NANOBODY^® (a registered trademark of Ablynx N.V) molecules (Nbs), also referred to as single domain-based VHHs, are antibody fragments derived from heavy-chain only IgG antibodies found in the Camelidae family. Due to their small size, simple structure, high antigen binding affinity, and remarkable stability in extreme conditions, Nbs possess the potential to overcome several of the limitations of conventional monoclonal antibodies. For many years, Nbs have been of great interest in a wide variety of research fields, particularly in the diagnosis and treatment of diseases. This has culminated with the approval of the world’s first Nb based drug (Caplacizumab) in 2018 with others following, and more on the way. This review will provide an overview, with examples, of (i) the structure and advantages of Nbs compared to conventional monoclonal antibodies, (ii) methods used to generate and produce antigen-specific Nbs, (iii) applications for diagnostics, and (iv) ongoing clinical trials for Nb therapeutics as well as promising candidates for clinical development.

The cytokine interleukin (IL)-1β is a key mediator of the inflammatory response and has been implicated in the pathophysiology of acute and chronic inflammation. IL-1β is synthesized in response to many stimuli as an inactive pro–IL-1β precursor protein that is further processed by caspase-1 into mature IL-1β, which is the secreted biologically active form of the cytokine. Although stimulation of membrane-bound Toll-like receptors (TLRs) up-regulates pro–IL-1β expression, activation of caspase-1 is believed to be mainly initiated by cytosolic Nod-like receptors. In this study, we show that polyinosinic:polycytidylic acid (poly[I:C]) and lipopolysaccharide stimulation of macrophages induces pro–IL-1β processing via a Toll/IL-1R domain–containing adaptor-inducing interferon-β–dependent signaling pathway that is initiated by TLR3 and TLR4, respectively. Ribonucleic acid interference (RNAi)–mediated knockdown of the intracellular receptors NALP3 or MDA5 did not affect poly(I:C)-induced pro–IL-1β processing. Surprisingly, poly(I:C)- and LPS-induced pro–IL-1β processing still occurred in caspase-1–deficient cells. In contrast, pro–IL-1β processing was inhibited by caspase-8 peptide inhibitors, CrmA or vFLIP expression, and caspase-8 knockdown via RNAi, indicating an essential role for caspase-8. Moreover, recombinant caspase-8 was able to cleave pro–IL-1β in vitro at exactly the same site as caspase-1. These results implicate a novel role for caspase-8 in the production of biologically active IL-1β in response to TLR3 and TLR4 stimulation.

Antigen variation is a successful defense system adopted by several infectious agents to evade the host immune response. The principle of this defense strategy in the African trypanosome paradigm involves a dense packing of variant surface glycoproteins (VSG) exposing only highly variable and immuno-dominant epitopes to the immune system, whereas conserved epitopes become inaccessible for large molecules. Reducing the size of binders that target the conserved, less-immunogenic, cryptic VSG epitopes forms an obvious solution to combat these parasites. This goal was achieved by introducing dromedary Heavy-chain antibodies. We found that only these unique antibodies recognize epitopes common to multiple VSG classes. After phage display of their antigen-binding repertoire, we isolated a single domain antibody fragment with high specificity for the conserved Asn-linked carbohydrate of VSG. In sharp contrast to labeled concanavalin-A that stains only the flagellar pocket where carbohydrates are accessible because of less dense VSG packing, the single domain binder stains the entire surface of viable parasites, irrespective of the VSG type expressed. This corroborates the idea that small antibody fragments, but not larger lectins or conventional antibody fragments, are able to penetrate the dense VSG coat to target their epitope. The diagnostic potential of this fluorescently labeled binder was proven by the direct, selective, and sensitive detection of parasites in blood smears. The employment of this binder as a molecular recognition unit in immuno-toxins designed for trypanosomosis therapy becomes feasible as well. This was illustrated by the specific trypanolysis induced by an antibody::β-lactamase fusion activating a prodrug.

