Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations
2019 Design and evaluation of single-dose polyanhydride nanovaccines against bacterial respiratory pathogens Danielle Ann Wagner Iowa State University
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by
Danielle Ann Wagner
A dissertation submitted to the graduate faculty
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Major: Immunobiology
Program of Study Committee: Michael Wannemuehler, Co-major Professor Balaji Narasimhan, Co-major Professor Bryan Bellaire Marian Kohut Randy Sacco
The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this dissertation. The Graduate College will ensure this dissertation is globally accessible and will not permit alterations after a degree is conferred.
Iowa State University
Ames, Iowa
2019
Copyright © Danielle Wagner, 2019. All rights reserved. ii
DEDICATION
I dedicate this dissertation to my mother, Denise.
For instilling in me a love of science and the determination to chase my dreams. iii
TABLE OF CONTENTS
Page
LIST OF FIGURES ...... v
LIST OF TABLES ...... vii
ACKNOWLEDGMENTS ...... viii
ABSTRACT ...... ix
CHAPTER 1. LITERATURE REVIEW ...... 1 Rational Vaccine Design ...... 1 Adjuvants: An Immunologist’s Secret Weapon ...... 3 Bacterial Respiratory Diseases and Prevention Strategies ...... 9 Trained Innate Immunity ...... 14 Humoral Immune Response ...... 16 References ...... 25
CHAPTER 2. ROOM TEMPERATURE STABLE PSPA-BASED NANOVACCINE INDUCES PROTECTIVE IMMUNITY...... 40 Abstract ...... 40 Introduction ...... 41 Materials and Methods ...... 46 Results ...... 50 Discussion ...... 57 Acknowledgments ...... 63 Conflict of Interest Statement ...... 63 References ...... 63
CHAPTER 3. SINGLE-DOSE COMBINATION NANOVACCINE INDUCES BOTH RAPID AND LONG-LIVED PROTECTION AGAINST PNEUMONIC PLAGUE ...... 70 Abstract ...... 70 Introduction ...... 71 Material and Methods ...... 75 Results ...... 82 Discussion ...... 95 Acknowledgements ...... 100 Author Contributions ...... 100 Conflict of Interest Statement ...... 101 References ...... 101 iv
CHAPTER 4. COMBINATION NANOVACCINE PROVIDES PROTECTION AGAINST YERSINIA PESTIS IN MICE WITH IMMUNOLOGICAL DEFICIENCIES ...... 106 Introduction ...... 106 Materials and Methods ...... 108 Results ...... 112 Discussion ...... 119 References ...... 123
CHAPTER 5. CO-ADJUVANT APPROACHES TO EARLY-ONSET PROTECTION ...... 126 Introduction ...... 126 Materials and Methods ...... 129 Results ...... 133 Discussion ...... 137 References ...... 140
CHAPTER 6. DISCUSSION ...... 142 References ...... 145
APPENDIX: DOCK2 MUTATION PRESENCE IN ENVIGO FACILITIES ...... 147 v
LIST OF FIGURES
Page
Figure 2.1 PspA nanovaccine characterization...... 51
Figure 2.2 PspA nanovaccines are capable of initiating protective immune responses...... 53
Figure 2.3 Nanovaccines with 25-fold reduction in total protein provided protection against lethal challenge...... 56
Figure 2.4 Room temperature-stored PspA nanovaccine maintains efficacy after 60 days...... 58
Figure 3.1 Characterization of F1-V Nanovaccine...... 83
Figure 3.2 Combination Nanovaccine provides rapid protective immunity against lethal Y. pestis challenge...... 85
Figure 3.3 Combination Nanovaccine provides long-lived protective immunity against lethal Y. pestis challenge...... 88
Figure 3.4. Combination Nanovaccine provides long-lived antibody responses to F1-V...... 89
Figure 3.5 Combination Nanovaccine induces class-switched antibody responses to F1-V...... 90
Figure 3.6 Combination Nanovaccine induces long-lived antibody responses in BAL fluid against F1-V...... 91
Figure 3.7 Combination Nanovaccine and CDN Vaccine provide broad antibody responses to potentially protective linear epitopes...... 93
Figure 3.8 Long-lived antibody responses to F1-V originate from long-lived plasma cells in the bone marrow...... 94
Figure 3.9 Long-lived anti-F1-V IgG1 and IgG2c antibody secreting cell responses in bone marrow and spleen...... 95
Figure 4.1 Efficacy of various F1-V containing vaccine regimen against lethal Y. pestis CO92 challenge...... 114
Figure 4.2 Long-Term Protection Observed in Adjuvanted Vaccine Formulations...... 116 vi
Figure 4.3 Varying Humoral Immune Response Observed Between Differentially- Sourced Mice...... 118
Figure 5.1 Protection against lethal Y. pestis challenge at 5 and 10 DPI...... 135
Figure 5.2 Neutralization of IFNγ and TNFα decreases overall survival...... 136
Figure 5.3 Analysis of serum cytokine expression...... 137 vii
LIST OF TABLES
Page
Table 1.1 List of known Yop proteins important for Yersinia pestis virulence ...... 14
Table 2.1 PspA Vaccine Formulations...... 53
Table 2.2 Dose-sparing PspA vaccine formulations...... 55
Table 4.1 Vaccine formulations...... 113
Table 5.1 F1-V vaccine formulations...... 134
viii
ACKNOWLEDGMENTS
I would like to thank my co-major professors Dr. Michael Wannemuehler and Dr.
Balaji Narasimhan, as well as my committee members Dr. Bryan Bellaire, Dr. Marian
Kohut, and Dr. Randy Sacco for their guidance and support over the duration of my graduate career, both in the classroom and in my research.
Additionally, I would like to thank my friends and colleagues in the
Wannemuehler and Narasimhan research groups, both past and present. A special thanks to the Bellaire lab group for their help in developing assays and running challenge experiments over the years.
For their unconditional encouragement and support, I would like to thank my family and close friends, who have all been instrumental to my success.
I would especially like to thank my esteemed colleague and great friend Sean
Kelly, without whom none of this work would have been possible.
Lastly, I would like to acknowledge financial support from NIH-NIAID (R01
AI111466 and R56-AI075026) and the Nanovaccine Institute.
ix
ABSTRACT
Design of a high-quality vaccine requires more than just initiation of a robust immune response, as there are several other factors that should be considered during development. An ideal vaccine should be efficacious and cost-effective, with minimal side effects and reactogenicity. Additionally, it is important to consider required number of doses and storage requirements.
Streptococcus pneumoniae is the major causative agent of bacterial pneumonia, a debilitating disease and the leading cause of death in children under the age of five worldwide. There are several existing vaccines against S. pneumoniae, though none are protective across all serotypes. Pneumococcal surface protein A (PspA), a key virulence factor of S. pneumoniae, shows promise as a target antigen for future vaccines, to address challenges presented by diversity of the capsular antigens.
Yersinia pestis, the causative agent of bubonic, septicemic, and pneumonic plague, induces a highly lethal infection if left untreated. There are no currently existing licensed vaccines against Y. pestis. New research using the recombinant fusion protein
F1-V, in vaccine formulations against pneumonic plague, have shown protection across several species.
Biodegradable polyanhydrides have been studied as a nanoparticle-based vaccine
(i.e., nanovaccine) platform for stabilization of labile proteins and enhanced shelf storage.
Nanovaccines have also been shown to have inherent adjuvanticity and induce humoral and cell-mediated immune responses. The STimulator of INterferon Genes (STING) agonists cyclic dinucleotides (CDNs) have been shown to be promising next-generation adjuvants capable of initiating rapid, protective humoral responses. x
Vaccine formulations, described herein, were comprised of either polyanhydride nanoparticles alone or in conjunction with the CDN R,R-CDG, against Y. pestis or S. pneumoniae. Immunization with a single-dose of these nanovaccine formulations, induced protective immunity against lethal challenge at both early (14 days post- immunization) and late (180 days post-immunization) time points, and maintained efficacy following shelf-storage at 25˚C for 60 days. The ability of these vaccine formulations to be efficacious following a single immunization, induce rapid and durable protective humoral responses, and maintain potency after shelf storage, highlight the potential of polyanhydride nanoparticles as a promising next-generation adjuvant and vaccine delivery platform. 1
CHAPTER 1. LITERATURE REVIEW
The introduction of vaccination, as a strategy against infectious disease, is largely considered one of the most influential developments in medicine. Vaccination has been responsible for the reduction in prevalence of diseases that were once the cause of severe morbidity and mortality worldwide (e.g., rinderpest, smallpox, polio). Beginning with the first observations of Edward Jenner in the 18th century, that inoculation with the cowpox variant of Variola virus conferred protection against smallpox, and leading to the development of what would be the strategy of live-attenuated vaccination, vaccines remain, to date, the most efficacious form of preventative medicine1.
New discoveries over the past decades have led to the development of many new antigens, adjuvants, and delivery platforms that allow modern-day scientists to further develop novel vaccination strategies. Coupled with furthered understanding of the immune system and pathogenesis of disease, we are better equipped now more than ever to design next-generation vaccines against infectious diseases.
Rational Vaccine Design
Designing a high-quality vaccine is about more than just initiating an immune response, as there are several other factors that should or need to be considered during development. An ideal vaccine should be efficacious and cost-effective, with minimal side effects and reactogenicity. Additionally, it is important to consider ease of administration, required number of doses, route of immunization, age and health status of immunized individuals, and storage requirements.
Storage of vaccine doses is of major concern in the health care community, as it is can be difficult to maintain cold storage and many existing vaccines are exposed to 2 temperatures, either above or below recommended temperature range, which may impact their potency2,3. Cold-chain storage of vaccines is so hindering, that many companies have started to develop solar-powered refrigerators for use in the field and in developing countries4. Development of shelf-stable vaccines would be beneficial both for global dissemination and reducing cost associated with storage.
Patient compliance can also be an obstacle to overcome when attempting to achieve proper herd immunity, as many individuals do not return for booster immunizations or the trained health care personnel are not available5,6. From this standpoint, design of single-dose efficacious vaccines is imperative. In addition to increasing percent of compliance, administering a single dose can also help to significantly reduce costs associated with vaccination, and allow for production of more doses, which is of extreme importance in the event of an outbreak. In addition to these concerns, single-dose immunization strategies may also confer better overall protection, particularly against those pathogens whose primary correlate of protection is opsonizing antibody. It has recently been demonstrated that repeat immunizations of human subjects can cause further class-switching of IgG1 to IgG4, which is known to be a poor opsonin7.
Route of immunization may also play a key role in the efficacy of a vaccine.
While oral immunization induces the production of local antigen-specific IgA and IgM, it has been recently reported that subcutaneous vaccination routes lead to the presence of higher levels of IgG systemically, as well as in the lungs8,9. Additionally, recombinant protein and inactivated/killed mucosal vaccines have been shown to be poor activators of cell-mediated immunity, showing decreased CD8+ T cell activation and proliferation as compared to systemic immunizations10. Therefore, when considering the rational design 3 of a vaccine against respiratory pathogens, it is important to consider that route of immunization could play a key role in conferring overall protection, especially for those pathogens where the correlate of protection is opsonizing antibody.
Adjuvants: An Immunologist’s Secret Weapon
Unlike many live-attenuated vaccines, those based on the use of subunit proteins, such as recombinant F1-V, are poorly immunogenic when administered alone11. In order to initiate a protective immune response to a vaccine, researchers must consider the incorporation of adjuvants into these formulations. Adjuvants are substances designed to enhance the body’s immune response to a given immunogen. Commonly used in vaccine formulations, they serve to improve efficacy through the induction of inflammation and pathogen-mimicking signals, helping to initiate an immune response to the target antigen12. They can also serve to better enhance delivery or stability of a vaccine formulation. The ideal adjuvant is inflammatory enough to initiate a proper immune response, without causing overt inflammation and administration-site tissue reactogenicity, and is able to provide additional stability to antigen of choice in the vaccine formulation13.
Licensed Adjuvants
Aluminum salts
One of the oldest and most widely-used adjuvants in vaccine development is aluminum salts (alum). First discovered in the 1920’s, aluminum salt-based vaccine adjuvants are the most prevalently used adjuvants in currently licensed vaccines14.
Antigens administered with alum are adsorbed to the adjuvant through anionic ligand exchange and electrostatic attraction, which may impact how antigens are seen by antigen presenting cells (APCs), specifically dendritic cells (DCs), in the early adaptive 4 response15–17. Vaccines adjuvanted with alum include formulations against: diphtheria, tetanus, pertussis, hepatitis A, hepatitis B, influenza, and pneumococcal pneumonia11.
These adjuvants have been shown to be strong initiators of Th2 immune responses, characterized by abundant antibody production, with little to no activation of cellular immune responses18. Current mechanism of action is not fully elucidated, but there have been numerous publications indicating that depot formation at the site of immunization created by alum leads to prolonged inflammatory signals that also contribute to the magnitude on the resulting immune response18,19.
AS04
Another FDA-approved adjuvant, currently used in the Ceravarix® vaccine,
Adjuvant System 04 (AS04) is the first combination adjuvant to be approved for use in the United States. Comprised of monophosphoryl lipid A (MPLA) and alum, AS04 has been shown to confer better cell-mediated immunity than formulations adjuvanted with alum alone, generating protective memory cytotoxic T cells in addition to long-lasting antigen-specific antibody11,20. MPLA is isolated from a deep rough mutant of Salmonella minnesota, and has been shown to have adjuvant properties similar to lipopolysaccharide
(LPS) as it relates to the stimulation of host cells through TLR4 in a pathogen-mimicking manner21. Combination of these two adjuvants (alum and MPLA) has been shown to increase the overall magnitude of the antigen-specific antibody titers, as compared to either component alone23.
MF59
MF59, an oil-in-water emulsion comprised of squalene droplets in aqueous solution, has been approved for use in the United States since 2015 as the adjuvant in
Fluad® seasonal influenza vaccine11. MF59 has been touted as a strong inducer of both 5 humoral and cellular immune responses, particularly in the elderly population22. Though the complete mechanism of action surrounding MF59 is still being fully elucidated, several studies have shown the production of pro-inflammatory cytokines (i.e. IL-2 and
IFN-γ) at the site of immunization, which led to the differentiation of DCs and have been shown to prolong germinal center persistence post-immunization22–24. While the last 30 years have yielded few new potent adjuvants (e.g., MPLA, AS04), further research is warranted to identify promising next-generation adjuvants.
Next-Generation Adjuvants
Cyclic dinucleotides
Cyclic dinucleotides (CDNs) are a novel class of adjuvants comprised of small molecules that act as second messengers. CDNs can be host-produced or bacterial in origin, signaling through STING (TMEM173) pathway, upstream of nuclear factor-κB
(NFκB) and interferon regulatory factor 3/7 (IRF-3/7)25–27. STING is a transmembrane protein located on the endoplasmic reticulum, which dimerizes and translocates to the perinuclear Golgi region after interaction with cytosolic DNA bound to cyclic GMP-
AMP synthase (cGAS) or dsDNA, recruiting TANK-binding kinase 1 (TBK-1), which phosphorylates STING and IRF-326,28.
Activation of STING by pathogen-secreted CDNs or host cytosolic DNA leads to the expression of type I interferons (IFNs) and pro-inflammatory cytokines highlighting the potential for CDNs as a potent immune activator29,30. Type I IFNs have been associated with increased antigen persistence within DCs by reducing endosomal and lysosomal acidification rates31. Type I IFNs have also been associated with the broadening of TCR specificity, augmenting proteasome peptidase activity allowing for a differential array of peptides to be presented on MHC I32. STING pathway signaling can 6 be initiated with host DNA that is the product of naturally-occurring DNA damage, leading to the production of type I IFNs that may serve to prime innate immunity for rapid responses to new pathogens25,33.
Of known CDNs, cyclic di-GMP (CDG) is the most common, produced by most gram-negative bacteria, and has been of interest as a potential vaccine adjuvant.
Administration of CDG as part of a vaccine formulation has been shown to induce balanced humoral immune responses, characterized by equal production of IgG1 and
IgG234. Additionally, it also activates cellular immune responses, leading to the production of IL-2, IL-4, IL-6, IL-12p40, IFN-γ, TNF-α, and increased lymphocytic proliferation34,35. Studies evaluating CDG as a potential adjuvant have largely focused on the induction of cell-mediated immunity and shown it to be a potent stimulator of cytotoxic T cell proliferation29. The ability of CDNs to facilitate the induction of both humoral and cellular immunity supports to inclusion of CDNs as a potent immunostimulatory adjuvant in vaccine formulations.
Polymer-based adjuvants
Biodegradable polymer-based vaccines are on the forefront of next-generation adjuvant and vaccine delivery platform technologies. Polymer-based biomaterials have been extensively studied as drug delivery platforms and biodegradable devices with surgical applications, but also show promise as potential adjuvants due to their biocompatible degradation products, potential ability to stabilize labile antigens, prolonged and customizable release kinetics, and their breadth of storage capabilities. Of the many researched polymer classes, the two that remain the most promising in potential applications are polyesters and polyanhydrides. 7
PLGA
Poly(lactic-co-glycolic acid) (PLGA) is the most successful polyester-based biodegradable polymer that has been studied to date. Upon hydrolysis, PLGA breaks down into easily metabolized monomers of lactic and glycolic acid and has a generally low phlogistic profile36. Current licensed applications of PLGA include biodegradable surgical sutures and microparticle drug delivery products37. PLGA can be fabricated into either micro- or nanoparticles containing antigen and are being extensively studied as vaccine delivery platforms38.
PLGA particles exhibit bulk-eroding degradation characteristics wherein water easily penetrates the polymer, sometimes resulting in quick release of the antigenic payload, and have a glass transition temperature (Tg) of 37˚C, making them very glassy and rigid at ambient temperatures36,39. PLGA-based nano- and microparticle vaccines have been associated with Th1 immune responses and the production of IFN-γ, IL-12p40 and antigen-specific IgG240–42. Recent studies have shown that a single vaccination with
PLGA microspheres containing tetanus toxoid initiated a comparable humoral immune response as a multiple-dose formulation of antigen adsorbed to alum43. PLGA is an appealing potential vaccine delivery candidate due to its history of FDA-approval, biocompatible degradation products, and ability to induce both humoral and cellular immune responses.
Polyanhydrides
Polyanhydride-based polymers have also been recently researched as potential vaccine candidates, with some polymers already being used in the FDA-approved drug delivery product Gliadel® wafer, composed of 1,3-bis-(p-carboxyphenoxy)propane and sebacic acid44. Comprised of varying ratios of sebacic acid (SA), 1,6-bis(p- 8 carboxyphenoxy)hexane (CPH) and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane
(CPTEG), the resultant co-polymers are biodegradable and have biocompatible dicarboxylic acid breakdown products and degrade through surface erosion45. Each individual polymer exhibits different degradation rates, with CPTEG exhibiting the highest rate of erosion, followed by sebacic acid at days to weeks before full degradation, with CPH having the slowest rate at months to possibly years45. Due to these varying erosion rates, altering the ratios of each monomer in the polymer formation allows for tailoring of the payload release kinetics46.
Following release, polyanhydride polymers have been shown to confer protein stability, conserving the functional activity of encapsulated proteins for up to four months47. Additionally, polyanhydride-based nanovaccines have been shown to maintain efficacy and induce protective immunity following storage at room temperature for two months48. Immunization with polyanhydride nanoparticles has demonstrated lower levels of administration site reactogenicity and inflammation as compared to more traditional adjuvants, such as alum and Freund’s Incomplete Adjuvant49.
Polyanhydride nanovaccines have shown to initiate protective immunity against a wide variety of bacterial and viral pathogens including Streptococcus pneumoniae,
48,50–53 Yersinia pestis, BRSV, and influenza virus . These formulations have also been shown to exhibit inherent adjuvanticity, capable of enhancing both cellular and humoral immunity, and contribute to dose-sparing capacity making them very promising adjuvants for expensive subunit protein vaccines48,50,51,54. Due to their characteristics of tailored payload release kinetics, protein shelf stability, adjuvanticity, biocompatibility, low injection reactogenicity, dose-sparing capabilities, and ability to induce protective 9 immunity following a single immunization, polyanhydride-based nanovaccines remain a promising next-generation vaccine delivery platform49,55,56.
Bacterial Respiratory Diseases and Prevention Strategies
Respiratory Infections
The mucosal surface of the respiratory tract is one of the body’s first lines of defense against infectious disease pathogens. Disease tolerance of the respiratory tract is mediated through physical barriers, innate immune activation, and the adaptive immune response. Physical barriers of defense are mediated by the production of mucus secreted by goblet cells, continual propelling of mucus by ciliated cells, club cells, and basal cells57,58. Cellular innate immunity in the respiratory tract is primarily mediated by alveolar macrophages and neutrophils, which rapidly produce type I interferons, antimicrobial peptides, and reactive oxygen species upon contact with pathogens, and secretion of naturally occurring antibody from innate-like B1a cells59–61. Components of adaptive immunity in the airway also include cytotoxic T cells, tissue resident memory T cells, and the local production of IgA and IgG62,63.
Infections of the respiratory tract manifest as either upper respiratory infections, such as those caused by the common cold, or lower respiratory infections, such as bronchitis and pneumonia64. Pneumonia occurs when there is significant swelling of the airway and the alveoli fill with interstitial fluid or inflammatory cells, and can be bacterial, viral, or fungal in origin65. Community-acquired pneumonia is the leading cause of death from infectious disease worldwide, leading to hospitalization in over 45% of all cases65. Though community-acquired pneumonia can be the result of many different pathogenic infections, the main causative agent is Streptococcus pneumoniae66. 10
Streptococcus pneumoniae
Streptococcus pneumoniae is a gram-positive bacterium, which colonizes the mucosal surfaces of the upper respiratory tract and is one of the most common bacterial respiratory pathogens worldwide67. Septicemic pneumococcal infections account for approximately 25% of all deaths in children under the age of 568. Colonization of the nasopharyngeal tract typically occurs within the first 6 months of life, with approximately
53% of children under the age of 6 being identified as carriers, as well as 4-11% of the adult population69,70. Streptococcus pneumoniae colonization is very common in healthy individuals and active infections occur primarily as the result of opportunistic infection following existing viral infection71,72. Other risk factors that have been associated with increased prevalence of infection include age (< 5 or > 65 years of age), primary or secondary immunodeficiencies, smoking, and colonization of a new serotype73. There are
95 known serotypes of S. pneumoniae, characterized by variations in the capsular polysaccharide74. Of the existing S. pneumoniae serotypes, 40 are known to be potentially pathogenic75.
Protection from S. pneumoniae infection is conferred primarily through protective antibody76. During natural infection, B1a cells play a significant role in conferring protection by traveling from the pleural space to the parenchyma from the lung, where secretion of polyreactive IgM has been shown to provide protection against acute S. pneumoniae infection77. Complement activation has also been shown to play a central role in reducing bacterial replication following infection, highlighting the importance of the innate immune system in controlling early infection78.
Current preventative strategies against S. pneumoniae include the use of PCV7,
PCV13, and PPSV23 vaccines. PCV7 and PCV13 are both pneumococcal conjugate 11 vaccines (PCV), where capsular polysaccharide from either 7 or 13 serotypes is conjugated to a carrier protein (diphtheria CRM197 Protein)79. PPSV23 is a polyvalent pneumococcal vaccine containing purified capsular polysaccharides from 23 serotypes, with 57-75% efficacy against homologous serotypes79,80. All existing vaccines initiate a similar humoral immune response and have been shown to significantly reduce incidence and severity of pneumococcal infection81. Though these vaccines have been successful, there are still limitations, as they only confer protection across homologous serotypes76.
Recent studies also have shown a shift in serotype prevalence in infectious cases towards those not included in the existing vaccines suggesting vaccination may be skewing infectious serotype prevalence73. New vaccination strategies against S. pneumoniae include the use of the recombinant subunit protein pneumococcal surface protein A
(PspA), which is found on most serotypes and plays a significant role in colonization and transmission82,83.
S. pneumoniae has several key virulence factors including pneumolysin, two neuraminidases, and PspA. PspA a choline binding protein, is one of the most abundant proteins located on the pneumococcal cell surface, and plays a critical role in facilitating nasopharyngeal colonization through inhibiting host complement activation82. Unlike the large number of capsular polysaccharide types, there appears to be only six clades of
PspA with the majority of strains in clades 1 and 2. Anti-PspA antibodies induced by immunization with a combination of PspA from clades 1 and 2 have been shown to be cross-protective against S. pneumoniae strains across all six clades of PspA, and provide protection against septic S. pneumoniae challenge when passively transferred to naïve 12 mice84–87. The ability of anti-PspA antibodies to provide protection across many serotypes of S. pneumoniae highlights its potential as a vaccine target.
Yersinia pestis
Yersinia pestis (Y. pestis), is a gram-negative, non-motile, facultative, intracellular, bacterium with a notorious reputation. As the causative agent of plague, Y. pestis has been responsible for over 200 million deaths historically including multiple pandemics often referred to as Justina’s plague and the ‘Black Death’ of the Middle
Ages88. Plague is a disease primarily afflicting rodents, with humans serving as an incidental or dead-end host to Y. pestis89. In the human, there are three manifestations of plague infection: bubonic, septicemic, and pneumonic.
Bubonic plague develops after an individual is bitten by an infected flea. The bacterium disseminates from the wound site to the draining lymph node where it undergoes rapid proliferation90. Following proliferation, the bacterium may disseminate systemically (i.e., septicemic plague) resulting in sepsis which may lead to septic shock and death90. Y. pestis can be transmitted from an infected individual through inhalation of respiratory droplets leading to the development of pneumonic plague91. Pneumonic plague is the deadliest manifestation of Y. pestis infection, with mortality rates near 100% if left untreated92. There are currently no existing FDA-approved vaccines against Y. pestis, though recent research has led to some limited success with subunit vaccines (e.g.,
F1-V antigen), live-attenuated and live-vectored Y. pestis vaccines93.
Yersinia pestis is a highly lethal pathogen, partly due to its various mechanisms of immune evasion. Upon passage from the flea to human host, the temperature transition causes a shift in surface lipopolysaccharide from hexa-acylated to tetra-acylated, causing it to no longer be detectable by TLR4 preventing initiation of 13 innate immunity94. This transition in LPS and the presence of outer membrane protein Ail has also been shown to provide Y. pestis with resistance to complement-mediated killing95. Following infection, Y. pestis preferentially infects and proliferates insides host macrophages, evading the host’s immune system and allowing it time to upregulate
Yersinia effector proteins (Yops)96.
There are currently six known Yop proteins, each serving a unique role and being critical to Y. pestis virulence and survival in the extracellular space (Table 1.1).
Utilizing a type III secretion system (TTSS), Y. pestis, directly delivers Yops to the cytosol of target cells97. LcrV is a key virulence factor of Y. pesits. Found at the tip of the
TTSS, it is critical for forming a pore in the cell membrane to allow for the injection of
Yops98. Y. pestis also expresses the capsule-like antigen fraction 1 (F1) in the human host, which acts as a virulence factor by inhibiting phagocytosis99.
Currently, there is no FDA-approved vaccine against Y. pestis, making it critical to develop a protective vaccine for application in potential outbreak scenarios. Many vaccination strategies have focused on developing strong humoral immunity, characterized by neutralizing antibodies against the V antigen and opsonizing antibodies against the F1 capsule100–103. In mice, anti-F1 IgG antibodies have been shown to correlate with protection100. Additionally, mAb 7.3, which binds to the V antigen, has been shown to neutralize Y. pestis in vitro and provide protection against lethal challenge following passive transfer101,104.
14
Table 1.1 List of known Yop proteins important for Yersinia pestis virulence
Yop Function Mechanism of action protein Inactivates Rho family GTPases, hindering actin YopE Anti-phagocytic cytoskeleton rearrangement195 Targets tyrosine-phosphorylated proteins, dephosphorylating (Fyn Binding protein) FyB YopH Anti-phagocytic and Src kinase-associated phosphoprotein 2 (Skap2) in macrophages196–198 Restricts activation of pyrin inflammasome – YopM Inhibits inflammasome mechanism largely unknown199 Inhibits Gαq signaling through phosphorylation YpkA Anti-phagocytic of the α subunit200 Acts as a ubiquitin-like protease to initiate Apoptotic apoptosis201 YopJ Inhibits NFκB signaling Blocks phosphorylation and activation of pathway mitogen-activated family kinases and NFκB202 Cleaves Rho protein, inactivating GTPases and YopT Anti-phagocytic hindering actin cytoskeleton rearrangement203
The United States Army Medical Research Institute of Infectious Diseases
(USAMRIID) developed a recombinant fusion protein F1-V, containing both full length proteins of both F1 and V, which has been of recent interest as a target antigen105. Using a prime-boost regimen, it was recently demonstrated that F1-V adjuvanted with alum provided complete protection against intranasal challenge with Y. pestis CO92 in mice, guinea pigs, and macaques, highlighting its potential as a protective immunogen in the development of vaccines against Y. pestis106.
Trained Innate Immunity
The innate immune system is the body’s first line of defense against invading microbial pathogens in a non-specific manner, through the ability to distinguish self from non-self. Innate immunity functions through the recognition of conserved pathogen- 15 associated features, such as microbial-associated molecular patterns (MAMPs) or danger- associated molecular patterns (DAMPs) through pattern-recognition receptors (PRRs).
There are five families of PRRs consisting of: Toll-like receptors (TLRs), C-type lectin receptors (CLRs), nucleotide-oligomerization domain (NOD)-like receptors (NLRs),
RIG-I-like receptors (RLRs), and AIM2-like receptors107,108. The families of PRRs together allow for the recognition of microorganisms both extra- and intracellularly107.
The recognition of MAMPs and DAMPs activates the innate immune system allowing it to respond quickly to foreign bodies and induce complement activation, inflammation, and recruitment of phagocytic cells. The cellular response associated with innate immunity is comprised primarily of monocytes, granulocytes, and dendritic cells, which facilitate the killing of microorganisms109. Additionally, activated macrophages contribute by inducing local inflammation, through the secretion of pro-inflammatory cytokines including TNFα, IFNγ, IL-1β, IL-6, IL-12 and IL-23110. This inflammatory response further attracts monocytes and neutrophils to the site of infection111. The combined efforts of the innate immune response are critically important for the control of infection in the first 24-48 hours post exposure. However, more recent data suggests that priming of the innate immune system by infection or vaccination could have longer lasting effects, up to three months, through the process of trained innate immunity112.
Trained innate immunity exhibits adaptive characteristics, initiated through the process of epigenetic reprogramming in neutrophils, monocytes, and natural killer (NK) cells, that alters immune signaling pathways and cellular metabolism113. Such involved mechanisms of epigenetic reprogramming include histone modification, chromatin reconfiguration, and DNA methylation114. Increased responsiveness to stimulation is also 16 conferred through a metabolic shift from oxidative phosphorylation to aerobic glycolysis in monocytes, allowing for increased proliferative and cytokine production capacity114.
Trained monocytes, with histone modifications, show significantly increased transcription of genes encoding IL-6 and TNFα, and ex vivo were found to have increased reactive oxygen species production for up to three weeks following non-specific challenge115,116. These mechanisms allow the innate immune responses to retain a certain degree of immunological memory, which can help to confer protection to secondary non- related infections in a B and T cell-independent manner116.
