Dual roles for the ER membrane protein complex in flavivirus infection: viral entry and protein biogenesis.

Hundreds of cellular host factors are required to support dengue virus infection, but their identity and roles are incompletely characterized. Here, we identify human host dependency factors required for efficient dengue virus-2 (DENV2) infection of human cells. We focused on two, TTC35 and TMEM111, which we previously demonstrated to be required for yellow fever virus (YFV) infection and others subsequently showed were also required by other flaviviruses. These proteins are components of the human endoplasmic reticulum membrane protein complex (EMC), which has roles in ER-associated protein biogenesis and lipid metabolism. We report that DENV, YFV and Zika virus (ZIKV) infections were strikingly inhibited, while West Nile virus infection was unchanged, in cells that lack EMC subunit 4. Furthermore, targeted depletion of EMC subunits in live mosquitoes significantly reduced DENV2 propagation in vivo. Using a novel uncoating assay, which measures interactions between host RNA-binding proteins and incoming viral RNA, we show that EMC is required at or prior to virus uncoating. Importantly, we uncovered a second and important role for the EMC. The complex is required for viral protein accumulation in a cell line harboring a ZIKV replicon, indicating that EMC participates in the complex process of viral protein biogenesis.

Antibodies to a Conserved Influenza Head Interface Epitope Protect by an IgG Subtype-Dependent Mechanism.

Vaccines to generate durable humoral immunity against antigenically evolving pathogens such as the influenza virus must elicit antibodies that recognize conserved epitopes. Analysis of single memory B cells from immunized human donors has led us to characterize a previously unrecognized epitope of influenza hemagglutinin (HA) that is immunogenic in humans and conserved among influenza subtypes. Structures show that an unrelated antibody from a participant in an experimental infection protocol recognized the epitope as well. IgGs specific for this antigenic determinant do not block viral infection in vitro, but passive administration to mice affords robust IgG subtype-dependent protection against influenza infection. The epitope, occluded in the pre-fusion form of HA, is at the contact surface between HA head domains; reversible molecular "breathing" of the HA trimer can expose the interface to antibody and B cells. Antigens that present this broadly immunogenic HA epitope may be good candidates for inclusion in "universal" flu vaccines.

Influenza Antigen Engineering Focuses Immune Responses to a Subdominant but Broadly Protective Viral Epitope.

Viral glycoproteins are under constant immune surveillance by a host's adaptive immune responses. Antigenic variation including glycan introduction or removal is among the mechanisms viruses have evolved to escape host immunity. Understanding how glycosylation affects immunodominance on complex protein antigens may help decipher underlying B cell biology. To determine how B cell responses can be altered by such modifications, we engineered glycans onto the influenza virus hemagglutinin (HA) and characterized the molecular features of the elicited humoral immunity in mice. We found that glycan addition changed the initially diverse antibody repertoire into an epitope-focused, genetically restricted response. Structural analyses showed that one antibody gene family targeted a previously subdominant, occluded epitope at the head interface. Passive transfer of this antibody conferred Fc-dependent protection to influenza virus-challenged mice. These results have potential implications for next-generation viral vaccines aimed at directing B cell responses to preferred epitope(s).

Influenza-Mediated Lung Infection Models.

Laboratory rodent influenza infection models have been and continue to be a critical tool for understanding virus-host interactions during infection. The incidence of seasonal influenza infections coupled with the need for novel therapeutics and universal vaccines highlights the need to uncover novel mechanisms of pathogenesis and protection. Mouse models are extremely useful for the evaluation of influenza vaccines and provide an invaluable tool to probe the immune response. This chapter describes the technique of intranasal inoculation of male C57BL/6J mice with an H1N1 strain of influenza (A/Puerto Rico/8/1934) and methods for assessing the optimum dose for infection, viral titers in lung tissue, and severity of disease.

Memory B Cells that Cross-React with Group 1 and Group 2 Influenza A Viruses Are Abundant in Adult Human Repertoires.

