Noninvasive Immuno-PET Imaging of CD8+ T Cell Behavior in Influenza A Virus-Infected Mice.

Immuno-positron emission tomography (immuno-PET) is a noninvasive imaging method that enables tracking of immune cells in living animals. We used a nanobody that recognizes mouse CD8α and labeled it with 89Zr to image mouse CD8+ T cells in the course of an infection with influenza A virus (IAV). The CD8+ signal showed a strong increase in the mediastinal lymph node (MLN) and thymus as early as 4 days post-infection (dpi), and as early as 6 dpi in the lungs. Over the course of the infection, CD8+ T cells were at first distributed diffusely throughout the lungs and then accumulated more selectively in specific regions of the lungs. These distributions correlated with morbidity as mice reached the peak of weight loss over this interval. CD8+ T cells obtained from control or IAV-infected mice showed a difference in their distribution and migration when comparing their fate upon labeling ex vivo with 89Zr-labeled anti-CD8α nanobody and transfer into infected versus control animals. CD8+ T cells from infected mice, upon transfer, appear to be trained to persist in the lungs, even of uninfected mice. Immuno-PET imaging thus allows noninvasive, dynamic monitoring of the immune response to infectious agents in living animals.

Converting an Anti-Mouse CD4 Monoclonal Antibody into an scFv Positron Emission Tomography Imaging Agent for Longitudinal Monitoring of CD4+ T Cells.

Immuno-positron emission tomography (PET), a noninvasive imaging modality, can provide a dynamic approach for longitudinal assessment of cell populations of interest. Transformation of mAbs into single-chain variable fragment (scFv)-based PET imaging agents would allow noninvasive tracking in vivo of a wide range of possible targets. We used sortase-mediated enzymatic labeling in combination with PEGylation to develop an anti-mouse CD4 scFv-based PET imaging agent constructed from an anti-mouse CD4 mAb. This anti-CD4 scFv can monitor the in vivo distribution of CD4+ T cells by immuno-PET. We tracked CD4+ and CD8+ T cells in wild-type mice, in immunodeficient recipients reconstituted with monoclonal populations of OT-II and OT-I T cells, and in a B16 melanoma model. Anti-CD4 and -CD8 immuno-PET showed that the persistence of both CD4+ and CD8+ T cells transferred into immunodeficient mice improved when recipients were immunized with OVA in CFA. In tumor-bearing animals, infiltration of both CD4+ and CD8+ T cells increased as the tumor grew. The approach described in this study should be readily applicable to convert clinically useful Abs into the corresponding scFv PET imaging agents.

Nanobodies in cancer.

For treatment and diagnosis of cancer, antibodies have proven their value and now serve as a first line of therapy for certain cancers. A unique class of antibody fragments called nanobodies, derived from camelid heavy chain-only antibodies, are gaining increasing acceptance as diagnostic tools and are considered also as building blocks for chimeric antigen receptors as well as for targeted drug delivery. The small size of nanobodies (∼15 kDa), their stability, ease of manufacture and modification for diverse formats, short circulatory half-life, and high tissue penetration, coupled with excellent specificity and affinity, account for their attractiveness. Here we review applications of nanobodies in the sphere of tumor biology.

In vivo detection of antigen-specific CD8+ T cells by immuno-positron emission tomography.

The immune system's ability to recognize peptides on major histocompatibility molecules contributes to the eradication of cancers and pathogens. Tracking these responses in vivo could help evaluate the efficacy of immune interventions and improve mechanistic understanding of immune responses. For this purpose, we employ synTacs, which are dimeric major histocompatibility molecule scaffolds of defined composition. SynTacs, when labeled with positron-emitting isotopes, can noninvasively image antigen-specific CD8+ T cells in vivo. Using radiolabeled synTacs loaded with the appropriate peptides, we imaged human papillomavirus-specific CD8+ T cells by positron emission tomography in mice bearing human papillomavirus-positive tumors, as well as influenza A virus-specific CD8+ T cells in the lungs of influenza A virus-infected mice. It is thus possible to visualize antigen-specific CD8+ T-cell populations in vivo, which may serve prognostic and diagnostic roles.

