Aluminum-based adjuvants are the most widely used adjuvants in human vaccines. A comprehensive understanding of the mechanism of action of aluminum adjuvants at the molecular level, however, is still elusive. Here, we unravel the effects of aluminum hydroxide Al(OH)3 by a systems-wide analysis of the Al(OH)3-induced monocyte response. Cell response analysis by cytokine release was combined with (targeted) transcriptome and full proteome analysis. Results from this comprehensive study revealed two novel pathways to become activated upon monocyte stimulation with Al(OH)3: the first pathway was IFNβ signaling possibly induced by DAMP sensing pathways like TLR or NOD1 activation, and second the HLA class I antigen processing and presentation pathway. Furthermore, known mechanisms of the adjuvant activity of Al(OH)3 were elucidated in more detail such as inflammasome and complement activation, homeostasis and HLA-class II upregulation, possibly related to increased IFNγ gene expression. Altogether, our study revealed which immunological pathways are activated upon stimulation of monocytes with Al(OH)3, refining our knowledge on the adjuvant effect of Al(OH)3 in primary monocytes. Aluminum salts are the most used adjuvants in human vaccines but a comprehensive understanding of the working mechanism of alum adjuvants at the molecular level is still elusive. Our Systems Vaccinology approach, combining complementary molecular biological, immunological and mass spectrometry-based techniques gave a detailed insight in the molecular mechanisms and pathways induced by Al(OH)3 in primary monocytes. Several novel immunological relevant cellular pathways were identified: type I interferon secretion potentially induced by TLR and/or NOD like signaling, the activation of the inflammasome and the HLA Class-I and Class-II antigen presenting pathways induced by IFNγ. This study highlights the mechanisms of the most commonly used adjuvant in human vaccines by combing proteomics, transcriptomics and cytokine analysis revealing new potential mechanisms of action for Al(OH)3.

Nowadays, mumps is re-emerging in highly vaccinated populations. Waning of vaccine-induced immunity plays a role, but antigenic differences between vaccine and mumps outbreak strains could also contribute to reduced vaccine effectiveness. CD8⁺ T cells play a critical role in immunity to viruses. However, limited data are available about sequence variability in CD8⁺ T cell epitope regions of mumps virus (MuV) proteins. Recently, the first set of naturally presented human leukocyte antigen Class I (HLA-I) epitopes of MuV was identified by us. In the present study, sequences of 40 CD8⁺ T cell epitope candidates, including previously and newly identified, obtained from Jeryl–Lynn mumps vaccine strains were compared with genomes from 462 circulating MuV strains. In 31 epitope candidates (78%) amino acid differences were detected, and in 17 (43%) of the epitope candidates the corresponding sequences in wild-type strains had reduced predicted HLA-I-binding compared to the vaccine strains. These findings suggest that vaccinated persons may have reduced T cell immunity to circulating mumps viruses due to antigenic differences.

Aluminum-based adjuvants have widely been used in human vaccines since 1926. In the absence of antigens, aluminum-based adjuvants can initiate the inflammatory preparedness of innate cells, yet the impact of antigens on this response has not been investigated so far. In this study, we address the modulating effect of vaccine antigens on the monocyte-derived innate response by comparing processes initiated by Al(OH)₃ and by Infanrix, an Al(OH)₃-adjuvanted trivalent combination vaccine (DTaP), containing diphtheria toxoid (D), tetanus toxoid (T) and acellular pertussis (aP) vaccine antigens. A systems-wide analysis of stimulated monocytes was performed in which full proteome analysis was combined with targeted transcriptome analysis and cytokine analysis. This comprehensive study revealed four major differences in the monocyte response, between plain Al(OH)₃ and DTaP stimulation conditions: (I) DTaP increased the anti-inflammatory cytokine IL-10, whereas Al(OH)₃ did not; (II) Al(OH)₃ increased the gene expression of IFNγ, IL-2 and IL-17a in contrast to the limited induction or even downregulation by DTaP; (III) increased expression of type I interferons-induced proteins was not observed upon DTaP stimulation, but was observed upon Al(OH)₃ stimulation; (IV) opposing regulation of protein localization pathways was observed for Al(OH)₃ and DTaP stimulation, related to the induction of exocytosis by Al(OH)₃ alone. This study highlights that vaccine antigens can antagonize Al(OH)₃-induced programming of the innate immune responses at the monocyte level.

Vaccines undergo stringent batch-release testing, most often including in-vivo assays for potency. For combination vaccines, such as diphtheria-tetanus-pertussis (DTaP), chemical modification induced by formaldehyde inactivation, as well as adsorption to aluminum-based adjuvants, complicates antigen-specific in-vitro analysis. Here, a mass spectrometric method was developed that allows the identification and quantitation of DTaP antigens in a combination vaccine. Isotopically labeled, antigen-specific internal standard peptides were employed that permitted absolute quantitation of their antigen-derived peptide counterparts and, consequently, the individual antigens. We evaluated the applicability of the method on monovalent non-adjuvanted antigens, on final vaccine lots and on experimental vaccine batches, where certain antigens were omitted from the drug product. Apart from the applicability for final batch release, we demonstrated the suitability of the approach for in-process control monitoring. The peptide quantification method facilitates antigen-specific identification and quantification of combination vaccines in a single assay. This may contribute, as part of the consistency approach, to a reduction in the number of animal tests required for vaccine-batch release.