While mechanisms surrounding the development of trained innate immunity are still being fully elucidated, several transcription factors have been identified that may be crucial in determining epigenetic and transcriptional programming117. Other research indicates inhibiting synthesis of cyclic adenosine monophosphate (cAMP) or mTOR during the primary response eliminates the protective effect of trained innate immunity following secondary infection in human and murine models117,118.
Humoral Immune Response
The whole of the adaptive immune response can be organized into either cellular
(i.e., cell-mediated) immunity or humoral immunity. Humoral immunity is responsible for immunity in the extracellular space and is primarily mediated by the production of and secretion of antibodies. Antibodies contribute to immunity through several mechanisms including marking pathogens for phagocytosis and elimination
(opsonization), prevention of pathogen entrance into cells (neutralization), immune exclusion at mucosal surfaces, and complement activation via the classical pathway119.
Opsonization (from Greek – prepare for eating) entails the coating of microbial surfaces with an opsonin, such as antibodies and complement C3b, enhancing phagocytic 17 uptake and playing a crucial role in the control of many diseases120. Phagocytic uptake via antibody-mediated opsonization is facilitated through the binding of the Fab portion to its cognate epitope on target pathogen surface and interaction with Fc receptors present on the phagocyte121. Antigen-specific antibodies can also contribute to control of infection through the process of neutralization, by which antibodies can coat microbial surfaces, bind to pathogen ligands, inhibit virulence factors or toxins, and prevent further spread of pathogens by preventing fusion/entry into host cells119,122. Additionally, antibodies can activate the complement system by the classical pathway.
There are three pathways upon which complement can be activated, the classical, lectin-mediated, and alternative pathways. The lectin pathway relies upon the recognition of MAMPs or DAMPs on the pathogen surface, by the pattern recognition molecules
(PRMs) mannose-binding lectin (MBL), collectins (collectin-10, collectin-11), or ficolins
(ficolin-1, ficolin-2, ficolin-3)123. The PRMs complex with the mannose-binding ligand- associated serine proteases (MASPs)123,124. Upon binding of PRMs, MASP-2 can initiate cleavage of C4 and C2 complement components, forming the C3 convertase C4bC2a124.
MASP-1 can also cleave C2 enhancing convertase formation and has been shown to activate MASP-2123.
The classical pathway is initiated by the binding of the C1 complex, consisting of
C1q and the serine proteases C1r and C1s, to the Fc portion of IgM and IgG antigen:antibody complexes clustering on the pathogen surface127. Binding of C1q causes a conformational change, activating C1r and leading to cleavage of C1s. Activated C1s will cleave C4 and C2 complement components, leading to the development of the C3 convertase C4bC2a, similar to the lectin pathway. 18
The C3 convertase (C4bC2a or C3bBb) cleaves C3 and leads to binding of C3b onto the target surface and the release of the anaphylatoxin C3a, which recruits phagocytic cells to the site128. C3 is rapidly cleaved by the C3 convertase, leading to the development of more C3 convertases and the C5 convertase (C4bC2aC3b or
C3bBb3b)129. The C5 convertase cleaves C5 into C5a and C5b and can lead to the development of the major attack complex (MAC)124. The MAC, comprised of C5b, C6,
C&, C8, and several C9 complement components, forms a transmembrane pore and disrupts the bilipid membrane leading to cell lysis128.
Of the five major isotypes of immunoglobulin (IgA, IgD, IgE, IgG, IgM) that exist in the human immune system, IgG is by far the most prevalent and is one of the most abundant proteins found in human serum, accounting for approximately 10-20 % of total plasma protein130. Different classes of antibodies are generated following B cell activation in a process called class switch recombination, through which the constant heavy (CH) gene is excised causing a switch from Cµ to a different CH gene resulting in an isotypic switch from IgM131,132.
Human IgG is very similar to the other isotypes of antibody in terms of its overall structure, consisting of two identical γ heavy (H) chains and two identical κ or λ light (L) chains, linked by disulfide bonds. The H chains are comprised of a variable domain (VH) and three constant domains (CH1, CH2, CH3), with a hinge region between CH1 and
CH2133. The light chain is also comprised of variable chain (VL) and constant domain
(CL). The fragment antigen binding or “Fab” portion of the antibody consists of the light chain associating with the VH and CH1 domains133. IgG responses to different antigen types lead to the skewing towards different subclasses of which there are four: IgG1, 19
IgG2, IgG3 and IgG4. Each of these subclasses has its own unique properties and functions within the immune system, and though they share 90% sequence identity in their constant domains, there is great variation in their hinge regions, leading to differences in their stability, flexibility, complement binding, and antigen binding capability130.
IgG1 is the most abundant subclass of IgG in humans, accounting for approximately 60% of all serum IgG130. Predominantly induced in response to soluble and membrane-bound proteins/antigens, IgG1 is a strong activator of complement via the classical pathway134,135. IgG2 antibodies are the second most abundant subclass and are primarily initiated by T-independent carbohydrate antigens, in extrafollicular responses, and have been shown to be a major isotype necessary for anti-bacterial antibody responses136. IgG2 shows poor complement fixation135. IgG3 antibodies, like IgG1, are produced primarily following interaction with T-dependent protein antigens and are also capable of initiating the complement cascade, with the strongest C1q binding of any IgG subclass135. Unlike IgG1, IgG3 antibodies are very short-lived with the lowest half-life of all IgG subclasses, at approximately 7 days137. IgG4 antibodies are induced primarily following chronic antigenic stimulation and are unable to activate complement135,138,139.
Mature B cells can be divided into three subsets: follicular (B2), marginal zone, and B1 cells. Marginal zone B cells, found in the spleen, consist of both memory B
140 (BMEM) cells and mature naïve B cells . Within the follicular B (B2) cell subpopulation, located in the lymphoid follicles, there are phenotypically-unique subsets, characterized by specific surface markers that include transitional, germinal center (GC), and BMEM 20 cells, which are responsible for the bulk of adaptive humoral immune response.
Additionally, there exists a minor subset of more innate-like B cells, termed B1 B cells.
B1 cells are found primarily in the spleen, bone marrow, and peritoneal and pleural cavity with very little presence in circulation or lymph nodes and account for the production of approximately 80-90% of serum IgM141. Within the B1 cell subset, there are two distinct populations of cells. B1a (CD5+) cells, which are more innate-like and spontaneously produce “natural antibody”, typically low-affinity and polyreactive, which could be crucial in maintaining protection against pathogens in the time prior to germinal center formation, and B1b (CD5-) cells which function as a part of the adaptive immune response142–144. B1b cells have been shown to induce long-lived T cell-independent IgM
145,146 BMEM cells, which provide rapid protection against natural infections .
B cells that interact with their cognate antigen and become activated in secondary lymphoid organs and receive T cell co-stimulation in the T zone, differentiate into plasmablasts in the medullary cords of the lymph node or extrafollicular foci of the spleen, or migrate to follicles, differentiating into germinal center B cells that proliferate and begin to form GCs, where long-lived plasma cells (LLPCs) and BMEM cells are formed. Plasmablasts proliferating in the extrafollicular space, have an average lifespan of 2-3 days and rapidly differentiate into short-lived plasma cells147. Short-lived plasma cells (SLPCs) initiated in the extrafollicular space have been shown to rapidly produce antibody and can persist for 3-4 weeks following initial infection/antigen interaction148,149.
Extrafollicular B cell activation and humoral responses can be initiated in both T- dependent (TD) and T-independent (TI) manners. TD extrafollicular responses can 21 induce class-switched antibody in a Bcl6-dependent manner150. While T follicular helper
(TFH) cells are required for most class-switched antibody production, TI antigens, such as bacterial capsular polysaccharides, can initiate extrafollicular B cell humoral responses by activating marginal zone B cells to differentiate into plasmablasts and SLPCs. These
TI antibody responses do not often induce GC formation or BMEM/LLPC differentiation, and are associated with production of rapidly appearing and low-affinity antibodies149.
Germinal centers are distinct structures which form within secondary lymphoid organs within 5-7 days after introduction of a foreign, TD antigen, either via immunization or infection151. Consisting of a both a light zone (LZ) and dark zone (DZ), germinal centers are critical for the development of long-lived humoral immunity and memory B cell populations. Within the light zone, signals are provided that sustain the
GC B cell response. These signals are derived from B cell interactions with cognate antigen presented by follicular dendritic cells (FDCs) and co-stimulation provided by TFH cells. The GC is also where positive selection occurs for high-affinity clones152. In the dark zone of the GC, antigen-specific B cells undergo somatic hypermutation (SHM), during which the genes encoding the B cell receptor (BCR) are altered to allow for increased affinity for cognate antigen (i.e., somatic hypermutation). These DZ B cells then travel back to the LZ, where preferential activation leads to the positive selection of high-affinity clones, through affinity maturation153. High-affinity GC B cells are preferentially selected and differentiate into either BMEM or antibody-secreting cells
(ASCs). Two types of ASCs are initiated, both SLPCs and LLPCs. Long-lived plasma cells migrate to the bone marrow and continue to produce high-affinity antibodies, while 22
SLPCs maintain residence in the draining lymph node or spleen and produce large amounts of antigen-specific antibodies154.
Class switch recombination (CSR) and somatic hypermutation (SH), in the LZ of the GC, require the presence of activation-induced cytidine deaminase (AID)132,155. AID is upregulated in activated B cells, following CD40/CD40L interaction and in the presence of IL-4, and acts by deaminating cytosine in the target DNA and converting it to uracil. During somatic hypermutation, this deamination event induces a mismatch which results in DNA diversification either through the introduction of transition mutations or the removal of uracil by uracil DNA glycosylase (UNG) prior to replication, leading to point mutations in the variable region156–158. In CSR, the deamination occurs upstream of the CH region of the immunoglobulin in the switch region. Uracil base pairs, introduced into both the donor and acceptor switch regions, are then excised by UNG causing double-strand breaks, which are then recombined to promote different CH through end- joining recombination, resulting in the production of different classes of antibody158,159.
Following immunization with TD antigens, antigen-specific B cells become activated and interact with TFH cells in the T zone through a process called linked recognition, in which the CD40/CD40L interaction provides necessary co-stimulation for
160 B cell differentiation and maturation . TFH cells are located within the lymph node follicle and upon interaction with foreign antigen, begin to downregulate CXCR9 on their cell surface, and upregulate CXCR5 expression, as well as CD40L and inducible T-cell
161,162 costimulator (ICOS) . TFH cell differentiation is largely regulated by B-cell lymphoma 6 (Bcl6), a transcription factor, that is necessary for the induction of TFH memory cell proliferation following rechallenge163–165. 23
This alteration in chemokine receptor expression (CXCR9 → CXCR5) stimulates the migration of T cells to the B cell follicle and GC LZ, to allow for interaction between
166 B and TFH cells . The CD40/CD40L interaction induces CSR. TFH cells secrete IL-21, which is a key regulatory molecule that enhances immunoglobulin production and GC formation167. Lack of IL-21 during early stages of the humoral immune response, has been associated with increased levels of IgE and impaired IgG responses; however, there is no impact on the extrafollicular antibody response168,169. Additionally, loss of IL-21 has been shown to lead to shorten TFH and GC B cell lifespans, and presence of IL-21 has been linked to increased Bcl6 expression indicating that IL-21 plays a crucial role in the development of long-lived humoral immunity170–173.
Located within the B cell follicle, FDCs are a major source of B cell activating factor (BAFF), IL-6, and IL-10 which are all critical for the differentiation of B cells within the germinal center174. Presence of BAFF during early germinal center formation has also been associated with higher overall B cell proliferation and improved B cell survival175. FDCs serve as the primary reservoir of antigen inside the B cell follicle, where they internalize and recycle complement-coated immune complexes and periodically display antigen on the cell surface to stimulate surrounding antigen-specific
B cells176.
Rapid development of a new population of SLPCs, following booster immunization or encounter with the target pathogen, is initiated by antigen-specific BMEM cells and IL-9, which is preferentially expressed on BMEM cells and upregulated following activation. IL-9 acts in an autocrine manner to upregulate proliferation and terminal
178,179 differentiation of BMEM cells into antibody-secreting cells . BMEM cells, following 24 interaction with their cognate antigen, are responsible for initiating rapid germinal center formation, repopulating the existing BMEM pool, and differentiation into antigen-specific
180 ASCs in as little at 3-5 days after initiation of the recall response . BMEM cells, both class-switched and IgM+, have been shown to persist in the spleen of mice for over 400 days post-immunization, and this response is independent of BAFF or a proliferation inducing ligand (APRIL)181,182. Characterized by their surface expression of CD38,
CD19, and B220, antigen-specific BMEM cells are critical for initiating rapid recall antibody responses following interaction with their cognate antigen.
Long-lived plasma cell maintenance in the bone marrow is not dependent on the presence of BMEM cells, antigen-loaded antigen-presenting dendritic cells, or secondary lymphoid structures183. It has been suggested that bone marrow CTLA4+ T regulatory
(Treg) cells may play an important role in LLPC maintenance, with LLPC number decreasing by 50% when Treg cells are absent184.
It has been shown that survival of long-lived ASCs in the bone marrow is dependent on presence of either BAFF and APRIL182. BAFF and APRIL are both members of the tumor-necrosis factor (TNF) family, TNFSF13B and TNFSF13, respectively. BAFF is primarily produced by monocytes, DCs and macrophages and production is upregulated in the presence of IFN-γ and IL-10185–189. A TNF family receptor member, B cell maturation antigen (BCMA) provides necessary survival signals by binding to its ligands BAFF and APRIL190. Studies on auto-immunity indicate that long-lived plasma cells numbers peak after about 12 weeks after activation, in the bone marrow191. As ASCs differentiate into long-lived plasma cells they downregulate FasR,
CD19 and CD20, and upregulate CD138 and IL6R on their cell surface192. LLPCs have 25 been shown to be metabolically unique from SLPCs in their ability to import higher rates of glucose for use in glycolysis and antibody glycosylation193. LLPCs express a wide variety of immunoglobulin production, though primarily produce IgA and IgG, and are most predominately found in the femur and lamina propria183,194.
Long-term protection against pathogens, either conferred by vaccination or following natural infection, is largely facilitated through the continued presence of circulating high-affinity antibodies produced by LLPCs and through the rapid induction of new ASCs following infection (i.e., secondary immune response). When considering development of an efficacious vaccine that is able to induce both short and long-lived protective humoral immune responses, it is important to consider the activation of not only short-lived ASCs, but also BMEM cell populations, that are able to quickly respond to reinfection, and LLPCs that are able to produce antigen-specific antibodies over extended periods of time. It may be possible to identify promising new vaccine adjuvants by evaluating upregulation of IL-10 and IFN-γ and in the draining lymph node following immunization, as a way of predicting induction of BAFF/APRIL expression associated with LLPC maintenance.
References
1. Riedel, S. Edward Jenner and the history of smallpox and vaccination. Baylor Univ. Med. Cent. Proc. 18, 21–25 (2017).
2. Hanson, C. M., George, A. M., Sawadogo, A. & Schreiber, B. Is freezing in the vaccine cold chain an ongoing issue? A literature review. Vaccine 35, 2127–2133 (2017). 26
3. Thakker, Y. & Woods, S. Storage of vaccines in the community: weak link in the cold chain? BMJ 304, 756–758 (2009).
4. Robertson, J., Franzel, L. & Maire, D. Innovations in cold chain equipment for immunization supply chains. Vaccine 35, 2252–2259 (2017).
5. Ventola, C. L. Immunization in the United States: Recommendations, barriers, and measures to improve compliance: Part 1: Childhood vaccinations. P T 41, 426–36 (2016).
6. Stern, A. M. & Markel, H. The history of vaccines and immunization: Familiar patterns, new challenges. Health Aff. 24, 611–621 (2005).
7. Diavatopoulos, D. A. & Edwards, K. M. What is wrong with pertussis vaccine immunity? Cold Spring Harb. Perspect. Biol. 9, a029553 (2017).
8. Ruedl, C., Frühwirth, M., Wick, G. & Wolf, H. Immune response in the lungs following oral immunization with bacterial lysates of respiratory pathogens. Clin. Diagn. Lab. Immunol. 1, 150–4 (1994).
9. Reynolds, H. Immunoglobulin G and its function in the human respiratory tract. Mayo Clin. Proc. 63, 161–174 (1988).
10. Belyakov, I. M. & Ahlers, J. D. What role does the route of immunization play in the generation of protective immunity against mucosal pathogens? J. Immunol. 183, 6883–6892 (2009).
11. Reed, S. G., Orr, M. T. & Coler, R. N. Vaccine adjuvants. in The Vaccine Book 1, 67–76 (Elsevier, 2016).
12. Del Giudice, G., Rappuoli, R. & Didierlaurent, A. M. Correlates of adjuvanticity: A review on adjuvants in licensed vaccines. Semin. Immunol. (2018). doi:10.1016/J.SMIM.2018.05.001
13. Pasquale, A., Preiss, S., Silva, F. & Garçon, N. Vaccine adjuvants: from 1920 to 2015 and beyond. Vaccines 3, 320–343 (2015).
14. Brunner, R., Jensen-Jarolim, E. & Pali-Schöll, I. The ABC of clinical and experimental adjuvants-A brief overview. Immunology Letters 128, 29–35 (2010).
15. Lindblad, E. B. Aluminium compounds for use in vaccines. Immunol. Cell Biol. 82, 497–505 (2004).
16. Heimlich, J. M., Regnier, F. E., White, J. L. & Hem, S. L. The in vitro displacement of adsorbed model antigens from aluminium-containing adjuvants by interstitial proteins. Vaccine 17, 2873–2881 (1999). 27
17. De Gregorio, E., Tritto, E. & Rappuoli, R. Alum adjuvanticity: Unraveling a century old mystery. European Journal of Immunology 38, 2068–2071 (2008).
18. Kool, M., Fierens, K. & Lambrecht, B. N. Alum adjuvant: Some of the tricks of the oldest adjuvant. J. Med. Microbiol. 61, 927–934 (2012).
19. Hogenesch, H., O’hagan, D. T. & Fox, C. B. Optimizing the utilization of aluminum adjuvants in vaccines: you might just get what you want. 3, 51 (2018).
20. David, A. et al. Vaccine adjuvants aluminum and monophosphoryl lipid A provide distinct signals to generate protective cytotoxic memory CD8 T cells. Proc. Natl. Acad. Sci. 108, 7914–7919 (2011).
21. Gregg, K. A. et al. Rationally designed TLR4 ligands for vaccine adjuvant discovery. MBio 8, e00492-17 (2017).
22. Weinberger, B. Adjuvant strategies to improve vaccination of the elderly population. Current Opinion in Pharmacology 41, 34–41 (2018).
23. Brito, L. A. & O’Hagan, D. T. Designing and building the next generation of improved vaccine adjuvants. J. Control. Release 190, 563–579 (2014).
24. Brito, L. A., Malyala, P. & O’Hagan, D. T. Vaccine adjuvant formulations: A pharmaceutical perspective. Seminars in Immunology 25, 130–145 (2013).
25. Danilchanka, O. & Mekalanos, J. J. Cyclic dinucleotides and the innate immune response. Cell 154, 962–70 (2013).
26. Ishikawa, H. & Barber, G. N. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 455, 674–678 (2008).
27. Jenal, U., Reinders, A. & Lori, C. Cyclic di-GMP: Second messenger extraordinaire. Nat. Rev. Microbiol. 15, 271–284 (2017).
28. Burdette, D. L. et al. STING is a direct innate immune sensor of cyclic di-GMP. Nature 478, 515–518 (2011).
29. Dubensky, T. W., Kanne, D. B. & Leong, M. L. Rationale, progress and development of vaccines utilizing STING-activating cyclic dinucleotide adjuvants. Ther. Adv. Vaccines 1, 131–143 (2013).
30. Ma, Z. & Damania, B. The cGAS-STING defense pathway and its counteraction by viruses. Cell Host and Microbe 19, 150–158 (2016).
31. Barber, G. N. STING: Infection, inflammation and cancer. Nature Reviews Immunology 15, 760–770 (2015). 28
32. Vatner, R. E. & Janssen, E. M. STING, DCs and the link between innate and adaptive tumor immunity. (2017). doi:10.1016/j.molimm.2017.12.001
33. Lienenklaus, S. et al. DNA damage primes the Type I interferon system via the cytosolic DNA sensor STING to promote anti-microbial innate immunity. Immunity 42, 332–343 (2015).
34. Ebensen, T. et al. The bacterial second messenger cyclic diGMP exhibits potent adjuvant properties. Vaccine 25, 1464–1469 (2007).
35. Chen, W., KuoLee, R. & Yan, H. The potential of 3′,5′-cyclic diguanylic acid (c- di-GMP) as an effective vaccine adjuvant. Vaccine 28, 3080–3085 (2010).
36. Makadia, H. K. & Siegel, S. J. Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier. Polymers (Basel). 3, 1377–1397 (2011).
37. Danhier, F. et al. PLGA-based nanoparticles: an overview of biomedical applications. J. Control. Release 161, 505–22 (2012).
38. Sharma, S., Parmar, A., Kori, S. & Sandhir, R. PLGA-based nanoparticles: A new paradigm in biomedical applications. Trends Anal. Chem. 80, 30–40 (2015).
39. Kamaly, N., Yameen, B., Wu, J. & Farokhzad, O. C. Degradable controlled- release polymers and polymeric nanoparticles: Mechanisms of controlling drug release. Chemical Reviews 116, 2602–2663 (2016).
40. Fairley, S. J. et al. Chlamydia trachomatis recombinant MOMP encapsulated in PLGA nanoparticles triggers primarily T helper 1 cellular and antibody immune responses in mice: a desirable candidate nanovaccine. Int. J. Nanomedicine 8, 2085–99 (2013).
41. Silva, A. L., Soema, P. C., Slütter, B., Ossendorp, F. & Jiskoot, W. PLGA particulate delivery systems for subunit vaccines: Linking particle properties to immunogenicity. Human Vaccines and Immunotherapeutics 12, 1056–1069 (2016).
42. Allahyari, M. & Mohit, E. Peptide/protein vaccine delivery system based on PLGA particles. Human Vaccines and Immunotherapeutics 12, 806–828 (2016).
43. Mir, M., Ahmed, N. & Rehman, A. ur. Recent applications of PLGA based nanostructures in drug delivery. Colloids and Surfaces B: Biointerfaces 159, 217– 231 (2017).
44. Perry, J., Chambers, A., Spithoff, K. & Laperriere, N. Gliadel wafers in the treatment of malignant glioma: a systematic review. Curr. Oncol. 14, 189–94 (2007). 29
45. Torres, M. P., Vogel, B. M., Narasimhan, B. & Mallapragada, S. K. Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms. J. Biomed. Mater. Res. - Part A 76, 102–110 (2006).
46. Torres, M. P., Determan, A. S., Anderson, G. L., Mallapragada, S. K. & Narasimhan, B. Amphiphilic polyanhydrides for protein stabilization and release. Biomaterials 28, 108–116 (2007).
47. Petersen, L. K., Phanse, Y., Ramer-Tait, a. E., Wannemuehler, M. J. & Narasimhan, B. Amphiphilic polyanhydride nanoparticles stabilize bacillus anthracis protective antigen. Mol. Pharm. 9, 874–882 (2012).
48. Wagner-Muñiz, D. A., Haughney, S. L., Kelly, S. M., Wannemuehler, M. J. & Narasimhan, B. Room temperature stable PspA-based nanovaccine induces protective immunity. Front. Immunol. 9, (2018).
49. Huntimer, L. et al. Evaluation of Biocompatibility and Administration Site Reactogenicity of Polyanhydride-Particle-Based Platform for Vaccine Delivery. Adv. Healthc. Mater. 2, 369–378 (2013).
50. Haughney, S. L., Ross, K. a., Boggiatto, P. M., Wannemuehler, M. J. & Narasimhan, B. Effect of nanovaccine chemistry on humoral immune response kinetics and maturation. Nanoscale 6, 13770–13778 (2014).
51. Ulery, B. D. et al. Design of a protective single-dose intranasal nanoparticle-based vaccine platform for respiratory infectious diseases. PLoS One 6, 1–8 (2011).
52. Ross, K. et al. Combination nanovaccine demonstrates synergistic enhancement in efficacy against influenza. ACS Biomater. Sci. Eng. 2, 368–374 (2016).
53. McGill, J. L. et al. Efficacy of mucosal polyanhydride nanovaccine against respiratory syncytial virus infection in the neonatal calf. Sci. Rep. 8, 1–15 (2018).
54. Vela Ramirez, J. E. et al. Polyanhydride nanovaccines induce germinal center B cell formation and sustained serum antibody responses. J. Biomed. Nanotechnol. 12, 1303–1311 (2016).
55. Vela-Ramirez, J. E. et al. Safety and biocompatibility of carbohydrate- functionalized polyanhydride nanoparticles. AAPS J. 17, 256–67 (2015).
56. Adler, A. F. et al. High throughput cell-based screening of biodegradable polyanhydride libraries. Comb. Chem. High Throughput Screen. 12, 634–45 (2009).
57. Rokicki, W., Rokicki, M., Wojtacha, J. & Dżeljijli, A. The role and importance of club cells (Clara cells) in the pathogenesis of some respiratory diseases. Kardiochirurgia i torakochirurgia Pol. = Polish J. cardio-thoracic Surg. 13, 26– 30 (2016). 30
58. Zanin, M., Baviskar, P., Webster, R. & Webby, R. The interaction between respiratory pathogens and mucus. Cell Host and Microbe 19, 159–168 (2016).
59. Iwasaki, A., Foxman, E. F. & Molony, R. D. Early local immune defences in the respiratory tract. Nature Reviews Immunology 17, 7–20 (2017).
60. Clarke, T. B. Early Innate immunity to bacterial infection in the lung is regulated systemically by the commensal microbiota via NOD-like receptor ligands. Infect. Immun. 82, 4596–4606 (2014).
61. Sibille, Y., Marchandise, F. X., Godding, V., Vaerman, J.-P. & Sibille, Y. Pulmonary immune cells in health and disease: polymorphonuclear neutrophils. Eur. Respir. J. 6, 1529–43 (1993).
62. Curtis, J. L. Cell-mediated adaptive immune defense of the lungs. Proc. Am. Thorac. Soc. 2, 412–416 (2005).
63. Renegar, K. B., Small, P. A., Boykins, L. G. & Wright, P. F. Role of IgA versus IgG in the control of influenza viral infection in the murine respiratory tract. J. Immunol. 173, 1978–1986 (2014).
64. Dasaraju, P. V. & Liu, C. Infections of the Respiratory System. Medical Microbiology (University of Texas Medical Branch at Galveston, 1996).
65. Kolditz, M. & Ewig, S. Community-acquired pneumonia in adults. Dtsch. Aerzteblatt Online 6, 14–26 (2017).
66. Musher, D. M. & Thorner, A. R. Community-acquired pneumonia. N. Engl. J. Med. 17, 1619–1647 (2014).
67. Vestjens, S. M. T. et al. Changes in pathogens and pneumococcal serotypes causing community-acquired pneumonia in The Netherlands. Vaccine 35, 4112– 4118 (2017).
68. O’Brien, K. L. et al. Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates. Lancet 374, 893–902 (2009).
69. Regev‐Yochay, G. et al. Nasopharyngeal carriage of Streptococcus pneumoniae by adults and children in community and family settings. Clin. Infect. Dis. 38, 632– 639 (2004).
70. Hamaluba, M. et al. A cross-sectional observational study of pneumococcal carriage in children, their parents, and older adults following the introduction of the 7-valent pneumococcal conjugate vaccine. Med. (United States) 94, e335 (2015). 31
71. Hament, J. M., Kimpen, J. L. ., Fleer, A. & Wolfs, T. F. . Respiratory viral infection predisposing for bacterial disease: A concise review. FEMS Immunol. Med. Microbiol. 26, 189–195 (1999).
72. Bosch, A. A. T. M., Biesbroek, G., Trzcinski, K., Sanders, E. A. M. & Bogaert, D. Viral and bacterial interactions in the upper respiratory tract. PLoS Pathogens 9, e1003057 (2013).
73. Mehr, S. & Wood, N. Streptococcus pneumoniae - a review of carriage, infection, serotype replacement and vaccination. Paediatric Respiratory Reviews 13, 258– 264 (2012).
74. Zafar, M. A., Hamaguchi, S., Zangari, T., Cammer, M. & Weiser, J. N. Capsule type and amount affect shedding and transmission of Streptococcus pneumoniae. MBio 8, (2017).
75. Musher, D. M. Infections caused by streptococcus clinical spectrum , pneumoniae : and treatment. Most 14, 801–807 (2010).
76. Pichichero, M. E., Khan, M. N. & Xu, Q. Next generation protein based Streptococcus pneumoniae vaccines. Human Vaccines and Immunotherapeutics 12, 194–205 (2016).
77. Weber, G. F. et al. Pleural innate response activator B cells protect against pneumonia via a GM-CSF-IgM axis. J. Exp. Med. 211, 1243–1256 (2014).
78. Kadioglu, A. & Andrew, P. W. The innate immune response to pneumococcal lung infection: The untold story. Trends in Immunology 25, 143–149 (2004).
79. Pallotta, A. & Rehm, S. J. Navigating pneumococcal vaccination in adults. Cleve. Clin. J. Med. 83, 427–433 (2016).
80. Highlights of prescribing information - PNEUMOVAX 23. (2014).
81. Lindley, M. C. et al. Use of 13-Valent Pneumococcal Conjugate Vaccine and 23- Valent Pneumococcal Polysaccharide Vaccine Among Adults Aged ≥65 Years: Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR. Morbidity and mortality weekly report 63, (2014).
82. Khan, N. & Jan, A. T. Towards identifying protective B-cell epitopes: The PspA story. Front. Microbiol. 8, 1–8 (2017).
83. Mirza, S. et al. The effects of differences in PspA alleles and capsular types on the resistance of Streptococcus pneumoniae to killing by apolactoferrin. Microb. Pathog. 99, 209–219 (2016). 32
84. Kristian, S. A. et al. Generation and improvement of effector function of a novel broadly reactive and protective monoclonal antibody against pneumococcal surface protein A of Streptococcus pneumoniae. PLoS One 11, 1–25 (2016).
85. Piao, Z. et al. Protective properties of a fusion pneumococcal surface protein A (PspA) vaccine against pneumococcal challenge by five different PspA clades in mice. Vaccine 32, 5607–5613 (2014).
86. Darrieux, M. et al. Recognition of pneumococcal isolates by antisera raised against PspA fragments from different clades. J. Med. Microbiol. 57, 273–278 (2008).
87. Goulart, C. et al. Selection of family 1 PspA molecules capable of inducing broad- ranging cross-reactivity by complement deposition and opsonophagocytosis by murine peritoneal cells. Vaccine 29, 1634–1642 (2010).
88. Ligon, B. L. Plague: A review of its history and potential as a biological weapon. Semin. Pediatr. Infect. Dis. 17, 161–170 (2006).
89. Butler, T. Plague history: Yersin’s discovery of the causative bacterium in 1894 enabled, in the subsequent century, scientific progress in understanding the disease and the development of treatments and vaccines. (2014). doi:10.1111/1469- 0691.12540
90. Gonzalez, R. J. & Miller, V. L. A deadly path: Bacterial spread during bubonic plague. (2016). doi:10.1016/j.tim.2016.01.010
91. Raoult, D., Mouffok, N., Bitam, I., Piarroux, R. & Drancourt, M. Plague: History and contemporary analysis. J. Infect. 66, 18–26 (2013).
