Recent epidemics of Zika virus (ZIKV) have been associated with congenital malformation during pregnancy and Guillain-Barré syndrome. There are two ZIKV lineages (African and Asian) that share >95% amino acid identity. Little is known regarding the ability of neutralizing antibodies elicited against one lineage to protect against the other. We investigated the breadth of the neutralizing antibody response following ZIKV infection by measuring the sensitivity of six ZIKV strains to neutralization by ZIKV-confirmed convalescent human serum or plasma samples. Contemporary Asian and early African ZIKV strains were similarly sensitive to neutralization regardless of the cellular source of virus. Furthermore, mouse immune serum generated after infection with African or Asian ZIKV strains was capable of neutralizing homologous and heterologous ZIKV strains equivalently. Because our study only defines a single ZIKV serotype, vaccine candidates eliciting robust neutralizing antibody responses should inhibit infection of both ZIKV lineages, including strains circulating in the Americas.

Flaviviruses infect hundreds of millions of people annually, and no antiviral therapy is available. We performed a genome-wide CRISPR/Cas9-based screen to identify host genes that, when edited, resulted in reduced flavivirus infection. Here, we validated nine human genes required for flavivirus infectivity, and these were associated with endoplasmic reticulum functions including translocation, protein degradation, and N-linked glycosylation. In particular, a subset of endoplasmic reticulum-associated signal peptidase complex (SPCS) proteins was necessary for proper cleavage of the flavivirus structural proteins (prM and E) and secretion of viral particles. Loss of SPCS1 expression resulted in markedly reduced yield of all Flaviviridae family members tested (West Nile, Dengue, Zika, yellow fever, Japanese encephalitis, and hepatitis C viruses), but had little impact on alphavirus, bunyavirus, or rhabdovirus infection or the surface expression or secretion of diverse host proteins. We found that SPCS1 dependence could be bypassed by replacing the native prM protein leader sequences with a class I major histocompatibility complex (MHC) antigen leader sequence. Thus, SPCS1, either directly or indirectly via its interactions with unknown host proteins, preferentially promotes the processing of specific protein cargo, and Flaviviridae have a unique dependence on this signal peptide processing pathway. SPCS1 and other signal processing pathway members could represent pharmacological targets for inhibiting infection by the expanding number of flaviviruses of medical concern.

There is increasing evidence that many solid tumors are hierarchically organized with the bulk tumor cells having limited replication potential, but are sustained by a stem-like cell that perpetuates the tumor. These cancer stem cells have been hypothesized to originate from transformation of adult tissue stem cells, or through re-acquisition of stem-like properties by progenitor cells. Adenosquamous carcinoma (ASC) is an aggressive type of lung cancer that contains a mixture of cells with squamous (cytokeratin 5+) and adenocarcinoma (cytokeratin 7+) phenotypes. The origin of these mixtures is unclear as squamous carcinomas are thought to arise from basal cells in the upper respiratory tract while adenocarcinomas are believed to form from stem cells in the bronchial alveolar junction. We have isolated and characterized cancer stem-like populations from ASC through application of selective defined culture medium initially used to grow human lung stem cells. Homogeneous cells selected from ASC tumor specimens were stably expanded in vitro. Primary xenografts and metastatic lesions derived from these cells in NSG mice fully recapitulate both the adenocarcinoma and squamous features of the patient tumor. Interestingly, while the CSLC all co-expressed cytokeratins 5 and 7, most xenograft cells expressed either one, or neither, with <10% remaining double positive. We also demonstrated the potential of the CSLC to differentiate to multi-lineage structures with branching lung morphology expressing bronchial, alveolar and neuroendocrine markers in vitro. Taken together the properties of these ASC-derived CSLC suggests that ASC may arise from a primitive lung stem cell distinct from the bronchial-alveolar or basal stem cells.

