Immunization and TLR8
Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that play a vital role in shaping adaptive immunity. DC maturation begins when endogenous or exogenous danger molecules are recognized by pattern recognition receptors (eg, Toll-like receptors [TLRs]), triggering upregulation of costimulatory molecules and production of immune-polarizing cytokines. Of note, human newborn DCs demonstrate impaired TH1 responses and particularly low production of TNF and IL-12p70, which are important for vaccine-induced protection against intracellular pathogens. Accordingly, development of novel adjuvanted vaccine formulations that enhance the maturation and functionality of human neonatal DCs might enable a new generation of early-life vaccines. Unlike agonists of most TLRs that elicit reduced TH1 cytokine production by newborn leukocytes, agonists of the endosomal TLR8, such as the synthetic imidazoquinoline (IMQ) CL075 (TLR8/7 agonist), induce robust TH1-polarizing responses from both neonatal and adult DCs.
Recent advances in the nascent field of immunoengineering might guide vaccine design by enabling targeting of DCs17 through vaccine delivery systems that mimic the size, shape, and surface chemistry of pathogens.18 Block copolymers of poly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-bl-PPS) are new macromolecular amphiphiles capable of forming a wide range of self-assembled configurations when dispersed in water and might present distinct advantages for human vaccine development. PEG-bl-PPS aggregates can form a wide variety of stable morphologies in aqueous solution, including spherical or cylindrical micelles (filomicelles), as well as liposome-like vesicles referred to as polymersomes. Polymersomes are significantly more stable than liposomes and can be engineered for bioresponsive intracellular payload delivery,16 which is highly advantageous for the specific targeting of endosomal receptors. Polymersomes are effective adjuvant and antigen-delivery systems, particularly for the induction of T cell–mediated immunity and the encapsulation of IMQ-derived adjuvants. Induction of durable TH1-type T-cell immunity might strengthen newborns’ defenses, thereby reducing the morbidity and mortality associated with intracellular pathogens, such as respiratory syncytial virus, HIV, tuberculosis, and malaria.
Immunoengineering-based rational vaccine design approach to develop and characterize a novel polymersome nanocarrier encapsulating CL075, which was designated CL075-PS. CL075 has low water solubility and is a highly potent immunostimulant that can be systemically toxic. Accordingly, a targeted delivery system is likely beneficial for minimization of systemic toxicity and translational application. The in vitro immunostimulatory activities of CL075-PS on human newborn and adult monocyte-derived dendritic cells (MoDCs) were benchmarked against conventional adjuvants and human vaccines, including the live attenuated BCG vaccine, which elicits moderate TH1 immunity in neonates26 and is safe and effective at birth.
Characterize the BCG-induced human DC cytokine and costimulatory molecule expression compared with conventional alum-adjuvanted vaccines and demonstrate that CL075-PS matches or exceeds this BCG-associated DC response.28 Compared with BCG, CL075-PS induced greater production of IL-12p70, a cytokine that enhances TH1-polarized immune responses and promotes cytotoxic T-cell proliferation and survival. The additional loading of the model HIV-1-Gag protein into CL075-PS did not negatively influence this response, triggering a similar protection-associated pattern of immune responses as induced by the whole bacterial vaccine BCG in newborn human DCs and greatly exceeding BCG-induced production of IL-12p70. When coloaded with the Mycobacterium tuberculosis antigen 85B (Ag85B) peptide 25 (p25), CL075:Ag85Bp25-PS induces Ag85B-specific CD4+ T cells in humanized TLR8 neonatal mice in vivo.
Strong potential for CL075-PS to serve as a dual antigen/adjuvant vaccine delivery system for human neonatal vaccines. Moreover, our age- and species-specific approach for formulation development through benchmarking new adjuvant formulations with respect to DC activation and potential reactogenicity profiles of licensed vaccine formulations presents a new in vitro methodology for the rational design and testing of human newborn vaccines.
- C57BL/6 wild-type (WT) dams and heterozygous humanized TLR8 (huTLR8Tg) male mice on a C57BL/6 background were bred to produce mixed litters. All littermates from one litter were immunized with the same formulations to eliminate the possibility of misidentification. Mice were immunized on day 7 of life and killed 14 days later.
- Genotyping was performed with tail clippings at 21 days of life. Mice were immunized subcutaneously at the scruff of the neck with either 0.2 × 106 colony-forming units of BCG (Organon Teknika/Merck, Durham, NC) or CL075:Ag85Bp25-PS containing approximately 8 to 10 μg of Ag85B/p25 (Biomatik, Cambridge, Ontario, Canada) and approximately 0.1 mg/kg CL075 in a total volume of 25 μL. For flow cytometric analysis, spleens were collected, and a single-cell suspension was generated and blocked with FcR blocking anti-mouse CD16/CD32 mAb 2.4G2 at 4°C for 10 minutes and then stained for 1 hour at room temperature with PE-conjugated I-A(b) M tuberculosis Ag85B precursor 280-294 (FQDAYNAAGGHNAVF) or human class II–associated invariant chain peptide (PVSKMRMARPLLMQA) tetramers (kindly produced by the National Institutes of Health MHC Tetramer Core Facility, Em ory University, Atlanta, Ga), as previously described
- Surface staining was performed at 4°C for 30 minutes with FITC-conjugated anti-CD3 (clone 17A2), allophycocyanin-conjugated anti-CD4 (clone RMA-5), BV421-conjugated anti-CD44 (clone IM7), and PE-conjugated anti-CD62L (clone SB/199, all from BD Biosciences). Samples were also labeled with Live/Dead Fixable Near IR Dead Cell Stain Kit, according to the manufacturer’s instructions (Invitrogen, Carlsbad, Calif). CD44+ tetramer-positive (Tet+) cells were identified after gating for singlets, forward scatter × side scatter lymphocyte characteristics, live cells, and CD4 expression. About 106 cells were stored for each sample acquired on the LSR II flow cytometer by using BD FACSDiva software (BD Biosciences, San Jose, Calif); data analysis was performed with FlowJo software (TreeStar, Ashland, Ore).
- Guiducci, C., Gong, M., Cepika, A.M., Xu, Z., Tripodo, C., Bennett, L. et al. RNA recognition by human TLR8 can lead to autoimmune inflammation. J Exp Med. 2013; 210: 2903-2919
- Prota, G., Christensen, D., Andersen, P., Medaglini, D., and Ciabattini, A. Peptide-specific T helper cells identified by MHC class II tetramers differentiate into several subtypes upon immunization with CAF01 adjuvanted H56 tuberculosis vaccine formulation. Vaccine. 2015; 33: 6823-6830