development of a multivalent recombinant protein vaccine

Development of a flagellin-based multivalent vaccine against Pseudomonas aeruginosa

by

Eric T. Weimer

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

Microbiology and Immunology

August 2009

Winston-Salem, NC

Approved by: Steven B. Mizel, Ph.D., Advisor __________________________ Examining Committee: Mark O. Lively, Ph.D., Chairman __________________________ Jason M. Grayson, Ph.D. __________________________ W. Edward Swords, Ph.D. __________________________ Rajender Deora, Ph.D. __________________________ Daniel J. Wozniak, Ph.D. __________________________

ACKNOWLEDGEMENTS

To my advisor, Dr. Mizel, I would like to thank you for all your time,

commitment, enthusiasm, and advice throughout my time in your lab. I would not be the

scientist I am today without your mentoring.

To my classmates: Kristen, Mary Jo, Cheraton, and Cynthia, I will miss all of you

and wish all of the success that you have worked so hard for. We have shared some of

the best moments. Kristen, you have been a terrific lab mate as well as a great friend.

My deepest thanks to all the members of the Department of Microbiology and

Immunology. It was an honor to be in a department that is so supportive of its students

and always looks out for their best interest. To Aaron Graff, James Phipps, and Dr. John

Bates, I want to thank you for all the help and advice. Lab would not have been the same

without you.

To Mom and Dad, I’m eternally gratefully for all your guidance and support. I

truly couldn’t have done this without you. To Shane, the best brother I could have asked

for, thank you for all your support and just being there when I needed you. Know there is

nothing you can’t achieve in this world if you want it bad enough.

To my love, Kristin, you have been the voice of reason through the storm. I can’t

say enough how much your love and support has meant to me. My weakness is your

strength and I appreciate your patience with me.

ii

Table of Contents

ACKNOWLEDGEMENTS................................................................................................ ii

LIST OF FIGURES ........................................................................................................... iv

LIST OF TABLES............................................................................................................. vi

ABBREVIATIONS .......................................................................................................... vii

ABSTRACT....................................................................................................................... ix

INTRODUCTION .............................................................................................................. 1

CHAPTER I: A Fusion Protein Containing OprF Epitope 8, OprI, and Type A and B Flagellins Promotes Enhanced Clearance of Nonmucoid Pseudomonas aeruginosa ...... 12

CHAPTER II: Immunization of young African green monkeys with OprF epitope 8-OprI-type A- and B-flagellin fusion proteins promotes the production of protective antibodies against nonmucoid Pseudomonas aeruginosa................................................. 49

DISCUSSION................................................................................................................... 69

REFERENCE LIST .......................................................................................................... 79

SCHOLASTIC VITAE................................................................................................... 101

iii

LIST OF FIGURES

Figure 1. Illustration of constructs used in this study …………………………...16

Figure 2. TLR5-specific signaling activity of P. aeruginosa type A and B flagellins and

OprF311-341–OprI Flagellins .…………………………………………..23

Figure 3. Immunization with OprF311-341–OprI–Flagellins promotes a potent antigen-

specific humoral response ...…………………………………………26

Figure 4. Generation of antigen-specific plasma cells and MBC by OprF311-341–OprI–

Flagellins immunization …………..……………………………………....29

Figure 5. OprF311-341–OprI–Flagellins immunization generates high affinity antigen-

specific IgG …………..……………………………………....32

Figure 6. Complement-activating activity of OprI, OprF, and type A and B flagellin-

specific IgG antibodies ….………………………………....…….35

Figure 7. Antibody-dependent complement (Comp)-mediated killing of P. aeruginosa by

OprF311-341–OprI–Flagellins-immunized mouse plasma ..........................................39

Figure 8. OprF311-341–OprI–Flagellins-immunized mice display enhanced rate of

clearance following pulmonary P. aeruginosa challenge ………...….…......44

Figure 9. OprF311-341–OprI–flagellin-immunized mice are protected against severe lung

pathology during pulmonary P. aeruginosa challenge ..........................................47

Figure 10. Intramuscular immunization of African green monkeys with OprF311-341-OprI-

Flagellins promotes a potent humoral immune response ..................55

Figure 11. OprF311-341-OprI-Flagellins elicit high-affinity antigen-specific IgG. ......58

iv

Figure 12. Complement activation and complement-mediated killing by OprI, OprF, and

type A and B-flagellin-specific IgG ......................................................61

Figure 13. Passive immunization of mice with immune monkey plasma enhances

clearance of nonmucoid P. aeruginosa ......................................................66

v

LIST OF TABLES

Table 1. Bacterial strains used in these studies ...........………………………..….14

Table 2. Complement-mediated killing of additional P. aeruginosa strains .….41

vi

ABBREVIATIONS CF cystic fibrosis

CFTR cystic fibrosis transmembrane conductance regulator

P. aeruginosa Pseudomonas aeruginosa

ABC ATP-Binding Cassette

Cl chloride

ENaC epithelium sodium channel

F phenylalanine

Na sodium

DNA deoxyribonucleic acid

ExoS, T, U, Y exotoxins S, T, U, and Y

AHL acyl homoserine lactones

C3 complement component 3

C5 complement component 5

LPS lipopolysaccharide

TLR toll-like receptor

TNF-α tumor necrosis factor alpha

IL-6 interleukin-6

NO nitric oxide

Opr outer membrane protein

IgG immunoglobulin G

PilA type IV pili protein

LD50 lethal dose 50

vii

DC dendritic cell

PRR pattern-recognition receptor

MHC major histocompatibility complex

ssRNA single-stranded ribonucleic acid

LB Luria-Bertaini broth

LBNS Luria-Bertaini broth lacking NaCl

PCR polymerase chain reaction

LAL limulus amebocyte lysate

ELISA enzyme-linked immunosorbent assay

NaSCN sodium thiocyanate

Avertin 2, 2, 2-tribromoethanol

ELISPOT enzyme-linked immunosorbent spot assay

FITC fluorescein isothiocyanate

H & E hematoxylin and eosin

MBC memory B cells

BM bone marrow

WT wild-type

Y. pestis Yersinia pestis

H. influenzae Haemophilus influenzae

S. aureus Staphylococcus aureus

S. pneumoniae Streptococcus pneumoniae

MMR Measles, mumbs, rubella vaccine

DTaP Diphtheria, tetanus, acellular pertussis toxin

viii

ABSTRACT

Although chronic Pseudomonas aeruginosa infection is the major cause of morbidity and

mortality in cystic fibrosis (CF) patients, there is no approved vaccine for human use

against P. aeruginosa. The goal of my research was to establish whether a multivalent

vaccine containing P. aeruginosa type A and B flagellins as well as the outer membrane

proteins OprF and OprI would promote enhanced clearance of P. aeruginosa. To test this

I utilized two animal models, mice and 4-6 month old African green monkeys.

Intramuscular immunization of mice with flagellins + OprI (separate), or OprI-Flagellins

fusion proteins generated significant anti-flagellin IgG responses. However, only OprI-

Flagellins fusion generated OprI-specific IgG. Although immunization of young African

green monkeys with OprF311-341-OprI-Flagellins promoted a high level of antigen-specific

IgG ten days post-boost, there was significant reduction in IgG three months later.

Immunization of mice and young African green monkeys with OprF311-341-OprI-

Flagellins elicited high affinity flagellins, OprI, and OprF-specific antibodies that

individually promoted extensive deposition of C3 on P. aeruginosa. Although these

antibodies exhibited potent antibody-dependent complement-mediated killing of

nonmucoid bacteria, they were significantly less effective with mucoid isolates. Mice

immunized with OprF311-341-OprI-Flagellins had a significantly lower bacterial burden

three days post-challenge and cleared the infection at a significantly faster rate than

OprF-OprI immunized mice. In addition, mice that were passively immunized with

OprF311-341-OprI-Flagellins monkey immune plasma had significantly less bacteria,

inflammation, and lung damage throughout the infection compared to control immunized

ix

mice. Based on my results, OprF311-341-OprI-A- and B-flagellin fusion proteins have

substantial potential as a vaccine against nonmucoid P. aeruginosa, which appears to be

the phenotype that initially colonizes CF patients.

x

INTRODUCTION Cystic fibrosis (CF) is a multisystem disease affecting the digestive system and

respiratory tract. The progressive lung disease is the major cause of morbidity and

mortality in CF patients. CF is an autosomal recessive disease caused by mutation in the

gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein

{1,2}. CFTR belongs to the ABC (ATP-Binding Cassette) family of proteins, a large

group of related proteins that share transmembrane transport functions. The protein

encodes six membrane-spanning regions, two intracellular nucleotide-binding folds, and

a highly charged “R” domain that contains phosphorylation motifs. Normally, CFTR

functions as a regulated chloride (Cl) ion transporter and to regulate epithelial sodium

channel (ENaC) {3}. More than 1,500 different mutations have been identified in the

CFTR protein and are divided into six different classes {4}. Class I mutations result in a

defect in protein production typically by nonsense, frameshift, or splice-site mutations.

Class II mutations are generally the result in defects in protein processing or transport.

The most common class II mutation is a deletion of phenylalanine (F) at position 508

referred to as ∆F508 {5,6}. The ∆F508 mutation causes the CFTR protein to misfold and

be retained within the Golgi {7,8}. Subsequent studies revealed that the ∆F508 CFTR is

found in extremely small quantities at the apical plasma membrane of epithelial cells and

transport very low or undetectable levels of Cl {8,9}. Thus, the ∆F508 mutation disrupts

CFTR function by two distinct mechanisms, by reducing surface protein levels and

reducing capacity to transport chloride ions. Class III mutations affect the regulation of

CFTR by preventing ATP hydrolysis at the nucleotide binding domains. Class IV

mutations result in a normal amount of CFTR at the membrane but reduced function and

1

class V mutations affect the synthesis of active CFTR. Unlike Class I, class VI mutations

produce functional CFTR but reduce the stability of the protein. Class I, II, III, and VI

mutations are generally associated with severe CF phenotypes. Class IV and V mutations

are typically associated with pancreatic exocrine sufficiency and milder disease {10}.

Defects in epithelial sodium (Na) and Cl transport, and accompanying

abnormalities in fluid secretion underlie many of the disease pathology observed in CF.

In CF patients, the lack of CFTR-mediated regulation of ENaC leads to abnormal sodium

transport while the defect in CFTR leads to abnormal chloride ion transport. The result

of this ion imbalance is the airway surface is depleted of water (i.e. thickened mucus) and

mucus plugging occurs in the upper respiratory tract {11,12}. Increased levels of

chloride ions in the respiratory tract of CF patients may inhibit the antimicrobial activity

of beta-defensins and neutrophils. In addition, increased fluid viscosity, impaired

mucociliary clearance, and dehydrated mucosal surfaces lead to the inability to clear

pathogens such as Haemophilus influenzae, Staphylococcus aureus, Burkholderia

cepacia, and eventually the Gram-negative, opportunistic pathogen Pseudomonas

aeruginosa {13}.

CF patients are particularly susceptible to P. aeruginosa infections due in part to

defects in normal respiratory tract function. The progressive mucus buildup within the

respiratory tract traps the bacteria in the hypoxic environment generated by increased

oxygen consumption by epithelial cells {14}. Within the low oxygen environment, P.

aeruginosa undergoes a distinct phenotypic change through accumulations of mutations

in mucA and the conversion to a mucoid phenotype {15-17}. The mucoid form of P.

aeruginosa is characterized by the over-production of the exopolysaccharide alginate and

2

loss of motility (i.e. loss of flagellin expression). There is a well-documented inverse

relationship between alginate and flagellin expression {18,19}. Alginate-overproducing

P. aeruginosa is rarely the initial infecting strain {20-25}. The appearance of mucoid P.

aeruginosa occurs over approximately a 10-year span and is a poor prognosis indicator in

CF patients {20,22,23,25}. Once infection has been established, neither the massive

infiltration of neutrophils nor the heavy use of antibiotics are capable of controlling the

bacteria. Recruited neutrophils consequently release elastase at concentrations that

overwhelm the protective capacity of lung and contribute to tissue destruction {26-28}.

As a result, large amounts of free DNA are released by degranulating neutrophils,

contributing to the increased viscosity of the airway mucus {15}. The accumulation of

free DNA acts as a scaffold for P. aeruginosa biofilm formation – a major virulence

factor of P. aeruginosa {29,30}. Over-production of alginate and eventual biofilm

development allows P. aeruginosa to evade the immune system and also increases

antibiotic resistance, making it extremely difficult to eradicate the infection {22,23}.

