Peli1 signaling blockade attenuates congenital zika syndrome.

Zika virus (ZIKV) infects pregnant women and causes devastating congenital zika syndrome (CZS). How the virus is vertically transmitted to the fetus and induces neuronal loss remains unclear. We previously reported that Pellino (Peli)1, an E3 ubiquitin ligase, promotes p38MAPK activation in microglia and induction of lethal encephalitis by facilitating the replication of West Nile virus (WNV), a closely related flavivirus. Here, we found that Peli1 expression was induced on ZIKV-infected human monocytic cells, peripheral blood mononuclear cells, human first-trimester placental trophoblasts, and neural stem cell (hNSC)s. Peli1 mediates ZIKV cell attachment, entry and viral translation and its expression is confined to the endoplasmic reticulum. Moreover, Peli1 mediated inflammatory cytokine and chemokine responses and induced cell death in placental trophoblasts and hNSCs. ZIKV-infected pregnant mice lacking Peli1 signaling had reduced placental inflammation and tissue damage, which resulted in attenuated congenital abnormalities. Smaducin-6, a membrane-tethered Smad6-derived peptide, blocked Peli1-mediated NF-κB activation but did not have direct effects on ZIKV infection. Smaducin-6 reduced inflammatory responses and cell death in placental trophoblasts and hNSCs, and diminished placental inflammation and damage, leading to attenuated congenital malformations in mice. Collectively, our results reveal a novel role of Peli1 in flavivirus pathogenesis and suggest that Peli1 promotes ZIKV vertical transmission and neuronal loss by mediating inflammatory cytokine responses and induction of cell death. Our results also identify Smaducin-6 as a potential therapeutic candidate for treatment of CZS.

An attenuated Zika virus NS4B protein mutant is a potent inducer of antiviral immune responses.

Live attenuated vaccines (LAVs) are one of the most important strategies to control flavivirus diseases. The flavivirus nonstructural (NS) 4B proteins are a critical component of both the virus replication complex and evasion of host innate immunity. Here we have used site-directed mutagenesis of residues in the highly conserved N-terminal and central hydrophobic regions of Zika virus (ZIKV) NS4B protein to identify candidate attenuating mutations. Three single-site mutants were generated, of which the NS4B-C100S mutant was more attenuated than the other two mutants (NS4B-C100A and NS4B-P36A) in two immunocompromised mouse models of fatal ZIKV disease. The ZIKV NS4B-C100S mutant triggered stronger type 1 interferons and interleukin-6 production, and higher ZIKV-specific CD4+ and CD8+ T-cell responses, but induced similar titers of neutralization antibodies compared with the parent wild-type ZIKV strain and a previously reported candidate ZIKV LAV with a 10-nucleotide deletion in 3'-UTR (ZIKV-3'UTR-Δ10). Vaccination with ZIKV NS4B-C100S protected mice from subsequent WT ZIKV challenge. Furthermore, either passive immunization with ZIKV NS4B-C100S immune sera or active immunization with ZIKV NS4B-C100S followed by the depletion of T cells affords full protection from lethal WT ZIKV challenge. In summary, our results suggest that the ZIKV NS4B-C100S mutant may serve as a candidate ZIKV LAV due to its attenuated phenotype and high immunogenicity.

A Structure-Informed Atlas of Human-Virus Interactions.

While knowledge of protein-protein interactions (PPIs) is critical for understanding virus-host relationships, limitations on the scalability of high-throughput methods have hampered their identification beyond a number of well-studied viruses. Here, we implement an in silico computational framework (pathogen host interactome prediction using structure similarity [P-HIPSTer]) that employs structural information to predict ∼282,000 pan viral-human PPIs with an experimental validation rate of ∼76%. In addition to rediscovering known biology, P-HIPSTer has yielded a series of new findings: the discovery of shared and unique machinery employed across human-infecting viruses, a likely role for ZIKV-ESR1 interactions in modulating viral replication, the identification of PPIs that discriminate between human papilloma viruses (HPVs) with high and low oncogenic potential, and a structure-enabled history of evolutionary selective pressure imposed on the human proteome. Further, P-HIPSTer enables discovery of previously unappreciated cellular circuits that act on human-infecting viruses and provides insight into experimentally intractable viruses.

Peli1 facilitates virus replication and promotes neuroinflammation during West Nile virus infection.