Improved GPCR ligands from nanobody tethering.

Antibodies conjugated to bioactive compounds allow targeted delivery of therapeutics to cell types of choice based on that antibody's specificity. Here we develop a new type of conjugate that consists of a nanobody and a peptidic ligand for a G protein-coupled receptor (GPCR), fused via their C-termini. We address activation of parathyroid hormone receptor-1 (PTHR1) and improve the signaling activity and specificity of otherwise poorly active N-terminal peptide fragments of PTH by conjugating them to nanobodies (VHHs) that recognize PTHR1. These C-to-C conjugates show biological activity superior to that of the parent fragment peptide in vitro. In an exploratory experiment in mice, a VHH-PTH peptide conjugate showed biological activity, whereas the corresponding free peptide did not. The lead conjugate also possesses selectivity for PTHR1 superior to that of PTH(1-34). This design approach, dubbed "conjugation of ligands and antibodies for membrane proteins" (CLAMP), can yield ligands with high potency and specificity.

Recognition of Class II MHC Peptide Ligands That Contain ß-Amino Acids.

Proteins are composed of α-amino acid residues. This consistency in backbone structure likely serves an important role in the display of an enormous diversity of peptides by class II MHC (MHC-II) products, which make contacts with main chain atoms of their peptide cargo. Peptides that contain residues with an extra carbon in the backbone (derived from ß-amino acids) have biological properties that differ starkly from those of their conventional counterparts. How changes in the structure of the peptide backbone affect the loading of peptides onto MHC-II or recognition of the resulting complexes by TCRs has not been widely explored. We prepared a library of analogues of MHC-II-binding peptides derived from OVA, in which at least one α-amino acid residue was replaced with a homologous ß-amino acid residue. The latter contain an extra methylene unit in the peptide backbone but retain the original side chain. We show that several of these α/ß-peptides retain the ability to bind tightly to MHC-II, activate TCR signaling, and induce responses from T cells in mice. One α/ß-peptide exhibited enhanced stability in the presence of an endosomal protease relative to the index peptide. Conjugation of this backbone-modified peptide to a camelid single-domain Ab fragment specific for MHC-II enhanced its biological activity. Our results suggest that backbone modification offers a method to modulate MHC binding and selectivity, T cell stimulatory capacity, and susceptibility to processing by proteases such as those found within endosomes where Ag processing occurs.

Internalization of Influenza Virus and Cell Surface Proteins Monitored by Site-Specific Conjugation of Protease-Sensitive Probes.

Commonly used methods to monitor internalization of cell surface structures involve application of fluorescently or otherwise labeled antibodies against the target of interest. Genetic modification of the protein of interest, for example through creation of fusions with fluorescent or enzymatically active protein domains, is another approach to follow trafficking behavior. The former approach requires indirect methods, such as multiple rounds of cell staining, to distinguish between a target that remains surface-disposed and an internalized and/or recycled species. The latter approach necessitates the creation of fusions whose behavior may not accurately reflect that of their unmodified counterparts. Here, we report a method for the characterization of protein internalization in real time through sortase-mediated, site-specific labeling of single-domain antibodies or viral proteins with a newly developed, cathepsin-sensitive quenched-fluorophore probe. Quenched probes of this type have been used to measure enzyme activity in complex environments and for different cell types, but not as a sensor of protein movement into living cells. This approach allows a quantitative assessment of the movement of proteins into protease-containing endosomes in real time in living cells. We demonstrate considerable variation in the rate of endosomal delivery for different cell surface receptors. We were also able to characterize the kinetics of influenza virus delivery to cathepsin-positive compartments, showing highly coordinated arrival in endosomal compartments. This approach should be useful for identifying proteins expressed on cells of interest for targeted endosomal delivery of payloads, such as antibody-drug conjugates or antigens that require processing.

Nanobody-Antigen Conjugates Elicit HPV-Specific Antitumor Immune Responses.

