Signature redacted Signature redacted


May 14, 2015 - Encapsulation of CDN in PEGylated liposomes enhanced its ... Thank you for always expecting the best out of me, for teaching me little ...

Enhancement of HIV vaccine efficacy via lipid nanoparticle-based

adjuvants

ARGIVES MASSACHUSETTS INSTITUTE OF rECHNOLOLGY

Melissa C. Hanson Bachelor of Science, Biomedical Engineering University of Utah, Salt Lake City, Utah, 2009

MAY 14 2015 LIBRARIES

Submitted to the Department of Biological Engineering In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the Massachusetts Institute of Technology December 2014

Z

I

C 2014 Massachusetts Institute of Technology All rights reserved

Signature of Author:

Signature redacted Melissa C. Hanson Department of Biological Engineering

Certified by:

Signature redacted Professor Darrell J. Irvine Department of Biological Engineering and Materials Science Thesis Supervisor

Accepted by:

Signature redacted Forest M. White Associate Professor of Biological Engineering Chairman, Graduate Program Committee for Biological Engineering

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Members of Thesis Committee Darrell J. Irvine Professor of Biological Engineering and Materials Science & Engineering Massachusetts Institute of Technology Thesis Advisor

Jianzhu Chen Professor of Biology MassachusettsInstitute of Technology

K. Dane Wittrup Professor of Biological Engineering and Chemical Engineering Massachusetts Institute of Technology Thesis Committee Chair

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Enhancement of HIV vaccine efficacy via lipid nanoparticle-based adjuvants by

Melissa Hanson Submitted to the Department of Biological Engineering On January 8, 2014 in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biological Engineering Adjuvants are immunomodulators and/or formulations/delivery vehicles which enhance immune responses to vaccines. The lack of progress in the development of an HIV humoral vaccine is due, in part, to the absence of available adjuvants which can be sufficiently potent with minimal adverse side effects. The main goal of this thesis was to develop nanoparticles as HIV vaccine adjuvants. Building upon previous work in the Irvine lab, we determined the potency of lipid-coated microparticles was due in part to the in situ generation of antigendisplaying liposomes. Synthetic liposomes were nearly as potent as lipid-coated microparticles, but with a 10-fold greater antigen conjugation efficiency. We subsequently optimized unilamellar liposomes as delivery vehicles for surface-displayed HIV antigens. For vaccines with a recombinant gpl20 monomer (part of the HIV envelope trimer), immunization at 0 and 6 weeks with 65 nm or 150 nm diameter liposomes with 7.5 pmol gpl20 was found to induce strong anti-gp120 titers which competed with the broadly-neutralizing antibody VRC01. The second HIV antigen used was a peptide derived from the membrane proximal external region (MPER) of the gp41 protein. High-titer IgG responses to MPER required the presentation of MPER on liposomes and the inclusion of molecular adjuvants such as monophosphoryl lipid A. Anti-MPER humoral responses were further enhanced optimizing the MPER density to a mean distance of -10-15 nm between peptides on the liposomes surfaces. Lastly, we explored the adjuvant potential of cyclic dinucleotides (CDNs) with MPER liposome vaccines. Encapsulation of CDN in PEGylated liposomes enhanced its accumulation in draining lymph nodes (dLNs) 15-fold compared to unformulated cyclic dinucleotide. Liposomal CDN robustly induced type I interferon in dLNs, and promoted durable antibody titers comparable to a 30-fold larger dose of unformulated CDN without the systemic toxicity of the latter. This work defines several key properties of liposome formulations that promote durable, high-titer antibody responses against HIV antigens and demonstrates the humoral immunity efficacy of nanoparticulate delivery of cyclic dinucleotides, which is an approach broadly applicable to small molecule immunomodulators of interest for vaccines and immunotherapy. Thesis Supervisor: Darrell J. Irvine Title: Professor of Biological Engineering and Materials Science & Engineering

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Acknowledgements It's hard to believe that five years have flown by and that I'm at the end of my graduate work; many people have helped and encouraged me, both scientifically and personally. It is a deep pleasure of mine to be able to acknowledge these contributions here in permanent form. First and foremost, I would like to thank my thesis advisor, Dr. Darrell Irvine, for your constant support of this work and for your immeasurable patience with me and optimism for my results. I would also like to thank you for doing the hard work of writing grants and managing the laboratory funds. It was a luxury to be able to concentrate solely on the science. By working in your lab, I fulfilled my goal for my PhD, which was to learn how to be a scientist while working to develop an HIV vaccine; thank you for taking me onboard! Secondly, I would like to thank my committee members, Dr. Jianzhu Chen and Dr. K. Dane Wittrup. You both helped me "see the forest through the trees", so to speak, and your insights and perspectives helped direct my project and prioritize the research goals. In addition, I'd like to thank Dr. Wittrup for your role as my academic advisor in my first year at MIT. Your honesty and openness were of great help as I was looking for an advisor and trying to navigate my first year classes. I want to extend a huge thank you to Dr. Anna Bershteyn, who supervised my first six months in the Irvine lab and has continued to provide mentorship ever since. I really don't know how to convey how much I appreciate your patience, your teaching, and your kindness. Thank you. I would also like to thank Dr. Jordi Mata-Fink from the Wittrup lab. Your cheerful optimism and thoughtful scientific analysis were huge assets to our project and your encouragement in my early graduate school years were immensely helpful. There are so many current and former members of the Irvine lab to thank, I could write a thesis alone on all of it. First and foremost, thank you all for your friendship. You've made the Irvine lab a great place to work. Dr. Talar Tokatlian, Chyan-Ying Ke and Dr. Tyson Moyer: thanks for being my liposome vaccine buddies and for having sympathetic ears to my griping and worries. Dr. Greg Szeto: thank you for all of the scientific help and personal advice. Dr. Bonnie Huang: thank you so much for your mentorship. More than anyone, you've challenged me to critically review my own work and your advice at the end of my PhD was invaluable. Dr. Matthias Stephan: Thank you for looking out for me, in your own way. I also need to thank four key people from the University of Utah. Dr. Patrick Kiser, thank you so very much for you constant support. Dr. Meredith Clark, thank you for all of the scientific training, for encouraging me to go to graduate school, and for your cheerful friendship. Azadeh Poursaid, thank you for being my sister-in-engineering, for remembering 4

when homework was due, and for going canyoneering! Dr. Greg Owens, thank you for challenging me about my medical school plans and for encouraging me to try out research. To all of my friends, thank you so much for the encouragement, for listening to tales of bleeding mice, and for tons of fun and laughter over the years. BE-2009, Simmons GRTs, and Martha House - Thank you! Dr. Dana Foarta, Carrie Margulies, and Rebecca Lescarbeau: thank you so much for going through grad school with me and for always lending your ears.

It's impossible to fully thank my family for all of their love and support of the years which helped me get to and then through graduate school. But I will try. Mom, you deserve my first thank you. Thank you for always expecting the best out of me, for teaching me little arithmetic tricks in 2 "d grade (I still use them), for answering when I call, and so many more things. Dad, thank you for your steadfast belief in me, for encouraging me to try bioengineering, and for life lessons on long skiing road trips. Uncle Steve, thank you for always making me laugh. To my brothers, Matthew, Garrett, and Alex: thank you for protecting me, for challenging me, and for allowing me to find my own way. Nicolas, thank you for your love. Thank you for Caf6 6 teas, for my bike fenders, for correcting my mispronunciations (in French and English), for taking the time to understand my science, for listening to me and for chasing away my doubts. Thank you. Now finally, I want to say thank you to my team, who has been there "in the trenches" with me day after day: Stephanie Chen, Wuhbet Abraham and Dr. Monica Crespo. Steph, thank you for your cheerful willingness to help, even with the gross stuff. Monica, thank you for being there at the beginning, working on the lipid-coated microparticles. But thank you even more for coming back at the end, and helping us to develop and complete the complicated experiments of the last chapter of this thesis. Most importantly, thank you for your constant friendship throughout the whole process. Wuhbet, you have become a second mother to me. Thank you for questioning me, for your absolute faith in me, and for the years of hard work that are now summed up as chapters 3 and 4.

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Table of Contents 1.Backgroundand scope of thesis

10

1.1.

Necessity of HIV vaccine adjuvant development

10

1.2.

Conventional vaccines and adjuvants

11

1.3.

Motivation for nanoparticle-based adjuvant systems

14

1.4.

Scope and outline of thesis

17

2.In situ vesicle shedding mediates antigen delivery by lipid-coated_

19

2.1.

Introduction

19

2.2.

Materials and Methods

20

2.2.1.

Materials

2.2.2.

Synthesis of lipid-coatedparticlesand liposomes

2.2.3. 2.2.4. 2.2.5. 2.2.6. 2.2.7. 2.2.8.

Antigen conjugation onto lipid-enveloped particlesand liposomes Analysis of lipiddelaminationfrom LCMPs Size characterizationof delaminatedlipid vesicles In vivo immunization studies Antibody titer analysis Statisticalanalysis

2.3. 2.3.1. 2.3.2. 2.3.3.

2.4.

20 21 . 21 - 22 - 22

23 23 _ 23

23

Rest Its and Discussion

23 27 30

Delaminationkinetics of lipid-coatedmicroparticles Immunogenicity ofdelaminated vesicles and lipid-coatedmicroparticles Immunogenicity of lipid-stabilizedmicroparticles

34

Con clusions

3. Exploration of liposomes with surface-displayedHIV protein as vaccines

35

3.1.

Introduction

35

3.2.

Materials and Methods_

36

3.2.1 3.2.2 3.2.3 3.2.4 3.2.5

3.3.

Materials Synthesis of gp120 antigen-displayingliposomes In vivo immunization studies Antibody titer analysis_ Statisticalanalysis

37 37 .37 38

38

Results and Discussion

3.3.1.

Immunogenicity ofgp120 liposomes in comparison to traditionalvaccine formulations

3.3.2.

Optimization of liposome formulation and immunization parameters

3.3.3.

VRC01-competing and class-switchingcapabilitiesof liposome-inducedanti-gpl20 sera.

3.4.

36

Conclusions

38

39 ___

42

43

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4. Optimization of liposomes with surface-displayedHIVpeptides as vaccines

44

4.1.

Introduction

44

4.2.

Materials and Methods

45

4.2.1. 4.2.2. 4.2.3. 4.2.4. 4.2.5. 4.2.6.

4.3.

Materials Synthesis and characterizationof liposomes In vivo immunization studies Immune response analyses Histology Statisticalanalysis

45 46 47 47 48 48

Results and Discussion Immunogenicity of liposomes in comparison to traditionaladjuvants

48

4.3.1.

4.3.2. 4.3.3.

Role of liposomephysicalpropertiesin vaccine immunogenicity Relationship between antigen density and immunogenicity

4.3.4.

Optimization of T-helperpeptide delivery

50 53 53

4.4.

48

Conclusions

57

5. Development of cyclic-di-nucleotidesas potent humoraladjuvants

58

5.1.

Introduction

58

5.2.

Materials and Methods_

60

5.2.1.

5.2.2. 5.2.3. 5.2.4.

Materials NP-MPER and NP-cdGMP synthesis In vivo immunization studies CDN characterizationstudies_61

60

60 61

5.2.5.

OT-II T-cell adoptive transferand ex vivo

5.2.6.

Antibodies and Flow cytometry

5.2.7.

5.2.8.

QuantitativePCR analysis Plate and bead-basedELISAs

5.2.9.

Statisticalanalysis

5.3.

restimulationstudies

62 62 62

_63

63

Results and Discussion

64 in lymph

5.3.1.

Lipid nanoparticlesconcentrate cdGMP

5.3.2.

NP-cdGMPinduces type I IFN directly in lymph nodes and elicits greaterAPC activation than

node APCs

64

soluble CDN

67

5.3.3. Nanoparticledelivery of cdGMP enhances expansion of helper T-cells and promotes germinal center induction 70 5.3.4. cdGMP nanoparticlespromote strong humoral responses while avoiding systemic cytokine induction 72

5.3.5.

Vaccine responses are independent ofplasmacytoiddendritic cells

5.3.6.