Chronic P. aeruginosa infection is the major contributor to respiratory failure in CF

patients.

Eventually, patients with CF succumb from respiratory failure because of a loss of

lung function. This results from the chronic inflammatory process triggered by persistent

P. aeruginosa infections {31-34}. Whole genome analysis of P. aeruginosa isolates

from CF patients revealed several virulence factors that are required to initiate acute

infections and are subsequently selected against during chronic infections. For example,

the exopolysaccharide Psl is important for attachment as well as biofilm initiation and

maintenance {35-37}. Many of these virulence factors allow P. aeruginosa to evade or

3

inhibit the immune response {38}. The type III secretion system is activated upon contact

with epithelial cells and is a major determinant of virulence {39}. P. aeruginosa secretes

four known effector proteins via type III secretion system: ExoS, ExoT, ExoU, and

ExoY. Secretion of the exotoxin S (ExoS) through the type III secretion system has been

shown to inhibit the activation of caspase-1 {40}. ExoT, possess an N-terminal GTPase

activating domain and a C-terminal adenosine diphosphate (ADP) ribosyltransferase

domain. ExoU is a phospholipase and ExoY is an adenylate cyclase {41,42}.

In contrast to invasive properties of type III secretion system, quorum sensing

regulates a complex circuit involving cell to cell signaling {43}. Quorum-sensing

molecules are typically acyl homoserine lactones (AHL), which are freely diffusible.

When a threshold AHL concentration is reached, AHL binds LasR/RhlR transcriptional

activators to induce expression of certain genes. P. aeruginosa predominately makes two

autoinducers: N-3-oxododecanoyl homoserine lactone and N-butyryl-L homoserine

lactone {44}. Activation of the quorum sensing signaling cascade promotes biofilm

formation and persistence in the lung {45}. In addition, quorum-sensing molecules can

directly modulate the immune response. Several investigators have shown that AHL

induces cyclooxygenase-2 and prostaglandin-E2, which inhibit the induction of adaptive

immune responses {44}. Outer membrane protein F (OprF) has also been shown to bind

interferon gamma and activate expression of quorum sensing virulence genes {46}.

Unlike quorum-sensing, the function of several virulence factors is inhibition of the

immune response.

Evasion of the complement cascade is mediated by binding and sequestering the

complement regulatory protein Factor H by bacterially expressed Tuf {47}. In addition,

4

P. aeruginosa secrete enzymes such as alkaline protease and elastase, which degrade

complement components and thus limit the role of complement in the clearance of early

pulmonary P. aeruginosa infections {48}. The critical role of complement in the

clearance of P. aeruginosa is evidenced by the observation that C3 and C5 KO mice were

unable to clear P. aeruginosa after challenge {49,50}. Also, P. aeruginosa express

lipopolysaccharide (LPS) variants that interfere with C3b deposition {51}.

The flagellum is an important virulence factor for P. aeruginosa, as non-

flagellated P. aeruginosa strains are less virulent {52}. P. aeruginosa strains that

initially colonize CF patients are generally flagella-positive, composed of ‘‘A’’ and/or

‘‘B’’ flagellin subtypes {53-55}. Both types of flagellin are crucial for establishing

infections in CF patients, as well as being involved in chemotaxis, motility, adhesion and

inflammation {56}. Type-A has a more variable molecular mass (45-52 kDa), while

type-B has an invariant molecular mass of approximately 50 kDa {57}. Both flagellins

are glycosylated {57,58}. Glycosylation of flagellin occurs in the domain outside the

region required for toll-like receptor-5 (TLR5) signaling {59}. Interaction with TLR5

induces the secretion of proinflammatory cytokines tumor necrosis factor alpha (TNF-α)

and interleukin-6 (IL-6) {60-62}. However, flagellin binding to TLR4/5 heteromeric

complexes is required for the induction of nitric oxide (NO) synthesis {62}. Also, P.

aeruginosa, Salmonella typhimurium, and Listeria monocytogenes flagellin has been

shown to interact with the intracellular cytoplasmic protein Ipaf {63-68}. Binding of

flagellin to Ipaf promotes activation of caspase-1, release of IL-1β, and subsequent cell

death in macrophages {69}. Delivery of flagellin to the cytoplasm and activation of Ipaf

of cells occurs via type III secretion system {40}. Flagellin, type III secretion system,

5

ExoS, LPS, alginate as well as other virulence factors play a key role in the establishing

and maintaining persistent P. aeruginosa infections in CF patients.

Initial efforts to develop a P. aeruginosa vaccine focused primarily on live

attenuated strains or components such as LPS, or alginate {70-79}. Several investigators

have utilized polysaccharide conjugate vaccines with carrier proteins keyhole limpet

hemocyanin, tetanus toxoid, or exotoxin A {70,74,79-81}. A live, attenuated vaccine

strain was employed by deletion of the aroA gene that is required for synthesis of

aromatic amino acids. This approach has been utilized to generate several other

attenuated strains, e.g. Salmonella species {82}. An attenuated aroA mutant of

Salmonella enterica serovar Typhimurium was used to express OprF-OprI as well as the

O-antigen from P. aeruginosa {72,83}. However, single aroA deletion mutants in S.

enterica retain significant virulence and thus make them unacceptable as human vaccines.

In contrast, P. aeruginosa aroA mutants are intrinsically less virulent and strains

expressing O-antigen have been evaluated in mice as a potential vaccine {84,85}. The

vaccine elicited high titers of O-antigen specific IgG that provide protection from

homologous but not heterologous challenge {84}.

Testing of LPS conjugate vaccines began in the late 1960s, when several

investigators evaluated a LPS vaccine in burn and CF patients. Although the vaccine

showed promise, it was ultimately stopped due to adverse side effects {86-92}.

Following modifications to the lipid A portion of LPS, an octavalent O-antigen

conjugated to exotoxin A was developed and tested in burn patients. In addition, the

vaccine was well tolerated and elicited high affinity antibodies that reduced the incidence

of infection in CF patients {74,93}. The results from this study were not able to obtain

6

regulatory approval due to lack of controls. A subsequent controlled study demonstrated

no significant difference between placebo and vaccine immunized CF patients {94}. As

indicated above, the major limitation of LPS vaccines has been toxicity and an inability

to provide protection against multiple P. aeruginosa serotypes {77,81,95,96}. In

contrast, the exopolysaccharide alginate is structurally less diverse than LPS. Alginate is

a linkage of β1-4 D-mannuronic acid and L-guluronic acid residues {97}. In humans and

mice, antibodies elicited by alginate immunization were mostly nonopsonic {78,98}.

Additional studies revealed the immunization with only the high molecular weight

fraction and at least 100µg of alginate elicited opsonic antibodies in a limited number of

healthy volunteers. However, the opsonic antibodies that were generated were not long-

lived, returning to background levels by 28 days post-boost {78,98}. Expression of

alginate is very low on nonmucoid strains of P. aeruginosa {99,100}. Given the low

expression level of alginate on initial infecting strains, investigators have focused on type

IV pili protein (PilA), type III secretion system protein, PcrV, ExoA, flagellin, and outer

membrane proteins I and F (OprI and OprF), as protective antigens. There is high level

of expression of flagellin, OprI, OprF, on nonmucoid P. aeruginosa as well as a high

degree of sequence conservation among CF isolates {72,101-117}. In deed, real-time

PCR using oprI-specific gene primers is often used to identify P. aeruginosa infection

{101,118,119}.

Immunization with the OprI antigen of P. aeruginosa and an appropriate adjuvant

elicited a protective response in mice that correlates with the titer of OprI-specific IgG

{103}. Importantly, Eckhardt et. al. {120} determined that the presence of OprI-specific

IgG that mediated complement activation as the best in vitro correlate of protection

7

compared to total IgG response and antibody affinity. In related studies, administration

of OprI-specific monoclonal antibodies mediated protection {104}. Four intramuscular

immunizations of healthy volunteers with 50µg of OprI absorbed on alum (aluminum

hydroxide) elicited elevated antibodies levels that remained high for 4 months post-

immunization {121,122}. In addition, an adenovirus expressing epitope 8 (amino acids

311-341) (Epi8) of OprF provided protection against acute P. aeruginosa infection

{116,117}. DNA vaccination with oprF elicited antibodies that were opsonic and

reduced the appearance of lesion during P. aeruginosa infection {113,123}. From the

success of these studies, several investigators began focusing on a fusion peptide

containing OprF and OprI as a potential vaccine candidate. Although large amounts of

this protein were required for an optimal response, immunization with an OprF-OprI

fusion protein resulted in a 95-fold increase in the lethal dose-50 (LD50) for mice. A

subsequent studies in burn, chronic obstructive pulmonary disease, and CF patients

revealed that 1mg of a OprF-OprI fusion protein was required to elicit a 3-fold increase in

IgG titers {110,124,125}. However, it is not known whether antibodies to OprF-OprI

mediate opsonization and killing of P. aeruginosa strains. In addition, investigators have

used type IV pili coupled to either tetanus toxoid or exotoxin A. Immunization of rabbits

with either conjugate vaccine elicited antibodies that reduced adherence and enhanced

clearance of P. aeruginosa following pulmonary infection {126,127}. In light of any

vaccine that leads to a substantial reduction in the incidence of infection, investigators to

use multivalent vaccines. For example, Saha et. al. {106} used DNA vaccination with

PcrV, PilA, OprF, and OprI to demonstrate the effectiveness of multivalent immunization

at enhancing the clearance of P. aeruginosa. A phase III clinical trial of P. aeruginosa

8

flagellins in CF patients demonstrated that the vaccine was well tolerated and caused a

30% reduction in the incidence of infection {128}. Several investigators have shown that

passive immunization with anti-flagellin IgG antibodies mediates protection from P.

aeruginosa infection {56,129,130}.

The use of adjuvants has been shown to enhance adaptive immune responses.

Currently, the only FDA-approved adjuvant is alum. Until recently, the adjuvant effect

of alum was not understood. Kool and colleagues {131,132} demonstrated that alum

particles activate inflammatory dendritic cell (DC) and promote release of uric acid that

elicits an inflammatory response. Alum has been shown to activate the cytoplasmic

NOD-like receptor called NLRP3 or Nalp3, which associates with ASC and caspase-1 to

form a protein complex called inflammasome. The activation of the inflammasome leads

to the secretion of several proinflammatory cytokines such as IL-1β and IL-18

{132,133}. NOD receptors are cytoplasmic form of pattern-recognition receptors (PRR).

TLR are membrane bound PRR. PRR on DC function to alert the immune system to

“danger”. PRR are a collection of receptors that recognize pathogen-associated

molecular patterns (PAMPs) on a diverse group of pathogens. Surface and endosomal

recognition of bacterial, viral, and fungal pathogens occurs through TLR. Upon TLR

stimulation, DC undergo a maturation process leading to increased expression of co-

stimulatory molecules CD40, CD80, and CD86 as well as the secretion of cytokines such

as IL-2, IL-12, and type I interferon. DC downregulate phagocytosis and increase

antigen processing and presentation of antigen peptides in the context of MHC class II or

I. DC are potent activators of CD8+ and CD4+ T cells, providing a key link between

innate and adaptive immunity. TLR agonists function not only as adjuvants but also as

9

antigens. TLR agonists fused to proteins are more effective than the separate proteins

{115,134-138}. CpG (TLR9), ssRNA (TLR7 & TLR8), LPS (TLR4), poly I:C (TLR3)

and flagellin (TLR5) have all been shown to function as adjuvants {61,134,136,138-

140,140-148}.

The conserved regions of flagellin are responsible for binding directly to a leucine

rich repeat on the extracellular domain of TLR5 {149}. Schirm et al. {59} demonstrated

that amino acids 88-97 were required for type A flagellin signaling via TLR5. Signaling

via TLR5 is dependent on IRAK-1 {150} and MyD88 {151}. Honko and Mizel {60}

demonstrated the in vivo stimulatory effects of flagellins from P. aeruginosa and

Salmonella. Intratracheal (i.t.) administration of flagellin induced maximal neutrophilic

infiltration within 12 hours. Consistent with the neutrophilic infiltration, cytokines TNF-

α and IL-6 as well as chemokines macrophage inflammatory protein-1 alpha and

keratinocyte-derived chemokine were upregulated four-fold in flagellin treated mice

{60}.

