Prolonged delivery of HIV-1 vaccine nanoparticles from hydrogels

Immunization is a straightforward concept but remains for some pathogens like HIV-1 a challenge. Thus, new approaches towards increasing the efficacy of vaccines are required to turn the tide. There is increasing evidence that antigen exposure over several days to weeks induces a much stronger and more sustained immune response compared to traditional bolus injection, which usually leads to antigen elimination from the body within a couple of days. Therefore, we developed a poly(ethylene) glycol (PEG) hydrogel platform to investigate the principal feasibility of a sustained release of antigens to mimic natural infection kinetics. Eight-and four-armed PEG macromonomers of different MWs (10, 20, and 40 kDa) were end-group functionalized to allow for hydrogel formation via covalent cross-linking. An HIV-1 envelope (Env) antigen in its trimeric (Envtri) or monomeric (Envmono) form was applied. The soluble Env antigen was compared to a formulation of Env attached to silica nanoparticles (Env-SiNPs). The latter are known to have a higher immunogenicity compared to their soluble counterparts. Hydrogels were tunable regarding the rheological behavior allowing for different degradation times and release timeframes of Env-SiNPs over two to up to 50 days. Affinity measurements of the VCR01 antibody which specifically recognizes the CD4 binding site of Env, revealed that neither the integrity nor the functionality of Envmono-SiNPs (Kd = 2.1 ± 0.9 nM) and Envtri-SiNPs (Kd = 1.5 ± 1.3 nM), respectively, were impaired after release from the hydrogel (Kd before release: 2.1 ± 0.1 and 7.8 ± 5.3 nM, respectively). Finally, soluble Env and Env-SiNPs which are two physico-chemically distinct compounds, were co-delivered and shown to be sequentially released from one hydrogel which could be beneficial in terms of heterologous immunization or single dose vaccination. In summary, this study presents a tunable, versatile applicable, and effective delivery platform that could improve vaccination effectiveness also for other infectious diseases than HIV-1

Dynamic Imine Bonding Facilitates Mannan Release from a Nanofibrous Peptide Hydrogel

Recently, there has been increased interest in using mannan as an immunomodulatory bioconjugate. Despite notable immunological and functional differences between the reduced (R-Man) and oxidized (O-Man) forms of mannan, little is known about the impact of mannan oxidation state on its in vivo persistence or its potential controlled release from biomaterials that may improve immunotherapeutic or prophylactic efficacy. Here, we investigate the impact of oxidation state on the in vitro and in vivo release of mannan from a biocompatible and immunostimulatory multidomain peptide hydrogel, K2(SL)6K2 (abbreviated as K2), that has been previously used for the controlled release of protein and small molecule payloads. We observed that O-Man released more slowly from K2 hydrogels in vitro than R-Man. In vivo, the clearance of O-Man from K2 hydrogels was slower than O-Man alone. We attributed the slower release rate to the formation of dynamic imine bonds between reactive aldehyde groups on O-Man and the lysine residues on K2. This imine interaction was also observed to improve K2 + O-Man hydrogel strength and shear recovery without significantly influencing secondary structure or peptide nanofiber formation. There were no observed differences in the in vivo release rates of O-Man loaded in K2, R-Man loaded in K2, and R-Man alone. These data suggest that, after subcutaneous injection, R-Man naturally persists longer in vivo than O-Man and minimally interacts with the peptide hydrogel. These results highlight a potentially critical, but previously unreported, difference in the in vivo behavior of O-Man and R-Man and demonstrate that K2 can be used to normalize the release of O-Man to that of R-Man. Further, since K2 itself is an adjuvant, a combination of O-Man and K2 could be used to enhance the immunostimulatory effects of O-Man for applications such as infectious disease vaccines and cancer immunotherapy

