Development of a TLR7/8 agonist adjuvant formulation to overcome early life hyporesponsiveness to DTaP vaccination

Abstract

Infection is the most common cause of mortality early in life, yet the broad potential of immunization is not fully realized in this vulnerable population. Most vaccines are administered during infancy and childhood, but in some cases the full benefit of vaccination is not realized in-part. New adjuvants are cardinal to further optimize current immunization approaches for early life. However, only a few classes of adjuvants are presently incorporated in vaccines approved for human use. Recent advances in the discovery and delivery of Toll-like receptor (TLR) agonist adjuvants have provided a new toolbox for vaccinologists. Prominent among these candidate adjuvants are synthetic small molecule TLR7/8 agonists. The development of an effective infant Bordetella pertussis vaccine is urgently required because of the resurgence of pertussis in many countries, contemporaneous to the switch from whole cell to acellular vaccines. In this context, TLR7/8 adjuvant based vaccine formulation strategies may be a promising tool to enhance and accelerate early life immunity by acellular B. pertussis vaccines. In the present study, we optimized (a) the formulation delivery system, (b) structure, and (c) immunologic activity of novel small molecule imidazoquinoline TLR7/8 adjuvants towards human infant leukocytes, including dendritic cells. Upon immunization of neonatal mice, this TLR7/8 adjuvant overcame neonatal hyporesponsiveness to acellular pertussis vaccination by driving a T helper (Th)1/Th17 biased T cell- and IgG2c-skewed humoral response to a licensed acellular vaccine (DTaP). This potent immunization strategy may represent a new paradigm for effective immunization against pertussis and other pathogens in early life.

Immune profiling of age and adjuvant-specific activation of human blood mononuclear cells in vitro

“…These observations highlight the potential of TLR7/8a as a candidate adjuvant to enhance immunogenicity of candidate vaccines for vulnerable populations. TLR7/8a-mediated abundance of NK cells in innate IFNγ + MNCs compartment TLR7/8a may enhance early life immunogenicity as an adjuvant and overcomes neonatal hyperresponsiveness to acellular pertussis vaccine 11 as well as pneumococcal conjugate vaccine 15 by inducing robust Th1 cytokines. After seeing the robust induction of Th1 related cytokine IFNγ by TLR7/8a R848 (Figure 5), we tried to capture the predominant source of IFNγ production mediated by major innate cell lineages among MNCs.…”

“…Cells were plated at 4 × 10 6 cells/ml in 24-well plates and stimulated with aqueous formulations of selected PRRa (Supplementary Table 3) at the concentrations noted in the figure legends. Concentration of PRRa for in vitro stimulations were chosen empirically based on either the results of previous 11,15 studies or according to the manufacturer's (InvivoGen) instruction. After 10h of incubation in a humidified incubator at 37°C (in 5% CO2), GolgiPlug (containing Brefeldin A) and GolgiStop (containing Monensin) were added according to the manufacturer's (BD Biosciences) instruction to all the wells to block the cytokines production and facilitate optimal intracellular mass cytometry analysis.…”

“…Results were considered significant at p values indicated in each figure legend. Sample size for in vitro stimulations were chosen empirically based on the results of previous studies 11,15 . In addition, for each CyTOF experiment (a total of 10 independent assays with BMCs from 30 participants, Supplementary Note 1) matching representatives of newborn, adult and elder samples were prepared to reduce batch effects and each experiment was performed as a single assay.…”

“…TLR agonists have been used in vaccine development to help stimulate the immune system, but selection of such adjuvant should be ontogeny-specific to avoid inflammaging in elders 13 . Notably, innate IFN responses to TLR agonist adjuvant stimulation have been correlated with increased vaccine immunogenicity at the extremes of age 11,[14][15][16][17] .…”

Immune-profiling of T helper 1 (Th1), Th2 and Th17 signatures in murine splenocytes by targeting intracellular cytokines

“…This 17-parameter spectral flow cytometry panel was developed to assess the functional cytokines production by conventional T cells which shapes the host immune responses upon natural infection or vaccinations (1). We previously demonstrated antigen specific responses or memory recall responses using microbial proteins (2,3) or peptides (4,5) and immunophenotyped functional cytokines in murine splenic T cell compartment after vaccination. Here, we expanded the flow cytometry panel using spectral flow technology by adding B cells, NK cells, and γδT cell markers, explored the memory T cell compartment and re-validated the panel upon mitogen stimulation using adult C57BL/6 and BALB/c mice strains (Figure 1, Table 2, Online Table 5 and Online Figure 8).…”