supernates were harvested

on day 3 and concentrated using a filter with a 10kDa cutoff (Millipore). The his-tagged

proteins were purified using a Ni-NTA Superflow column (Qiagen). Proteins were then

dialyzed into PBS pH 7.0 (L1R) or PBS pH 5.9 (B5R). To remove endotoxin and DNA

contaminants, the proteins were run through Acrodisc units with Mustang E (B5R) or Q

(L1R) filters (Pall Corporation). Endotoxin levels were <20pg/μg of protein (limulus

amoebocyte assay (Cape Cod Inc.)).

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Generation of flagellin and flagellin-poxvirus fusion proteins

FliC (hereafter referred to as flagellin) from Salmonella enterica serovar

Enteritidis (68) was prepared as previously described (47,68). The 229 truncation mutant

of flagellin contains only amino acids 297 – 471 of the hypervariable region, and

therefore does not signal via TLR5 (47,68,78) Two approaches were used to generate

fusion proteins containing flagellin and L1R or B5R or flagellin, L1R, and B5R.

Plasmids containing L1R and B5R cDNA were kindly provided by J.W. Hooper

(USAMRIID, Ft. Detrick, MD). To generate a flagellin-B5R (FB) fusion, a cDNA

encoding the ectodomain of B5R (defined as nucleotides 57–837) was cloned into the

fliC gene lacking the majority of the hypervariable region (lacking nucleotides 586-

1134). The L1R-flagellin (LF) fusion protein was generated by inserting the cDNA

encoding the ectodomain of L1R (nucleotides 1-543) at the N-terminus of full-length

fliC. These constructs are illustrated in Figure 1. Each construct was cloned into the

pET29a expression vector (Novagen).

Purification of proteins

Plasmids were transformed into E. coli BL-21 (DE3) for protein production. All

proteins used in this study contained a 6-His tag to facilitate rapid purification using

Talon metal affinity resin (Clontech) as previously described. To remove endotoxin and

nucleic acid contaminants, the purified proteins were passed through Acrodisc Mustang

Q filters (Pall Corporation). LPS levels (measured by Limulus Amoebocyte Assay, Cape

Cod Inc.) were <20pg/μg of protein.

13

Figure 1 Proteins used in this study and their TLR5-stimulating activity. Chimeric

proteins were produced containing the ectodomains of L1R and B5R fused to flagellin.

Diagrams illustrate antigen placement. L1R is fused to the N-terminus of full length

flagellin. B5R substitutes the hypervariable domain of flagellin. TLR5-stimulating

activity is calculated as units/mg. A unit is the inverse of the concentration yielding a

half maximal response. This is then standardized to units/mg.

14

15

TLR5-specific signaling activity of flagellin and flagellin fusion proteins

In vitro TNFα production was used as a measure of flagellin signaling activity

(17,18,47). To insure that the signaling was TLR5 specific, we assessed activity in

cultures of RAW 264.7 cells, a mouse macrophage cell line which does not express TLR5

and in cultures of RAW 424 cells, RAW 264.7 cells that stably express TLR5. These cell

lines were stimulated for 4 hours with flagellin-containing proteins and supernatants were

harvested for analysis of TNF by ELISA using a commercial kit (BD Biosciences) as per

the manufacturer’s instructions. Units are calculated as the inverse of the concentration

giving a 50% maximal response, which are then standardized to units per milligram.

Immunizations

For intranasal (i.n.) immunizations, mice were anesthetized with Avertin (2,2,2-

tribromoethanol (Sigma)) and 10-15μL of vaccine mixture was administered dropwise to

alternating nares. For intramuscular (i.m.) immunizations, groups of at least 7 mice were

anesthetized with Avertin and then 20 μl of vaccine mixture was injected into the right

rear calf. Groups of 7 mice received 2 immunizations on days 0 and 28, 3 immunizations

on days 0, 28, and 42, or 4 immunizations on days 0, 28, 42, and 56. Blood samples were

collected via tail vein bleeding 10-14 days after each immunization and the plasma

prepared for analysis of antibody titers.

Determination of plasma IgG titers by ELISA

ELISA plate wells (Immunosorb 96-well plates, NUNC) were coated with 100μL

of 10μg/mL recombinant antigen (L1R or B5R) in PBS overnight at 4°C. Plates were

16

blocked with PBS + 10% newborn calf serum, and then plasma samples were added to

triplicate wells. The plates were incubated overnight at 4°C, washed, and then rabbit

anti-mouse IgG conjugated to horseradish peroxidase was added and the plates incubated

for 2 hours at room temperature, followed by a 30-minute incubation with 3,3’,5,5’-

tetramethylbenzidine (TMB) liquid substrate. The reaction was stopped by the addition

of 2N H2SO4, and absorbances were read at 450nm. Titers were defined as the inverse

dilution of plasma which yields an absorbance of 0.1 over background (established using

naïve plasma).

Respiratory challenge with vaccinia virus

The 50% maximally tolerated dose (MTD50; equivalent to LD50) for BALB/c

mice was determined for each vaccinia virus preparation using non-linear regression.

Groups of 7 mice were anesthetized with avertin and 10μL of virus was administered

dropwise to alternating nares. Mice were observed daily for signs of disease. Symptoms

were scored according to the following disease index: Hunched Posture (no = 0, yes =

1), respiratory stress (no = 0, mild-moderate = 1, severe = 2). Conjunctivitis and lethargy

were evaluated on the same scale as respiratory stress. A mouse which received a disease

score of 2 for any symptom or lost 30% of its initial weight was sacrificed.

Vaccinia Virus Neutralization

A flow cytometry-based assay was used to assess vaccinia virus neutralization

(29). An eGFP-expressing vaccinia virus was diluted in DMEM with various dilutions of

heat-inactivated mouse plasma. HeLa cells (seeded in 96-well plates at 1x105 cells/well)

17

were infected at an MOI of 0.25 and incubated for 6 hours. The cells were then treated

with trypsin and fixed with PFA before FACs analysis. Virus incubated in medium only

was used as a control (0% neutralization).

Depletion of CD8+ T cells

Mice were treated with 0.3mg of monoclonal anti-CD8 antibody (clone 2.43)

injected i.p. three times on days -5, -3, and -1 in relation to challenge.

