Biomaterials-Based Vaccination Strategies for the Induction of CD8+T

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Materials-based Vaccination Strategies for the Induction of CD8+T cell Responses Charles B. Chesson, Shaunte Ekpo-Otu, Janice J. Endsley, and Jai S Rudra ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00412 • Publication Date (Web): 18 Oct 2016 Downloaded from http://pubs.acs.org on October 25, 2016

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ACS Biomaterials Science & Engineering

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Biomaterials-based Vaccination Strategies for the Induction of CD8+T cell Responses

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Charles B. Chesson1,3, Shaunte Ekpo-Otu1,3, Janice J. Endsley2,3, Jai S. Rudra1,3

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Department of Pharmacology & Toxicology, 2Department of Microbiology & Immunology, Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, TX 77555 3

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Key Words: biomaterial, CD8+T cell, cellular immunity, polymer, vaccine

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Address correspondence and reprint requests to Jai S. Rudra, Ph.D. Department of Pharmacology and Toxicology, Sealy Center for Vaccine Development, 301 University Blvd, Basic Sciences Building 3.308, Galveston, TX 77555. E-mail address: [email protected]

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ACS Paragon Plus Environment


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Abstract

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Natural and synthetic biomaterials are increasingly being used for the development of vaccines

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and immunotherapies as alternatives to traditional live-attenuated formulations due to their

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improved safety profiles and no risk of reversion to virulence. Polymeric materials in particular

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enjoy attention due to the ease of fabrication, control over physicochemical properties, and their

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wide range of immunogenicity. While the majority of studies focus on inducing protective

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antibody responses, in recent years, materials-based strategies for the delivery of antigens and

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immunomodulators to improve CD8+T cell immunity against infectious and non-infectious

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diseases has gained momentum. Notably, platforms based on polymeric nanoparticles,

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liposomes, micelles, virus-like particles, self-assembling peptides and peptidomimetics, and

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multilayer thin films show considerable promise in preclinical studies. In this review article, we

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first introduce the concepts of CD8+T cell activation, effector and memory functions, and

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cytotoxic activity followed by vaccine design for eliciting robust and protective long-lived

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CD8+T cell immunity. We then discuss different materials-based vaccines developed in the last

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decade to elicit CD8+T cell responses based on molecular composition or fabrication methods

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and conclude with a summary and glimpse of the future trends in this area.

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1. Introduction

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Programming the immune system through active vaccination to protect against disease

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has had an unparalleled impact on public health in the last century1. Although currently used

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vaccines are successful at preventing a variety of human diseases via induction of long-lasting

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antibody responses, a number of major hurdles remain2-4. Specifically, attempts at vaccinating

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against malaria, HIV, tuberculosis, hepatitis C, and cancer, are more problematic because host

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protection against these diseases requires the generation of quality memory CD8+T cells4.

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CD8+T cells possess the ability to specifically recognize malignant cells or cells infected

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with pathogens, leading to activation and differentiation into memory and effector cells which

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then exert cytotoxic effector functions to eliminate them5,6. The empirical strategies employed to

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generate antibody and CD4+T cell-mediated immunity through vaccination are clearly

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inadequate for targeting potent CD8+T cell recall. The unresolved challenges for development of

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CD8+T cell-based vaccines against HIV, despite a decade of intense research efforts, highlight

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this point and underscore the need for rational design2,7. A back-to- basics approach is needed

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that incorporates the current state of knowledge for how antigen-specific CD8+T cell memory is

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activated. Immunization strategies must target transcriptional programs in naïve CD8+T cells that

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are activated by endogenously presented antigen in the context of optimized environmental

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stimuli. Biomaterials have great potential to not only deliver antigen through the appropriate

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antigen processing pathways, but also to provide the environmental context that is difficult to

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achieve outside of live vaccines. The challenge for novel immunization strategies is to safely

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drive differentiation of CD8+T cells toward memory subsets programmed for rapid expansion

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and cytotoxic effector function upon subsequent pathogen exposure.

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2.