Protégé was a phase 3, randomized, double-blind, parallel, placebo-controlled 2-year study of three intravenous teplizumab dosing regimens, administered daily for 14 days at baseline and again after 26 weeks, in new-onset type 1 diabetes. We sought to determine efficacy and safety of teplizumab immunotherapy at 2 years and to identify characteristics associated with therapeutic response. Of 516 randomized patients, 513 were treated, and 462 completed 2 years of follow-up. Teplizumab (14-day full-dose) reduced the loss of C-peptide mean area under the curve (AUC), a prespecified secondary end point, at 2 years versus placebo. In analyses of prespecified and post hoc subsets at entry, U.S. residents, patients with C-peptide mean AUC >0.2 nmol/L, those randomized ≤6 weeks after diagnosis, HbA1c <7.5% (58 mmol/mol), insulin use <0.4 units/kg/day, and 8-17 years of age each had greater teplizumab-associated C-peptide preservation than their counterparts. Exogenous insulin needs tended to be reduced versus placebo. Antidrug antibodies developed in some patients, without apparent change in drug efficacy. No new safety or tolerability issues were observed during year 2. In summary, anti-CD3 therapy reduced C-peptide loss 2 years after diagnosis using a tolerable dose.

West Nile virus is a mosquito-borne flavivirus closely related to the human epidemic-causing dengue, yellow fever and Japanese encephalitis viruses. In establishing infection these icosahedral viruses undergo endosomal membrane fusion catalysed by envelope glycoprotein rearrangement of the putative receptor-binding domain III (DIII) and exposure of the hydrophobic fusion loop. Humoral immunity has an essential protective function early in the course of West Nile virus infection. Here, we investigate the mechanism of neutralization by the E16 monoclonal antibody that specifically binds DIII. Structurally, the E16 antibody Fab fragment engages 16 residues positioned on four loops of DIII, a consensus neutralizing epitope sequence conserved in West Nile virus and distinct in other flaviviruses. The E16 epitope protrudes from the surface of mature virions in three distinct environments, and docking studies predict Fab binding will leave five-fold clustered epitopes exposed. We also show that E16 inhibits infection primarily at a step after viral attachment, potentially by blocking envelope glycoprotein conformational changes. Collectively, our results suggest that a vaccine strategy targeting the dominant DIII epitope may elicit safe and effective immune responses against flaviviral diseases.

During cell entry of flaviviruses, low endosomal pH triggers the rearrangement of the viral surface glycoproteins to a fusion-active state that allows the release of the infectious RNA into the cytoplasm. In this work, West Nile virus was complexed with Fab fragments of the neutralizing mAb E16 and was subsequently exposed to low pH, trapping the virions in a pre-fusion intermediate state. The structure of the complex was studied by cryo-electron microscopy and provides the first structural glimpse of a flavivirus fusion intermediate near physiological conditions. A radial expansion of the outer protein layer of the virion was observed compared to the structure at pH 8. The resulting approximately 60 A-wide shell of low density between lipid bilayer and outer protein layer is likely traversed by the stem region of the E glycoprotein. By using antibody fragments, we have captured a structural intermediate of a virus that likely occurs during cell entry. The trapping of structural transition states by antibody fragments will be applicable for other processes in the flavivirus life cycle and delineating other cellular events that involve conformational rearrangements.

Bispecific antibodies capable of redirecting the lytic potential of immune effector cells to kill tumor targets have long been recognized as a potentially potent biological therapeutic intervention. Unfortunately, efforts to produce such molecules have been limited owing to inefficient production and poor stability properties. Here, we describe a novel Fv-derived strategy based on a covalently linked bispecific diabody structure that we term dual-affinity re-targeting (DART). As a model system, we linked an Fv specific for human CD16 (FcgammaRIII) on effector cells to an Fv specific for mouse or human CD32B (FcgammaRIIB), a normal B-cell and tumor target antigen. DART proteins were produced at high levels in mammalian cells, retained the binding activity of the respective parental Fv domains as well as bispecific binding, and showed extended storage and serum stability. Functionally, the DART molecules demonstrated extremely potent, dose-dependent cytotoxicity in retargeting human PBMC against B-lymphoma cell lines as well as in mediating autologous B-cell depletion in culture. In vivo studies in mice demonstrated effective B-cell depletion that was dependent on the transgenic expression of both CD16A on the effector cells and CD32B on the B-cell targets. Furthermore, DART proteins showed potent in vivo protective activity in a human Burkitt's lymphoma cell xenograft model. Thus, DART represents a biologically potent format that provides a versatile platform for generating bispecific antibody fragments for redirected killing and, with the selection of appropriate binding partners, applications outside of tumor cell cytotoxicity.