Complement Depletion

Mice were treated with 5μg (1 unit) of Cobra Venom Factor (CVF) (Calbiochem)

i.v. 18 hours before challenge with vaccinia virus. A second treatment of 5μg of CVF is

administered 4 days after the initial injection (3 days post-infection). In preliminary

experiments, we established that this protocol depletes complement levels for 5 days with

complement levels returning to ~50% on day 6.

Determination of Circulating Numbers of Neutrophils

Whole blood was collected in heparinized tubes 1 day after mice had been

immunized two or three times. After transfer to FACs staining tubes, a uniform number

of FluoSphere microspheres (Invitrogen F8836) were added to the tubes to allow for later

standardization of cell numbers. Samples were stained for F4/80 (monocytes; Caltag

Laboratories MF48020) and GR-1 (neutrophils; eBioscience 51-5931-80).

18

Measurement of C-reactive Protein

Plasma samples were collected from mice 1 day after mice had been immunized

two or three times. Plasma was prepared as described above and stored at -80°C until

analysis. C-reactive protein was measured by an ELISA kit (Kamiya Biomedical KT-

095) as per the manufacturer’s instructions. Plasma was diluted 1:5 instead of the

recommended 1:10.

Statistical analysis

Statistical analyses were performed using Prism 5 (GraphPad). Mouse titer data

was assumed to be non-parametric, thus the Mann-Whitney Test was utilized to evaluate

differences in titers. Survival data was evaluated using the integrated survival analysis

tool.

19

CHAPTER I

GENERATING RESPONSES TO WEAKLY IMMUNOGENIC PROTEINS

USING FLAGELLIN AS AN ADJUVANT

20

RESULTS

CHAPTER I: Generating Responses to Weakly Immunogenic Proteins Using

Flagellin as an Adjuvant

Immunization with flagellin and pox antigens results in a robust humoral

response and protection against respiratory challenge with vaccinia virus. A number of

studies using recombinant L1R and B5R proteins (36,37) or DNA encoding these

proteins (46,49-51) have demonstrated that recombinant forms of these antigens are

only weakly immunogenic . To evaluate whether flagellin could promote a more robust

response to these antigens, mice were immunized i.n. on 2, 3, or 4 occasions with 10μg of

purified Baculovirus-expressed L1R and B5R plus 1μg of flagellin and assessed for

plasma titers of anti-L1R and B5R IgG 10-14 days after each immunization. As shown in

Figures 2A and B, the IgG titers against L1R and B5R increased with increasing numbers

of immunizations. Although a significant number of mice responded after two

immunizations, additional immunizations were required to achieve high titers in most of

the animals. In the case of the anti-L1R response, all of the mice responded after 4

immunizations. However, even with 4 immunizations, 4/10 mice did not exhibit

significant titers of anti-B5R IgG. These results are in striking contrast to the response

to flagellin + the Y. pestis F1 and V antigens (6,48,75). In the case of the pestis antigens,

two immunizations results in IgG titers that are generally ≥ 5 x 105.

To evaluate the protection conferred by i.n. vaccination, the mice which had

received 0, 2, 3, or 4 immunizations were challenged i.n. with 10 MTD50 (equivalent to

10 LD50) of vaccinia virus. Immunizations were staggered so that all mice were

challenged at the same time. Mice losing 30% of their initial weight were sacrificed. All

21

Figure 2 Intranasal immunization with pox antigens + flagellin confers protection after 3

or more immunizations. Groups of 10 BALB/c mice were intranasally immunized with

10μg of L1R + 10μg of B5R + 1μg of Flagellin 2-4 times and plasma was collected after

each immunization. Titers of antibodies specific for pox antigens B5R (a) and L1R (b)

were determined by ELISA. Each symbol represents an individual mouse, and the

dashed line at 103 is the limit of detection for the assay. Numbers below the dashed line

indicate the number of mice whose titers were below the limit of detection (non-

responders). A single asterisk indicates that the titer is significantly higher than the titer

following a single immunization. Other groups which are significantly different are

indicated by bars and multiple asterisks. (c)Three weeks after the final immunization,

mice were intranasally challenged with 20 MTD50 of vaccinia virus and survival was

documented. (d) As an estimate of morbidity associated with challenge, weights were

recorded daily following infection.

22

23

of the mice that received 3-4 immunizations survived challenge (Fig. 2C), whereas only

about 50% of the animals that received 2 immunizations survived. Assessment of weight

loss revealed that even the mice that received 4 immunizations lost a significant amount

of weight (approximately 15%). However, these mice did not exhibit any other severe

signs of disease (for example, difficulty in breathing, conjunctivitis, or hunched posture).

Protective effect of a flagellin-adjuvanted poxvirus vaccine is dependent on B

cells, but not on CD8+ T cells. To evaluate which branches of adaptive immunity are

required for flagellin-mediated protection against poxvirus infection IgH-/- mice were

immunized three times i.n. with 10μg each of L1R and B5R and 1μg of flagellin. IgH-/-

mice lack the immunoglobulin heavy chain gene, and therefore cannot rearrange a stable

B cell receptor. Therefore, the mice do not develop mature B cells. As seen in figure 3,

when these mice were challenged with 10 MTD50 vaccinia virus all of them succumbed

to infection. We conclude from this that the humoral response to the flagellin + pox

antigen vaccine is absolutely required for the protective effect of the vaccine.

To evaluate the role of CD8+ T cells, BALB/c mice were immunized as described

above. Beginning five days prior to challenge, these mice were injected with a depleting

rat anti-mouse CD8 IgG antibody (clone 2.43). Mice were injected i.p. with 0.3mg on

days -5, -3, and -1 relative to challenge. Figure 4a shows the depletion that results from

this anti-CD8 IgG treatment, and figure 4b shows survival after challenge. There was no

difference in survival of mice which were treated with the CD8-depleting antibody.

Therefore, CD8+ T cells are not required for the protective effect of the flagellin-based

vaccine. However, this experiment does not rule out the possibility that CD8+ T cells

may still contribute to survival.

24

Figure 3 Flagellin-mediated protection requires B cells. BALB/c or IgH-/- mice were

immunized 3 times i.n. with 10μg each of L1R and B5R and 1μg of flagellin. Three

weeks after the final immunization mice were challenged with 10 MTD50 vaccina virus

WR. Survival was monitored for two weeks.