Flagellin has been proven to be an effective adjuvant in mice {60,61,134-

136,143,152,153} as well as cynomolgus and African green monkeys {61,154}. Arnon

and colleagues first demonstrated that flagellin could function as an adjuvant by

enhancing the influenza response by intranasal immunization with recombinant flagellin

expressing influenza peptides. Importantly, prior exposure to flagellin does not inhibit

the effectiveness of flagellin {143,152,155,156}. A recent study, demonstrated the

adjuvant effect of flagellin is mediated by tlr5+CD11c+ cells {157}. Flagellin is an

effective adjuvant for CD4+ T cells in vivo and in vitro {135,140,144}. The efficacy of

flagellin to promote a protective response has been demonstrated for bacteria and viral

10

pathogens when used with recombinant proteins as antigens {134-136,145,155}. Several

investigators have reported protection against P. aeruginosa using flagellin as an antigen

and adjuvant {115,128,145}.

Although experimental P. aeruginosa vaccines have shown promise in initial

preclinical and clinical trials, none have achieved the level of response required for

protection against P. aeruginosa in CF patients. A limitation of the previous vaccines

was the age of the CF patients when they were immunized. Given the pathophysiologic

events associated with CF in combination with the pathogenic mechanisms associated

with P. aeruginosa infection, I hypothesize that vaccine efficacy will be greatly enhanced

if administered to CF patients at a very young age. In addition, although the magnitude

of the humoral response was determined in previous studies, the functional quality of the

induced antibodies was not addressed, e.g., antibody affinity and the ability to activate

complement or promote opsonization {115,120,121,158,159}.

After critical review of the literature, I have identified several features that are

important for an effective P. aeruginosa vaccine: the presence of a potent adjuvant, the

ability to induce high titer antigen-specific IgG that exhibits a high degree of functional

activity (for example, complement activation), multivalency, and the ability to induce a

robust memory response. To that end, I generated a multivalent vaccine containing type

A and B-flagellin, OprF, and OprI and have evaluated its immunogenicity and protective

potential in mice as well as 4-6 month old African green monkeys.

11

Chapter I

A Fusion Protein Containing OprF Epitope 8, OprI, and Type A and B Flagellins

Promotes Enhanced Clearance of Nonmucoid Pseudomonas aeruginosa

E.T. Weimer, H. Lu, N. D. Kock, D.J. Wozniak, S.B. Mizel

The following manuscript was published in Infection and Immunity, volume 77, issue 6, pages 2355-2366, 2009 and is reprinted with permission. Stylistic variations are due to the requirement of the journal. E.T. Weimer performed the experiments and prepared the manuscript. Dr. Steven B. Mizel acted in an advisory and editorial capacity. Copyright © American Society for Microbiology 2009.

12

MATERIALS AND METHODS

Strains and plasmids. Bacterial strains and plasmids used in this study are

described in Table 1. Escherichia coli cultures were maintained at 37ºC in Luria-Bertaini

(LB; 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) broth, while P. aeruginosa was

cultured in LB broth lacking NaCl (LBNS; 10 g/L tryptone, 5 g/L yeast extract). Solid

media were prepared by adding 1.0-1.5% select agar (Gibco-BRL). Plasmids in E. coli

were selected using media supplemented with antibiotics at the following concentrations:

carbenicillin (Cb 100 µg ml-1), gentamicin (Gm 10 µg ml-1). Plasmids in P. aeruginosa

were selected on media containing Cb (300 µg ml-1), Gm (100 µg ml-1), and irgasan (Irg

25 µg ml-1). E. coli strain JM109 was used for all cloning procedures while E. coli SM10

was used to transfer plasmids into P. aeruginosa by bi-parental mating {160}. The P.

aeruginosa strains used were PAO1 and its derivatives WFPA850, WFPA852,

WFPA854, WFPA860, WFPA862, WFPA864, and WFPA866. Vectors pEX18Gm,

pEX18Ap or derivatives were used for cloning and gene replacements (Table 1).

Construction of non-polar deletion mutations in fliC, oprF, and oprI. To

engineer unmarked, non-polar deletion mutations in fliC, oprF, and oprI, we utilized a

previously described method {161}. Internal fragments of coding sequences within each

gene were deleted using a modified PCR technique termed splicing by overlap extension

{162}. In this assay, four gene-specific primers were employed in three separate PCR

reactions to generate DNA fragments with a defined in-frame deletion of coding

sequences within the fliC,

13

Table 1. Bacterial Strains used in this study Strain Description Source PAK WT {19} PAO1 WT {19}

WFPA850 In-frame fliC deletion in PAO1

This study

WFPA852 In-frame oprF deletion in PAO1

This study

WFPA854 In-frame oprI deletion in PAO1

This study

WFPA860 In-frame fliC and oprI deletions in PAO1

This study

WFPA862 In-frame fliC and oprF deletions in PAO1

This study

WFPA864 In-frame fliC, oprF, and oprI deletions in PAO1

This study

WFPA866 In-frame oprF and oprI deletions in PAO1

This study

T69833 Mucoid CF isolate Wozniak unpublished 1286 Nonmucoid CF isolate Wozniak unpublished

PDO300M Mucoid PAO1 {163} PDO300NM Nonmucoid PDO300

deficient in alginate production

{163}

14

oprF, or oprI genes. The primers were also designed such that the final amplicon,

harboring the specified deletion allele harbored restriction sites to allow direct cloning

into pEX18Ap or pEX18Gm resulting in plasmid pHL150 (∆fliC), pHL153 (∆oprF), or

pHL155 (∆oprI). The mutant alleles were introduced into the PAO1 chromosome as

outlined {164}. The merodiploids were resolved by growing on sucrose-containing

media and introduction of the deletion allele, which was verified by PCR.

Recombinant proteins. DNA encoding full-length type A-flagellin of P.

aeruginosa strain PAK and DNA encoding full-length type B-flagellin of strain PAO1

were each amplified by PCR and ligated into pET29a. DNA encoding the mature OprI

antigen of P. aeruginosa strain PAO1 (amino acids 21-83) was amplified by PCR and

ligated into pET29a or to the 5’ end of type A and B-flagellin genes in pET29a

generating constructs that encode OprI-type A Flagellin and OprI-type B flagellin. DNA

encoding the OprF epitope 8 (amino acids 311-341) of P. aeruginosa strain PAO1 was

amplified by PCR and ligated into pET29a or to the 5’ end of oprI-A-flagellin and oprI-

B-flagellin. The structure of each of the final proteins are presented in diagrammatic

form in Figure 1.

All expressed proteins were purified by metal ion affinity chromatography as

previously described {61,165}. Acrodisc Q membranes were used to deplete endotoxin

and nucleic acids. Endotoxin levels were <10pg/µg for all of the proteins (as detected by

QCL-1000 chromogenic Limulus amebocyte lysate (LAL) test kit, Cambrex Corporation

(East Rutherfrod, NJ)).

15

Figure 1. Illustration of the constructs used in this study.

16

ELISA for TNF-α and antigen-specific IgG. TNF-α levels in cultures of RAW 424

(TLR5+) or RAW 264.7 (TLR5-) cells were measured using a commercial ELISA kit

(OptiEIA ELISA, Becton Dickinson) according to the manufacturer's instructions. Data

represent three independent experiments with triplicate samples in each experiment.

Titers of antigen-specific IgG were measured using MaxiSorb plates coated with

100µl of antigen (A-flagellin, B-flagellin, OprI, or OprF) at 10 µg/ml in sterile PBS. The

plates were incubated overnight at 4°C and then blocked with 10% newborn calf serum in

PBS. Plasma samples (in triplicate) were added, and the plates incubated overnight at

4°C, followed by secondary anti-Ig antibodies (Roche Diagnostics) for 2h at room

temperature. Peroxidase activity was detected with 3,3’,5,5’-tetramethylbenzidine

(TMB) liquid substrate system (Sigma-Aldrich) and stopped with 2 N H2SO4. Endpoint

dilution titers were defined as the inverse of the lowest dilution that resulted in an

absorbance value (at 450 nm) of 0.1 over that of naive plasma. Groups of at least 7 mice

were used. To determine relative antibody affinities, the ELISA assay was conducted as

described above with the addition of a 15 min incubation with sodium thiocyanate

(NaSCN) (Sigma) solution as described previously {165,166}.

Mice. 6-8 week old BALB/c and DBA/2 mice were purchased from Charles

River Laboratories. All animals were maintained under pathogen-free conditions. All

research performed on mice in this study complied with federal and institutional

guidelines set forth by the Wake Forest University Animal Care and Use Committee.

Intramuscular (i.m.) immunization of mice. Groups of 7 mice were anesthetized

with Avertin (2, 2, 2-tribromoethanol [Sigma]; tert-amyl alcohol [Fisher]) by

intraperitoneal injection. Small volumes (20µL total) containing antigen and adjuvant in

18

PBS were injected using a 29 ½ G needle into the right calf of mice. Mice were boosted

at 4 weeks via the same route, and bled two weeks later. Plasma was prepared and stored

at -70°C until analysis.

ELISPOT assay. The frequency of antigen-specific plasma cells was determined

using limiting dilution analysis as previously described {167}. Briefly, Immobulin-P

high-affinity protein binding ELISPOT plates (Millipore) were coated with 100µL of A-

flagellin, B-flagellin, OprI, or OprF (10µg/mL) in sterile PBS. Bone marrow and spleen

were collected 45 days post boost, single cell suspensions were prepared, and dilutions of

the cells (5x105/well) were added to the antigen-coated wells. Plates were then incubated

at 37°C for 5 hours, washed, and probed with goat anti-mouse (4°C overnight). Plates

were developed using HRP-Avidin D diluted 1:1000 (Southern Biotechnology) and 3-

amino-9-ethylcardbazole (AEC) and dried overnight. Spots were enumerated using a

dissecting microscope. Only wells that contained ≥ 4 spots were counted for analysis.

Total spleen cell plasma cell numbers were calculated by multiplying the number of cells

in the spleen by the number of spots per million spleen cells. Total bone marrow plasma

cell numbers were calculated in the same manner with an additional multiplication by 7.9

to compensate for total bone marrow {168}.

To determine the frequency of antigen-specific memory B cells, the bone marrow

and spleen cells were incubated in vitro for 5 days in the presence of 1µg/mL OprF311-341-

OprI-Flagellins and then plated as described above. The number of memory B cells was

determined by subtracting the number of plasma cells from the 5 hr incubation from the

total number of plasma cells after the 5-day culture. Results are shown for two

independent experiments.

19

Antigen-specific IgG binding to P. aeruginosa. P. aeruginosa strains were

incubated with heat-inactivated control or immune mouse plasma for 1 h at 4°C prior to

staining with AlexaFlour647-conjugated anti-mouse IgG (Invitrogen) for 1 hr at 4°C.

Data are representative of two experiments with triplicate samples in each experiment.

Antigen-specific IgG-mediated complement activation. Control and immune

mouse plasma were diluted 1:10 and heat-inactivated at 56°C for 1 hour prior to use. P.

aeruginosa strains were grown in LANS broth to an OD600 of 0.5 (~108 cfu/mL), washed

2 times with sterile PBS, and then incubated with mouse plasma for 1 hr. The bacteria

were then washed and incubated for 1 hr at 37°C with 5% rabbit serum (Innovative

Research) as a source of complement. Finally, the bacteria were stained with goat anti-

rabbit C3-FITC (MP Biomedical). Flow cytometric analysis was performed using a BD

FACSCaliber and data analyzed with FloJo software (Tree Star, Inc., Ashland, OR).

Representative histograms of three experiments are shown. Complement-mediated

killing was performed as described above with the exception that the bacteria were

incubated for 4 hr with rabbit serum. A time-course experiment revealed minimal killing

at 1 hour with rabbit serum (data not shown). Percent bacteria killed was quantitated by

(number of input bacteria – number of recovered bacteria) / (number of input bacteria) X

100.

Respiratory challenge with agar-embedded P. aeruginosa. P. aeruginosa strains

were grown in LBNS to 108 cfu/mL. One-part bacteria were added to 9 parts warm

(52°C) 1.5% trypticase soy agar. After five minutes, the agar:bacteria mixture was

injected into rapidly spinning warm heavy mineral oil using a 22 gauge needle. The

20

suspension was then mixed for 6 min. The agar beads were then cooled on ice for 20 min

and washed 3 times with sterile PBS. The final volume was adjusted to approximately

5mL. To determine the number of cfu/mL, the agar:bacteria beads were homogenized

and bead size determined by comparison to 100-150µm chromatography beads. Mice (6-

7 per group) were anesthetized with Avertin by intraperitoneal injection and then 50µL of

agar-embedded bacteria were instilled intra-tracheally using a sterile gel-loading tip.