The structural flexibility or 'breathing' of the envelope (E) protein of flaviviruses allows virions to sample an ensemble of conformations at equilibrium. The molecular basis and functional consequences of virus conformational dynamics are poorly understood. Here, we identified a single mutation at residue 198 (T198F) of the West Nile virus (WNV) E protein domain I-II hinge that regulates virus breathing. The T198F mutation resulted in a ~70-fold increase in sensitivity to neutralization by a monoclonal antibody targeting a cryptic epitope in the fusion loop. Increased exposure of this otherwise poorly accessible fusion loop epitope was accompanied by reduced virus stability in solution at physiological temperatures. Introduction of a mutation at the analogous residue of dengue virus (DENV), but not Zika virus (ZIKV), E protein also increased accessibility of the cryptic fusion loop epitope and decreased virus stability in solution, suggesting that this residue modulates the structural ensembles sampled by distinct flaviviruses at equilibrium in a context dependent manner. Although the T198F mutation did not substantially impair WNV growth kinetics in vitro, studies in mice revealed attenuation of WNV T198F infection. Overall, our study provides insight into the molecular basis and the in vitro and in vivo consequences of flavivirus breathing.

Flaviviruses are controlled by adaptive immune responses but are exquisitely sensitive to interferon-stimulated genes (ISGs). How coinfections, particularly simian immunodeficiency viruses (SIVs), that induce robust ISG signatures influence flavivirus clearance and pathogenesis is unclear. Here, we studied how Zika virus (ZIKV) infection is modulated in SIV-infected nonhuman primates. We measured ZIKV replication, cellular ZIKV RNA levels, and immune responses in non-SIV-infected and SIV-infected rhesus macaques (RMs), which we infected with ZIKV. Coinfected animals had a 1- to 2-day delay in peak ZIKV viremia, which was 30% of that in non-SIV-infected animals. However, ZIKV viremia was significantly prolonged in SIV-positive (SIV+) RMs. ISG levels at the time of ZIKV infection were predictive for lower ZIKV viremia in the SIV+ RMs, while prolonged ZIKV viremia was associated with muted and delayed adaptive responses in SIV+ RMs.IMPORTANCE Immunocompromised individuals often become symptomatic with infections which are normally fairly asymptomatic in healthy individuals. The particular mechanisms that underlie susceptibility to coinfections in human immunodeficiency virus (HIV)-infected individuals are multifaceted. ZIKV and other flaviviruses are sensitive to neutralizing antibodies, whose production can be limited in HIV-infected individuals but are also sensitive to type I interferons, which are expressed at high levels in HIV-infected individuals. Data in this study highlight how individual components of the innate and adaptive immune responses which become perturbed in HIV-infected individuals influence ZIKV infection.

Cleavage of the flavivirus premembrane (prM) structural protein during maturation can be inefficient. The contribution of partially mature flavivirus virions that retain uncleaved prM to pathogenesis during primary infection is unknown. To investigate this question, we characterized the functional properties of newly-generated dengue virus (DENV) prM-reactive monoclonal antibodies (mAbs) in vitro and using a mouse model of DENV disease. Anti-prM mAbs neutralized DENV infection in a virion maturation state-dependent manner. Alanine scanning mutagenesis and cryoelectron microscopy of anti-prM mAbs in complex with immature DENV defined two modes of attachment to a single antigenic site. In vivo, passive transfer of intact anti-prM mAbs resulted in an antibody-dependent enhancement of disease. However, protection against DENV-induced lethality was observed when the transferred mAbs were genetically modified to inhibit their ability to interact with Fcγ receptors. These data establish that in addition to mature forms of the virus, partially mature infectious prM+ virions can also contribute to pathogenesis during primary DENV infections.