The E3 ubiquitin ligase Pellino 1 (Peli1) is a microglia-specific mediator of autoimmune encephalomyelitis. Its role in neurotropic flavivirus infection is largely unknown. Here, we report that mice deficient in Peli1 (Peli1-/-) were more resistant to lethal West Nile virus (WNV) infection and exhibited reduced viral loads in tissues and attenuated brain inflammation. Peli1 mediates chemokine and proinflammatory cytokine production in microglia and promotes T cell and macrophage infiltration into the CNS. Unexpectedly, Peli1 was required for WNV entry and replication in mouse macrophages and mouse and human neurons and microglia. It was also highly expressed on WNV-infected neurons and adjacent inflammatory cells from postmortem patients who died of acute WNV encephalitis. WNV passaged in Peli1-/- macrophages or neurons induced a lower viral load and impaired activation in WT microglia and thereby reduced lethality in mice. Smaducin-6, which blocks interactions between Peli1 and IRAK1, RIP1, and IKKε, did not inhibit WNV-triggered microglia activation. Collectively, our findings suggest a nonimmune regulatory role for Peli1 in promoting microglia activation during WNV infection and identify a potentially novel host factor for flavivirus cell entry and replication.

Zika, dengue and yellow fever viruses induce differential anti-viral immune responses in human monocytic and first trimester trophoblast cells.

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with severe neonatal birth defects, but the causative mechanism is incompletely understood. ZIKV shares sequence homology and early clinical manifestations with yellow fever virus (YFV) and dengue virus (DENV) and are all transmitted in urban cycles by the same species of mosquitoes. However, YFV and DENV have been rarely reported to cause congenital diseases. Here, we compared infection with a contemporary ZIKV strain (FSS13025) to YFV17D and DENV-4 in human monocytic cells (THP-1) and first-trimester trophoblasts (HTR-8). Our results suggest that all three viruses have similar tropisms for both cells. Nevertheless, ZIKV induced strong type 1 IFN and inflammatory cytokine and chemokine production in monocytes and peripheral blood mononuclear cells. Furthermore, ZIKV infection in trophoblasts induced lower IFN and higher inflammatory immune responses. Placental inflammation is known to contribute to the risk of brain damage in preterm newborns. Inhibition of toll-like receptor (TLR)3 and TLR8 each abrogated the inflammatory cytokine responses in ZIKV-infected trophoblasts. Our findings identify a potential link between maternal immune activation and ZIKV-induced congenital diseases, and a potential therapeutic strategy that targets TLR-mediated inflammatory responses in the placenta.

West Nile Virus Infection in the Central Nervous System.

West Nile virus (WNV), a neurotropic single-stranded flavivirus has been the leading cause of arboviral encephalitis worldwide.  Up to 50% of WNV convalescent patients in the United States were reported to have long-term neurological sequelae.  Neither antiviral drugs nor vaccines are available for humans.  Animal models have been used to investigate WNV pathogenesis and host immune response in humans.  In this review, we will discuss recent findings from studies in animal models of WNV infection, and provide new insights on WNV pathogenesis and WNV-induced host immunity in the central nervous system.

Subcapsular sinus macrophages limit dissemination of West Nile virus particles after inoculation but are not essential for the development of West Nile virus-specific T cell responses.

Macrophages encounter flaviviruses early after injection by arthropod vectors. Using in vivo imaging of mice inoculated with firefly luciferase-expressing single-cycle flavivirus particles (FLUC-SCFV), we examined the initial dissemination of virus particles in the presence or absence of lymph node (LN)-resident macrophages. Higher luciferase activity, indicating higher SCFV gene expression, was detected in the footpad of macrophage-depleted mice after 24h post infection (hpi). Moreover, FLUC-SCFV particles disseminated to the spleen within 14 hpi in macrophage-depleted, but not control mice. Although macrophages presented SCFV to naïve T cells in vitro, depletion of subcapsular sinus (SCS) macrophages did not alter the magnitude or effector function of the WNV-specific CD8(+) T cell response. Together, these results indicate that SCS macrophages play a role in limiting the dissemination of SCFV early in infection but are not required for the generation of a polyfunctional WNV-specific CD8(+) T cell response in the draining LN.

TLR3- and MyD88-dependent signaling differentially influences the development of West Nile virus-specific B cell responses in mice following immunization with RepliVAX WN, a single-cycle flavivirus vaccine candidate.