Histology. Lungs were harvested and transferred to 10% formalin for 24 h. The

tissue then was trimmed, embedded in paraffin, cut at 4 µm, and stained with

hematoxylin and eosin by routine methods. For histological examination, groups of four

mice were used for each condition. Slides were scored as blind groups on an increasing

severity index that incorporates values for consolidation, bronchiolar and vascular

degenerative changes, and edema (range for each factor: 0 to 4). Total inflammation

score was calculated by the sum of all categories. Representative images are shown from

4 animals/group with 3 sections by animal.

Statistical analyses. Statistical analysis was performed using SigmaStat 3.10

(Systat Software, Inc., Point Richmond, CA). For normally distributed data sets,

significance was determined using the Student's t-test. The significance of data sets,

which were not normally distributed, or were of unequal variance were determined using

the Mann–Whitney rank sum test. Where applicable a two-way ANOVA test was

applied. P values of less than 0.05 were considered significant.

21

RESULTS

TLR5-specific signaling activity of P. aeruginosa A- and B-flagellin and OprF311-341-

OprI-Flagellins. In order to generate antigens with flagellin as the adjuvants, we

generated several constructs as shown in Figure 1. In view of the insertion of the OprF

and OprI sequences at the N-terminus of flagellin, it was important to determine if this

addition would have a negative impact on the ability of each flagellin, i.e., type A or B, to

signal via TLR5. To test the ability of P. aeruginosa flagellins either alone or as part of a

tri-fusion with OprF and OprI (see Fig 1), to signal via TLR5, RAW 424 (TLR5+) or

RAW 264.7 (TLR5-) cells were incubated with 1pM-1nM of each protein and production

of TNF-α was assessed. Stimulation of RAW 424 cells with P. aeruginosa type A or B-

flagellin, OprI-type A or B-flagellin, or OprF311-341-OprI-type A or B-flagellin resulted in

a concentration-dependent increase in TNF-α (Fig 2A-C). In contrast, none of these

proteins induced TNF-α production in cultures of TLR5- RAW 264.7 cells. Consistent

with previous results with the P. aeruginosa flagellins, the half-maximal stimulation

occurred at 16 pM for A-flagellin and 40 pM for B-flagellin {169}. There was no

significant difference in the half-maximal stimulation between type A or B flagellin,

OprI-type A or B-flagellins, or OprF311-341-OprI-type A or B-flagellins. Thus, the

presence of OprF-OprI at the N-terminus of type A or B-flagellin does not alter

recognition and signaling via TLR5.

Immunization with OprF311-341-OprI-Flagellins promotes a potent antigen-specific

humoral response. To assess the ability of OprF311-341-OprI-A and B-

22

Figure 2. TLR5-specific signaling activity of P. aeruginosa A- and B-flagellin and

OprF311-341-OprI-Flagellins. RAW 424 (TLR5+) and RAW 264.7 (TLR5-) were

stimulated with 10-9 to 10-12 M of protein. At 4 hours post stimulation, supernatants were

harvested and the amount of TNF-α was determined by ELISA. A) A-flagellin and B-

flagellin B) OprI-Flagellins fusion C) OprF311-341-OprI-Flagellins fusion. Data represent

the results of three independent experiments done in triplicate.

23

flagellin to promote an antigen-specific humoral response, groups of 7 BALB/c or

DBA/2 mice were immunized with 5µg of each type A and B-flagellin + 10µg OprI, 5µg

OprI-type A and B-flagellin fusion proteins, or 5µg OprF311-341-OprI-type A and B-

flagellin fusion proteins. Prior experiments established that immunization of BALB/c

mice with 5µg OprI-Flagellins generated a maximal IgG response to flagellin and OprI

(data not shown). Control mice received either OprI or OprF311-341-OprI at equivalent

molar doses. DBA/2 mice were used because previous studies identified DBA/2 mice as

more susceptible to P. aeruginosa infection than BALB/c and C57BL/6 mice {32,34}.

Four weeks later, mice were boosted in an identical manner. Two-weeks after the boost

the mice were bled and plasma was prepared for analysis of circulating antigen-specific

IgG. Mice immunized with OprI-Flagellins or OprF311-341-OprI-Flagellins exhibited a

robust OprI-specific IgG response (Fig 3). In contrast, there was no significant OprI-

specific IgG in mice given only OprI or type A and B-flagellin + OprI. In all cases,

flagellin-specific responses were extremely robust. Mice immunized with OprF311-341-

OprI-A- and B-flagellin exhibited a high-level of OprF-specific IgG as well as flagellin

and OprI-specific IgG.

In addition to determining the titers of antigen-specific IgG following

immunization with OprF311-341-OprI-A- and B-flagellin, we also evaluated IgG isotypes

and IgE. Plasma was prepared from immune mice as described above and antigen-

specific IgG subclasses and IgE were determined by ELISA. Immunization of mice with

OprF311-341-OprI-Flagellins did not elicit any detectable antigen-specific IgE (data not

shown). This finding is consistent with our prior

25

Figure 3. Immunization with OprF311-341-OprI-Flagellins promotes a potent

antigen-specific humoral response. BALB/c or DBA/2 mice were immunized

intramuscularly with 5µg of A-flagellin and B-flagellin + 10µg OprI (white bars), 5µg

OprI-A-flagellin + 5µg OprI-B-flagellin (grey bars), or 5µg OprF311-341-OprI-A-flagellin

+ 5µg OprF311-341-OprI-B-flagellin (black bars). At 4 wks post-immunization, animals

were boosted and 2 wks post-boost blood was collected and antigen-specific total IgG

was determined by ELISA. Data represent at least 7 mice per group in triplicate. * =

P<0.05, using a Mann–Whitney rank sum test.

26

work demonstrating that flagellin does not promote antigen-specific IgE responses {61}.

Although high titers of antigen-specific IgG2a were induced, the overall response to

OprI-A- and B-flagellins or OprF311-341-OprI-A- and B-flagellin was biased towards IgG1

(data not shown). This finding is consistent with our prior work on flagellin as an

adjuvant in a Y. pestis vaccine {61}.

Generation of antigen-specific plasma and memory B cells (MBC) in response to

OprF311-341-OprI-A- and B-flagellin. In view of the robust antigen-specific IgG

response, we evaluated the frequency of antigen-specific plasma and memory B cells

generated in response to OprF311-341-OprI-A- and B-flagellin. Mice were immunized with

5µg of OprF311-341-OprI-A- and B-flagellin as described above and 45 days post-boost,

bone marrow (BM) and spleen were harvested and the frequency of antigen-specific

plasma and memory B cells were determined by ELISPOT. Antigen-specific plasma

cells were determined following 5 hr incubation. Eighty-five percent of antigen-specific

plasma cells were found in the BM and 15% in the spleen. Consistent with the IgG titer

data (Fig 3), there were more plasma cells for type A- and B-flagellin (~200/106 BM

cells) than OprI (42) and OprF (30) (Fig 4A). No plasma cells were detected in wells that

contained cells from non-immune mice. Although significantly more antigen-specific

plasma cells were found in the bone marrow, a substantial number of plasma cells

remained in the spleen (Fig 4B). The retention of antigen-specific plasma cells in the

spleen correlated with the immunogenicity of each antigen.

28

Figure 4. Generation of antigen-specific plasma and memory B cells by OprF311-341-

OprI-Flagellins immunization. DBA/2 mice were immunized intramuscularly with 5µg

of OprF311-341-OprI-Flagellins. Bone marrow and spleens were harvested 40-days post-

boost and analyzed for antigen-specific plasma and memory B cells by ELISPOT. (A)

Frequency of antigen-specific plasma and MBC cells. (B) Total number of plasma and

MBC cells. Results are the average of 2 independent experiments on 5 mice.

29

In contrast to plasma cells, the generation of MBC was equivalent across flagellins and

OprI (Fig 4A). The fewer number of OprF-specific memory B cells (108 MBC/106 cells)

was not unexpected given the presence of only a single epitope. Nonetheless, our results

clearly establish that OprF311-341-OprI-A- and B-flagellin elicits not only a significant

numbers of plasma cells, but also a substantial pool of memory B cells.

OprF311-341-OprI-Flagellin immunization generates high-affinity antigen-specific IgG.

Since antigen affinity plays a critical role in the functional activity of an antibody, we

evaluated the relative affinity of the IgG generated following immunization with OprF311-

341-OprI-A- and B-flagellin. The relative affinity of antibodies can be assessed in an

ELISA by determining the concentration of sodium thiocyanate required to reduce

antibody binding by 50%. As shown in Fig 5, immunization with OprI-Flagellins or

OprF311-341-OprI-Flagellins generated IgG with equivalent relative affinities for the three

antigens. For comparative purposes, a flagellin + Y. pestis F1 antigen vaccine generated

F1-specific IgG requiring 3M sodium thiocyanate for 50% reduction in binding {165}.

Given the observation that these antibodies provide complete protection against

respiratory challenge with Y. pestis {61,154}, we have defined high affinity IgG as those

antibodies requiring 2-3 M sodium thiocyanate for 50% reduction in antigen binding.

OprF311-341-OprI-A-and B-flagellin immune plasma had an average relative IgG affinity

approaching 3 M for flagellin, OprI, and OprF (Fig 5). Thus,

31

Figure 5. OprF311-341-OprI-Flagellins immunization generates high-affinity antigen-

specific IgG. Plasma samples from mice that received OprI-Flagellins or OprF-OprI-

Flagellins were used to determine relative antibody affinity for A-flagellin, B-flagellin,

OprI, and OprF. Antigen-specific IgG affinity was determined by ELISA using dilutions

of sodium thiocyanate (NaSCN). Data are presented as molar concentration of NaSCN

required to reduce absorbance 50%. Samples from the same mice were used in the

experiments presented in Figures 2 and 3. There were 7 mice per group with each sample

done in triplicate.

32

the data are consistent with the conclusion that OprF311-341-OprI-A- and B-flagellin elicits

high-affinity antigen-specific IgG.

Complement activating activity of antibodies specific for OprI, OprF, and type A and

B-flagellins. To assess the functional activity of each of the antigen-specific IgG, it was

first necessary to generate P. aeruginosa mutants lacking one or more of these antigens

(see Table 1 and Material and Methods). P. aeruginosa type-B-flagellin expressing strain

PAO1 was used as the genetic background for the mutants. Each mutant exhibited

growth kinetics that were similar to that of the wild-type strain (data not shown). P.

aeruginosa strains were incubated with immune or control mouse plasma at 4°C for 1

hour and then stained for the presence of IgG. As shown in Fig 6A, wild-type P.

aeruginosa bound significant amounts of IgG specific for flagellin, OprI, and OprF.

Furthermore, mutants positive for only flagellin, OprF, or OprI also bound high levels of

IgG. Experiments using a type-A-flagellin expressing strain, PAK, demonstrated similar

results (data not shown). These results demonstrate that the antibodies generated against

the recombinant fusion protein recognize these antigens in their cell-associated forms.

This is particularly important in the case of OprF, since only a single epitope was present

in the OprF311-341-OprI-flagellin fusion proteins.

Having established the ability of the individual populations of IgG to recognize

the cell-associated antigens, we next evaluated the potential of these antibodies to

activate complement. Previous work has clearly established the

34

Figure 6. Complement activating activity of OprI, OprF, and type A and B-

flagellin-specific IgG. Plasma samples from OprF311-341-OprI-Flagellins were incubated

with P. aeruginosa and IgG binding and C3 deposition was determined by flow

cytometry. The antigens expressed by each P. aeruginosa strain are shown. (A)

Antigen-specific IgG binding to P. aeruginosa. Left column – Plasma samples from

OprI-Flagellins immunized mice. Filled regions refer to control strain WFPA860

(∆fliC∆oprI) which lacks flagellin and OprI. Right column - Plasma samples from OprF-

OprI-Flagellins immunized mice. WT, wild-type; B-Flagellin+, fliC+∆oprI∆oprF;

OprI+, oprI+∆fliC∆oprF; OprF+, oprF+∆fliC∆oprI. Filled regions refer to control

WFPA864 (∆fliC∆oprI∆oprF) strain lacking all three antigens. (B) C3 deposition on P.

aeruginosa strains. (C) Percent C3 positive of the data shown in B. *=P<0.05, ** =

P<0.001, compared to WFPA860 (∆fliC∆oprI) (top) or WFPA864 (∆fliC∆oprI∆oprF)

(bottom). Statistics were performed using Student’s t-test. Data are from 2 independent

experiments performed in triplicate.

