Aluminum Adjuvants-'Back to the Future'.

Aluminum-based adjuvants will continue to be a key component of currently approved and next generation vaccines, including important combination vaccines. The widespread use of aluminum adjuvants is due to their excellent safety profile, which has been established through the use of hundreds of millions of doses in humans over many years. In addition, they are inexpensive, readily available, and are well known and generally accepted by regulatory agencies. Moreover, they offer a very flexible platform, to which many vaccine components can be adsorbed, enabling the preparation of liquid formulations, which typically have a long shelf life under refrigerated conditions. Nevertheless, despite their extensive use, they are perceived as relatively 'weak' vaccine adjuvants. Hence, there have been many attempts to improve their performance, which typically involves co-delivery of immune potentiators, including Toll-like receptor (TLR) agonists. This approach has allowed for the development of improved aluminum adjuvants for inclusion in licensed vaccines against HPV, HBV, and COVID-19, with others likely to follow. This review summarizes the various aluminum salts that are used in vaccines and highlights how they are prepared. We focus on the analytical challenges that remain to allowing the creation of well-characterized formulations, particularly those involving multiple antigens. In addition, we highlight how aluminum is being used to create the next generation of improved adjuvants through the adsorption and delivery of various TLR agonists.

Multiplex Assays for Analysis of Antibody Responses to South Asian Plasmodium falciparum and Plasmodium vivax Malaria Infections.

Malaria remains a major global health challenge, causing over 0.6 million yearly deaths. To understand naturally acquired immunity in adult human populations in malaria-prevalent regions, improved serological tools are needed, particularly where multiple malaria parasite species co-exist. Slide-based and bead-based multiplex approaches can help characterize antibodies in malaria patients from endemic regions, but these require pure, well-defined antigens. To efficiently bypass purification steps, codon-optimized malaria antigen genes with N-terminal FLAG-tag and C-terminal Ctag sequences were expressed in a wheat germ cell-free system and adsorbed on functionalized BioPlex beads. In a pilot study, 15 P. falciparum antigens, 8 P. vivax antigens, and a negative control (GFP) were adsorbed individually on functionalized bead types through their Ctag. To validate the multiplexing powers of this platform, 10 P. falciparum-infected patient sera from a US NIH MESA-ICEMR study site in Goa, India, were tested against all 23 parasite antigens. Serial dilution of patient sera revealed variations in potency and breadth of antibodies to various parasite antigens. Individual patients revealed informative variations in immunity to P. falciparum versus P. vivax. This multiplex approach to malaria serology captures varying immunity to different human malaria parasite species and different parasite antigens. This approach can be scaled to track the dynamics of antibody production during one or more human malaria infections.

Merozoite surface protein 2 adsorbed onto acetalated dextran microparticles for malaria vaccination.

Malaria remains a global health threat, with significant morbidity and mortality worldwide despite current interventions. The human disease is caused by five different parasitic species, with Plasmodium falciparum being the deadliest. As a result, vaccine research against P. falciparum is a global priority. Merozoite surface protein 2 (MSP2) is a promising vaccine antigen as MSP2-specific antibodies have been shown previously to be protective against malaria infection. In this study, the formulation of an MSP2 vaccine was explored to enhance antigen uptake and achieve both an antibody and Th1 immune response by adsorbing MSP2 antigen onto a biomaterial carrier system. Specifically, MSP2 antigen was adsorbed onto acetalated dextran (Ace-DEX) microparticles (MPs). IgG and IgG2a titers elicited by the Ace-DEX MP platform were compared to titer levels elicited by MSP2 adsorbed to an FDA-approved alum adjuvant, MSP2 alone, and PBS alone. Both adsorption of MSP2 to Ace-DEX MPs and to alum elicited antibody responses in vivo, but only the formulation containing Ace-DEX MPs was able to elicit a significant Th1-biased response needed to combat the intracellular pathogen. As such, MSP2 adsorbed to Ace-DEX MPs demonstrates promise as a malaria vaccine.

Adsorption onto aluminum hydroxide adjuvant protects antigens from degradation.