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26

Figure 4 Flagellin-mediated protection does not require CD8+ T cells. Groups of 5

BALB/c mice were immunized 3 times i.n. with 10μg of L1R and B5R plus 1μg of

flagellin. One group of mice was depleted of CD8+ cells by depletion with an anti-CD8

antibody (clone 2.43) starting 5 days before challenge. Depleted mice were injected i.p.

with 0.3mg of antibody on days -5, -3, and -1 relative to challenge. (A) Depletion of

CD8+ cells in lymph node (LN) and spleen (S). Numbers indicate the percentage of

recovered cells within a given quadrant. (B) Survival following i.n. challenge with 10

MTD50 VV-WR.

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28

Enhanced immunogenicity of flagellin-poxvirus fusion proteins. Several recent

studies have demonstrated that flagellin fusion proteins enhance responses to strong

antigens such as influenza virus epitopes (10,12,53,56,62,70), the F1 and V antigens of Y.

pestis (48,75), and West Nile Virus’s EIII (69). Since these fusion proteins promoted a

more robust response than separate antigen(s) plus flagellin, we tested whether the

immunogenicity of recombinant L1R and B5R could be enhanced by genetically linking

them to flagellin. Schematics for the flagellin/pox antigen fusion proteins are shown in

Figure 1. To determine if the fusion proteins retained the ability to signal via TLR5, we

assessed their ability to stimulate TNFα production in cultures of TLR5+ RAW 424 cells.

To control for TLR5-independent signaling, we also assayed TNFα production in

cultures of TLR5-negative RAW 264.7 cells. As shown in Figure 1, the LF and the FB

retained full TLR5 signaling activity relative to flagellin.

Having established that the fusion proteins retained TLR5 signaling activity, we

next compared the ability of the fusion proteins with that of the separate proteins

(flagellin + L1R +B5R) to induce IgG responses against L1R and B5R. Groups of 7 mice

were immunized i.n. or i.m. with equimolar doses of L1R (1.3μg) + B5R (2.4μg) +

flagellin (3.7μg), or LF (5μg) + FB (5μg). As shown in Figure 5A and 5B, the fusion

proteins LF and FB elicited IgG that recognized recombinant L1R and B5R. This

response was dramatically more robust than the response to separate proteins + flagellin

administered i.m. This difference was most evident with the B5R response.

Furthermore, the variability in response within the fusion protein groups was far less than

that observed when separate proteins were used for immunization.

29

Figure 5 Fusion proteins are more effective at promoting antigen-specific humoral

responses than separate proteins. Groups of 7 BALB/c mice were immunized 2 times

i.m. with 5μg of flagellin-antigen fusion proteins LF and FB or equimolar doses of the

separate proteins. B5R (A) and L1R (B) specific titers are shown as determined by

ELISA. Asterisks indicate significant differences in titer (P < 0.05).

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31

Comparison of i.n. and i.m. administration of flagellin-poxvirus fusion proteins.

With highly immunogenic antigens such as the F1 and V antigens of Y. pestis, there is not

a dramatic difference in the induced antibody titers when administered with flagellin i.n.

vs. i.m. (48). To evaluate the effect of route of administration on the humoral response to

the significantly less immunogenic recombinant L1R and B5R, groups of 7 mice were

immunized three times (i.n. or i.m.) with LF and FB and evaluated for plasma anti-L1R

and B5R IgG titers. As shown in Figure 6A and 6B, immunization via the i.m. route

elicited significantly higher titers of anti-L1R and B5R IgG than obtained using the i.n.

route of administration. The circulating titers of anti-L1R and B5R in mice immunized

i.m. were about two logs higher than those in mice immunized i.m.. Furthermore, the

variability in the response was more limited when the i.m. route was used. Thus, the

response to recombinant L1R and B5R can be markedly increased by using flagellin

fusions and by using the i.m. route of administration.

B5R-specific IgG2a titer correlates with protection from vaccinia virus challenge.

A recent study (13) found that passive immunization with a B5R-specific monoclonal

antibody confers protection against vaccinia virus challenge. To assess whether IgG2a

titer correlated with protection we tested archived plasma samples from mice which had

been immunized various times with separate proteins plus flagellin and seroconverted in

response to immunization. The plasma samples were taken before challenge, and mice

were grouped according to survival following vaccinia virus challenge. Pooled plasma

from convalescent mice was used as a positive control. There was no significant

difference between survivors and non-survivors in L1R-specific IgG2a titer (Fig. 7).

However, the anti-B5R IgG2a titers were significantly lower in mice that were not

32

Figure 6 Intramuscular immunization is more effective at promoting antigen-specific

humoral responses than intranasal immunization. Groups of 7 BALB/c mice were

immunized 2 times i.m. or i.n. with 5μg of flagellin-antigen fusion proteins LF and FB.

B5R (A) and L1R (B) specific titers are shown as determined by ELISA. Asterisks

indicate significant differences in titer (P < 0.05).

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34

Figure 7 Mice who are protected during challenge have significantly higher antigen-

specific IgG2a titers than mice who succumb to challenge. Archived plasma samples

from immunized BALB/c mice were collected prior to challenge. Samples were

separated into mice who went on to survive challenge, or those who succumbed to

infection. Pooled plasma from convalescent mice was used as a positive control.

Significant differences (P < 0.05) are indicated by asterisks.

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36

protected (Fig. 7A). These findings are consistent with the hypothesis that IgG2a plays a

role in antibody-mediated protection against vaccinia virus.

Immunization with LF and FB promotes the production of antigen-specific IgG1

and IgG2a. The previous experiment used plasma samples from mice immunized with

L1R + B5R + flagellin. To evaluate whether fusion proteins can promote IgG2a titers

comparable to those seen in survivors immunized with separate proteins, we immunized

mice 2, 3, or 4 times i.m. with 5μg each of LF and FB (Figure 8). Total IgG, IgG1, and

IgG2a antibody titers were determined by ELISA. Total anti-L1R and B5R IgG titers as

well as IgG1 and IgG2a increased with increasing numbers of immunization. Similarly,

the number of responding mice also increased with each additional immunization. To

determine if a higher dose of LF and FB would alter the balance of antigen-specific IgG1

and IgG2a, BALB/c mice were given 3 immunizations with 20μg of LF and FB (Figure

9). Although the variability in response was markedly reduced, the IgG1 and IgG2a titers

were not significantly different in mice which were given 20 μg of the fusions compared

to 5 μg.

