Influenza A virus replicates productively in primary human kidney cells and induces factors and mechanisms related to regulated cell death and renal pathology observed in virus-infected patients.

Introduction: Influenza A virus (IAV) infection can cause the often-lethal acute respiratory distress syndrome (ARDS) of the lung. Concomitantly, acute kidney injury (AKI) is frequently noticed during IAV infection, correlating with an increased mortality. The aim of this study was to elucidate the interaction of IAV with human kidney cells and, thereby, to assess the mechanisms underlying IAV-mediated AKI. Methods: To investigate IAV effects on nephron cells we performed infectivity assays with human IAV, as well as with human isolates of either low or highly pathogenic avian IAV. Also, transcriptome and proteome analysis of IAV-infected primary human distal tubular kidney cells (DTC) was performed. Furthermore, the DTC transcriptome was compared to existing transcriptomic data from IAV-infected lung and trachea cells. Results: We demonstrate productive replication of all tested IAV strains on primary and immortalized nephron cells. Comparison of our transcriptome and proteome analysis of H1N1-type IAV-infected human primary distal tubular cells (DTC) with existing data from H1N1-type IAV-infected lung and primary trachea cells revealed enrichment of specific factors responsible for regulated cell death in primary DTC, which could be targeted by specific inhibitors. Discussion: IAV not only infects, but also productively replicates on different human nephron cells. Importantly, multi-omics analysis revealed regulated cell death as potential contributing factor for the clinically observed kidney pathology in influenza.

HIV-1 Infection of Long-Lived Hematopoietic Precursors In Vitro and In Vivo.

Latent reservoirs in human-immunodeficiency-virus-1 (HIV-1)-infected individuals represent a major obstacle in finding a cure for HIV-1. Hematopoietic stem and progenitor cells (HSPCs) have been described as potential HIV-1 targets, but their roles as HIV-1 reservoirs remain controversial. Here we provide additional evidence for the susceptibility of several distinct HSPC subpopulations to HIV-1 infection in vitro and in vivo. In vitro infection experiments of HSPCs were performed with different HIV-1 Env-pseudotyped lentiviral particles and with replication-competent HIV-1. Low-level infection/transduction of HSPCs, including hematopoietic stem cells (HSCs) and multipotent progenitors (MPP), was observed, preferentially via CXCR4, but also via CCR5-mediated entry. Multi-lineage colony formation in methylcellulose assays and repetitive replating of transduced cells provided functional proof of susceptibility of primitive HSPCs to HIV-1 infection. Further, the access to bone marrow samples from HIV-positive individuals facilitated the detection of HIV-1 gag cDNA copies in CD34+ cells from eight (out of eleven) individuals, with at least six of them infected with CCR5-tropic HIV-1 strains. In summary, our data confirm that primitive HSPC subpopulations are susceptible to CXCR4- and CCR5-mediated HIV-1 infection in vitro and in vivo, which qualifies these cells to contribute to the HIV-1 reservoir in patients.

Tumours involving the retrobulbar space in cats: 37 cases.

OBJECTIVES: The aim of this multicentre retrospective study was to describe the clinical presentation, imaging findings, diagnosis and outcomes of cats with retrobulbar neoplasia. METHODS: A total of 37 cats that were diagnosed with retrobulbar neoplasia and underwent advanced imaging were recruited from searches of the clinical records of two referral hospitals. All cats had neoplasia confirmed via cytology or histopathology. Data relating to the signalment, presentation, results of investigations, treatment and outcome were recorded. A review of imaging studies was performed where possible. RESULTS: In total, 23 cases (62%) were presented with respiratory signs. Exophthalmos was the most common ophthalmological examination finding, present in 18 cases (49%). Thirty-two cases (86%) had secondary extension of neoplasia to the retrobulbar space (most commonly from the nasal cavities), present in 20 cases (54%), of which 12 were lymphoma. In cases where contrast was administered, 28/35 (80%) had contrast-enhancing masses. Orbital extension was detected in 21 cases (57%), exophthalmos in 22 (59%), globe deformation in 12 (32%) and local lymphadenomegaly in 22 (61%). In total, 36 (97%) retrobulbar tumours were malignant. Thoracic imaging, where it was performed, was concerning for metastasis in 8/25 cases (31%), with abdominal imaging suggestive of metastasis in 5/12 (42%). The most common diagnosis was lymphoma with 19 cases (51%), with nasal lymphoma representing 12 of these, followed by carcinoma in 10 (27%). The median survival time, for cases where death was recorded, was 85 days (range 1-263 days). CONCLUSIONS AND RELEVANCE: To the authors' knowledge, this is the largest study of neoplasia affecting the feline retrobulbar space. Retrobulbar tumours in cats are overwhelmingly malignant, and commonly due to secondary extension of tumours originating elsewhere. Lymphoma, particularly arising from the nasal cavities, was the most common cause. Cats presenting with signs suggestive of retrobulbar disease should be assessed for disease affecting any of the structures of the head.