CD8+T cell Memory Development and Effector Function: Raising the Army

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Protective immunity by CD8+T cells following natural infection is due to generation of

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long-lived memory populations that patrol tissue and peripheral sites. The mechanisms regulating

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CD8+T cell activation and memory differentiation have been the subject of several thorough

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reviews, and thus here we present an overview relevant to vaccine design8-10. After infection,

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CD8+T cells recognize endogenous antigen presented through major histocompatibility complex

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(MHC) class I by antigen-presenting cells (APCs). Optimal responses occur via T cell interactions

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with APCs that have undergone maturation programming which augments antigen processing and

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presentation machinery, expression of co-stimulatory and tissue migration receptors and ligands,

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and production of pro-inflammatory cytokines. Priming of naïve T cells is most efficiently carried

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out by dendritic cells (DC) while memory recall responses can be activated by other APCs such as

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macrophages. Activation of transcriptional programs that direct differentiation of memory and

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function require three important signals through APC and T cell interactions: 1) presentation of

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MHC I-bound peptide antigen to the cognate T cell receptor (TCR); 2) binding of co-stimulatory

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molecules (especially CD28 and B7), and 3) production of, and activation through, pro-

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inflammatory cytokines. The strength and nature of these three signals direct the magnitude,

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phenotype, function, and tissue localization of antigen-specific CD8+T cell populations that persist

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as memory pools (Fig. 1).

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Following priming, memory populations are identified based on relative expression of

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surface markers and transcription factors associated with defined functions or characteristics.

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Mouse memory CD8+T cell populations can be classified into effector or central memory

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subsets, based on relative expression of surface markers including CD11a, CD62L, and CD12711.

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Further assessment of killer cell lectin-like receptor G1, KLRG1, and CD27 molecules, as well

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as the transcription factors Eomesodermin or (eomes), and Tbet, allows assignment to short lived

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effector cell (SLEC) in which the ratio of Tbet to Eomes is high and decreases as differentiation

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progresses into memory precursor effector cell (MPEC), both of which have critical roles in

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disease clearance and long term protective immunity.12,13. Human CD8+T cell memory

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populations can be similarly assigned to effector, or central memory populations based on

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relative expression of CD45RA and CCR7 with supporting evidence provided by expression of

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CD127 and CD62L14,15. Patterns of CD27, CD28, Eomes, and Tbet expression further identify

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effector and late effector memory cell populations that are analogous to murine SLEC and

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MPECs, respectively16-18. The relative proportion of SLEC and MPEC populations generated by

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immunization are important predictors of the development of long-lived memory pools that form

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the basis for protective immunity.

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Effector functions mediated by CD8+T cells are an important feature of the protective recall

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response and can also serve as a correlate or surrogate of vaccine efficacy. Polyfunctional cytokine

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production (IL-2, TNF-α, and IFN-γ) is an important prognostic indicator of memory cell function

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for both CD4+ and CD8+T cells. The primary effector molecules used by CD8+T cells to limit

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infection, however, are IFN-γ and cytotoxic proteins19. IFN-γ induces an antimicrobial state in

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infected cells, restricting microbial proliferation through multiple innate mechanisms and

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activating APC antigen presentation and co-stimulatory function. The granule exocytosis pathways

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that occur following synapse-directed release of lytic molecules against infected targets primarily

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mediate cytotoxicity by CD8+T cells. The individual cytotoxic proteins (e.g. perforin, granzyme

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B, and in humans, granulysin) stored in the lytic granules of CTLs exert pathogen-specific effector

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functions and are differentially regulated by activation stimuli. Perforin functions to increase

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membrane permeability of target cells and facilitates killing by other cytotoxic proteins20.

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Granzymes promote clearance and limit inflammation by inducing apoptosis of infected or

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transformed cells21. Granulysin is an important part of the CTL armament of non-rodent species

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and mediates direct anti-microbial activity against multiple pathogens22-26.

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During natural infection, effector and effector memory T cell populations primarily carry

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out cytotoxic functions6,27,28. Both effector T cells (Te) and effector memory (Tem) cells display

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immediate cytotoxic capabilities in response to antigen, or cytotoxicity promoting cytokines (e.g.

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IL-2, IL-12), in the absence of antigen. Central memory T cells (Tcm) primarily reside/recirculate

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through the lymph nodes, and function to rapidly proliferate and generate effector T cells upon

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secondary antigenic encounters.

Tissue resident memory (Trm) CD8+T cells patrol sites of

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pathogen entry (e.g. lung, gut) that respond rapidly due to early detection of infection. The goal for

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vaccination is the generation of memory pools, though the subset, functional repertoire, and tissue

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homing properties best suited to protect the host will vary by pathogen.