Recognition of conserved pathogen-associated molecular patterns (PAMPs) by host pattern recognition receptors (PRRs) results in the activation of innate signaling pathways that drive the innate immune response and ultimately shape the adaptive immune response. RepliVAX WN, a single-cycle flavivirus (SCFV) vaccine candidate derived from West Nile virus (WNV), is intrinsically adjuvanted with multiple PAMPs and induces a vigorous anti-WNV humoral response. However, the innate mechanisms that link pattern recognition and development of vigorous antigen-specific B cell responses are not completely understood. Moreover, the roles of individual PRR signaling pathways in shaping the B cell response to this live attenuated SCFV vaccine have not been established. We examined and compared the role of TLR3- and MyD88-dependent signaling in the development of anti-WNV-specific antibody-secreting cell responses and memory B cell responses induced by RepliVAX WN. We found that MyD88 deficiency significantly diminished B cell responses by impairing B cell activation, development of germinal centers (GC), and the generation of long-lived plasma cells (LLPCs) and memory B cells (MBCs). In contrast, TLR3 deficiency had more effect on maintenance of GCs and development of LLPCs, whereas differentiation of MBCs was unaffected. Our data suggest that both TLR3- and MyD88-dependent signaling are involved in the intrinsic adjuvanting of RepliVAX WN and differentially contribute to the development of vigorous WNV-specific antibody and B cell memory responses following immunization with this novel SCFV vaccine.

Intrinsic adjuvanting of a novel single-cycle flavivirus vaccine in the absence of type I interferon receptor signaling.

Type I interferons (IFNs) are critical for controlling pathogenic virus infections and can enhance immune responses. Hence their impact on the effectiveness of live-attenuated vaccines involves a balance between limiting viral antigen expression and enhancing the development of adaptive immune responses. We examined the influence of type I IFNs on these parameters following immunization with RepliVAX WN, a single-cycle flavivirus vaccine (SCFV) against West Nile virus (WNV) disease. RepliVAX WN-immunized mice produced IFN-α and displayed increased IFN-stimulated gene transcription in draining lymph nodes (LN). SCFV gene expression was over 100 fold-higher on days 1-3 post-infection in type I IFN receptor knockout mice (IFNAR(-/-)) compared to wild-type (wt) mice indicating a profound IFN-mediated suppression of SCFV gene expression in the wt animals. IFNAR(-/-) mice produced nearly equivalent levels of WNV-specific serum IgG and WNV-specific CD4(+) T cell responses compared to wt mice. However, significantly higher numbers of WNV-specific CD8(+) T cells were produced by IFNAR(-/-) mice and a significantly greater percentage of these T cells from IFNAR(-/-) mice produced only IFN-γ following antigen-specific re-stimulation. This altered cytokine expression was not associated with increased antigen load suggesting the loss of type I IFN receptor signaling was responsible for the altered quality of the CD8(+) effector T cell response. Together, these results indicate that although type I IFN is not essential for the intrinsic adjuvanting of RepliVAX WN, it plays a role in shaping the cytokine secretion profiles of CD8(+) effector T cells elicited by this SCFV.

Analyses of mutations selected by passaging a chimeric flavivirus identify mutations that alter infectivity and reveal an interaction between the structural proteins and the nonstructural glycoprotein NS1.

We previously described a single-cycle dengue vaccine (RepliVAX D2) engineered from a capsid (C) gene-deleted West Nile virus (WNV) expressing dengue virus serotype 2 (DENV2) prM/E genes in place of the corresponding WNV genes. That work demonstrated that adaptation of RepliVAX D2 to grow in WNV C-expressing cells resulted in acquisition of non-synonymous mutations in the DENV2 prM/E and WNV NS2A/NS3 genes. Here we demonstrate that the prM/E mutations increase the specific infectivity of chimeric virions and the NS2A/NS3 mutations independently enhance packaging. Studies with the NS2A mutant demonstrated that it was unable to produce a larger form of NS1 (NS1'), suggesting that the mutation had been selected to eliminate a ribosomal frame-shift "slippage site" in NS2A. Evaluation of a synonymous mutation at this slippage site confirmed that genomes that failed to make NS1' were packaged more efficiently than WT genomes supporting a role for NS1/NS1' in orchestrating virion assembly.

Enhancing the utility of a prM/E-expressing chimeric vaccine for Japanese encephalitis by addition of the JEV NS1 gene.

Recently, we demonstrated that a single-cycle West Nile virus (WNV) named RepliVAX WN could be used to produce a chimeric Japanese encephalitis (JE) vaccine (RepliVAX JE) by replacing the WNV prM/E genes with those of JEV. Here, we tested if replacement of WNV NS1 gene in RepliVAX JE with that of JEV (producing TripliVAX JE) could produce a superior vaccine. TripliVAX JE elicited higher anti-E immunity and displayed better efficacy in mice than RepliVAX JE. Furthermore, TripliVAX JE displayed reduced immune interference caused by pre-existing anti-NS1 immunity. Thus, we propose prM/E/NS1 chimerization as a new strategy for flavivirus vaccine development.

RepliVAX WN, a single-cycle flavivirus vaccine to prevent West Nile disease, elicits durable protective immunity in hamsters.