Leishmania major parasites reside and multiply in late endosomal compartments of host phagocytic cells. Immune control of Leishmania growth absolutely requires expression of inducible Nitric Oxide Synthase (iNOS/NOS2) and subsequent production of NO. Here, we show that CD11b⁺ CD11c⁺ Ly-6C⁺ MHC-II⁺ cells are the main iNOS-producing cells in the footpad lesion and in the draining lymph node of Leishmania major-infected C57BL/6 mice. These cells are phenotypically similar to iNOS-producing inflammatory DC (iNOS-DC) observed in the mouse models of Listeria monocytogenes and Brucella melitensis infection. The use of DsRed-expressing parasites demonstrated that these iNOS-producing cells are the major infected population in the lesions and the draining lymph nodes. Analysis of various genetically deficient mouse strains revealed the requirement of CCR2 expression for the recruitment of iNOS-DC in the draining lymph nodes, whereas their activation is strongly dependent on CD40, IL-12, IFN-γ and MyD88 molecules with a partial contribution of TNF-α and TLR9. In contrast, STAT-6 deficiency enhanced iNOS-DC recruitment and activation in susceptible BALB/c mice, demonstrating a key role for IL-4 and IL-13 as negative regulators. Taken together, our results suggest that iNOS-DC represent a major class of Th1-regulated effector cell population and constitute the most frequent infected cell type during chronic Leishmania major infection phase of C57BL/6 resistant mice. Leishmania spp. are protozoan parasites infecting a variety of mammals, including humans and mice. Much information has been gleaned from murine models of Leishmania major infection. The control of L. major infection by resistant C57BL/6 mice requires the secretion of type 1 (Th1) cytokines (i.e. IFN-γ) by T cells as well as the expression of inducible nitric oxide synthase (iNOS) by phagocytic cells. Conversely, susceptible BALB/c mice are unable to control infection and develop a type 2 (Th2) immune response characterized by the secretion of IL-4 and IL-13 cytokines. In this study, we showed that the main iNOS-producing cells in the lesion and the draining lymph node are phenotypically similar to iNOS-producing “inflammatory” dendritic cells (DC), which are already described in the mouse models of Listeria monocytogenes and Brucella melitensis infection. Our data also highlighted a strong association between the recruitment and activation of these inflammatory DC and the resistance to L. major infection. In addition, we showed that iNOS production by these inflammatory DC is positively regulated by Th1 response and negatively by Th2 response. Taken together, our results provide new insights into how innate and adaptive immune responses fight L. major infection. A better understanding of the mechanisms regulating inflammatory DC recruitment and activation could lead to new therapeutic strategies against Leishmania infection.

African trypanosomes of the Trypanosoma brucei species are extra-cellular parasites that cause human African trypanosomiasis (HAT) as well as infections in game animals and livestock. Trypanosomes are known to evade the immune response of their mammalian host by continuous antigenic variation of their surface coat. Here, we aim to demonstrate that in addition, trypanosomes (i) cause the loss of various B cell populations, (ii) disable the hosts' capacity to raise a long-lasting specific protective anti-parasite antibody response, and (iii) abrogate vaccine-induced protective response to a non-related human pathogen such as Bordetella pertussis. Using a mouse model for T. brucei, various B cell populations were analyzed by FACS at different time points of infection. The results show that during early onset of a T. brucei infection, spleen remodeling results in the rapid loss of the IgM⁺ marginal zone (IgM⁺MZ) B cell population characterized as B220⁺IgM^HighIgD^Int CD21^HighCD23^LowCD1d⁺CD138⁻. These cells, when isolated during the first peak of infection, stained positive for Annexin V and had increased caspase-3 enzyme activity. Elevated caspase-3 mRNA levels coincided with decreased mRNA levels of the anti-apoptotic Bcl-2 protein and BAFF receptor (BAFF-R), indicating the onset of apoptosis. Moreover, affected B cells became unresponsive to stimulation by BCR cross-linking with anti-IgM Fab fragments. In vivo, infection-induced loss of IgM⁺ B cells coincided with the disappearance of protective variant-specific T-independent IgM responses, rendering the host rapidly susceptible to re-challenge with previously encountered parasites. Finally, using the well-established human diphtheria, tetanus, and B. pertussis (DTPa) vaccination model in mice, we show that T. brucei infections abrogate vaccine-induced protective responses to a non-related pathogen such as B. pertussis. Infections with T. brucei parasites result in the rapid loss of T–cell independent IgM⁺MZ B cells that are normally functioning as the primary immune barrier against blood-borne pathogens. In addition, ongoing trypanosome infections results in the rapid loss of B cell responsiveness and prevent the induction of protective memory responses. Finally, trypanosome infections disable the host's capacity to recall vaccine-induced memory responses against non-related pathogens. In particular, these last results call for detailed studies of the effect of HAT on memory recall responses in humans, prior to the planning of any mass vaccination campaign in HAT endemic areas. African trypanosomes are extracellular parasites that cause the deadly disease sleeping sickness in humans, and nagana in cattle. The control of infection is believed to be largely dependent on the host antibody response. We postulate here that protective anti-trypanosome responses mainly involve splenic marginal zone B cells, as they are implicated in the production of antibodies against blood-borne pathogens. In this work, we show that trypanosome infections induce the rapid loss of these marginal zone B cells, coinciding with the loss of the splenic marginal zone itself. While the infection does result in the induction of plasma cell differentiation and antibody secretion, the loss of the marginal zone B cell population results in the loss of specific protective responses. In addition, we also show that host memory responses are destroyed during infection, even affecting unrelated vaccine-induced memory responses such as those induced by the commercially available DTPa vaccine. The latter finding is crucial for the evaluation of mass vaccination approaches in African regions where trypanosome infections are prevalent.