Biomaterial Strategies for Immunomodulation

Strategies to enhance, suppress, or qualitatively shape the immune response are of importance for diverse biomedical applications, such as the development of new vaccines, treatments for autoimmune diseases and allergies, strategies for regenerative medicine, and immunotherapies for cancer. However, the intricate cellular and molecular signals regulating the immune system are major hurdles to predictably manipulating the immune response and developing safe and effective therapies. To meet this challenge, biomaterials are being developed that control how, where, and when immune cells are stimulated in vivo, and that can finely control their differentiation in vitro. We review recent advances in the field of biomaterials for immunomodulation, focusing particularly on designing biomaterials to provide controlled immunostimulation, targeting drugs and vaccines to lymphoid organs, and serving as scaffolds to organize immune cells and emulate lymphoid tissues. These ongoing efforts highlight the many ways in which biomaterials can be brought to bear to engineer the immune system.Bill & Melinda Gates FoundationUnited States. Army Research Office. Institute for Soldier Nanotechnologies (Contract W911NF-13-D-0001)Ragon Institute of MGH, MIT and HarvardCancer Research Institute (New York, N.Y.) (Irvington Postdoctoral Fellowship)National Institutes of Health (U.S.) (Awards AI104715, CA172164, CA174795, and AI095109

Optimizing the Development of an Acetalated Dextran Microparticulate Subunit Broadly Reactive Influenza Vaccine

Despite having an approved influenza virus vaccine in place for nearly 80 years, this highly contagious respiratory virus can still cause 1 billion infections and inflicts major economic losses globally per annum. Currently licensed vaccines elicit short-lived immune responses that are strain specific. However, influenza is a rapidly mutating virus which requires annual reformulation of a vaccine that often leads to low efficacy. To improve vaccine efficacy and remove the need for annual reformulation, vaccines should be designed to induce long-lasting broad immunity. Furthermore, current vaccines do not offer any controlled delivery mechanisms when it comes to eliciting a desired immune response. To this end, the work done here utilizes a subunit vaccine strategy where adjuvants that can stimulate long-lasting immunity and antigens that can provide broad protection are evaluated. The adjuvant explored here is the cyclic guanosine-adenosine monophosphate (cGAMP) dinucleotide which can stimulate the production of type 1 interferons through the induction of the Stimulation of Interferon Genes (STING) pathway. Here, cGAMP was encapsulated into polymeric microparticles to enhance its adjuvanticity. One antigen that can provide universal protection is the conjugated matrix protein 2 ectodomain (M2e) and stable trimeric hemagglutinin (HA) stem protein (4900-M2e). Here, the humoral response was enhanced for M2e when delivered in conjugation with 4900 compared to when they were delivered nonconjugated. Another antigen explored here was the computationally optimized broadly reactive antigen (COBRA) HA. COBRA HAs for two subtypes of the influenza virus were assessed in a bivalent vaccine platform. Additionally, the delivery of the two COBRA HAs were optimized when encapsulated in polymeric microparticles to enhance the delivery of the antigen. The polymeric microparticles are composed of the biocompatible and acid-sensitive material acetalated dextran (Ace-DEX). Ace-DEX MPs were used to explore the delivery of cGAMP and COBRA HAs in various release rates to optimize and modulate the stimulation of the immune response. The work offered here demonstrates that the timing and delivery of an adjuvant and antigen can have profound effects on their protective responses.Doctor of Philosoph

Engineering synthetic vaccines using cues from natural immunity

Vaccines aim to protect against or treat diseases through manipulation of the immune response, promoting either immunity or tolerance. In the former case, vaccines generate antibodies and T cells poised to protect against future pathogen encounter or attack diseased cells such as tumours; in the latter case, which is far less developed, vaccines block pathogenic autoreactive T cells and autoantibodies that target self tissue. Enormous challenges remain, however, as a consequence of our incomplete understanding of human immunity. A rapidly growing field of research is the design of vaccines based on synthetic materials to target organs, tissues, cells or intracellular compartments; to co-deliver immunomodulatory signals that control the quality of the immune response; or to act directly as immune regulators. There exists great potential for well-defined materials to further our understanding of immunity. Here we describe recent advances in the design of synthetic materials to direct immune responses, highlighting successes and challenges in prophylactic, therapeutic and tolerance-inducing vaccines.United States. Dept. of Defense (contract W911NF-13-D-0001)United States. Dept. of Defense (contract W911NF-07-D-0004)National Institutes of Health (U.S.) (AI095109)Bill & Melinda Gates FoundationRagon Institute of MGH, MIT, and HarvardNational Institutes of Health (U.S.) (AI091693)Howard Hughes Medical Institute (Investigator)Carigest S