Changes in retinal vascular diameters in senior and geriatric cats in association with variation in systemic blood pressure.

OBJECTIVES: Early diagnosis of arterial hypertension is essential to prevent target organ damage. In humans, retinal arteriolar narrowing predicts hypertension. This blinded prospective observational study investigated the retinal vessel diameters in senior and geriatric cats of varying systolic blood pressure (SBP) status and evaluated retinal vascular changes in hypertensive cats after treatment. METHODS: Cats with a median age of 14 years (range 9.1-22 years) were categorised into five groups: group 1, healthy normotensive (SBP <140 mmHg; n = 40) cats; group 2, pre-hypertensive (SBP 140-160 mmHg; n = 14) cats; group 3, cats with chronic kidney disease (CKD) and normotensive (n = 26); group 4, cats with CKD and pre-hypertensive (n = 13); and group 5, hypertensive cats (SBP >160 mmHg, n = 15). Colour fundus images (Optibrand ClearView) were assessed for hypertensive lesions. Retinal vascular diameters and bifurcation angles were annotated and calculated using the Vascular Assessment and Measurement Platform for Images of the Retina annotation tool (VAMPIRE-AT). When available, measurements were obtained at 3 and 6 months after amlodipine besylate treatment. RESULTS: Ten hypertensive cats had retinal lesions, most commonly intraretinal haemorrhages and retinal exudates. Arteriole and venule diameters decreased significantly with increasing age (-0.17 ± 0.05 pixels/year [P = 0.0004]; -0.19 ± 0.05 pixels/year). Adjusted means ± SEM for arteriole and venule diameter (pixels) were 6.3 ± 0.2 and 8.9 ± 0.2 (group 1); 7.6 ± 0.3 and 10.1 ± 0.4 (group 2); 6.9 ± 0.2 and 9.5 ± 0.3 (group 3); 7.4 ± 0.3 and 10.0 ± 0.4 (group 4); and 7.0 ± 0.3 and 9.8 ± 0.4 (group 5). Group 1 arteriole and venule diameters were significantly lower than those of groups 2 and 4. Group 2 arteriole bifurcation angle was significantly narrower than those of groups 1 and 3. Post-treatment, vessel diameters decreased significantly at 3 and 6 months in seven hypertensive cats. CONCLUSIONS AND RELEVANCE: Increased age was associated with reduced vascular diameters. Longitudinal studies are required to assess if vessel diameters are a risk indicator for hypertension in cats.

Lactobacilli Expressing Broadly Neutralizing Nanobodies against HIV-1 as Potential Vectors for HIV-1 Prophylaxis?

