A Dual-Approach Strategy to Optimize the Safety and Efficacy of Anti-Zika Virus Monoclonal Antibody Therapeutics.

Antibody-dependent enhancement of infection (ADE) is clinically relevant to Dengue virus (DENV) infection and poses a major risk to the application of monoclonal antibody (mAb)-based therapeutics against related flaviviruses such as the Zika virus (ZIKV). Here, we tested a two-tier approach for selecting non-cross-reactive mAbs combined with modulating Fc glycosylation as a strategy to doubly secure the elimination of ADE while preserving Fc effector functions. To this end, we selected a ZIKV-specific mAb (ZV54) and generated three ZV54 variants using Chinese hamster ovary cells and wild-type (WT) and glycoengineered ΔXF Nicotiana benthamiana plants as production hosts (ZV54CHO, ZV54WT, and ZV54ΔXF). The three ZV54 variants shared an identical polypeptide backbone, but each exhibited a distinct Fc N-glycosylation profile. All three ZV54 variants showed similar neutralization potency against ZIKV but no ADE activity for DENV infection, validating the importance of selecting the virus/serotype-specific mAbs for avoiding ADE by related flaviviruses. For ZIKV infection, however, ZV54CHO and ZV54ΔXF showed significant ADE activity while ZV54WT completely forwent ADE, suggesting that Fc glycan modulation may yield mAb glycoforms that abrogate ADE even for homologous viruses. In contrast to the current strategies for Fc mutations that abrogate all effector functions along with ADE, our approach allowed the preservation of effector functions as all ZV54 glycovariants retained antibody-dependent cellular cytotoxicity (ADCC) against the ZIKV-infected cells. Furthermore, the ADE-free ZV54WT demonstrated in vivo efficacy in a ZIKV-infection mouse model. Collectively, our study provides further support for the hypothesis that antibody-viral surface antigen and Fc-mediated host cell interactions are both prerequisites for ADE, and that a dual-approach strategy, as shown herein, contributes to the development of highly safe and efficacious anti-ZIKV mAb therapeutics. Our findings may be impactful to other ADE-prone viruses, including SARS-CoV-2.

Plant-Produced Anti-Zika Virus Monoclonal Antibody Glycovariant Exhibits Abrogated Antibody-Dependent Enhancement of Infection.

Monoclonal antibodies (mAb) against the envelope (E) protein of Zika virus (ZIKV) have shown great potential as therapeutics against the Zika epidemics. However, their use as a therapy may predispose treated individuals to severe infection by the related dengue virus (DENV) via antibody-dependent enhancement of infection (ADE). Here, we generated a broadly neutralizing flavivirus mAb, ZV1, with an identical protein backbone but different Fc glycosylation profiles. The three glycovariants, produced in wild-type (WT) and glycoengineered ΔXF Nicotiana benthamiana plants and in Chinese hamster ovary cells (ZV1WT, ZV1ΔXF, and ZV1CHO), respectively, showed equivalent neutralization potency against both ZIKV and DENV. By contrast, the three mAb glycoforms demonstrated drastically different ADE activity for DENV and ZIKV infection. While ZV1CHO and ZV1ΔXF showed ADE activity upon DENV and ZIKV infection, ZV1WT totally forwent its ADE. Importantly, all three glycovariants exhibited antibody-dependent cellular cytotoxicity (ADCC) against virus-infected cells, with increased potency by the fucose-free ZV1ΔXF glycoform. Moreover, the in vivo efficacy of the ADE-free ZV1WT was demonstrated in a murine model. Collectively, we demonstrated the feasibility of modulating ADE by Fc glycosylation, thereby establishing a novel approach for improving the safety of flavivirus therapeutics. Our study also underscores the versatile use of plants for the rapid expression of complex human proteins to reveal novel insight into antibody function and viral pathogenesis.

Quantification of West Nile Virus by Plaque-Forming Assay.