Human B cell antigen-receptor (BCR) repertoires reflect repeated exposures to evolving influenza viruses; new exposures update the previously generated B cell memory (Bmem) population. Despite structural similarity of hemagglutinins (HAs) from the two groups of influenza A viruses, cross-reacting antibodies (Abs) are uncommon. We analyzed Bmem compartments in three unrelated, adult donors and found frequent cross-group BCRs, both HA-head directed and non-head directed. Members of a clonal lineage from one donor had a BCR structure similar to that of a previously described Ab, encoded by different gene segments. Comparison showed that both Abs contacted the HA receptor-binding site through long heavy-chain third complementarity determining regions. Affinities of the clonal-lineage BCRs for historical influenza-virus HAs from both group 1 and group 2 viruses suggested that serial responses to seasonal influenza exposures had elicited the lineage and driven affinity maturation. We propose that appropriate immunization regimens might elicit a comparably broad response.

A robust microparticle platform for a STING-targeted adjuvant that enhances both humoral and cellular immunity during vaccination.

Most FDA-approved adjuvants for infectious agents boost humoral but not cellular immunity, and have poorly-understood mechanisms. Stimulator of interferon genes (STING, also known as MITA, MPYS, or ERIS) is an exciting adjuvant target due to its role in cyclic dinucleotide (CDN)-driven anti-viral immunity; however, a major hindrance is STING's cytosolic localization which requires intracellular delivery of its agonists. As a result, STING agonists administered in a soluble form have elicited suboptimal immune responses. Delivery of STING agonists via particle platforms has proven a more successful strategy, but the opportunity for improved formulations and bioactivity remains. In this study we evaluated the adjuvant activity of the potent STING agonist, CDN 3'3'-cGAMP (cGAMP), encapsulated in acid-sensitive acetalated dextran (Ace-DEX) polymeric microparticles (MPs) which passively target antigen-presenting cells for intracellular release. This formulation was superior to all particle delivery systems evaluated and maintained its bioactivity following a sterilizing dose of gamma irradiation. Compared to soluble cGAMP, the Ace-DEX cGAMP MPs enhanced type-I interferon responses nearly 1000-fold in vitro and 50-fold in vivo, caused up to a 104-fold boost in antibody titers, increased Th1-associated responses, and expanded germinal center B cells and memory T cells. Furthermore, the encapsulated cGAMP elicited no observable toxicity in animals and achieved protective immunity against a lethal influenza challenge seven months post-immunization when using CDN adjuvant doses up to 100-fold lower than previous reports. For these reasons, Ace-DEX MP-encapsulated cGAMP represents a potent vaccine adjuvant of humoral and cellular immunity.

The effect of antipyretics on immune response and fever following receipt of inactivated influenza vaccine in young children.

BACKGROUND: Antipyretics reduce fever following childhood vaccinations; after inactivated influenza vaccine (IIV) they might ameliorate fever and thereby decrease febrile seizure risk, but also possibly blunt the immune response. We assessed the effect of antipyretics on immune responses and fever following IIV in children ages 6 through 47 months. METHODS: Over the course of three seasons, one hundred forty-two children, receiving either a single or the first of 2 recommended doses of IIV, were randomized to receive either oral acetaminophen suspension (n = 59) or placebo (n = 59) (double-blinded) or ibuprofen (n = 24) (open-label) immediately following IIV and every 4-8 h thereafter for 24 h. Blood samples were obtained at enrollment and 4 weeks following the last recommended IIV dose. Responses to IIV were assessed by hemagglutination inhibition assay (HAI). Seroprotection was defined as an HAI titer ≥1:40 and seroconversion as a titer ≥1:40 if baseline titer <1:10 or four-fold rise if baseline titer ≥1:10. Participants were monitored for fever and other solicited symptoms on the day of and day following IIV. RESULTS: Significant differences in seroconversion and post-vaccination seroprotection were not observed between children included in the different antipyretic groups and the placebo group for the vaccine antigens included in IIV over the course of the studies. Frequencies of solicited symptoms, including fever, were similar between treatment groups and the placebo group. CONCLUSIONS: Significant blunting of the immune response was not observed when antipyretics were administered to young children receiving IIV. Studies with larger sample sizes are needed to definitively establish the effect of antipyretics on IIV immunogenicity.

Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination.