Immunization with LF and FB confers protection against respiratory challenge

with vaccinia virus. To determine if fusion proteins decrease the number of

immunizations required for protection against vaccinia virus, mice were immunized two,

three, or four times i.m. with 5μg each of LF and FB and then challenged i.n. with 20

MTD50 vaccinia virus. As shown in Figure 10, despite significantly higher poxvirus

specific antibody titers, fusion protein vaccination still required at least three

immunizations to achieve 100% protection. Although mice given three or four

immunizations survived challenge, they still lost a significant amount of weight (Fig.

37

Figure 8 IgG, IgG1, and IgG2a responses following i.m. immunization with 5μg of LF

and FB. Mice were immunized i.m. with 5μg of LF and 5μg of FB. (a-b) Total IgG

titers, (c-d) IgG1 titers, and (e-f) IgG 2a titers after each immunization were determined

by ELISA. Each symbol represents an individual mouse, and the dashed line at 103 is the

limit of detection for the assay. Numbers below the dashed line indicate the number of

mice whose titers were below the limit of detection (non-responders). In (a-d) all groups

are significantly different unless noted by n.s. (e-f) significant differences are indicated

by asterisks.

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39

Figure 9 IgG, IgG1, and IgG2a responses following i.m. immunization with 20μg of LF

and FB. Mice were immunized i.m. with 20μg of LF and 5μg of FB. (a-b) Total IgG

titers, (c-d) IgG1 titers, and (e-f) IgG 2a titers after each immunization were determined

by ELISA. Each symbol represents an individual mouse, and the dashed line at 103 is the

limit of detection for the assay. Numbers below the dashed line indicate the number of

mice whose titers were below the limit of detection (non-responders). In (a-d) all groups

are significantly different unless noted by n.s. (e-f) significant differences are indicated

by asterisks.

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41

Figure 10 Challenge after immunization with 5μg of LF and FB. Groups of 10 BALB/c

mice who received 2-4 i.m. immunizations of 5μg of LF and FB were challenged with 20

MTD50 vaccinia virus. (a) Survival, (b) weight loss, and (c) disease index were measured

following infection.

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43

Figure 11 Challenge after immunization with 20μg of LF and FB. Groups of 10

BALB/c mice who received 3 i.m. immunizations of LF and FB at the indicated doses

were challenged with 20 MTD50 vaccinia virus. (a) Survival, (b) weight loss, and (c)

disease index were measured following infection.

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45

10B). To evaluate disease symptoms other than weight loss, we used a disease index

score that evaluated hunched posture, respiratory distress, conjunctivitis, and lethargy.

As shown in Figure 10C, mice that received 3 or 4 immunizations exhibited only minimal

disease symptoms. In contrast, mice that were immunized only twice had disease

symptom scores that approached those of animals that were not immunized. Since 40%

of the mice immunized twice with the fusion proteins did not produce anti-B5R IgG2a, it

was possible that the lower level of survival might be associated with this subgroup of

animals. However, analysis of individual animals did not reveal a correlation between

anti-B5R IgG2a titer and survival (data not shown). It seems likely that the very high

titers of anti-B5R IgG1 obtained following immunization with the fusion proteins i.m. (as

opposed to separate proteins and i.n. immunization (Fig. 7)) may compensate for the

absence of IgG2a in some of the mice.

Complement plays a role in the protective effect of LF + FB. Since IgG2a is

known to be an efficient activator of complement in mice and monoclonal anti-B5R

IgG2a protects against vaccinia virus challenge (13), we evaluated whether complement-

mediated immunity was critical to the protective response generated in response to LF

and BF immunization. BALB/c mice were immunized 3 times i.m. with 5μg each of LF

and FB. One group of immunized mice was depleted of complement by i.v.

administration of CVF 18 hr before challenge with 20 MTD50 of vaccinia virus. A

second treatment of CVF occurred on day 3 post-infection. In preliminary experiments,

we established that two treatments with CVF dramatically reduces the level of circulating

C3. Six days after treatment, the level of C3 is only about 50% of that in untreated mice

(data not shown). All of the mice that were immunized, but not treated with CVF

46

Figure 12 Complement plays a role in the protective effect of a flagellin-poxvirus

antigen vaccine. BALB/c mice were immunized 3 times i.m. with LF and FB. Six mice

were treated with cobra venom factor (CVF) on days -1 and 3 relative to challenge with

20 MTD50 vaccinia virus. (a) Survival, (b) weight loss, and (c) disease index were

measured following infection.

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48

survived infection. However, 50% of the immunized mice that were treated with CVF

succumbed to infection (Fig. 12A). Although the degree of weight loss between the two

groups was similar (Fig. 12B), the complement-depleted mice had higher disease scores

(Fig. 12C). These results are consistent with the conclusion that complement component

C3 plays a role in LF + BF-induced protection.

Virus neutralization titers of plasma from immunized mice. To evaluate the virus

neutralization titer of antibodies generated by immunization with LF + FB, in vitro

vaccinia virus neutralization was performed using pooled heat-inactivated plasma

collected from mice prior to challenge. Plasma from immunized mice was incubated

with eGFP-expressing vaccinia virus for 1 hour prior to incubation with HeLa cells for 6

hours. Virus neutralization was defined as a 50% reduction in eGFP expression

compared to virus that was not incubated with plasma. All samples from immunized

mice neutralized significantly more virus than plasma from mock-immunized animals

(Fig. 13). There was a significant (~ 3-fold) difference in neutralization between two

and three immunizations with 5μg of the fusion proteins LF and FB. Mice immunized by

a single exposure to vaccinia virus (convalescent) had titers that were two-fold higher

than any of the LF + FB immunized mice. It is interesting to note that the neutralizing

titers correlated with the severity of disease. Naïve mice all succumbed to infection and

had neutralizing titers under 100. Mice immunized twice were partially protected, lost

weight, and had elevated disease scores and had an average neutralization titer of

approximately 300. The mice that received 3 or 4 immunizations were protected and had

low disease scores, but lost significant weight. Their average neutralizing titers were

approximately 900. The mice that received a sublethal dose of vaccinia virus exhibited

49

Figure 13 Plasma from immunized mice is capable of neutralizing vaccinia virus in

vitro. Pooled plasma from mice receiving 2-4 immunizations with 5μg of LF and FB or 3

immunizations with 20μg of LF and FB was incubated with a GFP-expressing vaccinia

virus for at 37°C at the indicated dilution. The plasma-treated virus was added to HeLa

cells and allowed to infect for 6 hours. Infected cells were detected by flow cytometry

and percent of neutralization was calculated by standardizing to mock infected cells

(100%) or cells infected with virus that was not treated with plasma (0% neutralization).