In the absence of an active prophylactic vaccine against HIV-1, passively administered, broadly neutralizing antibodies (bnAbs) identified in some chronically infected persons were shown to prevent HIV-1 infection in animal models. However, passive administration of bnAbs may not be suited to prevent sexual HIV-1 transmission in high-risk cohorts, as a continuous high level of active bnAbs may be difficult to achieve at the primary site of sexual transmission, the human vagina with its acidic pH. Therefore, we used Lactobacillus, a natural commensal in the healthy vaginal microbiome, to express bn nanobodies (VHH) against HIV-1 that we reported previously. After demonstrating that recombinant VHHA6 expressed in E. coli was able to protect humanized mice from mucosal infection by HIV-1Bal, we expressed VHHA6 in a soluble or in a cell-wall-anchored form in Lactobacillus rhamnosus DSM14870. This strain is already clinically applied for treatment of bacterial vaginosis. Both forms of VHHA6 neutralized a set of primary epidemiologically relevant HIV-1 strains in vitro. Furthermore, VHHA6 was still active at an acidic pH. Thus, lactobacilli expressing bn VHH potentially represent an attractive vector for the passive immunization of women in cohorts at high risk of HIV-1 transmission.

Advances in Antibody-Based HIV-1 Vaccines Development.

Despite the great success of antiretroviral therapy, both in the treatment and prevention of HIV-1 infection, a vaccine is still urgently needed to end the epidemic [...].

The Hard Way towards an Antibody-Based HIV-1 Env Vaccine: Lessons from Other Viruses.

Although effective antibody-based vaccines have been developed against multiple viruses, such approaches have so far failed for the human immunodeficiency virus type 1 (HIV-1). Despite the success of anti-retroviral therapy (ART) that has turned HIV-1 infection into a chronic disease and has reduced the number of new infections worldwide, a vaccine against HIV-1 is still urgently needed. We discuss here the major reasons for the failure of "classical" vaccine approaches, which are mostly due to the biological properties of the virus itself. HIV-1 has developed multiple mechanisms of immune escape, which also account for vaccine failure. So far, no vaccine candidate has been able to induce broadly neutralizing antibodies (bnAbs) against primary patient viruses from different clades. However, such antibodies were identified in a subset of patients during chronic infection and were shown to protect from infection in animal models and to reduce viremia in first clinical trials. Their detailed characterization has guided structure-based reverse vaccinology approaches to design better HIV-1 envelope (Env) immunogens. Furthermore, conserved Env epitopes have been identified, which are promising candidates in view of clinical applications. Together with new vector-based technologies, considerable progress has been achieved in recent years towards the development of an effective antibody-based HIV-1 vaccine.

Lectin Affinity Plasmapheresis for Middle East Respiratory Syndrome-Coronavirus and Marburg Virus Glycoprotein Elimination.

BACKGROUND/AIMS: Middle East respiratory syndrome coronavirus (MERS-CoV) and Marburg virus (MARV) are among the World Health Organization's top 8 emerging pathogens. Both zoonoses share nonspecific early symptoms, a high lethality rate, and a reduced number of specific treatment options. Therefore, we evaluated extracorporeal virus and glycoprotein (GP) elimination by lectin affinity plasmapheresis (LAP). METHODS: For both MERS-CoV (pseudovirus) as well as MARV (GPs), 4 LAP devices (Mini Hemopurifiers, Aethlon Medical, San Diego, CA, USA) and 4 negative controls were tested. Samples were collected every 30 min and analyzed for reduction in virus infectivity by a flow cytometry-based infectivity assay (MERS-CoV) and in soluble GP content (MARV) by an immunoassay. RESULTS: The experiments show a time-dependent clearance of MERS-CoV of up to 80% within 3 h (pseudovirus). Up to 70% of MARV-soluble GPs were eliminated at the same time. Substantial saturation of the binding resins was detected within the first treatment hour. CONCLUSION: MERS-CoV (pseudovirus) and MARV soluble GPs are eliminated by LAP in vitro. Considering the high lethality and missing established treatment options, LAP should be evaluated in vivo. Especially early initiation, continuous therapy, and timed cartridge exchanges could be of importance.

Clinical outcome of concomitant vs interrupted BRAF inhibitor therapy during radiotherapy in melanoma patients.

This corrects the article DOI: 10.1038/bjc.2017.85.

Optimization of the EC26-2A4 Epitope in the gp41 Membrane Proximal External Region Targeted by Neutralizing Antibodies from an Elite Controller.