Aluminum based adjuvants are widely used in commercial vaccines, since they are known to be safe and effective with a variety of antigens. The effect of antigen adsorption onto Aluminum Hydroxide is a complex area, since several mechanisms are involved simultaneously, whose impact is both antigen and formulation conditions dependent. Moreover, the mode of action of Aluminum Hydroxide is itself complex, with many mechanisms operating simultaneously. Within the literature there are contrasting theories regarding the effect of adsorption on antigen integrity and stability, with reports of antigen being stabilized by adsorption onto Aluminum Hydroxide, but also with contrary reports of antigen being destabilized. With the aim to understand the impact of adsorption on three recombinant proteins which, following in vivo immunization, are able to induce functional bactericidal antibodies against Neisseria meningitidis type B, we used a range of physico-chemical tools, such as DSC and UPLC, along with in vitro binding of antibodies that recognize structural elements of the proteins, and supported the in vitro data with in vivo evaluation in mice studies. We showed that, following exposure to accelerated degradation conditions involving heat, the recombinant proteins, although robust, were stabilized by adsorption onto Aluminum Hydroxide and retain their structural integrity unlike the not adsorbed proteins. The measure of the Melting Temperature was a useful tool to compare the behavior of proteins adsorbed and not adsorbed on Aluminum Hydroxide and to predict protein stability.

Formulation and preclinical studies with a trivalent rotavirus P2-VP8 subunit vaccine.

More effective rotavirus vaccines are essential for preventing extensive diarrheal morbidity and mortality in children under five years of age in low-resource regions. Nonreplicating rotavirus vaccines (NRRV) administered parenterally provide an alternate vaccination method to the current licensed oral vaccine. Live attenuated vaccines and may generate increased efficacy in low-resource settings because the parenteral administration route bypasses some of the challenges associated with oral administration, including differences in intestinal environments. Work described here supports development of a trivalent NRRV vaccine for parenteral administration to avoid complications of the gastrointestinal route. Recombinant VP8* subunit proteins representing some of the most prevalent strains of rotavirus infecting humans - DS-1 (P[4]), 1076 (P[6]), and Wa (P[8]) - were combined with an aluminum adjuvant and the P2 epitope of tetanus toxoid to enhance the immune response to this NRRV antigen. Vaccine formulation development included selection of aluminum hydroxide (Alhydrogel®) as an appropriate adjuvant as well as an optimal buffer to maintain antigen stability and optimize antigen binding to the adjuvant. Characterization assays were used to select the lead vaccine formulation and monitor formulation stability. The NRRV liquid formulation was stable for one year at 2°C to 8°C and four weeks at 37°C. Immunogenicity of the NRRV formulation was evaluated using a guinea pig model, where we demonstrated that the adjuvant provided a 20-fold increase in neutralization titer against a homologous antigen and that the P2-fusion also enhanced the serum neutralizing antibody responses. This vaccine candidate is currently being evaluated in human clinical trials.

Use of a quartz crystal microbalance platform to study protein adsorption on aluminum hydroxide vaccine adjuvants: Focus on phosphate-hydroxide ligand exchanges.

Aluminum hydroxide (AH) salts are widely used as vaccine adjuvants and controlling antigen-AH interactions is a key challenge in vaccine formulation. In a previous work, we have developed a quartz crystal microbalance (QCM) platform, based on stable AH-coated sensors, to explore the mechanisms of model antigen adsorption. The QCM study of bovine serum albumin (BSA) adsorption at different pH and ionic strength (I) values showed that protein adsorption on AH adjuvant at physiological pH cannot be explained mainly by electrostatic interactions, in contrast with previous reports. Here, we exploit further the developed QCM platform to investigate the role of phosphate-hydroxyl ligand exchanges in the adsorption mechanism of BSA, human serum albumin (HSA) and ovalbumin (OVA) on two commercial AH adjuvants. BSA adsorption decreased on immobilized AH particles previously treated with KH2PO4, highlighting the role of exchangeable sites on AH particles in the adsorption process. BSA and OVA were dephosphorylated by treatment with an acid phosphatase to decrease their phosphate content by about 80% and 25%, respectively. Compared to native BSA, adsorption of dephosphorylated BSA decreased significantly on one AH adjuvant at pH 7. Adsorption of dephosphorylated OVA was comparable to the one of native OVA. Further QCM assays showed that phospho-amino acids (PO4-serine and PO4-threonine) displaced previously adsorbed BSA and OVA from AH particles in conditions that were depending on the protein and the AH. Taken together, these observations suggest that phosphate-hydroxyl ligand exchange is an important adsorption mechanism of proteins on AH. These results moreover confirm that the developed AH-coated QCM sensors offer a new platform for the study of antigen adsorption, to the benefit of vaccine formulation.