Nanovaccine Delivery Approaches and Advanced Delivery Systems for the Prevention of Viral Infections: From Development to Clinical Application

open access articleViral infections causing pandemics and chronic diseases are the main culprits implicated in devastating global clinical and socioeconomic impacts, as clearly manifested during the current COVID-19 pandemic. Immunoprophylaxis via mass immunisation with vaccines has been shown to be an efficient strategy to control such viral infections, with the successful and recently accelerated development of different types of vaccines, thanks to the advanced biotechnological techniques involved in the upstream and downstream processing of these products. However, there is still much work to be done for the improvement of efficacy and safety when it comes to the choice of delivery systems, formulations, dosage form and route of administration, which are not only crucial for immunisation effectiveness, but also for vaccine stability, dose frequency, patient convenience and logistics for mass immunisation. In this review, we discuss the main vaccine delivery systems and associated challenges, as well as the recent success in developing nanomaterials-based and advanced delivery systems to tackle these challenges. Manufacturing and regulatory requirements for the development of these systems for successful clinical and marketing authorisation were also considered. Here, we comprehensively review nanovaccines from development to clinical application, which will be relevant to vaccine developers, regulators, and clinicians

Molecular design of nanoparticle-based delivery vehicles for pneumonic plague

The work described in this dissertation focuses on the design of polyanhydride nanoparticles that function as both adjuvants and long-term antigen delivery vehicles in order to improve vaccination, specifically for biodefense-related applications. Chapter 1 is an introduction into the threat of bioterrorism, polymer-based controlled delivery systems and challenges associated with vaccine design. Chapter 2 is a detailed literature review of topics related to the research conducted in this dissertation. Areas covered include basic immunology, vaccine design, degradable polymer-based adjuvant engineering, plague (Yersinia pestis) biology, and vaccines that confer protection against plague. Chapter 3 overviews the research objectives and the specific aims of this work. Chapter 4 describes the effect polymer chemistry has on uptake of polyanhydride nanoparticles by THP-1 human monocytic cells. Nanoparticles of similar size, regardless of poly[1,6-bis(p-carboxyphenoxy)hexane-co-sebacic acid] (CPH:SA) copolymer chemistry, were fabricated using a novel anti-solvent precipitation technique. Confocal microscopy revealed that less hydrophobic nanoparticles (SA-rich) were more readily internalized and trafficked by monocytes. Interestingly, exposure to nanoparticles of any chemistry enhanced soluble protein uptake by monocytes over cells exposed to soluble protein alone. Chapter 5 utilizes the combination of population and individual analyses in order to better understand the effect chemistry has on nanoparticle uptake and subsequent activation of dendritic cells. Nanoparticles composed of CPH:SA and poly[1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane-co-CPH] (CPTEG:CPH) were used for this study in order to investigate a wide range of chemistries. Nanoparticles composed of less hydrophobic chemistry were able to activate cell surface marker expression where as nanoparticles composed of more hydrophobic chemistry were able to cause the enhanced secretion of cytokines. Using confocal microscopy it was determined that less hydrophobic nanoparticles were more readily internalized and degraded where as more hydrophobic nanoparticles maintained their size intracellularly. 50:50 CPTEG:CPH nanoparticles possessed characteristics of both less hydrophobic and more hydrophobic chemistries. Also, 50:50 CPTEG:CPH nanoparticles were intracellularly aggregated in vesicles which is similar to how dendritic cells treat bacteria. This pathogen-like behavior may explain the activation capacity of nanoparticles of this chemistry. Chapter 6 describes the capacity of a single-dose, antigen-loaded 50:50 CPTEG:CPH nanoparticle-based vaccine to convey long-lived protection against live Yersinia pestis challenge. While the combination of a commercial adjuvant (MPLA) or unloaded 50:50 nanoparticles with soluble antigen was able to convey some protection at 6 weeks post-vaccination, only a combination of soluble antigen with antigen-loaded 50:50 CPTEG:CPH nanoparticles was able to convey 100% protection at 6 and 23 weeks post-vaccination. For mice vaccinated with soluble antigen plus antigen-loaded nanoparticles, bacteria burden and histopathological analyses showed no presence of bacteria and no pathological damage, respectively. Chapter 7 details the conclusions of this dissertation and the future directions of this research. Antigen modification, nanoparticle optimization, novel polymer chemistry and new intracellular imaging tools are all topics covered in this chapter