92. Inglesby, T. V. et al. Plague as a biological weapon. JAMA 283, 2281 (2000).
93. Sun, W. & Singh, A. K. Plague vaccine: Recent progress and prospects. npj Vaccines 4, 11 (2019).
94. Montminy, S. W. et al. Virulence factors of Yersinia pestis are overcome by a strong lipopolysaccharide response. Nat. Immunol. 7, 1066–1073 (2006).
95. Bartra, S. S. et al. Resistance of Yersinia pestis to complement-dependent killing is mediated by the Ail outer membrane protein. Infect. Immun. 76, 612–622 (2008).
96. Li, B. & Yang, R. Interaction between Yersinia pestis and the host immune system. Infect. Immun. 76, 1804–1811 (2008).
97. Pechous, R. D., Sivaraman, V., Price, P. A., Stasulli, N. M. & Goldman, W. E. Early host cell targets of Yersinia pestis during primary pneumonic plague. PLoS Pathog. 9, e1003679 (2013). 33
98. Anisimov, A. P. et al. Amino acid and structural variability of Yersinia pestis LcrV protein. Infect. Genet. Evol. 10, 137–145 (2010).
99. Sun, W. & Curtiss, R. Rational Considerations about development of live attenuated Yersinia pestis vaccines. Curr. Pharm. Biotechnol. 14, 878–886 (2014).
100. Williamson, E. D. et al. An IgG1 titre to the F1 and V antigens correlates with protection against plague in the mouse model. Clin. Exp. Immunol. 116, 107–114 (1999).
101. Ivanov, M. I., Hill, J. & Bliska, J. B. Direct neutralization of type III effector translocation by the variable region of a monoclonal antibody to Yersinia pestis LcrV. Clin. Vaccine Immunol. (2014). doi:10.1128/CVI.00013-14
102. Hill, J., Leary, S. E. C., Griffin, K. F., Diane Williamson, E. & Titball, R. W. Regions of Yersinia pestis V antigen that contribute to protection against plague identified by passive and active immunization. Infect. Immun. 65, 4476–4482 (1997).
103. Xiao, X. et al. Human anti-plague monoclonal antibodies protect mice from Yersinia pestis in a bubonic plague model. PLoS One 5, (2010).
104. Hill, J. et al. Synergistic protection of mice against plague with monoclonal antibodies specific for the F1 and V antigens of Yersinia pestis. Infect. Immun. 71, 2234–2238 (2003).
105. Heath, D. G. et al. Protection against experimental bubonic and pneumonic plague by a recombinant capsular F1-V antigen fusion protein vaccine. Vaccine 16, 1131– 1137 (1998).
106. Quenee, L. E., Ciletti, N. A., Elli, D., Hermanas, T. M. & Schneewind, O. Prevention of pneumonic plague in mice, rats, guinea pigs and non-human primates with clinical grade rV10, rV10-2 or F1-V vaccines. Vaccine 29, 6572– 6583 (2011).
107. Brubaker, S. W., Bonham, K. S., Zanoni, I. & Kagan, J. C. Innate immune pattern recognition: A cell biological perspective. Annu. Rev. Immunol. 33, 257–290 (2015).
108. Akira, S., Uematsu, S. & Takeuchi, O. Pathogen recognition and innate immunity. Cell 124, 783–801 (2006).
109. D’Osualdo, A. et al. Cellular innate immunity: An old game with new players. J. Innate Immun. 9, 111–125 (2016).
110. Duque, G. A. & Descoteaux, A. Macrophage cytokines: Involvement in immunity and infectious diseases. Front. Immunol. 5, 1–12 (2014). 34
111. Elkon, K. B. & Rhiannon, J. J. Innate immunity. in Scleroderma: From Pathogenesis to Comprehensive Management 125, 191–197 (NIH Public Access, 2012).
112. Song, W. M. & Colonna, M. Immune training unlocks innate potential. Cell 172, 3–5 (2018).
113. Netea, M. G., Quintin, J. & Van Der Meer, J. W. M. Trained immunity: A memory for innate host defense. Cell Host and Microbe 9, 355–361 (2011).
114. Netea, M. G. et al. Trained immunity: A program of innate immune memory in health and disease. Science (80-. ). 352, aaf1098-aaf1098 (2016).
115. Quintin, J. et al. Candida albicans infection affords protection against reinfection via functional reprogramming of monocytes. Cell Host Microbe 12, 223–232 (2012).
116. Blok, B. A., Arts, R. J. W., van Crevel, R., Benn, C. S. & Netea, M. G. Trained innate immunity as underlying mechanism for the long-term, nonspecific effects of vaccines. J. Leukoc. Biol. 98, 347–356 (2015).
117. Saeed, S. et al. Epigenetic programming of monocyte-to-macrophage differentiation and trained innate immunity. Science (80-. ). 345, 1251086– 1251086 (2014).
118. Cheng, S.-C. et al. mTOR- and HIF-1 -mediated aerobic glycolysis as metabolic basis for trained immunity. Science (80-. ). 345, 1250684–1250684 (2014).
119. Warrington, R., Watson, W., Kim, H. L. & Antonetti, F. R. An introduction to immunology and immunopathology. Allergy, Asthma Clin. Immunol. 7, S1 (2011).
120. Shishido, S. N., Varahan, S., Yuan, K., Li, X. & Fleming, S. D. Humoral innate immune response and disease. Clinical Immunology 144, 142–158 (2012).
121. Bournazos, S., Wang, T. T., Dahan, R., Maamary, J. & Ravetch, J. V. Signaling by Antibodies: Recent Progress. Annu. Rev. Immunol. 35, 285–311 (2017).
122. Forthal, D. N. Functions of Antibodies. in Antibodies for Infectious Diseases 25– 48 (2014). doi:10.1128/microbiolspec.aid-0019-2014
123. Garred, P. et al. A journey through the lectin pathway of complement—MBL and beyond. Immunological Reviews 274, 74–97 (2016).
124. Hein, E. & Garred, P. The lectin pathway of complement and biocompatibility. Adv. Exp. Med. Biol. 865, 77–92 (2015).
125. Harboe, M. & Mollnes, T. E. The alternative complement pathway revisited. Journal of Cellular and Molecular Medicine 12, 1074–1084 (2008). 35
126. Nesargikar, P., Spiller, B. & Chavez, R. The complement system: History, pathways, cascade and inhibitors. Eur. J. Microbiol. Immunol. 2, 103–111 (2012).
127. Noris, M. & Remuzzi, G. Overview of complement activation and regulation. Semin. Nephrol. 33, 479–492 (2013).
128. Sarma, J. V. & Ward, P. A. The complement system. Cell Tissue Res. 343, 227– 235 (2011).
129. Noris, M. & Remuzzi, G. Overview of complement activation and regulation. Semin. Nephrol. 33, 479–92 (2013).
130. Vidarsson, G., Dekkers, G. & Rispens, T. IgG subclasses and allotypes: From structure to effector functions. Front. Immunol. 5, (2014).
131. Stavnezer, J. Antibody Class Switching. Adv. Immunol. 61, 79–146 (1996).
132. Muramatsu, M. et al. Class switch recombination and hypermutation require Activation-Induced Cytidine Deaminase (AID), a potential RNA editing enzyme. Cell 102, (2000).
133. Schroeder, H. W., Cavacini, L. & Cavacini, L. Structure and function of immunoglobulins. J. Allergy Clin. Immunol. 125, S41-52 (2010).
134. Schumaker, V. N., Calcott, M. A., Spiegelberg, H. L. & Miüller-Eberhard, H. J. Ultracentrifuge studies of the binding of IgG of different subclasses to the Clq subunit of the first component of complement. Biochemistry 15, 5175–5181 (1976).
135. Tao, M. H., Smith, R. I. F. & Morrison, S. L. Structural features of human immunoglobulin G that determine isotype- specific differences in complement activation. J. Exp. Med. 178, 661–667 (1993).
136. Clynes, R. Antitumor Antibodies in the Treatment of Cancer: Fc receptors link opsonic antibody with cellular immunity. Hematol. Oncol. Clin. North Am. 20, 585–612 (2006).
137. Guy, A. J. et al. Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases. Mol. Immunol. 67, 171–182 (2015).
138. Bruhns, P. et al. Specificity and affinity of human Fc receptors and their polymorphic variants for human IgG subclasses. Blood 113, 3716–3725 (2009).
139. Howie, H. L. et al. Affinity of human IgG subclasses to mouse Fc gamma receptors. MAbs 9, 767–773 (2017). 36
140. Tierens, A., Delabie, J., Michiels, L., Vandenberghe, P. & De Wolf-Peeters, C. Marginal-zone B cells in the human lymph node and spleen show somatic hypermutations and display clonal expansion. Blood 93, 226–234 (1999).
141. Rothstein, T. L., Griffin, D. O., Holodick, N. E., Quach, T. D. & Kaku, H. Human B-1 cells take the stage. Ann. N. Y. Acad. Sci. 1285, 97–114 (2013).
142. Hardy, R. R. B-1 B cell development. J. Immunol. 177, 2749–2754 (2006).
143. Aziz, M., Holodick, N. E., Rothstein, T. L. & Wang, P. The role of B-1 cells in inflammation. Immunologic Research 63, 153–166 (2015).
144. Rauch, P. J. et al. Innate response activator B cells protect against microbial sepsis. Science (80-. ). 335, 597–601 (2012).
145. Alugupalli, K. R. et al. B1b lymphocytes confer T cell-independent long-lasting immunity. Immunity 21, (2004).
146. Hsu, M.-C., Toellner, K.-M., Vinuesa, C. G. & MacLennan, I. C. M. B cell clones that sustain long-term plasmablast growth in T-independent extrafollicular antibody responses. Proc. Natl. Acad. Sci. 103, 5905–5910 (2006).
147. Smith, K. G. C., Hewitson, T. D., Nossal, G. J. V. & Tarlinton, D. M. The phenotype and fate of the antibody-forming cells of the splenic foci. Eur. J. Immunol. 26, 444–448 (1996).
148. MacLennan, I. C. M., de Vinuesa, C. G., Sze, D. M.-Y., Taylor, D. R. & Toellner, K.-M. Intrinsic constraint on plasmablast growth and extrinsic limits of plasma cell survival. J. Exp. Med. 192, 813–822 (2002).
149. Vinuesa, C. G., Toellner, K. M. & Papa, I. Extrafollicular antibody responses. in Encyclopedia of Immunobiology 3, 208–215 (Elsevier, 2016).
150. Lee, S. K. et al. B cell priming for extrafollicular antibody responses requires Bcl- 6 expression by T cells. J. Exp. Med. 208, 1377–1388 (2011).
151. Victora, G. D. & Nussenzweig, M. C. Germinal centers. Annu. Rev. Immunol. 30, 429–457 (2012).
152. Butt, D. et al. Differentiation of germinal center B cells into plasma cells is initiated by high-affinity antigen and completed by Tfh cells. J. Exp. Med. 214, 1259–1267 (2017).
153. Okada, T. et al. Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells. PLoS Biol. (2005). doi:10.1371/journal.pbio.0030150 37
154. Bortnick, A. & Allman, D. What is and what should always have been: long-lived plasma cells induced by T cell-independent antigens. J. Immunol. 190, 5913–8 (2013).
155. Meyers, G. et al. Activation-induced cytidine deaminase (AID) is required for B- cell tolerance in humans. Proc. Natl. Acad. Sci. 108, 11554–11559 (2011).
156. Chaudhuri, J., Evans, T., Kumar, R. & DiMenna, L. Biological function of activation-induced cytidine deaminase (AID). Biomed. J. 37, 269 (2014).
157. Sablitzky, F., Wildner, G. & Rajewsky, K. Somatic mutation and clonal expansion of B cells in an antigen-driven immune response. EMBO J. 4, 345–350 (2018).
158. Stavnezer, J. & Schrader, C. E. IgH chain class switch recombination: mechanism and regulation. J. Immunol. 193, 5370–8 (2014).
159. Stavnezer, J., Guikema, J. E. J. & Schrader, C. E. Mechanism and regulation of class switch recombination. Annu. Rev. Immunol. 26, 261–292 (2008).
160. Ise, W. Development and function of follicular helper T cells. Bioscience, Biotechnology and Biochemistry 80, 1–6 (2016).
161. Breitfeld, D. et al. Follicular B helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and support immunoglobulin production. J. Exp. Med. 192, 1545–52 (2000).
162. Kim, C. H. et al. Subspecialization of CXCR5+ T cells: B helper activity is focused in a germinal center-localized subset of CXCR5+ T cells. J. Exp. Med. 193, 1373–81 (2001).
163. Okada, T. et al. Memory B cells contribute to rapid Bcl6 expression by memory follicular helper T cells. Proc. Natl. Acad. Sci. 111, 11792–11797 (2014).
164. Ebert, L. M., Horn, M. P., Lang, A. B. & Moser, B. B cells alter the phenotype and function of follicular-homing CXCR5+ T cells. Eur. J. Immunol. 34, 3562–3571 (2004).
165. Donnarumma, T. et al. Opposing development of cytotoxic and follicular helper CD4 T Cells controlled by the TCF-1-Bcl6 nexus. Cell Rep. 17, 1571–1583 (2016).
166. Schaerli, P. et al. CXC chemokine receptor 5 expression defines follicular homing T cells with B cell helper function. J. Exp. Med. 192, 1553–62 (2000).
167. Tarlinton, D. M. et al. IL-21 regulates germinal center B cell differentiation and proliferation through a B cell–intrinsic mechanism. J. Exp. Med. 207, 365–378 (2010). 38
168. Ozaki, K. et al. A critical role for IL-21 in regulating immunoglobulin production. Science (80-. ). 298, 1630–1634 (2002).
169. Liu, S. M. & King, C. IL-21-Producing Th cells in immunity and autoimmunity. J. Immunol. 191, 3501–3506 (2013).
170. Sarkar, S. et al. Interleukin-21 is a critical cytokine for the generation of virus- specific long-lived plasma cells. J. Virol. 87, 7737–7746 (2013).
171. Rigby, R. J. et al. IL-21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses. J. Exp. Med. 207, 353–363 (2010).
172. Bessa, J., Kopf, M. & Bachmann, M. F. Cutting edge: IL-21 and TLR signaling regulate germinal center responses in a B cell-intrinsic manner. J. Immunol. 184, 4615–4619 (2010).
173. Rankin, A. L. et al. IL-21 receptor is critical for the development of memory B cell responses. J. Immunol. 186, 667–674 (2011).
174. Garin, A. et al. Article Toll-like Receptor 4 signaling by follicular dendritic cells is pivotal for germinal center onset and affinity maturation. Immunity 33, 84–95 (2010).
175. Plummer, J. R., McGettigan, J. P., Haley, S. L., Meuwissen, S. & Tzvetkov, E. P. Targeting vaccine-induced extrafollicular pathway of B cell differentiation improves rabies postexposure prophylaxis. J. Virol. 91, (2017).
176. Kirchhausen, T. et al. Endocytosis and recycling of immune complexes by follicular dendritic cells enhances B cell antigen binding and activation. Immunity 38, 1164–1175 (2013).
177. Chappell, C. P., Draves, K. E., Giltiay, N. V & Clark, E. A. Extrafollicular B cell activation by marginal zone dendritic cells drives T cell–dependent antibody responses. J. Exp. Med. 209, 1825–1840 (2012).
178. Renauld, J.-C. et al. IL-9 receptor signaling in memory B cells regulates humoral recall responses. Nat. Immunol. 19, 1025–1034 (2018).
179. Nakaya, H. I. et al. Defining antigen-specific plasmablast and memory B cell subsets in human blood after viral infection or vaccination. Nat. Immunol. 17, 1226–1234 (2016).
180. Thouvenel, C. D. et al. Somatically hypermutated plasmodium-specific IgM+ memory B cells are rapid, plastic, early responders upon malaria rechallenge. Immunity 45, 402–414 (2016). 39
181. Jones, D. D., Wilmore, J. R. & Allman, D. cellular dynamics of memory B cell populations: IgM + and IgG + memory B cells persist indefinitely as quiescent cells. J. Immunol. 195, 4753–4759 (2015).
182. Benson, M. J. et al. Cutting Edge: The dependence of plasma cells and independence of memory B cells on BAFF and APRIL. J. Immunol. 180, 3655– 3659 (2014).
183. Holden, L. A. et al. Plasma cell survival in the absence of B cell memory. Nat. Commun. 8, (2017).
184. Wolf, A. I. et al. T regulatory cells support plasma cell populations in the bone marrow. Cell Rep. 18, 1906–1916 (2017).
185. Hahne, M. et al. APRIL, a new ligand of the tumor necrosis factor family, stimulates tumor cell growth. J. Exp. Med. 188, 1185–90 (1998).
186. Schneider, P. et al. BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth. J. Exp. Med. 189, 1747–1756 (1999).
187. Mackay, F. & Browning, J. L. BAFF: A fundamental survival factor for B cells. Nat. Rev. Immunol. 2, 465–475 (2002).
188. Chu, V. T. et al. Eosinophils promote generation and maintenance of immunoglobulin-A-expressing plasma cells and contribute to gut immune homeostasis. Immunity 40, 582–93 (2014).
189. Fillatreau, S. et al. Eosinophils are required for the maintenance of plasma cells in the bone marrow. Nat. Immunol. 12, 151–159 (2011).
190. Coquery, C. M. & Erickson, L. D. Regulatory roles of the tumor necrosis factor receptor BCMA. Crit. Rev. Immunol. 32, 287–305 (2013).
191. Taddeo, A. et al. Long-lived plasma cells are early and constantly generated in New Zealand Black/New Zealand White F1 mice and their therapeutic depletion requires a combined targeting of autoreactive plasma cells and their precursors. Arthritis Res. Ther. 17, 39 (2015).
192. Medina, F., Segundo, C., Campos-Caro, A., González-García, I. & Brieva, J. A. The heterogeneity shown by human plasma cells from tonsil, blood, and bone marrow reveals graded stages of increasing maturity, but local profiles of adhesion molecule expression. Blood 99, 2154–2161 (2002).
193. Corcoran, L. M. & Nutt, S. L. Long-lived plasma cells have a sweet tooth. Immunity 45, 3–5 (2016).
194. Chang, H. D., Tokoyoda, K. & Radbruch, A. Immunological memories of the bone marrow. Immunological Reviews 283, 86–98 (2018). 40
CHAPTER 2. ROOM TEMPERATURE STABLE PSPA-BASED NANOVACCINE INDUCES PROTECTIVE IMMUNITY
Modified from a manuscript published in Frontiers in Immunology
Danielle A Wagner-Muñiz1, Shannon L Haughney2, Sean M Kelly2, Michael J
Wannemuehler1,3, and Balaji Narasimhan2,3
1Department of Veterinary Microbiology and Preventive Medicine, Iowa State
University, Ames, IA 50011, USA 2Department of Chemical and Biological Engineering,
Iowa State University, Ames, IA 50011, USA 3Nanovaccine Institute, Iowa State
University, Ames, IA 50011, USA
Abstract
Streptococcus pneumoniae is a major causative agent of pneumonia, a debilitating disease particularly in young and elderly populations, and is the leading worldwide cause of death in children under the age of five. While there are existing vaccines against S. pneumoniae, none are universally protective across all serotypes. Pneumococcal surface protein A (PspA), a key virulence factor of S. pneumoniae, is an antigen that may be incorporated into future vaccines to address the immunological challenges presented by the diversity of capsular antigens. PspA has been shown to be immunogenic and capable of initiating a humoral immune response that is reactive across approximately 94% of pneumococcal strains. Biodegradable polyanhydrides have been studied as a nanoparticle-based vaccine (i.e., nanovaccine) platform to stabilize labile proteins, to provide adjuvanticity, and enhance patient compliance by providing protective immunity in a single dose. In this study, we designed a room temperature stable PspA-based 41 polyanhydride nanovaccine that eliminated the need for a soluble protein component (i.e.,
100% encapsulated within the nanoparticles). Mice were immunized once with the lead nanovaccine and upon challenge, presented significantly higher survival rates than animals immunized with soluble protein alone, even with a 25-fold reduction in protein dose. This lead formulation performed similarly to protein adjuvanted with Alum, however, with much less tissue reactogenicity at the site of immunization. By eliminating the free PspA from the nanovaccine formulation, the lead nanovaccine was efficacious after being stored dry for 60 days at room temperature, breaking the need for maintaining the cold chain. Altogether, this study demonstrated that a PspA-based single dose nanovaccine against S. pneumoniae induced protective immunity and provided thermal stability when stored at room temperature for at least 60 days.
Introduction
Community-acquired pneumonia is a debilitating disease and is globally responsible for 16% of deaths in children under the age of five annually 1. Streptococcus pneumoniae, a causative agent of bacterial pneumonia, is a Gram-positive bacterial pathogen of humans, which colonizes the upper respiratory tract and leads to the development of potentially life-threatening diseases, such as otitis media, sinusitis, and pneumonia, with pneumonia being the most serious of these conditions 2,3. While otitis media and sinusitis are not as severe, they still account for seven million cases in the
United States annually 3. S. pneumoniae is asymptomatically carried in approximately
25% of healthy individuals and is spread between individuals through respiratory droplets, with children serving as the primary transmission source to adults (Althouse et al., 2017; Mendy et al., 2017; Quintero et al., 2011; Wyllie et al., 2017). Pneumococcal 42 infections are most prevalent in infants, young children, and the elderly, accounting for over $3 billion spent annually in direct healthcare spending; in addition, 60 to 87% of all pneumococcal bacteremia cases are associated with pneumonia in adults 3. While control of S. pneumoniae by antibiotics has been beneficial, up to 50% of all strains are resistant to erythromycin, with 97% of those also being resistant to azithromycin, making intervention strategies such as vaccination a necessary component of health care management 8. To date, the single most effective advance in the field of pneumonia prevention has been through immunization, though existing vaccines are still not completely effective 9,10.
Current preventive strategies involve the use of pneumococcal polysaccharide vaccines (PPVs) and pneumococcal conjugate vaccines (PCVs). PPVs (T-independent antigens) are comprised of capsular polysaccharides, which are poorly immunogenic in very young and elderly individuals, while PCVs (T-dependent antigens) are more effective in these at-risk populations because of the adjoined protein component. While introduction of PPV23, PCV7, and PCV13 have had significant contributions in reducing the overall global burden of pneumococcal pneumonia, there is still room for improvement 11. As it currently stands, infection with strains not covered by vaccine serotype infections account for approximately 50% of all deaths in individuals over 55 years of age 12. Additionally, it has been observed that introduction of new vaccine strategies against S. pneumoniae, such as the introduction of PCV10 immunization program for children in the Netherlands or use of PCV7 in Spain, may be directly correlated to an increased emergence of disease caused by non-vaccine serotype strains cases 13–15. It was demonstrated that PCV7 immunization, which contains the 19F 43 serotype antigen, actually caused an increase in pneumococcal infections by the the closely-related 19A serotype 16. Globally, this is of particular concern when considering the development of a universal vaccine, as it is now clear that the prevalence of certain serotypes differs greatly between age, geographic region, and ethnicity (Jedrzejas, 2001;
Kristian et al., 2016; Rolo et al. 2009). PPV23 encompasses the most pneumococcal serotypes of the commercially available vaccines. However, it is not recommended for use in children under the age of 18 months because of their poor antibody response to T- independent polysaccharide antigens, explaining why PCV13 is recommended for this age group. While the use of PCV and PPV vaccine formulations have effectively reduced the incidence of pneumococcal pneumonia, there is a need for newer vaccines to protect against the emergence of non-vaccine strains of S. pneumoniae 19. Coupled with the phase variation in expression of capsular and surface protein antigens, broadly protective vaccines against colonization and invasive pneumococcal disease are likely to require the inclusion of more conserved antigens that will also facilitate the induction of antigen- specific CD4+ T cells 20,21.
S. pneumoniae has several key virulence factors that play a critical role in colonization, transmission, and tissue damage including pneumolysin, two neuraminidases, and pneumococcal surface protein A (PspA). PspA is a choline binding protein, one of the most abundant proteins located on the pneumococcal cell surface, and has been shown to be of particular importance in facilitating nasopharyngeal colonization through inhibiting host complement responses 22. When considering the development of new vaccines against S. pneumoniae, it is important to consider a target immunogen that will be capable of inducing a broad, serotype-independent, protective immune response. 44
PspA has been of recent interest as a potential vaccine candidate due to its location on the cell surface of all 95 strains of S. pneumoniae currently known and its critical role in pneumococcal pathogenesis 2,23,24. Anti-PspA antibodies to clades 1 and 2 of PspA have been shown to be cross-protective against S. pneumoniae strains encompassing all six clades of PspA, and provide protection when passively transferred to naïve mice as a therapeutic intervention following septic S. pneumoniae challenge 17,25–27. The ability to provide protection across many serotypes of S. pneumoniae using only two clades of
PspA allows for the design of a broadly cross-protective vaccine when compared to vaccines containing numerous capsular polysaccharides.
Polyanhydride nanoparticle-based vaccines (i.e., nanovaccines) represent next generation vaccine platforms against pathogens such as S. pneumoniae. These nanovaccines are formulated using random copolymers based on 1,8-bis-(p- carboxyphenoxy)-3,6-dioxaoctane (CPTEG), 1,6-bis-(p-carboxyphenoxy)hexane (CPH), and sebacic acid (SA) and have been extensively studied as a nanovaccine platform against infectious pathogens such as influenza virus, Yersinia pestis, and Bacillus anthracis 28–31.
Polyanhydrides provide vaccine delivery benefits and adjuvant properties that make them well suited as a vaccine delivery platform. These materials exhibit high biocompatibility with minimal injection site reactivity 32–34 (i.e., tenderness, swelling, and pain) in comparision to traditional adjuvants, such as Alum, which has been associated with immunization site tenderness and pain, and are currently FDA-approved for use to treat malignant gliomas in the brain 35,36. In addition, polyanhydrides are hydrophobic and exhibit surface erosion characteristics, which helps stabilize labile proteins, protects 45 protein denaturation from enzymatic cleavage and acidic degradation products, and allows for the prolonged release of antigen 28,37–41. The sustained release of antigen allows for enhanced bioavailability of antigen to drive adaptive immune responses and allows for single dose administration and dose-sparing capabilities 29,42–44. Previous work has shown that mice immunized with a single dose of nanovaccine encapsulating Y. pestis fusion protein F1-V were completely protected against Y. pestis lethal challenge after at least 280 days post-immunization (DPI) 31. Varying polyanhydride copolymer composition has shown to modulate internalization and persistence within APCs in vitro, as well as induction of both cellular and humoral immune responses in vivo indicating the ability to tailor polymer chemistry in order to rationally design nanovaccines that optimally inducing antigen-specific protective immunity 31,42,45–54.
Previous work from our laboratories has demonstrated that PspA encapsulated into nanoparticles maintained its stability, conformational structure, as well as biological activity upon release, which was measured using an apolactoferrin binding assay 38. In this same work, mice immunized with a single dose of the nanovaccine generated robust antibody titer and avidity to PspA, equivalent to that of antigen adjuvanted with MPLA.
However, the protective efficacy of a PspA-based nanovaccine against lethal challenge has not been demonstrated. In this study, the ability of various polyanhydride nanovaccine chemistries as vaccine candidates was evaluated and it was demonstrated that a lead PspA-based nanovaccine protected animals against lethal challenge with a 25- fold reduction in overall PspA dose. Further, this lead formulation was shown to be capable of storage at room temperature for at least two months without any loss of efficacy. 46
Materials and Methods
Materials
Chemicals used for monomer and polymer synthesis included sodium hydroxide, hydrobenzoic acid, dibromohexane, 1-methyl-2-pyrrolidinone, sebacic acid monomer, and triethylene glycol purchased from Sigma Aldrich (St. Louis, MO). Acetic anhydride, ethyl ether, petroleum ether, chloroform, methylene chloride, hexane, acetone, sulfuric acid, potassium carbonate, dimethyl formamide, toluene, acetonitrile, N,N- dimethylacetamide, and acetic acid were purchased from Fisher Scientific (Fairlawn, NJ);
4-p-fluorobenzonitrile was purchased from Apollo Scientific (Cheshire, UK). For 1H
NMR analysis of the copolymers, deuterated chloroform and dimethyl sulfoxide were purchased from Cambridge Isotope Laboratories (Andover, MA). The N-terminal region of a recombinant PspA (UAB055, PspA/Rx1 AA1 to 303, clade 2 PspA of the PspA family 1) was produced by Dr. David McPherson (University of Alabama at
Birmingham) as described previously 55. Prior to immunization, endotoxin was removed from the protein using endotoxin removal beads (Miltenyi Biotec, Bergisch Gladbach,
Germany) according to the manufacturer’s instructions followed by dialysis and lyophilization. The final endotoxin content of the protein was determined to be less than
1.9 EU/mg as determined by a limulus amebocyte lysate (LAL) chromogenic endotoxin quantification kit (Thermo Fisher Scientific).
Copolymer and Nanoparticle Synthesis
Monomers of CPH and CPTEG were synthesized as previously described 28,47. A
50:50 molar composition copolymer of CPH and CPTEG and a 20:80 molar composition copolymer of CPH and SA was synthesized using melt condensation as described by
Torres et al. 28. PspA protein, 1% (w/w), was encapsulated into polyanhydride 47 nanoparticles using solid/oil/oil nanoprecipitation, as previously described 38. Excess buffer was removed from PspA protein solution using a 5 kDa MWCO dialysis microcentrifuge tube and the resulting protein solution was lyophilized overnight at -40 ̊C under vacuum. The lyophilized protein and the respective copolymer was then dissolved in methylene chloride at a concentration of 20 mg/mL of solvent. The solution was sonicated using the VibraCell ultrasonic probe (Sonics & Materials, Inc., Newton, CT) to ensure complete dissolution and homogenization of the protein and the copolymer. The resulting solution was then added to pentane at a 1 to 200 (v/v) ratio of methylene chloride to pentane at 20°C for the 20:80 CPH:SA nanoparticle formulation or at a 1 to
250 (v/v) ratio of methylene chloride to pentane at -40°C and for the 50:50 CPTEG:CPH nanoparticle formulation. Nanoparticles were collected using vacuum filtration. Surface charge of representative 20:80 CPH:SA and 50:50 CPTEG:CPH nanoparticle formulations were measured using quasi-elastic light scattering (QELS, Zetasizer Nano,
Malvern Instruments Ltd., Worcester, UK).
