Weifeng Zhang

Immune responses to vaccines involving a combined antigen–nanoparticle mixture and nanoparticle-encapsulated antigen formulation

Many physicochemical characteristics significantly influence the adjuvant effect of micro/nanoparticles; one critical factor is the kinetics of antigen exposure to the immune system by particle-adjuvanted vaccines. Here, we investigated how various antigen-nanoparticle formulations impacted antigen exposure to the immune system and the resultant antigen-specific immune responses. We formulated antigen with poly(lactic-co-glycolic acid) (PLGA) nanoparticles by encapsulating antigen within nanoparticles or by simply mixing soluble antigen with the nanoparticles. Our results indicated that the combined formulation (composed of antigen encapsulated in nanoparticles and antigen mixed with nanoparticles) induced more powerful antigen-specific immune responses than each single-component formulation. Mice immunized with the combined vaccine formulation displayed enhanced induction of antigen-specific IgG antibodies with high avidity, increased cytokine secretion by splenocytes, and improved generation of memory T cell. Enhanced immune responses elicited by the combined vaccine formulation might be attributed to the antigen-depot effect at the injection site, effective provision of both adequate initial antigen exposure and long-term antigen persistence, and efficient induction of dendritic cell (DC) activation and follicular helper T cell differentiation in draining lymph nodes. Understanding the effect of antigen-nanoparticle formulations on the resultant immune responses might have significant implications for rational vaccine design.

pH-Responsive Poly(D,L-lactic-co-glycolic acid) Nanoparticles with Rapid Antigen Release Behavior Promote Immune Response.

In the quest to treat intracellular infectious diseases and virus infection, nanoparticles (NPs) have been considered to be efficient tools for inducing potent immune responses, specifically cellular immunity. Antigen processing and presenting by antigen presenting cells (APCs) could influence immune response, especially the priming of T-cell-mediated cellular immunity. Here, we fabricated pH-responsive poly(D,L-lactic-co-glycolic acid) (PLGA) NPs with rapid antigen intracellular release behavior in APCs. The NPs, which had thin shells and large inner space, contain ammonium bicarbonate (NH4HCO3), which could regulate release in endosomes and lysosomes, acting as an antigen release promoter in dendritic cells (DCs), and were coencapsulated with antigen (ovalbumin, OVA). Hydrogen ions (H(+)) in DC endosomes and lysosomes (pH ∼5.0 and 6.5) could react with NH4HCO3 to generate NH3 and CO2, which broke NPs and released antigens. After uptake by DCs, antigens encapsulated in pH-responsive PLGA NPs could escape from lysosomes into the cytoplasm and be cross-presented. Moreover, the NPs induced up-regulation of co-stimulatory molecules and stimulated cytokine production. Mouse immunization with pH-responsive PLGA NPs induced greater lymphocyte activation, more antigen-specific CD8(+) T cells, stronger cytotoxic capacity (IFN-γ and granzyme B), enhanced antigen-specific IgG antibodies, and higher serum IgG2a/IgG1, indicating cellular immunity. The NPs also improved generation of memory T cells to protect against reinfection. Thus, pH-responsive PLGA NPs, which induced strong cellular immune responses and offered antibody protection, could be potentially useful as effective vaccine delivery and adjuvant systems for the therapy of intracellular infectious diseases and virus infection.

Surface hydrophobicity of microparticles modulates adjuvanticity

Polymeric microparticles are promising adjuvants and they exhibit various physicochemical characteristics that can regulate the immune response, including hydrodynamic size, morphology, and surface properties, among others. Surface hydrophobicity is also a key microparticle characteristic, but how it affects microparticle adjuvanticity remains unknown. To study the correlation between microparticle hydrophobicity and adjuvanticity in-depth, we prepared poly(d,l-lactic acid) (PLA)-, poly(d,l-lactic-co-glycolic acid) (PLGA)-, and poly(monomethoxypolyethylene glycol-co-d,l-lactide) (mPEG-PLA, PELA)-based microparticles by premix membrane emulsification, which were similar in size and morphology but differed in surface hydrophobicity. We then systematically evaluated their ability to induce immune responses in vitro and in vivo. Increased surface hydrophobicity on PLA-based microparticles greatly promoted antigen internalization into dendritic cells (DCs) as well as MHC II and CD86 expression on DCs in vitro. Similarly, in vivo studies showed that increased microparticle surface hydrophobicity significantly elevated cytokine secretion levels by splenocytes harvested from vaccinated mice. Adhesion force measurements confirmed that increased surface hydrophobicity enhanced the physical interaction between microparticles and cell membranes, a condition favorable for promoting microparticle internalization into cells. Taken together, these results indicated that microparticle hydrophobicity is an important factor that determines the magnitude of immune responses elicited by vaccination with different particulate systems.