Type I IFN and TNF-aplay complementary roles following NP-cdGMP vaccination

78

5.3.7.

Discussion

81

5.4.

_76

Conclusions

84

6. Conclusions andfuture work

85 7

_6.1. Lipid nanoparticlesas vaccine adjuvants

85

_6.2. Potentialfuturework

86

7.Appendix

87

_7.1.Appendix A: Supplementary Figures

87

7.1.1.Gating strategyfor macrophage identificationvia flow cytometry _87 7.1.2. Identification of plasmacytoiddendriticcells and confirmation of plasmacytoiddendritic cell depletion 88 in vivo 7.1.3. Stability of palm-MPER on liposomes

89

-7.2. Appendix B: Protocols 7.2.1. ELISA

to quantify the loading of ovalbumin on liposomes

7.2.2. ELISA for the detection of anti-gp120(4G) antibodies 7.2.3. ELISA to quantify the 7.2.4. ELISA

loading of gp120 on liposomes

to determine VCRO1-competition ability of anti-gp120 sera

7.2.5. UV-based quantificationof CDN encapsulationefficiency into liposomes 8. References

89

89

90 90 91

91 93

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List of Figures Schematic 1-1. Classification of adjuvants with respect to their depot/carrier and immunostimulatory properties...........................................................................................................................................................12 Schematic 1-2. TLR trafficking and signaling. .................................................................................................. 13 Figure 2-1. Delamination of lipid vesicles from lipid-coated nanoparticles.................................................... 25 Figure 2-2. Kinetics of lipid delamination from lipid-coated microparticles. ................................................ 27 Figure 2-3. Immunogenicity of lipid coated microparticles and delaminated lipid vesicles............................29 Figure 2-4. LCMPs prepared with high-TM lipids show reduced vesicle shedding and weaker antibody responses in vivo................................................................................................................................................31 Figure 2-5. Cholesterol was included in the lipid bilayer of LCMPs to decrease delamination of the envelope. ............................................................................................................................................................................ 33 Schematic 3-1: Representation of 4G gp120 protein and 4G liposomes...........................................................36 Figure 3-1. Liposomes induce strong, class-switched gp120-specific antibody responses ............................... 39 Figure 3-2. Optimization of formulation and vaccination parameters to enhance antibody responses.............41 Figure 3-3. Liposomal Fc-4G elicits VRC01-competing antibodies. ................................................................. 42 Schematic 4-1. Display of gp4l-derived MPER peptide on unilamellar liposomes ........................................ 45 Figure 4-1. Humoral responses against MPER peptides are promoted by liposomal delivery and molecular adjuvants. .......................................................................................................................................................... 49 Figure 4-2. Anti-MPER humoral responses are independent of PEGylation..................................................51 Figure 4-3. Anti-MPER humoral responses are shaped by particle size and liposome composition..............52 Figure 4-4. Anti-MPER humoral responses are maximized by liposomes carrying peptide with a mean spacing near 10 nm . .......................................................................................................................................... 53 Figure 4-5. Liposomes carrying encapsulated bilayer-anchored helper peptide stimulate both Thi and Th2 cytokine production from antigen-specific T-cells.....................................................................................55 Figure 4-6. Liposomes with surface-displayed pMPER and encapsulated HIV30 promote strong B-cell responses against MPER while minimizing off-target responses against the helper epitope................56 Figure 5-1. NP-cdGMP enhances lymph node uptake of cyclic dinucleotides..................................................66 Figure 5-2. NP-cdGMP potently activates antigen presenting cells. ................................................................. 69 Figure 5-3. NP-cdGMP promotes antigen-specific CD4' T-cell expansion. ..................................................... 71 Figure 5-4: Primary plasmablast and germinal center formation is enhanced with NP-cdGMP.................72 Figure 5-5. NP-cdGMP promotes robust humoral immunity while minimizing systemic cytokine induction..74 Figure 5-6. NP-cdGMP elicits durable class-switched humoral responses and synergizes with MPLA to adjuvant M PER vaccines.................................................................................................................................76 Figure 5-7. NP-cdGMP-adjuvanted vaccine responses are independent of plasmacytoid dendritic cells. (A-B) ............................................................................................................................................................................ 78 Figure 5-8. Type I IFN shapes early activation of antigen-presenting cells (APCS) while TNF-a is critical for IgG production following cdGMP-adjuvanted immunization..................................................................80 Supplemental Figure SS-1: Gating to identify lymph node macrophages........................................................87 Supplemental Figure SS-2: Flow cytometry gating and depletion of plasmacytoid dendritic cells...............88 Supplemental Figure S6-1: Kinetics of palm-MPER loss from liposomes...................................................... 89

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1. Background and scope of thesis

1.1. Necessity of HIV vaccine adjuvant development In 2012, it was estimated that 35.3 million people were living with HIV, 2.3 million people became newly infected, and 1.6 million people died of HIV1' 2 . Although antiretroviral therapy has significantly extended the life expectancy of HIV-infected individuals, it is prohibitively expensive in sub-Saharan Africa and other developing regions. Despite the increased availability of antiretroviral therapy in developing countries in the last decade, only 7.6 million of the 21.2 million of those in need of antiretroviral therapy are actually are receiving antiretroviral therapy in Africa 3 . The lack of curative treatment options and the economic, societal, and personal destruction caused by the HIV epidemic makes it imperative that a prophylactic method to prevent HIV infection be developed.

.

Since the development of the smallpox vaccine by Edward Jenner in the 1790s, vaccines have revolutionized global health, decreasing infant and childhood mortality from diseases such as smallpox, polio, measles, and diphtheria. The large-scale development and implementation of vaccines is considered by the US Center for Disease Control to be the greatest public health achievement of the twentieth century4 . In addition, the cost-effectiveness of vaccination is indisputable5 '6 . In comparison to other HIV prophylactics such as microbicides, abstinence & monogamy, or condoms, vaccines also do not require any longterm user adherence or lifestyle change in order to be effective. Hence, development of an HIV vaccine is a major goal of the global health community7 Although HIV vaccine research has been ongoing for more than 25 years, the scientific community has failed thus far to develop a protective vaccine due to several biological hurdles unique to HIV 7 -9 . These factors include the high mutation rate of HIV replication, which results in extraordinary antigenic diversity, as well as the rapid development of a latent reservoir of HIV-infected cells and the fact that HIV infects CD4' T-cells, which are critical for development of adaptive immune responses1o'". HIV vaccine concepts typically fall into one of two approaches, with goals of generating either cytotoxic T lymphocytes (CTLs) or neutralizing antibodies against HIV1 0 . Hypothetically, in a CTL-based approach, memory Tcells induced by vaccination rapidly expand after HIV infection and the subsequently produced CTLs kill virus-infected cells. Although a CTL based vaccine could kill infected cells, it wouldn't prevent the initial infection. Complete protection from viral infection is mediated by the elicitation of neutralizing antibodies; these antibodies are the body's primary defense against an infection. Neutralizing antibodies act by opsonizing viral particles and inducing complement activation and viral clearance before cellular infection can be established. In addition, at mucosal surfaces, neutralizing antibodies block entry of the virus into the tissue. 10

Based on studies in large animal models, induction of high and durable levels of broadly neutralizing antibody (BNAbs) should provide sterilizing immunity against HIV", although thus far, strategies to induce such antibodies by vaccination remain elusive

3

Antibodies that neutralize a diverse array of HIV- 1 strains, known as broadly neutralizing Abs (BNAbs), are very rare, but have been isolated from HIV-1 infected patients. These antibodies react against the HIV envelope at the membrane proximal external region (MPER)

of gp4l (2F5, 4E10, Z13E1), the gpl20 CD4-binding site (B 12, VRC01, VRC02), a complex of glycans on the surface of gpl2O (2G12), or a set of variable loops of gpl20 trimer (PG9 and PG16) 14-21. A successful humoral vaccine against HIV will need to generate antibodies similar to these patient-isolated antibodies, and therefore, both gp4l and gpl20 have been the subjects of considerable study as model antigens for an HIV vaccine. Unfortunately, a BNAb-inducing antigen has not been elucidated due to several factors, including the highly variable nature of HIV, low density of Env protein trimers on each viral particle, the heavy glycosylation of the HIV envelope, and the masking of vulnerable epitopes either by glycosylation, virion lipids, or by conformation' 2. All currently identified broadly neutralizing antibodies arise only after several years of infection and are typically highly mutated from germline antibodies; this is indicative of the highly variant nature of the virus, forcing the immune system to constantly 3 try and develop new antibodies against the viral escape mutants . These findings suggest that a vaccine platform which encourages somatic hypermutation may be critical for the development of antibodies capable of neutralizing the HIV virus. As a member of both the Ragon Institute and the Collaboration for AIDS Vaccine Discovery, the Irvine lab is fortunate to have access to some of the most-promising gpl20 and gp4l -based antigens currently in development. However, it is becoming apparent that even the most sophisticated HIV envelope protein antigen designs will be unable to generate sufficiently high titers of neutralizing antibodies and memory B-cell levels. All of the HIV vaccines tested thus far in humans elicit only short-lived antibody responses, exhibiting half-lives of roughly eight weeks. Therefore, it is imperative that vaccine platforms be developed to help translate the antigenicity of current protein constructs into immunogenicity which results in long-lasting titers of neutralizing antibodies.

1.2. Conventional vaccines and adjuvants The development of structure-based vaccinology employing well-defined subunit antigens has enabled the development of vaccines with excellent safety profiles, but this increase in safety has been accompanied by a decrease in immunogenicity compared to traditional live attenuated vaccines. This lack of immunogenicity is mainly because induction of the adaptive immune response is dependent on the initial activation of the innate immune system, which is triggered by danger signals not present on engineered antigens. These signal include pathogen11

associated molecular patterns (PAMPs) which are recognized by the pattern-recognition receptors (PRRs) of innate immune cells, however the links between innate and adaptive immunity are still not fully understood26- 28. An adjuvant can be defined as a substance that enhances the body's immune response to an antigen; adjuvants have become critical components of subunit vaccines. Traditionally, adjuvants were broadly separated into immunopotentiators (agonists of PRRs, saponins, cytokines, bacterial toxins) and vehicles/delivery systems (aluminum salts, 29 31 oil-in-water emulsions, virus-like particles (VLPs), and liposomes) - . As our understanding of the mechanisms of action for adjuvants has expanded, this separation has merged into spectrum, varying from purely delivery-based adjuvants to purely immunostimulatory adjuvants, as summarized in schematic 1- 132. Neutral

liposomes

Microspheres

Mneral salts,

atLn

Cationic

liposomnes

ISCOMs

W/o

emulwsons

O/W

emulsions

PRR/TLR agonists Saponins

00~

(__

0

T,,2 Depot /carrier

T,1

T1i

T4 2

Tk2

Ts1

T1 or T1 /T,,2

kmmunostimulation

Immunostimulation and carrier

Schematic 1-1. Classification of adjuvants with respect to their depot/carrier and immunostimulatory properties. ISCOMs: immunostimulating complexes; W/O: water-in-oil emulsion; 01W: oil-in-water emulsion; PRR: Pattern-recognitionreceptor; TLR: Toll-like recptor; THJ/TH2: T helper 1- or 2- biased responses. Adapted from 32 Traditional adjuvants such as aluminum salts and water/oil emulsions have set the standard for safety and efficacy in vaccine development but fail to elicit effective immune responses to many candidate antigens, and diverse new adjuvant formulations have been pursued in both academic and industrial vaccine research 3 3 . Alum is an aluminum salt-based adjuvant currently in use for human vaccines against diphtheria, tetanus, Hepatitis B and Hepatitis A, to name a

.

few 2 9. Unfortunately, it is incapable of inducing long-lived humoral responses against HIV envelope antigens34. While water-in-oil emulsions are considered too toxic for prophylactic use in humans3 5 , oil-in-water emulsion have a better safety profile and have been licensed for several vaccines (MF59 by Novartis and ASO3 by GSK) 36 Toll-like receptors (TLRs) were the first set of PRRs to be identified and they are subsequently the most well characterized set as well. Currently, 10 functional human and 12 functional mouse TLRs have been identified in total. TLRs are transmembrane proteins, expressed by innate immune cells, which contain an ectodomain to recognize PAMPs as well as a cytosolic Toll-IL-1 receptor (TIR) domain to initiate downstream signaling. TLRs 12

recognize a variety of PAMPs from bacteria, mycobacteria, parasites, fungi, and viruses.