Neutralizing titer is defined as the dilution at which 50% of virus is neutralized, and is

calculated by non-linear regression. A reference line is provided at 50% neutralization,

and calculated values for neutralization titer are in the legend.

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51

complete immunity and did not exhibit any disease symptoms or weight loss. These mice

had titers ≥2,000.

Analysis of inflammatory effectors following immunization. Although the changes

in neutralizing titer may account for the difference in protection observed between two

and three immunizations with LF + FB, one or more additional factors may be involved.

We evaluated the possibility that three immunizations with LF + BF might trigger the

production of liver-derived C-reactive protein, an acute phase reactant with complement-

activating and opsonic activities, and thus compensate for insufficient levels of IgG2a.

To evaluate this possibility, mice were immunized i.m. 2 or 3 times with 5μg of LF and

FB or PBS, and plasma samples were collected 24 hours after the final immunization.

Plasma C-reactive protein levels were determined by ELISA. There was no change in

circulating CRP levels of mice immunized with the flagellin fusion proteins compared to

PBS-injected mice (Fig. 14A).

We also considered the possibility that after three immunizations, flagellin might

induce sufficient titers of GM-CSF (19) that would increase the numbers of circulating

neutrophils and monocytes available for opsonization of antibody-coated virions. To

evaluate this possibility, we immunized mice as described for the analysis of C-reactive

protein levels and collected whole blood after 2 and 3 immunizations with LF and FB.

Cells were stained for the markers F4/80 (monocyte marker) and GR-1(neutrophil

marker) and analyzed by flow cytometry. The addition of a known number of fluorescent

microspheres allowed us to back-calculate numbers of cells per mL of blood. Monocyte

counts remained constant in all tested conditions (Fig. 14B). The drop in neutrophil

levels 24 hours after a second immunization with LF and FB is consistent with

52

Figure 14 Effect of repeated immunization on circulating levels of neutrophils,

monocytes, and C-reactive protein. BALB/c mice were immunized two or three times

i.m. with 5μg each of LF and FB. (A) Plasma samples were collected 24 hours after the

final immunization and analyzed for C-reactive protein levels by ELISA. (B) Twenty

four hours post-immunization whole blood samples were taken and stained for

neutrophils (GR-1+, F4/80-/lo) and monocytes/macrophages (GR-1-, F4/80+). Addition of

a known number of fluorescent microspheres allowed for the quantitation of neutrophils

and monocytes per mL of blood.

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54

neutrophils being recruited to the site of injection (6,48). After a third immunization with

LF and FB, the level of neutrophils in the blood approached that of control mice. Since

neutrophilic recruitment to the site of injection would occur after the third immunization,

it is likely that the increased level of circulating neutrophils in this group is due to

enhanced bone marrow production of these cells. However, additional studies are clearly

required to formally test this hypothesis.

55

CHAPTER II

LESSONS ABOUT FLAGELLIN AS AN ADJUVANT

AND A VACCINE VECTOR

56

RESULTS

CHAPTER II: Lessons about Flagellin as an Adjuvant and a Vaccine Vector

The constructs used in chapter one were not the only constructs generated in this

project, and with these other proteins shown in figure 15 we learned some valuable

lessons about the strengths and weaknesses of flagellin. In the currently published

studies using flagellin as an adjuvant the target antigens are strong immunogens such as

F1 and V from Yersinia pestis (48,75). In model systems with strong immunogens the

limitations of flagellin are masked by the inherent immunogenicity of the antigen. These

are the limitations we discovered using weakly immunogenic poxvirus antigens.

Variability among antigens for optimal placement within a flagellin fusion

protein. The proteins generated in figure 15 were evaluated for their ability to induce

antigen-specific humoral responses. BALB/c mice were immunized three times i.n. with

the indicated protein. Fourteen days after the final immunization, plasma samples were

collected and tested for antigen-specific IgG. FB and LF induce robust antigen-specific

humoral responses. However, FL, with L1R replacing the hypervariable region of

flagellin, was a very poor inducer of L1R-specific IgG in immunized mice. From this it

is evident that individual antigens are limited in the locations they can be inserted into

flagellin, but each antigen will be different. This certainly is a limitation of flagellin, but

since there are 3 locations (N-terminus, C-terminus, and hypervariable region) in flagellin

capable of inserting proteins without disrupting bioactivity it is likely that a site can be

found for most antigens that will not compromise immunogenicity.

57

Figure 15: Flagellin-pox antigen fusion proteins and their TLR5-stimulating activity. Chimeric proteins were produced containing the ectodomains of L1R and B5R fused to

flagellin. Diagrams illustrate antigen placement. TLR5-stimulating activity is calculated

as units/mg. A unit is the inverse of the concentration yielding a half maximal response.

This is then standardized to units/mg.

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59

Figure 16 Variability among antigens for optimal placement within a flagellin fusion

protein. BALB/c mice were immunized three times i.n. with 5μg each of FB, LF, or FL.

B5R (A) and L1R (B) titers were determined by ELISA. The dashed line represents the

limit of detection for the assay, and the number below that line indicates the number of

mice whose antigen-specific titers were below the limit of detection.

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Antigenic competition is observed when LF and FB administered together.

Groups of 7 BALB/c mice were immunized intramuscularly on three occasions with 5μg

of both LF and FB or only a single fusion protein. Plasma samples were assayed for B5R

and L1R-specific IgG titers. As shown in Figure 17, the combination vaccine induced

titers of anti-L1R and B5R IgG that were significantly less than the titers using only a

single fusion protein.

We considered two possible explanations for this effect. It was possible that L1R

and B5R were involved in some form of antigenic competition and thus the combination

of the two would result in a reduced response to both antigens. Alternatively, the reduced

response might be due in some way to an inhibitory effect of the flagellin component of

each fusion protein. However, based on our prior results (47), we know that flagellin

doses up to at least 15 μg do not result in a diminished humoral response to an

immunogen.

The humoral response to a flagellin-antigen fusion protein can be reduced by

addition of excess flagellin. Thus we considered the possibility that the flagellin

components of each fusion were competing for a limiting amount of TLR5 to facilitate

efficient uptake of each fusion protein. If this were the case, then co-administration of

LF or FB with increasing doses of flagellin should result in a reduced response to the

poxvirus antigen. To test this notion, groups of mice were immunized with FB alone or

FB with increasing doses of flagellin and then the induced titers of anti-B5R IgG were

measured. The results (Figure 18) clearly demonstrate that as the dose of flagellin was

increased, the resultant titer of anti- B5R was significantly reduced.