Enhanced humoral and cell-mediated immune responses generated by cationic polymer-coated PLA microspheres with adsorbed HBsAg.

Surface-engineered particulate delivery systems for vaccine administration have been widely investigated in experimental and clinical studies. However, little is known about charge-coated microspheres as potential recombinant subunit protein antigen delivery systems in terms of adsorption and related immune responses. In the present study, cationic polymers, including chitosan (CS), chitosan chloride (CSC), and polyethylenimine (PEI), were used to coat PLA microspheres to build positively charged surfaces. Antigen adsorption capacity was enhanced with increased surface charge of coated microspheres. In macrophages, HBsAg adsorbed on the surface of cationic microspheres specifically enhanced antigen uptake and augmented CD86, MHC I, and MHC II expression and IL-1β, IL-6, TNF-α, and IL-12 release. Antigens were more likely to localize independent of lysosomes after phagocytosis in antigen-attached cationic microsphere formulations. After intraperitoneal immunization, cationic microsphere-based vaccine formulations generated a rapid and efficient humoral immune response and cytokine release as compared with aluminum-adsorbed vaccine and free antigens in vivo. Moreover, microspheres coated with cationic polymers with relatively high positive charges and higher antigen adsorption exhibited strong stimulation of the Th1 response. In conclusion, PLA microspheres coated with cationic polymers may be a potential recombinant antigen delivery system to induce strong cell and humoral immune responses.

Comparison of PLA Microparticles and Alum as Adjuvants for H5N1 Influenza Split Vaccine: Adjuvanticity Evaluation and Preliminary Action Mode Analysis

PurposeTo compare the adjuvanticity of polymeric particles (new-generation adjuvant) and alum (the traditional and FDA-approved adjuvant) for H5N1 influenza split vaccine, and to investigate respective action mode.MethodsVaccine formulations were prepared by incubating lyophilized poly(lactic acid) (PLA) microparticles or alum within antigen solution. Antigen-specific immune responses in mice were evaluated using ELISA, ELISpot, and flow cytometry assay. Adjuvants’ action modes were investigated by determining antigen persistence at injection sites, local inflammation response, antigen transport into draining lymph node, and activation of DCs in secondary lymphoid organs (SLOs).ResultsAlum promoted antigen-specific humoral immune response. PLA microparticles augmented both humoral immune response and cell-mediated-immunity which might enhance cross-protection of influenza vaccine. With regard to action mode, alum adjuvant functions by improving antigen persistence at injection sites, inducing severe local inflammation, slightly improving antigen transport into draining lymph nodes, and improving the expression of MHC II on DCs in SLOs. PLA microparticles function by slightly improving antigen transport into draining lymph nodes, and promoting the expression of both MHC molecules and co-stimulatory molecules on DCs in SLOs.ConclusionsConsidering the adjuvanticity and side effects (local inflammation) of both adjuvants, we conclude that PLA microparticles are promising alternative adjuvant for H5N1 influenza split vaccine.

Microspheres and Microcapsules for Protein Delivery: Strategies of Drug Activity Retention

With the recent progress in biotechnology and genetic engineering, a variety of proteins have formed a very important class of therapeutic agents. However, most proteins have short half-lives in vivo requiring multiple treatments to provide efficacy. In order to overcome this limitation, sustained release systems as hydrophilic microspheres and hydrophobic microcapsules have received extensive attention in recent years. As therapeutic proteins delivery systems, it is necessary to maintain protein bioactivity during microspheres or microcapsules formation as much as possible. This paper reviews different influencing factors that are closely involved in protein denaturation during the preparation of hydrophilic polymer microspheres and hydrophobic polymer microcapsules. The various strategies usually employed for overcoming these obstacles are described in detail. Both processing and formulation parameters can be modified for improving protein stability. The maximum or full protein stability retention within the microspheres or microcapsules might be achieved by individual or combined optimized strategies. In addition, the common techniques for proteins stability determination are also briefly reviewed.

Engineering biomaterial-associated complement activation to improve vaccine efficacy.

