Aluminum salts, developed almost a century ago, remain the most commonly used adjuvant for licensed human vaccines. Compared to more recently developed vaccine adjuvants, aluminum adjuvants such as Alhydrogel are heterogeneous in nature, consisting of 1–10 micrometer-sized aggregates of nanoparticle aluminum oxyhydroxide fibers. To determine whether the particle size and aggregated state of aluminum oxyhydroxide affects its adjuvant activity, we developed a scalable, top-down process to produce stable nanoparticles (nanoalum) from the clinical adjuvant Alhydrogel by including poly(acrylic acid) (PAA) polymer as a stabilizing agent. Surprisingly, the PAA:nanoalum adjuvant elicited a robust TH1 immune response characterized by antigen-specific CD4+ T cells expressing IFN-γ and TNF, as well as high IgG2 titers, whereas the parent Alhydrogel and PAA elicited modest TH2 immunity characterized by IgG1 antibodies. ASC, NLRP3 and the IL-18R were all essential for TH1 induction, indicating an essential role of the inflammasome in this adjuvant’s activity. Compared to microparticle Alhydrogel this nanoalum adjuvant provided superior immunogenicity and increased protective efficacy against lethal influenza challenge. Therefore PAA:nanoalum represents a new class of alum adjuvant that preferentially enhances TH1 immunity to vaccine antigens. This adjuvant may be widely beneficial to vaccines for which TH1 immunity is important, including tuberculosis, pertussis, and malaria.

For nearly a century, aluminum salts have been the most widely used vaccine adjuvant formulation, and have thus established a history of safety and efficacy. Nevertheless, for extremely challenging disease targets such as tuberculosis or HIV, the adjuvant activity of aluminum salts may not be potent enough to achieve protective efficacy. Adsorption of TLR ligands to aluminum salts facilitates enhanced adjuvant activity, such as in the human papilloma virus vaccine Cervarix®. However, some TLR ligands such as TLR7/8 agonist imidazoquinolines do not efficiently adsorb to aluminum salts. The present report describes a formulation approach to solving this challenge by developing a lipid-based nanosuspension of a synthetic TLR7/8 ligand (3M-052) that facilitates adsorption to aluminum oxyhydroxide via the structural properties of the helper lipid employed. In immunized mice, the aluminum oxyhydroxide-adsorbed formulation of 3M-052 enhanced antibody and TH1-type cellular immune responses to vaccine antigens for tuberculosis and HIV.

Pulmonary tuberculosis (TB) remains one of the leading causes of infectious disease death despite widespread usage of the BCG vaccine. A number of new TB vaccines have moved into clinical evaluation to replace or boost the BCG vaccine including ID93+GLA-SE, an adjuvanted subunit vaccine. The vast majority of new TB vaccines in trials are delivered parenterally even though intranasal delivery can augment lung-resident immunity and protective efficacy in small animal models. Parenteral immunization with the adjuvanted subunit vaccine ID93+GLA-SE elicits robust TH1 immunity and protection against aerosolized Mycobacterium tuberculosis in mice and guinea pigs. Here we describe the immunogenicity and efficacy of this vaccine when delivered intranasally. Intranasal delivery switches the CD4 T cell response from a TH1 to a TH17 dominated tissue-resident response with increased frequencies of ID93-specific cells in both the lung tissue and at the lung surface. Surprisingly these changes do not affect the protective efficacy of ID93+GLA-SE. Unlike intramuscular immunization, ID93+GLA does not require the squalene-based oil-in-water emulsion SE to elicit protective CD4 T cells when delivered intranasally. Finally we demonstrate that TNF and the IL-17 receptor are dispensable for the efficacy of the intranasal vaccine suggesting an alternative mechanism of protection.

With over eight million cases of tuberculosis each year there is a pressing need for the development of new vaccines against Mycobacterium tuberculosis. Subunit vaccines consisting of recombinant proteins are an attractive vaccine approach due to their inherent safety compared to attenuated live vaccines and the uniformity of manufacture. Addition of properly formulated TLR agonist-containing adjuvants to recombinant protein vaccines enhances the antigen-specific CD4+ T cell response characterized by IFN-γ and TNF, both of which are critical for the control of TB. We have developed a clinical stage vaccine candidate consisting of a recombinant fusion protein ID93 adjuvanted with the TLR4 agonist GLA-SE. Here we examine whether ID93+GLA-SE can be improved by the addition of a second TLR agonist. Addition of CpG containing DNA to ID93+GLA-SE enhanced the magnitude of the multi-functional TH1 response against ID93 characterized by co-production of IFN-γ, TNF, and IL-2. Addition of CpG also improved the protective efficacy of ID93+GLA-SE. Finally we demonstrate that this adjuvant synergy between GLA and CpG is independent of TRIF signaling, whereas TRIF is necessary for the adjuvant activity of GLA-SE in the absence of CpG.

BackgroundAdjuvants have the potential to increase the efficacy of protein-based vaccines but need to be maintained within specific temperature and storage conditions. Lyophilization can be used to increase the thermostability of protein pharmaceuticals; however, no marketed vaccine that contains an adjuvant is currently lyophilized, and lyophilization of oil-in-water nanoemulsion adjuvants presents a specific challenge. We have previously demonstrated the feasibility of lyophilizing a candidate adjuvanted protein vaccine against Mycobacterium tuberculosis (Mtb), ID93 + GLA-SE, and the subsequent improvement of thermostability; however, further development is required to prevent physicochemical changes and degradation of the TLR4 agonist glucopyranosyl lipid adjuvant formulated in an oil-in-water nanoemulsion (SE).Materials and methodsIn this study, we took a systematic approach to the development of a thermostable product by first identifying compatible solution conditions and stabilizing excipients for both antigen and adjuvant. Next, we applied a design-of-experiments approach to identify stable lyophilized drug product formulations.ResultsWe identified specific formulations that contain disaccharide or a combination of disaccharide and mannitol that can achieve substantially improved thermostability and maintain immunogenicity in a mouse model when tested in accelerated and real-time stability studies.ConclusionThese efforts will aid in the development of a platform formulation for use with other similar vaccines.