PAMPs recognized by TLRs include lipopolysaccharide (TLR-4), flagellin (TLR-5), cytosineguanine dinucleotide DNA (TLR-9), single-stranded RNA (TLR-7,-8), double stranded RNA (TLR-3), and lipopeptides (TLR-1,-2,-6)3 7 . After recognition of their respective PAMPS, TLRs initiate signaling cascades through TIR-containing adaptor proteins such as MyD88 and TRIF. These signaling cascases, as summarized in schematic 1-2, result in the secretion of inflammatory cytokines and type 1 interferon, which induces innate immune responses such

.

as neutrophil recruitment and macrophage activation as well as the maturation of dendritic cells (which is a key step in initiating adaptive immunity)3 7

Schematic 1-2. TLR trafficking and signaling. Illustration of the location of TLR receptors and their signalingpathways. TLRs 1, 2, 4, 5 & 6 are expressed on the cell-surface while TLRs 3, 7, 8, & 9 are localized to intracellularvesicles. Recognition of respective PAMP by a TLR induces TIR-dependent signaling cascades which culminate in type 1 interferon expression (TLRs 2, 3, 4, 7, 8 & 9) and/or inflammatory cytokine secretion (TLRs 1, 2, 4, 5, 6, 7, 8 & 9.) Adaptedfrom ".

13

Adjuvants developed for TLRs include monophosphoryl lipid A (MPLA), CpG ODN, R848, Poly I:C, and Pam3Cys; these are agonists to TLR 4, 9, 7 and 8, and 1,2 and 6, respectively 38 . At present, MPLA is the only agonist licensed for human use 39 . TLR adjuvants have been applied to HIV vaccine development in mice and non-human primate studies, with an improvement in immune responses over non-adjuvanted controls, however they will probably require additional adjuvant help via a delivery system3 8 . Indeed, in the Irvine lab, TLR-agonists with soluble antigen generate very weak immune responses in comparison to .

TLR-agonists loaded onto particulate delivery systems4 0'41

In addition to TLR agonists, in recent years other small molecule immunomodulators have been explored as potential adjuvants, including agonists of nucleotide-binding oligomerization domain-like receptors (NLRs), retinoic acid-inducible gene (RIG)- 1-like receptors (RLRs) and STING agonists 4 2 ,4 3 . The cytosolic nucleotide sensor STING (stimulator of interferon genes)

.

localizes to the endoplasmic reticulum and is a potent inductor of type I interferons in response to sensing cyclic dinucleotides (CDNs)4 2. The cyclic dinucleotides recognized by STING are small molecule second messengers used by all phyla of bacteria44, and are also produced as endogenous products of the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) 45-47 The canonical bacterial CDN, cyclic-di-guanosine monosphophate (cdGMP), has been shown to directly bind STING and subsequently initiate IRF3- and NF-KB-dependent immune responses41-50.

1.3. Motivation for nanoparticle-based adjuvant systems Nanoparticle-based adjuvants are very appealing for a number of reasons. First and foremost, synthetic nanoparticles enable vaccines to mimic virus particles while minimizing side effects 51. Virus-like particles (VLPs) are a prominent example of this approach and are discussed in further depth below. Particulate viral-mimicry offers vaccines three distinct advantages. First, in comparison to small soluble antigen, nanoparticle-linked antigen is efficiently taken up by antigen presenting cells, which is a crucial step to induce adaptive immune responses 1 52 . Secondly, nanoparticle-delivered antigen undergoes cross-presentation more efficiently than soluble antigen, resulting in MHC I presentation and greater induction of CD8+ T-cell responses5 2,53 . In addition, nanoparticles that are 20-100 nm in diameter efficiently enter the lymphatic system in free form, without requiring specialized transportation via dendritic cells 54. Free draining of vaccine to the lymph node enables antigen to directly interact with B-cells, which generates potent immune responses 5 1. In addition, by surface-displaying antigens on the surfaces of particulate carriers such as liposomes or polymer particles, antigen delivery can be modulated at the single-cell level. Surface display has been shown to enhance immune responses, likely by increasing the degree of B-cell receptor crosslinking and .

subsequent B-cell activation 5 -61

14

.

In addition to the viral mimicry benefits, there are several additional motivations that nanoparticles offer. In particular, nanoparticles serve not only as adjuvants, but as platforms which can be further engineered. The prime example of this is the inclusion of TLR-agonist adjuvants on nanoparticles to additionally enhance the immune responses4 0 4 1 62 . Secondly, certain nanoparticle formulations, such as lipid-coated PLGA particles, generate sustained levels of serum IgG antibodies at very low doses (10 ng of ovalbumin) of antigen40 Conversely, vaccines adjuvanted with TLR agonist and alum at such low antigen doses fail to generate immune responses. Potency at such low doses strongly suggests the potential of nanoparticles as vaccine adjuvants.

Virus-like particles, as mentioned above, are an excellent example of the viral mimicry benefits that nanoparticles may offer. VLPs consist of antigen-functionalized viral capsid components which self-assemble into nanoparticles that induce both innate and adaptive immune responses 63. Virosomes-which are semi-synthetic virus-like particles-are in use in several non-American markets for hepatitis A and influenza vaccines and the VLP-based

.

vaccine Gardasil* is FDA-approved for prevention of human papillomavirus infection 6 '. In addition to their uptake by APCs and efficient drainage to lymph nodes, surface display of antigen on VLPs is attributed to greater degrees of B-cell receptor crosslinking and subsequent B-cell activation 5 . Virus-like particles have been developed for delivery of gpl20 antigens, albeit without sufficient immunogenicity, which may be attributed to the low rate of gp120 incorporation into VLPs and thus work is ongoing to increase this incorporation efficiency 66' 67 In addition to the low immunogenicity of VLPs for HIV antigens, designing VLPs is a nontrivial process which can make varying the antigen-of-study cumbersome. Although many different nanoparticles have been developed for drug and vaccine delivery, liposomes are a particularly attractive option. Effective yet safe, liposomes have been recognized for more than 35 years for their potential as immunological adjuvants 6 . Non-toxic themselves, liposomes are efficacious by presenting antigen in particulate form, which prolongs antigen half life 69. In addition, small unilamellar liposomes (40-150 nm) are excellent virus-mimics in terms of APC uptake, cross-presentation, and lymphatic draining. Liposomal antigen may either be encapsulated or surface-displayed, the latter of which is achieved either by specific chemical linkage, non-specific adsorption by electrostatic interactions, or the

inclusion of a hydrophilic tail. During liposome formation, antigen with a hydrophilic tail will insert itself into the phospholipid bilayer. Cell-targeting of lipids can be achieved by inclusion in the lipid bilayer of antibodies to specific cell receptors 7 0 . Liposomes also confer the ability to deliver both hydrophobic molecules (in the lipid bilayer) and hydrophilic molecules (encapsulated or surface-displayed). A major benefit of liposomes is their elimination of

MPLA's solubility issues. MPLA is a lipopolysaccharide-derived TLR-4 agonist and a potent inducer of both humoral and cellular immunity, however, it is prone to aggregation with

15

.

decreases its bioavailability. Inclusion of MPLA in the lipid bilayer of liposomes eliminates aggregation events while maintaining its adjuvant abilities2 9 There are several liposomal vaccines currently approved or in development. A lyophilized liposomal formulation, Stimuvax®, is currently being investigated for treatment of non-small cell lung cancer". The liposomal adjuvant ASO1, developed by GlaxoSmithKline, consists of liposomes with MPLA and QS21 (a saponin) and is in development as an adjuvant for a malaria vaccine. In Phase III trials, ASOl elicited high antibody titers and prevented a substantial number of malaria cases71 ' 7 2 . ASO 1 is also in development as an HIV vaccine adjuvant. A phase I clinical trial indicated superior CD4' T-cell activation in the ASO 1 group over the two other .

adjuvants used, ASO2v and ASO2A (oil-in-water emulsions)73

As discussed in Chapter 1.1, the membrane proximal external region of gp4l (MPER) is actively being pursued as a possible HIV antigen because of the isolation of broadly neutralizing antibodies which bind to it. A key element to consider when designing MPERbased vaccines is that MPER is closely associated with the HIV virion lipid membrane and this lipid membrane association holds MPER in a conformation which may be essential to elicit broadly neutralizing antibodies like 2F5 and 4E10 74 . Surface display of MPER on liposomes is a simple and effective method to maintain the lipid-specific confirmation of 2F5/4E10binding liposomes. Stealth liposomes are liposomes whose surfaces are shielded by poly(ethylene glycol); they were originally developed to extend the half-life of drug-loaded liposomes after intravenous injection to improve drug delivery to tumor sites 69. Application of PEGylated liposomes as vaccine adjuvants is less common. For CTL-based vaccine approaches, stealth liposomes with encapsulated antigen have been shown to both decrease and enhance CD8+ T-cell activation in comparison to non-PEGylated liposomes 75- 7 7 . In a humoral-based vaccine study, 20kDa PEGylated liposomes were used for delivery of surface-displayed gp4l and induced higher immune responses than non-PEGylated liposomes, although this was not maintained over time7 . The effect of PEGylation on humoral immune responses against MPER is explored in Chapter 4. Despite the well-established adjuvant properties of liposomes, there is no clearly defined optimal liposome composition. This lack of clarity is due in part to the vast array of liposomal vaccine strategies. Indeed, the numerous options in lipid composition, size, membrane fluidity and surface charge, immunization route, immunization schedule, adjuvant/small molecule inclusion, and antigen loading strategies, as well as the choice of antigen itself, manifests in the limitation that currently only general assumptions can be inferred from the literature. To mitigate these current limitations, chapters 3 and 4 systematically study liposomes to design the optimal liposome-based HIV vaccine

16

adjuvant/delivery vehicle for both a model HIV peptide antigen (MPER) and a model HIV protein antigen (gpl20).

1.4.Scope and outline of thesis In this thesis, we designed lipid particles loaded with immunomodulators as adjuvant/delivery systems for surface-displayed HIV antigens. After a comparison of synthetic unilamellar liposomes with lipid-coated microparticles which generate liposomes in situ, synthesis liposomes were chosen as the delivery vehicle of two HIV immunogens: a peptide (MPER) derived from the gp4l protein and an engineered recombinant gpl20 protein. The adjuvant capabilities of nanoparticle-loaded immunomodulators was explored, particularly

with the STING agonist cyclic-di-GMP. Chapter 2 describes the work with lipid-coated microparticles from which the rest of this thesis evolved from. Lipid-coated poly(lactide-co-glycolide) microparticles (LCMPs) consist of a solid polymer core wrapped by a surface lipid bilayer and previous studies demonstrated LCMPs elicit potent humoral immune responses in mice. Here we characterized the spontaneous delamination of the lipid envelope and subsequent in situ production of antigendisplaying liposomes. We then compared the immunogenicity profiles of LCMPs, bilayerstabilized LCMPs and synthetic liposomes. Chapter 3 was done in collaboration of Dr. Jordi Mata-Fink, who designed a recombinant gpl20 monomer as an HIV antigen. In this chapter, we explored the immunogenicity profile of this gpl20 construct with respect to display on liposomes or formulated in aluminum saltor emulsion-based adjuvants. Furthermore, we optimized formulation and vaccination parameters to enhance anti-gpl20 antibody responses. In Chapter 4 we systematically explored how the structure and composition of liposomes displaying MPER peptides impacts the strength and durability of humoral responses to this antigen as well as helper T-cell responses in mice. We compared liposomes to traditional adjuvant delivery systems. To characterize the impact of density of a surface-displayed antigen, we determined anti-vaccine responses as a function of the number of MPER peptides per liposome. We then explored the how size, PEGylation, and composition of liposomes impacts immunogenicity. CD4' T-helper peptides were included in the vaccines to provide CD4' T-cell help and we thus systematically explored how various methods of T-helper peptide delivery impacted humoral immunogenicity. Chapter 5 explores the adjuvant capability of STING agonists (cyclic dinucleotides (CDNs)) when encapsulated in PEGylated liposomes and co-delivered with MPER liposomes. 17

We determined the kinetics of drainage to the lymphatic system or blood stream, the kinetics of activation of lymph node antigen-presenting cells, and the impact on humoral immune responses for both soluble and nanoparticulate CDN. Furthermore, we explored the dependence of CDN-induced responses on the presence of plasmacytoid dendritic cells, TNF-

a signaling, and type I interferon signaling. Chapter 6 provides an overall summary and the broad conclusions of this thesis work as well as possible future directions. Chapter 7 details several key methods which were developed in for this work, as well as supplemental data. Chapter 8 is a bibliography of the literature cited in this thesis.