High-risk human papillomavirus-associated cancers express viral oncoproteins (e.g., E6 and E7) that induce and maintain the malignant phenotype. The viral origin of these proteins makes them attractive targets for development of a therapeutic vaccine. Camelid-derived single-domain antibody fragments (nanobodies or VHHs) that recognize cell surface proteins on antigen-presenting cells (APC) can serve as targeted delivery vehicles for antigens attached to them. Such VHHs were shown to induce CD4+ and CD8+ T-cell responses against model antigens conjugated to them via sortase, but antitumor responses had not yet been investigated. Here, we tested the ability of an anti-CD11b VHH (VHHCD11b) to target APCs and serve as the basis for a therapeutic vaccine to induce CD8+ T-cell responses against HPV+ tumors. Mice immunized with VHHCD11b conjugated to an H-2Db-restricted immunodominant E7 epitope (E749-57) had more E7-specific CD8+ T cells compared with those immunized with E749-57 peptide alone. These CD8+ T cells acted prophylactically and conferred protection against a subsequent challenge with HPV E7-expressing tumor cells. In a therapeutic setting, VHHCD11b-E749-57 vaccination resulted in greater numbers of CD8+ tumor-infiltrating lymphocytes compared with mice receiving E749-57 peptide alone in HPV+ tumor-bearing mice, as measured by in vivo noninvasive VHH-based immune-positron emission tomography (immunoPET), which correlated with tumor regression and survival outcome. Together, these results demonstrate that VHHs can serve as a therapeutic cancer vaccine platform for HPV-induced cancers. Cancer Immunol Res; 6(7); 870-80. ©2018 AACR.

An RNA nanoparticle vaccine against Zika virus elicits antibody and CD8+ T cell responses in a mouse model.

The Zika virus (ZIKV) outbreak in the Americas and South Pacific poses a significant burden on human health because of ZIKV's neurotropic effects in the course of fetal development. Vaccine candidates against ZIKV are coming online, but immunological tools to study anti-ZIKV responses in preclinical models, particularly T cell responses, remain sparse. We deployed RNA nanoparticle technology to create a vaccine candidate that elicited ZIKV E protein-specific IgG responses in C57BL/6 mice as assayed by ELISA. Using this tool, we identified a unique H-2Db-restricted epitope to which there was a CD8+ T cell response in mice immunized with our modified dendrimer-based RNA nanoparticle vaccine. These results demonstrate that this approach can be used to evaluate new candidate antigens and identify immune correlates without the use of live virus.

Annexin A2 antibodies but not inhibitors of the annexin A2 heterotetramer impair productive HIV-1 infection of macrophages in vitro.

During sexual transmission of human immunodeficiency virus (HIV), macrophages are initial targets for HIV infection. Secretory leukocyte protease inhibitor (SLPI) has been shown to protect against HIV infection of macrophages through interactions with annexin A2 (A2), which is found on the macrophage cell surface as a heterotetramer (A2t) consisting of A2 and S100A10. Therefore, we investigated potential protein-protein interactions between A2 and HIV-1 gp120 through a series of co-immunoprecipitation assays and a single molecule pulldown (SiMPull) technique. Additionally, inhibitors of A2t (A2ti) that target the interaction between A2 and S100A10 were tested for their ability to impair productive HIV-1 infection of macrophages. Our data suggest that interactions between HIV-1 gp120 and A2 exist, though this interaction may be indirect. Furthermore, an anti-A2 antibody impaired HIV-1 particle production in macrophages in vitro, whereas A2ti did not indicating that annexin A2 may promote HIV-1 infection of macrophages in its monomeric rather than tetrameric form.

Isoaspartylation appears to trigger small cell lung cancer-associated autoimmunity against neuronal protein ELAVL4.

Autoantibodies against SCLC-associated neuronal antigen ELAVL4 (HuD) have been linked to smaller tumors and improved survival, but the antigenic epitope and mechanism of autoimmunity have never been solved. We report that recombinant human ELAVL4 protein incubated under physiological conditions acquires isoaspartylation, a type of immunogenic protein damage. Specifically, the N-terminal region of ELAVL4, previously implicated in SCLC-associated autoimmunity, undergoes isoaspartylation in vitro, is recognized by sera from anti-ELAVL4 positive SCLC patients and is highly immunogenic in subcutaneously injected mice and in vitro stimulated human lymphocytes. Our data suggest that isoaspartylated ELAVL4 is the trigger for the SCLC-associated anti-ELAVL4 autoimmune response.