35

importance of the complement system in the clearance of P. aeruginosa {49-51,120}. To

assess the ability of antibodies specific for OprF, OprI, type A and B-flagellin IgG to

activate complement, we measured the extent of IgG-dependent C3 deposition on P.

aeruginosa. The various P. aeruginosa strains were incubated with a 1:10 dilution of

heat-inactivated immune mouse plasma for 1 hour, and then 5% rabbit complement was

added for an additional hour. The bacteria were then stained with FITC-labelled C3-

specific antibody and the extent of C3-deposition was determined by flow cytometry. A

time course revealed that 1-hour incubation with serum was optimal for C3 deposition

and yielded minimal cell death (data not shown). As a control, we used a P. aeruginosa

strain lacking flagellin, OprI, and OprF. OprI-A- and B-flagellin or OprF311-341-OprI-A-

and B-flagellin immune plasma promoted significant C3 deposition on the surface of

wild-type P. aeruginosa (Fig 6A). By using mutants that lack one or more of the eliciting

antigens, we found that IgG with specificity for each of the eliciting antigens promoted

robust C3 deposition (Fig 6B-C). When all three antigens were present, there was a

synergistic increase in the level of C3 deposition.

These results indicate that OprF311-341-OprI-A- and B-flagellin immunization

generated antigen-specific IgG that exhibited a high-degree of functional activity and that

the combination of flagellins, OprI, and OprF-specific IgG triggered the highest level of

C3 deposition.

Antibody-dependent, complement-mediated killing of P. aeruginosa by OprF311-341-

OprI-A- and B-flagellin immune plasma. In view of the robust ability of OprF, OprI,

and type A and B-flagellin-specific IgG to promote C3 deposition, we next examined the

37

ability of those antibodies to promote complement-mediated killing of P. aeruginosa.

Bacteria were incubated with heat-inactivated immune plasma for 1 hr and then 5%

rabbit complement was added for an additional 4 hr at 37°C. It is important to note that

like wild-type bacteria, the P. aeruginosa mutants were not susceptible to significant non-

specific killing by normal serum (data not shown). Approximately 90% of wild-type,

nonmuciod P. aeruginosa (PAO1, PAK, and 1286) as well as strains expressing B-

flagellin, OprI, or OprF were susceptible to antibody-dependent complement mediated

killing (Fig 7 and Table 2). In contrast, only 18% of mucoid P. aeruginosa (T68933 and

PD0300M) were susceptible to killing (Table 2). This result is not unexpected, given the

presence of a large amount of alginate exopolysaccharide in the mucoid strains that

would likely mask OprI and OprF. In support of this conclusion, we found that a strain

of PAO1 (PDO300NM) deficient in alginate production (and thus nonmucoid) was quite

sensitive to killing (Table 2). In addition, the generally applicable inverse relationship

between flagella and alginate expression {19} would also limit the effectiveness of the

flagellin-specific IgG. The antigen-dependence of the killing was evidenced by the very

low level of killing with bacteria lacking all three of the eliciting antigens. When the

source of complement was heat-inactivated, only background levels of killing were

observed. Taken together, these findings clearly demonstrate that the antibodies

generated in response to OprF311-341-OprI-A- and B-flagellin exhibit potent antigen

binding, complement activating activity, and killing of nonmucoid, but not mucoid P.

aeruginosa.

38

Figure 7. Antibody-dependent complement-mediated killing P. aeruginosa by

OprF311-341-OprI-Flagellins immunized mouse plasma. Plasma samples from OprF311-

341-OprI-Flagellins immunized mice were diluted 1:10 and heat-inactivated at 56°C for 1

hr. Samples were then supplemented with 5% rabbit complement for 4 hr at 37°C. Data

are from 4 samples over two experiments, each sample done in duplicate. * P= <0.05,

using Student’s t-test.

39

Table 2. Complement-mediated killing of additional P. aeruginosa strains

Strain Percent Killed

PAK (WT) 86.5 ±0.2%

1286 (nonmucoid CF isolate) 84.3 ±0.8%

PD0300M (mucoid PAO1) 15.2 ±1%

PD0300NM (alginate deficient PDO300) 83.4±0.4%

T68933 (mucoid CF isolate) 24.8 ±0.3%

41

Enhanced clearance of P. aeruginosa in OprF311-341-OprI-Flagellins immunized mice.

Nonmucoid P. aeruginosa does not cause a chronic infection in healthy mice as it does in

CF patients. If large doses of bacteria are used, the mice quickly succumb to bacteremia

(Weimer, Wozniak, and Mizel, unpublished observations). With small doses, the mice

rapidly clear the bacteria. In view of the lack of a suitable animal model that closely

mimics the situation in CF patients, i.e., chronic infection, investigators have evaluated

agar-embedded mucoid P. aeruginosa as a way to infect mice such that rapid septic

shock is avoided and the time of infection is lengthened {34,170}. For example,

Stevenson and colleagues {32,34} used the agar bead model to demonstrate that DBA/2

mice were more susceptible to mucoid P. aeruginosa than were BALB/c or C57BL/6

mice. However, since the initial P. aeruginosa infection in CF patients is mediated by

nonmucoid strains {22,23,25}, we felt it was more appropriate to use nonmucoid bacteria

in the agar bead model. Preliminary results revealed that unimmunized mice did not

succumb when infected intra-tracheally with up to 3.5 x 106 cfu of nonmucoid P.

aeruginosa embedded in agar beads, but the mice did exhibit substantial morbidity. In

view of the finding that DBA/2 mice are more susceptible to P. aeruginosa, we used this

strain to evaluate the ability of OprF311-341-OprI-Flagellins immunization to promote

enhanced clearance of P. aeruginosa embedded in agar. DBA/2 mice were immunized as

described above and infected intratracheally with 3.5x106 cfu agar-embedded PAO1.

Lungs were harvested 1, 3, and 5 days post-infection and bacteria enumerated by serial

dilutions on LANS plates. One day after challenge, immunized mice displayed a marked

decrease in bacterial burden compared to control mice (Fig 8). After 3 days, 5 of 6 mice

immunized with OprF311-341-OprI-Flagellins had cleared the infection. In contrast, the

42

control mice had large numbers of bacteria in the lungs. Although the control mice

cleared the infection by day 5, our results clearly demonstrate that immunization with

OprF311-341-OprI-Flagellins had a dramatic effect on the rate of bacterial clearance. It is

important to emphasize that the ability of mice immunized with OprF-OprI to clear the

infection by day 5 reflects a limitation of this model and not the efficacy of the OprF311-

341-OprI-Flagellins vaccine.

Reduced lung pathology following pulmonary P. aeruginosa challenge in OprF311-341-

OprI-Flagellin immunized mice. In addition to determining bacterial burden following

challenge, we also evaluated the histopathology of lungs from mice immunized with

OprF311-341-OprI-Flagellin or OprF-OprI. Lungs were harvested 1, 3, and 5 days after P.

aeruginosa challenge. One day after P. aeruginosa challenge, alveolar walls from

OprF311-341-OprI-Flagellin immunized mice displayed slight thickening owing to

congestion and increased numbers of inflammatory cells. In contrast, lungs from mice

immunized with OprF-OprI developed bronchopneumonia, with airway-oriented

neutrophils, edema, and abundant visible bacteria (Fig 9A). After 3 days, immune mice

exhibited only minor inflammatory changes in the lung, whereas more severe pneumonia

with diffuse consolidation was present in the control animals. After 5 days, the lungs of

immune mice were normal, while those of the controls had thickened alveolar walls, a

result of congestion and inflammatory cells (Fig 9B). In summary, mice immunized with

OprF311-341-OprI-Flagellin displayed minimal lung pathology which completely resolved

by day 5 post challenge. The absence of lung pathology in the immune mice not only

43

Figure 8. OprF311-341-OprI-Flagellins immunized mice display enhanced rate of

clearance following pulmonary P. aeruginosa challenge. DBA/2 mice were

immunized twice with 5µg of OprF311-341-OprI-Flagellins and challenged intratracheally

with 3.5x106 cfu of agar-embedded PAO1. The right lungs were harvested 1, 3, and 5

days post-infection and bacterial burden was assessed by counting. Data are the average

of 6-7 mice per group. Dotted line indicates the limit of detection. #P=0.053,

**P=0.002, using a Mann–Whitney rank sum test.

44

demonstrates the efficacy of the vaccine in promoting bacterial clearance, but also the

ability of the vaccine to promote clearance without inducing secondary tissue damage. In

striking contrast, mice immunized with OprF-OprI demonstrated severe pneumonia

which only partially resolved by day 5 (Fig 9A). In conjunction with the results of in

vitro experiments (Figs 5 and 6), it is clear that OprF311-341-OprI-Flagellin immunization

promotes an adaptive immune response that promotes the generation of antigen-specific

IgG that exhibits robust functional activity, facilitates rapid clearance, and prevents the

development of severe pneumonia following P. aeruginosa infection.

46

Figure 9. OprF311-341-OprI-Flagellins immunized mice are protected against severe

lung pathology during pulmonary P. aeruginosa challenge. The left lung of identical

mice used in Figure 8 was evaluated for histology. Lungs were fixed in 10% formalin for

24 hours, paraffin embedded, and 4µm sections were cut. Slides were stained with H&E.

Representative images are shown from 6 sections/animal. (A) OprF-OprI immunized

mice. A1-3 magnified 4x. A4 magnified 40x to show bacteria. A5-6 magnified 20x (B)

OprF311-341-OprI-Flagellins immunized mice. B1-3 magnified 4x. B4-6 magnified 20x.

(C) Slides were blindly scored for consolidation, bronchiolar and vascular degenerative

changes, alveolar wall thickness, and edema. The score for inflammation was determined

by the sum for each category. Dotted line indicates lowest score possible. *P<0.05,

using Student’s t-test.

47

Chapter II

Immunization of young African green monkeys with OprF epitope 8-OprI-type A- and B-

flagellin fusion proteins promotes the production of protective antibodies against

nonmucoid Pseudomonas aeruginosa

E.T. Weimer, S.E. Ervin, D.J. Wozniak, S.B. Mizel

The following manuscript was submitted to Vaccine, May 27, 2009. Stylistic variations

are due to the requirement of the journal. E.T. Weimer performed the experiments and

prepared the manuscript. Dr. Steven B. Mizel acted in an advisory and editorial capacity.

49

MATERIALS AND METHODS

Strains and plasmids. Escherichia coli was cultured in Luria-Bertaini (LB; 10

g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) broth at 37oC and P. aeruginosa in LB

broth lacking NaCl (LBNS; 10 g/L tryptone, 5 g/L yeast extract). Solid media was

prepared by adding 1.0-1.5% select agar (Gibco-BRL). Plasmid selection in E. coli was

done using media supplemented with antibiotics at the following concentrations:

carbenicillin (Cb; 100 µg/ml), gentamicin (Gm; 10 µg/ml) and in P. aeruginosa using

media containing Cb (300 µg/ml), Gm (100 µg/ml), and irgasan (Irg; 25 µg/ml). E. coli

strain JM109 was used for all cloning procedures. E. coli SM10 was used to transfer

plasmids into P. aeruginosa by bi-parental mating {160}. The P. aeruginosa strains used

were PAO1 and its derivatives: WFPA850, WFPA852, WFPA854, WFPA860,

WFPA862, WFPA864, and WFPA866. These non-polar deletion mutants were

constructed by overlap extension as previously described {115}.

Recombinant proteins. OprF311-341-OprI-Flagellins were purified by metal ion

affinity chromatography as previously described {61,115,165}. Acrodisc Q membranes

were used to remove endotoxin and nucleic acids. Endotoxin levels were <10pg/µg for

all of the proteins (as detected by QCL-1000 chromogenic LAL test kit, Cambrex

Corporation (East Rutherfrod, NJ)).