18

2. In situ vesicle shedding mediates antigen delivery by lipid-coated

2.1. Introduction Subunit antigen vaccines have increased safety but decreased potency profiles in comparison to traditional live attenuated microbe vaccines. To increase the immunogenicity of subunit vaccines, adjuvants thus play an important role in vaccine development. As discussed in Chapter 1.2, adjuvants are materials that enhance immune responses elicited by vaccines either by providing inflammatory signals (e.g., ligands for Toll-like receptors 79 ), modulating the delivery of antigen to immune cells, or both3 2 . For example, antigen delivery can be altered by providing a depot for long-term antigen release from a vaccination site. Long-term biomolecule release is often achieved by encapsulation of the cargo into a biodegradable polymer matrix, such as poly(lactide-co-glycolide) (PLGA), which is often employed due to its history of safe use in humans, efficient encapsulation of hydrophobic materials and tunable drug release behavior 80 . However, delivery of protein antigens encapsulated in PLGA microor nano-particles is challenging due to low antigen encapsulation efficiency and denaturation/aggregation of proteins during encapsulation and release 8 1 -83 . Surface-display of antigen on a lipid bilayer is an attractive alternative to encapsulation PLGA as it enhances immune responses and can be readily achieved by chemically linking the antigen to lipids containing reactive groups. For example, phospholipid headgroups functionalized with maleimide can readily link to cysteine-containing antigens. Furthermore, incorporation into lipid particles has previously been shown to be an effective delivery method of lipophilic adjuvants such as MPLA8 4' 5 . Despite the disadvantages of degradable polymers for use with protein antigen, these polymers remain attractive for the slow-release co-delivery of inflammatory adjuvant compounds that could shape the immune response over time In order to combine surface-display of antigen with a biodegradable core in which we could ultimately co-deliver additional adjuvant molecules, in work led by Dr. Anna Bershteyn, we previously described an approach for synthesis of lipid-enveloped polymer microparticles and nanoparticles that present antigen bound to a surface lipid bilayer 90 . A self-assembled lipidbilayer coat surrounding a PLGA core was achieved by using lipids as the surfactant component of an emulsion/solvent evaporation-based PLGA particle synthesis. The lipid bilayer was observed to be a two-dimensionally fluid surface that tightly envelops the polymer core. We employed these lipid-coated microparticles (LCMPs) as vaccine delivery agents by conjugating protein antigens to PEGylated lipids anchored in the bilayer coating, and coincorporating adjuvant compounds such as the TLR agonist monophosphoryl lipid A (MPLA) or a-galactosyl ceramide in the particles, LCMPs elicited high, durable humoral immune responses in response to injection of as little as 2.5 ng of the model antigen ovalbumin (OVA) surfaced-displayed on LCMPs 40. In addition, these particles triggered antigen-specific 19

proliferation of both CD4' and CD8' T-cells and production of Thl-biased cytokines from Tcells in vivo". When formulated as nanoparticles and functionalized with a candidate malaria antigen VMPOO01 and MPLA, LCMPs were shown to induce germinal center formation and elicited higher, more durable antigen-specific titers IgG antibodies of diverse isotypes .

compared to vaccination with soluble VMP001 and MPLA9 1

Despite the efficacious nature of these lipid-coated particles, it was unclear how they presented antigen to the immune system, particularly in the case of LCMPs, because these microparticles (diameter: 2.6 1.2 pm) did not freely drain to lymph nodes 92. However, during initial cryo-TEM characterization studies on the LCMPs, we observed that over time, lipid bilayers at the surface of the biodegradable particles begin to delaminate from the polymer core90 . This observation of delamination suggested that the lipid bilayer might not be stable on the PLGA particle cores over time. Since antigen was conjugated to the lipid bilayer, we hypothesized that delamination of the lipid envelope could play a role in the adjuvant characteristics of LCMPs. In this chapter, we directly evaluated the stability of the bilayer coating of LCMPs and examined the role of bilayer delamination in the immunogenicity of this particulate vaccine system. We found that under physiological conditions, LCMPs exhibit rapid bilayer delamination, leading to the release of antigen-bearing lipid vesicles. We evaluated the kinetics of bilayer shedding and the resulting effects on the immunogenicity of LCMPs in vivo. In addition, we explored the kinetic dependence of lipid delamination on the presence of lipid/serum in the surrounding environment. To test the hypothesis that delamination impacts immunogenicity, stabilized-bilayer LCMPs were developed either by the inclusion in the lipid bilayer of cholesterol or lipids with saturated carbon chains. Mice immunized with OVALCMPs generated higher anti-OVA titers than mice immunized with stabilized-bilayer OVALCMPs or OVA on delaminated lipid vesicles (DLVs) alone. These results suggest that the in situ release of delaminated lipid vesicles enhances humoral immune responses to surfacedisplayed antigen, with LCMPs acting as a source of in situ-generated antigen-bearing liposomes following injection. The majority of this work has been published in a first author publication, Biomacromolecules (2014);15(7):2475-813.

2.2. Materials and Methods 2.2.1.

Materials

All lipids-1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero3-phoshpo-(1'-rac-gylcerol) (DOPG), 1,2, distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2 distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene

glycol)2000] 20

(DSPE-PEG2K-maleimide), B sulfonyl) rhodamine

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine 1,2-distearoyl-sn-glycero-3(14:0 Liss-Rhod-DOPE),

phospohethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-DSPE) and cholesterolwere purchased from Avanti Polar Lipids (Alabaster, Alabama). Poly(lactic-co-glycolic acid) (PLGA) with a 50:50 ratio of lactic acid and glycolic acid and an inherent viscosity of 0.42 dL/G was purchased from Evonik Corporation (Birmingham, Alabama). Monophosphoryl lipid A (MPLA,from Salmonella enterica serotype minnesota Re 595 cat. no. L6895) and solvents were purchased from Sigma Aldrich (St. Louis, Missouri). n-succinimidyl sacetyl(thiotetraethylene glycol) (SAT(PEG) 4) was purchased from Pierce Biotechnology (Rockford, Illinois). Purified ovalbumin (OVA) was purchased from Worthington Biochemical (Lakewood, New Jersey) and subsequently passed through detoxi-gel endotoxin removing columns (Pierce Biotechnology, Rockford, Illinois) to remove any trace endotoxin. 2.2.2. Synthesis of lipid-coatedparticlesand liposomes Microparticles consisting of a PLGA core and lipid bilayer envelope were synthesized as

previously reported. 4 0 90 Briefly, 5mg of lipid in a 72:18:10 DOPC:DOPG:DSPE-PEG2Kmaleimide molar ratio (for DOPC-LCMPs) or a 75:16:9 DSPC:DOPG:DSPE-PEG2Kmaleimide molar ratio (for DSPC-LCMPs) was dried under nitrogen followed by incubation under vacuum at 25'C for 18 hr. The resulting lipid film was dissolved in dichloromethane (DCM) containing PLGA for a final polymer:lipid weight ratio of 16:1. This organic solution was emulsified into distilled deionized ultrapure water by homogenization at a ratio of 8:1 aqueous phase:organic phase and stirred for 12 hr at 25'C to remove DCM by evaporation and passed through a 40 pm filter. Microparticles were isolated from the resulting polydisperse samples by two centrifugation steps at 1,100 RCF for 1 min each, with removal of the supernatant and resuspension into pH 7.4 PBS following each centrifugation step. Particle size distributions were determined using the Horiba Partica LA-950V2 Laser Diffraction Particle Size Analysis System.

Liposomes prepared with a 72:18:10 molar ratio of DOPC:DOPG:DSPE-PEG2Kmaleimide were used for immunization studies and vesicles with an 80:20 molar ratio of DOPC:DOPG were used for in vitro lipid delamination studies. Lipid films dried as described above were resuspended in pH 7.4 PBS, vortexed for 30 seconds every 10 min for 1 hr, subjected to 6 freeze-thaw cycles in liquid nitrogen and a 37 0 C water bath, and extruded for 21 passes through a 200 nm pore polycarbonate membrane (Whatman Inc, Sanford, Maine). Vesicle sizes were determined by dynamic light scattering. (Brookhavenn 90Plus Particle Size Analyzer, Worcetershire, UK). Liposomes were stored at 4'C until use.

2.2.3. Antigen conjugation onto lipid-en veloped particles and liposomes Thiolated ovalbumin was conjugated to the surface of maleimide-functionalized lipidenveloped particles or liposomes as previously described 40 . In brief, endotoxin-free ovalbumin 21

was functionalized with the heterobifunctional cross-linker SAT(PEG) 4 (Pierce Biotechnology, Rockford, Illinois), which was then deacetylated to expose sulfhydryl groups

following the manufacturer's instructions. Following buffer exchange into 10 mM EDTA (pH=7.4), via 7000 mwco Zeba spin desalting columns (Pierce Biotechnology, Rockford, Illinois), thiolated OVA (5 mg/mL) was incubated with particles (70 mg/mL) or liposomes (3 mg/mL) at 25'C for 4 hr (for particles) or overnight (for liposomes). To remove unbound antigen, particles were washed three times by centrifugation for 5 min at 10,000 rcf with pH 7.4 PBS and liposomes were washed three times by centrifugation in 30 KDa MWCO Vivaspin columns (Vivaproducts, Littleton, Massachusetts). The amount of OVA coupled was determined by solubilizing lipids from the particles/vesicles in 30 mM Triton-X100 and measuring the quantity of OVA by enzyme-linked immunosorbent assay (ELISA). Particles and liposomes were stored at 4'C until use, which was within 4 hours for immunization experiments and 48 hours for in vitro experiments.

2.2.4. Analysis of lipid delaminationfrom LCMPs Particles were synthesized as described above, incorporating 2 mole % of 14:0 Rhod-

DOPE (for DOPC-LCMPs) or NBD-DSPE (for DSPC-LCMPs) in the lipid composition. For characterization of the delamination of protein antigen displayed on the lipid envelope, OVA was conjugated to lipid-enveloped particles as described above. Post-synthesis, particles were washed 3 times by centrifugation at 5,000 r.c.f. for 5 minutes and subsequent suspension in pH 7.4 PBS. After the third wash, particles were suspended at 12 mg/mL in pH 7.4 PBS, fetal

bovine serum, or 10 mM 80:20 DOPC:DOPG liposomes in pH 7.4 PBS, divided into 150 uL aliquots in separate eppendorf tubes for each time point/replicate and incubated with rotation at 37 0 C. At each time point, replicate aliquots were centrifuged for 20 min at 16,100 r.c.f. and the resulting supernatant collected for analysis. Lipid release from the LCMPs was determined by adding 30 mM Triton-X100 to the supernatants, measuring rhod-DOPE fluorescence in a fluorescence plate reader (Tecan Infinite M200 Pro, Mannedorf, Switzerland), and normalized to the total amount of fluorescent lipid present. OVA released from particles was determined by anti-ovalbumin ELISA on the supernatants of the particle aliquots and normalized to the total amount of OVA-lipid present. This total amount of lipid per aliquot was determined in fluorescently-tagged samples by addition of Triton to three or four standard aliquots, incubated at 55'C and subsequently vortexed and sonicated for 1 min each prior to centrifugation for 15 min at 16,100 r.c.f. and fluorescent-based quantification of the supernatant. To determine the total amount of the antigen, ovalbumin, released from DOPC-LCMPs particles, the same procedure as above was employed minus the 55 0 C incubation step and with ELISA-based quantification. The 55'C incubation step is unnecessary for lipid delamination from DOPCLCMPs and therefore was omitted to prevent any degradation of the ovalbumin protein.

