Characterization of a major neutralizing epitope on the yellow fever virus envelope protein using human recombinant monoclonal antibody fragments generated by phage display

Yellow fever virus (YFV) is a mosquito-transmitted, enveloped, positive stranded RNA virus belonging to the genus flavivirus, which causes hemorrhagic fever in humans in Africa and South America. The YFV is responsible for 200 000 clinical infections per year including 40 000 deaths. Despite the presence of a highly effective YF vaccine called 17D vaccine, this disease is now strongly re-emerging and has to be considered as a public health problem. The present live attenuated 17D vaccine has two major drawbacks: 1) the ancient production method by inoculating viable embryonated eggs which limits the vaccine production capacity and, therefore, impairs attempts to control the disease and may contribute to vaccine supply shortage. 2) this vaccine is a non clonal vaccine which is constituted of heterogenous virion sub-populations. Furthermore, recent reports of several cases of viscerotropic and neurotropic disease associated with 17D vaccination have raised the obvious question of vaccine safety. Taken together, these data show that it appears essential to design a new clonal vaccine which could be based on infectious cDNA clone and produced in animal cell culture. For this purpose, the knowledge of YFV neutralizing epitopes is essential. Because YFV immunity is mainly antibody-mediated, we wanted to isolate human neutralizing antibodies specific for YFV and use them as a tool to characterize the neutralizing epitopes of YFV. The phage display technology provides one of the most convenient systems to isolate such neutralizing recombinant antibody fragments. We generated YF patient-derived antibody phage libraries which were screened against purified virions of the YFV-204-WHO vaccine strain. This step led to the isolation of several single-chain antibody fragments (scFv) which recognized conformational and pH sensitive epitopes in the envelope E protein. Three genetically closely-related and competing scFvs were found to be able to neutralize in vitro the 17D vaccine strain and five wild-type African strains of YFV. To map their epitopes, neutralization escape variants of the YFV-17D-204-WHO were generated using one high-affinity scFv (scFv-7A). Amino acids (aa) E-153, E-154 and E-155 in domain I and aa E-71 in domain II of the E protein were shown to be the critical components of one complex neutralizing epitope. These aa do not form a contiguous epitope on the monomeric E protein, but are in close vicinity in the dimeric form the E protein is predicted to adopt, based on the crystal structures of related flaviviruses. The neutralizing epitope is thus predicted to be formed by contribution of aa from domain I and II of opposing E monomers. The nature of this epitope was supported by the analysis of one wild-type YFV strain (Senegal 90) which is naturally resistant to neutralization by scFv-7A. Microneutralization assays using sera from YFV-infected patients and 17D-immunized travelers confirm the importance of E-71 in YFV neutralization but also showed that those escape variants, originally present in the vaccine lot, do not carry a risk of neutralization escape in persons who are immunized with the 17D vaccine. The potential neutralization mechanism by which these scFvs act, particularly by preventing the fusion process, and their potential use as a therapeutical tool are discussed. The structural complexity of the epitope identified in this work has implications for understanding the mechanism of antibody-mediated neutralization of YFV and these data may be useful for the design of a new recombinant yellow fever vaccine based on a cDNA-derived infectious clone

On Perception and Consciousness in HPPD:A Systematic Review

Hallucinogen-persisting perception disorder (HPPD) features as a diagnostic category in the DSM-5, ICD-11, and other major classifications, but our knowledge of the phenomenology of the perceptual symptoms involved and the changes in consciousness during the characteristic “flashbacks” is limited. We systematically evaluated original case reports and case series on HPPD to define its phenomenology, associated (psycho)pathology, and course. Our search of PubMed and Embase yielded 66 relevant publications that described 97 people who, together, experienced 64 unique symptoms of HPPD. Of these, 76% concerned symptoms characteristic of Alice in Wonderland syndrome, over 50% non-visual symptoms, and 38% perceptual symptoms not clearly linked to prior intoxication states. This is in contrast with the DSM-5 diagnostic criteria for HPPD. Even though less than half of the patients showed a protracted disease course of over a year, a third achieved remission. However, in patients with co-occurring depression (with or without anxiety) HPPD symptoms persisted longer and treatment outcomes were more often negative. Thus, unlike the acute stages of psychedelic drug intoxication, which may be accompanied by altered states of consciousness, HPPD is rather characterized by changes in the content of consciousness and an attentional shift from exogenous to endogenous phenomena. Since HPPD is a more encompassing nosological entity than suggested in the DSM-5, we recommend expanding its diagnostic criteria. In addition, we make recommendations for clinical practice and future research

Alice in Wonderland Syndrome as a Presenting Manifestation of Creutzfeldt-Jakob Disease