Meta-analysis of evidence level I to IV studies.

West Nile virus (WNV) has become the principal cause of viral encephalitis in North America since its introduction in New York in 1999. This emerging virus is transmitted to humans via the bite of an infected mosquito. While there have been several candidates in clinical trials, there are no approved vaccines or WNV-specific therapies for the treatment of WNV disease in humans. From studies with small animal models and convalescent human patients, a great deal has been learned concerning the immune response to infection with WNV. Here, we provide an overview of a subset of that information regarding the humoral and antibody response generated during WNV infection.

West Nile virus (WNV), a member of the Flavivirus genus, is a leading cause of viral encephalitis in the United States1. The development of neutralizing antibodies against the flavivirus envelope (E) protein is critical for immunity and vaccine protection2. Previously identified candidate therapeutic mouse and human neutralizing monoclonal antibodies (mAbs) target epitopes within the E domain III lateral ridge and the domain I-II hinge region, respectively3. To explore the neutralizing antibody repertoire elicited by WNV infection for potential therapeutic application, we isolated ten mAbs from WNV-infected individuals. mAb WNV-86 neutralized WNV with a 50% inhibitory concentration of 2 ng ml-1, one of the most potently neutralizing flavivirus-specific antibodies ever isolated. WNV-86 targets an epitope in E domain II, and preferentially recognizes mature virions lacking an uncleaved form of the chaperone protein prM, unlike most flavivirus-specific antibodies4. In vitro selection experiments revealed a neutralization escape mechanism involving a glycan addition to E domain II. Finally, a single dose of WNV-86 administered two days post-infection protected mice from lethal WNV challenge. This study identifies a highly potent human neutralizing mAb with therapeutic potential that targets an epitope preferentially displayed on mature virions.

The re-emergence of Zika virus (ZIKV) caused widespread infections that were linked to Guillain-Barré syndrome in adults and congenital malformation in fetuses, and epidemiological data suggest that ZIKV infection can induce protective antibody responses. A more detailed understanding of anti-ZIKV antibody responses may lead to enhanced antibody discovery and improved vaccine designs against ZIKV and related flaviviruses. Here, we applied recently-invented library-scale antibody screening technologies to determine comprehensive functional molecular and genetic profiles of naturally elicited human anti-ZIKV antibodies in three convalescent individuals. We leveraged natively paired antibody yeast display and NGS to predict antibody cross-reactivities and coarse-grain antibody affinities, to perform in-depth immune profiling of IgM, IgG, and IgA antibody repertoires in peripheral blood, and to reveal virus maturation state-dependent antibody interactions. Repertoire-scale comparison of ZIKV VLP-specific and non-specific antibodies in the same individuals also showed that mean antibody somatic hypermutation levels were substantially influenced by donor-intrinsic characteristics. These data provide insights into antiviral antibody responses to ZIKV disease and outline systems-level strategies to track human antibody immune responses to emergent viral infections.

The emergence of ZIKV infection has prompted a global effort to develop safe and effective vaccines. We engineered a lipid nanoparticle (LNP) encapsulated modified mRNA vaccine encoding wild-type or variant ZIKV structural genes and tested immunogenicity and protection in mice. Two doses of modified mRNA LNPs encoding prM-E genes that produced virus-like particles resulted in high neutralizing antibody titers (∼1/100,000) that protected against ZIKV infection and conferred sterilizing immunity. To offset a theoretical concern of ZIKV vaccines inducing antibodies that cross-react with the related dengue virus (DENV), we designed modified prM-E RNA encoding mutations destroying the conserved fusion-loop epitope in the E protein. This variant protected against ZIKV and diminished production of antibodies enhancing DENV infection in cells or mice. A modified mRNA vaccine can prevent ZIKV disease and be adapted to reduce the risk of sensitizing individuals to subsequent exposure to DENV, should this become a clinically relevant concern.

