A glycan-based adjuvant expands the breadth and duration of protection of mRNA-based vaccines.
Inflammation initiated in response to a microbial encounter, or deliberately induced upon vaccination, is central for the activation of adaptive lymphocytes. The stimulation of pattern recognition receptors (PRRs) orchestrates the inflammatory programs that regulate adaptive immunity. Here, we took advantage of an mRNA-based vaccine generated against the ancestral spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to test whether tuning inflammation via PRR stimulation enhances the efficacy of mRNA vaccines. We combined mRNA-based vaccine with mannadjuvant, a formulation of fungal mannan and aluminum hydroxide that targets Dectin-2, a C-type lectin PRR. In mice and non-human primates, mannadjuvant increased the magnitude and durability of the response elicited by the mRNA-based vaccine, and it also led to the induction of neutralizing antibodies directed against variants of concern with a high escape capacity, overcoming antigenic imprinting. Mechanistically, we demonstrated that prolonged type I interferon production and potentiated interleukin-1 signaling are necessary and sufficient to exert the activities of mannadjuvant in mice and in human myeloid cells.  Overall, our data demonstrate that an anti-fungal PRR can be harnessed to create more potent and durable mRNA-based vaccines. Overall design: We performed single-cell RNA sequencing (scRNA-seq) coupled with single-cell B cell receptor sequencing (scBCR-seq) in mice that received Comirnaty alone or Comirnaty formulated with mannadjuvant. As an additional group for direct comparison of B cell responses, mice were immunized with a monovalent Comirnaty vaccine developed against the spike protein of XBB.1.5 (hereafter referred to as XBB.1.5-Comirnaty). Briefly, C57BL/6 mice were immunized intramuscularly (i.m.) in the thigh muscle with Comirnaty alone or Comirnaty plus mannadjuvant. At day 56 after priming, the draining lymph nodes were collected and processed into single-cell suspensions by enzymatic digestion. A total of 1–2 × 106 cells were stained with 0.5 µg of cell hashing antibodies (BioLegend Total-Seq™ C antibodies, Hashtags 1–12) following Fc receptor blocking with TruStain FcX™ PLUS (anti-mouse CD16/32; BioLegend, Cat. #156604), according to the manufacturer's protocol. After incubation with hashing antibodies, cells were washed, stained with surface antibodies, and processed for fluorescence-activated cell sorting (FACS). Spike-positive germinal center (GC) B cells expressing B cell receptors (BCRs) specific for either WA1 or XBB.1.5 spike protein were sorted and subjected to scRNA-seq and scBCR-seq. Single-cell suspensions of FACS-sorted GC B cells from four biological samples were processed using the Chromium GEM-X Single Cell 5' Reagent Kit v3 (Cat. #1000699, 10x Genomics) and the Single Cell 5' Gel Bead Kit v3 (Cat. #1000700, 10x Genomics). Cells (27,000–35,000 per channel) were loaded onto a Chromium X Series instrument (10x Genomics) to generate single-cell gel beads-in-emulsion (GEMs), following the manufacturer's instructions. The recovery rate was approximately 45–55%. Captured cells were lysed within individual GEMs, and released RNA was barcoded during reverse transcription. 5' gene expression (GEX) libraries, single-cell V(D)J libraries (Cat. #1000255, 10x Genomics), and cell surface protein libraries were prepared according to the manufacturer's protocols. Library quality was assessed using an Agilent 2200 TapeStation, and final libraries were quantified by qPCR using the KAPA Library Quantification Kit for Illumina platforms. Libraries were diluted to 2 nM and pooled into two separate batches. A total of 650 pM of pooled libraries was mixed with 5% PhiX control library prior to sequencing. Sequencing was performed in two independent runs on an Illumina NextSeq 2000 platform using the P4 XLEAP-SBS Reagent Kit (200 cycles), with paired-end reads configured as follows: 28 cycles for read 1, 10 cycles for the i7 index, 10 cycles for the i5 index, and 90 cycles for read 2. Sequencing achieved a median depth of approximately 32,000 reads per cell for gene expression, 55,000 reads per cell for BCR libraries, and 5,000 reads per cell for hashtag (ADT) libraries.