Background: Alice in Wonderland syndrome (AIWS) is a rare neurological disorder characterized by distortions of visual perception (metamorphopsias), the body image, and the experience of time, along with derealization and depersonalization. Some 85% of patients present with perceptual distortions in a single sensory modality, e.g., only visual or only somesthetic in nature. Moreover, the majority experience only a single type of distortion, e.g., only micropsia or only macropsia. AIWS has many different etiologies, and hence an extensive differential diagnosis. Its amenability to treatment depends on the underlying pathological process, which in children is mostly encephalitis, and in adults, migraine. In the literature, no more than 180 “clinical” cases of AIWS have been described (i.e., cases in need of medical attention). Of them, some 50% showed a favorable prognosis. However, non-clinical cases (i.e., fleeting, transient cases of AIWS for which no professional help is needed) have been described in up to 30% of the general population. This indicates that AIWS is perhaps not as rare as traditionally assumed, and has led some authors to conclude that, prognostically, AIWS is usually harmless.Methods: From our own clinical practice, we describe the first known case of Creutzfeldt-Jakob Disease (CJD, Heidenhain variant) that presented with symptoms of AIWS.Results: In our patient, disease onset was sudden and rapidly progressive, starting with isolated visual symptoms. Symptoms of AIWS comprised akinetopsia, chloropsia, micropsia, macropsia, zoom vision, and time distortions (quick-motion phenomenon and protracted duration). Soon, these were complicated by paraesthesias, gait instability, aphasia, expressive amusia, cognitive decline, and behavioral changes in the form of agitation and emotional lability. The diagnosis of probable sporadic CJD was confirmed with the aid of a head MRI and cerebrospinal fluid (14-3-3 protein). In the absence of any treatment options, our patient was discharged home and died within 2 months after his visual symptoms had begun. Autopsy consent was not obtained.Conclusion: We conclude that AIWS is not always as harmless as sometimes suggested, and that CJD, although extremely rare, must be part of its extensive differential diagnosis, notably in the presence of rapid cognitive decline

Transcriptional profiling of vaccine-induced immune responses in humans and non-human primates

There is an urgent need for pre-clinical and clinical biomarkers predictive of vaccine immunogenicity, efficacy and safety to reduce the risks and costs associated with vaccine development. Results emerging from immunoprofiling studies in non-human primates and humans demonstrate clearly that (i) type and duration of immune memory are largely determined by the magnitude and complexity of the innate immune signals and (ii) genetic signatures highly predictive of B-cell and T-cell responses can be identified for specific vaccines. For vaccines with similar composition, e.g. live attenuated viral vaccines, these signatures share common patterns. Signatures predictive of vaccine efficacy have been identified in a few experimental challenge studies. This review aims to give an overview of the current literature on immunoprofiling studies in humans and also presents some of our own data on profiling of licensed and experimental vaccines in non-human primates

HIV-1 Conserved Mosaics Delivered by Regimens with Integration-deficient, DC-targeting Lentivirus Induce Robust T Cells

Background: To be effective against HIV-1, vaccine-induced T cells must selectively target functionally conserved and, at the same time, protective epitopes present on the majority of currently circulating and reactivated HIV-1 strains, and rapidly reach protective frequencies upon exposure to the virus. Heterologous prime-boost regimens using virally vectored vaccines are currently the most promising strategy towards achieving this goal, nevertheless, induction of robust longterm memory remains challenging. To this end, lentiviral vectors induce high frequencies of memory cells due to their low-inflammatory nature, while typically inducing only low antivector immune responses. Methods: We describe construction of novel candidate vaccines ZVex.tHIVconsv1 and ZVex.tHIVconsv2, which are based on an integration-deficient lentiviral vector platform with preferential transduction of human dendritic cells and express bivalent mosaic of conserved-region T-cell immunogens with a high global HIV-1 match. Results: Each of the two mosaics was individually immunogenic and together in heterologous prime-boost regimens with nonreplicating simian (chimpanzee) adenovirus or non-replicating poxvirus MVA vaccines induced very high frequencies of plurifunctional and broadly cross-reactive T cells in BALB/c and outbred CD1-SWISS mice. Conclusions: These data support further development of this vaccine concept

Mandibular Overdentures Supported by 6-mm Dental Implants:A 1-Year Prospective Cohort Study