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3.

Materials-based CD8+T cell Vaccine Development

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A key issue in vaccine development is balancing immunogenicity with safety; vaccine

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safety gets more public attention than vaccine efficacy29. Many of the successful vaccines we

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have today are based on live-attenuated or inactivated pathogens or live vectors such as non-

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replicating bacteria or viruses expressing subunit antigens that induce robust humoral and

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cellular immunity30. However, anti-vector immunity and safety concerns associated with live

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vectors complicate their use in infants, the elderly and in those with compromised immunity. At

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the other end of the spectrum, recombinant antigens based on purified subunit proteins and

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peptides hold high potential for CD8+T cell vaccine development against intracellular pathogens

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and cancer, but their co-dependency on adjuvants has remained a significant barrier to clinical

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translation31-33. Due to the toxicity and reactogenicity associated with most plant- or pathogen-

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derived adjuvants currently under development, those clinically approved in the U.S. are limited

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to alum (aluminum hydroxide) and alum-based derivatives, which elicit strong humoral

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responses but poor CD8+T cell responses33,34.

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In the last decade, cross-fertilization of ideas across the fields of materials science,

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nanotechnology, and immunology has laid the foundation for the emerging new field of

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‘immunobioengineering’, which aims to design materials to both manipulate the immune system

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and better understand it35-37. Biomaterials, both natural and synthetic, are attractive platforms for

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the development of CD8+T cell vaccines due to their intrinsic immunomodulatory properties and

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chemical versatility for further modification with immune signals that polarize T cells for short-

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term effector function and long-term memory responses37-39. The generation of protective

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immunity after vaccination is dependent on various factors amongst which, antigen availability,

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delivery of the vaccine to antigen-presenting cells, and activation of innate immunity are key.

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Versatility in the design and engineering of materials offers a distinct advantage, where

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activation of appropriate innate immune pathways, spatial and temporal control of antigen

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release, targeted delivery for enhanced cross-presentation, and polarizing the profile of cytokines

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produced can be controlled not only for optimal induction of effector CD8+T cell responses and

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immunological memory against infectious diseases and cancers, but also to suppress them in

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autoimmunity35,37. Therefore, optimism is high for the potential of materials-based vaccination

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strategies to overcome the current limitations of live-attenuated formulations and combined with

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future advances in cellular and molecular immunology, will lead to the development of rationally

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designed CD8+T cell vaccines that protect against pathogens and cancers. In this review, we

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introduce the reader to the development of novel materials-based vaccines intent on generating

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protective immunity through CD8+T cell expansion and memory cell formation in the last

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decade and discuss the current challenges and future expectations.

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3.1

Liposomes and Lipid-based Materials

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Liposomes are dispersions of lamellar liquid crystals that have been extensively studied

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as drug and vaccine carriers due to their ability to deliver a wide range of molecules by either

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surface-adsorption, direct conjugation, integration within the lipid bilayer, or encapsulation

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within the aqueous core40. They are a useful platform for sustained and targeted release of

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antigens and immunomodulatory molecules. Studies demonstrate that conjugation of peptide

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antigens to the liposome surface, or encapsulation of antigens within liposomes, leads to uptake

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by antigen-presenting cells via pinocytosis, enhances cross-presentation and CD8+T cell

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responses, and induces cellular immunity that protects against disease41.

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The choice of lipids used in liposome preparation significantly affects their physical

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properties and immunological activity and cationic liposomes are becoming increasingly favored

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over neutral and anionic liposomes for CD8+T cell vaccine development42. This is due to their

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efficient uptake by antigen-presenting cells and induction of cross-presentation of antigens

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through the MHC-I pathway43. Liposomal cationic adjuvant formulation 01 (CAF01) is a novel

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two-component liposomal adjuvant system composed of a cationic liposome vehicle

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(dimethyldioctadecyl-ammonium (DDA)) stabilized with a glycolipid immunomodulator

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(trehalose 6,6-dibehenate (TDB)), a component of the mycobacterial cell wall44,45. In addition to

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acting as an immunomodulator, TDB also ensures long-term stability of the DDA liposomes.

