Padma Malyala

Uptake of particulate vaccine adjuvants by dendritic cells activates the NALP3 inflammasome

Many currently used and candidate vaccine adjuvants are particulate in nature, but their mechanism of action is not well understood. Here, we show that particulate adjuvants, including biodegradable poly(lactide-co-glycolide) (PLG) and polystyrene microparticles, dramatically enhance secretion of interleukin-1β (IL-1β) by dendritic cells (DCs). The ability of particulates to promote IL-1β secretion and caspase 1 activation required particle uptake by DCs and NALP3. Uptake of microparticles induced lysosomal damage, whereas particle-mediated enhancement of IL-1β secretion required phagosomal acidification and the lysosomal cysteine protease cathepsin B, suggesting a role for lysosomal damage in inflammasome activation. Although the presence of a Toll-like receptor (TLR) agonist was required to induce IL-1β production in vitro, injection of the adjuvants in the absence of TLR agonists induced IL-1β production at the injection site, indicating that endogenous factors can synergize with particulates to promote inflammasome activation. The enhancement of antigen-specific antibody production by PLG microparticles was independent of NALP3. However, the ability of PLG microparticles to promote antigen-specific IL-6 production by T cells and the recruitment and activation of a population of CD11b+Gr1− cells required NALP3. Our data demonstrate that uptake of microparticulate adjuvants by DCs activates the NALP3 inflammasome, and this contributes to their enhancing effects on innate and antigen-specific cellular immunity.

Rational design of small molecules as vaccine adjuvants

Small-molecule immune potentiators can be engineered to be potent adjuvants with localized innate immune activation and short in vivo residence times. Better Adjuvants Through Chemistry Vaccine development has come a long way since Jenner first noticed that cowpox protected against smallpox. And yet, many vaccines do not work well alone; adjuvants are included with the vaccine to boost the immune response. Despite the critical role of adjuvants in vaccine efficacy, new adjuvant development has been empirical. Now, Wu et al. report the rational optimization of small-molecule immune potentiators (SMIPs) as adjuvants. These SMIPs were engineered to have limited bioavailability and remain localized, inducing temporally and spatially restricted inflammation. This systematic approach to optimizing adjuvant properties may allow for improved immune responses to vaccines with fewer side effects. Adjuvants increase vaccine potency largely by activating innate immunity and promoting inflammation. Limiting the side effects of this inflammation is a major hurdle for adjuvant use in vaccines for humans. It has been difficult to improve on adjuvant safety because of a poor understanding of adjuvant mechanism and the empirical nature of adjuvant discovery and development historically. We describe new principles for the rational optimization of small-molecule immune potentiators (SMIPs) targeting Toll-like receptor 7 as adjuvants with a predicted increase in their therapeutic indices. Unlike traditional drugs, SMIP-based adjuvants need to have limited bioavailability and remain localized for optimal efficacy. These features also lead to temporally and spatially restricted inflammation that should decrease side effects. Through medicinal and formulation chemistry and extensive immunopharmacology, we show that in vivo potency can be increased with little to no systemic exposure, localized innate immune activation and short in vivo residence times of SMIP-based adjuvants. This work provides a systematic and generalizable approach to engineering small molecules for use as vaccine adjuvants.

Enhancing the therapeutic efficacy of CpG oligonucleotides using biodegradable microparticles

Oligonucleotides, with specific sequence surrounding CpG motifs, appear to be very effective for the induction of a potent Th1 responses. This molecule represents pathogen-associated molecular patterns (PAMPs) that allows the pathogen recognition receptors (PRRs) present on innate immune cells to recognize them and become activated. PAMPs and related compounds are often labelled as immunopotentiators, allowing a clear distinction between them and particulate delivery systems such as emulsions, liposomes, virus-like particles and microparticles. Microparticles prepared from biodegradable, biocompatible polyesters, and poly (lactide co-glycolide) (PLG). They have been proven to be a good particulate delivery system for the co-delivery of antigens and adjuvants. PLG has been used in humans for many years as a resorbable suture material and controlled-release drug delivery systems. It has been demonstrated that antigen presenting cells (APCs) efficiently uptake the PLG microparticles ( approximately 1 microm) both in vivo and in vitro. After uptake, the PLG subsequently induces an antigen specific CTL response in rodents. Several groups, including our group, have evaluated CpG as an immunopotentiator in various formulations and delivery systems (i.e. emulsions and particulate systems). This review will discuss in detail the work conducted so far with CpG using PLG microparticles as a delivery system. We will also discuss the advantages and enhancement of immune properties of formulating CpG (soluble, adsorbed, and encapsulated forms) with PLG microparticles along with future directions for these microparticles with CpG.