ELISA for antigen-specific IgG. Titers of antigen-specific IgG were measured

using MaxiSorb plates coated with 100µl of purified recombinant antigen (A-flagellin, B-

flagellin, OprI, or OprF) at 10 µg/ml in sterile PBS. The plates were incubated overnight

at 4°C and then blocked with 10% newborn calf serum in PBS. Plasma samples (in

triplicate) were added and the plates incubated overnight at 4°C. Secondary anti-Ig

50

antibodies (Roche Diagnostics) were then added for 2h at room temperature. Peroxidase

activity was detected with 3,3’,5,5’-tetramethylbenzidine (TMB) liquid substrate system

(Sigma-Aldrich). Endpoint dilution titers were defined as the inverse of the lowest

dilution that resulted in an absorbance value (at 450 nm) of 0.1 over that of pre-

immunization plasma. To determine relative antibody affinities, the ELISA assay was

conducted as described above with the addition of a 15 min incubation with sodium

thiocyanate (NaSCN) (Sigma) solution as described previously {115,165,166}.

Monkeys. Immunization studies with 4-6 month old African green monkeys

(Chlorocebus aethiops SK) (approximately 1 kg) were conducted at the Behavioral

Science Foundation, St. Kitts, using a protocol approved by the foundation-associated

Animal Care and Use Committee. The animals were bled several days prior to

immunization to obtain control plasma. Groups of 7 monkeys were immunized with 1, 3,

10, or 30µg of OprF311-341-OprI-Flagellins (in a volume of approximately 160 µl) in the

gluteus maximus muscle on two occasions separated by 28 days. Ten days and three

months after the boost, blood was collected and plasma was prepared for analysis of

antigen-specific IgG titers.

Antigen-specific IgG-mediated complement activation. Antibody-specific

complement activation was determined using flow cytometry as previously described

{115}. Complement-mediated killing was determined by incubating bacteria with serial

dilutions of plasma samples for 4 hr and then plating aliquots {115}. Percent bacteria

killed was quantified by [(number of input bacteria – number of recovered bacteria) /

(number of input bacteria)] X 100.

51

Passive immunization and respiratory challenge with agar-embedded P.

aeruginosa. Groups of 5-7 week old female DBA/2 mice (6 mice per group) were given

100µL of pre-immunization or post-immunization monkey plasma intra-peritoneally and

then two days later were challenged intratracheally with 2x106 colony forming units (cfu)

of agar-embedded PAO1 {34,115-117,171}. The agar-embedded PA01 were prepared

and given intratracheally as previously described {115}.

Histology. Lungs were harvested and placed in 10% formalin for 24 h. The

tissue then was trimmed, embedded in paraffin, cut at 4 µm, and stained with

hematoxylin and eosin by routine methods. Three sections per lung from each of 4 mice

in control and immune groups were evaluated. Representative images are presented.

Statistical analyses. Statistical analysis was performed using GraphPad Prism

5.02 for Windows (GraphPad Software, San Diego California USA). The significance of

data sets was determined using the Mann–Whitney rank sum test. Where applicable a

one-way ANOVA test was applied. P values of less than 0.05 were considered

significant.

52

RESULTS

OprF311-341-OprI-Flagellins promote a robust humoral immune response in young

African green monkeys. In view of our previous study indicating that OprF311-341-OprI-

Flagellins promote the production of protective IgG in mice {115}, we evaluated the

ability of OprF311-341-OprI-Flagellins to elicit a humoral immune response in 4-6 month

old African green monkeys. We chose monkeys at this age because we favor the view

that protection against P. aeruginosa in CF patients will be maximal if immunity is

established at an early age prior to P. aeruginosa infection. To that end, groups of seven

4-6 month old African green monkeys (~ 1 kg) were immunized intramuscularly with 1,

3, 10, or 30µg of total protein (equal amounts of each fusion protein). Four weeks later,

the monkeys were boosted via the same route, plasma was prepared ten days post-boost,

and evaluated by ELISA for titers of anti-OprF, OprI, and type A and B flagellin IgG.

Pre-immunization plasma from each animal was used as a control. The results using the

30µg dose are shown in Figure 10. As might be expected, the flagellin-specific IgG

responses were extremely strong. However, the magnitude of the OprI and OprF-specific

IgG titers were also quite high, generally exceeding 105. Enhanced antigen-specific IgG

production was also observed in monkeys immunized with the 1, 3, and 10 µg doses (data

not shown). Although some of the monkeys had low pre-immunization titers against

each antigen, immunization with OprF311-341-OprI-Flagellins induced a dramatic increase

in the plasma IgG titers against all of the eliciting antigens (Fig 10). Importantly, the

induced IgG was not due to reactivation of memory B cells, as immunization with OprF-

OprI fusion protein, without flagellin, did not elicit any increase in plasma IgG (data not

shown).

53

To determine the stability of antigen-specific IgG following immunization with

OprF311-341-OprI-Flagellins, 4-6 month old African green monkeys were immunized as

described above. Three months post-boost, plasma was prepared and antigen-specific

IgG was determined by ELISA. Pre-immunization plasma was used as a control.

Surprisingly, there was a substantial reduction in the level of IgG specific for flagellin,

OprI and OprF (Fig 10). However, the decrease in antigen-specific IgG was not from

reduced stability of the plasma IgG as the calculated half-life was 26.9 days (data not

shown). Reduced antigen-specific IgG production was also observed in monkeys

immunized with the 1, 3, and 10 µg doses (data not shown). Based on nonlinear

regression analysis, the animals possessed protective levels of IgG out to 2.5 months after

immunization (Fig 10B). Thus, ten days post-immunization plasma was used for all

subsequent experiments.

OprF311-341-OprI-Flagellins elicit high affinity IgG against flagellin, OprI, and OprF.

Given that antigen affinity plays a critical role in the overall functional activity of an

antibody, we evaluated the relative affinity of the IgG generated following immunization

with OprF311-341-OprI-A- and B-flagellin. The relative affinity of antibodies was assessed

by determining the concentration of sodium thiocyanate required to reduce antibody

binding by 50% in an ELISA. Samples were pooled from the 30µg pre- and ten days

post-immunizations.

54

Figure 10. Intramuscular immunization of African green monkeys with OprF311-341-

OprI-Flagellins promotes a potent humoral immune response. African green

monkeys were immunized twice intramuscularly with 30µg of OprF311-341-OprI-

Flagellins. Ten-days and three months post-boost, plasma was prepared and ELISA used

to determine antigen-specific IgG titers. (A) Open circles represent the pre-immunization

titers for each animal. Filled squares represent individual monkeys ten days post-

immunization IgG titer. Filled triangles represent individual monkeys three months post-

immunization (7 monkeys per group in triplicate). (B) Average nonlinear regression

analysis on IgG titers from individual moneys immunized with 30µg of OprF311-341-OprI-

Flagellins. Using one-way ANOVA analysis, post-immunization IgG titers were

significantly higher than pre-immunization IgG titers (P<0.05).

55

For comparative purposes, in mice immunized with OprF311-341-OprI-Flagellins 3M

NaSCN was required to reduce immune complex formation by 50% for type A and B

flagellins and 2M for OprI and OprF {115}. Given the observation that these antibodies

provide enhanced clearance of P. aeruginosa, we have classified high affinity antibodies

as requiring 2-3 M sodium thiocyanate for 50% reduction in antigen binding. As shown

in Fig 11, immunization with OprF311-341-OprI-Flagellins generated IgG with relatively

high-affinity for flagellin, OprI, and OprF. OprF311-341-OprI-A-and B-flagellin immune

plasma had an average IgG affinity approaching 3M sodium thiocyanate for A- and B-

flagellin and OprI, and 2M for OprF (Fig 11). In contrast, pre-immunization IgG

exhibited significantly lower affinities than post-immunization IgG. For example, pre-

immunization anti-OprI IgG had a relative affinity of 600 mM as opposed to 3M for

immunization-induced anti-OprI IgG (Fig. 11A). The presence of high affinity anti-A-

flagellin IgG prior to immunization is most likely a result of exposure to environmental

P. aeruginosa. However, immunization with OprF311-341-OprI-Flagellins significantly

enhanced the affinity of A-flagellin-specific IgG (Fig 11). These results are consistent

with the conclusion that OprF311-341-OprI-A- and B-flagellin elicits high-affinity, antigen-

specific IgG.

57

Figure 11. OprF311-341-OprI-Flagellins elicit high-affinity antigen-specific IgG.

Pooled plasma samples from monkeys that received 30µg of OprF311-341-OprI-Flagellins

were used to determine relative antibody affinity for A-flagellin, B-flagellin, OprI, and

OprF. Antigen-specific IgG affinity was determined by ELISA using dilutions of sodium

thiocyanate (NaSCN). Data are presented as percent maximal absorbance relative to

PBS. Dotted line indicates the 50% maximal absorbance, which is an indicator of

antibody affinity. Samples were obtained from the monkeys used in Fig 10.

58

IgG specific for flagellin, OprI, and OprF mediate complement activation and killing

of nonmucoid P. aeruginosa. To assess the functional activity of each of the antigen-

specific IgG, we utilized several P. aeruginosa mutants lacking one or more of the

eliciting antigens {115}. P. aeruginosa type-B-flagellin expressing strain PAO1 was

used as the genetic background for the mutants. Using these mutant strains, we evaluated

the ability of flagellin, OprI, and OprF-specific IgG antibodies to individually mediate C3

deposition on P. aeruginosa. The various P. aeruginosa strains were incubated with

dilutions of heat-inactivated pooled immune monkey plasma for 1 hour, and then 5%

rabbit complement was added for an additional hour. The bacteria were then stained with

FITC-labelled C3-specific antibody and the extent of C3-deposition was determined by

flow cytometry. A mutant P. aeruginosa strain that was unable to synthesize flagellin,

OprI, and OprF was used as a control. Consistent with previous observations in mice

{115}, antibodies specific for flagellin, OprI, and OprF mediated high levels of

complement deposition on wild-type P. aeruginosa strain PAO1 (Fig. 12A). The

magnitude of the effect was dependent on the concentration of immune plasma and was

detectable at a dilution as low as 1:1,000 with wild-type PAO1 as the target. The antigen

specificity of the effect was evidenced by the ability of immune plasma to promote

significantly greater C3 deposition on mutant PAO1 strains expressing only one of the

eliciting antigens than on bacteria lacking flagellin, OprI, and OprF expression (shaded

peaks in Fig. 12A). The benefit of having a multivalent vaccine containing flagellin,

OprI, and OprF is evidenced by the observation that C3 deposition on

60

Figure 12. Complement activation and complement-mediated killing by OprI,

OprF, and type A and B-flagellin-specific IgG. Pooled plasma samples from the

monkeys immunized with 30 μg OprF311-341-OprI-Flagellins were incubated with wild

type and mutant P. aeruginosa PAO1. (A) C3-depostion was determined by flow

cytometry after bacteria had been incubated with plasma for 1 hr and rabbit serum for an

additional hr. WT, wild-type; B-Flagellin+, fliC+∆oprI∆oprF; OprI+, oprI+∆fliC∆oprF;

OprF+, oprF+∆fliC∆oprI. The shaded peak was obtained using the WFPA864 strain

(∆fliC∆oprI∆oprF) lacking all three antigens. (B) 1:10, 1:100, 1:1000, and 1:10,000

dilutions of plasma samples were incubated with PAO1 (WT) for 4 hrs and complement-

dependent killing was determined by plating and enumerating bacteria. (C) Mutant

strains of P. aeruginosa were used to determine the ability of flagellin, OprI, and OprF-

specific IgG to mediate complement-dependent killing of the bacteria. In each case, a

1:100 dilution of plasma was used. (D) Effect of immune plasma on PAO1, as well as

nonmucoid (1286) and mucoid (T68933) P. aeruginosa clinical isolates. Data represents

3 independent experiments performed in triplicate. * P<0.05, compared to WFPA864

(∆fliC∆oprI∆oprF) strain lacking all three antigens.

61

PAO1 expressing all three antigens was still measurable at a 1:1,000 dilution, whereas C3

deposition was only minimally evident at this dilution when individual PAO1 mutants

were used—a finding that is consistent with the notion that the individual populations of

anti-type A and B flagellin, OprI, and OprF synergize in promoting C3 deposition.