Animals
CBA/CaHN-Btkxid/J (CBA/N) mice were purchased from Jackson Laboratory
(Bar Harbor, ME). These mice, which have a mutation in their Bruton’s tyrosine kinase gene, were used for the immunization studies. Due to this mutation, these mice are unable to initiate a proper antibody response to Type II thymic-independent antigens (e.g., capsular polysaccharides), and are thus able to better mimic natural antibody responses in at-risk elderly or young populations. Mice were housed under specific pathogen-free conditions where all bedding, caging, water, and feed were sterilized prior to use. All studies were conducted with the approval of the Iowa State University Institutional
Animal Care and Use Committee. 48
Immunization Protocols
Groups of 8-10 CBA/N mice were immunized subcutaneously at the nape of the neck with 0 to 20 µg PspA, 0 to 5 µg PspA encapsulated into 50 to 500 µg polyanhydride nanoparticles, and/or 50 µL of Imject Alum Adjuvant (ThermoFisher Scientific) and delivered in a total volume of 100 µL. Immunization groups for the individual studies
(see Tables 1 and 2) were comprised of: i) 25 µg soluble PspA alone, ii) 25 µg PspA administered with 50 µL Imject Alum, iii) 5 µg PspA encapsulated into 250 µg 50:50
CPTEG:CPH nanoparticles + 20 µg soluble PspA, iv) 5 µg PspA encapsulated into 250
µg 20:80 CPH:SA nanoparticles + 20 µg soluble PspA, v) 1 µg soluble PspA alone, vi) 1
µg PspA administered with 50 µL Imject Alum, vii) 1 µg PspA encapsulated into 50 µg
50:50 CPTEG:CPH nanoparticles, viii) 0.5 µg PspA encapsulated into 25 µg 50:50
CPTEG:CPH nanoparticles + 25 µg blank 50:50 CPTEG:CPH nanoparticles + 0.5 µg soluble PspA, or ix) saline control. Blood was collected from mice via saphenous vein and serum was isolated following centrifugation (10,000 rcf for 10 min) at days 17 or 21
DPI. Serum was stored at -20°C until analysis could be performed.
S. pneumoniae Challenge
S. pneumoniae strain A66.1, of PspA family 1, clade 2 was used for challenge and grown as previously described 56,57. Briefly, the bacteria was grown at 37°C for 18 hours in filter-sterilized Todd-Hewitt broth with 0.5% yeast extract, and stored at -80°C at a concentration of 1 x 108 CFU/mL. Inoculum was diluted to a concentration of 2.5 x 104
CFU/mL in sterile PBS and 100 µL was administered intravenously at 24 DPI. Mice were monitored three times per day for the duration of the challenge (14 days). Body temperatures and body condition scores were also recorded once per day. Mice were sacrificed when determined to be moribund or at 14 days post-challenge. 49
Anti-PspA Antibody Titer
Anti-PspA antibody titers were determined via an ELISA as described previously
38. Briefly, high-binding Costar 590 EIA/RIA microtiter plates (Corning) were coated overnight at 4 °C with 0.5 µg/mL PspA. The blocking buffer for this assay was comprised of 2.5% (w/v) powdered milk dissolved in PBS-T and incubated for two hours at 56°C to inactivate any endogenous phosphatase activity. PspA-coated plates were incubated with blocking solution for two hours at room temperature before being washed three times with PBS-T. Serum obtained from immunized mice was added to the first well of a row at a dilution of 1:200 and serially diluted in PBS-T containing 1% (v/v) goat serum. Each serum sample was tested in duplicate. Following incubation overnight at 4°C, plates were washed three times with PBS-T after which secondary antibody was added at a dilution of 1 µg/mL. The secondary antibody used in these studies was alkaline phosphatase-conjugated goat anti-mouse IgG heavy and light chain (Jackson
ImmunoResearch) and was incubated on the plates for two hours at room temperature.
Plates were then washed three times with PBS-T and alkaline phosphatase substrate
(Fisher Scientific, Pittsburgh, PA) was added at a concentration of 1 mg/mL dissolved in
50 mM sodium carbonate, 2 mM magnesium chloride buffer at pH 9.3 for colorimetric development. Plates were analyzed after 30 min using a SpectraMax M3 microplate reader at a wavelength of 405 nm. Titer is reported as the reciprocal of serum dilution at which optical density value was at least twice that of the saline group average plus two standard deviations.
Shelf Stability of Stored PspA Nanovaccine
Shelf stability of the PspA nanovaccine was assessed via ELISA and lethal challenge. 50:50 CPTEG:CPH nanoparticles encapsulating PspA were taken out of the 50 freezer (-80 °C) and stored as a dry powder formulation at 25 °C (room temperature) in a glass desiccator containing drierite for 60 days prior to immunization. Soluble PspA was stored at 4°C and combined with Imject Alum prior to immunization. A separate group of nanoparticles were kept in the freezer over the course of this experiment as a freezer- stored standard control. Following room temperature storage, vaccine efficacy was evaluated by immunizing mice with a nanovaccine dose equivalent to 1 μg PspA per animal from one of the storage conditions above, soluble PspA adsorbed to Alum, or the saline control and assessing antibody titer via ELISA 21 DPI (see ‘Anti-PspA Antibody
Titer’ section for details). Following lethal challenge with 2500 CFU of S. pneumoniae
(see S. pneumoniae challenge section for details), survival was assessed over the next 24 days.
Statistical Analyses
Statistical analyses were performed using the Gerhan-Breslow-Wilcoxon test and the one-way analysis of variance (ANOVA) using Tukey’s multiple comparisons test. A p-value ≤ 0.05 was considered statistically significant. All analyses were performed using
GraphPad Prism v. 7.0 software.
Results
Synthesis and characterization of PspA-encapsulated polyanhydride nanoparticles
The nanoparticle chemistries chosen for the current study were based on 50:50
CPTEG:CPH and 20:80 CPH:SA copolymers, consistent with our previous work, in which these chemistries were shown to provide protein structural stability and maintain the functional activity of PspA following release 38. The nanoparticles were synthesized using solid/oil/oil emulsion 48. The PspA release kinetics from these formulations are described in our previous work 38. Scanning electron photomicrographs of the 51 nanoparticles revealed similar sizes for the two formulations, with sizes of 455 ± 175 nm and 422 ± 163 nm for 50:50 CPTEG:CPH and 20:80 CPH:SA nanoparticles, respectively
(Figure 2.1). Zeta potential measurements of empty 20:80 CPH:SA and 50:50
CPTEG:CPH nanoparticles revealed that the particles had a negative surface charge, with zeta potentials of -41.9 ± 0.9 mV and -33.1 ± 5.1 mV, respectively, consistent with previous publications 49,53,58.
Figure 2.1 PspA nanovaccine characterization. Representative scanning electron photomicrographs of (A) 2% PspA-loaded (w/w) 20:80 CPH:SA nanoparticles and (B) 2% PspA-loaded (w/w) 50:50 CPTEG:CPH nanoparticles (scale bar = 5 μm). (C) Particle size distribution of 2% PspA-loaded (w/w) 20:80 CPH:SA nanoparticles (422 ± 163 nm). (D) Particle size distribution of 2% PspA-loaded (w/w) 50:50 CPTEG:CPH nanoparticles (455 ± 175 nm).
Immunization with PspA-based nanovaccines induces protective antibody response
Previously, it was demonstrated that mice vaccinated with PspA-based nanovaccines induced a robust humoral response, however protective efficacy was not 52 evaluated 38. In the current studies, separate groups of CBA/CaHN mice (n = 8 per group) were immunized subcutaneously as follows: i) 25 µg soluble PspA alone, ii) 25 µg PspA administered with Imject Alum, iii) PspA-containing 50:50 CPTEG:CPH nanoparticles , iv) PspA-containing
20:80 CPH:SA nanoparticles, or v) saline (Table 2.1). Both the nanovaccine formulations contained 5 µg of encapsulated PspA along with 20 µg of soluble protein, as shown in Table 1.
Anti-PspA total IgG titers were evaluated at 17 DPI and there were no significant differences in titer between vaccinated groups (Figure 2.2A). Mice were challenged with a lethal dose of S. pneumoniae strain A66.1 at 24 DPI and survival was assessed over a period of two weeks. All
PspA naïve (saline) mice succumbed to infection within four days, while all the immunized animals showed prolonged survival following challenge. At 14 days post-challenge, only 25% of the (non-adjuvanted) soluble PspA-immunized animals survived, whereas adjuvanting the protein with alum provided 100% protection. Immunization with the 20:80 CPH:SA nanovaccine resulted in 87% survival, while 100% of the animals immunized with 50:50 CPTEG:CPH nanovaccine were protected, as shown in Figure 2.2B. The survival data, in correlation with the antibody titers indicate that not only is PspA an effective immunogen, but induces protective immunity when delivered with an adjuvant. Though both nanoparticle chemistries protected animals from lethal challenge compared to saline, the 50:50 CPTEG:CPH PspA nanovaccine performed significantly (p ≤ 0.008) better than sPspA alone. Based on these results, we selected the 50:50 CPTEG:CPH nanovaccine as our lead candidate for further evaluation.
Table 2.1 PspA Vaccine Formulations. 25 µg PspA containing single dose vaccine treatments groups. Separate groups of mice were immunized s.c. and all formulations were delivered in 100 µL total volume. Soluble PspA is represented as sPspA, and nanoparticle-encapsulated PspA is represented as ePspA.
Formulation sPspA (µg) ePspA (µg) 50:50 CPTEG:CPH PspA Nanovaccine 20 5 20:80 CPH:SA PspA Nanovaccine 20 5 sPspA + Alum 25 0 sPspA 25 0 Saline 0 0
53
Figure 2.2 PspA nanovaccines are capable of initiating protective immune responses. (A) Serum was collected from immunized mice (see Table 1) at 17 DPI and analyzed for total anti-PspA IgG (data is represented as log10) using ELISA. Dashed line represents background anti-PspA IgG levels in serum from the saline-immunized mice. (B) S. pneumoniae challenge was performed at 24 DPI by i.v. administration of 2500 CFUs of A66.1 strain and survival was monitored for 14 days post-challenge. * indicates significance (p ≤ 0.0025) compared to sPspA alone and # indicates significance (p ≤ 0.001) compared to saline treated mice. Data is representative of single experiment containing n = 8 mice per treatment group. 54
PspA nanovaccine demonstrates dose-sparing capabilities
Rational design of vaccines using subunit proteins involves considering total cost of the end product, as well as protein availability that potentially impacts vaccine shortages. Our previous work has shown that encapsulating a lowered dose of antigen into polyanhydride nanoparticles can induce a robust immune response 44. In the current studies, the ability of the nanovaccine to provide dose-sparing (i.e., 25-fold reduction) was evaluated by immunizing separate groups of CBA/N mice (n = 8 per group; see
Table 2.2) subcutaneously using the following regimens: i) 1 µg PspA encapsulated into
50:50 CPTEG:CPH nanoparticles (PspA Nanovaccine), ii) 0.5 µg PspA encapsulated into
50:50 CPTEG:CPH nanoparticles plus 0.5 µg soluble PspA (PspA Nanovaccine +
Soluble PspA), iii) 1 µg soluble PspA administered with Imject Alum, iv) 1 µg soluble
PspA alone, and v) saline alone (i.e., naïve control). Serum was collected at 21 DPI and analyzed for total anti-PspA IgG. Mice immunized with the nanovaccines or PspA +
Alum showed significantly (p ≤ 0.008) higher titers to PspA compared to soluble PspA alone (Figure 2.3A). Survival rates following lethal challenge showed significantly (p ≤
0.001) higher survival (87.5%) in mice immunized with both the nanovaccine formulations compared to animals immunized with soluble PspA (12%) (Figure 2.3B).
Animals immunized with PspA adjuvanted with Alum performed similarly to the animals receiving the nanovaccine formulations. There were no observable differences in disease severity between the immunized animals, as measured by body condition scores and temperature (data not shown). Additionally, soluble PspA administered in conjunction with 100 µg of empty 50:50 CPTEG:CPH nanoparticles, performed similarly to other adjuvanted groups, further indicating the ability for PspA to initiate a protective immune response when adjuvanted properly (data not shown). No significant differences in 55 survival were observed between mice immunized with PspA fully encapsulated within the nanoparticles (i.e., PspA Nanovaccine) and animals that received half of the protein as a soluble bolus (i.e., PspA Nanovaccine + Soluble PspA). Eliminating the use of soluble PspA in the nanovaccine is beneficial when designing a shelf-stable dry powder formulation for the long-term storage of a PspA nanovaccine.
Table 2.2 Dose-sparing PspA vaccine formulations. One µg PspA containing single dose vaccine treatments groups. Separate groups of mice were immunized s.c. and all formulations were delivered in 100 µL total volume. Soluble PspA is represented as sPspA, and nanoparticle-encapsulated PspA is represented as ePspA.
Formulation sPspA (µg) ePspA (µg) PspA Nanovaccine 0 1 PspA Nanovaccine + sPspA 0.5 0.5 sPspA + Alum 1 0 sPspA 1 0 Saline 0 0
PspA nanovaccine maintains efficacy following room temperature storage
To evaluate the potential for a room temperature stored (i.e., non-refrigerated) PspA nanovaccine, 1 µg of PspA was encapsulated within 50 µg of 50:50 CPTEG:CPH nanoparticles (i.e., 2 wt.% loading) and stored at room temperature (25°C) and standard storage conditions (-80°C), as detailed in the methods section and as previously described
28. The PspA encapsulated nanovaccine was stored at -80° C (freezer stored) or at 25°C for 60 days.
56
Figure 2.3 Nanovaccines with 25-fold reduction in total protein provided protection against lethal challenge. (A) Serum was collected from immunized mice (see Table 3) at 21 DPI and analyzed for total anti-PspA IgG (data is represented as log10) using ELISA. Dashed line represents background anti-PspA IgG levels in the serum from the saline-immunized mice. * indicates significance (p ≤ 0.008) compared to sPspA alone. (B) S. pneumoniae challenge was performed at 24 DPI by i.v. administration of 2500 CFUs of A66.1 strain and survival was monitored for 14 days. * indicates significance (p ≤ 0.003) compared to sPspA alone and # indicates significance (p ≤ 0.0002) compared to saline treated mice. Data is representative of single experiment containing n = 8 mice per treatment group. 57
The soluble PspA was stored at 4°C and mixed with Alum prior to immunization.
Following storage, separate groups of mice (n= 8-10 per group) were immunized subcutaneously with the PspA nanovaccine stored at the different conditions or with alum-adjuvanted PspA and serum was collected and analyzed at 21 DPI. Storage conditions did not have a significant effect on anti-PspA IgG titers, with all of the PspA nanovaccines performing similarly (Figure 2.4A). Mice were then challenged with a lethal dose of S. pneumoniae at 24 DPI and survival was monitored for two weeks. All of the non-immunized mice succumbed to challenge within four days post challenge, while the nanovaccine-immunized mice presented with 60-70% survival with a single dose
(Figure 2.4B). Once again, no significant differences were observed between the survival rates of mice immunized with the PspA nanovaccine stored at various conditions and those immunized with soluble PspA adjuvanted with Alum. This data indicates that the
PspA nanovaccine is efficacious when stored both in the freezer or at room temperature for time periods up to 60 days and performs similarly to refrigerated PspA adjuvanted with Alum.
Discussion
Streptococcus pneumoniae is a causative agent of debilitating bacterial pneumonia. While many existing vaccines have proven to be valuable in reducing overall global disease burden, none are without limitations. Newly developed vaccine strategies against S. pneumoniae have identified several novel antigens, including PspA. Anti-PspA antibodies have been observed, following natural colonization or infection in children
59,60. Other studies have shown success, against lethal intranasal or septic S. pneumoniae challenge by vaccinating with PspA which further indicates its potential as an efficacious vaccine immunogen 22,61.
58
Figure 2.4 Room temperature-stored PspA nanovaccine maintains efficacy after 60 days. (A) Serum was collected from groups of immunized mice at 21 DPI with vaccine stored at different conditions as indicated and analyzed for total anti-PspA IgG (data is represented as log10) using ELISA. Dashed line represents background anti-PspA IgG levels in the serum from the saline-immunized mice. (B) S. pneumoniae challenge was performed at 24 DPI by i.v. administration of 2500 CFUs of A66.1 strain and survival was monitored for 14 days. * indicates significance (p ≤ 0.001) compared to saline treated mice. Data is representative of single experiment containing n = 8-10 mice per treatment group. 59
Aluminum hydroxide (i.e., Alum)-based adjuvants are widely used because of their ability to promote Th2 immune responses 62. However, Alum-based vaccines are associated with higher levels of immunization site tissue reactogenicity (Chen and
Kristensen, 2009; Levy et al. 2013). Immunization-site tenderness and pain are the most commonly reported symptoms following immunization with vaccines containing Alum.
Prevalence of these adverse reactions has been found to be directly associated with increased protein dose administered and decreased age of the vaccinated individuals
(Brady et al., 2009; Petrovsky 2015; Willhite et al. 2014). Previous work with polyanhydride nanovaccines have shown low-level tissue site inflammation and toxicity making them a safe alternative to traditional vaccine adjuvants 34,41,68. Therefore, nanovaccines may represent a safe and efficacious alternative to Alum-based vaccines, which have limitations associated with administration site reactogenicity and only promote humoral immune responses against PspA.
Work from other laboratories has shown limited success using nanotechnology vaccine platforms with PspA in various animal models though, to date, none of these studies have shown protection against lethal challenge with single-dose immunization 69–
71. Previous work from our laboratories has indicated that encapsulation of PspA into polyanhydride nanoparticles is capable of retaining protein structure and function, which enabled initiation of a humoral immune response 38. Building upon these studies, PspA was encapsulated within both 50:50 CPTEG:CPH and 20:80 CPH:SA nanoparticle formulations in the current work. Both formulations produced nanoparticles of similar size and size distribution (Figure 2.1). The size, morphology and size distribution of these 60 particles were similar to those previously reported for these formulations28,29,38,42.
Following subcutaneous immunization of mice with a single dose of either formulation, anti-PspA IgG antibodies were produced, in agreement with our previous findings 38.
Following lethal septic challenge, both nanovaccine formulations (containing 25 µg
PspA) were capable of conferring protective immunity in mice, with 87.5% survival in
20:80 CPH:SA nanovaccine immunized mice and 100% protection in those that received the 50:50 CPTEG:CPH nanovaccine (Figure 2.2). Based on the increased protection and higher antibody titers observed following immunization with a single dose of the 50:50
CPTEG:CPH nanovaccine formulation, this chemistry was selected as the lead candidate nanovaccine.
Reducing the amount of protein necessary per dose can greatly reduce the cost associated with using a recombinant protein vaccine, thereby allowing for the production and storage of more doses. Most of the published reports evaluating PspA-based vaccine success immunized mice with a minimum of 5 µg of antigen plus an adjuvant and required multiple boosts 56,61,72,73. Indeed, a recent report demonstrated complete protection in mice receiving 0.5 µg of PspA adjuvanted with poly(I:C) intranasally; however, the immunization schedule required three doses to provide protection 73. The need for a single dose vaccination regimen cannot be overstated from a patient compliance standpoint because according to the WHO, an estimated 29% of children aged one month to five years die each year from vaccine preventable diseases, citing non- compliance with the immunization regimen as a key reason for lack of successful national vaccination programs 74. Therefore, single-dose vaccines can aid in improving patient compliance, reduce cost, and improve national immunization rates worldwide. 61
Recent work from our laboratories has demonstrated that 25 µg of ovalbumin encapsulated into polyanhydride microparticles was able to elicit robust antibody titers equivalent to a 16x larger soluble dose of ovalbumin while 25 µg of soluble ovalbumin alone was unable to elicit detectable antibody responses 44. In order to corroborate these results with PspA, the dose of antigen evaluated was reduced 25-fold to 1 µg.
Immunization with the 1 µg PspA-based 50:50 CPTEG:CPH nanovaccine formulation, both with and without soluble PspA, induced production of high anti-PspA IgG titers
(Figure 2.3A), with very little reduction in overall antibody titer compared to the previous study with a 25 µg dose of PspA (i.e., data in Figure 2.2). Antibody titers were similar between mice immunized with 1 µg of soluble PspA administered with Alum or with the nanovaccine formulations, with the highest overall titers observed in mice immunized with the fully-encapsulated nanovaccine formulation. When compared to soluble protein adjuvanted with Alum, mice immunized with the 50:50 CPTEG:CPH nanovaccine were similarly protected from lethal challenge. Furthermore, 87.5% of the animals challenged with the reduced dose PspA nanovaccine (with or without soluble PspA) were protected
(Figure 2.3B). These data demonstrate that reducing the protein dose by 25-fold had little effect on survival, with both the fully encapsulated PspA nanovaccine and the nanovaccine administered with a soluble bolus providing significantly higher survival following lethal challenge, compared to soluble protein alone.
By eliminating the soluble protein component from vaccine formulations, nanovaccines can be stored as a dry powder outside of refrigeration with the potential for long-term shelf storage, thereby circumventing the cold chain. As it currently stands, all prequalified WHO vaccines require refrigeration 74. Nanovaccines may enable next- 62 generation vaccines to be stored without the need for refrigeration (i.e., eliminating the cold chain), allowing for vaccines to be sent to remote locations in need for vaccination, resulting in reduced medical costs and productivity losses associated with disease 75.
Previous work from our laboratories demonstrated that nanovaccines encapsulating PspA were able to retain protein structure and activity following release, and a nanovaccine encapsulating the Bacillus anthracis protective antigen (PA) released biologically and immunologically functional protein following storage at both room temperature and above after four months 28,38. In contrast, aluminum salt-based adjuvants have been reported to alter the structure of proteins and decrease thermal stability following adsorption 76, and previous work from our laboratories has shown that PA adsorbed to alum lost its functional activity when stored for four months at refrigerated conditions or above 28. Following storage at room temperature for 60 days, the PspA nanovaccine was shown to be efficacious, as measured by antibody titer and survival (Figure 2.4). PspA adsorbed to alum performed similarly, however, because PspA is a stable protein with coiled structure, it is likely that differences may be observed between the efficacy of the nanovaccine and alum-based formulations following storage for an extended period of time, or at elevated temperature, and should be evaluated in future studies 22. As such, this work indicates that the PspA nanovaccine formulation can be stored as a dry powder for at least 60 days while maintaining its immunogenicity.
In summary, this work describes the design of a safe next-generation PspA-based pneumonia nanovaccine with dose-sparing, protective immunity, and enhanced room temperature storage. These studies are promising first steps with respect to establishing the polyanhydride nanovaccine platform as an alternative to traditional vaccines in terms 63 of reduced cost (enabled by the dose sparing and single dose capabilities) and difficulties associated with transportation and storage of vaccines in developing countries (enabled by the superior room temperature storage capabilities).
Acknowledgments
The authors thank Dr. David McPherson of the University of Alabama at
Birmingham for producing the recombinant PspA used in this work. The authors acknowledge financial support from NIH-NIAID (R56-AI075026) and the Nanovaccine
Institute. B.N. acknowledges the support of the Vlasta Klima Balloun Faculty Chair.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
References
1. O’Brien, K. L. et al. Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates. Lancet 374, 893–902 (2009).
2. Jedrzejas, M. J. Pneumococcal Virulence Factors : Structure and Function Pneumococcal Virulence Factors : Structure and Function. Microbiol. Mol. Biol. Rev. 65, 187–207 (2001).
3. Huang, S. S. et al. Healthcare utilization and cost of pneumococcal disease in the United States. Vaccine 29, 3398–3412 (2011).
4. Mendy, A. L. et al. Kinetics of antibodies against pneumococcal proteins and their relationship to nasopharyngeal carriage in the first two months of life. 4, 1–15 (2017).
5. Quintero, B. et al. Epidemiology of Streptococcus pneumoniae and Staphylococcus aureus colonization in healthy Venezuelan children. Eur. J. Clin. Microbiol. Infect. Dis. 30, 7–19 (2011).
6. Wyllie, A. L. et al. Sequencing of the variable region of rpsB to discriminate between Streptococcus pneumoniae and other streptococcal species. Open Biol. 7, 170074 (2017). 64
7. Althouse, B. M. et al. Identifying transmission routes of Streptococcus pneumoniae and sources of acquisitions in high transmission communities. Epidemiol. Infect. 145, 2750–2758 (2017).
8. Song, J.-H. et al. Macrolide resistance and genotypic characterization of Streptococcus pneumoniae in Asian countries: a study of the Asian Network for Surveillance of Resistant Pathogens (ANSORP). J. Antimicrob. Chemother. 53, 457–463 (2004).
9. Jacobs, D. M., Yung, F., Hart, E., Nguyen, M. N. H. & Shaver, A. Trends in pneumococcal meningitis hospitalizations following the introduction of the 13- valent pneumococcal conjugate vaccine in the United States. Vaccine 35, 6160– 6165 (2017).
10. Grabenstein, J. D. & Musey, L. K. Differences in serious clinical outcomes of infection caused by specific pneumococcal serotypes among adults. Vaccine 32, 2399–2405 (2014).
11. Bonten, M. J. M. et al. Polysaccharide Conjugate Vaccine against Pneumococcal Pneumonia in Adults. N. Engl. J. Med. 372, 1114–1125 (2015).
12. Marrie, T. J., Tyrrell, G. J., Majumdar, S. R. & Eurich, D. T. Effect of Age on the Manifestations and Outcomes of Invasive Pneumococcal Disease in Adults. Am. J. Med. (2017). doi:10.1016/j.amjmed.2017.06.039
13. Vestjens, S. M. T. et al. Changes in pathogens and pneumococcal serotypes causing community-acquired pneumonia in The Netherlands. Vaccine 35, 4112– 4118 (2017).
14. Guevara, M. et al. Changing epidemiology of invasive pneumococcal disease following increased coverage with the heptavalent conjugate vaccine in Navarre, Spain. Clin. Microbiol. Infect. 15, 1013–1019 (2009).
15. Pletz, M. W., Maus, U., Krug, N., Welte, T. & Lode, H. Pneumococcal vaccines: mechanism of action, impact on epidemiology and adaption of the species. Int. J. Antimicrob. Agents 32, 199–206 (2008).
16. Hausdorff, W. P., Hoet, B. & Schuerman, L. Do pneumococcal conjugate vaccines provide any cross-protection against serotype 19A? BMC Pediatr. 10, 4 (2010).
17. Kristian, S. A. et al. Generation and improvement of effector function of a novel broadly reactive and protective monoclonal antibody against pneumococcal surface protein A of Streptococcus pneumoniae. PLoS One 11, 1–25 (2016).
18. Rolo, D., Ardanuy, C., Fleites, A., Martín, R. & Liñares, J. Diversity of pneumococcal surface protein A (PspA) among prevalent clones in Spain. BMC Microbiol. 9, 80 (2009). 65
19. Weil-Olivier, C., van der Linden, M., de Schutter, I., Dagan, R. & Mantovani, L. Prevention of pneumococcal diseases in the post-seven valent vaccine era: A European perspective. BMC Infect. Dis. 12, 1–12 (2012).
20. Pichichero, M. E. Pneumococcal whole-cell and protein-based vaccines: changing the paradigm. Expert Rev. Vaccines 16, 1181–1190 (2017).
21. Khan, M. N., Xu, Q. & Pichichero, M. E. Protection against Streptococcus pneumoniae Invasive Pathogenesis by a Protein-Based Vaccine Is Achieved by Suppression of Nasopharyngeal Bacterial Density during Influenza A Virus Coinfection. Infect. Immun. 85, e00530-16 (2017).
22. Khan, N. & Jan, A. T. Towards identifying protective B-cell epitopes: The PspA story. Front. Microbiol. 8, 1–8 (2017).
23. Mehr, S. & Wood, N. Streptococcus pneumoniae - a review of carriage, infection, serotype replacement and vaccination. Paediatr. Respir. Rev. 13, 258–264 (2012).
24. Zafar, M. A., Hamaguchi, S., Zangari, T., Cammer, M. & Weiser, J. N. Capsule Type and Amount Affect Shedding and Transmission of Streptococcus pneumoniae. MBio 8, e00989-17 (2017).
25. Piao, Z. et al. Protective properties of a fusion pneumococcal surface protein A (PspA) vaccine against pneumococcal challenge by five different PspA clades in mice. Vaccine 32, 5607–5613 (2014).
26. Darrieux, M. et al. Recognition of pneumococcal isolates by antisera raised against PspA fragments from different clades. J. Med. Microbiol. 57, 273–278 (2008).
27. Goulart, C. et al. Selection of family 1 PspA molecules capable of inducing broad- ranging cross-reactivity by complement deposition and opsonophagocytosis by murine peritoneal cells. Vaccine 29, 1634–1642 (2010).
28. Petersen, L. K., Phanse, Y., Ramer-Tait, a. E., Wannemuehler, M. J. & Narasimhan, B. Amphiphilic polyanhydride nanoparticles stabilize bacillus anthracis protective antigen. Mol. Pharm. 9, 874–882 (2012).
29. Ulery, B. D. et al. Design of a protective single-dose intranasal nanoparticle-based vaccine platform for respiratory infectious diseases. PLoS One 6, 1–8 (2011).
30. Ross, K. et al. Combination Nanovaccine Demonstrates Synergistic Enhancement in Efficacy against Influenza. ACS Biomater. Sci. Eng. 2, 368–374 (2016).
31. Ulery, B. D. et al. Rational design of pathogen-mimicking amphiphilic materials as nanoadjuvants. Sci. Rep. 1, 198 (2011). 66
32. Huntimer, L. et al. Evaluation of Biocompatibility and Administration Site Reactogenicity of Polyanhydride-Particle-Based Platform for Vaccine Delivery. Adv. Healthc. Mater. 2, 369–378 (2013).
33. Vela-Ramirez, J. E. et al. Safety and Biocompatibility of Carbohydrate- Functionalized Polyanhydride Nanoparticles. AAPS J. 17, 256–67 (2015).
34. Adler, A. F. et al. High throughput cell-based screening of biodegradable polyanhydride libraries. Comb. Chem. High Throughput Screen. 12, 634–45 (2009).
35. Brem, H. et al. Biocompatibility of a biodegradable, controlled-release polymer in the rabbit brain. Sel. Cancer Ther. 5, 55–65 (1989).
36. Brem, H. et al. Brain biocompatibility of a biodegradable controlled release polymer consisting of anhydride copolymer of fatty acid dimer and sebacic acid. J. Control. Release 19, 325–329 (1992).
37. Torres, M. P., Vogel, B. M., Narasimhan, B. & Mallapragada, S. K. Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms. J. Biomed. Mater. Res. - Part A 76, 102–110 (2006).
38. Haughney, S. L. et al. Retention of structure, antigenicity, and biological function of pneumococcal surface protein A (PspA) released from polyanhydride nanoparticles. Acta Biomater. 9, 8262–8271 (2013).
39. Ross, K. A. et al. Structural and antigenic stability of H5N1 hemagglutinin trimer upon release from polyanhydride nanoparticles. J. Biomed. Mater. Res. Part A 102, 4161–4168 (2014).
40. Renukaradhya, G. J., Narasimhan, B. & Mallapragada, S. K. Respiratory nanoparticle-based vaccines and challenges associated with animal models and translation. J. Control. Release 219, 622–631 (2015).
41. Vela Ramirez, J. E. et al. Carbohydrate-functionalized nanovaccines preserve HIV-1 antigen stability and activate antigen presenting cells. J. Biomater. Sci. Polym. Ed. 25, 1387–406 (2014).
42. Haughney, S. L., Ross, K. a., Boggiatto, P. M., Wannemuehler, M. J. & Narasimhan, B. Effect of nanovaccine chemistry on humoral immune response kinetics and maturation. Nanoscale 6, 13770–13778 (2014).
43. Vela Ramirez, J. E. et al. Polyanhydride Nanovaccines Induce Germinal Center B Cell Formation and Sustained Serum Antibody Responses. J. Biomed. Nanotechnol. 12, 1303–1311 (2016). 67
44. Huntimer, L. et al. Single immunization with a suboptimal antigen dose encapsulated into polyanhydride microparticles promotes high titer and avid antibody responses. J Biomed Mater Res B Appl Biomater 101, 91–98 (2013).