The plaque-forming assay is a gold standard technique to determine the concentration of infectious viral particles. In this assay, lytic viruses infect and lyse the cells but are immobilized due to the presence of an agarose-containing overlay medium. The progeny viruses can only spread locally to and kill the adjacent cells and finally form a clear zone or plaque after staining the live cells. The number of plaques formed can be theoretically considered as the initial number of the infectious viral particles present in the sample and hence can be expressed as plaque-forming units (PFU) in a volume of the sample. Here, we provide a step-by-step method to carry out a plaque-forming assay to determine the titer of West Nile virus in a cell culture medium, which also can be adapted to other lytic viruses of eukaryotic cells.

Mouse Trophoblast Cells Can Provide IFN-Based Antiviral Protection to Embryonic Stem Cells via Paracrine Signaling.

The blastocyst is the preimplantation stage embryo that consists of two major components: the inner cell mass (ICM) and the trophectoderm (TE). The ICM gives rise to the fetus and some extraembryonic tissues whereas the TE contributes to development of the placenta. Previous studies have demonstrated that both human and mouse embryonic stem cells (ESCs) derived from the ICM are deficient in expressing type I IFNs in response to viral infection. In this study, we investigated the IFN response in mouse trophoblast stem cells (TSCs) and their in vitro differentiated trophoblasts (TSC-TBs). In this study, we report that, unlike ESCs, TSCs have a functional IFN system. They can express type I IFNs in response to viral stimuli and express IFN-stimulated genes in response to type I IFNs. TSC-TBs have a further developed IFN system and acquired the ability to express specialized type III IFN-λ. Furthermore, TSCs and TSC-TBs can provide ESCs with antiviral activity against Chikungunya, West Nile, and Zika virus infection, as demonstrated with a novel coculture model that simulates the temporal and spatial relationship between the ICM and the TE in a blastocyst. Taken together, our data demonstrate that mouse ESCs can respond to type I IFNs and gain IFN-based antiviral protection from TSCs and TSC-TBs via paracrine signaling mechanisms even though they themselves are unable to express type I IFNs.

Murine Trophoblast Stem Cells and Their Differentiated Cells Attenuate Zika Virus In Vitro by Reducing Glycosylation of the Viral Envelope Protein.

Zika virus (ZIKV) infection during pregnancy can cause devastating fetal neuropathological abnormalities, including microcephaly. Most studies of ZIKV infection in pregnancy have focused on post-implantation stage embryos. Currently, we have limited knowledge about how a pre-implantation stage embryo deals with a viral infection. This study investigates ZIKV infection on mouse trophoblast stem cells (TSCs) and their in vitro differentiated TSCs (DTSCs), which resemble the cellular components of the trophectoderm layer of the blastocyst that later develops into the placenta. We demonstrate that TSCs and DTSCs are permissive to ZIKV infection; however, ZIKV propagated in TSCs and DTSCs exhibit substantially lower infectivity, as shown in vitro and in a mouse model compared to ZIKV that was generated in Vero cells or mouse embryonic fibroblasts (MEFs). We further show that the low infectivity of ZIKV propagated in TSCs and DTSCs is associated with a reduced level of glycosylation on the viral envelope (E) proteins, which are essential for ZIKV to establish initial attachment by binding to cell surface glycosaminoglycans (GAGs). The decreased level of glycosylation on ZIKV E is, at least, partially due to the low-level expression of a glycosylation-related gene, Hexa, in TSCs and DTSCs. Furthermore, this finding is not limited to ZIKV since similar observations have been made as to the chikungunya virus (CHIKV) and West Nile virus (WNV) propagated in TSCs and DTSCs. In conclusion, our results reveal a novel phenomenon suggesting that murine TSCs and their differentiated cells may have adapted a cellular glycosylation system that can limit viral infectivity by altering the glycosylation of viral envelope proteins, therefore serving as a unique, innate anti-viral mechanism in the pre-implantation stage embryo.

Interleukin-17A Facilitates Chikungunya Virus Infection by Inhibiting IFN-α2 Expression.