In view of the robust complement activation mediated by immune monkey

plasma, we assessed whether the plasma was equally effective in promoting complement-

mediated killing of P. aeruginosa. Aliquots of wild-type PAO1 were incubated for 4 hr

with varying dilutions of heat-inactivated pooled immune plasma and then supplemented

with rabbit serum as a source of complement prior to plating {115}. To control for non-

specific effects, WPA864, a mutant strain lacking type B flagellin, OprF, and OprI

expression was also evaluated. As shown in Fig 12B, pooled immune plasma promoted

high level, concentration-dependent killing of wild-type P. aeruginosa PAO1. As

expected, the low level of killing observed with WPA864 was similar to that observed in

the absence of complement (Table 2) or pre-immunization plasma (data not shown) and

thus demonstrates that the observed effect of the pooled immune monkey plasma is

antigen-specific. Consistent with the ability of anti-flagellin, OprI and OprF IgG to

individually induce C3 deposition, high level complement-mediated killing was observed

when the PAO1 mutants expressing only one of the eliciting antigens were used as targets

(Fig. 12C).

In a prior study {115}, we found that plasma from mice immunized with OprF311-

341-OprI-Flagellins promoted robust complement-mediated killing of nonmucoid, but not

mucoid strains of P. aeruginosa. The diminished ability of immune plasma to kill

mucoid strains is most likely due to the absence of flagella on mucoid P. aeruginosa

63

{172} and interference of antibody and/or complement deposition by alginate

exopolysaccharide. To determine if pooled immune plasma from monkeys also exhibits

activity against non-mucoid, but not mucoid strains of P. aeruginosa, we incubated wild-

type PAO1, strain 1286, a nonmucoid CF isolate, strain T68933, a mucoid CF isolate,

and PAO1 WPA864 (∆fliC∆oprI∆oprF) with a 1:100 dilution of pooled immune monkey

plasma for 4 hr prior to addition of rabbit serum as a source of complement. Aliquots of

the bacteria were then plated and surviving bacteria were enumerated. As shown in Fig

12D, the pooled immune plasma promoted high level killing of nonmucoid wild-type

PAO1 and 1286 clinical isolate, but had little if any effect on the mucoid isolate T68933.

Passive immunization of mice with plasma from OprF311-341-OprI-Flagellins

immunized monkeys enhances clearance of P. aeruginosa following pulmonary

challenge. To test the ability of antibodies from immunized monkeys to mediate

clearance of nonmucoid P. aeruginosa, we passively immunized mice with pooled pre-

immunization or immune monkey plasma. Due to the development of a lethal systemic

Arthus –like reaction in DBA/2 mice given a volume of monkey plasma in excess of 100

µl (unpublished observations), we were forced to limit the passive immunization volume

to 100 µl. Using this volume of monkey plasma and intra-peritoneal injection, there was

no observable morbidity or mortality in any of the mice. Therefore, groups of 5-6 female

DBA/2 mice were given 100 µl of pooled pre-immunization or post-immunization

plasma intra-peritoneally and then after two days the mice were challenged

intratracheally with 2x106 cfu of agar-embedded PAO1 {115}. Animals were sacrificed

three days post-infection and one lung was harvested for bacterial enumeration and the

64

other for hematoxylin and eosin (H & E) staining. As shown in Figure 13A, mice that

received immune plasma had approximately two-log lower numbers of bacteria than mice

that received pre-immunization plasma. Indeed, two of the mice that received immune

plasma had no detectable bacteria.

Since some of the mice that received immune monkey plasma had no detectable

bacteria in their lungs, we considered the possibility that these mice may have had higher

circulating IgG of the transferred immune monkey plasma as a result of differences in the

kinetics of distribution of the monkey plasma during the 48 hr after intraperitoneal

injection. Thus, some mice were better able to clear the bacteria following challenge. To

evaluate this possibility, we prepared plasma at the time of sacrifice (day 3 after

challenge) from the same mice shown in Fig 13A determined the titers of circulating

antigen-specific monkey IgG. The results of this analysis are presented in Figure 13B.

Recipient mice had type A and B flagellin-specific IgG titers approaching 105, whereas

OprI and OprF-specific IgG titers were in the range of 1-4 x 104. The IgG titers represent

a 30-fold dilution of the transferred plasma and are consistent with a blood volume of

approximately 3 ml. In contrast, mice that received pre- immunization plasma had

antigen-specific IgG titers below the limit of detection (data not shown). Importantly, the

level of total antigen-specific IgG (sum of flagellin, OprI, and OprF-specific IgG) three

days post-infection inversely correlated (r2 = 0.89) with the bacterial load in a given

animal. Based on these results, it is quite likely that all of the mice receiving immune

plasma would have had undetectable bacterial loads if their antigen-specific monkey IgG

titers had been above 4-5 x 105—the equivalent of undiluted immune monkey plasma

(Fig. 13B, right panel).

65

Figure 13. Passive immunization of mice with immune monkey plasma enhances

clearance of nonmucoid P. aeruginosa. Mice were passively immunized

intraperitoneally with 100µL of pre- or post-immunization monkey plasma. Two days

post-injection mice were intratracheally challenged with agar-embedded PAO1 [16].

Three days post-infection, the mice were sacrificed and lungs harvested for bacterial

counts and H+E staining. (A) Lung bacterial burden three days post-infection. * P<0.05.

(B) Flagellin, OprI, and OprF-specific IgG titers three days post-infection were

determined by ELISA. The sum of flagellin, OprI, and OprF-specific IgG (Total antigen-

specific IgG) inversely correlates with bacterial burden r2=0.89. (C) 4µm thick lung

sections were stained with H+E. Left panel is representative of mice that received pre-

immunization plasma. Center panel is representative of mice that received post-

immunization plasma that had >102 bacteria. Right panel is representative of mice that

received post-immunization plasma with undetectable (<102) bacteria.

66

These findings are consistent with the conclusion that the titers of anti-flagellins, OprI,

and OprF in the immune monkeys may be of sufficient magnitude to promote enhanced

clearance of P. aeruginosa in these animals.

Mice immunized with OprF311-341-OprI-Flagellins are spared from the serious

inflammation generated in response to respiratory infection with P. aeruginosa {115}.

To determine if mice receiving pooled immune monkey plasma are also spared from

serious lung inflammation following challenge with agar-embedded P. aeruginosa

PAO1, H & E lung sections from mice receiving pre-immunization or post-immunization

monkey plasma were examined (Fig. 13C). As might be expected from the variability in

the bacterial load in lungs (Fig. 13A) and circulating titers of antigen-specific IgG (Fig.

13B) mice that received immune monkey plasma displayed variable levels of lung

inflammation 3 days post-challenge. The mice that received immune monkey plasma and

had sufficient circulating titers of monkey IgG to rapidly and completely clear the

infection had no signs of lung pathology (Fig. 13C right panel), whereas mice that had

detectable bacteria had signs of lung inflammation (Fig. 13C middle panel). However, it

is important to emphasize that the extent of lung inflammation in these mice was

significantly less than observed in mice that received pre-immunization plasma (Fig. 13C

left panel). The latter group of mice clearly had severe bronchial pneumonia that was

characterized by consolidation, alveolar wall thickening, and edema.

68

DISCUSSION

The goal of my thesis studies was two-fold: to establish a set of criteria for a

vaccine against P. aeruginosa and then to develop and test a vaccine based on these

criteria in adult mice as well as very young African green monkeys. Based on my results,

I conclude that the OprF311-341-OprI-A- and B-flagellins vaccine meets all of the proposed

criteria: the vaccine contains flagellin, a potent adjuvant, is multivalent, generates high-

titer antigen-specific IgG that exhibits a high degree of functional activity, generates a

robust memory response, and enhances clearance of nonmucoid P. aeruginosa without

secondary tissue damage. In addition, OprF311-341-OprI-A- and B-flagellins vaccine

promotes a humoral immune response in 4-6 month old monkeys that induces not only a

high level of antigen-specific IgG but also high affinity IgG that mediate complement-

dependent killing of nonmucoid P. aeruginosa. Although the antigen-specific IgG

induced by this vaccine did not promote complement-mediated killing of mucoid P.

aeruginosa (Table 2), it is important to emphasize that longitudinal studies of CF patients

have clearly demonstrated that the initial P. aeruginosa infection is mediated by

nonmucoid bacteria {22,23,25}. The overexpression of alginate on mucoid P.

aeruginosa may prevent antibodies from binding to antigen or inhibit the deposition on

C3 on the surface of the bacteria. The latter hypothesis is consistent with the

observations that S. pneumoniae capsule polysaccharide prevents the Fc region of

antibodies from binding to Fc receptors on phagocytic cells as well as reduces the amount

of C3 deposited on the surface of the bacteria {173,174}. I wish to emphasize that

although the OprF311-341-OprI-Flagellins has many of the features of an efficacious

69

vaccine, the evaluation of its potential for use in CF patients is limited at this time by the

lack of a suitable animal model that closely mirrors the situation in CF patients.

In addition to promoting high-level antigen-specific IgG production (Fig 3 and

10), flagellin facilitated the generation of significant numbers of antigen-specific memory

B cells (Fig 4). In view of these potent actions of flagellin, it is likely that the very

limited reduction in the incidence of P. aeruginosa infections in CF patients immunized

with sheared flagella may be due to a low level of functional adjuvant activity of the

material {128}. Although the adjuvants Pam3Cys and Pam2Ser {175} , alum {81,176},

adenovirus {116,117}, activated DC {171}, and B-cell activating factor {177} were

found to enhance the response to P. aeruginosa antigens, it appears that flagellin is far

more effective as evidenced by the dramatic difference in the amount of antigen required

and the resultant titers of antigen-specific IgG.

Multivalency not only promotes synergistic activity of individual antibodies in

activating complement but also enhances vaccine coverage against nonmucoid P.

aeruginosa strains (Fig 6). The value of multivalency was also demonstrated by Saha et.

al. {106} who used multivalent DNA vaccination with OprF-OprI, PilA, and PcrV

antigens.

The titers of flagellins, OprI, and OprF-specific IgG following immunization with

OprF311-341-OprI-Flagellins are in most cases two logs higher than those reported in other

studies {81,103,106,116,117,128,145,178}. For example, von Specht et. al. {179}

required three immunizations with 70-fold more antigen to achieve equivalent antibody

responses. In addition, Sorichter et. al. {124} reported that intranasal immunization of

CF patients with 1mg of OprF-OprI fusion protein promoted a 3-fold increase in IgG

70

titers. The difference may be due to the extraordinary potency of flagellin as an adjuvant,

as well as the use of fusion proteins that enhance the efficiency of antigen delivery to

dendritic cells via the binding of the associated flagellin to TLR5 on these cells {157}.

The finding that immunization with OprF311-341-OprI-Flagellins promotes the generation

of large numbers of plasma cells is consistent with the very high titers of induced IgG.

Furthermore, the generation of immunologic memory is evidenced by the relatively high

frequency of antigen-specific MBC.

In line with prior work on flagellin, OprF, and OprI antigens, I have shown that

all three antigens significantly affect the clearance of P. aeruginosa in mice. In addition,

antibodies specific for each antigen activate complement and mediate killing of P.

aeruginosa (Figs 6, 7). In this regard, Von Specht and colleagues {120} found that the

level of complement-activating OprI-specific IgG correlated with the level of protection

against P. aeruginosa. In addition, several investigators have demonstrated that high-

affinity IgG that exhibits a high degree of functional activity can mediate protection

against P. aeruginosa {80,180}. My results clearly support these conclusions. The

finding that intramuscular immunization with OprF311-341-OprI-Flagellins promotes

clearance is consistent with the conclusion that protection is not dependent on the

availability of antigen-specific IgA. In this regard, Pier et al. {181} demonstrated using

LPS-based vaccination that intraperitoneal immunization could prevent mucosal

colonization by P. aeruginosa.

A number of studies {182-185} have presented evidence in support of the

conclusion that heightened production of Th2 cell-derived cytokines such as IL-4, IL-10,

and IL-13 is associated with a poor prognosis in CF patients. In view of the notion that

71

isotype switching to IgG1 is Th2 cell-driven, the question arises as to whether the

observed IgG1 bias (relative to IgG2a) of the humoral response to OprF311-341-OprI-

Flagellins might exacerbate the pathology in CF patients. First, the linkage between Th

bias and prognosis in CF patients is based on cytokine production and not IgG isotype.