Background: The extremely resorbed edentulous mandible, with a bone height of 8 mm or less, is still a challenge in implant dentistry. Recently, dental implants of 6 mm in length have been developed. Purpose: The purpose of this 1-year prospective cohort study was to evaluate treatment outcome of mandibular overdentures supported by four 6-mm dental implants. Materials and Methods: Twelve edentulous patients with a mandibular height between 6 and 8 mm participated. The patients were treated with an overdenture supported by four 6-mm OsseoSpeed (TM) dental implants (Astra Tech AB, Molndal, Sweden). Clinical and radiographic parameters were evaluated 1 year after completion of the prosthetic treatment. Patients' satisfaction was scored before implant surgery and 1 year after prosthetic treatment. Results: One-year implant survival rate was 96% (two implants were lost). One patient had a fracture of the mandible in the region of one of the implants 3 weeks after implant surgery. Mean scores for plaque, calculus, gingiva, bleeding, and pocket probing depth were low. Patients' satisfaction was high. Conclusion: One-year follow-up data revealed that four 6-mm dental implants inserted in an extremely resorbed edentulous mandible provided a solid basis for a bar-retained overdenture

HIV-1 Conserved Mosaics Delivered by Regimens with Integration-Deficient DC-Targeting Lentiviral Vector Induce Robust T Cells

To be effective against HIV type 1 (HIV-1), vaccine-induced T cells must selectively target epitopes, which are functionally conserved (present in the majority of currently circulating and reactivated HIV-1 strains) and, at the same time, beneficial (responses to which are associated with better clinical status and control of HIV-1 replication), and rapidly reach protective frequencies upon exposure to the virus. Heterologous prime-boost regimens using virally vectored vaccines are currently the most promising vaccine strategies; nevertheless, induction of robust long-term memory remains challenging. To this end, lentiviral vectors induce high frequencies of memory cells due to their low-inflammatory nature, while typically inducing only low anti-vector immune responses. Here, we describe construction of novel candidate vaccines ZVex.tHIVconsv1 and ZVex.tHIVconsv2, which are based on an integration-deficient lentiviral vector platform with preferential transduction of human dendritic cells and express a bivalent mosaic of conserved-region T cell immunogens with a high global HIV-1 match. Each of the two mosaic vaccines was individually immunogenic. When administered together in heterologous prime-boost regimens with chimpanzee adenovirus and/or poxvirus modified vaccinia virus Ankara (MVA) vaccines to BALB/c and outbred CD1-Swiss mice, they induced a median frequency of over 6,000 T cells/10^6splenocytes, which were plurifunctional, broadly specific, and cross-reactive. These results support further development of this vaccine concept

Design of E. coli expressed stalk domain immunogens of H1N1 HA that protect mice from lethal challenge

The hemagglutinin protein (HA) on the surface of influenza virus is essential for viral entry into the host cells. The HA1 subunit of HA is also the primary target for neutralizing antibodies. The HA2 subunit is less exposed on the virion surface and more conserved than HA1. We have previously designed an HA2 based immunogen derived from the sequence of the H3N2 A/HK/68 virus. In the present study we report the design of an HA2 based immunogen from the H1N1 subtype (PR/8/34). This immunogen (H1HA0HA6) and its circular permutant (H1HA6) were well folded and provided complete protection against homologous viral challenge. Anti-sera of immunized mice showed cross-reactivity with HA proteins of different strains and subtypes. Although no neutralization was observable in a conventional neutralization assay, sera of immunized guinea pigs competed with a broadly neutralizing antibody CR6261 for binding to recombinant Viet/04 HA protein suggesting that CR6261 like antibodies were elicited by the immunogens. Stem domain immunogens from a seasonal H1N1 strain (A/NC/20/99) and a recent pandemic strain (A/Cal/07/09) provided cross-protection against A/PR/8/34 viral challenge. HA2 containing stem domain immunogens therefore have the potential to provide subtype specific protection

Fabrication and replication of high efficiency blazed gratings with grayscale electron beam lithography and UV nanoimprint lithography

In a waveguide-type display for augmented reality, the image is injected in the waveguide and extracted in front of the eye appearing superimposed on the real world scene. An elegant and compact way of coupling these images in and out is by using blazed gratings, which can achieve high diffraction efficiencies, thereby reducing stray light and decreasing the required power levels. This study investigates the fabrication of blazed gratings with grayscale electron beam lithography and the subsequent replication of the realized 3D grating structures in a polymer material with ultraviolet nanoimprint lithography. As such, diffractive elements are realized on a waveguide sheet, with very good control over the dimensions and the profile of the printed features. Blazed gratings are designed for green light (λ= 543 nm) and a diffraction angle of 43°. Making use of a PMMA resist and by carefully optimizing the electron-beam parameters, electron dose distributions and development step, blazed gratings with a pitch of 508 nm and a fill factor of 0.66 are achieved. Finally, a master is realized with two blazed gratings, 3 cm apart, which are replicated using ultraviolet nanoimprint lithography onto a waveguide sheet. The in- and outcoupling of an image through these two blazed gratings is shown, appearing sharp and non-distorted in the environment, and a throughput efficiency of 17.4% is confirmed