2.2.5.

Size characterizationof delaminatedlipid vesicles

22

DOPC-LCMPs were prepared and incubated at 37'C in pH 7.4 PBS for 7 days, after which the microparticles were pelleted via a 30 minute centrifugation step at 16,100 r.c.f. The size distribution of DLVs in the supernatant was determined by laser diffraction as described above. 2.2.6.

In vivo immunization studies

All animal experiments were conducted under an IUCAC-approved protocol in accordance with local, state, and NIH animal care and use guidelines. Immunizations were carried out on female BALB/c mice, 6-7 weeks of age, purchased from Jackson Laboratories. Immediately prior to immunization, 1.3 ig of the TLR-4 agonist MPLA per 50 pL was mixed with 10 ng OVA conjugated to LCMPs, DLVs, or liposomes in sterile pH 7.4 PBS, following postsynthesis insertion techniques described previously 40,94. Mice were immunized by injection of 50 ptL solutions s.c. at the tail base, and boosted 14 days later. Serum samples were collected on a weekly basis for analysis of serum antibody titers.

2.2.7. Antibody titer analysis Serum total IgG titers, isotype IgGi and IgG2A titers, and avidity indices were determined as previously described. 4 0 Briefly, 96-well plates were coated with OVA and blocked with bovine serum albumin, then incubated with serially diluted serum and detected with HRPlabeled anti-mouse IgG, IgGi or IgG2A (Bio-Rad), followed by development and measurement of optical absorbance at 450 nm. Antibody titer is reported as reciprocal serum dilution at an absorbance of 0.5. For avidity indices, duplicate serum dilutions were prepared for each sample and for one set of dilutions, wells were incubated for 10 min with 6 M urea prior to detection with the respective anti-mouse secondary antibody. The avidity index is reported as the ratio of the titers of the urea treated sample to the non-urea treated sample.

2.2.8. Statistical analysis Statistical analyses were performed using GraphPad Prism software. Comparisons of formulations over time were performed using two-way ANOVA tests and comparisons of multiple formulations at a single time point were performed using one-way ANOVA tests. Two-tailed unpaired t-tests were used to determine statistical significance between two experimental groups for all other data.

2.3. Results and Discussion 2.3.1. Delamination kinetics of lipid-coated microparticles

23

We previously reported that phospholipids incorporated into PLGA particles during an emulsion/solvent evaporation synthesis segregate to the surface of nascent particles, selfassembling into a lipid envelope surrounding the polymer core (Fig. 2-1A). When these particles were incubated in pH 7.4 PBS at 37'C for 7 days to permit partial hydrolysis of the biodegradable particle core, cryoTEM imaging revealed evidence of delamination of lipid bilayers from the particle surfaces, which was observed even in the absence of added MPLA, suggesting that adjuvant incorporation did not induce this effect90 . This finding suggested that

lipids might be shed from LCMPs by "budding" of lipid bilayers from the particles over time (Fig. 2-1A). This might be particularly promoted in vivo, since serum albumin and lipoproteins are known to extract lipid from fluid bilayers 95- 97. To directly test this hypothesis, LCMPs with a diameter of 2.54 0.95 pm (Fig. 2-1B) were incubated in PBS at 37'C for one week. After this incubation time, the PLGA particle cores were still macroscopically intact'o, and the size distribution of the particles recovered by centrifugation were essentially unchanged from the starting material (data not shown). However, analysis of the supernatant by laser diffraction to detect released lipid vesicles revealed nanoparticles with a mean size of 176 6 nm in the LCMP supernatants (Fig. 2-iC). These particles were not PLGA fragments, as neat PLGA nanoparticles of this size prepared independently were pelleted by the centrifugation step used to remove LCMPs from the sups in this experiment. To verify that these nanoparticles in the LCMP supernatant were in fact lipid vesicles, we prepared particles containing a rhodaminetagged lipid tracer in the bilayer coating. Fluorescence measurements on the supernatant collected from LCMPs incubated 7 days in PBS at 37 0 C showed the release of 54 11% of the total lipid tracer into the supernatant, confirming the release of delaminated lipid vesicles (DLVs) from the microparticles over time.

24

Antigen PEG

B

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0

0

2

4 6 8 Diameter (pm)

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Figure 2-1. Delamination of lipid vesicles from lipid-coated nanoparticles. (A) Schematic structure of as-synthesized LCMPS with surface-conjugatedprotein antigen and vesicles "budded"from PLGA polymer core, forming delaminated-lipidvesicles (DLVs). (B) Size distributionoffreshly synthesized microparticlespost-wash steps determined by laser diffraction. (C) Size distributionof delaminatedvesicles releasedfrom LCMPs after 7 days in PBS at 3 7'C determined by laser diffraction. We next characterized the kinetics of DLV shedding from LCMPs. Microparticles containing rhodamine-labeled lipid were incubated in PBS and DLVs released into the supernatants over time were detected by fluorescence spectroscopy. As shown in Fig. 2-2A, although lipids remained stably associated with LCMPs at 4'C, vesicles were rapidly shed from the particles at 37'C in PBS, with delamination reaching a plateau after 24 hrs. To test the effect of serum on lipid delamination kinetics, LCMPs containing rhodamine-conjugated 25

lipid were incubated in either serum or PBS and delamination was quantified as before. Fig. 22A shows that serum increased the fraction of delaminated lipid by 1.6-fold, with substantial vesicle shedding within 4 hours that continued slowly through 48 hours. We hypothesized that interactions of the lipid surface layers with lipid droplets in serum may be a major contributor to vesicle delamination, as the adsorption of lipids by serum lipoprotein particles is essential for lipid transport in vivo 98. Previous studies have shown that liposomes are destabilized in the presence of serum due to the transfer of phospholipids to lipoproteins 95,96 99 . To model interactions of LCMPs with lipids in serum, a group of microparticles was incubated in PBS containing 10 mM of 200 nm-diameter synthetic 4:1 DOPC:DOPG liposomes. The results indicate that the inclusion of liposomes in the aqueous buffer replicates the kinetics of lipid delamination in serum (Fig. 2-2B), thus suggesting the presence of environmental lipid promotes DLV delamination from LCMPs. LCMPs carrying protein antigen covalently linked to the membrane (e.g., as illustrated in Fig. 2-lA) elicit robust humoral immune responses in vivo*0' 9 1. To test whether antigen conjugated to the lipid coat is transferred to delaminating vesicles, thiol-functionalized ovalbumin (OVA) was conjugated to maleimide-functionalized PEG chains incorporated into the particle bilayer coating, and its release over time into serum at 37'C was quantified by ELISA. As expected, lipid-conjugated OVA was shed from the LCMPs with kinetics matching rhodamine-labeled lipid delamination (Fig. 2-2C). Altogether, these data suggest that LCMPs rapidly shed submicron liposomes in conditions mimicking interstitial fluid to which the particles would be exposed to in vivo during immunization.

26

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Figure 2-2. Kinetics of lipid delamination from lipid-coated microparticles. Kinetics of lipid delaminationfrom LCMPs in vitro determined by monitoring release of fluorescently-labeled lipid tracer (A-C) or PEG-lipid-conjugated OVA (C) into the supernatants of particles over time. (A) Release of rhodamine-lipid into the supernatantof LCMPparticles was assessedas afunction of temperaturein pH 7.4 PBS or 100%fetal bovine serum. (p < 0.0001 comparing 37'C serum to 37C PBS, 4'C serum to 4'C PBS, 37C serum to 4'C serum, and 37 0 C PBS to 4 0C PBS. (B) Lipid release kinetics for rhodamine-lipidlabeled LCMPs incubated in PBS containing 10 mM unlabeled DOPC/DOPG liposomes at 37 0 C. (C) LCMPs conjugated with OVA protein were incubated in 100% fetal bovine serum at 37C and OVA accumulation in the supernatantwas assessedover time by ELISA analysis of LCMP supernatants.

2.3.2. Immunogenicity of delaminated vesicles and lipid-coated microparticles Given the rapid shedding of liposomes from LCMPs in the presence of serum, we hypothesized that vesicles spontaneously released from the microparticles following injection could play an important role in the immunogenicity of LCMP vaccines. To explore this possibility, we prepared OVA-conjugated LCMPs and incubated a fraction of the particles at 37 0C in PBS to induce delamination, followed by collection of the supernatant containing shed vesicles. The concentration of antigen in the shed vesicle preparation was measured by ELISA, and mice were then immunized with MPLA mixed with 10 ng OVA carried by purified delaminated vesicles or the parent (non-delaminated) particle fraction. In addition, a third group of mice were immunized with OVA-conjugated pure liposomes prepared with the same lipid composition as the LCMPs, to control for possible changes in the lipid structure or composition occurring during "budding" of vesicles from the PLGA-core particles. Each group of mice was boosted on day 14 with identical formulations, and serum was collected over time for analysis of titers of anti-ovalbumin IgG. As shown in Figs. 2-3A and B, DLVs and the control synthetic liposomes elicited essentially identical OVA-specific IgG responses. Both liposomal vaccines were somewhat less immunogenic than intact LCMPs, eliciting average 27

antibody titers 2-fold lower than LCMPs. However, DLVs were still capable of priming a strong immune response to this low dose of OVA, which elicited undetectable anti-OVA titers in 3 out of 4 animals when administered as a soluble vaccine mixed with MPLA (Figure 23A). Titers in all 3 particle immunization groups were maintained over at least 70 days post

priming (Fig. 2-3B). Although DLVs elicited weaker OVA-specific IgGi and IgG2A antibodies than parent LCMPs (Fig. 2-3C), both groups exhibited identical IgG2A/IgGl ratios (Fig. 2-3D). As IgG2A is considered indicative of "Thl-like" responses and IgGi "Th2-like" responses, this result suggests both the lipid-coated microparticles and shed liposomes primed balanced Thl/Th2 responses, and that the small difference in titers comparing LCMPs and shed vesicles reflects a difference in strength of priming rather than different Th-biasing of the antibody response. Altogether, these data suggest that delamination of antigen-bearing liposomes plays a critical role in the immune response primed by LCMPs carrying surface-bound antigens.

28

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29

2.3.3. Immunogenicity of lipid-stabilizedmicroparticles Although Fig. 3 demonstrates that in vitro-generatedDLVs induced slightly lower antibody titers than parental LCMPs, it remained unclear whether in vivo budding of antigen-carrying vesicles from the microparticles was necessary for the high immunogenicity of the lipid-coated microparticles. If in vivo delamination were essential, then LCMPs that failed to undergo lipid delamination would be expected to prime weaker immune responses. To test this hypothesis, we sought to prepare LCMPs with lipid envelopes stabilized against delamination. We tested two strategies to create such stabilized-envelope particles: incorporation of high-TM lipid and incorporation of cholesterol into the lipid coating. Phospholipids with high melting temperatures have few/no unsaturated bonds in their acyl tails, allowing the lipids to pack tightly and favoring formation of liquid crystalline gel phases. In addition, the removal of double bonds in the acyl chains of phospholipids in vesicles reduces the rates of lipid transfer from liposomes to serum lipoproteins by four fold10 0 . Therefore,

DSPC (TM = 55C) was used in place of DOPC (TM = -20'C), to generate "high-TM" DSPCLCMPs. In vitro, inclusion of high-TM lipid did not block vesicle shedding completely but did lower the fraction of lipid lost from the particles in the presence of serum by 33%, as shown in Fig. 2-4A. We tested whether the reduced shedding of vesicles would impact the immunogenicity of these particles compared to DOPC-based LCMPs. Balb/c mice were

immunized s.c. on day 0 and day 14 with MPLA mixed with DOPC-LCMPs or DSPC-LCMPs each carrying 10 ng OVA. As shown in Fig. 2-4B, despite the modest reduction in lipid shedding exhibited by DSPC-LCMPs, the immunogenicity of these particles was significantly altered, as mice immunized with DSPC-LCMPs showed a 3.5-fold reduction in titers of OVAspecific antibodies compared to DOPC-LCMPs at one week post-boost (Fig. 2-4B). Futhermore, DSPC-LCMPs elicited 64 10% lower total OVA-specific IgG titers (p = 0.0023, Fig. 2-4C) and lower serum IgGi titers at the post-boost peak on day 21 (p = 0.0067, Fig. 24D) when compared to DOPC-LCMPs. Interestingly, the avidity index of the antibody response elicited by DSPC-LCMPs was higher than that of the response primed by to DOPCLCMPs from day 28 onward (p = 0.0491 comparing the two groups over time). However, the overall strength of the antibody response elicited by LCMPs was reduced when vesicle shedding was impeded by incorporation of high-TM lipids.