Indeed, two studies revealed that elevated levels of alginate-specific IgG2 and IgG3, and

not IgG1, are associated with a poor prognosis in CF patients {186,187}. It is also

important to note that class switching to IgE is also promoted by Th2 cells. Yet we have

never observed any increase in IgE in response to OprF311-341-OprI-Flagellins or any

flagellin-based vaccine {61}. Thus, we believe that the humoral response driven by the

adjuvant activity of flagellin does not fit the paradigm of a classic Th2 response and thus

the possibility that a flagellin-based P. aeruginosa vaccine might cause substantial

secondary tissue damage is unlikely. This conclusion is clearly supported by the

observation (Fig 9) that the lungs of mice immunized with OprF311-341-OprI-Flagellins did

not exhibit any evidence of residual tissue damage following clearance of the bacteria.

In contrast to the protective, inflammation-sparing effect of immunization with

OprF311-341-OprI-Flagellins, P. aeruginosa infection in CF patients generates a humoral

immune response against P. aeruginosa antigens such as LPS, exotoxin A, and alginate

that is ineffective in promoting clearance of the bacteria. High levels of P. aeruginosa-

specific IgG have been associated with increased inflammation and lung damage most

likely from increased immune complex formation {23,188,189}. The P. aeruginosa-

specific antibodies exhibit mixed affinity or low functional activity {25,190}, in part to

the fragmentation of these antibodies by released elastase{191}. Importantly, Tosi et. al.

{25} found that the opsonic activity declined as chronic P. aeruginosa infection was

72

established. The generation of low affinity antibodies during chronic P. aeruginosa

infection reduces the ability of CF patients to clear immune complexes and results in

increased inflammation {192}. In addition, IgG isolated from CF patients airway is

fragmented, explaining why the high levels of P. aeruginosa IgG exhibit low functional

activity. The fragmentation of the IgG was directly correlated with the elastase levels

{191}. Since CF patients have a progressive increase in elastase concentrations,

vaccination at an early age would circumvent fragmentation of the antibodies by elastase.

In contrast to infection elicited IgG, vaccine-induced antibodies generally display

a high affinity. For example, Bruderer et. al. {193} demonstrated that vaccination

promoted a 100-fold increase in antibody affinity compared to naturally acquired

antibodies of the same isotype. Given the chronic nature of P. aeruginosa infections, it is

quite likely that the B and T cells undergo “exhaustion”. Constant antigen stimulation in

T cells leads to reduced proliferation, differentiation, and function {194,195}. Thus, the

possible lack of sufficient CD4+ T cell help may explain the reduced antibody affinity

seen in CF patients. Alternatively, the inability of CF patients to generate a high quality

IgG response following P. aeruginosa infection may be due to a combination of several

factors, most importantly virulence factors expressed by P. aeruginosa and the abnormal

respiratory environment.

Although OprF311-341-OprI-A- and B-flagellin elicits a humoral immune response

that promotes rapid clearance of bacteria, it remains to be determined if this vaccine will

be efficacious in CF patients. The pathophysiologic events that occur in CF patients as

well as the virulence factors expressed by P. aeruginosa {196} clearly represent a

significant challenge for any vaccine. The presence of a defective CFTR leads to

73

abnormal ion transport and water depletion in the airway of CF patients that ultimately

results in an increase in fluid viscosity, impaired mucociliary clearance, increased

bacterial trapping in the mucus layer facilitating chronic infection, and a reduction in

antibody penetration {33}. In addition, infection by S. aureus and H. influenzae, and

later P. aeruginosa contribute to the development of a hyper-inflammatory environment

in the respiratory tract {13}. All of these events contribute to a vicious cycle of infection

and inflammation that ultimately causes severe pathology. Chronic inflammation can

adversely affect vaccine efficacy due to the high level production of prostaglandins that

exert an inhibitory effect on lymphocytes {197}. In addition, chronic inflammation

promotes the continual recruitment of neutrophils to the lung that in turn release elastase

that induces tissue damage and release of DNA—a substance that provides a scaffold for

P. aeruginosa biofilms {29,30}.

The major cause of chronic inflammation in CF patients is persistent P.

aeruginosa infection. Over-production of alginate and subsequent, biofilm development

allows P. aeruginosa to evade the immune system and also increases antibiotic

resistance, making it extremely difficult to eradicate the infection {22,23}. Mucoid

conversion is also associated with a significant decrease in lung function in CF patients

{198}. Our findings indicate the best time to vaccinate CF patients using OprF311-341-

OprI-A- and B-flagellins would be prior to mucoid conversion, since complement-

mediated killing occurred only in nonmucoid bacteria (Fig 7, 12, and Table 2).

The pathophysiologic events associated with CF in combination with the

pathogenic mechanisms associated with P. aeruginosa represent a significant challenge

to the elements of protective immunity. Thus, it is important to evaluate the efficacy of

74

OprF311-341-OprI-Flagellins in a hyper-inflammatory environment that closely mimics CF.

To that end, studies have been initiated in the laboratory of Dr. Steven Mizel using CFTR

KO mice (on a C57BL/6 background) to assess the ability of the vaccine to elicited a

protective antibody response in the lungs of mice undergoing progressive damage caused

by excessive inflammation. C57BL/6 CFTR KO mice display increased susceptibility to

P. aeruginosa as well as a progressive lung disease that resembles CF {13,199}.

However, no P. aeruginosa vaccine has been evaluated in these animals. I hypothesize

that there is an inverse correlation between vaccine efficacy and age in the C57BL/6

CFTR KO mice. It is quite likely that vaccine efficacy will be most effective at a very

early stage in the disease process. Perhaps the limited success of previous vaccine

studies was not solely due to the limited inherent efficacy of the vaccines, but rather to

the timing of immunization. In addition, previous clinical trials focused primarily on the

level of antigen-specific IgG induced, instead of the quality or functional activity of the

IgG {115,120,121,158,159}.

Given that the timing of immunization and the quality of the immune response

play important roles in determining vaccine efficacy, I tested OprF311-341-OprI-Flagellins

in 4-6 month old African green monkeys. In line with my observations in mice, I found

that the vaccine elicited a high level of antigen-specific IgG at low antigen doses (Fig

10). Consistent with a previous report {61}, the presence of pre-existing flagellin IgG

does not affect vaccines efficacy (Fig 10). Individual antigen-specific IgG was capable

of mediating complement activation and complement-dependent killing of nonmucoid P.

aeruginosa strains (Fig 12). Importantly, passive immunization of mice with immune

monkey plasma enhanced the clearance of nonmucoid P. aeruginosa (Fig 13). Thus, I

75

believe that this study provides a strong foundation for future studies in infant CF

patients.

Children receive numerous vaccines including diphtheria, tetanus, acellular

pertussis toxin (DTaP), inactivated poliovirus, H. influenzae type B, measles, mumps,

rubella (MMR), and Streptococcus pneumoniae. Each of these vaccines is clearly

effective at reducing the incidence of infection. Children receive the vaccines over a

period of 18-months. Most childhood vaccines are given at least 3 times to achieve

protective antibody levels {200}. Importantly, lowering the number of immunizations

required to generate high levels of antibodies will reduce the cost of immunization and

number of doctor visits. My results with very young African green monkeys, OprF311-341-

OprI-Flagellins demonstrate that this vaccine promotes high levels of antigen-specific

IgG with only two immunizations using small amounts of antigen (Fig 10). The finding

that OprF311-341-OprI-Flagellins induces a high level of antibody production with only

two immunizations once again demonstrates the extraordinary potency of flagellin as an

adjuvant. However, I did observe a substantial decrease in the antigen-specific IgG three

months post-boost (Fig 10B). This result is consistent with what has been reported for

early life vaccination against H. influenzae type B, S. pneumoniae, and MMR {201-204}.

For example, infants immunized with a conjugate H. influenzae polysaccharide vaccine

had high antigen-specific IgG one month post-immunization but the IgG level fell 84%

by seven month post-immunization {205,206}. This is in contrast to what has been

observed for adult animals, in which the antigen-specific IgG titer remains high for at

least 3-6 months {207-209}. However, recent evidence suggests that the infant immune

system doesn’t support development of long-lived plasma cells {210}. At a young age,

76

the bone marrow lacks sufficient secretion of key plasma cells survival factors such as a

proliferation-inducing ligand, IL-6, and stromal cell-derived factor 1 to promote long-

lived plasma cells {211-216}. Thus, I hypothesis that the lack of sustainable IgG titers in

the young monkeys is due to the inability of the young immune system to support the

generation of long-lived plasma cells.

Although OprF311-341-OprI-Flagellins immunized monkeys have reduced antigen-

specific IgG titers three months after immunization, I hypothesize that the vaccine

induces the production of a large population of memory B cells. This hypothesis is based

on my results with mice in which large numbers of antigen-specific memory B cells were

generated after immunization with OprF311-341-OprI-Flagellins (Fig 4). Previous studies

have demonstrated that infants are biased towards generation of memory B cells

{217,218}. In addition, antigen-specific memory B cells can survive for extended

periods and upon reactivation rapidly generate a high titer of protective antibodies

{209,219-223}. Thus, it will be important to determine how the antigen-specific memory

B cells levels change over time in African green monkeys immunized with OprF311-341-

OprI-Flagellins.

The development of vaccines that are effective at generating a high quality long-

lasting immune response in very young CF patients may not only prevent the initial P.

aeruginosa infection, but may also prevent much of the inflammation induced by chronic

mucoid P. aeruginosa infection. However, it is quite likely that pathogens such as S.

aureus and H. influenzae would continue to be a significant concern in these patients. In

view of the flexibility of flagellin for use as an adjuvant and carrier, it may be that a

cocktail of flagellin fusion proteins targeting a range of CF pathogens may afford broad

77

immunity and thus spare the respiratory system from significant pathogen-induced

inflammatory damage.

The use of cocktail immunizations is an emerging concept in the field of

vaccinology. Several clinical trials have focused on reducing the number of

immunizations children receive by combining vaccines {224-226}. Many of the current

childhood vaccines are polysaccharide conjugate vaccines. The use of flagellin as a

carrier for polysaccharides has not been extensively studied. In view of the protective

efficacy of polysaccharides from S. pneumoniae, H. influenzae, and P. aeruginosa, it

would be beneficial to determine how conjugation of polysaccharides to flagellin

modulates the humoral immune response. Psl, a polysaccharide that is important for

attachment and biofilm formation and maintenance {35-37,227,228}, is an excellent

candidate for evaluation as a component of a flagellin conjugate vaccine. Conjugation of

Psl to flagellin would demonstrate a “proof of concept” that flagellin linked

polysaccharide vaccines are effective in promoting long-lived plasma and memory B

cells.

My studies represent a significant step forward for P. aeruginosa vaccine

development. Importantly, I have established a set of criteria for an effective vaccine and

have demonstrated the validity of these criteria. In addition, I have demonstrated the

efficacy of OprF311-341-OprI-Flagellins in adult mice as well as very young African green

monkeys.

78

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SCHOLASTIC VITAE NAME: Eric T. Weimer ADDRESS: Department of Microbiology and Immunology Wake Forest University Health Sciences Medical Center Boulevard Winston-Salem, North Carolina 27157-1064 Telephone - (336) 716-4138 Email – [email protected] EDUCATION: 2001-2005 East Carolina University Greenville, NC B.S. (Biology) Magna Cum Laude 2005-2009 Wake Forest University School of Medicine Winston Salem, NC Ph.D. (Microbiology & Immunology)

"Development of a multivalent flagellin-based Pseudomonas aeruginosa vaccine"

Steven B. Mizel, Ph.D., Advisor PROFESSIONAL MEMBERSHIPS: American Association of Immunologists BIBLIOGRAPHY: E.T. Weimer, S. Ervin, D. Wozniak, S. Mizel. Immunization of young African green

monkeys with OprF epitope 8-OprI-type A- and B-flagellin fusion proteins promotes the production of protective antibodies against nonmucoid Pseudomonas aeruginosa. Submitted to Vaccine, May 2009.

E.T. Weimer, H. Lu, N. Kock, D. Wozniak, S. Mizel. A fusion protein vaccine

containing OprF epitope 8-OprI-type A- and B-flagellin promotes enhanced

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clearance of nonmucoid Pseudomonas aeruginosa. Infect Immun. June 2009 77:2356-2366.

R.D. Yammani, S. Pejawar-Gaddy, T.G. Gurley, E.T. Weimer, E.M. Hiltbold, M.A.

Alexander-Miller. Regulation of maturation and activating potential in CD8+ versus CD8- dendritic cells following in vivo infection with vaccinia virus. Virology. 2008 Aug 15;378(1):142-50.

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development of a multivalent recombinant protein vaccine

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