Quantitative Analysis of Vaccine Antigen Adsorption to Aluminum Adjuvant Using an Automated High-Throughput Method.

Aluminum-containing adjuvants have been widely used in vaccine formulations to safely and effectively potentiate the immune response. The examination of the extent of antigen adsorption to aluminum adjuvant is always evaluated during the development of aluminum adjuvant containing vaccines. A rapid, automated, high-throughput assay was developed to measure antigen adsorption in a 96-well plate format using a TECAN Freedom EVO® (TECAN). The antigen adsorption levels at a constant adjuvant concentration for each sample were accurately measured at 12 antigen/adjuvant (w/w) formulation ratios. These measurements were done at aluminum adjuvant concentrations similar to normal vaccine formulations, unlike previous non-automated and automated adjuvant adsorption studies. Two high-sensitivity analytical methods were used to detect the non-absorbed antigens. The antigen-to-adjuvant adsorption curves were fit to a simple Langmuir adsorption model for quantitatively analyzing the antigen to the adjuvant adsorption level and strength. The interaction of two model antigens, bovine serum albumin and lysozyme, with three types of aluminum adjuvant, were quantitatively analyzed in this report. Automated, high-throughput methodologies combined with sensitive analytical methods are useful for accelerating practical vaccine formulation development.LAY ABSTRACT: Vaccines are probably the most effective public health method to prevent epidemics of many infectious diseases. Many of the most effective vaccines contain aluminum adjuvant. This report describes novel technology that can be used to better optimize the efficacy and stability of aluminum adjuvant-containing vaccines.

Methods to Prepare Aluminum Salt-Adjuvanted Vaccines.

Many human vaccines contain certain insoluble aluminum salts such as aluminum oxyhydroxide and aluminum hydroxyphosphate as vaccine adjuvants to boost the immunogenicity of the vaccines. Aluminum salts have been used as vaccine adjuvants for decades and have an established, favorable safety profile. However, preparing aluminum salts and aluminum salt-adjuvanted vaccines in a consistent manner remains challenging. This chapter discusses methods to prepare aluminum salts and aluminum salt-adjuvanted vaccines, factors to consider during preparation, and methods to characterize the vaccines after preparation.

Elucidating the mechanisms of protein antigen adsorption to the CAF/NAF liposomal vaccine adjuvant systems: effect of charge, fluidity and antigen-to-lipid ratio.

The reverse vaccinology approach has recently resulted in the identification of promising protein antigens, which in combination with appropriate adjuvants can stimulate customized, protective immune responses. Although antigen adsorption to adjuvants influences vaccine efficacy and safety, little is generally known about how antigens and adjuvants interact at the molecular level. The aim of this study was to elucidate the mechanisms of interactions between the equally sized, but oppositely charged model protein antigens α-lactalbumin and lysozyme, and i) the clinically tested cationic liposomal adjuvant CAF01 composed of cationic dimethyldioctadecylammonium (DDA) bromide and trehalose-6,6'-dibehenate (TDB) or ii) the neutral adjuvant formulation NAF01, where DDA was replaced with zwitterionic distearoylphosphatidylcholine (DSPC). The effect of liposome charge, bilayer rigidity, isoelectric point and antigen-to-lipid ratio was investigated using dynamic light scattering, transmission electron microscopy, differential scanning calorimetry, intrinsic fluorescence and Langmuir monolayers. The net anionic α-lactalbumin adsorbed onto the cationic liposomes, while there was no measureable attractive interaction with the zwitterionic liposomes. In contrast, the net cationic lysozyme showed very little interaction with either types of liposome. Adsorption of α-lactalbumin altered its tertiary structure, affected lipid membrane packing below and above the phase transition temperature, and neutralized the liposomal surface charge, resulting in reduced colloidal stability and liposome aggregation. Langmuir studies revealed that α-lactalbumin was not squeezed out of DDA monolayers upon compression, which suggests additional hydrophobic interactions. Such interactions are thus likely to affect the way vaccine antigens are presented to antigen-presenting cells, and may play an important role for the efficacy of the vaccine-induced immune response. These studies thus exemplify the importance of characterizing the molecular interactions between the vaccine antigen and adjuvant along with immunogenicity and efficacy studies.