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Vaccinating humans with CAF01 liposomes bearing antigens specific to Mycobacterium

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tuberculosis, led to the activation of CD4+T cells with a Th1 cytokine bias measured out to 3

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years post vaccination suggesting a robust memory immune response46. However, when TDB

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was replaced with monomycoloyl glycerol (MMG)-1, a synthetic analogue of a mycobacterial

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cell wall lipid, and combined with polyI:C, an agonist for the innate immune receptor toll-like

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receptor 3 (TLR3) known as formulation 09 (CAF09) liposomes, were shown to induce antigen-

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specific CD8+T cells and reduce the growth of already established subcutaneous E7-expressing

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TC-1 tumors in approximately 40% of mice. Within the same study in a corresponding

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prophylactic model, 100% of the mice were protected (n=8)47. In addition to CD8+T cell

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expansion, other studies investigated vaccine formulations based on the cationic lipid DOTAP

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(1,2-dioleoyl-3-trimethylammonium-propane) and reported that cationic liposomes induced

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higher levels of reactive oxygen species in DCs compared to neutral liposomes, which may in

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part be a driving mechanism behind the immune response48. Mechanistic studies revealed that

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DOTAP liposomes also upregulated the levels of chemokines CCL2, CCL3 and CCL4 in DC

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cultures, and CCL2 induction was mediated through the extracellular-signal-regulated kinase

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(ERK) pathway, which was confirmed using specific inhibitors of the ERK pathway and siRNA

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approaches49 facilitating rational design of CD8+T cell vaccines based on cationic lipids.

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Vaccination with antigenic peptides linked to DOTAP has been shown to induce robust CD8+T

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cell responses leading to protective immunity against infectious cancers50. Owing to their charge

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properties, cationic liposomes also serve as excellent carriers for plasmid DNA antigens and

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complexing negatively charged TLR agonists (TLR3 and TLR9) to activate innate immunity and

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powerful therapeutic T cell responses against cancer47,51. Zaks et al. demonstrated this with

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liposome-Ag-nucleic acid complexes (LANAC) to elicit CD8+T cell responses against cancer

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and tuberculosis. Antigen-specific CD8+T cells elicited by LANAC liposomes were found to be

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functionally active and to persist for a long time in the tissues of vaccinated animals52. Notably,

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LANAC containing TLR3 or TLR9 agonists effectively cross-primed CD8+T cell responses even

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when low doses of antigens were used. This effect was independent of CD4+T cell help making

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the platform attractive for vaccination in immunocompromised individuals. A number of other

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cationic liposomal and lipid-polymer hybrid particles have been shown to induce potent cell-

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mediated immunity with associated protection from viral or bacterial challenge, or therapeutic

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reduction or elimination of tumors. Fan et al. reported on a liposome-hyaluronic acid (HA)

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hybrid nanoparticle which incorporated the cationic lipids 1,2-dioleoyl-3-trimethylammonium

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propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) with the anionic

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HA polymer and surface conjugated PEG53. These polymer-coated liposomes were found to have

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increased stability, solubility, and bioavailability and induced a balanced Th1/Th2 response with

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significant expansion of antigen-specific CD8+T using both the model antigen OVA, and F1-V, a

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recombinant fusion of two key Yersinia pestis antigens53.

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While most liposomes are unilamellar and comprised of a single lipid bilayer, Irvine and

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co-workers pioneered the development of multi-lamellar liposomal vaccines stabilized via inter-

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bilayer cross-linking, called inter-bilayer cross-linked multi-lamellar vesicles (ICMVs) (Figure

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2A, 2B)54. ICMVs were found to be significantly more efficient at antigen encapsulation

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compared to traditional liposomes or PLGA nanoparticles and elicited robust CD8+T cell

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responses when injected into mice. The multilayer structure of ICMVs also facilitates the

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sequestration of lipophilic adjuvants within the lipid bilayers or on the surface, and addition of

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MPLA (TLR4 agonist) to ICMVs had a more striking effect on the CD8+ T-cell response to

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vaccination. Interestingly, in terms of the degree of antigen-specific T-cell expansion, persistence

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of memory cells, and IFN-γ functionality, this is one of the strongest endogenous T-cell

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responses ever reported for a protein vaccine, comparable to strong live vectors such as

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recombinant viruses54. While most liposomal formulations are tested via parenteral

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administration, local Trm CD8+T cell memory populations play an important protective role at

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mucosal sites of pathogen entry. Mucosal immunity is best induced via vaccination through