Zika virus (ZIKV) has recently emerged as a pandemic associated with severe neuropathology in newborns and adults. There are no ZIKV-specific treatments or preventatives. Therefore, the development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins. Here we demonstrate that a single low-dose intradermal immunization with lipid-nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) encoding the pre-membrane and envelope glycoproteins of a strain from the ZIKV outbreak in 2013 elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 µg of nucleoside-modified ZIKV mRNA-LNP protected mice against ZIKV challenges at 2 weeks or 5 months after vaccination, and a single dose of 50 µg was sufficient to protect non-human primates against a challenge at 5 weeks after vaccination. These data demonstrate that nucleoside-modified mRNA-LNP elicits rapid and durable protective immunity and therefore represents a new and promising vaccine candidate for the global fight against ZIKV.

Modified Vaccinia Ankara Virus Vaccination Provides Long-Term Protection against Nasal Rabbitpox Virus Challenge.

Modified vaccinia Ankara virus (MVA) is a smallpox vaccine candidate. This study was performed to determine if MVA vaccination provides long-term protection against rabbitpox virus (RPXV) challenge, an animal model of smallpox. Two doses of MVA provided 100% protection against a lethal intranasal RPXV challenge administered 9 months after vaccination.

γδ T Cells Play a Protective Role in Chikungunya Virus-Induced Disease.

UNLABELLED: Chikungunya virus (CHIKV) is an alphavirus responsible for causing epidemic outbreaks of polyarthralgia in humans. Because CHIKV is initially introduced via the skin, where γδ T cells are prevalent, we evaluated the response of these cells to CHIKV infection. CHIKV infection led to a significant increase in γδ T cells in the infected foot and draining lymph node that was associated with the production of proinflammatory cytokines and chemokines in C57BL/6J mice. γδ T cell(-/-) mice demonstrated exacerbated CHIKV disease characterized by less weight gain and greater foot swelling than occurred in wild-type mice, as well as a transient increase in monocytes and altered cytokine/chemokine expression in the foot. Histologically, γδ T cell(-/-) mice had increased inflammation-mediated oxidative damage in the ipsilateral foot and ankle joint compared to wild-type mice which was independent of differences in CHIKV replication. These results suggest that γδ T cells play a protective role in limiting the CHIKV-induced inflammatory response and subsequent tissue and joint damage. IMPORTANCE: Recent epidemics, including the 2004 to 2007 outbreak and the spread of CHIKV to naive populations in the Caribbean and Central and South America with resultant cases imported into the United States, have highlighted the capacity of CHIKV to cause explosive epidemics where the virus can spread to millions of people and rapidly move into new areas. These studies identified γδ T cells as important to both recruitment of key inflammatory cell populations and dampening the tissue injury due to oxidative stress. Given the importance of these cells in the early response to CHIKV, this information may inform the development of CHIKV vaccines and therapeutics.

Isolation and Characterization of Broad and Ultrapotent Human Monoclonal Antibodies with Therapeutic Activity against Chikungunya Virus.

Chikungunya virus (CHIKV) is a mosquito-transmitted RNA virus that causes acute febrile infection associated with polyarthralgia in humans. Mechanisms of protective immunity against CHIKV are poorly understood, and no effective therapeutics or vaccines are available. We isolated and characterized human monoclonal antibodies (mAbs) that neutralize CHIKV infectivity. Among the 30 mAbs isolated, 13 had broad and ultrapotent neutralizing activity (IC50 < 10 ng/ml), and all of these mapped to domain A of the E2 envelope protein. Potent inhibitory mAbs blocked post-attachment steps required for CHIKV membrane fusion, and several were protective in a lethal challenge model in immunocompromised mice, even when administered at late time points after infection. These highly protective mAbs could be considered for prevention or treatment of CHIKV infection, and their epitope location in domain A of E2 could be targeted for rational structure-based vaccine development.

Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation.

Oxidative tissue injury often accompanies viral infection, yet there is little understanding of how it influences virus replication. We show that multiple hepatitis C virus (HCV) genotypes are exquisitely sensitive to oxidative membrane damage, a property distinguishing them from other pathogenic RNA viruses. