Tailoring the immune response by targeting C-type lectin receptors on alveolar macrophages using “pathogen-like” amphiphilic polyanhydride nanoparticles
Introduction
Acute respiratory infections cause 4.25 million deaths worldwide every year [1]. A critical need exists for the development of efficacious intranasal vaccines against respiratory pathogens capable of inducing robust and protective mucosal immunity. In this regard, there is growing interest in the development of vaccines that can be easily administered to the site of infection in order to elicit both local and systemic immune responses [2], [3], [4], [5].
The study of alveolar macrophages (AMϕ), a type of antigen presenting cell (APC) in the respiratory tract, is central to the development of intranasal vaccines. AMϕ constitute more than 80% of the total cells obtained by bronchoalveolar lavage of a healthy individual and they constitutively migrate from the lung to the draining lymph nodes (DLN) [6], [7], [8]. Indeed, AMϕ containing bacteria appear in the pulmonary DLN prior to the onset of pathogen-induced DC migration, thereby making them integral to the establishment of protective pulmonary immune responses [6]. AMϕ are equipped to detect pathogens with the aid of pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) [9]. One family of PRRs found on AMϕ, known as C-type lectin receptors (CLRs), recognize conserved carbohydrate structures, including mannose and galactose, found on the surface of many respiratory pathogens, such as Yersinia pestis, Mycobacterium tuberculosis, Streptococcus pneumoniae and influenza viruses [10], [11], [12], [13], [14]. CLRs also function as phagocytic receptors and include members of the mannose receptor family and DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) [15]. Depending on the specific CLR, ligand binding initiates downstream signaling cascades that promote immune cell migration to the DLN as well as antigen processing and presentation via MHC I and/or MHC II to prime naïve T cells [16], [17], [18], [19], [20].
Several research groups have explored CLR targeting as a vaccine design strategy to promote efficient delivery of cargo to intracellular compartments responsible for antigen processing and presentation [21], [22], [23], [24], [25], [26], [27], [28], [29]. Many studies have demonstrated the effectiveness of using antibodies [22] or mannoproteins from pathogens [26] to target CLRs and activate APCs. However, only a limited number report the use of carbohydrate-functionalized vaccine carriers as part of an improved adjuvant for intranasal vaccines [21], [24]. Work published by Jiang et al. indicated that alveolar macrophages could recognize mannosylated chitosan microparticles when delivered intranasally [24]. Unfortunately, mechanistic studies demonstrating the engagement of the mannose receptor on AMϕ by these particles were not performed. Here, we describe functionalization of polyanhydride nanoparticles with two conserved carbohydrate structures commonly found on the surface of respiratory pathogens, di-mannose and galactose. We also investigate the mechanisms by which these functionalized polyanhydride nanoparticles are internalized by and influence the activation of AMϕ.
Section snippets
Materials
The chemicals needed for monomer synthesis, polymerization and nanoparticle fabrication included 1,6-dibromohexane, triethylene glycol, 4-p-hydroxybenzoic acid, and 1-methyl-2-pyrrolidinone; these were purchased from Sigma–Aldrich (St. Louis, MO); 4-p-fluorobenzonitrile was obtained from Apollo Scientific (Cheshire, UK); toluene, sulfuric acid, acetonitrile, dimethyl formamide, acetic anhydride, methylene chloride, pentane, and potassium carbonate were obtained from Fisher Scientific (Fairlawn,
Synthesis and characterization of functionalized polyanhydride nanoparticles
Amphiphilic 50:50 CPTEG:CPH copolymer was synthesized as described previously [31]. The molecular weight (Mw) of the copolymer was 8000 Da and 1H NMR spectra of the copolymer were consistent with previously published data [31], [33], [35]. Particle morphology was evaluated by SEM and was found to be consistent with previously published results ([21], [33], [43] and data not shown). The average diameter of the non-functionalized nanoparticles was 163 ± 24 nm with a ζ-potential of −23 ± 2.5 mV
Discussion
In the present work, we have designed an approach to targeted antigen delivery by functionalizing the surface of polyanhydride nanoparticles with specific carbohydrates to enable the nanoparticles to engage C-type lectin receptors on AMϕ. Our rationale is that receptor-mediated engagement of nanoparticles will enhance their uptake and the activation of AMϕ, leading to the induction of robust immune responses in the respiratory tract.
Co-culture of functionalized nanoparticles with AMϕ
Conclusions
The approach outlined in this present work demonstrates that rational design of efficacious vaccine adjuvants can be achieved by targeting CLRs on APCs. Specifically, we describe the functionalization of polyanhydride nanoparticles with two conserved carbohydrate structures commonly found on the surface of respiratory pathogens, di-mannose and galactose. The addition of these carbohydrates significantly enhanced the intrinsic adjuvant activity of our polyanhydride nanovaccine platform by
Acknowledgments
The authors would like to thank the United States Army Medical Research and Materiel Command for financial support (Grant No. W81XWH-10-1-0806). The authors are grateful to Shawn Rigby for his expertise in flow cytometry and to Dr. Mary Ann McDowell of the University of Notre Dame for generously providing the MMR−/− mice. BN acknowledges the Balloun Professorship in Chemical and Biological Engineering and NLBP acknowledges the Wilkinson Professorship of Interdisciplinary Engineering.
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