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mucosal surfaces and injected vaccines typically elicit poor mucosal immunity55. Furthermore,

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the microenvironment and cellular populations of mucosal lymphoid tissues differ from

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peripheral lymph nodes and are therefore distinct as an inductive site for priming of acquired

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immune responses56. Li et al. investigated the delivery of ICMVs via mucosal routes and

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demonstrated that pulmonary vaccination with ICMVs leads to longer antigen persistence in the

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lungs and draining lymph nodes (Figure 2C) and higher frequency of T cells expressing the

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mucosal homing integrin α4β7 (Figure 2D)57. Following recall, strong CD8+T cell responses in

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the lungs, draining lymph nodes, spleen, and peripheral blood were observed and induced

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disseminated effector memory-biased immunity (Figure 2E, 2F). Protective efficacy following

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pulmonary vaccination with ICMVs was demonstrated in both therapeutic tumor (Figure 2G) and

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prophylactic viral challenge models (Figure 2H)57. In a novel approach, Nguyen et al. utilized

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large-scale screening to identify lipids capable of complexing with immunostimulatory RNA to

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form nanoparticles termed ‘lipidoids’. These lipidoid-RNA nanoparticles were shown to be

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effective at enhanced delivery of the cargo and distinct from conventional liposomes in their

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endosomal retention and cell targeting properties and induced strong antigen-specific CD8+T cell

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responses in mice.

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Other lipid-based biomaterials currently gaining popularity for eliciting CD8+T cell

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responses are cubosomes58, bacteriosomes59, and virosomes60. These liposome-based

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nanostructures are composed of highly ordered lipid bilayers with intercalated bacterial or viral

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envelope proteins. Cubosomes are particles formed through solvent evaporation of phyantriol,

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pluronic F127, and propylene glycol, and have a unique nanostructure composed of a highly

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twisted lipid bilayer and two non-intersecting water channels. When compared to the simple

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liposomes, cubosomes offer increased encapsulation of antigens and adjuvants due to the high

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surface area imparted by the greater proportion of lipid comprising the particle61. Rizwan et al.

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demonstrated cubosomes to be effective sustained release vehicles for antigens and TLR agonists

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to induce CD8+T cell immune responses62. Bacteriosomes based on Escherichia coli (E. coli) are

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a novel unilamellar liposome modality prepared from membrane phospholipids isolated from E.

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coli bacterial membranes63. These ‘escheriosomes’ readily fuse with cell membranes,

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presumably due to their higher content of anionic phospholipids, to deliver encapsulated

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antigens, which are processed for MHC class I presentation. Escheriosome-based delivery of

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parasitic antigens has been demonstrated to elicit cellular immunity in animal models without the

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need for added adjuvants64. Virosomes are viral liposomes synthesized through the inclusion of

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purified viral envelope proteins into a mixture of liposomes comprised of phosphatidylcholine

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(PC), phosphatidylethanolamine (PE) and other viral lipids. Virosomes lack the infectious

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genetic material of viruses but retain their cell entry and membrane fusion characteristics and are

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taken up by cells via receptor-mediated endocytosis65. Encapsulation of peptide antigens inside

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the liposomal vectors has been shown to be an effective strategy to elicit strong CD8+T cell

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responses directed against infectious diseases as well as cancer models, and virosomes based on

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influenza proteins have been registered for use in humans66.

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3.2

Polymeric Micelles

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Polymeric micelles (PMs) consist of a core and shell structure formed in an aqueous

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solution containing amphiphilic block copolymers typically composed of a water-soluble

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segment and an ionic segment67. Poly(ethylene glycol) (PEG) is most commonly the water

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soluble segment since it is FDA approved, however poly(N-vinyl-2-pyrrolidone) (PVP) and

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poly(acrylic acid) (PAA) are frequently used as alternatives67. Micellar preparations with ionic

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segments composed of poly(propylene oxide) (PPO)68, poly(lactic acid) (PLA)69, poly(amino

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acids)70,71, PLGA, and polyethyleneamine (PEI)72 have been described.. When the concentration

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of the block copolymer increases above the critical micelle concentration (CMC), the

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hydrophobic segments start to associate leading to the formation of micelles typically in the

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range of tens to hundreds of nanometers in size. Micelles remain in equilibirium where

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individual monomers can escape from the micelles and insert their tails into other hydrophobic