Human Immunodeficiency Virus Immune Cell Receptors, Coreceptors, and Cofactors: Implications for Prevention and Treatment.

In the last three decades, extensive research on human immunodeficiency virus (HIV) has highlighted its capability to exploit a variety of strategies to enter and infect immune cells. Although CD4(+) T cells are well known as the major HIV target, with infection occurring through the canonical combination of the cluster of differentiation 4 (CD4) receptor and either the C-C chemokine receptor type 5 (CCR5) or C-X-C chemokine receptor type 4 (CXCR4) coreceptors, HIV has also been found to enter other important immune cell types such as macrophages, dendritic cells, Langerhans cells, B cells, and granulocytes. Interestingly, the expression of distinct cellular cofactors partially regulates the rate in which HIV infects each distinct cell type. Furthermore, HIV can benefit from the acquisition of new proteins incorporated into its envelope during budding events. While several publications have investigated details of how HIV manipulates particular cell types or subtypes, an up-to-date comprehensive review on HIV tropism for different immune cells is lacking. Therefore, this review is meant to focus on the different receptors, coreceptors, and cofactors that HIV exploits to enter particular immune cells. Additionally, prophylactic approaches that have targeted particular molecules associated with HIV entry and infection of different immune cells will be discussed. Unveiling the underlying cellular receptors and cofactors that lead to HIV preference for specific immune cell populations is crucial in identifying novel preventative/therapeutic targets for comprehensive strategies to eliminate viral infection.

Secretory Leukocyte Protease Inhibitor Expression and High-Risk HPV Infection in Anal Lesions of HIV-Positive Patients.

OBJECTIVE: The aim of this study was to evaluate secretory leukocyte protease inhibitor (SLPI) expression in anal biopsies from HIV-positive (HIV+) individuals, and compare that to anal intraepithelial neoplasia (AIN) diagnoses and human papillomavirus (HPV) status. DESIGN: This is a cross-sectional study of a cohort of 54 HIV+ (31 males and 23 females) from an AIDS clinic in Rio de Janeiro, Brazil. METHODS: The study material consisted of anorectal tissue biopsies obtained from HIV+ subjects, which were used to construct tissue microarray paraffin blocks for immunohistochemical analysis of SLPI expression. Biopsies were evaluated by an expert pathologist and classified as low-grade AIN1, high-grade AIN2/3, or normal squamous epithelium. In addition, DNA from the biopsies was extracted and analyzed for the presence of low- or high-risk HPV DNA. RESULTS: Histologically, normal squamous epithelium from the anorectal region showed strong positive SLPI staining in 17/20 (85%) samples. In comparison, 9/17 (53%) dysplastic squamous epithelial samples from AIN1 patients showed strong SLPI staining, and only 5/17 (29%) samples from AIN2/3 patients exhibited strong SPLI staining, which both were significantly fewer than those from normal tissue (P = 0.005). Furthermore, there was a significantly higher proportion of samples in which oncogenic high-risk HPV genotypes were detected in low SLPI-expressing tissues than that in tissues with high SLPI expression (P = 0.040). CONCLUSIONS: Taken together these results suggest that low SLPI expression is associated with high-risk HPV infections in the development of AIN.

T cell ignorance is bliss: T cells are not tolerized by Langerhans cells presenting human papillomavirus antigens in the absence of costimulation.

Human papillomavirus type 16 (HPV16) infections are intra-epithelial, and thus, HPV16 is known to interact with Langerhans cells (LCs), the resident epithelial antigen-presenting cells (APCs). The current paradigm for APC-mediated induction of T cell anergy is through delivery of T cell receptor signals via peptides on MHC molecules (signal 1), but without costimulation (signal 2). We previously demonstrated that LCs exposed to HPV16 in vitro present HPV antigens to T cells without costimulation, but it remained uncertain if such T cells would remain ignorant, become anergic, or in the case of CD4+ T cells, differentiate into Tregs. Here we demonstrate that Tregs were not induced by LCs presenting only signal 1, and through a series of in vitro immunizations show that CD8+ T cells receiving signal 1 + 2 from LCs weeks after consistently receiving signal 1 are capable of robust effector functions. Importantly, this indicates that T cells are not tolerized but instead remain ignorant to HPV, and are activated given the proper signals.