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Figure 2-4. LCMPs prepared with high-Tm lipids show reduced vesicle shedding and weaker antibody responses in vivo. (A) Kinetics of in vitro lipid releasefrom high-TM lipid-enveloped microparticles(DSPCLCMPs) or regular, low-TM lipid microparticles (DOPC-LCMPs) in serum, determined by following fluorescent lipid tracer released into particle supernatants upon incubation with FBS at 37 0 C (p < 0.0001). (B-E) Balbic mice (n = 5) were immunized on days 0 and 14 with 1.3 pg MPLA mixed with DOPC-LCMPsor DSPC-LCMPs, each conjugatedwith 10 ng OVA. (B) OVA-specific antibodies detected by ELISA as a function of serum dilution at the postboost peak, day 21. (p < 0.0001) (C) Mean OVA-specific IgG serum titers on days 21, 38 and 45. (DOPC LCMPs vs DSPE LCMPs over time, p = .0023) (D) OVA-specific IgGi and IgG2A isotype serum titers at day 21. (DOPC IgGi vs. DSPE IgGi, * = p value of 0.0067) (E) Mean OVA-specific IgG avidity indices as afunction of time. (p = 0.0491)

To further test the idea that vesicle "budding" from antigen-conjugated LCMPs is important for their immunogenicity, we also tested a second strategy for inhibiting liposome shedding from the microparticles. Cholesterol is a major component of cell membranes and is often used as a stabilizing agent in lipid vesicle preparations because it orders and condenses fluid-phase bilayers' 0 1 -0 3 . Thus, we hypothesized that cholesterol, like high TM lipids, could also act to stabilize the lipid bilayer of LCMPs. We prepared DOPC-LCMPs incorporating 0, 1, or 10 mg cholesterol per 80 mg PLGA. In vitro, DLV formation was not inhibited completely 31

by the inclusion of cholesterol but delamination did decrease with increasing cholesterol quantity. As shown in Figs. 2-5A and B, increasing the amount of cholesterol incorporated in the particles lowered the fraction of lipid shed into solution from LCMPs, although the effect was less pronounced in serum than in PBS, perhaps due to cholesterol absorption by lipoprotein particles in serum. As with the high-TM lipid LCMPs, we tested the immunogenicity of LCMPs with cholesterol by vaccinating balb/c mice. A plot of mean ova-specific IgG endpoint titers shows decreased immunogenicity (up to a 2.5-fold average drop in titers) with increasing cholesterol content (Fig. 2-5C) (Comparison of 10 mg vs 1 mg titer over time, p

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Figure 5-1. NP-cdGMP enhances lymph node uptake of cyclic dinucleotides. (A) Groupsof balb/c mice were injectedwith dGMPDY547 and CDN in draininglymph nodes (top panel, 2 pg cdGMPDy547/mouse, n=3/group) or serum (lower panel, 5 pg cdGMPDY547/mouse, n=5/group)were traced byfluorescence spectroscopy. (B) Representative flow cytometry plots of CDN fluorescence in APCs 24 hrs following s.c. injection of 2 pg cdGMPDY547or NP- cdGMPDY547. (C) Mean percentagesof cdGMPDY547+ of APCs in inguinal or axillary lymph nodes at 24 hrs after immunization of NP- or soluble cdGMPD547 (n=3/group). *, p < 0.05; as determined byANOVA followed by Tukey's multiple comparison test. (D) Kinetics of cdGMP DY547 releasefrom NPs incubatedin PBS containing10% serum at 37'C. (E) Representative images of DC2.4 cells after 2 hour incubations with NP- or soluble cdGMPDY547. (F) Percent OVAAF647+ of dendritic cells over time following injection of NPcdGMP and NP-OVAAF647 (n=4/group). 66

5.3.2. NP-cdGMP induces type I IFN directly in lymph nodes and elicits greaterAPC activation than soluble CDN To determine the impact of enhanced lymph node delivery on the adjuvant activity of cyclic dinucleotides, we evaluated CDNs as adjuvants for a poorly immunogenic vaccine antigen, the membrane proximal external region (MPER) from HIV gp4l. The MPER antigen, which as part of the HIV envelope trimer is thought to reside in juxtaposition to the viral membrane 123,124, was formulated as a palmitoyl-anchored peptide displayed on the surface of liposomes that also contained a helper epitope derived from gpl20 (denoted HIV30 15 ) tethered by PEG to the inner leaflet of the vesicles (Figure 5-2A). We have previously shown that this nanoparticle MPER formulation (NP-MPER) elicits extremely weak anti-MPER humoral responses in the absence of co-administered molecular adjuvants13 6 . Thus, we compared NPMPER vaccines adjuvanted with soluble cdGMP or NP-cdGMP (Figure 5-2A). We first assessed the activation of antigen presenting cells (APCs) in dLNs following cdGMP/NP-MPER immunizations. Twenty hours after NP-MPER + NP-cdGMP vaccination, both CD86 and MHC-II were strongly upregulated on CD8a' and CD8a- dendritic cells (Figure 5-2B-C), while NP-MPER + soluble cdGMP elicited much weaker CD86 and MHC-II expression. CD86 and CD69 were also upregulated on a large proportion of B-cells following NP-cdGMP immunization, while soluble cdGMP induced these activation markers on a minority of these cells (Figure 5-2D). Tracking the frequencies of CD86+ APCs over time revealed distinct dynamics for APC activation induced by soluble vs. nanoparticle cdGMP: DC activation was low and peaked at 48 hr following NP-MPER vaccination with soluble cdGMP as adjuvant. By contrast, vaccination with NP-cdGMP as adjuvant elicited -4-fold higher frequencies of activated DCs, which remained elevated for 48 hr before decaying toward baseline (Figure 5-2E). In addition, the peak frequency of macrophage activation was 3-fold higher in NP-cdGMP-adjuvanted vaccination than in soluble cdGMP-adjuvanted vaccination. As expected, non-adjuvanted NP-MPER vaccines elicited lower frequencies of activated APCs than either of the cdGMP-adjuvanted groups. The induction of activation markers on a majority of lymph node APCs following NPcdGMP immunization indicated that many more APCs were activated than directly acquired cdGMP, indicating strong in trans activation of B-cells and DCs by cdGMP+ cells. To distinguish between direct action of CDNs locally in dLNs vs. stimulation of lymph nodes remotely via cytokines produced at the vaccine injection site, we evaluated the expression of IFN-, a signature product of STING activation by cdGMP18 2,1 83 . RT-PCR analysis of dLNs showed that NP-MPER + NP-cdGMP immunization induced robust expression of both IFNBJ and its downstream gene target RSAD2 that peaked 20 hr post-injection, reaching 35-fold higher levels relative to soluble cdGMP vaccination (Figure 5-2F). Non-adjuvanted vaccines showed minimal IFN-P expression at any time point. Thus, concentration of CDNs in LNs by 67

nanoparticle delivery induced by a much greater frequency of activated APCs and higher expression of activation markers on a per-cell basis compared to vaccination with unformulated CDNs, which correlated with evidence for direct activation of type I IFN expression in lymph nodes by the nanoparticle formulation.

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69

5.3.3. Nanoparticle delivery of cdGMP enhances expansion of helper T-cells and promotes germinal center induction Follicular helper T-cells (TFH Cells) provide critical signals to germinal center B-cells in support of humoral immunity184 . To test the impact of cdGMP adjuvants on the expansion of vaccine-specific helper T-cells and TFH cell differentiation, we used an adoptive transfer model employing OVA-specific OT-II TCR-transgenic CD4' T-cells and liposomal MPER vaccines incorporating the OT-II cognate peptide antigen instead of HIV30 as a helper epitope. CD90.1' OT-II CD4+ T-cells were adoptively transferred into C57B/6 recipients, which were immunized 24 hrs later with MPER vaccines carrying the OT-II epitope with or without addition of cdGMP or NP-cdGMP. One week post-immunization animals were sacrificed for flow cytometry analysis. As shown in Figure 5-3A, immunization with liposomes lacking Thelper peptide or using soluble cdGMP adjuvant failed to significantly expand OT-II' cells compared to unadjuvanted liposomes. By contrast, vaccination with MPER/OT-JI + NPcdGMP induced a substantial expansion of the transferred OVA-specific T-cells, with 5.1-fold more OT-II+ cells recovered on day 7 compared to soluble cdGMP-adjuvanted vaccines (Figure 5-3A-B). The greater increase in T-cell expansion for NP-cdGMP seen in total cell counts compared to cell frequencies was a result of substantially greater lymph node swelling and total LN cell numbers in NP-cdGMP-vaccinated mice (data not shown). The frequency of antigen-specific T-cells differentiating to a CXCR5+PD-1+ follicular helper phenotype was only slightly greater for NP-cdGMP- vs. soluble- cdGMP adjuvanted vaccines (Figure 5-3C), but the much greater overall expansion of the antigen-specific T-cell population in the former vaccines led to 5.3-fold more OT-II TFH cells (Figure 5-3D). In parallel, we assessed endogenous T-cell responses to NP-MPER/OT-II + CDN vaccines in C57B/6 mice (with no adoptive transfers). Splenocytes from all of the vaccine groups that received helper peptide produced IL-4 and IL-5 when restimulated with OT-II peptide ex vivo, but only animals vaccinated with NP-cdGMP as adjuvant also produced IFN-y and TNF-a (Figure 5-3E).

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Type I IFN and TNF-aplay complementary rolesfollowing NP-cdGMP

vaccination The discordance between the frequency of APCs directly taking up NP-cdGMP and the extent of APC activation elicited by this adjuvant suggests that cytokine signaling in trans plays a critical role in the adjuvant activity of CDN nanoparticles. Type I interferons and TNF-a are major downstream products following cdGMP-induced activation of STING" 5' 1 91 and given the strong upregulation of IFN-O expression in lymph nodes 20 hr after NP-cdGMP immunization, we first examined the role of type I IFN in the response to NP-cdGMP immunization. To test the role of type I IFN signaling, mice were administered a blocking anti78

IFN-a/p receptor antibody (anti-IFNAR1) or its isotype control and then immunized with NPMPER + NP-cdGMP. 24 hr post-immunization, animals treated with anti-IFNUR1 showed greatly reduced frequencies of CD86+ B-cells, macrophages, CD8aI+ DCs, and CD8t- DCs relative to the isotype control group (Figure 5-8A). However, this reduction in early APC activation had no influence on early antibody titers against the HIV30 helper peptide at day 14 (Figure 5-8B). Blaauboer et al. previously observed that antibody responses to mucosal vaccines adjuvanted with soluble cdGMP were independent of type I IFN but dependent on TNF-a 19 1 . Although Blaauboer et al. used TNFR1-/- mice to demonstrate the role of this cytokine on the adjuvant action of cdGMP, we chose to investigate this question by

administration of an anti-TNF-a blocking antibody, due to the absence of follicular dendritic cell networks observed in TNFR1-- mice 19 2 . Twenty-four hours post-immunization with NP-

MPER and NP-cdGMP, TNF-a-blocked mice exhibited marginal impairment in APC activation (Figure 5-8C), but at two weeks post-prime, TNF-a-blockade reduced vaccinespecific antibody titers 115-fold (Figure 5-8D). Thus, cdGMP targeted to lymph nodes via nanoparticle delivery acts through type I IFN and TNF-a to promote early APC activation and class-switched IgG production, respectively.

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80

5.3.7.