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environments, such as other micelles, or cell membranes. The hydrophobic inner core serves to

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encapsulate drugs and other biologics whereas the outer shell or corona protects the drug from

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the aqueous environment. Their small size and pegylated surface make them less susceptible to

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renal clearance and recognition in vivo by the reticuloendothelial system73. Furthermore,

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incorporating chemistries into block copolymers that sense their surrounding milieu and respond

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to stimuli can lead to the design of ‘smart micelles’ for enhanced biological performance such as

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intracellular or endosomal release of the payload74,75. All of these advantages related to PMs

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have led to their application as drug delivery systems for efficient encapsulation of drugs,

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controlled release in vivo, controlled tissue distribution, and intracellular trafficking. This in turn

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laid the foundation for PM-based antigen and adjuvant delivery for CD8+T cell vaccine

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development76.

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Stayton and colleagues first reported antigen delivery with poly(propylacrylic acid)

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(PPAA) conjugation for enhancing MHC class I presentation and T-cell activation. PPAA carrier

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particles in attractive electrostatic association with cationic poly(dimethylaminoethyl

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methacrylate) (PDMAEMA) were shown to have increased endosomolytic activity resulting in

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intracellular accumulation of antigen and significantly increased antigen-specific cells in the

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spleen with one dose77. Using poly(propylacrylic acid-co-pyridyldisulfide acrylate) (PPAA-

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PDSA) block copolymers, the model antigen ovalbumin was conjugated by disulfide exchange to

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make reversible conjugates that could be reduced by the glutathione redox system in the cytosol

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of antigen-presenting cells77. A more recent study reported the synthesis of a pH-responsive

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neutral micellar carrier consisting of N-(2-hydroxypropyl) methacrylamide corona block with

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pendent pyridyl disulfide groups for reversible conjugation of thiolated ovalbumin and 2-(N,N-

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diethylamino)ethyl methacrylate (DEAEMA) and butyl methacrylate (BMA) as an

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endoosmolytic core75. The diblock copolymers self-assembled into 25-30 nm diameter micellar

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nanoparticles and the membrane-interactive properties of the pH-responsive core block enhanced

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cytosolic delivery, promoted uptake by antigen-presenting cells in the draining lymph nodes,

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increased cross-presentation of conjugated OVA, and significantly enhanced antigen-specific

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CD8+ T cell responses in vivo. Wilson and co-workers built on this original design to synthesize

13

linear, hyperbranched, and cross-linked copolymers of DEAEMA and BMA using reversible

14

addition-fragmentation chain transfer (RAFT) polymerization78. The copolymer was chain

15

extended with a hydrophilic N,N-dimethylacrylamide (DMA) segment bearing thiol-reactive

16

pyridyl disulfide (PDS) groups linked to a thiolated protein antigen, ovalbumin. In aqueous

17

solution, the polymer chains assembled into 25 nm micellar nanoparticles, with the

18

hyperbranched and cross-linked polymer architectures exhibiting significantly higher hemolysis

19

at acidic pH (< 7.0) than the linear diblock. OVA delivery with the hyperbranched and cross-

20

linked polymer led to a 5-fold increase in MHC class I presentation relative to the cross-linked

21

architecture and the block co-polymer had no discernable effect compared to soluble OVA78.

22

These studies demonstrate the superior capacity of PM-based vaccination systems at enhancing

14


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1

cross-presentation and CD8+T cell responses and that MHC class I presentation can be

2

modulated via micellar architectures.

3

Hubbell and co-workers developed a poly(ethylene glycol) (PEG)-bl-poly(propylene

4

sulfide) (PPS) block copolymer that self-assembles into 25–35 nm micelles under aqueous

5

conditions and preliminary studies showed that these micelles were able to travel to draining

6

lymph nodes, where they preferentially interacted with APCs68. A new polymer was synthesized

7

with a terminal pyridyl disulfide (PDS) for conjugation of OVA. Mice vaccinated with a

8

combination of OVA-conjugated micelles and CpG (TLR9 agonist) adjuvant had 2.4-fold higher

9

levels of antigen-specific CD8+T cells in their peripheral blood. In addition, higher (1.7-fold)

10

levels of interferon-gamma from splenocytes were observed upon re-stimulation compared to