The analysis of patient derived HIV neutralizing antibodies (nAbs) and their target epitopes in the viral envelope (Env) protein provides important basic information for vaccine design. In this study we optimized an epitope, EC26-2A4, that is targeted by neutralizing antibodies from an elite controller (EC26) and localizes in the membrane-proximal external region from the gp41 transmembrane protein. Due to its overlap with the epitope of the first generation broadly neutralizing monoclonal Ab (mAb) 2F5 associated with autoreactivity, we first defined the minimal core epitope reacting with antibodies from EC26 plasma, but not with mAb 2F5. The optimized minimal epitope, EC26-2A4ΔM, was able to induce neutralizing antibodies in vaccinated mice. We further analyzed the frequency of antibodies against the EC26-2A4ΔM peptide in HIV-positive patient sera from a treated cohort and an untreated long-term nonprogressor (LTNP) cohort. Interestingly, 27% of the LTNP sera reacted with the peptide, whereas only 9% showed reactivity in the treated cohort. Although there was no association between the presence of antibodies against the EC26-2A4ΔM epitope and viral load or CD4 count in these patients, the CD4 nadir in the treated cohort was higher in patients positive for EC26-2A4ΔM antibodies, in particular in patients having such antibodies at an early and a late timepoint after infection.

Selection of nanobodies with broad neutralizing potential against primary HIV-1 strains using soluble subtype C gp140 envelope trimers.

Broadly neutralizing antibodies (bnAbs) against HIV-1 protect from infection and reduce viral load upon therapeutic applications. However no vaccine was able so far to induce bnAbs demanding their expensive biotechnological production. For clinical applications, nanobodies (VHH) derived from heavy chain only antibodies from Camelidae, may be better suited due to their small size, high solubility/stability and extensive homology to human VH3 genes. Here we selected broadly neutralizing nanobodies by phage display after immunization of dromedaries with different soluble trimeric envelope proteins derived from HIV-1 subtype C. We identified 25 distinct VHH families binding trimeric Env, of which 6 neutralized heterologous primary isolates of various HIV-1 subtypes in a standardized in vitro neutralization assay. The complementary neutralization pattern of two selected VHHs in combination covers 19 out of 21 HIV-1 strains from a standardized panel of epidemiologically relevant HIV-1 subtypes. The CD4 binding site was preferentially targeted by the broadly neutralizing VHHs as determined by competition ELISAs and 3D models of VHH-Env complexes derived from negative stain electron microscopy. The nanobodies identified here are excellent candidates for further preclinical/clinical development for prophylactic and therapeutic applications due to their potency and their complementary neutralization patterns covering the majority of epidemiologically relevant HIV-1 subtypes.

Biocompatibility of a Novel Microfistula Implant in Nonprimate Mammals for the Surgical Treatment of Glaucoma.

PURPOSE: The purpose of this study was to evaluate the ocular safety of a novel microfistula implant and its composite materials in an animal model. METHODS: The anterior chambers of 12 rabbit eyes were injected with either glutaraldehyde cross-linked porcine gelatin extract or balanced salt solution and were followed by serial slit lamp examinations over 3 days. The eyes of 18 canines underwent microfistula implantation or a sham procedure. The animals were monitored over the subsequent 12 months, using serial slit lamp examinations, indirect ophthalmoscopy, tonometry, specular microscopy, and high-resolution ultrasonography. Ocular tissues were examined histopathologically on postoperative days 7, 30, 90, 180, and 365. RESULTS: Glutaraldehyde cross-linked porcine gelatin did not induce significant intraocular inflammation in the rabbit model. The microfistula implant was well tolerated and did not stimulate significant tissue response in the canine eye. The microfistula tube did not undergo structural change or degradation over the course of the study. CONCLUSIONS: In nonprimate mammals, the material composing the microfistula implant and the implant itself do not induce significant inflammation or tissue reaction.

Cutaneous, gastrointestinal, hepatic, endocrine, and renal side-effects of anti-PD-1 therapy.