Current therapeutic vaccination and immunotherapy strategies for HPV-related diseases.

Carcinomas of the anogenital tract, in particular cervical cancer, remains one of the most common cancers in women, and represent the most frequent gynecological malignancies and the fourth leading cause of cancer death in women worldwide. Human papillomavirus (HPV)-induced lesions are immunologically distinct in that they express viral antigens, which are necessary to maintain the cancerous phenotype. The causal relationship between HPV infection and anogenital cancer has prompted substantial interest in the development of therapeutic vaccines against high-risk HPV types targeting the viral oncoproteins E6 and E7. This review will focus on the most recent clinical trials for immunotherapies for mucosal HPV-induced lesions as well as emerging therapeutic strategies that have been tested in pre-clinical models for HPV-induced diseases. Progress in peptide- and protein-based vaccines, DNA-based vaccines, viral/bacterial vector-based vaccines, immune checkpoint inhibition, immune response modifiers, and adoptive cell therapy for HPV will be discussed.

Herpes simplex virus downregulation of secretory leukocyte protease inhibitor enhances human papillomavirus type 16 infection.

Herpes simplex virus (HSV) was originally implicated in the aetiology of cervical cancer, and although high-risk human papillomavirus (HPV) is now the accepted causative agent, the epidemiological link between HSV and HPV-associated cancers persists. The annexin A2 heterotetramer (A2t) has been shown to mediate infectious HPV type 16 (HPV16) uptake by human keratinocytes, and secretory leukocyte protease inhibitor (SLPI), an endogenous A2t ligand, inhibits HPV16 uptake and infection. Interestingly, HSV infection induces a sustained downregulation of SLPI in epithelial cells, which we hypothesized promotes HPV16 infection through A2t. Here, we show that in vitro infection of human keratinocytes with HSV-1 or HSV-2, but not with an HSV-1 ICP4 deletion mutant that does not downregulate SLPI, leads to a >70% reduction of SLPI mRNA and a >60% decrease in secreted SLPI protein. Consequently, we observed a significant increase in the uptake of HPV16 virus-like particles and gene transduction by HPV16 pseudovirions (two- and 2.5-fold, respectively) in HSV-1- and HSV-2-infected human keratinocyte cell cultures compared with uninfected cells, whereas exogenously added SLPI reversed this effect. Using a SiMPull (single-molecule pulldown) assay, we demonstrated that endogenously secreted SLPI interacts with A2t on epithelial cells in an autocrine/paracrine manner. These results suggested that ongoing HSV infection and resultant downregulation of local levels of SLPI may impart a greater susceptibility for keratinocytes to HPV16 infection through the host cell receptor A2t, providing a mechanism that may, in part, provide an explanation for the aetiological link between HSV and HPV-associated cancers.

Immunostimulatory Activity of the Cytokine-Based Biologic, IRX-2, on Human Papillomavirus-Exposed Langerhans Cells.

Langerhans cells (LCs) are the antigen-presenting cells of the epithelial layer and are responsible for initiating immune responses against skin and mucosa-invading viruses. Human papillomavirus (HPV)-mediated suppression of LC function is a crucial mechanism of HPV immune evasion, which can lead to persistent infection and development of several human cancers, including cervical, anal, and head and neck cancers. The cell-derived cytokine-based biologic, IRX-2, consists of multiple well-defined cytokines and is broadly active on various immune cell subsets. In this study, we investigated primary human LC activation after exposure to HPV16, followed by treatment with IRX-2 in vitro, and evaluated their subsequent ability to induce HPV16-specific T cells. In contrast to its activity on dendritic cells, HPV16 alone is not sufficient to induce phenotypic and functional activation of LCs. However, IRX-2 induces a significant upregulation of antigen presentation and costimulatory molecules, T helper 1 (Th1)-associated cytokine release, and chemokine-directed migration of LCs pre-exposed to HPV16. Furthermore, LCs treated with IRX-2 after HPV16 exposure induced CD8(+) T-cell responses against specific HLA-A*0201-binding HPV16 T-cell epitopes. The present study suggests that IRX-2 is an attractive immunomodulator for assisting the immune response in eradication of HPV-infected cells, thereby potentially preventing HPV-induced cancers.