Discussion

Cyclic dinucleotides are a relatively new class of immunomodulatory compounds with the potential to promote protective immunity through a unique pathway employing the cytosolic The danger sensor STING and its downstream transcription factors NF-KB and IRF-3 42". publication of crystal structures demonstrating the structural basis for CDN sensing through STING 14 1'1 93 and the identification of endogenous CDNs as signaling products produced by cyclic GMP-AMP synthase sensing of double-stranded DNA 4 6 47"1 94 have provided both a rationale and mechanistic guidance for the development of cyclic dinucleotides as adjuvants acting through type I interferons and NF-kB activation in host cells. Indeed, several laboratories have recently demonstrated innate immune stimulatory 5 5 ,162 ,191 and adjuvant activities'0 with CDNs, particularly when applied as mucosal adjuvants. For example, Ebensen et al. first demonstrated that intranasal administration of soluble antigen and cdGMP induced higher antigen-specific serum IgG and mucosal IgA when using the model antigen pgalactosidase 5 6 . When administered intranasally with the S. pneumonia antigen pneumococcal surface protein A, cdGMP protected against S. pneumonia colonization 195 . The ability of structurally-related CDNs such as c-di-IMP and c-di-AMP to adjuvant mucosal immunizations 58 9 6

"

.

has also been reported

In contrast to the robust response to cyclic dinucleotides in mucosal immunization, the efficacy of CDNs as adjuvants in the setting of traditional parenteral immunization has been less clear, with much of the early published data focused on the use of CDNs to boost immunity against highly immunogenic model antigens and/or employing large doses of cyclic dinucleotides. As shown here, the limited potency of parenterally-administered CDNs reflects poor lymphatic uptake of these small molecule immunomodulators; they are instead cleared from tissues via the blood. This biodistribution issue is a property of molecular size: because the blood absorbs -10-fold more fluid from tissues than lymph, molecules small enough to .

permeate blood vessels (< -1 KDa) tend to show predominant clearance to the blood 9 7 Therefore, increasing CDN-mediated vaccine responses through increased dosing of unformulated CDNs is accompanied by parallel increases in systemic inflammatory toxicity. This pharmacokinetic behavior is shared by other small-molecule adjuvant compounds such as resiquimod (R848) and related imidazoquinoline TLR7/8 agonist compounds, muramyl dipeptides that trigger NLRs, and RNA oligonucleotide ligands of RIG-I 63 ,198 ,1 9 9. For example, parenteral injection of R848 is known to rapidly trigger systemic inflammatory cytokines similar to the systemic signature observed here for CDNs, and induces transient systemic

.

lymphopenia within hours of injection 172,200. To overcome these issues, a number of strategies have been explored to limit systemic exposure and/or re-focus molecular adjuvant delivery to lymph nodes, including conjugation of adjuvant compounds with lipid tails 200-202, directly coupling to large molecular weight antigens203,204, or encapsulating in nano/microparticles 62 ,86,1 68,1 72

81

Here we demonstrate that nanoparticle delivery using PEGylated liposomal carriers substantially enhances the potency of the canonical CDN cyclic di-GMP, eliciting high titer, durable humoral responses to a model weakly immunogenic antigen at low doses of NPcdGMP. These responses were only matched by -30-fold higher doses of unformulated CDNs that may not be translatable to large animals or humans, and which induced substantial systemic cytokine induction. This increase in potency was achieved by increased lymph node

accumulation of NP-cdGMP by 15-fold relative to soluble cdGMP injection. NP-cdGMP was avidly pinocytosed/endocytosed by dendritic cells in vitro, and despite the fact that these PEGylated nanoparticles were not designed to promote delivery of CDNs to the cytosol, we observed robust APC activation in vivo. However, it has recently been reported that STING is capable of sensing cyclic dinucleotides contained within impermeable vacuoles of host cells during Chlamydia trachomatis infection and it is hypothesized that the ER wraps around the vacuole, enabling ER-localized STING to detect vacuole-entrapped cdGMP 20 . Thus, APCs may harbor intrinsic mechanisms to sense endosomal CDNs. Type I interferons are signature downstream products of STING activation by CDNs in host cells 182 18 , 3 , and NP-cdGMP led to direct induction of type I IFN and downstream target gene expression in the draining lymph nodes. By contrast, soluble CDN administration induced no type I IFN in the dLNs, implying that its effects on APC activation are mediated by inflammatory cytokines produced at the injection site acting remotely on draining nodes. Such in transactivation of dendritic cells by inflammatory cytokines has been shown to elicit weaker priming of T-cell responses compared to cis activation of DCs directly through pathogen sensing receptors 206 ,207. Direct type I IFN induction in LNs by NP-cdGMP correlated with more robust upregulation of costimulatory and activation markers on LN APCs, which occurred earlier and was more sustained (over -48 hr) compared to soluble CDN vaccination. Coincident with enhanced APC activation, NP-cdGMP drove a 5-fold increase in vaccinespecific CD4+ T-cell expansion compared to unformulated cyclic dinucleotides. Thus, nanoparticle delivery of CDNs enhanced both early antigen presenting cell activation and subsequent T-cell responses. Our subsequent analyses focused on determining the impact of NP CDN delivery on humoral immunity, employing a liposomal gp4l peptide as a model poorly immunogenic vaccine antigen. First we examined the induction of antigen-specific follicular helper cells, because type I IFN induction in LNs by NP-cdGMP might be expected to drive TFH differentiation 20 1. Nanoparticle delivery of cdGMP only moderately affected the frequency of CD4' T-cells differentiating toward a follicular helper phenotype, but the much greater overall expansion of antigen-specific CD4' T-cells by NP-cdGMP compared to soluble CDN adjuvants in turn translated into 5.3-fold more vaccine-specific TFH cells. In line with their expected importance in class switching and germinal center formation18 4 , increased numbers of specific TFH were accompanied by enhanced germinal center B-cell differentiation, 11-fold

82

.

increased antigen-specific IgG titers, and humoral responses that were more durable than vaccines administered with the well-known TLR agonist MPLA. However, sera from these MPER-immunized animals did not neutralize HIV (data not shown), indicating that further structural refinements are required for this antigen to elicit antibodies capable of recognizing the functional stalk of the HIV envelope trimer 13 6 The presence of interferon expression suggesting STING activation directly in dLNs led us to explore the source of type I IFNs following NP-cdGMP vaccination and to quantify the relative importance of the key downstream products of STING activation on the observed humoral responses. Plasmacytoid DCs are major producers of type I IFN, but paradoxically these cells appeared to be strongly ablated following immunization with cdGMP. pDC depletion was detected by 20 hr post immunization, contemporaneous with peak interferon expression, suggesting these cells are not the source of the type I IFN induced by NP-cdGMP. This finding is in agreement with a prior study reporting minimal human pDC activation by CDNs 5. In further support of the idea that pDCs do not play an important role in the adjuvant function of CDNs, pDC depletion had no impact on either early APC activation or subsequent IgG production following NP-cdGMP immunization. While type I interferons are a characteristic product of STING activation, CDNs also trigger the production of TNF-a through NF-KB18 3,191 . Both type I IFN and TNF-a have been shown to be important regulators of humoral immunity1 74 ,2 0 ,210 but a recent study suggested that cdGMP applied intranasally adjuvants mucosal immunity in an type I IFN-independent, TNF-a-dependent manner191 . Here we found that following NP-cdGMP vaccination, both pathways of STING signaling are

involved, with early APC activation dependent on IFN-a signaling and early (day 14) classswitched antibody responses dependent on TNF-a. In the interest of translational relevance, we employed a PEGylated liposome formulation with a composition similar in nature to clinically approved liposomal drugs 211 . A limitation of our bench-scale formulation approach was the relatively low CDN loading efficiency (-35%). However, more advanced polymer or lipid nanoparticle carriers 4 0' 4 1 that achieve more efficient drug loading and/or lipophilic modifications to CDNs to promote liposome association (a strategy used successfully with imidazoquinoline adjuvants 200) can readily be envisioned to circumvent this issue. Delivery of pattern recognition receptor agonists as adjuvants has variously been reported to require co-encapsulation in the same particle as the antigen (in Cis) 212 or simply simultaneous delivery on separate particles (in trans)62, 17 2 . The ability of NP-cdGMP to adjuvant immune responses in trans to particulate antigen provides flexibility for vaccine production and facilitates inclusion of NP-cdGMP into existing vaccine platforms. Although many danger signals are effective in promoting immune responses in mice, type I IFN-inducing adjuvants have shown particular promise in non-human primate models 213 ,2 14 and humans 211,2 16 for promoting superior cellular and humoral immunity. The nanoparticle delivery strategy demonstrated here provides a simple means to promote both the

83

safety and efficacy of cyclic dinucleotides as a novel type I IFN-promoting adjuvant with potential relevance to human vaccine development. Altogether, these results suggest that lymph node-targeted CDNs can promote both strong antigen-specific T-cell priming and hightiter, durable antibody responses that outperform the strong benchmark adjuvant MPLA, suggesting these compounds are of interest for further development as candidate adjuvants.

5.4. Conclusions Cyclic dinucleotides (CDNs) are agonists of the innate immune receptor stimulator of interferon genes (STING) and are of interest as potential molecular vaccine adjuvants. However, cyclic di-GMP (cdGMP) injected subcutaneously showed minimal uptake into lymphatics/draining lymph nodes (dLNs) and instead distributed rapidly to the bloodstream, leading to systemic inflammation. To redirect cdGMP to dLNs, we encapsulated the CDN in PEGylated lipid nanoparticles, which blocked systemic dissemination of the compound and enhanced its accumulation in dLNs 15-fold compared to unformulated cyclic dinucleotide. Combining CDNs with a liposomal HIV gp4l peptide as a model poorly immunogenic vaccine antigen, nanoparticle-cdGMP (NP-cdGMP) robustly induced type I interferon in dLNs, induced 5-fold greater expansion of vaccine-specific CD4+ T-cells, and greatly increased the number of germinal center B-cells in dLNs compared to soluble CDN. Further, NP-cdGMP promoted durable antibody titers >10-fold higher than the well-studied TLR agonist monophosphoryl lipid A, and comparable to a 30-fold larger dose of unformulated cdGMP without the systemic toxicity of the latter. Thus, lymph node targeting of cyclic dinucleotides via nanoparticulate delivery simultaneously promoted enhanced safety and efficacy of CDNs, an approach broadly applicable to small molecule immunomodulators of interest for vaccines and immunotherapy.

84

6. Conclusions and future work 6.1.Lipid nanoparticles as vaccine adjuvants We have designed and characterized a system for vaccine delivery that utilizes several advantages of lipid nanoparticles: surface display of antigen, efficient lymphatic drainage, and versatile packaging of immunomodulating adjuvants and T-helper peptides. While liposomes have a long history of use as adjuvants, this thesis is uniquely comprehensive in its exploration of liposomes for both peptide and protein antigens, its characterization of how liposome and its use of liposome-loaded impact humoral immunogenicity, properties here are directly applicable to described immunomodulators as adjuvants. While the results HIV vaccine development, they also broadly applicable to vaccine and cancer immunotherapy development in general. Small, unilamellar liposomes were the focus of the bulk of this work after the investigation into the potency of lipid-coated microparticles (LCMPs) in Chapter 2. Here, we found that the lipid bilayer of LCMPs delaminated off of the PLGA core, and formed 180 nm diameter liposomes. We demonstrated that the in situ formation of these delaminated liposomes was responsible for the enhanced immunogenicity of LCMPs compared to synthetic liposomes or stabilized-bilayer LCMPs (whose bilayer does not delaminate). The decision to pursue synthetic liposomes rather than LCMPs was made due to the significantly better conjugation efficiency of antigen to liposomes and furthermore, in previous studies performed by Anna Bershteyn, MPER-loaded LCMPs elicited significantly weaker anti-MPER titers than MPERloaded liposomes (data not shown). In studies utilizing liposomes with surface-displayed gpl20 monomer, humoral antibody responses were independent of the size of liposomes, the dose of gpl20, and presentation of gp120 (Fc-4G or 4G). Conjugation of gp120 to liposomes was essential for humoral responses; soluble gpl20 and plain liposomes alone elicited no response. Interestingly, anti-MPER titers proved to be more sensitive to liposome parameters. Density of MPER peptides per liposome, the presence of higher-TM lipid (DMPC), and the size of liposome all affected MPER humoral responses. Due to the ease of MPER liposome synthesis (MPER peptide is produced synthetically in large quantities and MPER liposomes do not require purification), MPERliposomes developed into our primary tool for studying the adjuvant abilities of liposomes. As it became clear that we had optimized liposome parameters (size, composition, etc) and yet still had only moderate immune responses, we shifted focus to providing CD4' T-cell help and

PAMP-based help. Although no particular form of T helper peptide construct resulted in significantly stronger anti-MPER titers, the cleaved form of DSPE-S-S-HIV30 (present only on the inner bilayer of 85

liposomes) was an ideal construct because it generated strong Thl/Th2 cytokine responses and minimal off-target anti-HIV30 titers. With respect to PAMP-based help, we developed liposome-encapsulated cyclic dinucleotides (CDNs) as a potent co-adjuvant formulation. Soluble CDN is rapidly disseminated systemically while liposomal CDN efficiently traffics to draining lymph nodes, where is induces the activation of APCs, the secretion of type I interferon, expansion of germinal centers B cells and vaccine-specific CD4+ T-cells, and ultimately robust and long-lived anti-vaccine IgG responses. This TLR-independent adjuvant system is particularly attractive in its ability to synergize with TLR-based agonists (such as MPLA) and because CDN liposomes can co-delivered with any antigen formulation, which allows for rapid prototyping building off of existing vaccine systems.