Interferons (IFNs) are the key components of innate immunity and are crucial for host defense against viral infections. Here, we report a novel role of interleukin-17A (IL-17A) in inhibiting IFN-α2 expression thus promoting chikungunya virus (CHIKV) infection. CHIKV infected IL-17A deficient (Il17a-/- ) mice expressed a higher level of IFN-α2 and developed diminished viremia and milder footpad swelling in comparison to wild-type (WT) control mice, which was also recapitulated in IL-17A receptor-deficient (Il17ra-/- ) mice. Interestingly, IL-17A selectively blocked IFN-α2 production during CHIKV, but not West Nile virus (WNV) or Zika virus (ZIKV), infections. Recombinant IL-17A treatment inhibited CHIKV-induced IFN-α2 expression and enhanced CHIKV replication in both human and mouse cells. We further found that IL-17A inhibited IFN-α2 production by modulating the expression of Interferon Regulatory Factor-5 (IRF-5), IRF-7, IFN-stimulated gene 49 (ISG-49), and Mx1 expression during CHIKV infection. Neutralization of IL-17A in vitro leads to the increase of the expression of these antiviral molecules and decrease of CHIKV replication. Collectively, these results suggest a novel function of IL-17A in inhibiting IFN-α2-mediated antiviral responses during CHIKV infection, which may have broad implications in viral infections and other inflammatory diseases.

Zika virus infection causes widespread damage to the inner ear.

Zika virus (ZIKV) has been recently recognized as a causative agent of newborn microcephaly, as well as other neurological consequences. A less well recognized comorbidity of prenatal ZIKV infection is hearing loss, but cases of hearing impairment following adult ZIKV infection have also been recognized. Diminished hearing following prenatal ZIKV infection in a mouse model has been reported, but no cellular consequences were observed. We examined the effects of ZIKV infection on inner ear cellular integrity and expression levels of various proteins important for cochlear function in type I interferon receptor null (Ifnar1-/-) mice following infection at 5-6 weeks of age. We show that ZIKV antigens are present in cells within the cochlear epithelium, lateral wall, spiral limbus and spiral ganglion. Here we show that ZIKV infection alters cochlear expression of genes that signal cell damage (S100B), transport fluids (AQP1), are gaseous transmitters (eNOs) and modulate immune response (F4/80). Morphological analyses shows that not only are cochlear structures compromised by ZIKV infection, but damage also occurs in vestibular end organs. ZIKV produces a graded distribution of cellular damage in the cochlea, with greatest damage in the apex similar to that reported for cytomegalovirus (CMV) infection. The graded distribution of damage may indicate a differential susceptibility to ZIKV along the cochlear tonotopic axis. Collectively, these data are the first to show the molecular and morphological damage to the inner ear induced by ZIKV infection in adults and suggests multiple mechanisms contributing to the hearing loss reported in the human population.

Congenital Zika Virus Infection in Immunocompetent Mice Causes Postnatal Growth Impediment and Neurobehavioral Deficits.

A small percentage of babies born to Zika virus (ZIKV)-infected mothers manifest severe defects at birth, including microcephaly. Among those who appeared healthy at birth, there are increasing reports of postnatal growth or developmental defects. However, the impact of congenital ZIKV infection in postnatal development is poorly understood. Here, we report that a mild congenital ZIKV-infection in pups born to immunocompetent pregnant mice did not display apparent defects at birth, but manifested postnatal growth impediments and neurobehavioral deficits, which include reduced locomotor and cognitive deficits that persisted into adulthood. We found that the brains of these pups were smaller, had a thinner cortical layer 1, displayed increased astrogliosis, decreased expression of microcephaly- and neuron development- related genes, and increased pathology as compared to mock-infected controls. In summary, our results showed that even a mild congenital ZIKV infection in immunocompetent mice could lead to postnatal deficits, providing definitive experimental evidence for a necessity to closely monitor postnatal growth and development of presumably healthy human infants, whose mothers were exposed to ZIKV infection during pregnancy.

Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells.