Human papillomavirus-exposed Langerhans cells are activated by stabilized Poly-I:C.

Human papillomaviruses (HPV) establish persistent infections because of evolved immune evasion mechanisms, particularly HPV-mediated suppression of the immune functions of Langerhans cells (LC), the antigen presenting cells of the epithelium. Polyinosinic-polycytidilic acid (Poly-I:C) is broadly immunostimulatory with the ability to enhance APC expression of costimulatory molecules and inflammatory cytokines resulting in T cell activation. Here we investigated the activation of primary human LC derived from peripheral blood monocytes after exposure to HPV16 virus like particles followed by treatment with stabilized Poly-I:C compounds (s-Poly-I:C), and their subsequent induction of HPV16-specific T cells. Our results indicate that HPV16 particles alone were incapable of inducing LC activation as demonstrated by the lack of costimulatory molecules, inflammatory cytokines, chemokine-directed migration, and HPV16-specific CD8+ T cells in vitro. Conversely, s-Poly-I:C caused significant upregulation of costimulatory molecules and induction of chemokine-directed migration of LC that were pre-exposed to HPV16. In HLA-A*0201-positive donors, s-Poly-I:C treatment was able to induce CD8+ T cell immune responses against HPV16-derived peptides. Thus, s-Poly-I:C compounds are attractive for translation into therapeutics in which they could potentially mediate clearance of persistent HPV infection.

Langerhans cells from women with cervical precancerous lesions become functionally responsive against human papillomavirus after activation with stabilized Poly-I:C.

Human papillomavirus (HPV)-mediated suppression of Langerhans cell (LC) function can lead to persistent infection and development of cervical intraepithelial neoplasia (CIN). Women with HPV-induced high-grade CIN2/3 have not mounted an effective immune response against HPV, yet it is unknown if LC-mediated T cell activation from such women is functionally impaired against HPV. We investigated the functional activation of in vitro generated LC and their ability to induce HPV16-specific T cells from CIN2/3 patients after exposure to HPV16 followed by treatment with stabilized Poly-I:C (s-Poly-I:C). LC from patients exposed to HPV16 demonstrated a lack of costimulatory molecule expression, inflammatory cytokine secretion, and chemokine-directed migration. Conversely, s-Poly-I:C caused significant phenotypic and functional activation of HPV16-exposed LC, which resulted in de novo generation of HPV16-specific CD8(+) T cells. Our results highlight that LC of women with a history of persistent HPV infection can present HPV antigens and are capable of inducing an adaptive T cell immune response when given the proper stimulus, suggesting that s-Poly-I:C compounds may be attractive immunomodulators for LC-mediated clearance of persistent HPV infection.

A novel murine model for evaluating bovine papillomavirus prophylactics/therapeutics for equine sarcoid-like tumours.

Equine sarcoids are highly recurrent bovine papillomavirus (BPV)-induced fibroblastic neoplasms that are the most common skin tumours in horses. In order to facilitate the study of potential equine sarcoid prophylactics or therapeutics, which can be a slow and costly process in equines, a murine model for BPV-1 protein-expressing equine sarcoid-like tumours was developed in mice through stable transfection of BPV-1 E5 and E6 in a murine fibroblast tumour cell line (K-BALB). Like equine sarcoids, these murine tumour cells (BPV-KB) were of fibroblast origin, were tumorigenic and expressed BPV-1 proteins. As an initial investigation of the preclinical potential of this tumour model for equine sarcoids prophylactics, mice were immunized with BPV-1 E5E6 Venezuelan equine encephalitis virus replicon particles, prior to BPV-KB challenge, which resulted in an increased tumour-free period compared with controls, indicating that the BPV-KB murine model may be a valuable preclinical alternative to equine clinical trials.