Vaccine adjuvant formulations: A pharmaceutical perspective

Formulation science is an unappreciated and often overlooked aspect in the field of vaccinology. In this review we highlight key attributes necessary to generate well characterized adjuvant formulations. The relationship between the adjuvant and the antigen impacts the immune responses generated by these complex biopharmaceutical formulations. We will use 5 well established vaccine adjuvant platforms; alum, emulsions, liposomes, PLG, and particulate systems such as ISCOMS in addition to immune stimulatory molecules such as MPL to illustrate that a vaccine formulation is more than a simple mixture of component A and component B. This review identifies the challenges and opportunities of these adjuvant platforms. As antigen and adjuvant formulations increase in complexity having a well characterized robust formulation will be critical to ensuring robust and reproducible results throughout preclinical and clinical studies.

Polylactide-co-glycolide microparticles with surface adsorbed antigens as vaccine delivery systems.

Several groups have shown that vaccine antigens can be encapsulated within polymeric microparticles and can serve as potent antigen delivery systems. We have recently shown that an alternative approach involving charged polylactide co-glycolide (PLG) microparticles with surface adsorbed antigen(s) can also be used to deliver antigen into antigen presenting cell (APC). We have described the preparation of cationic and anionic PLG microparticles which have been used to adsorb a variety of agents, which include plasmid DNA, recombinant proteins and adjuvant active oligonucleotides. These PLG microparticles were prepared using a w/o/w solvent evaporation process in the presence of the anionic surfactants, including DSS (dioctyl sodium sulfosuccinate) or cationic surfactants, including CTAB (hexadecyl trimethyl ammonium bromide). Antigen binding to the charged PLG microparticles was influenced by several factors including electrostatic and hydrophobic interactions. These microparticle based formulations resulted in the induction of significantly enhanced immune responses in comparison to alum. The surface adsorbed microparticle formulation offers an alternative and novel way of delivering antigens in a vaccine formulation.

Analysis of immunoglobulin transcripts and hypermutation following SHIV(AD8) infection and protein-plus-adjuvant immunization.

Developing predictive animal models to assess how candidate vaccines and infection influence the ontogenies of Envelope (Env)-specific antibodies is critical for the development of an HIV vaccine. Here we use two nonhuman primate models to compare the roles of antigen persistence, diversity and innate immunity. We perform longitudinal analyses of HIV Env-specific B-cell receptor responses to SHIVAD8 infection and Env protein vaccination with eight different adjuvants. A subset of the SHIVAD8-infected animals with higher viral loads and greater Env diversity show increased neutralization associated with increasing somatic hypermutation (SHM) levels over time. The use of adjuvants results in increased ELISA titres but does not affect the mean SHM levels or CDR H3 lengths. Our study shows how the ontogeny of Env-specific B cells can be tracked, and provides insights into the requirements for developing neutralizing antibodies that should facilitate translation to human vaccine studies.

β7-integrin-independent enhancement of mucosal and systemic anti-HIV antibody responses following combined mucosal and systemic gene delivery

Vaccination strategies that can block or limit heterosexual human immunodeficiency virus (HIV) transmissions to local and systemic tissues are the goal of much research effort. Herein, in a mouse model, we aimed to determine whether the enhancement of antibody responses through mucosal and systemic immunizations, previously observed with protein‐based vaccines, applies to immunizations with DNA‐ or RNA‐based vectors. Intranasal (i.n.) followed by intramuscular (i.m.) immunizations (i.n./i.m.) with polylactide‐coglycolide (PLG)‐DNA microparticles encoding HIV‐gag (PLG‐DNA‐gag) significantly enhanced serum antibody responses, compared with i.m., i.n. or i.m. followed by i.n. (i.m./i.n.) immunizations. Moreover, while i.n./i.m., i.n. or i.m./i.n. immunizations with PLG‐DNA‐gag resulted in genital tract antibody responses, i.m. immunizations alone failed to do so. Importantly, β7‐deficient mice developed local and systemic antibody responses following i.n./i.m. immunization, or immunization via any other route, similar to those of wild‐type mice. To compare the DNA with an RNA delivery system, immunizations were performed with VEE/SIN‐gag replicon particles, composed of Venezuelan equine encephalitis virus (VEE) replicon RNA and Sindbis surface structure (SIN). i.n./i.m., compared with any other immunizations, i.n./i.m. immunization with VEE/SIN‐gag resulted in enhanced genital tract but not serum antibody responses. These data show for the first time that mucosal followed by systemic immunizations with gene delivery systems enhance B‐cell responses independent of the mucosal homing receptors α4β7 and αEβ7.