11

mice immunized with non-conjugated OVA and CpG68. Studies focusing on phagocytosis and

12

antigen-presentation in DCs demonstrated a link between the capacity of DCs to cross-present

13

antigen and the extended presence of an oxidative environment within their endosomes79. To

14

exploit this, Evan et al. reported the synthesis of self-assembled vesicular aggregates of block

15

copolymers called ‘polymerosomes’ with emphasis on oxidation-sensitive chemistries rather

16

than pH- or reduction-sensitive chemistries as NOX2-dependent reactive oxygen species have

17

been shown to be present in DCs in early endosomes and lysosomes80. Using the block co-

18

polymer PEG17-bl-PSS30 and thin-film rehydration methods, uni- and multi-lamellar

19

polymerosomes were synthesized for the delivery of adjuvants (TLR7 and TLR8 agonists) and

20

antigens (ovalbumin) to endosomal compartments of DCs80. In vitro studies demonstrated that

21

the polymerosomes released OVA in an oxidative environment. Co-culture assays using splenic

22

DCs and OT-I CD8+T cells (transgenic T cells specific to OVA) showed that OVA-loaded

23

polymersomes resulted in greater CD8+T cell proliferation and also a higher percentage of

15



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Page 16 of 56

1

CD8+T cells expressed IFN-γ compared to soluble controls80. Stano et al. compared cellular

2

immune responses induced by PPS-bl-PEG polymersomes (encapsulated antigen) with that of

3

PEG-stabilized PPS nanoparticles (NPs, solid-core structures with antigen conjugated to the

4

surface). Interestingly, antigen-conjugated NPs induced stronger CD8+T cell responses compared

5

to antigen-loaded polymerosomes, whereas antigen-loaded polymerosomes induced enhanced

6

frequencies of antigen-specific CD4+T cells compared to the NP formulation81. This study

7

highlights the potential of using different antigen-delivery systems for tuning cell-mediated

8

responses. Following this finding, Nembrini et al. investigated mucosal vaccination efficacy of

9

OVA-conjugated PPS nanoparticles that when administered in the lungs of mice admixed with

10

the immunostimulatory oligonucleotide CpG as an adjuvant. Strong CD8+T cell responses were

11

detected both in the lung and in the spleen and protected mice from challenge with influenza

12

virus encoding a CD8+ T-cell epitope from OVA, demonstrating strong CTL-mediated

13

protection82. Interestingly, conjugation of both OVA and CpG to PSS nanoparticles, as opposed

14

to simple mixing, induced a more potent cellular response at an early time, characterized by

15

increased degranulation and secretion of IFN-γ by CD8+ T cells and enhanced proportion of

16

multifunctional CD4+ T cells, at CpG doses as low as 4 µg83. Recalling the effector responses 60

17

days after vaccination led to enhanced clonal expansion of antigen-specific memory CD8+ T

18

cells and diminished the growth of B16-F10-OVA melanoma83. To overcome the limitations of

19

covalent attachment of vaccine components on polymer-based nanoparticles, Brubaker et al.

20

developed cationic micelles through self-assembly of a polyarginine-conjugated poly(ethylene

21

glycol)-b-poly(propylene sulfide) (PEG–PPS) diblock copolymer amphiphiles84. Antigen (OVA)

22

and adjuvant (MPLA and CpG) loading into the micelles was mediated by non-covalent

23

molecular encapsulation and electrostatic complexation. Micelle-mediated co-delivery of OVA

16


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1

antigen induced strong cellular responses with significantly increased populations of IFNγ+,

2

TNFα+, and polyfunctional IFNγ+ TNFα+ producing CD8+T cells. However, cytotoxic function

3

was not investigated in this study84.

4

Luo et al. reported self-assembled cationic micelles based on poly(ethylene glycol)-b-

5

poly(L-lysine)-b-poly(L-leucine) (PEG-PLL-PLLeu) hybrid polypeptides with high antigen

6

loading capacity and stability, and addition of poly I:C (TLR3 agonist) synergistically

7

augmented tumor specific cytotoxic T lymphocyte response70. In a novel approach, Huang and

8

coworkers developed PEG-CUR micelles as sensitizers against B16F10 melanoma tumors owing

9

to the mild anti-tumor effects of curcumin85. A Trp2-specific peptide vaccine containing calcium