45. Torres, M. P. et al. Polyanhydride microparticles enhance dendritic cell antigen presentation and activation. Acta Biomater. 7, 2857–2864 (2011).
46. Petersen, L. K., Xue, L., Wannemuehler, M. J., Rajan, K. & Narasimhan, B. The simultaneous effect of polymer chemistry and device geometry on the in vitro activation of murine dendritic cells. Biomaterials 30, 5131–5142 (2009).
47. Kipper, M. J., Wilson, J. H., Wannemuehler, M. J. & Narasimhan, B. Single dose vaccine based on biodegradable polyanhydride microspheres can modulate immune response mechanism. J. Biomed. Mater. Res. Part A 76A, 798–810 (2006).
48. Ulery, B. D. et al. Polymer chemistry influences monocytic uptake of polyanhydride nanospheres. Pharm. Res. 26, 683–690 (2009).
49. Goodman, J. T. et al. Nanoparticle Chemistry and Functionalization Differentially Regulates Dendritic Cell-Nanoparticle Interactions and Triggers Dendritic Cell Maturation. Part. Part. Syst. Charact. 31, 1269–1280 (2014).
50. Phanse, Y. et al. Functionalization of polyanhydride microparticles with di- mannose influences uptake by and intracellular fate within dendritic cells. Acta Biomater. 9, 8902–8909 (2013).
51. Carrillo-Conde, B. R., Ramer-Tait, A. E., Wannemuehler, M. J. & Narasimhan, B. Chemistry-dependent adsorption of serum proteins onto polyanhydride microparticles differentially influences dendritic cell uptake and activation. Acta Biomater. 8, 3618–3628 (2012).
52. Chavez-Santoscoy, A. V. et al. Tailoring the immune response by targeting C-type lectin receptors on alveolar macrophages using “pathogen-like” amphiphilic polyanhydride nanoparticles. Biomaterials 33, 4762–4772 (2012).
53. Carrillo-Conde, B. et al. Mannose-Functionalized “Pathogen-like” Polyanhydride Nanoparticles Target C-Type Lectin Receptors on Dendritic Cells. Mol. Pharm. 8, 1877–1886 (2011).
54. Petersen, L. K. et al. Activation of innate immune responses in a pathogen- mimicking manner by amphiphilic polyanhydride nanoparticle adjuvants. Biomaterials 32, 6815–6822 (2011).
55. Mirza, S. et al. The effects of differences in pspA alleles and capsular types on the resistance of Streptococcus pneumoniae to killing by apolactoferrin. Microb. Pathog. 99, 209–219 (2016). 68
56. Arulanandam, B. P., Lynch, J. M., Briles, D. E., Hollingshead, S. & Metzger, D. W. Intranasal Vaccination with Pneumococcal Surface Protein A and Interleukin- 12 Augments Antibody-Mediated Opsonization and Protective Immunity against Streptococcus pneumoniae Infection. Infect. Immun. 69, 6718–6724 (2001).
57. Briles, D. E. et al. Immunization of Humans with Recombinant Pneumococcal Surface Protein A (rPspA) Elicits Antibodies That Passively Protect Mice from Fatal Infection with Streptococcus pneumoniae Bearing Heterologous PspA. J. Infect. Dis. 182, 1694–1701 (2000).
58. Wafa, E. I., Geary, S. M., Goodman, J. T., Narasimhan, B. & Salem, A. K. The effect of polyanhydride chemistry in particle-based cancer vaccines on the magnitude of the anti-tumor immune response. Acta Biomater. 50, 417–427 (2017).
59. Melin, M. M. et al. Development of antibodies to PspA families 1 and 2 in children after exposure to Streptococcus pneumoniae. Clin. Vaccine Immunol. 15, 1529–35 (2008).
60. Lebon, A. et al. Natural antibodies against several pneumococcal virulence proteins in children during the pre-pneumococcal-vaccine era: the generation R study. Infect. Immun. 79, 1680–7 (2011).
61. Daniels, C. C. et al. The proline-rich region of pneumococcal surface proteins A and C contains surface-accessible epitopes common to all pneumococci and elicits antibody-mediated protection against sepsis. Infect. Immun. 78, 2163–2172 (2010).
62. Bungener, L. et al. Alum boosts TH2-type antibody responses to whole-inactivated virus influenza vaccine in mice but does not confer superior protection Alum boosts TH2-type antibody responses to influenza WIV. Vaccine 26, (2008).
63. Levy, O., Goriely, S. & Kollmann, T. R. Immune Response to Vaccine Adjuvants during the First Year of Life. doi:10.1016/j.vaccine.2012.10.016
64. Chen, D. & Kristensen, D. Opportunities and challenges of developing thermostable vaccines. Expert Rev. Vaccines 8, 547–557 (2009).
65. Brady, R. C. et al. Safety and immunogenicity of a subvirion inactivated influenza A/H5N1 vaccine with or without aluminum hydroxide among healthy elderly adults. Vaccine 27, 5091–5 (2009).
66. Willhite, C. C. et al. Systematic review of potential health risks posed by pharmaceutical, occupational and consumer exposures to metallic and nanoscale aluminum, aluminum oxides, aluminum hydroxide and its soluble salts. doi:10.3109/10408444.2014.934439
67. Petrovsky, N. Comparative safety of vaccine adjuvants: a summary of current evidence and future needs. doi:10.1007/s40264-015-0350-4 69
68. Huntimer, L. et al. Evaluation of Biocompatibility and Administration Site Reactogenicity of Polyanhydride-Particle-Based Platform for Vaccine Delivery. Adv. Healthc. Mater. 2, 369–378 (2013).
69. Fukuyama, Y. et al. Nanogel-based pneumococcal surface protein A nasal vaccine induces microRNA-associated Th17 cell responses with neutralizing antibodies against Streptococcus pneumoniae in macaques. (2015). doi:10.1038/mi.2015.5
70. Kunda, N. K. et al. Pulmonary dry powder vaccine of pneumococcal antigen loaded nanoparticles. Int. J. Pharm. 495, 903–912 (2015).
71. Gyu Kong, I. et al. Nanogel-based pspa intranasal vaccine prevents invasive disease and nasal colonization by streptococcus pneumoniae. Infect. Immun. 81, 1625–1634 (2013).
72. Tostes, R. O. et al. Protection elicited by nasal immunization with recombinant Pneumococcal surface protein A (rPspA) adjuvanted with whole-cell pertussis vaccine (wP) against co-colonization of mice with Streptococcus pneumoniae. PLoS One 12, 1–17 (2017).
73. Ezoe, H. et al. Intranasal vaccination with pneumococcal surface protein A plus poly(I:C) protects against secondary pneumococcal pneumonia in mice. Vaccine 29, 1754–1761 (2011).
74. Walters, A. A., Krastev, C., Hill, A. V. S. & Milicic, A. Next generation vaccines: Single-dose encapsulated vaccines for improved global immunisation coverage and efficacy. J. Pharm. Pharmacol. 67, 400–408 (2015).
75. Lee, B. Y. et al. Economic impact of thermostable vaccines. Vaccine 35, 3135– 3142 (2017).
76. Jones, L. S. et al. Effects of Adsorption to Aluminum Salt Adjuvants on the Structure and Stability of Model Protein Antigens. J. Biol. Chem. 280, 13406– 13414 (2005).
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CHAPTER 3. SINGLE-DOSE COMBINATION NANOVACCINE INDUCES BOTH RAPID AND LONG-LIVED PROTECTION AGAINST PNEUMONIC PLAGUE
Modified from a manuscript to be submitted to npj Vaccines
Danielle A Wagner-Muñiz1, Sean M Kelly2, Andrew C Petersen1, Nathan Peroutka-
Bigus1,3, Ross J Darling1, Bryan H Bellaire1,3,4, Michael J Wannemuehler1,4, and Balaji
Narasimhan2,4
1Department of Veterinary Microbiology and Preventive Medicine, Iowa State
University, Ames, IA, USA 2Department of Chemical and Biological Engineering, Iowa
State University, Ames, IA, USA 3Interdepartmental Microbiology, Iowa State
University, Ames, IA, USA 4Nanovaccine Institute, Iowa State University, Ames, IA,
USA
Abstract
Yersinia Pestis, the causative agent of pneumonic plague, induces a highly lethal infection if left untreated. Currently, there is no FDA-approved vaccine against this pathogen; however, USAMRIID has developed a recombinant fusion protein, F1-V, that has been shown to protect multiple animal species against pneumonic plague. Many F1-
V-based vaccine formulations require prime-boost immunization to achieve protective immunity, and there are no reports of rapid induction of protective immunity (i.e., 14 days post-immunization (DPI)). The STimulator of INterferon Genes agonists cyclic 71 dinucleotides (CDNs) have been shown to be promising vaccine adjuvants.
Polyanhydride nanoparticle-based vaccines (i.e., nanovaccines) have also been shown to enhance immune responses due to their dual functionality as both an adjuvant and a delivery platform. Utilizing the synergy provided by these two platforms, a combination nanovaccine was designed that comprised F1-V-loaded nanoparticles combined with the
CDN, dithio-RP,RP-cyclic di-guanosine monophosphate (R,R-CDG), to induce rapid and long-lived protective immunity against pneumonic plague. All mice immunized with a single dose combination nanovaccine were protected from Y. pestis lethal challenge within 14 DPI, and demonstrated significantly enhanced protection over a soluble bolus of F1-V adjuvanted with CDNs. In addition, 75 percent of mice were protected from challenge at 182 DPI, while maintaining high levels of antigen-specific serum IgG.
ELISPOT analysis of vaccinated animals at 218 DPI revealed the presence of F1-V- specific long-lived plasma cells in the bone marrow. Microarray analysis of linearly overlapping peptides revealed the presence of serum antibody with high affinity for a broad range of F1 and V linear epitopes. Altogether, these results demonstrate that combining the adjuvanticity of CDNs with a nanoparticle-based vaccine delivery system enables induction of both rapid and long-lived protective immunity against Y. pestis.
Introduction
Plague has relentlessly affected humans throughout history accounting for an estimated 200 million deaths 1,2 and continues to persist worldwide, with the latest outbreak occurring in Madagascar in 2017. Plague is caused by the non-motile, facultative intracellular, Gram-negative bacterium Yersinia Pestis 3. Pneumonic plague, the respiratory manifestation of Y. pestis infection, is transmitted through aerosolized 72 droplets 4. In the case of pneumonic plague, treatment with intravenous or oral ciprofloxacin and doxycycline over 48 hours is successful, though mortality rates quickly approach 90-100% if left untreated for 24-36 hours post-infection 1.
The threat of weaponization of Y. pestis is high, largely evidenced by its history of such use 2. Though the United States and 103 other countries co-signed an agreement to terminate biological weapons programs in 1972, Y. pestis remains listed as a Tier 1 Select
Agent. Currently, there is no FDA-approved vaccine against Y. pestis, making it critical to develop a protective vaccine with the capability to provide both rapid and long-lived immunity in the event of mass exposure of aerosolized Y. pestis to civilian or military populations. As Y. pestis is predominantly an extracellular pathogen, many vaccination strategies have focused on developing strong humoral immunity, characterized by neutralizing antibodies against the V antigen and opsonizing antibodies against the F1 capsule 5–8. In mice, anti-F1 IgG antibody titers have been shown to correlate with protection 5. Additionally, mAb 7.3, which binds to the V antigen, has been shown to neutralize Y. pestis in vitro and provides passive protection against lethal challenge 6,9.
The United States Army Medical Research Institute of Infectious Diseases
(USAMRIID) developed a recombinant fusion protein F1-V, containing both full length proteins of both F1 and V, and shows promise as a target antigen for Y. pestis 10. Using a
IM/SC prime-boost regimen, it was recently shown that F1-V adjuvanted with aluminum hydroxide (alum) provided complete protection against intranasal challenge with virulent
Y. pestis CO92 in mice, guinea pigs, and macaques 11. Additionally, serum collected from immunized macaques conferred passive protection to mice. Despite such success, this vaccine formulation requires a prime-boost regimen; in the event of mass exposure, it 73 would be ideal to use single dose formulations that can provide rapid immunity in as short a time frame as possible and that can maintain long-lived protective immunity.
Cyclic dinucleotides (CDNs), a class of small molecule adjuvants, are recognized as microbial-associated molecular patterns (MAMPs) by the pattern recognition receptor
(PRR) STING (STimulator of INterferon Genes) 12, resulting in the phosphorylation of transcription factors NFκB and IRF3 and the induction of type I interferons (IFNα and
IFNβ) which are associated with anti-pathogenic activity 13,14. Cyclic di-GMP (CDG), the most well-studied CDN to date, has been recognized as a universal secondary messenger molecule in Gram-negative bacteria, playing a role in bacterial development, motility, and virulence 15. Vaccines adjuvanted with CDG induce a balanced immune response, characterized by equal presence of IgG subclasses in serum 16, and have been shown to elicit significantly higher antibody titers than alum-adjuvanted formulations 17.
Biodegradable polyanhydrides, another novel class of adjuvants and delivery vehicles, possess a multitude of beneficial characteristics to address challenges faced by many current vaccines 18. Comprised of 1,6-bis(p-carboxyphenoxy)hexane (CPH) and
1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG), these materials are highly biocompatible, demonstrating minimal injection-site reactivity when administered as particles 19,20 and whose non-toxic dicarboxylic acid degradation products are safely excreted from the body 21. Their amphiphilic properties allow for stabilization of labile proteins 22–24, provide sustained release of encapsulated proteins via surface erosion kinetics 23,25–27, and allow for enhanced shelf stability of encapsulated proteins, even at elevated temperatures (e.g., 40 °C) 23. Additionally, polyanhydride particles display inherent adjuvanticity, demonstrating chemistry-dependent internalization, persistence, 74 and activation of antigen presenting cells in vitro, as well as the ability to prime humoral and cell-mediated immune responses in vivo 28,29,38–40,30–37. These properties have enabled the study of polyanhydride nanoparticle-based vaccines (i.e., nanovaccines) against multiple bacterial and viral pathogens 23,27,35,41,42.
Previous work from our laboratories demonstrated that encapsulation of F1-V into polyanhydride particles maintained F1-V structure and prolonged antigen bioavailability
22, and a single, intranasal dose of F1-V nanovaccine provided complete protection against lethal challenge of Y. pestis for at least 280 days post-immunization (DPI) 35. In addition to long-lived protective immunity, it is also important that vaccines induce rapid
(i.e., ≤ 14 days) protective immunity to counter acute outbreaks of disease. In this regard, to our knowledge there are no single dose plague vaccine formulations that can induce rapid protective immunity against Y. pestis. Designing next-generation vaccine platforms that provide both rapid and long-lived (i.e., > 1 year) immunity against highly lethal pathogens will likely require novel approaches, including developing new adjuvants or vaccine regimen with combination adjuvants to enhance both rapid and long-lived protective immunity. Herein, the design and evaluation of a combination nanovaccine, comprising of F1-V-containing polyanhydride nanoparticles and a non-canonical CDG
CDN adjuvant (containing 2’,5’-3’-5’ phosphate linkages) is described. This nanovaccine formulation synergistically combines the adjuvant properties of polyanhydride nanoparticles and CDNs to induce rapid immune responses and facilitate the induction of long-lived protective immunity against Y. pestis.
75
Material and Methods
Materials
Chemicals used for CPTEG and CPH diacid and polymer synthesis included 1,6- dibromohexane, triethylene 4-p-hydroxybenzoic acid, and 1-methyl-2-pyrrolidinone, purchased from Sigma-Aldrich (St. Louis, MO). Chloroform, petroleum ether, ethyl ether, hexanes, sodium hydroxide, toluene, sulfuric acid, acetonitrile, dimethyl formamide, acetic anhydride, methylene chloride, pentane, and potassium carbonate were purchased from Fisher Scientific (Fairlawn, NJ). 4-p-fluorobenzonitrile was purchased from Apollo Scientific (Cheshire, UK). Deuterated chloroform used for 1H NMR analysis was purchased from Cambridge Isotope Laboratories (Andover, MA). Dithio-RP,RP- cyclic di-guanosine monophosphate (R,R-Cyclic di-GMP (cdG)) was provided by Aduro
Biotech (Berkeley, CA). Complete cell culture medium reagents RPMI 1640 and penicillin-streptomycin were purchased from Mediatech (Herndon, VA); heat inactivated fetal calf serum was purchased from Atlanta Biologicals (Atlanta, GA). Y. pestis, strain
CO92 (NR-641) and the Y. pestis fusion protein F1-V (NR-4526) were obtained from the
Biodefense and Emerging Infections Repository (Manassas, VA).
Polyanhydride synthesis
CPTEG and CPH diacids were synthesized as previously described 26,33. 20:80
CPTEG:CPH copolymer synthesis was performed using melt polycondensation 26.
Copolymer composition and molecular weight were estimated using end group analysis of 1H NMR (DXR 500) spectra.
Nanoparticle synthesis
10 % (w/w) F1-V-loaded 20:80 CPTEG:CPH nanoparticles were synthesized using flash nanoprecipitation, as described previously 28. Briefly, F1-V and 20:80 76
CPTEG:CPH copolymer was dissolved in methylene chloride at 2 mg/mL and 20 mg/mL, respectively, sonicated at 30 Hz for approximately 30 seconds, and poured into pentane chilled to -20 °C at a methylene chloride:pentane ratio of 1:250. Nanoparticles were imaged using scanning electron microscopy (SEM; JEOL 840 A, JEOL Ltd.,
Tokyo, Japan), and nanoparticle mean size and size distribution were determined using
ImageJ (National Institutes of Health, Bethesda, MD). Nanoparticle zeta potential was measured using Zetasizer Nano (Malvern Instruments, Worcestershire, UK).
Protein release and encapsulation efficiency
To quantify released protein, approximately 2 mg of nanoparticles were suspended in 500 μL of PBS, sonicated for 15 seconds, and placed on a shaker plate at 37
°C. Periodically, 400 μL aliquots of sample supernatant were withdrawn and sample volumes were reconstituted with 400 μL of fresh PBS. After 32 days, 40 mM sodium hydroxide was used in place of PBS to catalyze the release of any remaining protein.
Protein released from F1-V-loaded 20:80 CPTEG:CPH nanoparticles was quantified using a microbichoninic acid (microBCA) assay (Thermo Fisher Scientific, Waltham,
MA), and percent by mass of protein released over time was calculated as the cumulative protein released from nanoparticles at each time point divided by total mass of protein released. To quantify protein encapsulation efficiency, approximately 1 mg of nanoparticles was suspended in 500 μL of 40 mM sodium hydroxide solution, sonicated for 15 seconds, and placed on a shaker plate at 37 °C. Frequently, 400 μL samples of supernatant were withdrawn and sample volumes were reconstituted with 400 μL of 40 mM sodium hydroxide. Protein released from F1-V-loaded 20:80 CPTEG:CPH nanoparticles was quantified using a microBCA assay, and the encapsulation efficiency 77 was calculated as the sum of the protein released from the nanoparticles divided by the initial mass of protein.
Animals
Seven to eight-week old female C57BL/6NCrl mice were purchased from Charles
River (Wilmington, MA). Mice were housed under specific pathogen-free conditions where all bedding, caging, water, and feed were sterilized prior to use. All studies were conducted with the approval of the Iowa State University Institutional Animal Care and
Use Committee (IACUC).
Immunization and serum collection
Groups comprised of C57BL/6 mice (n = 8-16/group) were immunized subcutaneously at the nape of the neck with either of the following: 50 µg F1-V + 35 µg
CDNs (R,R-CDG, Aduro Biotech), 36-50 µg F1-V encapsulated into 500 µg of 20:80
CPTEG:CPH nanoparticles, 36-50 µg F1-V encapsulated into 500 µg of 20:80
CPTEG:CPH nanoparticles + 35 µg CDNs, or saline in a total volume of 200 µL. Blood was collected from mice via saphenous vein and serum was separated following centrifugation (10,000 rcf for 10 min) at 13, 14, 36, 49, 79, 101, 121, 150, and 178 DPI.
An additional serum sample was obtained from mice sacrificed for ELISPOT analysis at
218 DPI via cardiac exsanguination. Serum was stored at -20°C until analysis.
Bronchoalveolar lavage (BAL) fluid was collected post-euthanization by introducing a catheter needle through a small incision made in the trachea and injecting/aspirating 700
µL of PBS thrice.
ELISA
Anti-F1-V antibody titers were determined via ELISA, as previously described 27.
Briefly, high-binding Costar 590 EIA/RIA microtiter plates (Corning) were coated 78 overnight with 100 µL of a 0.5 µg/mL solution of F1-V at 4 °C. After washing the wells, microtiter plates were blocked for two hours with a solution of 2.5% (w/v) powdered skim milk dissolved in PBS-Tween with 0.05% Tween 20, pH 7.4, that had been incubated for two hours at 56 °C to inactivate any endogenous phosphatase activity.
Following block, microtiter plates were washed thrice with PBS-T. Serum obtained from immunized mice was added at a dilution of 1:200 and serially diluted in PBS-T containing 1% (v/v) goat serum. Each sample was tested in duplicate. Following incubation overnight at 4°C, plates were washed thrice with PBS-T, after which secondary antibody was added at a dilution of 1 µg/mL. Secondary antibodies used in these studies were: alkaline phosphatase-conjugated goat anti-mouse IgG heavy and light chain, IgG1 and IgG2c (Jackson ImmunoResearch). Plates were incubated for two hours at room temperature and then washed three times with PBS-T. To each well, 100 µL of alkaline phosphatase substrate (Fisher Scientific, Pittsburgh, PA) was added at a concentration of 1 mg/mL dissolved in 50 mM sodium carbonate, 2 mM magnesium chloride buffer at pH 9.3 for colorimetric development. Plates were analyzed after 30 min using a SpectraMax M3 microplate reader at a wavelength of 405 nm. Titer is reported as the reciprocal of serum dilution at which the optical density (OD) value was at most 0.2, a conservative endpoint greater than the average OD of saline-mouse serum, at a 1:200 dilution, plus two standard deviations.
ELISPOT
MultiScreen 96-well plates (Millipore Sigma, Billerica, MA) were pretreated with
35 % ethanol for one minute, washed three times with PBS, and coated overnight at 4 °C with 0.5 µg/mL F1-V in PBS. The following day, plates were dumped and blocked with complete tissue culture medium consisting of RPMI 1640 (Gibco, Grand Island, NY), 79
10% (v/v) fetal calf serum, 1% (v/v) penicillin-streptomycin, and 1% (v/v) L-glutamine for at least two hours at 37 °C. Plates were dumped and single cell suspensions of bone marrow or splenic lymphocytes harvested from mice between 214-218 days post- immunization were added to the plates at 500,000 cells per well and placed in a 37 °C incubator for two hours. Plates were then washed three times with PBS, another three times with PBS-T, and alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) secondary antibody (Jackson ImmunoResearch) was added to the wells at a 1:500 dilution in 1% (v/v) goat serum PBS-T for two hours at room temperature. Plates were washed six times with PBS-T and 25 µL per well of BCIP/NBT liquid substrate
(Millipore Sigma) was added to the wells and developed for 15 min at room temperature.
Plates were emptied, the bottoms removed, and gently washed with nanopure water, after which they were left to dry completely. Spots were counted using an AID Multispot
Reader (AID, Strassberg, Germany). AID EliSpot software (Version 6.0) was used for data analysis.
Peptide microarray printing and analysis
Twenty seven 14- to 17-mer linear peptides (11 amino acid overlaps) spanning the full length of F1 antigen and fifty three 15- to 17-mer linear peptides (11 or 12 amino acid overlaps) spanning the full length of V antigen, as well as full length proteins F1-V,
Bacillus anthracis protective antigen (PA), and chicken egg ovalbumin (OVA) were printed onto Nexterion Slide AL (Schott, Louisville, KY) using a BioRobotics
MicroGRID II microarray printer (Genomic Solutions, Inc. Ann Arbor, MI). Peptides and proteins were dissolved into DMSO at 10 mg/mL to ensure dissolution, then diluted 10- fold in water to 1 mg/mL. The solution was diluted to bring the final concentration to 0.5 mg/mL in 1x print buffer (5% (v/v) DMSO, 137 mM NaCl, 9 mM KOH, 11.3 mM 80
NaH2PO4). The full length F1-V fusion protein was used as a positive control, while PA and OVA were used as negative controls. Slides were printed with 16 arrays per slide, each array containing 16x16 spots per array, with peptides printed in a serpentine pattern in triplicate. Following printing, slides were vacuum sealed and stored at -80 °C until further use.
Microarray slides were warmed to room temperature and then placed on a hot plate at 37 °C for 30 min to dry; the slides were then placed immediately in a blocking solution consisting of 1 % BSA (w/v) in PBS-T for one hour. Slides were subsequently blocked for one hour in 1 % (v/v) goat serum PBS-T, then thrice washed with PBS-T and placed into a 16-well incubation chamber (Nexterion IC-16) to separate individual arrays.
Serum was added at a 1:20 dilution for one hour at room temperature with gentle agitation. Slides were washed thrice with PBS-T and biotinylated goat anti-mouse IgG
(H+L) secondary antibody (Jackson ImmunoResearch) was added to wells at 1:1000 dilution for one hour at room temperature with gentle agitation. Slides were washed thrice with PBS-T. Streptavidin Alexa Fluor 555 conjugate was added to wells at a
1:1000 dilution for 30 min at room temperature with gentle agitation. Slides were removed from incubation chambers, washed thrice with PBS-T, followed by another three washes with PBS. Slides were spun dry by centrifugation and read on the Scanarray
5000 laser scanner (GSI Lumonics, Bedford, MA).
The scanned images of the microarray slides were analyzed using SoftWorRx
Tracker v2.8 software (Applied Precision, Inc., Issaquah, WA) to detect and quantify the fluorescence of each spot. R (v3.4.1) software was used to calculate the background- corrected fluorescence of each spot as the mean fluorescence intensity of each spot minus 81 the median background fluorescence intensity surrounding the spot. The mean fluorescence intensity for each peptide or protein was calculated as the average of the corresponding triplicate background-corrected fluorescence spot values, and a heat map was generated based upon the magnitude of individual mouse serum responses to each peptide. Saline-treated mouse serum was used as a negative control.
Challenge
Y. pestis CO92 (NR-641) was obtained from BEI Resources. Frozen stocks were prepared in advance of challenges. Y. pestis was initially grown on brain heart infusion
(BHI) agar at 28 °C for 72 h; a single colony was isolated and inoculated in BHI broth and cultivated for 24 h at 28 °C while shaking at 120 rpm. Glycerol solution was added to the broth to bring final glycerol concentration to 5% (v/v). Aliquots were snap frozen and stored at -72 °C. Prior to challenge experiments, aliquots of the frozen stock were thawed and cultured on tryptic soy agar (TSA) with 1% bovine hemoglobin at 37°C and 5% CO2 to check viability and CFU enumeration. For challenge experiments, frozen Y. pestis stocks were thawed and diluted in room temperature PBS. Groups of C57BL/6 mice immunized as described previously were anesthetized with an intraperitoneal injection of ketamine/xylazine cocktail and infected intranasally with 5,700, 7,000, or 10,000 CFU
(14 DPI) or 6500 CFU (218 DPI) of Y. pestis CO92 in a 50 µL volume. Y. pestis bacterial suspension was administered intranasally to nares of mice by a micropipette. CFU enumeration was performed on infectious inoculum preceding to and following infection of the animals to determine infectious dose administered. Animals were checked daily for morbidity and mortality over the course of two weeks. All activities were performed in an animal biosafety level 3 (ABSL-3) laboratory at Iowa State University with protocols approved by the IACUC and Institutional Biosafety Committee (IBC). 82
Statistical analyses
Statistical analyses on the ELISA data as well as the survival data were performed using the Mantel-Cox log rank test and the one-way analysis of variance (ANOVA) using
Tukey’s multiple comparisons test. Statistical significance in microarray serum responses to each peptide between vaccinated groups of animals was evaluated using Student’s t test. A p-value ≤ 0.05 was considered to be statistically significant. All analyses were performed using GraphPad Prism v. 7.0 (GraphPad, La Jolla, CA).
Results
F1-V nanovaccine characterization
Following synthesis, the F1-V nanovaccine was characterized for size distribution, surface charge, release kinetics, and encapsulation efficiency of encapsulated
F1-V. The size of the F1-V nanovaccine was consistent with previous work 35, with a mean diameter of 228 ± 78 nm and a polydispersity index of 0.12, indicating a narrow size distribution (Figure 3.1A and 3.1B). Zeta potential measurements of the F1-V nanovaccine demonstrated a negative surface charge, with zeta potentials of -34.8 ± 1.4 mV. In order to provide sufficient antigen to induce a robust immune response for rapid protection, while also sustaining the release of antigen to induce long-lived protective immunity, this nanoparticle formulation was designed with a 10% w/w loading of F1-V providing a biphasic release profile that was characterized by an initial burst of approximately 75% of the protein released within the first 24 h, followed by slow zero order release over more than two months (Figure 3.1C). The encapsulation efficiency of the F1-V within the various nanoparticle batches for each experiment was 51-72%. 83
Figure 3.1 Characterization of F1-V Nanovaccine. A) Representative scanning electron photomicrograph of F1-V nanovaccine (scale bar = 3 µm). B) Nanoparticle size distribution of the F1-V nanovaccine (228 ± 78 nm), determined via ImageJ analysis. C) Cumulative in vitro F1-V release profile from the nanovaccine. Nanoparticles were suspended in PBS (pH 7.4) and released protein was quantified via a microBCA assay.
Combination nanovaccine induces rapid protective immunity
In order to assess the potential of CDNs and nanovaccines to rapidly stimulate protective immunity, C57BL/6 mice (n=12-16 per group) were immunized subcutaneously with a single dose of one of the following vaccine formulations: i) soluble F1-V adjuvanted with CDNs (CDN Vaccine), ii) F1-V encapsulated into 20:80
CPTEG:CPH nanoparticles (Nanovaccine), iii) F1-V encapsulated into 20:80
CPTEG:CPH nanoparticles co-adjuvanted with CDNs (Combination Nanovaccine), or iv) saline alone. Blood samples were collected from immunized animals at 13 DPI (Figure 84
3.2A, C, E). Anti-F1-V total IgG antibody titers from CDN Vaccine- and Combination
Nanovaccine-immunized animals evaluated at 13 DPI were similar in magnitude and showed significantly (p ≤ 0.0001) higher titers than sera from animals immunized with the Nanovaccine alone.
Mice were challenged intranasally 14 DPI with escalating lethal doses of Y. pestis
CO92 in separate studies and survival was assessed over a period of two weeks (Figure
3.2B, D, F). All naïve (i.e., saline-treated) mice succumbed to infection within four days of challenge, while all Nanovaccine-immunized mice succumbed over a period of eight days following challenge. In contrast, immunization with Combination Nanovaccine yielded 100% protection against all challenge doses, while immunization with CDN
Vaccine showed decreasing levels of efficacy with increasing challenge doses. Both
Combination Nanovaccine and CDN Vaccine immunization regimens provided significantly (p ≤ 0.0001) superior protection when compared to Nanovaccine or saline regimens for challenges at 5,700, 7,000, and 10,000 CFU. Immunization with
Combination Nanovaccine formulation provided significantly better protection (p ≤
0.05), as compared to CDN Vaccine against challenges at 7,000 and 10,000 CFU.