6.2.Potential future work

.

Although 4G is an exciting potential HIV immunogen due to its ability to elicit VRCO1competing antibodies, we found that overall, the magnitude of antibody response elicited was too long to enable the purification of enough anti-gp120 antibodies to test for broadlyneutralizing antibodies when MPLA was the adjuvant. In future work, the potential of CDN liposomes to adjuvant gpl20 liposome vaccines should be explored. In general, since CDN liposomes worked very well with the weakly immunogenic MPER peptide, it would be interesting to explore the application of CDN liposomes with other peptide-based cancer or infectious disease vaccines 217 While we optimized the adjuvant and delivery parameters of the MPER vaccine system, there remains a fair amount of work to be done in antigen design. The palm-MPER induces antibodies focused primarily at the C-terminus (away from the palmitoyl anchor). As the bnAb 4E10 binds residues in the middle of the MPER peptide, the Ellis Reinherz group isactively working to find an MPER construct which elicits antibodies more 4E10-like. Furthermore, the stability of palm-MPER when exposed to serum is also an issue. Development of an improved anchor for MPER is in progress with a synthetic lipid strategy (Tyson Moyer of the Irvine Lab) and by utilizing the transmembrane region of gp41 (the Ellis Reinherz group). Lastly, although the CDN liposome formulation is a potent adjuvant, to our knowledge, this is the first report of CDN nanoparticles as an infectious disease adjuvant. This means that there is a lot of exciting, unexplored scientific space within the field of nanoparticulate CDN. For example, the ability of CDN nanoparticles to elicit CD8+ T-cell responses is unknown and the use of CDN (soluble or nanoparticulate) as a cancer vaccine adjuvant has thus far been limited to only a few studies 218 ,2 19 . In our studies, dendritic cells were major consumers of liposomal CDN; actively targeting DCs could enhance vaccine efficacy. Furthermore, we observed liposomal CDN uptake to be primarily in an endocytic manner; designing endosomal escape-capable CDN delivery vehicles could enable CDN dose-sparing

86

7. Appendix 7.1. Appendix A: Supplementary Figures

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Gating strategy for macrophage identification via flow cytometry

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Supplemental Figure S5-1: Gating to identify lymph node macrophages. Representative staining of naYve balbic mice for lymph node macrophages,which were defined as B220~NK1.J-CDJ1c-CD1JbLy6G-SSC"of single live cells. Foractivation studies, CD86* macrophageswere defined by histogram gating.

87

Identification of plasmacytoid dendritic cells and confirmation of plasmacytoid dendritic cell depletion in vivo

7.1.2.

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88

7.1.3.

Stability of palm-MPER on liposomes

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7.2. Appendix B: Protocols

Although not comprehensive, the protocols included here are techniques developed in this thesis work that are not currently used by other members in the Irvine lab, but could foreseeably

be useful in the future. Please don't hesitate to contact me with any questions or clarifications about these procedures.

7.2.1. ELISA to quantify the loading of ovalbumin on liposomes 1. Coat wells of MAXIsorp NUNC plates with 100 uL/well of l0ug/mL cc-chick egg albumin Ab (Sigma A6075, Clone OVA-14). Incubate O/N at RT. 2. Empty plates, add 300 uL/well of PBS 1% BSA. Incubate for at least 2 hrs at RT. 3. Now these plates can be sealed and stored until use. 4. Set up sample dilutions in a V-bottom plate. (Typically 1:100 initial diluation and 4x serial dilution.) 5. Prep 2 sets of serially diluted OVA at 1000 ng/mL as initial dilution for standard.

89

6. Wash a-OVA-coated plates 4x. Transfer 100 uL/well of sample dilutions to each plate. Incubate 1 hour @ RT. Don't rotate. 7. Wash 4x. Add 100 uL/well of 1:5000 a-ovalbumin-HRP (from Abcam) in PBS 1% BSA. Incubate 45 mins. 8. Wash 4x. Add 100 uL/well of TMB. Incubate 20 min. 1. Stop TMB development with 50 uL H 2 SO 4 . Read absorbance on plate reader at 450 nm, with background at 540 nm. 9. For surface-displayed ova on liposomes, it's unnecessary to use a surfactant (eg Tween 20 or Trition-100) to disrupt the liposomes prior to this ELISA. However, if quantifying an encapsulated protein, use of a surfactant is essential. Be sure to also include the surfactant in the serial dilution buffer and ova-standard buffer to account for possible signal alteration due to surfactant presence.

7.2.2. ELISA for the detection of anti-gp120 (4G) antibodies 1. Coat wells of MAXIsorp NUNC plates with 100 uL/well of poly-L-Lysine (Sigma, cat. no. P0879) at 0.5 mg/mL in PBS. Rotate for 4 hrs @ RT (or O/N) 2. Empty plates, add 200 uL/well of PBS 1% BSA. Incubate O/N @ 4'C. 3. Now these plates can be stored wrapped until use. 4. Wash 4x, add 100 uL of 50 nM 4G gpl20 (no Fc group). Rotate 2 hours at RT. 5. Meanwhile, set up serum dilutions. 6. Wash peptide-coated plates 4x. Transfer 100 uL/well of serum dilutions to each plate. Incubate 1.5 hours @ RT. 7. Wash 4x. Add 100 uL/well of 1:5000 a-mouse IgG-HRP in PBS 1% BSA. Incubate 1 hour. 8. Wash 4x. Add 100 uL/well of TMB. (Common stock in the door of MCH fridge.) Incubate 20 min. If you have more than 4 plates, stagger the addition of TMB by 2 mins each. Keep the plates in wash buffer till right before the TMB addition. 9. Stop TMB development with 50 uL H 2 SO4. Read absorbance on plate reader at 450 nm, with background at 540 nm.

7.2.3. ELISA to quantify the loading of gp120 on liposomes 1.

2.

Coat wells of MAXIsorp NUNC plates with 2.5 ug/mL mouse B12 (anti-gpl20) antibody in PBS for four hours. (Mouse-B 12 was produced by Jordi Mata-Fink, Wittrup Lab.) Block overnight with PBS 1% BSA.

90

3.

Prep serial dilutions of gpl20-liposome samples as well as standard gpl20 protein (starting at 20 nM).

4.

Wash mB12 plates 4x, add 100 uL/well of serial dilutions. Incubate 1 hr at RT.

5. 6.

Wash 4x, incubate 45 mins with 1:2000 anti-his HRP at RT, 100 ul/well. Wash 4x. Add 100 uI~well of TMB. Incubate 20 min.

7.

Stop TMB development with 50 uL H2SO4. Read absorbance on plate reader at 450 nm, with background at 540 nm.

7.2.4. ELISA to determine VCRO1-competition ability of anti-gp120 sera 8.

Coat wells of MAXIsorp NUNC plates with 100 uL/well of poly-L-Lysine (Sigma Aldrich, cat. no. P8954) at 0.5 mg/mL in PBS. Block, 4G @ 50nm, rotate for 2 hrs @ 4'C (or O/N)

9.

Empty plates, add 200 uL/well of PBS 1% BSA. Incubate O/N @ 4'C.

10. Now these plates can be stored wrapped until use.

11. Wash 4x, add 100 uL of 50 nM 4G gp120 (no Fc group). Rotate 2 hours at RT. 12. Set up 1:100 serum dilutions for each sample. Best to do in duplicate if possible. Include serum from naive mice as well. 13. Wash 4G-coated plates 4x. Transfer 100 uL/well of serum dilutions to 4G-coated plates. Incubate 1.5 hours @ RT. Do not rotate.

14. Empty plates (don't wash), and add 100 uL/well of 50 nM VRC01 in PBS 1% BSA 15. Incubate for 1 hour at room temp, do not rotate.

16. Wash 4x. Add 100 uL/well of 1:5000 a-human-Fc-IgG-HRP in PBS 1% BSA (which detects VRCO1 binding). Incubate 1 hr.

17. Wash 4x. Add 100 uIJwell of TMB. Incubate 20 min. 18. Stop TMB development with 50 uL H 2 SO 4 . Read absorbance on plate reader at 450 nm, with background at 540 nm. 19. Divide absorbance signal per each sample by the average naive serum signal (represents 100% binding) to determine the fraction of competed VCRO1 binding.

7.2.5. UV-based quantification of CDN encapsulation efficiency into liposomes Background: Although it's possible to detect cyclic dinucleotides via HPLC, we were unable to entirely remove trace lipids from the supernatant of Airfuge-pelleted liposomes, and found that these trace lipids over time built up in C18 columns, resulting in unreliable CDN peaks and were extremely difficult to remove from the columns. Therefore, we developed this method to quantify the encapsulation of CDN in liposomes via basic UV spectroscopy. The peak absorbance for c-di-GMP is 254 nm, and we found there is a 91

slight shift in signal due to lipid presence. Thus, this method first determines the amount of lipid present in the CDN liposome supernatant and then adds this amount of lipid to the CDN standard curve, thus standardizing the signal due to lipid presence. Due to variance in centrifugation efficacy via Airfuge, it is important to measure the lipid presence each time CDN liposomes are prepped. Preparationof StandardLipids Prepare plain liposome batch (10 mg total lipid) - 2:2:1 DOPC:DMPC:DOPG with 5% DSPE-PEG. Resuspend in 1200 uL PBS and synthesize as normally. 2. Airfuge liposomes for 30 mins and collect and store supernatant. 3. Prepare and store lipid standard curve (dilutions of supernatant) in PBS: 100%, 75%, 50%, 20%, 10%, and 0% lipid supernatant in PBS. 1.

Sample processing 1. Airfuge CDN liposomes for 1 hr. Measure initial volume (VI), supernatant volume (V2) and final CDN liposome volume (V3). 2. Add 80ul of CDN sup to cuvette, as well as 80ul of each standard from lipid standard curve. 3. Measure UV at 350nm. 4. Open UV template on excel and plot lipid standard curve. This template can be found on the DUMAS server: IRVINELAB - PROTOCOLS - Melissa. 5. Fill CDN UV and copy linear equation from fit on corresponding cell. 6. Under "vol" a number will appear, corresponding to the amount of plain liposome supernatant necessary to add to each standard to have the same lipid concentration as CDN supernatant. 7. Prepare 60, 40, 30, 20 and 10 ug/ml CDN standards. Take "volume of standard to add" from Excel sheet and add it to "vol" of plain liposome supernatant. 8. Prepare two lOx dilutions of CDN supernatant in PBS. 9. Transfer 80ul of samples and standards to cuvettes. 10. Read UV 254nm. 11. Add standard curve values on excel sheet, fill in CDN sup values and copy equation from linear fit of plot. 12. Make sure to fill in V1, V2, V3, and update original ug of CDN added, as well as mice needed to immunize to get final volumes needed.

92

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