West Nile virus (WNV) causes significant disease, yet no vaccines exist to prevent WN disease in humans. We have previously reported that RepliVAX WN is a safe and efficacious vaccine in mouse and hamster models of WN disease. Here, we report that vaccination of hamsters with RepliVAX WN induces antibody responses that remain stable for at least 6 months. Furthermore, animals challenged with virulent WNV 6 months after vaccination were protected from disease as well as those challenged 2 months post-vaccination, with no vaccinated animals succumbing to WNV challenge. These results indicate that RepliVAX is capable of inducing durable protective immunity after a single dose.

Construction and characterization of a single-cycle chimeric flavivirus vaccine candidate that protects mice against lethal challenge with dengue virus type 2.

We have previously described a novel flavivirus vaccine technology based on a single-cycle, capsid (C) gene-deleted flavivirus called RepliVAX. RepliVAX can be propagated in cells that express high levels of C but undergoes only a single cycle of infection in vaccinated hosts. Here we report that we have adapted our RepliVAX technology to produce a dengue vaccine by replacing the prM/E genes of RepliVAX WN (a West Nile virus [WNV] RepliVAX) with the same genes of dengue virus type 2 (DENV2). Our first RepliVAX construct for dengue virus (RepliVAX D2) replicated poorly in WNV C-expressing cells. However, addition of mutations in prM and E that were selected during blind passage of a RepliVAX D2 derivative was used to produce a second-generation RepliVAX D2 (designated D2.2) that displayed acceptable growth in WNV C-expressing cells. RepliVAX D2.2 grew better in DENV2 C-expressing cells than WNV C-expressing cells, but after several passages in DENV2 C-expressing cells it acquired further mutations that permitted efficient growth in WNV C-expressing cells. We tested the potency and efficacy of RepliVAX D2.2 in a well-described immunodeficient mouse model for dengue (strain AG129; lacking the receptors for both type I and type II interferons). These mice produced dose-dependent DENV2-neutralizing antibody responses when vaccinated with RepliVAX D2.2. When challenged with 240 50% lethal doses of DENV2, mice given a single inoculation of RepliVAX D2.2 survived significantly longer than sham-vaccinated animals, although some of these severely immunocompromised mice eventually died from the challenge. Taken together these studies indicate that the RepliVAX technology shows promise for use in the development of vaccines that can be used to prevent dengue.

Proteçäo fetal frente a desafio com o vírus da Diarréia Viral Bovina (BVDV) em ovelhas imunizadas com duas amostras de vírus modificadas experimentalmente / Fetal protection against challenge with bovine viral diarrhea virus (BVDV) in pregnant ewes immunized with two strains experimentally attenuated

Duas amostras do vírus da Diarréia Viral Bovina (BVDV) submetidas a múltiplas passagens em cultivo celular e exposiçäo à radiaçäo ultravioleta (UV) a cada passagem foram avaliadas como candidatos a vírus vacinais. As amostras foram testadas quanto à sua atenuaçäo para bezerros e fetos ovinos, reatividade antigênica contra isolados de campo, e capacidade de induzir proteçäo fetal em ovelhas prenhes. Inoculaçäo intramuscular (IM) dos vírus modificados em quatro bezerros produziu apenas uma elevaçäo discreta e passageira da temperatura corporal, seguida de produçäo de altos títulos de anticorpos neutralizantes. O vírus näo foi detectado em secreçöes nasais ou sangue nos dias seguintes à inoculaçäo. Porém, a inoculaçäo IM desses vírus em quatro ovelhas prenhes foi seguida de transmissäo transplacentária e infecçäo em todos os fetos. Para os testes de proteçäo fetal, ovelhas prenhes previamente imunizadas com duas doses vacinais, foram inoculadas por via intranasal com amostras de BVDV-1 (SV-126.8, n=6) ou BVDV-2 (SV-260, n=5). No dia do desafio (134 dias após a segunda dose), todos os animais apresentavam altos títulos de anticorpos neutralizantes (256 a >4096) contra os vírus vacinais; além de títulos variados (8 a >4096) contra várias isolados brasileiros de BVDV-1 e BVDV-2. Quinze dias após o desafio, as ovelhas foram sacrificadas e os tecidos fetais foram examinados para a presença de vírus. Todos os fetos das ovelhas controle näo-vacinadas apresentaram-se (n=4) positivos para os vírus utilizados no desafio. Em contraste, nenhum feto das ovelhas imunizadas (n=11) foi positivo para vírus, indicando que a resposta imunológica induzida pela vacinaçäo com os vírus modificados foi capaz de prevenir a infecçäo fetal. Estes resultados indicam que é possível obter-se forte resposta imunológica e proteçäo fetal contra o BVDV com o uso de vacinas vivas modificadas