Effect of the strength of adsorption of HIV 1 SF162dV2gp140 to aluminum-containing adjuvants on the immune response

The importance of the strength of antigen adsorption by aluminum-containing adjuvants on immunopotentiation was studied using HIV 1 SF162dV2gp140 (gp140), a potential HIV/AIDS antigen. The strengths of adsorption by aluminum hydroxide (AH) adjuvant and aluminum phosphate adjuvant, as measured by the Langmuir adsorptive coefficient, were 1900 and 400 mL/mg, respectively. The strength of adsorption by AH was modified by pretreatment of AH with two different concentrations of potassium dihydrogen phosphate to produce phosphate-treated aluminum hydroxide adjuvants having adsorptive coefficients of 1200 and 800 mL/mg. The four adjuvants were used to prepare vaccines containing either 1 or 10 μg of gp140 per dose. Antibody studies in mice revealed that the presence of an adjuvant increased the immune response in comparison with a solution of gp140 when the dose was 1 μg. Furthermore, the immune response was inversely related to the adsorptive coefficient. In contrast, no significant difference in immunopotentiation was observed between treatments in the presence or absence of an adjuvant when the dose of gp140 was 10 μg. Analysis of the binding of gp140 to CD4 and anti-gp140 monoclonal antibodies by surface plasmon resonance suggests that tight binding induced structural changes in the antigen.

Physicochemical and functional characterization of vaccine antigens and adjuvants

As novel vaccine antigens and adjuvants are being tested in humans, understanding of critical quality attributes essential for eliciting optimal vaccine response and vaccine antigen–adjuvant interactions is pivotal for vaccine safety and eliciting ‘protective’ immune responses. Therefore, the efforts to better characterize and evaluate vaccine antigen and antigen–adjuvant drug products need to begin very early during the discovery and development phase. In this review, we discuss the importance of characterization of physicochemical and functional properties in vaccine antigen, adjuvant and the final antigen–adjuvant drug product and emphasize the greater need for more extensive understanding of vaccine antigen–adjuvant interactions. We highlight the key parameters and quality attributes that are critical to measure during preclinical and clinical testing of the vaccine and discuss in some detail the technologies, and their limitations, used in analyzing the key physicochemical and functional attributes of vaccine antigen and antigen–adjuvant drug product.

Innate transcriptional effects by adjuvants on the magnitude, quality, and durability of HIV envelope responses in NHPs

Adjuvants have a critical role for improving vaccine efficacy against many pathogens, including HIV. Here, using transcriptional RNA profiling and systems serology, we assessed how distinct innate pathways altered HIV-specific antibody responses in nonhuman primates (NHPs) using 8 clinically based adjuvants. NHPs were immunized with a glycoprotein 140 HIV envelope protein (Env) and insoluble aluminum salts (alum), MF59, or adjuvant nanoemulsion (ANE) coformulated with or without Toll-like receptor 4 (TLR4) and 7 agonists. These were compared with Env administered with polyinosinic-polycytidylic acid:poly-L-lysine, carboxymethylcellulose (pIC:LC) or immune-stimulating complexes. Addition of the TLR4 agonist to alum enhanced upregulation of a set of inflammatory genes, whereas the TLR7 agonist suppressed expression of alum-responsive inflammatory genes and enhanced upregulation of antiviral and interferon (IFN) genes. Moreover, coformulation of the TLR4 or 7 agonists with alum boosted Env-binding titers approximately threefold to 10-fold compared with alum alone, but remarkably did not alter gene expression or enhance antibody titers when formulated with ANE. The hierarchy of adjuvant potency was established after the second of 4 immunizations. In terms of antibody durability, antibody titers decreased ∼10-fold after the final immunization and then remained stable after 65 weeks for all adjuvants. Last, Env-specific Fc-domain glycan structures and a series of antibody effector functions were assessed by systems serology. Antiviral/IFN gene signatures correlated with Fc-receptor binding across all adjuvant groups. This study defines the potency and durability of 8 different clinically based adjuvants in NHPs and shows how specific innate pathways can alter qualitative aspects of Env antibody function.