85
Figure 3.2 Combination Nanovaccine provides rapid protective immunity against lethal Y. pestis challenge. Groups of C57BL/6 mice (n=12-16 per group) were immunized subcutaneously with the following groups: F1-V + CDNs (CDN Vaccine), F1-V encapsulated into nanoparticles (Nanovaccine), F1-V encapsulated into nanoparticles + CDNs (Combination Nanovaccine) or saline. (A,C,E) Serum was collected at 13 DPI and evaluated via ELISA for total anti-F1-V IgG antibody titers. The dashed line represents anti-F1-V IgG (H+L) antibody titers from saline-treated mice as a negative control. * p ≤ 0.0001 compared to Nanovaccine and saline. Mice 86 were challenged at 14 DPI with (B) 5,700 CFU, (D) 7,000 CFU, or (F) 10,000 CFU Y. pestis CO92 and survival was monitored for two weeks post-challenge. * p ≤ 0.0001 compared to saline-treated mice. + p ≤ 0.0001 compared to Nanovaccine-immunized mice. # p ≤ 0.05 compared to CDN Vaccine-immunized mice. Combination nanovaccine enhances long-lived protective immunity
Previous work from our laboratories has shown the potential for a single dose nanovaccine, consisting of a soluble bolus of F1-V and F1-V containing nanoparticles, to enhance long-lived antibody responses for at least 280 DPI and provide protective immunity 27,35. To evaluate the potential for a single dose of the CDN vaccine and the two nanovaccines (with no soluble F1-V bolus) to induce long-lived protective immunity, separate groups of C57BL/6 mice (n=8-12 per group) were immunized subcutaneously with the same formulations as above and anti-F1-V total IgG titers were evaluated between 14-218 DPI (Figure 3.3A and Figure 3.4). At all timepoints, sera from both
Combination Nanovaccine-immunized and CDN Vaccine-immunized mice showed comparable and significantly (p ≤ 0.0001) higher titers than sera from Nanovaccine- immunized mice. Serum was analyzed at 14, 79 and 178 DPI for presence of class- switched anti-F1-V IgG1, IgG2c and IgG3 antibodies (Figure 3.5). All vaccination regimens produced detectable IgG1 at 14 and 79 DPI significant (p ≤ 0.0001) over background, with Combination Nanovaccine and CDN Vaccine groups also showing significant (p ≤ 0.04) responses at 178 DPI (Figure 3.5A). Additionally, Combination
Nanovaccine- and CDN Vaccine-immunized animals showed significantly (p ≤ 0.05) higher IgG1 titers compared to Nanovaccine-immunized mice at 79 DPI. Sera from animals immunized with both Combination Nanovaccine and CDN Vaccine exhibited significantly (p ≤ 0.0001) higher IgG2c antibody titers at all time points, and IgG3 at 14
DPI, compared to sera from Nanovaccine-immunized and Saline-treated mice (Figure 87
3.5B and C). Lastly, a subset of mice (n=2-4) were euthanized 214-218 DPI and bronchoalveolar lavage (BAL) fluid was collected for evaluation of anti-F1-V lung IgG
(Figure 3.6) and IgA. The BAL fluid recovered from the animals immunized with
Combination Nanovaccine and CDN Vaccine was found to have significantly (p ≤ 0.05) higher total anti-F1-V IgG compared to that from animals immunized with Nanovaccine or treated with Saline. No IgA was detected in the BAL fluid (data not shown).
Mice were challenged intranasally at 182 DPI with a lethal dose of Y. pestis CO92
(6500 CFU) and survival was assessed over a two-week period (Figure 3.3B). All naïve mice succumbed to infection within three days of challenge. However, all Nanovaccine- immunized mice succumbed over a period of seven days following challenge that was significantly (p ≤ 0.05) prolonged compared to naïve mice. Both the Combination
Nanovaccine and CDN Vaccine immunization regimens induced superior protection (p ≤
0.01) when compared to the Nanovaccine or saline treated mice. Specifically, immunization with the Combination Nanovaccine provided 75% protection from lethal challenge and immunization with the CDN Vaccine provided 62% protection.
88
Figure 3.3 Combination Nanovaccine provides long-lived protective immunity against lethal Y. pestis challenge. Groups of C57BL/6 mice (n=12-16 per group) were immunized subcutaneously with the following treatments: F1-V + CDNs (CDN Vaccine), F1-V encapsulated into nanoparticles (Nanovaccine), F1-V encapsulated into nanoparticles + CDNs (Combination Nanovaccine) or saline. (A) Serum samples were collected at 14, 36, 79, 121, and 178 DPI and analyzed for total anti-F1-V IgG (H+L) antibodies via ELISA. The dashed line represents the background anti-F1-V IgG (H+L) antibody response from naive mice. # p ≤ 0.05 compared to CDN Vaccine. * p ≤ 0.0001 compared to Nanovaccine-immunized and saline-treated mice. (B) Mice were challenged at 182 DPI with 6500 CFU Y. pestis CO92 and survival was monitored for two weeks post- challenge. # p ≤ 0.01 compared to Nanovaccine-immunized mice. * p ≤ 0.05 compared to Saline-treated mice. 89
Figure 3.4. Combination Nanovaccine provides long-lived antibody responses to F1-V. Groups of C57BL/6 mice (n=12-16 per group) were immunized subcutaneously with the following groups: 50 µg F1-V + 35 µg CDNs (CDN Vaccine), 36 µg F1-V encapsulated into 500 µg nanoparticles (Nanovaccine), 36 µg F1-V encapsulated into 500 µg nanoparticles + 35 µg CDNs (Combination Nanovaccine) or Saline. Serum samples were collected at 14, 36, 49, 79, 101, 121, 150, 178, and 218 DPI and analyzed for total anti- F1-V IgG (H+L) antibodies via ELISA. # p ≤ 0.05 compared to CDN Vaccine. * p ≤ 0.0001 compared to Nanovaccine-immunized and saline-treated mice. 90
Figure 3.5 Combination Nanovaccine induces class-switched antibody responses to F1-V. Groups of C57BL/6 mice (n=12-16 per group) were immunized subcutaneously with the following groups: 50 µg F1-V + 35 µg CDNs (CDN Vaccine), 36 µg F1-V encapsulated into 500 µg nanoparticles (Nanovaccine), 36 µg F1-V encapsulated into 500 µg nanoparticles + 35 µg CDNs (Combination Nanovaccine) or Saline. Serum samples were collected at 14, 79, and 178 DPI and analyzed for anti-F1-V IgG1, IgG2c, and IgG3 antibodies via ELISA. * p ≤ 0.0001 compared to saline-treated mice. # p ≤ 0.05 compared to Nanovaccine-immunized mice. 91
Figure 3.6 Combination Nanovaccine induces long-lived antibody responses in BAL fluid against F1-V. Groups of C57BL/6 mice (n=12-16 per group) were immunized subcutaneously with the following groups: 50 µg F1-V + 35 µg CDNs (CDN Vaccine), 36 µg F1-V encapsulated into 500 µg nanoparticles (Nanovaccine), 36 µg F1-V encapsulated into 500 µg nanoparticles + 35 µg CDNs (Combination Nanovaccine) or Saline. BAL fluid was collected 213-218 DPI and assessed for total anti-F1-V IgG (H+L) antibodies via ELISA. * p ≤ 0.0001 compared to Nanovaccine-immunized and saline- treated mice.
Combination nanovaccine provides broad antibody IgG recognition to F1-V linear epitopes
A linearly overlapping peptide array was performed to identify F1-V-specific IgG
(H+L) antibodies in serum, collected from mice immunized with either CDN Vaccine,
Nanovaccine, or Combination Nanovaccine and naïve controls at 14, 79, and 178 DPI, reactive to 14- to 17-mer linear peptides spanning the full length of F1 antigen, 15- to 17- mer linear peptides spanning the full length of V antigen, and F1-V fusion protein (Figure
3.7). The antibody response to F1-V and all linear epitopes in mouse serum collected at
14 DPI was noticeably lower than at 79 and 178 DPI. In addition, naïve mouse serum did not bind appreciably to either F1-V, or any F1 or V peptides, at any time point evaluated. 92
Serum from CDN Vaccine- and Combination Nanovaccine-immunized animals elicited antibodies that bound multiple linear peptides from F1 and V antigens across all time points, with responses observed to peptides F1-3, F11, and V45-46 at all time points evaluated (p < 0.05 compared to non-vaccinated mice). At 178 DPI, serum antibody from both CDN Vaccine- and Combination Nanovaccine-immunized animals continued to react with peptides F9, V18, V32, and V44 (Fig. 3.7, blue arrows). Serum from mice immunized with Nanovaccine possessed fewer F1- and V-peptide specific antibodies compared to CDN Vaccine- and Combination Nanovaccine-immunized animals, however, responses to peptides F1 and V44-45 were observed at all time points evaluated.
Combination nanovaccine induces long-lived plasma cells
As anti-F1-V-specific serum antibody was found to persist for at least 218 days,
ELISPOT analysis was performed on cells harvested from spleen and bone marrow at
214-218 DPI to demonstrate the presence of the long-lived antibody secreting cells
(ASCs). Analysis of bone marrow revealed a significantly (p < 0.05) higher number of
F1-V-specific ASCs in CDN Vaccine- and Combination Nanovaccine-immunized mice compared to non-vaccinated mice (Figure 3.8A).
93
Figure 3.7 Combination Nanovaccine and CDN Vaccine provide broad antibody responses to potentially protective linear epitopes. Serum samples collected from C57BL/6 mice (n=12-16 per group) immunized with either saline, CDN Nanovaccine, Nanovaccine, or Combination Nanovaccine 14, 79, and 178 DPI was analyzed for total anti-F1-V IgG antibodies against twenty-seven 14- to 17-mer linear peptides (11 amino acid overlaps) from the F1 antigen and fifty-three 15- to 17-mer linear peptides (11 or 12 amino acid overlaps) from the V antigen. The peptides were covalently bound to microarray slides as described in Materials and Methods. Each row corresponds to a specific peptide, the top row representing peptide F1 and the proceeding downward rows corresponding to each following linear peptide incrementally through peptide V53. Each column represents responses from a single mouse. The mean fluorescence intensity of serum responses to each peptide is represented by a range of color from white (no response) to purple (maximum response). The full-length F1-V fusion protein was used as a positive control, and Bacillus anthracis protective antigen (PA) and chicken egg ovalbumin (OVA) were used as negative controls. Black arrows indicate significance (p < 0.05) of Combination Nanovaccine and CDN Vaccine serum compared to naïve serum at all time points evaluated. Blue arrows indicate significance (p < 0.05) of Combination Nanovaccine and CDN Vaccine serum compared to naïve serum at 178 DPI. 94
There was no difference in the number of ASCs in the bone marrow of
Nanovaccine-immunized mice compared to non-vaccinated mice. Additionally, there was no difference in the number of ASCs detected in the spleens harvested from immunized mice versus naïve mice at 218 DPI (Figure 3.8B). Analysis of ASC-derived antibody IgG subclass expression revealed a trend of a greater number of F1-V-specific IgG2c ASCs present in the bone marrow of CDN Vaccine- and Combination Nanovaccine-immunized mice compared to non-vaccinated mice 218 DPI; however, this difference was not statistically significant (Figure 3.9). There was no difference in the number of F1-V- specific IgG2c ASCs in the spleen 218 DPI, nor was there a difference in the number of
F1-V-specific IgG1 ASCs between all vaccine groups in either spleen or bone marrow.
Figure 3.8 Long-lived antibody responses to F1-V originate from long-lived plasma cells in the bone marrow. Bone marrow and splenic lymphocytes were harvested at 213-218 DPI from groups (n=3-4/group) of C57BL/6 mice immunized with CDN Vaccine, Nanovaccine, Combination Nanovaccine regimens, or treated with saline, and were analyzed via ELISPOT for the number of F1-V-specific antibody secreting cells. Data represent the number of F1-V-specific antibody secreting cells per 500,000 bone marrow or spleen cells from individual mice from one experiment. * p ≤ 0.02 compared to saline- treated mice. # p ≤ 0.03 compared to Nanovaccine-immunized mice. 95
Figure 3.9 Long-lived anti-F1-V IgG1 and IgG2c antibody secreting cell responses in bone marrow and spleen. Groups of C57BL/6 mice (n=12-16 per group) were immunized subcutaneously with the following treatments: F1-V + CDNs (CDN Vaccine), F1-V encapsulated into nanoparticles (Nanovaccine), F1-V encapsulated into nanoparticles + CDNs (Combination Nanovaccine) or saline. Bone marrow and spleen lymphocytes were harvested at 213-218 DPI from groups (n=3-4/group) of mice and were analyzed via ELISPOT for the number of F1-V-specific IgG1 and IgG2c antibody secreting cells from spleen (panels A and C) or bone marrow (panels B and D). Data points represent the number of F1-V-specific antibody secreting cells per 500,000 bone marrow or spleen cells from individual mice from one experiment.
Discussion
With increasing concern of biological weaponization, it is of paramount importance to enhance preparedness with biodefense vaccines that can induce both rapid and long-lived protective immunity. To date, there is no FDA-approved vaccine against 96
Y. pestis, a weaponizable pathogen. An efficacious, single-dose vaccine would be vitally important to induce rapid and long-lived protective immunity both in the critical window of time immediately following an exposure event as well as for military personnel against potential exposure before deployment. Previous work on pneumonic plague vaccines from other laboratories has shown some success using aluminum hydroxide-based and
TLR-targeting adjuvants; however, many of these studies required prime-boost vaccination and none demonstrated the ability to induce protective immunity within 14
DPI 10,27,43,44.
The goal of this work was to incorporate the benefits of two novel adjuvants (i.e.,
CDNs and polyanhydride nanoparticles) in order to design a single-dose, combination vaccine that would provide both rapid and long-lived immunity. Previously, it was shown that a single dose of intranasally administered nanovaccine containing 40 µg soluble F1-
V antigen and 10 µg F1-V encapsulated into polyanhydride nanoparticles provided 100
% protection for at least 280 DPI 35. Additionally, it was previously demonstrated that intranasally administered 20:80 CPTEG:CPH nanoparticles elicited demonstrable F1-V- specific antibody titers as early as 14 DPI 28. Motivated by these studies, a combination nanovaccine including R,R-CDG CDNs and F1-V-containing 20:80 CPTEG:CPH nanoparticles was designed with rapid release of antigen (Figure 3.1) in order to initiate the adaptive immune response. This current work highlights the potent immunostimulatory properties of polyanhydride nanoparticle- and CDN-based combination adjuvants, which individually could not provide rapid protection when the challenge inoculum contained ≥ 7000 CFU Y. pestis; however, when administered together, rapid and complete protection against all three lethal doses of Y. pestis was 97 achieved (Figure 3.2). The results indicate that while the CDN vaccine induced a similar anti-F1-V antibody titer as did the Combination Nanovaccine formulation, only the latter formulation induced rapid protection (14 DPI) against all three challenge doses of Y. pestis CO92, suggesting a qualitative difference in the immune response induced by the
Combination Nanovaccine formulation. Therefore, it is possible that polyanhydride nanoparticles modulated immune responses beyond humoral immunity. Indeed, previous reports have demonstrated that vaccinated B cell-deficient µMT mice were protected from lethal challenge with the KimD27 strain of Y. pestis52 and that blocking IFN-ɣ and
TNF-α in F1-V-vaccinated C57BL/6 mice resulted in loss of protection against Y. pestis
CO9253. As previously mentioned, polyanhydride particles display inherent adjuvanticity demonstrating the ability to prime humoral and cell-mediated immune responses in vivo
28,29,38–40,54,30–37; therefore, the nanovaccine regimen may be activating cellular immune responses that contribute to the protection at higher challenge doses of Y. pestis CO92.
The focus on the importance of serum antibody to the immunity induced by plague vaccines is based largely on the following: 1) human patients infected with Y. pestis demonstrate long-lived antibody responses to F1 and V antigens with little to no demonstrable T cell responses 45, 2) monoclonal antibodies passively transferred to mice have been shown to be protective against lethal challenge 7–9, 3) direct neutralization of
LcrV has been shown to be necessary to prevent Yop translocation into macrophages in vitro 6, 4) murine challenge models have suggested that anti-F1 IgG1 antibody titer correlates with survival 5, and 5) mucosal and serum anti-V antigen antibodies are the best correlates for survival against pneumonic plague in mice 46. Regardless of the route of immunization, the induction or presence of neutralizing antibodies in the lungs is 98 likely critical for protection against Y. pestis; and, it has been demonstrated that intratracheally administered monoclonal antibody (mAb) 7.3 protected mice against lethal intranasal challenge 47 48,49. The results herein support this notion, as high IgG levels in peripheral blood and BAL fluid were observed 218 DPI (Supplementary Figures
3.1 and 3.3) following a single, subcutaneous administration of the Combination
Nanovaccine.
Elevated antibody responses observed in this work are in agreement with previously reported studies that demonstrated that the use of CDG as an adjuvant enhances antibody responses compared to traditional adjuvants 16,17,50,51. In this work, the resultant antibody responses induced by both the Combination Nanovaccine and CDN
Vaccine were characterized by high affinity, class-switched F1-V-specific IgG1 and
IgG2c by 14 DPI (Figure 3.2 and Figure 3.4). The antibody response was also characterized by a stable F1-V-specific antibody response over 218 DPI that was accompanied by evidence of F1-V-specific long-lived plasma cells in the bone marrow
(Figure 3.4 & Figure 3.8).
Using microarray technology, potential protective immunodominant peptide were identified that were consistent with previously published reports. Xiao et al. identified monoclonal antibodies (mAbs) which bound to peptides F1-2, V19-20, and V28 and together conferred protection against lethal challenge 8. In the current study, responses to
F1-3 were observed in sera from immunized mice at all time points. It has also been reported that a neutralizing mAb BA5 binds amino acids 196-225, corresponding to peptides V31-39, and protects mice against systemic challenge 55. Consistent with this study, serum antibody recognized V32 suggesting that the Combination Nanovaccine 99 elicited antibodies with potentially similar neutralizing capacity as mAb BA5. Khan et al. previously characterized peptides ‘b’ (a.a. 46-60) and ‘g’ (a.a. 256-270) as B cell epitopes56. Similar antibody responses were observed in this study to peptides F9 (a.a. 49-
65) and V44-46 (a.a. 258-286). Interestingly, there are no previous reports of antibody recognition of peptide F11 (a.a. 61-77) or V18 (a.a. 102-118), and, therefore, these regions may be of interest for further research. Together, these results suggest that both
CDN Vaccine and Combination Nanovaccine elicited a polyclonal antibody responses that recognized multiple linear epitopes.
Encapsulation of antigen into polyanhydride nanoparticles can provide many beneficial aspects for labile proteins including maintenance of protein structure, and adjuvant activity for multiple vaccine antigens 23,57–59, including F1-V 22. The surface eroding characteristics of these polymers enables chemistry-dependent release kinetics of antigen providing for single-dose (i.e., no booster) vaccination 27,35,41. Previously, polyanhydride nanoparticles have shown the ability to adjuvant pneumococcal surface protein A (PspA) resulting in the induction of complete protection of mice from a lethal challenge of S. pneumoniae that was comparable to that induced by alum-adjuvanted
PspA with markedly less injection site reactogenicity 19,41. In addition to the adjuvanticity of the polyanhydrides, previous reports have demonstrated that protein structure and functional activity of the labile recombinant protective antigen from Bacillus anthracis was maintained upon encapsulation and release from polyanhydride nanoparticles after storage for at least four months at temperatures up to 40 °C 23. Consequently, encapsulation of proteins into polyanhydride nanoparticles has the capability to both 100 enhance immune outcomes and provide extended shelf storage, both of which may be beneficial with respect to stockpiling biodefense vaccines such as plague vaccines.
In summary, these results indicate that the Combination Nanovaccine is a promising vaccine candidate against Y. pestis based on its ability to induce both rapid and long-lived protective immunity. Immune responses to F1-V were characterized by long- lived high antibody titers, increased breadth of antibody responses to a broader array of epitopes, and induction of long-lived plasma cells in the bone marrow for at least 218
DPI. With the ability to enhance immune responses to F1-V and potential for enhance shelf stability of labile proteins, the Combination Nanovaccine shows promise as a next- generation vaccine platform against weaponized Y. pestis.
Acknowledgements
The authors acknowledge financial support from NIH-NIAID (R01 AI111466) and the Nanovaccine Institute. The authors thank Drs. David Kanne, Chudi Ndubaku, and
Thomas Dubensky Jr at Aduro Biotech for providing the cdG used in the immunization experiments, as well as Dr. Chris Minion at Iowa State University for providing the
BioRobotics MicroGRID II microarray printer used for printing microarray slides. The authors also acknowledge Dr. Thomas Waldschmidt at the University of Iowa for his guidance in ELISPOT assay development. The authors would also like to thank Min
Zhang at Iowa State University for assistance with the statistical analyses. B.N. acknowledges the support of the Vlasta Klima Balloun Faculty Chair.
Author Contributions
DW-M and SK performed the experiments and analyzed the data. ACP and RJD helped with the development of the microarray experiments and data analysis. NPB and 101
BHB performed the challenge studies and analyzed the data. MW, BN, DW-M, and SK designed the experimental plan. All authors participated in the writing of the manuscript.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
References
1. Pechous, R. D., Sivaraman, V., Stasulli, N. M. & Goldman, W. E. Pneumonic Plague: The Darker Side of Yersinia Pestis. Trends Microbiol. 24, 190–197 (2016).
2. Riedel, S. Plague: from natural disease to bioterrorism. Proc. (Bayl. Univ. Med. Cent). 18, 116–124 (2005).
3. Ke, Y., Chen, Z. & Yang, R. Yersinia Pestis: mechanisms of entry into and resistance to the host cell. Front. Cell. Infect. Microbiol. 3, (2013).
4. Inglesby, T. V et al. Plague as a biological weapon: medical and public health management. JAMA 283, 2281–2290 (2000).
5. Williamson, E. D. et al. An IgG1 titre to the F1 and V antigens correlates with protection against plague in the mouse model. Clin. Exp. Immunol. 116, 107–114 (1999).
6. Ivanov, M. I., Hill, J. & Bliska, J. B. Direct neutralization of type III effector translocation by the variable region of a monoclonal antibody to Yersinia Pestis LcrV. Clin. Vaccine Immunol. 21, 667–673 (2014).
7. Hill, J., Leary, S. E. C., Griffin, K. F., Williamson, E. D. & Titball, R. W. Regions of Yersinia pestis V antigen that contribute to protection against plague identified by passive and active immunization. Infect. Immun. 65, 4476–82 (1997).
8. Xiao, X. et al. Human anti-plague monoclonal antibodies protect mice from Yersinia pestis in a bubonic plague model. PLoS One 5, 1–12 (2010).
9. Hill, J. et al. Synergistic protection of mice against plague with monoclonal antibodies specific for the F1 and V antigens of Yersinia Pestis. Infect. Immun. 71, 2234–2238 (2003). 102
10. Heath, D. G. et al. Protection against experimental bubonic and pneumonic plague by a recombinant capsular F1-V antigen fusion protein vaccine. Vaccine 16, 1131– 7 (1998).
11. Quenee, L. E., Ciletti, N. A., Elli, D., Hermanas, T. M. & Schneewind, O. Prevention of pneumonic plague in mice, rats, guinea pigs and non-human primates with clinical grade rV10, rV10-2 or F1-V vaccines. Vaccine 29, 6572– 6583 (2011).
12. Ma, Z. & Damania, B. The cGAS-STING Defense Pathway and Its Counteraction by Viruses. Cell Host Microbe 19, 150–158 (2016).
13. Danilchanka, O. & Mekalanos, J. J. Cyclic dinucleotides and the innate immune response. Cell 154, 962–70 (2013).
14. Ishikawa, H. & Barber, G. N. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 455, 674–678 (2008).
15. Jenal, U., Reinders, A. & Lori, C. Cyclic di-GMP: Second messenger extraordinaire. Nat. Rev. Microbiol. 15, 271–284 (2017).
16. Ebensen, T. et al. The bacterial second messenger cyclic diGMP exhibits potent adjuvant properties. Vaccine 25, 1464–1469 (2007).
17. Hu, D.-L. et al. c-di-GMP as a vaccine adjuvant enhances protection against systemic methicillin-resistant Staphylococcus aureus (MRSA) infection. Vaccine 27, 4867–4873 (2009).
18. Narasimhan, B., Goodman, J. T. & Vela Ramirez, J. E. Rational Design of Targeted Next-Generation Carriers for Drug and Vaccine Delivery. 18, 25–49 (2016).
19. Huntimer, L. et al. Evaluation of Biocompatibility and Administration Site Reactogenicity of Polyanhydride-Particle-Based Platform for Vaccine Delivery. Adv. Healthc. Mater. 2, 369–378 (2013).
20. Vela-Ramirez, J. E. et al. Safety and Biocompatibility of Carbohydrate- Functionalized Polyanhydride Nanoparticles. AAPS J. 17, 256–267 (2015).
21. Kumar, N., Langer, R. S. & Domb, A. J. Polyanhydrides: an overview. Adv. Drug Deliv. Rev. 54, 889–910 (2002).
22. Carrillo-Conde, B. et al. Encapsulation into amphiphilic polyanhydride microparticles stabilizes Yersinia Pestis antigens. Acta Biomater. 6, 3110–9 (2010). 103
23. Petersen, L. K., Phanse, Y., Ramer-Tait, a. E., Wannemuehler, M. J. & Narasimhan, B. Amphiphilic polyanhydride nanoparticles stabilize bacillus anthracis protective antigen. Mol. Pharm. 9, 874–882 (2012).
24. Vela Ramirez, J. E. et al. Carbohydrate-functionalized nanovaccines preserve HIV-1 antigen stability and activate antigen presenting cells. J. Biomater. Sci. Polym. Ed. 25, 1387–1406 (2014).
25. Huntimer, L. et al. Single immunization with a suboptimal antigen dose encapsulated into polyanhydride microparticles promotes high titer and avid antibody responses. J Biomed Mater Res B Appl Biomater 101, 91–98 (2013).
26. Torres, M. P., Vogel, B. M., Narasimhan, B. & Mallapragada, S. K. Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms. J. Biomed. Mater. Res. - Part A 76, 102–110 (2006).
27. Ulery, B. D. et al. Design of a protective single-dose intranasal nanoparticle-based vaccine platform for respiratory infectious diseases. PLoS One 6, 1–8 (2011).
28. Haughney, S. L., Ross, K. A., Boggiatto, P. M., Wannemuehler, M. J. & Narasimhan, B. Effect of nanovaccine chemistry on humoral immune response kinetics and maturation. Nanoscale 6, 13770–13778 (2014).
29. Kipper, M. J., Shen, E., Determan, A. & Narasimhan, B. Design of an injectable system based on bioerodible polyanhydride microspheres for sustained drug delivery. Biomaterials 23, 4405–4412 (2002).
30. Petersen, L. K. et al. Activation of innate immune responses in a pathogen- mimicking manner by amphiphilic polyanhydride nanoparticle adjuvants. Biomaterials 32, 6815–6822 (2011).
31. Torres, M. P. et al. Polyanhydride microparticles enhance dendritic cell antigen presentation and activation. Acta Biomater. 7, 2857–2864 (2011).
32. Petersen, L. K., Xue, L., Wannemuehler, M. J., Rajan, K. & Narasimhan, B. The simultaneous effect of polymer chemistry and device geometry on the in vitro activation of murine dendritic cells. Biomaterials 30, 5131–5142 (2009).
33. Kipper, M. J., Wilson, J. H., Wannemuehler, M. J. & Narasimhan, B. Single dose vaccine based on biodegradable polyanhydride microspheres can modulate immune response mechanism. J. Biomed. Mater. Res. - Part A 76, 798–810 (2006).
34. Ulery, B. D. et al. Polymer chemistry influences monocytic uptake of polyanhydride nanospheres. Pharm. Res. 26, 683–690 (2009).
35. Ulery, B. D. et al. Rational design of pathogen-mimicking amphiphilic materials as nanoadjuvants. Sci. Rep. 1, 198 (2011). 104
36. Goodman, J. T. et al. Nanoparticle chemistry and functionalization differentially regulates dendritic cell-nanoparticle interactions and triggers dendritic cell maturation. Part. Part. Syst. Charact. 31, 1269–1280 (2014).
37. Phanse, Y. et al. Functionalization of polyanhydride microparticles with di- mannose influences uptake by and intracellular fate within dendritic cells. Acta Biomater. 9, 8902–8909 (2013).
38. Carrillo-Conde, B. R., Ramer-Tait, A. E., Wannemuehler, M. J. & Narasimhan, B. Chemistry-dependent adsorption of serum proteins onto polyanhydride microparticles differentially influences dendritic cell uptake and activation. Acta Biomater. 8, 3618–28 (2012).
39. Chavez-Santoscoy, A. V. et al. Tailoring the immune response by targeting C-type lectin receptors on alveolar macrophages using “pathogen-like” amphiphilic polyanhydride nanoparticles. Biomaterials 33, 4762–4772 (2012).
40. Carrillo-Conde, B. et al. Mannose-functionalized ‘pathogen-like’ polyanhydride nanoparticles target C-type lectin receptors on dendritic cells. Mol. Pharm. 8, 1877–1886 (2011).
41. Wagner-Muñiz, D. A., Haughney, S. L., Kelly, S. M., Wannemuehler, M. J. & Narasimhan, B. Room Temperature Stable PspA-Based Nanovaccine Induces Protective Immunity. Front. Immunol. 9, (2018).
42. Ross, K. et al. Combination Nanovaccine Demonstrates Synergistic Enhancement in Efficacy against Influenza. ACS Biomater. Sci. Eng. 2, 368–374 (2016).
43. Tao, P. et al. A bivalent anthrax-plague vaccine that can protect against two Tier-1 bioterror pathogens, Bacillus anthracis and Yersinia Pestis. Front. Immunol. 8, (2017).
44. Mizel, S. B. et al. Flagellin-F1-V fusion protein is an effective plague vaccine in mice and two species of nonhuman primates. Clin. Vaccine Immunol. 16, 21–28 (2009).
45. Li, B. et al. Humoral and cellular immune responses to Yersinia Pestis infection in long-term recovered plague patients. Clin. Vaccine Immunol. 19, 228–234 (2012).
46. Reed, D. S. & Martinez, M. J. Respiratory immunity is an important component of protection elicited by subunit vaccination against pneumonic plague. Vaccine 24, 2283–2289 (2006).
47. Hill, J. et al. Administration of antibody to the lung protects mice against pneumonic plague. Infect. Immun. 74, 3068–70 (2006). 105
48. Glynn, A., Freytag, L. C. & Clements, J. D. Effect of homologous and heterologous prime-boost on the immune response to recombinant plague antigens. Vaccine 23, 1957–1965 (2005).
49. Uddowla, S., Freytag, L. C. & Clements, J. D. Effect of adjuvants and route of immunizations on the immune response to recombinant plague antigens. Vaccine 25, 7984–7993 (2007).
50. Blaauboer, S. M. et al. The mucosal adjuvant cyclic di-GMP enhances antigen uptake and selectively activates pinocytosis-efficient cells in vivo. Elife 2015, 1– 25 (2015).