10

phosphate, phosphopeptide, and CpG adjuvant encapsulated in an asymmetric lipid

11

membrane was used to synergize the anti-tumor responses. Administration of CUR–PEG and

12

Trp2 vaccine resulted in a 7-fold increase in CD8+T cell responses and IFN-γ production leading

13

to significant antitumor effects as compared to individual treatments. The combination therapy

14

significantly down-regulated immunosuppressive factors and enhanced pro-inflammatory

15

cytokines and numbers of CD8+T cells in the tumor microenvironment. A distinct M2 to M1

16

macrophage phenotype switch in the treated tumors was also observed85. In contrast to strategies

17

that rely on loading soluble antigens and adjuvants into micellar structures, Tirrell and co-

18

workers reported facile synthesis of amphiphilic micelles based on peptide-lipid conjugates86. A

19

dialkyl tail with two palmitic acid chains (DiC16) was conjugated to a cytotoxic T cell epitope

20

from the model tumor antigen ovalbumin. DiC16-OVA amphiphiles self-assembled into

21

cylindrical micelles ~ 8.0 nm in diameter with polydisperse length, and were able to elicit robust

22

prophylactic and therapeutic CD8+T cell immunity in vaccinated mice leading to tumor

17



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1

regression86. This study demonstrates that the assembly of antigens into micelles can impart self-

2

adjuvanting properties.

3

3.3

Page 18 of 56

Polymeric and Inorganic Nanoparticles

4

As the focus of vaccine development shifts from traditional whole cell vaccines to less

5

immunogenic “minimalist” compositions based on subunit antigens, strategies that boost

6

immunogenicity are required for enhancing vaccine efficacy. The use of nanoparticles for

7

formulating subunit vaccines allows not only improved antigen stability and immunogenicity,

8

but also targeted delivery and sustained release87. Furthermore, it is possible to functionalize the

9

nanoparticles with antibodies, peptides, aptamers, or polysaccharides for targeted delivery to

10

specific cells or organs87. Depending on their size and nature and the targeted cell type,

11

nanoparticles enter cells by passive penetration, or clathrin- or caveolin-mediated endocytosis,

12

macropinocytosis, or by phagocytosis88. Nanoparticle shape, size, and charge have been shown

13

to influence their distribution, kinetics, intracellular processing and subsequently their

14

immunogenicity36,90-92. For example, smaller nanoparticles (25 nm) can enter draining lymph

15

nodes via the lymphatic system whereas larger nanoparticles are mostly associated with dendritic

16

cells in the site of injection93. Numerous nanoparticle-based systems have been explored for co-

17

delivery of antigens with adjuvants leading to activation of innate immunity and targeted

18

delivery of cargo antigen-specific cells via surface modification strategies. This has enabled

19

rational vaccine design for the optimal expression of B and T cell effector function and the

20

differentiation and maintenance of antigen-specific memory CD4+ and CD8+T cells94.

21

Nanoparticle is a broad term used to describe particulates or molecular entities that are typically

22

between 1-100 nm in diameter fabricated from a variety of biological, inorganic, and hybrid

23

materials. The application of nanoparticles in vaccine delivery has been summarized in recent

18


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1

reviews87 and here we limit our discussion to nanoparticles fabricated from synthetic polymers

2

and inorganic nanoparticles.

3

Compared to natural polymers, nanoparticles fabricated from synthetic polymers have

4

unique advantages with respect to control over physicochemical properties such as rate of

5

degradation, permeability, and stimulus sensitivity. Nanoparticle formulations based on synthetic

6

polymers such as poly(lactic-co-glycolic acid) (PLGA)95,96, poly(γ-glutamic acid) (γ-PGA)97,

7

poly(styrene) (PS)98, poly(anhydride) (PA), poly(caprolactone) (PCL), and polyesters (PE) have

8

been the most extensively investigated for encapsulation and sustained or targeted delivery of a

9

variety of antigens and adjuvants and have demonstrated considerable promise in preclinical

10

models. In particular, PLGA nanoparticles have enjoyed significant attention due to their FDA-

11

approved status for use in the clinic, ease of synthesis, control over particle size and degradation

12

profiles, and versatility of surface modification for targeted delivery to antigen-presenting cells.

13

PLGA particles can be fabricated with varying size distributions, and nano-scale (

Biomaterials-Based Vaccination Strategies for the Induction of CD8+T

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