Hydrogel Biomaterials for Drug Delivery: Mechanisms, Design, and Drugs

Due to their unique physical and chemical properties, hydrogels have attracted significant attention in several medical fields, specifically, drug delivery applications in which gel-based nanocarriers deliver drug molecules to the region of interest in biological organs. For different drug delivery applications, hydrogel systems can be manipulated to provide passive and/or active delivery. Thus, several drug targeting, loading, and releasing mechanisms have been devised and reported in the literature. This chapter discusses these mechanisms and their efficacy with respect to different drug delivery applications. Furthermore, the drug dosage is dependent on the design and shape of the hydrogel systems, which in turn depend on the route of the drug administration. This chapter covers the types of hydrogel-based products applied via different routes of drug administration. Lastly, this chapter addresses different classifications of delivered drugs including small molecular weight drugs; therapeutic proteins and peptides; and vaccines

PHASE-SEPARATING MICROBUBBLES FUNCTIONING AS VACCINE DEPOTS

Failure in receiving a booster for specific vaccines contributes to incomplete seroconversion, particularly in the developing world. Single injection vaccine technology could potentially be a solution such that health care personnel would not need to meet patients multiple times at designated points in time thereafter. The main challenge for single injection vaccine systems to date is controlling the stability of the antigen. to maintain the antigenic protein structure while in the physiological environment. We engineered a novel phase-separating microbubble technology which could function as an injectable depot which we hypothesize will enable us to control the microenvironment of the antigen for the durations required, in addition to controlling the antigen release time. We have successfully accomplished the following Main Specific Aims and subaims: Main Specific Aim 1: Synthesize polymers for microbubbles formation and Engineer methods for stabilizing Microbubbles: 1A: Synthesize PCL and PLGA library at different molecular weights and characterizing the polymers 1B: Synthesize acrylate polymers for microbubbles 1C: Engineer stable microbubble through UV cure and lyophilization 1D: Engineering the microbubbles to be stationary for maintaining sphere shape during the curing process and the inject of the cargo 1E: Engineering the cargo to be stationary within the polymeric microbubble to maximize the release time 1F: Quantify the diameter of the microbubble by varying syringe pump rate and comparing diameter pre- and post-lyophilization 1G: Quantify the angle of the micromotor for injecting cargo into the center of the microbubbles 1H: Engineer a self-contained lyophilization-capable system for the microbubbles Main Specific Aim 2: Engineering cargo release time of the microbubbles: 2A: Quantify how different molecular weights of PCL affect release time of the microbubbles 2C: Quantify how varying the microbubbles’ thickness of the shell controls the release time Main Specific Aim 3: Quantify stability of HIV and Hepatitis B antigens: 3A: Quantify how the HIV gp120/41 and HBsAg ayw antigens are stable in time in an aqueous environment versus in a cryo-protectant context at varying temperatures Our novel phase-separating technology which can form microbubble vaccine depots is a promising method to alleviate stability issues which hinders the single injection vaccine field. Enhancement of antigen stability in the microbubbles will be determined in future work