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Figure 17 Competition between antigens when multiple flagellin fusion proteins are

administered. BALB/c mice were immunized 3 times i.m. with 5μg each of LF, FB, or

LF and FB. B5R (A) and L1R (B) titers were determined by ELISA.

63

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Figure 18 The humoral response to a flagellin-antigen fusion protein can be reduced by

addition of excess flagellin. Groups of 7 BALB/c mice were immunized 3 times i.n. with

FB alone or FB with excess soluble flagellin. Ratios on the x-axis indicate the

FB:Flagellin ratio. Asterisks indicate significant differences.

65

66

Although this finding is not a direct proof, it is consistent with the hypothesis that

competition is due to a rate-limiting level of TLR5 on antigen-presenting cells.

A trifusion protein that combines flagellin with two poxvirus antigens loses

immunogenicity. In view of the competition between fusion proteins and the need to

generate responses against IMV and EEV antigens, we generated a trifusion protein that

contained L1R at the amino terminus of flagellin and B5R as a replacement for the

majority of the hypervariable region of flagellin (Fig. 15). This protein retained full

TLR5 signaling activity as measured in the RAW cell assay for TNFα production. As

seen in figure 19, when groups of mice were immunized with the trifusion protein, the

anti-L1R IgG responses were similar to those obtained with L1R-flagellin (mean titer =

5.8x105). However, there was no detectable B5R-specific response.

B5R is not recognized by B5R-specific antibodies in the context of the LFB

trifusion. To determine if the absence of B5R-specific IgG was due to a change in the

conformation or availability of the B5R in the trifusion, we coated ELISA plates with

LFB and used plasma from mice immunized with B5R and alum as a source of anti-B5R

IgG. The B5R-specific plasma did not react with trifusion (Figure 20), indicating that the

conformation of B5R is altered in the trifusion, or that the B5R is not accessible to

antibodies. Thus, although flagellin exhibits substantial plasticity with regard to the

insertion of antigens, the antigens themselves can place limitations on the carrier function

of flagellin (as opposed to its adjuvant activity).

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Figure 19 A trifusion protein that combines flagellin with two poxvirus antigens loses

immunogenicity. Groups of 7 BALB/c mice were immunized three times i.m. with 5μg

of LF and FB or a molar equivalent (6.5μg) of LFB. B5R (A) and L1R (B) specific IgG

responses were determined by ELISA.

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69

Figure 20 B5R is not recognized by B5R-specific antibodies in the context of the LFB

trifusion. B5R-specific plasma was generated by immunizing mice twice i.m. with B5R

adsorbed on alum. ELISA plates were coated with baculovirus-produced B5R or LFB.

Wells were probed with the B5R-specific plasma and followed by a HRP-conjugated

anti-mouse IgG antibody.

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71

DISCUSSION

A major goal of these studies was to evaluate the efficacy of flagellin as an

adjuvant with proteins that are poorly immunogenic in their recombinant forms. Our

studies clearly demonstrate flagellin can be an effective adjuvant with weakly

immunogenic antigens. Although administration of L1R, B5R, and flagellin promoted

antigen-specific humoral responses, fusion proteins were clearly more potent. This was

especially evident in the B5R-specific response. Even after 4 immunizations with the

separate antigens 40% of mice did not respond to B5R (Figure 2). Alternatively, after 3

immunizations with the LF and FB fusion proteins all mice have B5R-specific IgG titers

(Figure 8). This observation indicates that the mice have the full potential for a humoral

response to B5R. One possible explanation for the low titers after immunization with

separate antigens is a low frequency of B5R-specific CD4 T cells to aid in B cell

activation and class switching. When the pox antigens are delivered in the form of fusion

proteins, flagellin-specific CD4 T cells can activate B5R-specific B cells via linked

recognition, thus overcoming a shortage of B5R-specific CD4 T cells.

Another factor that may play a role in the immunogenicity of a flagellin-antigen

fusion protein is stability. Antigens which persist longer have a greater potential to be

targeted for immune responses. It is possible that one of the benefits of flagellin fusion

proteins is that by linking the poxvirus antigens to the highly stable flagellin molecule the

stability of L1R and B5R is increased.

Even with the fusion proteins there was a requirement for at least three

immunizations with LF and FB for protection against respiratory challenge with vaccinia

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virus. Fogg et al. (36) found that four immunizations with recombinant poxvirus antigens

were required to achieve high antibody titers and protection against challenge. In

contrast to the more limited response to the recombinant poxvirus antigens, a single

exposure to vaccinia virus elicits high titers of anti-L1R and B5R antibodies. At the most

simple level, a sublethal dose of vaccinia virus may be more effective than immunization

with a small number of recombinant antigens due to the availability of a dramatically

larger number of potentially protective antigens during virus infection. However, it is

also possible that there is some feature of antigens such as L1R and B5R in the context of

the viral infection that cannot be replaced or overcome by flagellin or other adjuvants

(30,36,37,46,49-51).

The duration of antigen availability represents another major difference between

live virus vaccination and recombinant protein immunization. The persistence of antigen

in the viral infection, as opposed to the limited availability of the flagellin fusion proteins

following immunization provides a reasonable explanation for the difference in the level

of the induced antibody response. Dryvax immunization results in the shedding of live

vaccinia virus for 3-4 weeks (38,57). In contrast, flagellin administered i.m. is no longer

detectable in the draining lymph nodes after 24 hours (Bates and Mizel, unpublished

observations). In addition to antigen availability, the poxvirus-induced innate immune

response may promote a more robust activation of dendritic cells and thus enhanced

helper T cell activation and associated B cell responsiveness. Finally, it may be that the

conformations of the recombinant L1R and B5R do not precisely mirror those of the

same proteins in the context of a vaccinia virus infected cell.

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In contrast to immunization with a sublethal dose of vaccinia virus, significant

weight loss is observed in mice immunized with recombinant L1R and B5R. It is

interesting to note that this weight loss in the LF + FB immunized mice occurred in the

absence of other disease symptoms. In comparison to our protein-based vaccination

strategy, recombinant viruses such as VSV (C. Lambeth et al., submitted for publication)

and SV5 (K. Clark and G. D. Parks, unpublished observations) engineered to express

L1R and B5R also promote protection in the presence of significant weight loss. In the

case of SV5 and VSV, it may be that more limited activation of DC may be a more

important factor in the reversible weight loss than antigen persistence or conformation.