51. Karaolis, D. K. R. et al. Bacterial c-di-GMP is an immunostimulatory molecule. J. Immunol. 178, 2171–2181 (2007).
52. Parent, M. A. et al. Cell-Mediated Protection against Pulmonary Yersinia Pestis Infection. Infect. Immun. 73, 7304–7310 (2005).
53. Lin, J. S. et al. TNFα and IFNγ contribute to F1/LcrV-targeted immune defense in mouse models of fully virulent pneumonic plague. Vaccine 29, 357–362 (2010).
54. Huntimer, L. M. et al. Polyanhydride nanovaccine platform enhances antigen- specific cytotoxic T cell responses. Technology 02, 171–175 (2014).
55. Quenee, L. E. et al. Amino acid residues 196–225 of LcrV represent a plague protective epitope. doi:10.1016/j.vaccine.2009.11.076
56. Khan, A. A., Babu, J. P., Gupta, G. & Rao, D. N. Identifying B and T cell epitopes and studying humoral, mucosal and cellular immune responses of peptides derived from V antigen of Yersinia Pestis. Vaccine 26, 316–332 (2008).
57. Determan, A. S., Graham, J. R., Pfeiffer, K. a & Narasimhan, B. The role of microsphere fabrication methods on the stability and release kinetics of ovalbumin encapsulated in polyanhydride microspheres. J. Microencapsul. 23, 832–43 (2006).
58. Ross, K. A. et al. Hemagglutinin-based polyanhydride nanovaccines against H5N1 infuenza elicit protective virus neutralizing titers and cell-mediated immunity. Int. J. Nanomedicine 10, 229–243 (2015).
59. Haughney, S. L. et al. Retention of structure, antigenicity, and biological function of pneumococcal surface protein A (PspA) released from polyanhydride nanoparticles. Acta Biomater. 9, 8262–8271 (2013).
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CHAPTER 4. COMBINATION NANOVACCINE PROVIDES PROTECTION AGAINST YERSINIA PESTIS IN MICE WITH IMMUNOLOGICAL DEFICIENCIES
Danielle A Wagner-Muñiz1, Sean M Kelly2, Andrew C Petersen1, Nathan Peroutka-
Bigus1,3, Ross J Darling1, Bryan H Bellaire1,3,4, Michael J Wannemuehler1,4, and Balaji
Narasimhan2,4
1Department of Veterinary Microbiology and Preventive Medicine, Iowa State
University, Ames, IA, USA 2Department of Chemical and Biological Engineering, Iowa
State University, Ames, IA, USA 3Interdepartmental Microbiology, Iowa State
University, Ames, IA, USA 4Nanovaccine Institute, Iowa State University, Ames, IA,
USA
Introduction
Use of inbred mice as a biological model in scientific research is common across many different fields of biomedical research. Differences have been described between differentially sourced mice, and though they are rare, can have a significant impact on biological discoveries1,2. Though uncommon, it is possible for spontaneous genetic mutations to arise in certain mouse colonies, particularly those using the practice of backcrossing3. One such mutation, first described in 2016, entails a double variant mutation in the 28 and 29 exons of the gene Dock24.
Dock2, dedicator of cytokinesis 2, is a member of the Caenorhabditis elegans
Ced-5, mammalian DOCK180 and Drosophila melanogaster myoblast city (CDM) family of scaffolding proteins, that is highly expressed in leukocytes and is known to play 107 a key role in chemokine-related lymphocyte migration5–7. Other members of the CDM protein family, such as Dock180, have been shown to play a key role in the activation of the GTPase Rac, a regulator of the actin cytoskeleton8,9.
Loss of Dock2 functionality, in Dock2-/- mouse models, has been associated with decreased chemokine responsiveness, B and T cell migratory defects, loss of marginal zone B cell populations and malformities in the lymph node follicles, and impaired polarity/chemotaxis of plasmacytoid dendritic cells (pDCs).5,10 The absence of Dock2, however does not seem to effect neutrophil trafficking or monocyte activation in regards to chemokines5,11,12. Fukui et al. also described abnormal thymus architecture and reduced B and T cell homing to the spleen in mice lacking Dock2.
Though lymphocyte migration is significantly hindered in Dock2-/- mice, it is not entirely absent, operating at approximately 10% of normal capacity13. The small subset of
Dock2-/- lymphocytes that are capable of migrating, may be doing so in a PI3K- dependent manner5,14–16. Loss of Dock2 also results in lowered populations of CD4 T cells in the periphery and secondary lymphoid tissues, as well as the inability to maintain
CD4 T cells numbers following transfer, indicating Dock2 may also play role in cell survival13. Dock2-/- T cells express normal levels of integrins following activation and adhere to high endothelial venules inside secondary lymphoid organ (SLO), but fail to properly extravasate into lymphoid tissue despite the capability of normal transendothelial migration in the absence of Dock214,17
Unlike T cells, B cells show no integrin activation in the absence of Dock2 and do not adhere in SLO venules indicating a differential role for Dock2 in T and B cell chemokine-mediated migration. Total B cell precursor population numbers are largely 108 unchanged in the absence of Dock2, rather they experience a marked decrease in chemotactic responsiveness16. Reduced motility in the follicles and paracortex of lymph nodes has been observed in Dock2-/- mice and was coupled with a reduced egress of activated B and T cells from the peripheral lymph nodes15. Additionally, it was found that
CD8+ T cells, from Dock2-/- mice, express significantly lower levels of IFN-γ, granzyme
B, and perforin, due to decreased numbers of lymphocytes in lymphoid tissue18. Though uncommon, Dock2 mutations have been documented in a small subset of the human population, with clinical manifestations of early-onset invasive infections in children19.
Following correspondence with Envigo, about the source and genotype of the mice used for these studies, it was confirmed that they were from barriers/breeding facilities (locations 206, 208, or 231) where C57BL/6NHsd mice containing the Dock2 double variant mutation were present (Appendix). While the genotype of the mice in these studies has not yet been confirmed, we hypothesize that they did in fact contain a mutation. This conclusion is based on the observation that these mice presented with a reduced humoral response that is characteristic of mice with the Dock2 mutation as previously described4,20. The data herein describes the induction of a protective immune response in mice sourced from Envigo (C57BL/6NHsdENV) following immunization with a Combination Nanovaccine formulation, comprised of F1-V-encapsulated into polyanhydride nanoparticles and co-adjuvanted with cyclic dinucleotide cdG (CDNs).
Materials and Methods
Materials
Chemicals used for CPTEG and CPH diacid and polymer synthesis included 1,6- dibromohexane, triethylene 4-p-hydroxybenzoic acid, and 1-methyl-2-pyrrolidinone, purchased from Sigma-Aldrich (St. Louis, MO). Chloroform, petroleum ether, ethyl 109 ether, hexanes, sodium hydroxide, toluene, sulfuric acid, acetonitrile, dimethyl formamide, acetic anhydride, methylene chloride, pentane, and potassium carbonate were purchased from Fisher Scientific (Fairlawn, NJ). 4-p-fluorobenzonitrile was purchased from Apollo Scientific (Cheshire, UK). Deuterated chloroform used for 1H NMR analysis was purchased from Cambridge Isotope Laboratories (Andover, MA). Dithio-RP,RP- cyclic di-guanosine monophosphate (R,R-Cyclic di-GMP (cdG)) was provided by Aduro
Biotech (Berkeley, CA). Complete cell culture medium reagents RPMI 1640 and penicillin-streptomycin were purchased from Mediatech (Herndon, VA); heat inactivated fetal calf serum was purchased from Atlanta Biologicals (Atlanta, GA). Y. pestis, strain
CO92 (NR-641) and the Y. pestis fusion protein F1-V (NR-4526) were obtained from the
Biodefense and Emerging Infections Repository (Manassas, VA).
Polyanhydride synthesis
CPTEG and CPH diacids were synthesized as previously described 21,22. 20:80
CPTEG:CPH copolymer synthesis was performed using melt polycondensation 21.
Copolymer composition and molecular weight were estimated using end group analysis of 1H NMR (DXR 500) spectra.
Nanoparticle synthesis
10 % (w/w) F1-V-loaded 20:80 CPTEG:CPH nanoparticles were synthesized using flash nanoprecipitation, as described previously 23. Briefly, F1-V and 20:80
CPTEG:CPH copolymer was dissolved in methylene chloride at 2 mg/mL and 20 mg/mL, respectively, sonicated at 30 Hz for approximately 30 seconds, and poured into pentane chilled to -20 °C at a methylene chloride:pentane ratio of 1:250. Nanoparticles were imaged using scanning electron microscopy (SEM; JEOL 840 A, JEOL Ltd.,
Tokyo, Japan), and nanoparticle mean size and size distribution were determined using 110
ImageJ (National Institutes of Health, Bethesda, MD). Nanoparticle zeta potential was measured using Zetasizer Nano (Malvern Instruments, Worcestershire, UK).
Animals
Seven to eight-week old female mice were purchased, C57BL/6NHsd from
Envigo (Somerset, NJ) or C57Bl/6NCrlWT from Charles River (Wilmington, MA). Mice were housed under specific pathogen-free conditions where all bedding, caging, water, and feed were sterilized prior to use. All studies were conducted with the approval of the
Iowa State University Institutional Animal Care and Use Committee (IACUC).
Immunization and serum collection
Groups comprised of C57BL/6 mice (n = 8-16/group) were immunized subcutaneously at the nape of the neck with either of the following: 50 µg F1-V + 35 µg
CDNs (R,R-CDG, Aduro Biotech), 50 µg F1-V encapsulated into 500 µg of 20:80
CPTEG:CPH nanoparticles, 50 µg F1-V encapsulated into 500 µg of 20:80 CPTEG:CPH nanoparticles + 35 µg CDNs, or saline in a total volume of 200 µL. Blood was collected via saphenous vein and serum was separated following centrifugation (10,000 rcf for 10 min) at 12, 13, 42, 70, 100, and 114 DPI. Serum was stored at -20°C until analysis.
ELISA
Anti-F1-V antibody titers were determined via ELISA, as previously described 24.
Briefly, high-binding Costar 590 EIA/RIA microtiter plates (Corning) were coated overnight with 100 µL of a 0.5 µg/mL solution of F1-V at 4 °C. After washing the wells, microtiter plates were blocked for two hours with a solution of 2.5% (w/v) powdered skim milk dissolved in PBS-Tween with 0.05% Tween 20, pH 7.4, that had been incubated for two hours at 56 °C to inactivate any endogenous phosphatase activity.
Microtiter plates were washed thrice with PBS-T. Serum obtained from immunized mice 111 was added at a dilution of 1:200 and serially diluted in PBS-T containing 1% (v/v) goat serum. Samples were tested in duplicate. Following incubation overnight at 4°C, plates were washed thrice with PBS-T, after which secondary antibody was added at a dilution of 1 µg/mL. Secondary antibodies used in these studies were: alkaline phosphatase- conjugated goat anti-mouse IgG heavy and light chain (Jackson ImmunoResearch). Plates were incubated for two hours at room temperature and washed three times with PBS-T.
To each well, 100 µL of alkaline phosphatase substrate (Fisher Scientific, Pittsburgh, PA) was added at a concentration of 1 mg/mL dissolved in 50 mM sodium carbonate, 2 mM magnesium chloride buffer at pH 9.3 for colorimetric development. Plates were analyzed after 30 min using a SpectraMax M3 microplate reader at a wavelength of 405 nm. Titer is reported as the reciprocal of serum dilution at which the optical density (OD) value was at most 0.2.
Challenge
Y. pestis CO92 (NR-641) was obtained from BEI Resources. Frozen stocks were prepared in advance of challenges. Y. pestis was initially grown on brain heart infusion
(BHI) agar at 28 °C for 72 h; a single colony was isolated and inoculated in BHI broth and cultivated for 24 h at 28 °C while shaking at 120 rpm. Glycerol solution was added to the broth to bring final glycerol concentration to 5% (v/v). Aliquots were snap frozen and stored at -72 °C. Prior to challenge experiments, aliquots of the frozen stock were thawed and cultured on tryptic soy agar (TSA) with 1% bovine hemoglobin at 37°C and 5% CO2 to check viability and CFU enumeration. For challenge experiments, frozen Y. pestis stocks were thawed and diluted in room temperature PBS. Groups of C57BL/6 mice were anesthetized with an intraperitoneal injection of ketamine/xylazine cocktail and infected intranasally with 8,000-8,500 CFU (14 and 120 DPI) of Y. pestis CO92 in 50 µL volume. 112
Y. pestis bacterial suspension was administered intranasally to nares of mice by a micropipette. CFU enumeration was performed on infectious inoculum preceding to and following infection of the animals to determine infectious dose administered. Animals were checked daily for morbidity and mortality over the course of 12-14 days post- challenge. All activities were performed in an animal biosafety level 3 (ABSL-3) laboratory at Iowa State University with protocols approved by the IACUC and
Institutional Biosafety Committee (IBC).
Statistical analyses
Statistical analyses on the ELISA data as well as the survival data were performed using the Mantel-Cox log rank test and the one-way analysis of variance (ANOVA) using
Tukey’s multiple comparisons test. A p-value ≤ 0.05 was considered to be statistically significant. All analyses were performed using GraphPad Prism v. 7.0 (GraphPad, La
Jolla, CA).
Results
Combination Nanovaccine Provides Early-Onset Protection
In order to assess rapidly-induced protection, separate groups of
C57BL/6NHsdENV mice were immunized subcutaneously as described in Table 4.1.
Serum was collected from vaccinated mice at 12 days post-immunization (DPI) and analyzed via ELISA for anti-F1-V specific IgG (H+L) to charaterize the humoral immune response. Mice were challenged intranasally at 13 DPI with 8500 CFUs of Y. pestis strain
CO92. Mice immunized with Combination Nanovaccine formulation showed signifcantly
(p < .05) higher anti-F1-V-specific titers compared to that induced by the Nanovaccine alone (Figure 4.1A). Following challenge, immunization with the Combination
Nanovaccine induced 100 % survival which was signifcantly (p ≤ .025) better protection 113 than that indiced by any other vaccine regimen (Figure 4.1B). CDN Vaccine provided for
50% protection, but did not provide superior protection compared to other vaccinated groups. Nanovaccine alone provided 25% protection, while 12.5 % of the soluble F1-V protein alone and the saline treated mice survived the Y. pestis CO92 challenge.
Table 4.1Vaccine formulations. Vaccine formulations were comprised of soluble F1-V protein, F1-V encapsulated into 20:80 CPTEG:CPH nanoparticles at 10% w/w loaded, and soluble CDNs (cdG).
Long-Term Protection Observed in Adjuvanted Vaccine Formulations
To determine whether long-term protection was induced, C57BL/6NHsdENV mice were immunized subcutaneously as described in Table 4.1. Serum was collected at 12,
42, 70, 100, and 114 DPI and analyzed via ELISA for antigen-specific IgG (H+L) (Figure
4.2A). Regardless of the time point, Combination Nanovaccine-immunized mice showed significantly (p ≤ .04) increased titers compared to serum from mice immunized with soluble F1-V, and significantly (p ≤ .008) higher titers than Nanovaccine at 14 and 42
DPI, with highest titers observed at 14 DPI.
114
Figure 4.1 Efficacy of various F1-V containing vaccine regimen against lethal Y. pestis CO92 challenge. Serum from subcutaneously immunized mice (n = 8-16) was collected at 12 DPI and analyzed for total anti-F1-V-specific IgG (A). Animals were challenged intranasally with 8500 CFUs of Y. pestis at 14 DPI and survival was assessed for 14 days post- challenge (B). * indicates significant difference (p ≤ .05) as compared to F1-V group. # indicates significant difference (p ≤ .029) as compared to all formulation groups. 115
Mice immunized with CDN Vaccine showed significantly (p ≤ .04) higher titers than those immunized with F1-V alone at 42, 70, and 101 DPI, and significantly (p ≤ .02) higher titers than Nanovaccine at 42 DPI.
A challenge dose of 8000 CFUs of Y. pestis CO92 was administered intranasally at 120 DPI. All adjuvanted formulations (Combination Nanovaccine, Nanovaccine, and
CDN Vaccine) performed similarly providing 71-75% protection from lethal challenge, that was significantly (p ≤ .05) higher than the 12.5 to 15 % protection for the mice vaccinated with soluble F1-V alone or saline (Figure 4.2B). IgG class switching was evaluated for F1-V-specific IgG1 and IgG2c at all time points (Figure 4.2C). IgG1 was measurable in all vaccinated groups, with CDN Vaccine showing significantly (p ≤ .02) higher IgG1 titers than F1-V alone at all time points, and in mice receiving the
Nanovaccine at 14 and 79 DPI. IgG1 responses were also significantly (p ≤ .05) higher in
Combination Nanovaccine as compared to F1-V, at 14 and 79 DPI, and Nanovaccine at
14 DPI. F1-V-specific IgG2c was only observed in vaccine formulations adjuvanted with
CDNs (CDN Vaccine and Combination Nanovaccine), and was significantly (p ≤ .01) higher than F1-V or Nanovaccine at all time points indicating that addition of CDNs enhanced IgG class-switching to IgG2c. 116
Figure 4.2 Long-Term Protection Observed in Adjuvanted Vaccine Formulations. Serum from subcutaneously immunized mice (n=7-16) at 14, 42, 79, 101, and 114 DPI was analyzed for total anti-F1-V-specific IgG (A). Dashed line indicates titer of Saline control mice. Animals were challenged intranasally at 120 DPI and survival was assessed for 12 days post-challenge (B). IgG class switching was characterized at all time points and is reported as an OD value at a dilution of 1:500 (C/D). * indicates significant difference (p ≤ .04) as compared to F1-V group. # indicates significant difference (p ≤ .015) as compared to Nanovaccine.
117
Varying Humoral Immune Response Observed Between Differentially-Sourced Mice
Vaccination of both Envigo-sourced mice (C57BL/6NHsdENV) and Dock2 wildtype mice sourced from Charles River (C57Bl/6NCrlWT) showed varying levels of humoral immunity. Mice were immunized subcutaneously with vaccine formulations described in Table 4.1. Total serum anti-F1-V IgG was assessed via ELISA at 14, 49, and
70 DPI. Titers observed in C57Bl/6NCrlWT mice were significantly (p ≤ .05) higher (i.e, four to 10-fold increase) than those measured in C57BL/6NHsdENV mice when CDNs were included in the vaccine regimen (Combination Nanovaccine and CDN Vaccine) across all time points (Figure 4.3A). Comparison of the kinetics of the humoral response between C57BL/6NHsdENV and C57Bl/6NCrlWT mice immunized with the Combination
Nanovaccine showed peak antibody titers in C57Bl/6NENV mice at 14 DPI, while immunized C57Bl/6NCrlWT presented with increasing antibody titers up to at least 101
DPI (Figure 4.3B). Comparison of the IgG2c:IgG1 ratio between C57BL/6NHsdENV and
C57Bl/6NCrlWT demonstrated a higher IgG2c:IgG1 ratio in Combination Nanovaccine- immunized mice as compared to all other vaccinated groups in the Envigo-sourced mice, while in WT mice a higher ratio was observed in all groups adjuvanted with CDNs
(Figure 4.3C). 118
Figure 4.3 Varying Humoral Immune Response Observed Between Differentially- Sourced Mice. Serum from subcutaneously immunized C57BL/6NHsdENV and C57BL/6NCrlWT mice (n=7-16) at 14, 42, and 79 DPI was analyzed for total anti-F1-V- specific IgG (A). Comparison of the kinetics of the humoral immune response following immunization with Combination Nanovaccine, as measured by total serum IgG for C57BL/6NHsdENV compared to C57BL/6NCrlWT (B). IgG2c:IgG1 in immunized C57BL/6NHsdENV C57BL/6NCrlWT mice (C). 119
Discussion
Cell motility plays a critical role in the overall function of the immune system, contributing to cell trafficking, activation, proliferation, and the architecture of secondary lymphoid structures. While the actin cytoskeleton is primarily responsible for proper immune synapse formulation and the ability of cells to extravasate from the circulation to their appropriate locations, other factors such as chemokines, cell surface receptors/ligands, adhesion molecules, and the downstream signaling resulting from these interactions also play an equally important role. One such key player in cell motility is
Dock2.
In addition to reduced overall cell motility in the secondary lymphoid tissues, activation, and cell trafficking between tissues, mice with a double variant of the Dock2 gene have been found to be lacking marginal zone B cells4. Though the absence of this cell population primarily influences the spleen, in terms of secondary lymphoid organs, it is possible that this deficit in the B cell population could play some role in the decreased overall humoral immune response in these Dock2-/- mice. Removal of an entire subset of
B cells could also alter the overall functionality of other cell types, due to altered secondary tissue architecture or the decrease of certain cytokine/chemokine levels.
Because Dock2’s function in regulation of actin cytoskeleton rearrangement, it is possible that it plays a critical role in other functions not related to chemokine-mediated migration, such as immune synapse formation, initiation of signaling cascades, and apoptosis.
The data herein demonstrates that immunization of C57BL/6NHsdENV mice with
Combination Nanovaccine formulation was able to induce both rapid and long-lived 120 protection (at 14 and 120 DPI, respectively) against lethal Y. pestis CO92 challenge
(Figures 4.1 and 4.2), despite lower overall titers as compared to WT mice. Combination
Nanovaccine immunization conferred 100% protection against lethal challenge at 14 DPI
(Figure 4.1B). Additionally, all adjuvanted vaccine formulations (i.e. Combination
Nanovaccine, Nanovaccine, CDN Vaccine) were capable of conferring 71-75% protective immunity at 120 DPI (Figure 4.2B).
Class-switching to IgG2c was observed in both CDN Vaccine and Combination
Nanovaccine formulations, though Combination Nanovaccine showed higher ratios of
IgG2c:IgG1 (Figure 4.3). While loss of Dock2 functionality has been associated with decreased germinal center responses, it has been suggested that class-switching is determined between 7-10 days post-infection/-immunization and occurs prior to germinal center formation4,25,26. Therefore, it is possible the class-switching observed here is occurring in the absence of germinal center formation.
While the genotype of these C57BL/6NHsdENV mice cannot be confirmed, comparison of the humoral immune response following vaccination to WT mice shows a strikingly different immune phenotype. F1-V-specific IgG titers observed in WT vaccinated mice were 4-10 times higher and showed peak antibody titers at a later time point (101 DPI) as compared to those measured in serum from C57BL/6NHsdENV (Figure
4.3). Analysis of the kinetics of the humoral immune response in C57BL/6NHsdENV mice may seem to indicate a predominantly short-lived plasma cell or extrafollicular antibody response suggesting a decreased ability to form germinal centers in response to vaccination. 121
While Dock2-/- mice were shown to have severely atrophied T and B cell areas of the lymph nodes, they do form rudimentary white pulp cords that serve to maintain a modicum of lymphocyte populations17. It is hypothesized that this small subset of normally functioning immune cells, may migrate using a PI3K-dependent mechanism which would represent biological redundancy associated with cell motility. This may help to explain why a measurable humoral immune response following vaccination, though at a significantly lower level than that measured in WT mice receiving the same vaccine formulation.
Though impaired cell trafficking makes it unlikely that long-lived plasma cells
(LLPCs) induced by these vaccine formulations would be able to migrate to the bone marrow, it has been previously demonstrated that LLPCs do maintain small populations in the spleen and lymph nodes for up to 10 years following primary immunization27. It is possible that the long-lived protective immune response observed here could be due to long-lived antibody secreting cells present in the secondary lymphoid tissues.
Additionally, it has been described by Darling et al., that CDNs induce BAFF expression, which may contribute to the development and survival of LLPCs in the secondary lymphoid tissue.
In addition to impaired B cell development, Mahajan et al. also described the presence of an unusually large proportion of memory CD8+ T cells circulating in the periphery of double variant Dock2 mice. It is possible that this population of T cells could also be contributing to overall protection from Y. pestis infection through the production of IFNγ and activation of macrophages, which has been linked to protection against pneumonic plague28. 122
Though Dock2 mutations are uncommon, there are many other immunological deficiencies in the general population, which are associated with decreased overall humoral immune responses. One such population exhibiting decreased humoral responses are HIV infected individuals, which show significantly decreased antibody production in comparison to healthy individuals largely due to decreased CD4+ T cell populations29,30.
Actin cytoskeleton-associated immunodeficiencies, such as Wiskott-Aldrich Syndrome, mutations in Dock8 gene, autosomal-recessive hyper IgE syndrome (AR-HIES), and
Epidermodysplasia verruciformis, all exhibit decreased humoral immune responses as well31. In these instances, use of a vaccine that is capable of inducing strong humoral immune responses, such as those observed following Combination Nanovaccine immunization in both C57BL/6NHsdENV and WT mice, could be able to increase overall vaccine efficacy in immunodeficient individuals.
The development of next-generation vaccines, which can overcome immunological defects such as Dock2, is critically important when considering the vaccination of a biologically diverse population. While many vaccines do offer sufficient protection in healthy individuals, use of a vaccine that is better able to induce protective immune responses in immunodeficient individuals provides an opportunity for higher overall levels of protective immunity. The use of co-adjuvants, such as CDNs and polyanhydride nanoparticles, allows for a synergistic approach towards vaccine development which may be able to overcome such immune defects in the general population.
123
References
1. Simon, M. M. et al. A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains. Genome Biol. 14, (2013).
2. Mekada, K. et al. Genetic differences among C57BL/6 substrains. Exp. Anim 58, (2009).
3. Davisson, M. T., Bergstrom, D. E., Reinholdt, L. G. & Donahue, L. R. Discovery genetics - the history and future of spontaneous mutation research. Curr. Protoc. Mouse Biol. 2, 103–118 (2012).
4. Mahajan, V. S. et al. Striking immune phenotypes in gene-targeted mice are driven by a copy-number variant originating from a commercially available C57BL/6 strain. Cell Rep. 15, 1901–1909 (2016).
5. Fukui, Y. et al. Haematopoietic cell-specific CDM family protein DOCK2 is essential for lymphocyte migration. Nature 412, 826–831 (2001).
6. Nishihara, H. et al. Non-adherent cell-specific expression of DOCK2, a member of the human CDM-family proteins. Biochim. Biophys. Acta - Mol. Cell Res. 1452, 179–187 (1999).
7. Wang, L. et al. DOCK2 regulates cell proliferation through Rac and ERK activation in B cell lymphoma. (2010). doi:10.1016/j.bbrc.2010.03.148
8. Cote, J.-F. Identification of an evolutionarily conserved superfamily of DOCK180- related proteins with guanine nucleotide exchange activity. J. Cell Sci. 115, 4901– 4913 (2002).
9. Miyamoto, Y. & Yamauchi, J. Cellular signaling of Dock family proteins in neural function. Cellular Signalling 22, 175–182 (2010).
10. Gotoh, K. et al. Differential requirement for DOCK2 in migration of plasmacytoid dendritic cells versus myeloid dendritic cells. Blood 111, 2973–6 (2008).
11. Reif, K. & Cyster, J. G. The CDM protein DOCK2 in lymphocyte migration. Trends Cell Biol. 12, 368–373 (2002).
12. Kunisaki, Y. et al. DOCK2 is a Rac activator that regulates motility and polarity during neutrophil chemotaxis. J. Cell Biol. 174, 647–52 (2006).
13. Fukui, Y. et al. Haematopoietic cell-specific CDM family protein DOCK2 is essential for lymphocyte migration. Nature 412, 826–831 (2001).
14. Shulman, Z. et al. DOCK2 regulates chemokine-triggered lateral lymphocyte motility but not transendothelial migration. Blood 108, 2150–2158 (2006). 124
15. Nombela-Arrieta, C. et al. A central role for DOCK2 during interstitial lymphocyte motility and sphingosine-1-phosphate-mediated egress. J. Exp. Med. 204, 497–510 (2007).
16. Ward, S. G. & Marelli-Berg, F. M. Mechanisms of chemokine and antigen- dependent T-lymphocyte navigation. Biochem. J. 418, 13–27 (2009).
17. Nombela-Arrieta, C. et al. Differential requirements for DOCK2 and Phosphoinositide-3-Kinase γ during T and B lymphocyte homing. Immunity 21, 429–441 (2004).
18. Jiang, H. et al. Deletion of DOCK2, a regulator of the actin cytoskeleton in lymphocytes, suppresses cardiac allograft rejection. J. Exp. Med. 202, 1121–30 (2005).
19. Dobbs, K. et al. Inherited DOCK2 deficiency in patients with early-onset invasive infections. N. Engl. J. Med. 372, 2409–2422 (2015).
20. Wong, S.-Y. et al. B cell defects observed in Nod2 knockout mice are a consequence of a Dock2 mutation frequently found in inbred strains. J. Immunol. 201, 1442–1451 (2018).
21. Torres, M. P., Vogel, B. M., Narasimhan, B. & Mallapragada, S. K. Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms. J. Biomed. Mater. Res. - Part A 76, 102–110 (2006).
22. Kipper, M. J., Wilson, J. H., Wannemuehler, M. J. & Narasimhan, B. Single dose vaccine based on biodegradable polyanhydride microspheres can modulate immune response mechanism. J. Biomed. Mater. Res. Part A 76A, 798–810 (2006).
23. Haughney, S. L., Ross, K. a., Boggiatto, P. M., Wannemuehler, M. J. & Narasimhan, B. Effect of nanovaccine chemistry on humoral immune response kinetics and maturation. Nanoscale 6, 13770–13778 (2014).
24. Ulery, B. D. et al. Design of a protective single-dose intranasal nanoparticle-based vaccine platform for respiratory infectious diseases. PLoS One 6, 1–8 (2011).
25. Pape, K. A. et al. Visualization of the genesis and fate of isotype-switched B cells during a primary immune response. J. Exp. Med. J. Exp. Med. 197, 1677–1687 (2003).
26. Stavnezer, J., Guikema, J. E. J. & Schrader, C. E. Mechanism and regulation of class switch recombination. Annu. Rev. Immunol. 26, 261–292 (2008).
27. Holden, L. A. et al. Plasma cell survival in the absence of B cell memory. Nat. Commun. 8, (2017). 125
28. Lin, J. S. et al. TNFα and IFNγ contribute to F1/LcrV-targeted immune defense in mouse models of fully virulent pneumonic plague. Vaccine 29, 357–362 (2010).
29. Kernéis, S. et al. Long-term immune responses to vaccination in HIV-infected patients: A systematic review and meta-analysis. Clin. Infect. Dis. 58, 1130–1139 (2014).
30. Landrum, M. L. et al. Hepatitis B vaccine responses in a large U.S. military cohort of HIV-infected individuals: another benefit of HAART in those with preserved CD4 count. Vaccine 27, 4731–8 (2009).
31. Moulding, D. A., Record, J., Malinova, D. & Thrasher, A. J. Actin cytoskeletal defects in immunodeficiency. Immunol. Rev. 256, 282–99 (2013).
