performed experiments. linkers enhances long-lived B cell responses and can selectively direct antibodies toward protective neutralizing epitopes. == Main == The majority of current vaccines are thought to prevent disease through the induction of a protective antibody response1,2. For elicitation of protective humoral immunity by vaccination, B cells must be activated, enter germinal centers to undergo affinity maturation of their antigen receptors and then differentiate into either long-lived plasma cells that secrete antibody constitutively or memory B cells that participate in a recall response on re-exposure to the pathogen. Adjuvants help drive these immunological mechanisms, shaping vaccine protection3,4. The most prevalent adjuvant in licensed human vaccines is also one of the oldest known adjuvants, aluminum hydroxide (alum). The function of alum is complex and potentially dependent on induction of inflammatory cell death at the injection site, activation of innate immune-sensing pathways and production of chemokines and cytokines at the injection site and/or draining lymph nodes (LNs)57. Although effective in many vaccines, immune responses elicited by alum are often weaker than other adjuvants in clinical and preclinical studies810. Despite these limitations, the rigorous requirements for safety in vaccines make the successful development NMDA-IN-1 of new adjuvants a major challenge and alum remains an important gold standard benchmark for all adjuvants7. Here we aimed to understand immunological mechanisms of action of this classic adjuvant by evaluating whether the function of alum could be Colec11 enhanced by engineering the interaction between antigens and alum. Although alum is most commonly employed by adsorbing antigens to alum particles for administration, it NMDA-IN-1 is known that many antigens rapidly desorb from alum in the presence of serum or interstitial fluid1115. Additionally, the rate of antigen NMDA-IN-1 clearance in vivo is often unaffected by preadsorption to alum16,17. We thus designed immunogens for tight binding to alum through site-specific introduction of multivalent phosphoserine (pSer) peptidepolymer affinity tags, which undergo a ligand exchange reaction with the surface of alum to anchor immunogens in an oriented manner on alum particles. We then studied how this modification altered both the fate of these immunogens in vivo and the resulting immune responses. == Results == == Site-specific introduction of pSer affinity tags promotes stable immunogen binding to alum == We designed short peptide and polymer linkers that would mediate binding to alum by ligand exchange between phosphate groups and hydroxyls at the surface of alum particles. Peptides consisting of 112 consecutive pSer followed by a short poly(ethylene glycol) spacer and N-terminal maleimide functional group were prepared by solid-phase synthesis (Fig.1a). We first coupled linkers with one, two or four pSer groups to yeast cytochrome c, which has a free solvent-exposed cysteine. The pSer-modified cytochrome c exhibited steadily increasing binding to alum as the number of serine residues in the tag increased (Extended Data Fig.1a). To evaluate the role of pSer valency in achieving stable binding to alum, we modified the 240 kDa fluorescent NMDA-IN-1 protein phycoerythrin (PE) with 220 linkers, where each linker contained either one or four pSer residues (Extended Data Fig.1b). The pSer1- or pSer4-modified PE was adsorbed to alum, followed by incubation in buffer containing 10% serum, and protein bound to alum after this two-step process was measured by fluorescence spectroscopy. As shown in Extended Data Fig.1c, unmodified PE showed almost no retention on alum, but pSer tags NMDA-IN-1 promoted a majority of the protein to adhere to alum for >24 h. Only 24 pSer4linkers per protein were required to achieve the same level of binding as 1020 linkers that had a single pSer residue. Thus, even for very large proteins, modification with a few multivalent pSer tags promotes stable binding to alum in the presence of serum. == Fig. 1. Phosphoserine affinity tags enable tunable binding of immunogens to aluminum hydroxide adjuvant. == a, Chemical structure of pSer peptide-polyethylene glycol (PEG) affinity tags for site-specific antigen modification.b, Unmodified or pSer-conjugated fluorescent eOD protein (10 g ml1) was.