The complement system plays an important role in innate and adaptive immunity, which suggests that complement activation could be exploited as a potential strategy for vaccine adjuvants. Here we explored the potential of chitosan-based microparticles (CS-NH2 MPs) as a vaccine adjuvant with an active surface for complement activation due to the abundance of amino groups. In vaccination studies, using recombinant anthrax protective antigen as a model antigen, compared with the control microparticles (amino-cross-linked MPs), we found that microparticles (MPs) with abundant amino groups significantly enhanced higher antigen-specific IgG titers in vivo and enhanced the production of IL-4 and IFN-γ with ex vivo restimulation. Furthermore, proliferative responses of splenocytes to ex vivo antigen restimulation were enhanced following immunization with MPs with amino groups. Overall, these results indicated that CS-NH2 MPs with a high surface density of amino groups were favorable for complement activation and immune responses. Our data provide further design principles for studies on complement-activating MPs as a vaccine platform.

Immunopotentiator-Loaded Polymeric Microparticles as Robust Adjuvant to Improve Vaccine Efficacy

PurposeAdjuvants are required to ensure the efficacy of subunit vaccines. Incorporating molecular immunopotentiators within particles could overcome drawbacks of molecular adjuvants (such as solubility and toxicity), and improve adjuvanticity of particles, achieving stronger adjuvant activity. Aim of this study is to evaluate the adjuvanticity of immunopotentiator-loaded polymeric particles for subunit vaccine.MethodsPLGA microparticles (PMPs) and imiquimod (TLR-7 ligand)-loaded PLGA microparticles (IPMPs) were prepared by SPG premix membrane emulsification. In vitro and in vivo studies were performed to their adjuvant activity, using ovalbumin and H5N1 influenza split vaccine as antigens.ResultsIncorporating imiquimod into microparticles significantly improved the efficacy of PLGA microparticles in activating BMDCs and pMΦs, and antigen uptake by pMΦs was also promoted. IPMPs showed stronger adjuvanticity to augment OVA-specific immune responses than PMPs. IgG subclass profiles and cytokine secretion levels by splenocytes indicated that IPMPs elicited more Th1-polarized immune response, compared to PMPs. In vivo study using H5N1 influenza split vaccine as antigen also confirmed the effects of IPMPs on antigen-specific cellular immunity.ConclusionsConsidering adjuvanticity and safety profiles (PLGA and IMQ, both approved by FDA), we conclude that IMQ-loaded PLGA microparticles are promising robust adjuvant for subunit vaccines.

Adjuvanticity Regulation by Biodegradable Polymeric Nano/microparticle Size

Polymeric nano/microparticles as vaccine adjuvants have been researched in experimental and clinical studies. A more profound understanding of how the physicochemical properties regulate specific immune responses has become a vital requirement. Here we prepared poly(d,l-lactic-co-glycolic acid) (PLGA) nano/microparticles with uniform sizes (500 nm, 900 nm, 2.1 μm, and 4.9 μm), and the size effects on particle uptake, activation of macrophages, and antigen internalization were evaluated. Particle uptake kinetic studies demonstrated that 900 nm particles were the easiest to accumulate in cells. Moreover, they could induce macrophages to secrete NO and IL-1β and facilitate antigen internalization. Furthermore, 900 nm particles, mixed with antigen, could exhibit superior adjuvanticity in both humoral and cellular immune responses in vivo, including offering the highest antibody protection, promoting the maximum secretion levels of IFN-γ and IL-4 than particles with other sizes. Overall, 900 nm might be the optimum choice for PLGA particle-based vaccine adjuvants especially for recombinant antigens. Understanding the effect of particle size on the adjuvanticity based immune responses might have important enlightenments for rational vaccine design and applications.

Electron transfer driven highly valent silver for chronic wound treatment

Although silver is widely added to various chronic wounds to kill higher concentrations (107-108 CFU mL-1) of bacteria, overdose of silver remains a major cause of diverse side effects, such as cytotoxicity and tissue and organ damage. Here we showed that reducing the dose level of silver, additionally conferring electron transfer potential, could simultaneously achieve good biocompatibility and strong bactericidal ability without introducing extra chemical residuals for chronic wound treatment. A systematic investigation demonstrated that 1 ppm trivalent silver ions performed rapid (5 min) and effective antibacterial activities against pathogens while not significantly affecting cell viability which were equivalent to 20 ppm monovalent silver ions with cytotoxicity, and accelerated the healing process and improved the tissue quality of burn wounds. The killing effect is independent of material and is mainly controlled by the electron transfer potentials of trivalent silver ions, which disrupts the electron transport of bacteria membrane respiration and leads to the death of bacteria. Together, such trivalent silver opens up new possibilities for dispelling the concern of silver usage in biosafety and provides an avenue for designing antibiotics or other biomedical applications.