126
CHAPTER 5. CO-ADJUVANT APPROACHES TO EARLY-ONSET PROTECTION
Danielle A Wagner-Muñiz1, Sean M Kelly2, Andrew C Petersen1, Nathan Peroutka-
Bigus1,3, Ross J Darling1, Bryan H Bellaire1,3,4, Michael J Wannemuehler1,4, and Balaji
Narasimhan2,4
1Department of Veterinary Microbiology and Preventive Medicine, Iowa State
University, Ames, IA, USA 2Department of Chemical and Biological Engineering, Iowa
State University, Ames, IA, USA 3Interdepartmental Microbiology, Iowa State
University, Ames, IA, USA 4Nanovaccine Institute, Iowa State University, Ames, IA,
USA
Introduction
Successful control of Yersinia pestis (Y. pestis) infection is contingent upon activation of both innate and adaptive branches of the immune system. Following primary infection, the innate immune system is of extreme importance in controlling early bacterial replication. The majority of Y. pestis bacterium entering the host are immediately killed by neutrophils; however, many evade the immune system by preferentially infecting host macrophages. The adaptive humoral immune response contributes to the control of extracellular bacteria by neutralizing Y. pestis virulence factors (e.g., V antigen) and inhibiting infection of macrophages. Additionally, the cellular immune response and production of pro-inflammatory cytokines such as interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) can contribute to control of bacterial replication and overall protective immune response, particularly in pulmonary infections1,2. 127
Engulfment of micro-organisms by Ly-6G+ neutrophils is capable of controlling
Y. pestis growth and spread of infection in the first 48 hours post-infection3. At 72-96 hours post-infection, the bacterium is primarily found inside CD11b+ macrophages, which allows the pathogen to evade host immunity, as well as traffic to secondary lymphoid tissues4. It is there, inside host macrophages, Y. pestis begins to replicate and upregulate expression of the type III secretion system, and downregulates the macrophage-induced pro-inflammatory response5. Upon escape from the macrophage, Y. pestis can further evade the immune response by insertion of Yersinia effector proteins
(Yops) into the cytosol of target host cells6. It has been previously described that pre- treatment of macrophages in vitro with IFNγ and TNFα can help to overcome these mechanisms of immunosuppression3.
In order for a vaccine to overcome the Y pestis mechanisms of immune evasion, it is important to engage both the innate and adaptive immune response. Though the innate immune response is relatively short, in comparison to adaptive immunity, it is possible for it to have a large impact on overall infection outcome over a prolonged period of weeks to months through the process of trained innate immunity7,8.
Trained immunity is induced following a primary infection or vaccination through epigenetic reprogramming of monocytes, macrophages, and natural killer (NK) cells, which directly alter cellular metabolism and activation of immune signaling pathways9,10.
Metabolically, trained monocytes exhibit a shift from oxidative phosphorylation to glycolysis, and have epigenetic changes present in genes involved in the mammalian target of rapamycin (mTOR) pathway11.
The trained immune response primarily involves activation of neutrophils and 128 monocyte-derived macrophages, and has been shown to confer cross-protection against nonrelated secondary infections at 7 days following initial infection, in a T and B cell independent manner12,13. It has been observed that trained monocytes show increased histone methylation, as well as, increased transcription of genes encoding IL-6 and TNFα following secondary heterologous challenge12. Trained monocytes and macrophages stimulated ex vivo following infection, were shown to have larger capacity for IL-6 and
TNFα production, as compared to naïve cells for two weeks post-challenge, and had increased reactive oxygen species production for three weeks following infection14.
Though the mechanisms surrounding the development of trained innate immunity are still being fully elucidated, several transcription factors have been identified that may play a large impact on epigenetic and transcriptional programming. Of particular interest, it has been reported that inhibiting synthesis of cyclic adenosine monophosphate (cAMP) or mTOR during the primary response eliminates the protective effect of trained innate immunity following secondary infection in human and murine models11,15.
By incorporating adjuvants, that are capable of activating the innate immune responses and that induce trained immunity, into vaccine formulations, we may be better able to rationally design vaccines capable of providing rapid protection, prior to full activation of the humoral immune response, necessary in outbreak scenarios. Herein, the capacity of Combination Nanovaccine and CDN Vaccine formulations, previously described in Chapters 3 and 4, to induce protective immunity at both 5 and 10 days post- immunization (DPI) was evaluated. Additionally, the impact of the pro-inflammatory cytokines IFNγ and TNFα on vaccine-induced protective immunity against lethal pulmonary Y. pestis infection was evaluated at 14 DPI. 129
Materials and Methods
Materials
Chemicals used for CPTEG and CPH diacid and polymer synthesis included 1,6- dibromohexane, triethylene 4-p-hydroxybenzoic acid, and 1-methyl-2-pyrrolidinone, purchased from Sigma-Aldrich (St. Louis, MO). Chloroform, petroleum ether, ethyl ether, hexanes, sodium hydroxide, toluene, sulfuric acid, acetonitrile, dimethyl formamide, acetic anhydride, methylene chloride, pentane, and potassium carbonate were purchased from Fisher Scientific (Fairlawn, NJ). 4-p-fluorobenzonitrile was purchased from Apollo Scientific (Cheshire, UK). Deuterated chloroform used for 1H NMR analysis was purchased from Cambridge Isotope Laboratories (Andover, MA). Dithio-RP,RP- cyclic di-guanosine monophosphate (R,R-Cyclic di-GMP (cdG)) was provided by Aduro
Biotech (Berkeley, CA). Complete cell culture medium reagents RPMI 1640 and penicillin-streptomycin were purchased from Mediatech (Herndon, VA); heat inactivated fetal calf serum was purchased from Atlanta Biologicals (Atlanta, GA). Y. pestis, strain
CO92 (NR-641) and the Y. pestis fusion protein F1-V (NR-4526) were obtained from the
Biodefense and Emerging Infections Repository (Manassas, VA).
Polyanhydride synthesis
CPTEG and CPH diacids were synthesized as previously described 16,17. 20:80
CPTEG:CPH copolymer synthesis was performed using melt polycondensation 16.
Copolymer composition and molecular weight were estimated using end group analysis of 1H NMR (DXR 500) spectra.
Nanoparticle synthesis
10 % (w/w) F1-V-loaded 20:80 CPTEG:CPH nanoparticles were synthesized using flash nanoprecipitation, as described previously 18. Briefly, F1-V and 20:80 130
CPTEG:CPH copolymer was dissolved in methylene chloride at 2 mg/mL and 20 mg/mL, respectively, sonicated at 30 Hz for approximately 30 seconds, and poured into pentane chilled to -20 °C at a methylene chloride:pentane ratio of 1:250. Nanoparticles were imaged using scanning electron microscopy (SEM; JEOL 840 A, JEOL Ltd.,
Tokyo, Japan), and nanoparticle mean size and size distribution were determined using
ImageJ (National Institutes of Health, Bethesda, MD). Nanoparticle zeta potential was measured using Zetasizer Nano (Malvern Instruments, Worcestershire, UK).
Animals
Seven to eight-week old female C57BL/6NCrl mice were purchased from Charles
River (Wilmington, MA). Mice were housed under specific pathogen-free conditions where all bedding, caging, water, and feed were sterilized prior to use. All studies were conducted with the approval of the Iowa State University Institutional Animal Care and
Use Committee (IACUC).
Immunization and serum collection
Groups comprised of C57BL/6Crl mice (n = 8-16/group) were immunized subcutaneously (sc) at the nape of the neck with either of the following: 50 µg F1-V + 35
µg CDNs (R,R-CDG, Aduro Biotech), 50 µg F1-V encapsulated into 500 µg of 20:80
CPTEG:CPH nanoparticles + 35 µg CDNs, or saline in a total volume of 200 µL. Blood was collected via saphenous vein and serum was separated following centrifugation
(10,000 rcf for 10 min) at 4, 9, and 13 DPI. Serum was stored at -20°C until analysis.
ELISA
Anti-F1-V antibody titers were determined via ELISA, as previously described 19.
Briefly, high-binding Costar 590 EIA/RIA microtiter plates (Corning) were coated overnight with 100 µL of a 0.5 µg/mL solution of F1-V at 4 °C. After washing the wells, 131 microtiter plates were blocked for two hours with a solution of 2.5% (w/v) powdered skim milk dissolved in PBS-Tween with 0.05% Tween 20, pH 7.4, that had been incubated for two hours at 56 °C to inactivate any endogenous phosphatase activity.
Microtiter plates were washed thrice with PBS-T. Serum obtained from immunized mice was added at a dilution of 1:200 and serially diluted in PBS-T containing 1% (v/v) goat serum. Samples were tested in duplicate. Following incubation overnight at 4°C, plates were washed thrice with PBS-T, after which secondary antibody was added at a dilution of 1 µg/mL. Secondary antibodies used in these studies were: alkaline phosphatase- conjugated goat anti-mouse IgG heavy and light chain (Jackson ImmunoResearch). Plates were incubated for two hours at room temperature and washed three times with PBS-T.
To each well, 100 µL of alkaline phosphatase substrate (Fisher Scientific, Pittsburgh, PA) was added at a concentration of 1 mg/mL dissolved in 50 mM sodium carbonate, 2 mM magnesium chloride buffer at pH 9.3 for colorimetric development. Plates were analyzed after 30 min using a SpectraMax M3 microplate reader at a wavelength of 405 nm. Titer is reported as the reciprocal of serum dilution at which the optical density (OD) value was at most 0.2.
Challenge
Y. pestis CO92 (NR-641) was obtained from BEI Resources. Frozen stocks were prepared in advance of challenges. Y. pestis was initially grown on brain heart infusion
(BHI) agar at 28 °C for 72 h; a single colony was isolated and inoculated in BHI broth and cultivated for 24 h at 28 °C while shaking at 120 rpm. Glycerol solution was added to the broth to bring final glycerol concentration to 5% (v/v). Aliquots were snap frozen and stored at -72 °C. Prior to challenge experiments, aliquots of the frozen stock were thawed and cultured on tryptic soy agar (TSA) with 1% bovine hemoglobin at 37°C and 5% CO2 132 to check viability and CFU enumeration. For challenge experiments, frozen Y. pestis stocks were thawed and diluted in room temperature PBS. Groups of C57BL/6Crl mice were anesthetized with an intraperitoneal injection of ketamine/xylazine cocktail and infected intranasally with 7,000 to 10,000 CFU of Y. pestis CO92 in 50 µL volume at 4,
9, or 14 DPI. Y. pestis bacterial suspension was administered intranasally to nares of mice by a micropipette. CFU enumeration was performed on infectious inoculum preceding to and following infection of the animals to determine infectious dose administered.
Animals were checked daily for morbidity and mortality over the course of two weeks.
All activities using Y. pestis were performed in a biosafety level 3 (ABSL-3) laboratory at
Iowa State University with protocols approved by the IACUC and Institutional Biosafety
Committee (IBC).
Statistical Analyses
Statistical analyses on the ELISA data as well as the survival data were performed using the Mantel-Cox log rank test and the one-way analysis of variance (ANOVA) using
Tukey’s multiple comparisons test. A p-value ≤ 0.05 was considered to be statistically significant. All analyses were performed using GraphPad Prism v. 7.0 (GraphPad, La
Jolla, CA).
IFNγ and TNFα Cytokine Neutralization
Mice received 1 mg each anti-TNFα (rat IgG1, XT3.11) mAb and anti-IFNγ (rat
IgG1, XMG1.2) mAb delivered intraperitoneally in 500 µL PBS one day before challenge.
Serum Cytokine Analysis
Serum was collected at 4, 9, or 13 DPI, diluted 1:4, and analyzed for presence of IL-1β, IL-4, IL-6, TNF-α, IL-12p40, and IFN-γ using multiplex cytokine 133 assay as previously described20 and analyzed via Bio-Plex 200™ system (Bio-Rad,
Hercules, CA).
Results
Protection against lethal Y. pestis challenge at 5 and 10 days post-immunization
In order to assess the humoral and protective immune response at very early time points post-immunization (5 and 10 days), two independent experiments were conducted.
Mice were immunized sc with one of the formulations outlined in Table 5.1. Serum was collected at 4 or 9 days post-immunization (DPI) and analyzed for total IgG via ELISA
(Figure 5.1 A and C). There was no detectable anti-F1-V specific IgG (H+L) at 4 DPI
(Figure 5.1 A); however, by 9 DPI, there was a significant (p ≤ .05) increase in titer for each vaccine group, with the CDN Vaccine and the Combination Nanovaccine inducing similar titers (Figure 5.1 A and C). In the first set of experiments shown in Figure 5.1 panels A and B, mice were challenged intranasally at 5 or 10 DPI with 10,000 CFUs of
Yersinia pestis strain CO92. Vaccination with the Combination Nanovaccine provided
100 % protection at 10 DPI, performing significantly (p ≤ .01) better than all other vaccine formulas where all mice in these treatment groups succumbed to infection by four days post-challenge (Figure 5.1B). For the second experimental iteration, as shown in Figure 5.1 panels C and D, mice were challenged at 10 DPI with 7,000 CFUs of Y. pestis strain CO92. CDN Vaccine-immunized mice showed significantly (p ≤ .03) increased survival (i.e., 87.5 %) while only 37 % % of the mice treated with the
Combination Nanovaccine survived (Figure 5.1 D). 134
Table 5.1 F1-V vaccine formulations. Formulations used in this study were comprised of soluble F1-V protein, F1-V encapsulated into 20:80 CPTEG:CPH nanoparticles at 10% w/w loaded, and soluble CDNs (cdG)
Neutralization of IFNγ/TNFα impacts challenge outcome
In order to analyze the importance of pro-inflammatory cytokines IFNγ and TNFα on protection at 14 DPI, mice were immunized with Combination
Nanovaccine formulation outlined in Table 5.1. Anti-F1-V specific serum IgG was analyzed at 13 DPI via ELISA and was found to be similar across all immunized mice
(Figure 5.2 A). Cytokines (IFNγ and TNFα ) were neutralized one day prior to challenge using specific mAbs delivered intraperitoneally. Mice were challenged intranasally with
7000 CFUs of Y. pestis CO92 at 14 DPI and survival was monitored for 14 days post- challenge. Mice, in which IFNγ and TNFα were neutralized showed significantly (p ≤
.03) less survival than Combination Nanovaccine-immunized mice that did not have cytokines neutralized prior to challenge (Figure 5.2 B). Combination Nanovaccine and saline survival data shown here is also shown in Figure 3.2D.
135
Figure 5.1 Protection against lethal Y. pestis challenge at 5 and 10 DPI. Serum from immunized mice (n = 6-16) was collected at 4 (A) or 9 (A/C) DPI and analyzed for total anti-F1-V-specific IgG DPI. Mice were challenged intranasally with 10000 (B) or 7000 (D) CFU of Y. pestis at 5 (B) or 10 DPI (B/D). Survival was assessed for 14 days post-challenge 136
Figure 5.2 Neutralization of IFNγ and TNFα decreases overall survival. Mice were immunized subcutaneous with Combination Nanovaccine formulation. Total F1-V specific serum IgG was determined at 13 DPI via ELISA (A). Animals (n = 4-16) were challenged intranasally at 14 DPI with 7000 CFU of Y. pestis and survival was assessed for 14 days following challenge (B). *indicates significant (p ≤ .03) compared to IFNγ/TNFα neutralization. 137
Serum cytokine analysis
To evaluate potential cytokines that may impacting protection at early timepoints, serum was collected from mice immunized as part of the experiments outlined in Figure
5.1 A-D, at 4, 9, or 13 DPI. Serum was analyzed for presence of IL-1β, IL-4, IL-6, TNF-
α, IL-12p40, and IFN-γ using multiplex cytokine assay. The only detectable cytokine in the serum of immunized mice regardless of the time point and treatment group was IL-
12p40 (Figure 5.3). No significant differences were observed between groups.
Figure 5.3 Analysis of serum cytokine expression. Mice were immunized subcutaneously and serum was evaluated at 4, 9, or 13 DPI for presence of IL-1β, IL-4, IL-6, TNF-α, IFN-γ (data not shown) and IL-12p40 using multiplex cytokine assay.
Discussion
Development of efficacious vaccines against potential agents of biological warfare, such as Y. pestis, requires the induction of rapid protection following immunization. The data described herein, demonstrates the potential for both CDN
Vaccine and Combination Nanovaccine formulations to provide protection in as little as
10 DPI (Figure 5.1 B and D). Though the survival conferred is variable between 138 experiments, combined overall protection provided by Combination Nanovaccine and
CDN Vaccine was between 50-64%, which is still a significant improvement over unvaccinated animals who consistently succumb to challenge in as little as 3 days post- challenge.
Further analysis of the innate and humoral immune responses at these early time points may be better able to offer insight into the variability in protection observed.
Though F1-V specific IgG (H+L) titers were similar across vaccinated groups at 9 DPI
(Figure 5.1 A and C), it is possible that the titer measured is comprised of varying percentages of IgM and IgG subclasses, which may differentially contribute to neutralization and overall control of infection. Presence of a higher ratio of IgM to neutralizing IgG could be detrimental in overall protection conferred.
Additionally, though we did not detect IL-1β, IL-4, IL-6, TNFα, or IFNγ in the serum at 4, 9, or 13 DPI (data not shown), it is possible that these cytokines are being upregulated locally in the draining lymph node or would be detectable at earlier time points (24-48 hours) following immunization. We did find evidence of systemic secretion of IL-12p40 in all vaccinated mice at 4 and 9 DPI, and in Combination Nanovaccine and saline mice at 13 DPI (Figure 5.3).
IL-12p40, a component of IL-12p70 and IL-23, serves as a macrophage chemoattractant. IL-12p70 and IL-23 have been shown to promote IFNγ production by
CD8 T cells in vivo and mediate inflammatory responses and macrophage accumulation in lung tissue, indicating a possible key role in control of pulmonary infections21. It is possible that the presence of IL-12p40 observed in the serum at 9 and 13 DPI could be contributing to larger production of local IFNγ, though none was detectable systemically. 139
Evaluation of cytokines in the draining lymph node at these same timepoints could allow further insight into the correlates of protection involved in this early protective response.
Neutralization of TNFα and IFNγ significantly decreased the efficacy of
Combination Nanovaccine immunization against challenge at 14 DPI (Figure 5.2 B).
Consistent with these findings, previous work from Szaba et al. has shown that neutralization of TNFα and IFNγ, prior to challenge, significantly impairs survival induced by intranasal vaccination22. While neutralization of either TNFα or IFNγ reduced survival significantly, depletion of CD8+ T cells prior to challenge did not have as pronounced of an effect, suggesting that other cell types could be contributing to the production of these cytokines and overall protection22.
In order to evaluate other cell types that may be contributing to protection and production of pro-inflammatory cytokines, it may be of interest to perform flow cytometry on draining lymph node cell populations at these early time points.
Additionally, analysis of the bronchioalveolar lavage fluid between 10-14 DPI for presence of antigen-specific IgG could offer insight into the neutralizing capacity of antibodies induced by various vaccine formulations.
Though highly variable, the induction of protective immunity in as little as 10 DPI is promising for application in biodefense or outbreak scenarios. Further research into the quality of the humoral immune response and possible contributions that trained innate immunity provide during early DPI by these vaccine candidates may provide insight into co-adjuvant approaches to vaccine design. 140
References
1. Parent, M. A. et al. Gamma interferon, tumor necrosis factor alpha, and nitric oxide synthase 2, key elements of cellular immunity, perform critical protective functions during humoral defense against lethal pulmonary Yersinia pestis infection. Infect. Immun. 74, 3381–3386 (2006).
2. Parent, M. A. et al. Cell-mediated protection against pulmonary Yersinia pestis infection. Infect. Immun. 73, 7304–7310 (2005).
3. Lukaszewski, R. A. et al. Pathogenesis of Yersinia pestis infection in BALB/c mice: Effects on host macrophages and neutrophils. Infect. Immun. 73, 7142–7150 (2005).
4. Smiley, S. T. Immune defense against pneumonic plague. Immunological Reviews 225, 256–271 (2008).
5. Li, B. & Yang, R. Interaction between Yersinia pestis and the host immune system. Infect. Immun. 76, 1804–1811 (2008).
6. Pechous, R. D., Sivaraman, V., Price, P. A., Stasulli, N. M. & Goldman, W. E. Early host cell targets of Yersinia pestis during primary pneumonic plague. PLoS Pathog. 9, e1003679 (2013).
7. Cassone, A. The case for an expanded concept of trained immunity. MBio 9, e00570-18 (2018).
8. Song, W. M. & Colonna, M. Immune training unlocks innate potential. Cell 172, 3–5 (2018).
9. Netea, M. G., Quintin, J. & Van Der Meer, J. W. M. Trained immunity: A memory for innate host defense. Cell Host and Microbe 9, 355–361 (2011).
10. Rusek, P., Wala, M., Druszczyńska, M. & Fol, M. Infectious agents as stimuli of trained innate immunity. International Journal of Molecular Sciences 19, (2018).
11. Cheng, S.-C. et al. mTOR- and HIF-1 -mediated aerobic glycolysis as metabolic basis for trained immunity. Science (80-. ). 345, 1250684–1250684 (2014).
12. Quintin, J. et al. Candida albicans infection affords protection against reinfection via functional reprogramming of monocytes. Cell Host Microbe 12, 223–232 (2012).
13. Kik, S. V. et al. Mycobacterial growth inhibition is associated with trained innate immunity. J. Clin. Invest. 128, 1837–1851 (2018). 141
14. Netea, M. G., Blok, B. A., van Crevel, R., Benn, C. S. & Arts, R. J. W. Trained innate immunity as underlying mechanism for the long-term, nonspecific effects of vaccines. J. Leukoc. Biol. 98, 347–356 (2015).
15. Saeed, S. et al. Epigenetic programming of monocyte-to-macrophage differentiation and trained innate immunity. Science (80-. ). 345, 1251086– 1251086 (2014).
16. Torres, M. P., Vogel, B. M., Narasimhan, B. & Mallapragada, S. K. Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms. J. Biomed. Mater. Res. - Part A 76, 102–110 (2006).
17. Kipper, M. J., Wilson, J. H., Wannemuehler, M. J. & Narasimhan, B. Single dose vaccine based on biodegradable polyanhydride microspheres can modulate immune response mechanism. J. Biomed. Mater. Res. Part A 76A, 798–810 (2006).
18. Haughney, S. L., Ross, K. a., Boggiatto, P. M., Wannemuehler, M. J. & Narasimhan, B. Effect of nanovaccine chemistry on humoral immune response kinetics and maturation. Nanoscale 6, 13770–13778 (2014).
19. Ulery, B. D. et al. Design of a protective single-dose intranasal nanoparticle-based vaccine platform for respiratory infectious diseases. PLoS One 6, 1–8 (2011).
20. Torres, M. P. et al. Polyanhydride microparticles enhance dendritic cell antigen presentation and activation. Acta Biomater. 7, 2857–64 (2011).
21. Cooper, A. M. & Khader, S. A. IL-12p40: an inherently agonistic cytokine. Trends Immunol. 28, 33–38 (2007).
22. Szaba, F. M. et al. TNFα and IFNγ but not perforin are critical for CD8 T cell- mediated protection against pulmonary Yersinia pestis infection. PLoS Pathog. 10, e1004142 (2014).
142
CHAPTER 6. DISCUSSION
The development of high-quality novel vaccines is contingent not only upon their ability to be efficacious and provide protective immunity, but also on their potential for application. While many vaccines are capable of conferring protection in healthy individuals, general population protection still largely relies on herd immunity. The potential for a vaccine to induce protective immune responses in individuals who are typically “low-responders” or with immunological deficiencies would allow for a larger impact on global health. An ideal vaccine candidate should be efficacious in as few doses as possible, allowing for decreased cost associated with vaccination and better patient compliance. Additionally, an ideal vaccine formulation should be shelf-stable above refrigeration temperatures reducing the necessity for cold-chain storage.
The use of combinatorial adjuvants, such as AS04 (MPLA and alum) or
Combination Nanovaccine (polyanhydride nanoparticles and CDNs), may allow for the synergistic activation of many different aspects of the immune response. Antigen-specific humoral responses were induced by Combination Nanovaccine immunization, in as little as 10 DPI (Figure 5.1) and maintained through 218 DPI (Figure 3.3A and Figure 3.4).
Characterization of the humoral response induced by immunization with Combination
Nanovaccine or CDN Vaccine indicate no significant differences in overall magnitude of total antigen-specific IgG titer (Figure 3.2, 3.3, and 3.4), epitope specificity (Figure 3.7), presence of long-lived antibody-secreting cells (Figure 3.8), or isotype class-switching
(Figure 3.5) between formulations, however, differences in protection were observed at varying challenge doses (Figure 3.2). This varying level of protection observed between the two formulations, at 14 DPI, indicates that the humoral immune response may not be 143 the sole correlate of protection against Yersinia pestis pulmonary infections.
Further research into the quality of the humoral immune response at early time points, prior to 14 DPI, may provide further insight into the mechanisms of action conferring protection following immunization with Combination Nanovaccine formulation. Evaluation of germinal center formation, antigen-specific IgG in the bronchoalveolar fluid, and antibody-secreting cell presence in the spleen and lymph node at these early time points may highlight key differences between the early humoral immune response induced by each formulation. Additionally, the quality of the early humoral response in the draining lymph node, may be predictive of a durable, long-lived response.
Significantly decreased efficacy of Combination Nanovaccine immunization was observed at 14 DPI following neutralization of TNFα and IFNγ 24 hours prior to Y. pestis challenge (Figure 5.2 B). This data indicates that these pro-inflammatory cytokines play a critical/contributing role in the acute protective immune response following immunization. Additional research into the cell types involved in production of TNFα and IFNγ could allow for better understanding of the cellular and innate immune responses rapidly induced following immunization.
Immunization with Combination Nanovaccine formulation was also found to induce protective immunity at both early (14 DPI, Figure 4.1) and late (120 DPI, Figure
4.2) time points in C57BL/6NHsdENV mice, which exhibited decreased overall humoral responses as compared to wild type (Figure 4.3) and may have had a mutation in the
Dock2 gene. While Dock2 mutations are rare, many other disorders and deficiencies exist which exhibit lowered humoral responses to vaccination1–3. The potential for combined 144 adjuvants (CDNs and polyanhydride nanoparticles) in the Combination Nanovaccine formulation to overcome possible immunological deficiencies, makes it a promising vaccine candidate for use in the general population.
Storage of polyanhydride-based nanovaccine formulation encapsulating PspA, for
60 days at 25˚C, showed similar efficacy to more traditional cold-stored vaccine adjuvanted with alum (Figure 2.4). Removal of the soluble bolus from this formulation allowed for the storage of a dry powder formulation that was capable of maintaining efficacy and providing protection against lethal S. pneumoniae challenge, similar to those that were stored at freezer temperatures. This ability of the nanovaccine formulation to maintain efficacy following storage at temperatures above refrigeration, allows for a shift away from cold chain storage, currently recommended for all licensed vaccines4. Moving away from cold storage could be extremely beneficial in helping to facilitate easier dissemination of a vaccine, maintenance of vaccine efficacy, and decrease in cost associated with vaccine distribution and storage4–6.
The use of an efficacious single-dose vaccine is beneficial, not only from the standpoint of patient compliance and cost, but also the humoral response. In humans, it has recently been suggested that repeat immunizations may push isotype class-switching to IgG4, which is a poor opsonin7. This switch in antibody production, following repeat boosters, could be detrimental against pathogens where the primary correlate of protection is opsonizing antibody. Additionally, patient compliance remains a large concern when considering vaccination strategies, as over two thirds of all cases of non- compliance in children (age 19-35 months) are due to missed doses or mis-timed boosters8. Previous studies using F1-V have shown success in protection against lethal Y. 145 pestis challenge in macaques, guinea pigs, and mice, using a prime boost regimen9. It would be of interest, in future studies, to evaluate the potential for our Combination
Nanovaccine to provide protection in a single-dose using these expanded animal models.
Immunization with single-dose nanovaccine formulations against Y. pestis and S. pneumoniae, described herein, induced protective immunity against lethal challenge at both early (14 DPI) and late (180 DPI) timepoints following immunization, and maintained efficacy following room temperature shelf-storage. These characteristics, along with those previously described (i.e. tailored payload release kinetics10, protein shelf stability11, adjuvanticity12,13, biocompatibility14, low injection reactogenicity12, dose-sparing capabilities15), highlight the potential of polyanhydride nanoparticles as a promising next-generation adjuvant and vaccine delivery platform.
References
1. Landrum, M. L. et al. Hepatitis B vaccine responses in a large U.S. military cohort of HIV-infected individuals: another benefit of HAART in those with preserved CD4 count. Vaccine 27, 4731–8 (2009).
2. Kernéis, S. et al. Long-term immune responses to vaccination in HIV-infected patients: A systematic review and meta-analysis. Clin. Infect. Dis. 58, 1130–1139 (2014).
3. Moulding, D. A., Record, J., Malinova, D. & Thrasher, A. J. Actin cytoskeletal defects in immunodeficiency. Immunol. Rev. 256, 282–99 (2013).
4. Thakker, Y. & Woods, S. Storage of vaccines in the community: weak link in the cold chain? BMJ 304, 756–758 (2009).
5. Robertson, J., Franzel, L. & Maire, D. Innovations in cold chain equipment for immunization supply chains. Vaccine 35, 2252–2259 (2017).
6. Hanson, C. M., George, A. M., Sawadogo, A. & Schreiber, B. Is freezing in the 146
vaccine cold chain an ongoing issue? A literature review. Vaccine 35, 2127–2133 (2017).
7. Diavatopoulos, D. A. & Edwards, K. M. What Is Wrong with Pertussis Vaccine Immunity? Cold Spring Harb. Perspect. Biol. 9, a029553 (2017).
8. Luman, E. T., Shaw, K. M. & Stokley, S. K. Compliance with Vaccination Recommendations for U.S. Children. Am. J. Prev. Med. 34, 463–470.e2 (2008).
9. Quenee, L. E., Ciletti, N. A., Elli, D., Hermanas, T. M. & Schneewind, O. Prevention of pneumonic plague in mice, rats, guinea pigs and non-human primates with clinical grade rV10, rV10-2 or F1-V vaccines. Vaccine 29, 6572– 6583 (2011).
10. Torres, M. P., Vogel, B. M., Narasimhan, B. & Mallapragada, S. K. Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms. J. Biomed. Mater. Res. - Part A 76, 102–110 (2006).
11. Petersen, L. K., Phanse, Y., Ramer-Tait, a. E., Wannemuehler, M. J. & Narasimhan, B. Amphiphilic polyanhydride nanoparticles stabilize bacillus anthracis protective antigen. Mol. Pharm. 9, 874–882 (2012).
12. Huntimer, L. et al. Evaluation of Biocompatibility and Administration Site Reactogenicity of Polyanhydride-Particle-Based Platform for Vaccine Delivery. Adv. Healthc. Mater. 2, 369–378 (2013).
13. Haughney, S. L., Ross, K. a., Boggiatto, P. M., Wannemuehler, M. J. & Narasimhan, B. Effect of nanovaccine chemistry on humoral immune response kinetics and maturation. Nanoscale 6, 13770–13778 (2014).
14. Vela-Ramirez, J. E. et al. Safety and Biocompatibility of Carbohydrate- Functionalized Polyanhydride Nanoparticles. AAPS J. 17, 256–67 (2015).
15. Wagner-Muñiz, D. A., Haughney, S. L., Kelly, S. M., Wannemuehler, M. J. & Narasimhan, B. Room Temperature Stable PspA-Based Nanovaccine Induces Protective Immunity. Front. Immunol. 9, 325 (2018). 147
APPENDIX: DOCK2 MUTATION PRESENCE IN ENVIGO FACILITIES