Complement is known to be a part of the host response to vaccinia virus,

especially in promoting lysis of the extracellular enveloped virus (EEV) (65,66).

Clearance of EEV is very important as this form of the virus enters cells more rapidly

than the intracellular mature virion (IMV) form (28), and it is released earlier in infection

(before lysis of the host cell). In a recent study (13), Benhnia et. al. generated a series of

monoclonal anti-B5R antibodies and screened them for their ability to protect mice from

vaccinia virus. The only recovered monoclonal which conferred full protection at the

administered doses was of the IgG2a isotype--an isotype in mice that is particularly

efficient at activation of the classical complement pathway. Furthermore, Benhnia et. al.

(13) demonstrated that depletion of complement reduced the protective effect of passive

immunization with anti-B5R IgG2a. Our finding that the B5R-specific IgG2a response

was significantly higher in mice that survived challenge with vaccinia virus is consistent

with the findings of Benhnia et al. we found that 50% of the mice given the CVF

treatment survived vaccinia virus challenge. There are several possible explanations for

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this observation. First, it is possible that the return of complement levels in some of the

depleted mice may be more rapid, thus providing these mice with sufficient complement

to meet the needs of a protective response. Alternatively, other factors may contribute to

the observed protection. LF +BF immunized mice generate multiple antigen-specific IgG

isotypes. Although IgG1 is a less efficient activator of the classical complement pathway

than IgG2a in mice (83), it is quite likely that sufficiently high titers of antigen-specific

IgG1 may compensate for reduced inherent complement activating activity.

Additionally, the LF + FB-induced IgG may promote clearance of virus by opsonization

or direct neutralization.

One interesting observation was that in mice immunized with the fusion proteins

B5R-specific IgG2a titers no longer correlated with survival as we had seen in mice

immunized with separate pox antigens + flagellin (Figure 7). Of the mice challenged

after 2 immunizations there was no significant difference in B5R-specific IgG2a titers

between survivors and non-survivors (data not shown). This may be the result of

flagellin fusion proteins inducing more broad activation of the immune system which

would lessen the dependence on a single mechanism of protection. For example, very

high IgG1 titers may be able to activate complement to a level that compensates for

reduced IgG2a responses.

Despite the fact that i.m. immunization with fusion proteins generated greater

antigen-specific IgG responses to poxvirus antigens than the separate proteins, three

immunizations were still required for all of the mice to survive vaccinia virus challenge.

The finding that three immunizations results in a higher level of vaccinia virus

neutralizing activity than two immunizations (Figure 13), raises the possibility that

75

elevation of the neutralizing titer following a third immunization is a key event. Fogg et.

al. also observed an increase in neutralizing titer between two and three immunizations

(37).

Although a mechanism for neutralization of vaccinia virus is not known, it has

been hypothesized to be caused by steric hindrance (66). Steric hindrance could be

achieved by antibodies of very high affinity or that interact with epitopes that are near or

involved in virus-host cell interactions. With regard to antibody affinity, we found that

the average avidity of mice immunized 3 times i.m. with LF and FB was 2.1 times higher

than those immunized only twice (data not shown). It is also possible that the general

response to neutralizing epitopes comes up more slowly, possibly due to a lower

frequency of neutralizing epitope-specific B cells.

The avidity of an antibody response is refined in germinal centers where B cells

interact with helper T cells to proliferate and undergo somatic hypermutation, affinity

maturation, and isotype switching. The limited number of immune complexes presented

on follicular dendritic cells create strict competition for antigen, therefore only the

highest avidity clones can expand. There are several possible factors that could influence

the B cell response. First, if the flagellin-poxvirus antigen fusion proteins possess only a

limited ability to induce antigen-specific CD4+ T cell activation, then multiple rounds of

immunization would be required to achieve a sufficient number of these cells to facilitate

B cell activation and maturation. Thus the delay in the generation of high neutralization

titers may be due to not only the limited frequency of B cells with specificity for

neutralizing epitopes on L1R and B5R, but also a limited expansion of antigen-specific

CD4+ T cells.

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Our neutralization data may provide a guide to the minimal levels of neutralizing

IgG required for survival. With average neutralization titers of ~300, some mice survive

infection while others succumb. Mice that had mean neutralization titers ranging from

900-1300 all survived and showed little to no disease symptoms. However, mice in this

range did lose weight at the peak of the infection, but this weight loss was reversible.

When mice were immunized with sublethal doses of vaccinia virus the average

neutralization titer was ~2700. These mice do not lose weight when challenged with high

doses of vaccinia virus, thus exhibiting maximal protection. Thus it appears that a

neutralizing titer between approximately 300 and 900 is required for survival, whereas a

titer above 1300 is key for protection against weight loss.

Virus neutralization may only be part of the explanation for a difference in

survival after two versus three immunizations. In a toxicology study in rabbits done

according to currently accepted Good Laboratory Practices (Mizel, unpublished

observations), it was observed that two immunizations with 100, 250, or 500 μg of a

flagellin fusion containing the Y. pestis antigens F1 and V did not induce any significant

changes in blood chemistry. However, after a third immunization with 250 or 500 μg,

there was a significant increase in several liver enzymes indicating hepatic activation.

One result of liver activation is the release of the complement activating and opsonic

acute phase reactant, C-reactive protein. However, the circulating levels of CRP in the

blood did not change following immunization with flagellin.

Although flagellin did not increase circulating levels of CRP (Figure 14A), it

might have triggered an increase in neutrophils available for the clearance of antibody-

coated vaccinia virus (Fig. 14B). In this regard, a study comparing motile (flagellated)

77

and non-motile (non-flagellated) strains of Pseudomonas aeruginosa revealed that only

the strain expressing flagellin induced GM-CSF production in primary human lung cells

(19). After 7 days blood neutrophil levels were all equal in immunized and unimmunized

mice (data not shown), so it is unlikely that increased numbers of neutrophils (as a result

of a third immunization) are contributing to protection since mice were challenged with

vaccinia virus 3 weeks after the final immunization.

