Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.
In vivo electroporation (EP) is used to enhance the uptake of nucleic acids and its association with DNA vaccination greatly stimulates immune responses to vaccine antigens delivered through the skin. However, the effect of EP on cutaneous cell behavior, the dynamics of immune cell recruitment and local inflammatory factors, have not been fully described. Here, we show that intradermal DNA vaccination combined with EP extends antigen expression to the epidermis and the subcutaneous skin muscle in non-human primates. In vivo fibered confocal microscopy and dynamic ex vivo imaging revealed that EP promotes the mobility of Langerhans cells (LC) and their interactions with transfected cells prior to their migration from the epidermis. At the peak of vaccine expression, we detected antigen in damaged keratinocyte areas in the epidermis and we characterized recruited immune cells in the skin, the hypodermis and the subcutaneous muscle. EP alone was sufficient to induce the production of pro-inflammatory cytokines in the skin and significantly increased local concentrations of Transforming Growth Factor (TGF)-alpha and IL-12. Our results show the kinetics of inflammatory processes in response to EP of the skin, and reveal its potential as a vaccine adjuvant.
The development of new immunization strategies requires a better understanding of early molecular and cellular events occurring at the site of injection. The skin is particularly rich in immune cells and represents an attractive site for vaccine administration. Here, we specifically targeted vaccine antigens to epidermal Langerhans cells (LCs) using a fusion protein composed of HIV antigens and a monoclonal antibody targeting Langerin. We developed a fluorescence imaging approach to visualize, in vivo, the vaccine-targeted cells. Studies were performed in nonhuman primates (NHPs) because of their relevance as a model to assess human vaccines. We directly demonstrated that in NHPs, intradermally injected anti-Langerin-HIVGag specifically targets epidermal LCs and induces rapid changes in the LC network, including LC activation and migration out of the epidermis. Vaccine targeting of LCs significantly improved anti-HIV immune response without requirement of an adjuvant. Although the co-injection of the TLR-7/8 synthetic ligand, R-848 (resiquimod), with the vaccine, did not enhance significantly the antibody response, it stimulated recruitment of HLA-DR+ inflammatory cells to the site of immunization. This study allowed us to characterize the dynamics of early local events following the injection of a vaccine-targeted epidermal LCs and R-848. Keywords:Fluorescence imaging r Langerhans cell r Nonhuman primate r Skin r Vaccination Additional supporting information may be found in the online version of this article at the publisher's web-site Correspondence: Dr. Catherine Chapon e-mail: catherine.chapon@cea.fr IntroductionFluorescence imaging is a powerful and minimally invasive technology for real-time visualization in vivo of the dynamics of the behavior of immune cells in their natural microenvironment [1][2][3][4][5][6].C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 690Nina Salabert et al. Eur. J. Immunol. 2016. 46: 689-700 It also facilitates the characterization of the fate of vaccine antigens in the organism allowing unprecedented progress in the understanding of vaccine-induced immune response mechanisms [7,8]. Up to now, most in vivo studies have used transgenic mouse models expressing fluorescent molecules under specific promoters. However, relevance of mouse models to the study of human vaccines is questionable. Major differences between the two species have been reported, when comparing mature immune system [9][10][11][12][13][14]. Mice models turned out to be little predictive of vaccine immunogenicity and efficacy in humans, even when highly advanced transgenic and humanized animals were used [15]. Currently, due to their close genetic relatedness with humans, and immune compartment similarities in particular, nonhuman primates (NHP) are considered to be the most appropriate model species for predicting vaccine immunogenicity and efficacy in humans [16,17]. The development of new strategies for immunization [17,18] requires a better understanding of molecular and cellular change...
The pivotal role of DCs in initiating the immune responses led to their use as vaccine vectors. However, the relationship between DC subsets involved in antigen presentation and the type of elicited immune responses underlined the need for the characterization of the DCs generated in vitro. The phenotypes of tissue-derived APCs from a cynomolgus macaque model for human vaccine development were compared with ex vivo-derived DCs. Monocyte/macrophages predominated in bone marrow (BM) and blood. Myeloid DCs (mDCs) were present in all tested tissues and were more highly represented than plasmacytoid DCs (pDCs). As in human skin, Langerhans cells (LCs) resided exclusively in the macaque epidermis, expressing CD11c, high levels of CD1a and Langerin (CD207). Most DC subsets were endowed with tissue-specific combinations of PRRs. DCs generated from CD34+ BM cells (CD34-DCs) were heterogeneous in phenotype. CD34-DCs shared properties (differentiation and PRR) of dermal and epidermal DCs. After injection into macaques, CD34-DCs expressing HIV-Gag induced Gag-specific CD4+ and CD8+ T cells producing IFN-γ, TNF-α, MIP-1β or IL-2. In high responding animals, the numbers of polyfunctional CD8+ T cells increased with the number of booster injections. This DC-based vaccine strategy elicited immune responses relevant to the DC subsets generated in vitro.
DNA vaccines delivered with electroporation (EP) have shown promising results in preclinical models and are evaluated in clinical trials. In this study, we aim to characterize early mechanisms occurring in the skin after intradermal injection and EP of the auxoGTUmultiSIV DNA vaccine in nonhuman primates. First, we show that EP acts as an adjuvant by enhancing local inflammation, notably via granulocytes, monocytes/macrophages, and CD1a int-expressing cell recruitment. EP also induced Langerhans cell maturation, illustrated by CD86, CD83, and HLA-DR upregulation and their migration out of the epidermis. Second, we demonstrate the crucial role of the DNA vaccine in soluble factors release, such as MCP-1 or IL-15. Transcriptomic analysis showed that EP played a major role in gene expression changes postvaccination. However, the DNA vaccine is required to strongly upregulate several genes involved in inflammatory responses (e.g., Saa4), cell migration (e.g., Ccl3, Ccl5, or Cxcl10), APC activation (e.g., Cd86), and IFN-inducible genes (e.g., Ifit3, Ifit5, Irf7, Isg15, orMx1), illustrating an antiviral response signature. Also, AIM-2, a cytosolic DNA sensor, appeared to be strongly upregulated only in the presence of the DNA vaccine and trends to positively correlate with several IFN-inducible genes, suggesting the potential role of AIM-2 in vaccine sensing and the subsequent innate response activation leading to strong adaptive T cell responses. Overall, these results demonstrate that a combined stimulation of the immune response, in which EP and the auxoGTUmultiSIV vaccine triggered different components of the innate immunity, led to strong and persistent cellular recall responses.
Purpose We developed a new approach to visualize skin Langerhans cells by in vivo fluorescence imaging in nonhuman primates. Procedures Macaques were intradermally injected with a monoclonal, fluorescently labeled antibody against HLA-DR molecule and were imaged for up to 5 days by fibered confocal microscopy (FCFM). Results The network of skin Langerhans cells was visualized by in vivo fibered confocal fluorescence microscopy. Quantification of Langerhans cells revealed no changes to cell density with time. Ex vivo experiments confirmed that injected fluorescent HLA-DR antibody specifically targeted Langerhans cells in the epidermis. Conclusions This study demonstrates the feasibility of single-cell, in vivo imaging as a noninvasive technique to track Langerhans cells in nontransgenic animals.
In the version of this article initially published online, the labels identifying each plot in Figure 1b In the version of this article initially published online, the flow cytometric dots were missing in the middle plot of the leftmost column in Figure 2f. The error has been corrected in the print, PDF and HTML versions of this article.
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