Although divergent dengue viruses (DENVs) have been isolated in insects, nonhuman primates, and humans, their relationships to the four canonical serotypes (DENV 1-4) are poorly understood. One virus isolated from a dengue patient, DKE-121, falls between genotype and serotype levels of sequence divergence to DENV-4. To examine its antigenic relationship to DENV-4, we assessed serum neutralizing and protective activity. Whereas DENV-4-immune mouse sera neutralize DKE-121 infection, DKE-121-immune sera inhibit DENV-4 less efficiently. Passive transfer of DENV-4 or DKE-121-immune sera protects mice against homologous, but not heterologous, DENV-4 or DKE-121 challenge. Antigenic cartography suggests that DENV-4 and DKE-121 are related but antigenically distinct. However, DENV-4 vaccination confers protection against DKE-121 in nonhuman primates, and serum from humans immunized with a tetravalent vaccine neutralize DENV-4 and DKE-121 infection equivalently. As divergent DENV strains, such as DKE-121, may meet criteria for serotype distinction, monitoring their capacity to impact dengue disease and vaccine efficacy appears warranted.

Flaviviruses are positive-stranded RNA viruses that incorporate envelope (E) and premembrane (prM) proteins into the virion. Furin-mediated cleavage of prM defines a required maturation step in the flavivirus lifecycle. Inefficient prM cleavage results in structurally heterogeneous virions with unique antigenic and functional characteristics. Recent studies with dengue virus suggest that viruses produced in tissue culture cells are less mature than those produced in primary cells. In this study, we describe a Vero cell line that ectopically expresses high levels of human furin (Vero-furin) for use in the production of more homogenous mature flavivirus populations. Laboratory-adapted and clinical dengue virus isolates grow efficiently in Vero-furin cells. Biochemical and structural techniques demonstrate efficient prM cleavage in Vero-furin derived virus preparations. These virions also were less sensitive to neutralization by antibodies that bind efficiently to immature virions. This furin-expressing cell line will be of considerable utility for flavivirus neutralization and structural studies.

Neutralization of West Nile virus (WNV) in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Using random mutagenesis and yeast surface display, we defined individual contact residues of 14 newly generated monoclonal antibodies against domain III of the WNV E protein. Monoclonal antibodies that strongly neutralized WNV localized to a surface patch on the lateral face of domain III. Convalescent antibodies from individuals who had recovered from WNV infection also detected this epitope. One monoclonal antibody, E16, neutralized 10 different strains in vitro, and showed therapeutic efficacy in mice, even when administered as a single dose 5 d after infection. A humanized version of E16 was generated that retained antigen specificity, avidity and neutralizing activity. In postexposure therapeutic trials in mice, a single dose of humanized E16 protected mice against WNV-induced mortality, and may therefore be a viable treatment option against WNV infection in humans.

We previously developed a panel of neutralizing monoclonal antibodies against Dengue virus (DENV)-1, of which few exhibited inhibitory activity against all DENV-1 genotypes. This finding is consistent with reports observing variable neutralization of different DENV strains and genotypes using serum from individuals that experienced natural infection or immunization. Herein, we describe the crystal structures of DENV1-E111 bound to a novel CC' loop epitope on domain III (DIII) of the E protein from two different DENV-1 genotypes. Docking of our structure onto the available cryo-electron microscopy models of DENV virions revealed that the DENV1-E111 epitope was inaccessible, suggesting that this antibody recognizes an uncharacterized virus conformation. While the affinity of binding between DENV1-E111 and DIII varied by genotype, we observed limited correlation with inhibitory activity. Instead, our results support the conclusion that potent neutralization depends on genotype-dependent exposure of the CC' loop epitope. These findings establish new structural complexity of the DENV virion, which may be relevant for the choice of DENV strain for induction or analysis of neutralizing antibodies in the context of vaccine development.

Response to trastuzumab in metastatic breast cancer correlates with expression of the high binding variant (158V) of the activating Fcγ receptor IIIA (CD16A). We engineered MGAH22, a chimeric anti-HER2 monoclonal antibody with specificity and affinity similar to trastuzumab, with an Fc domain engineered for increased binding to both alleles of human CD16A.