It remains that the observation that neutrophil levels are increased immediately

after exposure to a pathogen-associated molecular pattern (PAMP) could enhance

clearance of a pathogen, even though it may not play a role in protection following

vaccination. However, it is possible that exposure to the virus acted as another danger

signal and itself induced an enhanced response as a result of the “priming” the host

received during vaccination.

Multiple exposures to danger signals such as flagellin and other PAMPs would

signal to the host that the efforts to clear the pathogen have failed. It seems logical that

individuals that possess enhanced immune responsiveness when frequently re-exposed to

a pathogen would have an evolutionary advantage. It would be of great interest to

determine if multiple PAMPs can promote this enhanced response to infectious agents,

and whether the types of responses would be tailored to the type of pathogen from which

the PAMP was derived.

Take together, these results establish that flagellin can promote robust responses

against weakly immunogenic poxvirus antigens. Although weight loss is observed, the

immunized mice do not exhibit other signs of disease and survival challenge. It may be

78

that the overall effectiveness of a flagellin/poxvirus fusion protein vaccine may be

enhanced by inclusion of additional target antigen/flagellin fusion proteins.

The results of an increasing number of studies clearly demonstrate that flagellin is

a potent adjuvant when delivered intransally, intramuscularly, or intradermally

(11,20,48,52,53,62,69,70,75,98). A particular strength of flagellin is that its adjuvant

properties are not inhibited if the host has pre-existing immunity to flagellin (10).

Flagellin elicits robust antigen-specific humoral responses without promoting deleterious

antigen-specific IgE responses or serious adverse inflammation (47,48). Flagellin is

especially potent at promoting antigen-specific responses when the antigen is genetically

linked to flagellin (7,11,12,48,52,53,56,62,69,70,75,105). Our studies reinforce this

concept. Administration of the LF and FB fusion proteins decreased the required molar

doses, raised total antigen-specific titers, increased the number of mice that

seroconverted, and decreased variability among animals. Similar trends were seen by

Weimer et. al. in the context of a flagellin-based fusion protein vaccine against

Pseudomonas aeruginosa (105).

Another benefit of flagellin is that it possesses substantial plasticity as an antigen

carrier. Depending on the antigen, target sequences can be inserted at the amino or

carboxy termini as well as within the hypervariable region without loss of TLR5

signaling and adjuvant activity (Figures 1 and 14 and (7,10-

12,20,52,53,56,60,62,70,75,105)). Furthermore, these flagellin fusion proteins can be

produced in bacteria and purified in large amounts under conditions that are compatible

with current Good Manufacturing Practices (75).

79

The purpose of our studies with flagellin and poxvirus antigens was to “test the

limits” of flagellin as an adjuvant by using L1R and B5R, two antigens that are poorly

immunogenic in their recombinant forms. Our studies clearly demonstrate that there are

limitations on the carrier function of flagellin. Although TLR5 signaling activity was

preserved in all of the flagellin fusion proteins used in this study, in some instances (FL

and LFB; Figure 14) the immunogenicity of the antigen was lost. There are two possible

explanations for the lack of IgG that recognized the native antigen. In these fusion

proteins, there may have been a deviation from the normal conformation of L1R in FL or

LFB. This could be from changes in folding or the pattern of glycosylation. An

alternative explanation is that a decrease in accessibility of epitopes to B cell receptors.

Since this problem is antigen-specific, it is essential that for each new flagellin-antigen

fusion protein, multiple insertion sites on flagellin need to be evaluated to obtain a fusion

that results in retention of TLR5 signaling activity as well as the production of antibodies

with specificity for the native antigen.

The use of flagellin as a carrier for weakly immunogenic proteins may be limited

by the potential for some form of antigenic competition (Figures 17 and 18). The limited

competition did not appear to have a negative effect on a vaccine formulation containing

only two fusion proteins. However, a problem may arise if a vaccine contains more than

two flagellin fusion proteins. There are many vaccines, such as those targeting

Streptococcus pneumoniae and Haemophilus influenzae, which are multivalent to provide

protection against a broad range of clinical isolates. For these pathogens, flagellin fusion

proteins might be limited in their efficacy unless successful multi-antigen fusion proteins

or conjugates could be generated.

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While we were unable to generate a flagellin-pox antigen trifusion protein that

retained immunogenicity of all components, such fusions have been made against other

targets (48,75,105). Again, this is an antigen-driven limitation. Each future vaccine

strategy involving flagellin-antigen fusion proteins will require testing of multiple

insertion sites and insertion order when generating fusions involving 3 or more proteins.

In summary, flagellin greatly enhances protective humoral immune responses

against weakly immunogenic proteins. The most efficient way to use flagellin with the

poxvirus antigens L1R and B5R is in the form of single flagellin-antigen fusion proteins

that are administered intramuscularly.

81

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SCHOLASTIC VITAE

Kristen N. Delaney

Work Address Department of Microbiology and Immunology Wake Forest University School of Medicine Medical Center Boulevard Winston-Salem, NC 27517 Work Phone 336-716-4138 Personal Phone 336-671-7434 E-mail kdelaney@wfubmc.edu FAX 336-716-9928 Citizenship United States Education 2005-present Wake Forest University Ph.D. Candidate 2000-2004 Eastern Kentucky University in Richmond, KY Bachelor’s of Science in Biology with a concentration in

Molecular, Cellular, and Microbiology Research2005-present Pre-doctoral student Department of Microbiology and Immunology Wake Forest University School of Medicine Flagellin as an adjuvant for weakly immunogenic antigens (Advisor: Steven B. Mizel, Ph.D.) 2004-2005 Laboratory Technician in the laboratory of Kevin D. Sarge, Ph.D. University of Kentucky Department of Molecular and Cellular Biochemistry 2002-2004 Student Researcher in the laboratory of Pat Callie, Ph.D. Eastern Kentucky University Department of Biology Awards 2005-2006 Graduate fellowship Wake Forest University Graduate School 2006-2008 Training Grant fellowship Wake Forest University School of Medicine Department of Microbiology and Immunology

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Publications Delaney KN, Phipps JP, and Mizel SB. A recombinant flagellin-poxvirus protein vaccine

elicits protection against respiratory challenge with vaccinia virus in mice. (submitted for publication).

Abstracts Delaney KN, Phipps JP, and Mizel SB. A vaccine containing recombinant poxvirus antigens and flagellin promotes protective immunity against vaccinia virus. The American Association of Immunologists Annual Meeting. Seattle, WA. May 2009.

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