Molecular determinants and mechanisms of arthropod-borne flavivirus transmission to the vertebrate host are poorly understood. In this study, we show for the first time that a cell line from medically important arthropods, such as ticks, secretes extracellular vesicles (EVs) including exosomes that mediate transmission of flavivirus RNA and proteins to the human cells. Our study shows that tick-borne Langat virus (LGTV), a model pathogen closely related to tick-borne encephalitis virus (TBEV), profusely uses arthropod exosomes for transmission of viral RNA and proteins to the human- skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed the presence of purified arthropod/neuronal exosomes with the size range of 30 to 200 nm in diameter. Both positive and negative strands of LGTV RNA and viral envelope-protein were detected inside exosomes derived from arthropod, murine and human cells. Detection of Nonstructural 1 (NS1) protein in arthropod and neuronal exosomes further suggested that exosomes contain viral proteins. Viral RNA and proteins in exosomes derived from tick and mammalian cells were secured, highly infectious and replicative in all tested evaluations. Treatment with GW4869, a selective inhibitor that blocks exosome release affected LGTV loads in both arthropod and mammalian cell-derived exosomes. Transwell-migration assays showed that exosomes derived from infected-brain-microvascular endothelial cells (that constitute the blood-brain barrier) facilitated LGTV RNA and protein transmission, crossing of the barriers and infection of neuronal cells. Neuronal infection showed abundant loads of both tick-borne LGTV and mosquito-borne West Nile virus RNA in exosomes. Our data also suggest that exosome-mediated LGTV viral transmission is clathrin-dependent. Collectively, our results suggest that flaviviruses uses arthropod-derived exosomes as a novel means for viral RNA and protein transmission from the vector, and the vertebrate exosomes for dissemination within the host that may subsequently allow neuroinvasion and neuropathogenesis.

Evidence of Chikungunya virus circulation in the Terai region of Nepal in 2014 and 2015.

Background: Chikungunya virus (CHIKV) infection is an emerging viral disease in Nepal, with the reported by our laboratories in 2013. Because of the similarity in signs and symptoms to dengue virus (DENV) and misdiagnosis, the under-reporting of CHIKV infection in DENV-endemic areas of Nepal is very common. The aim of the present study is to identify CHIKV infection among patients with febrile illness during the 2014-2015 outbreaks. Methods: A total of 219 serum samples were tested for CHIKV and DENV by using enzyme-linked immunosorbent assays (ELISAs) to detect immunoglobulin M (IgM) and IgG antibodies, with confirmation by a 50% focal reduction neutralization test (FRNT50). Results: ELISA results demonstrated that for CHIKV and DENV, 20.5% and 14.2% of samples, respectively, were positive for IgG and/or IgM and 9.6% and 5.9% were positive for IgM alone. Further, FRNT50 results confirmed that 7.3% of samples possessed neutralizing anti-CHIKV antibodies. Conclusion: We conclude that CHIKV infection is settling in Nepal as an endemic disease. It is highly recommended to strengthen the surveillance system for CHIKV and DENV to prevent possible outbreaks.

Serological and Entomological Study of Dengue in Dang and Chitwan Districts of Nepal.

A descriptive cross-sectional study was carried out among 264 suspected dengue patients in two districts (Dang and Chitwan) of Nepal from June 2013 to November 2013. The anti-dengue IgM positivity was found to be (51/264)19.31% by capture ELISA, of which 21 (41.2%) were male and 30 (58.8%) were female. Symptoms of seropositive cases were fever, anorexia, nausea, headache, retro-orbital pain, skin rashes, and myalgia. Hematological features like thrombocytopenia and leucopenia were found to be significantly associated with the dengue fever (DF). Discarded tires were found as the commonest breeding habitats for the dengue vectors. Higher sero-positivity was recorded from the area having higher Breteau index (BI). The pH, chloride ion concentration and the salinity of the water from breeding habitats were found to be ranging from 6.9±0.82 to 8, 103.33±17.52 mg/L to 140.65 mg/L, and 0.19±0.032 ppt to 0.25 ppt respectively. This study may be helpful for the health authorities and public health workers for early diagnosis of DF and for the improved preventive measures to be adopted in the epidemic and possible epidemic areas.

Persistent dengue emergence: the 7 years surrounding the 2010 epidemic in Nepal.