BACKGROUND: Anti-programmed cell death receptor-1 (PD-1) antibodies represent an effective treatment option for metastatic melanoma as well as for other cancer entities. They act via blockade of the PD-1 receptor, an inhibitor of the T-cell effector mechanisms that limit immune responses against tumours. As reported for ipilimumab, the anti-PD-1 antibodies pembrolizumab and nivolumab can induce immune-related adverse events (irAEs). These side-effects affect skin, gastrointestinal tract, liver, endocrine system and other organ systems. Since life-threatening and fatal irAEs have been reported, adequate diagnosis and management are essential. METHODS AND FINDINGS: In total, 496 patients with metastatic melanoma from 15 skin cancer centers were treated with pembrolizumab or nivolumab; 242 side-effects were described in 138 patients. In 116 of the 138 patients, side-effects affected the skin, gastrointestinal tract, liver, endocrine, and renal system. Rare side-effects included diabetes mellitus, lichen planus, and pancreas insufficiency due to pancreatitis. CONCLUSION: Anti-PD1 antibodies can induce a plethora of irAEs. The knowledge of them will allow prompt diagnosis and improve the management resulting in decreased morbidity.

Neurological, respiratory, musculoskeletal, cardiac and ocular side-effects of anti-PD-1 therapy.

BACKGROUND: Anti-programmed cell death 1 (PD-1) antibodies represent an effective treatment option for metastatic melanoma and other cancer entities. They act via blockade of the PD-1 receptor, an inhibitor of the T-cell effector mechanisms that limit immune responses against tumours. As reported for ipilimumab, the anti-PD-1 antibodies pembrolizumab and nivolumab can induce immune-related adverse events (irAEs). These side-effects can involve skin, gastrointestinal tract, liver, the endocrine system and other organ systems. Since life-threatening and fatal irAEs have been reported, adequate diagnosis and management are essential. METHODS AND FINDINGS: In total, 496 patients with metastatic melanoma from 15 skin cancer centres were treated with pembrolizumab or nivolumab. Two hundred forty two side-effects in 138 patients have been analysed. In 77 of the 138 patients side-effects affected the nervous system, respiratory tract, musculoskeletal system, heart, blood and eyes. Not yet reported side-effects such as meningo-(radiculitis), polyradiculitis, cardiac arrhythmia, asystolia, and paresis have been observed. Rare and difficult to manage side-effects such as myasthenia gravis are described in detail. CONCLUSION: Anti-PD-1 antibodies can induce a plethora of irAEs. The knowledge of them will allow prompt diagnosis and improve the management resulting in decreased morbidity.

Systemic therapy of metastatic melanoma.

For patients with metastatic melanoma, there are currently several effective therapeutic options. The BRAF inhibitors vemurafenib and dabrafenib are characterized by rapid tumor control and high response rates. In combination with one of the two MEK inhibitors trametinib and cobimetinib, they achieve response rates (CR + PR, complete plus partial remissions) of 70%, while delaying the development of treatment resistance, as well as a median overall survival of > 2 years with tolerable side effects. Showing long-term survival rates of approximately 20%, the anti-CTLA-4 antibody ipilimumab is the first substance that has led to a significant prolongation of overall survival in patients with metastatic melanoma. However, delayed treatment response and severe immune-mediated side effects may pose limitations to its therapeutic benefit. Usually well tolerated, anti-PD-1 antibody monotherapy using nivolumab and pembrolizumab has yielded response rates (CR + PR) of up to 45% and one-year survival rates of > 70%. The combination of ipilimumab and nivolumab has shown response rates of up to 58% and a median progression-free survival of > 11 months. While this combination is expected to result in a rapid and long-lasting response, this potential benefit comes at the expense of a high level of toxicity. Strategies for treatment sequencing and treatment combinations are currently being investigated in clinical studies. Overall, the prognosis for patients with metastatic melanoma has significantly improved. With long-term survival a possibility, not only acute but also long-term therapeutic side effects must be taken into account.

In Silico Prediction and Experimental Confirmation of HA Residues Conferring Enhanced Human Receptor Specificity of H5N1 Influenza A Viruses.