BACKGROUND: Dengue fever (DF) is an emerging public health problem in Nepal, and yet neither the magnitude of the DF burden or its epidemiological trends are well understood. METHODS: We conducted a sero-epidemiological, seasonal trend and demographic analysis of the trends in DF in the Terai region of Nepal, from 2007 to 2013. In that period, 2002 serum samples were collected from febrile patients suspected of dengue virus infection. Samples were screened by IgM antibody ELISA analysis for the presence of anti-dengue IgM antibodies. RESULTS: The overall prevalence of DF cases was found to range from 26.1 to 55.4%. Infection was found to be more common among adults and males. Seasonal trends revealed that cases peaked in October and November. CONCLUSIONS: The study offers perspective on the dengue fever burden before, during and after a major epidemic and can now be used as a basis for formulating strategies by policy makers that will enhance and develop relevant control and preventive measures against dengue fever. The findings of this study reinforce the perceived need for urgent dengue virus surveillance to enhance dengue control strategies that need to be developed for future preparedness. In the light of the recent earthquakes, future major outbreaks of vector borne disease are likely to recur.

Detection of Chikungunya Virus in Nepal.

Chikungunya virus (CHIKV) is an emerging alphaviral disease and a public health problem in South Asia including Nepal in recent years. In this study, sera were collected from patients presenting with fever, headache, muscular pain, fatigue, and joint pain of both upper and lower extremities. A total of 169 serum samples were tested for CHIKV and dengue virus (DENV) by using Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibody using enzyme-linked immunosorbent assay (ELISA) method during August to November 2013. Results showed that 3.6% and 27.8% samples were positive for CHIKV and DENV IgM positive, respectively. Similarly, results of IgG showed 3.0% samples were positive for CHIKV IgG and 29.0% were for DENV IgG positive. Further, a 50% focal reduction neutralization test (FRNT50) was performed to confirm the presence of CHIKV, which demonstrated that 8.9% of CHIKV IgM and/or IgG ELISA positive possessed neutralizing anti-CHIK antibodies. To our knowledge, this is the first report in which the presence of CHIKV is confirmed in Nepalese patients by FRNT50. Basic scientists and clinicians need to consider CHIKV as a differential diagnosis in febrile Nepalese patients, and policy makers should consider appropriate surveillance and actions for control strategies.

Re-emergence of susceptibility to conventional first line drugs in Salmonella isolates from enteric fever patients in Nepal.

INTRODUCTION: Enteric fever is endemic in Nepal and poses a significant public health burden. The first-line drugs ampicillin, chloramphenicol, and cotrimoxazole have not been part of empirical therapy for two decades due to the development of multidrug-resistant Salmonella strains. The objective of this study was to determine the antibiogram pattern of Salmonella serovars isolated from the blood of clinically suspected enteric fever patients. METHODOLOGY: A cross sectional study was carried out in a tertiary care hospital in Lalitpur, Nepal, between July 2011 and February 2012. Standard microbiological procedures were followed during collection and processing of blood samples, isolation and identification of Salmonella serotypes. The antimicrobial sensitivity of ampicillin, chloramphenicol, cotrimoxazole, nalidixic acid, and ciprofloxacin was determined using a modified Kirby-Bauer disk diffusion method as per the guidelines of the Clinical and Laboratory Standards Institute. RESULTS: Out of 86 Salmonella isolates, 56 (65.1%) were Salmonella Typhi and 30 (34.9%) were Salmonella Paratyphi A. Salmonella Typhi were 100% sensitive to chloramphenicol, cotrimoxazole, and ciprofloxacin and 98.2% sensitive to ampicillin. Similarly, Salmonella Paratyphi A isolates were 100% sensitive to ampicillin and cotrimoxazole and 96.7% sensitive to chloramphenicol and ciprofloxacin. More than 90.0% of isolates were nalidixic acid resistant and none of the Salmonella isolates were multi-drug resistant. CONCLUSIONS: This study revealed the increasing frequency of nalidixic acid-resistant Salmonella isolates, indicating the possibility of fluoroquinolone resistance in near future. Furthermore, re-emergence of susceptibility to conventional first-line drugs ampicillin, chloramphenicol, and cotrimoxazole supports the possibility of using these drugs in empirical therapy.