Newly emerging influenza A viruses (IAV) pose a major threat to human health by causing seasonal epidemics and/or pandemics, the latter often facilitated by the lack of pre-existing immunity in the general population. Early recognition of candidate pandemic influenza viruses (CPIV) is of crucial importance for restricting virus transmission and developing appropriate therapeutic and prophylactic strategies including effective vaccines. Often, the pandemic potential of newly emerging IAV is only fully recognized once the virus starts to spread efficiently causing serious disease in humans. Here, we used a novel phylogenetic algorithm based on the informational spectrum method (ISM) to identify potential CPIV by predicting mutations in the viral hemagglutinin (HA) gene that are likely to (differentially) affect critical interactions between the HA protein and target cells from bird and human origin, respectively. Predictions were subsequently validated by generating pseudotyped retrovirus particles and genetically engineered IAV containing these mutations and characterizing potential effects on virus entry and replication in cells expressing human and avian IAV receptors, respectively. Our data suggest that the ISM-based algorithm is suitable to identify CPIV among IAV strains that are circulating in animal hosts and thus may be a new tool for assessing pandemic risks associated with specific strains.

Catch Me If You Can--The Race Between HIV and Neutralizing Antibodies.

Broadly neutralizing antibodies represent the major protective mechanism of vaccines targeting pathogenic microbes in humans and animals. For HIV, broadly neutralizing antibodies have also been shown to be protective in experimental animal models. However, despite the identification of a respectable number of broadly neutralizing antibodies from chronically infected HIV-positive persons in recent years, attempts to induce such antibodies by vaccines have generally failed over the last decades. Though unsuccessful in view of achieving a protective vaccine against HIV, many of these studies have contributed significantly to the understanding of the generation of broadly neutralizing antibodies against HIV-1 as well as to the vulnerable sites they target on the surface of the virus. Here we review the most important features of patient-derived broadly neutralizing antibodies, the long and complex B-cell maturation pathways required for their production, and the resulting consequences for vaccine development. We further address characteristics of the epitopes targeted by broadly neutralizing antibodies on the virus surface as well as mechanisms of viral escape. Taken together, the identification of vaccine candidates able to induce broadly neutralizing antibodies against HIV-1 is the major challenge in HIV vaccine development. Mutual coevolution of rationally designed HIV vaccine candidates, with affinity maturation pathways of antibodies they induce upon vaccination, may best mimic the natural situation of chronically HIV-infected patients who are able to generate broadly neutralizing antibodies.

Genetic characterization of near full length SIVdrl genomes from four captive drills (Mandrillus leucophaeus).

We sequenced near full length SIVdrl genomes from four captive drills (Mandrillus leucophaeus). All four animals were born in captivity in German zoos. Although serologically SIV negative before acquisition in zoo A in 2008 and 2009, during a routine analysis all four animals were determined to be SIV antibody positive in 2011. Comparisons of the four new SIVdrl sequences showed high identity among each other (90.7-97.7% in env) and to the only published full length sequence SIVdrl FAO (90.5-92.8% in env), which is also derived from a captive drill. SIVdrl infections seem to be highly prevalent in captive drills, probably resulting from frequent animal transfers between the zoos in an effort to maintain this highly endangered species and its genetic diversity. This should be kept in mind as SIVdrl may be transmitted to uninfected animals in open groups and potentially also to animal keepers having contact with these nonhuman primates.

Functional and structural characterization of 2-amino-4-phenylthiazole inhibitors of the HIV-1 nucleocapsid protein with antiviral activity.

The nucleocapsid protein (NC) is a highly conserved protein in diverse HIV-1 subtypes that plays a central role in virus replication, mainly by interacting with conserved nucleic acid sequences. NC is considered a highly profitable drug target to inhibit multiple steps in the HIV-1 life cycle with just one compound, a unique property not shown by any of the other antiretroviral classes. However, most of NC inhibitors developed so far act through an unspecific and potentially toxic mechanism (zinc ejection) and are mainly being investigated as topical microbicides. In an effort to provide specific NC inhibitors that compete for the binding of nucleic acids to NC, here we combined molecular modeling, organic synthesis, biophysical studies, NMR spectroscopy, and antiviral assays to design, synthesize, and characterize an efficient NC inhibitor endowed with antiviral activity in vitro, a desirable property for the development of efficient antiretroviral lead compounds.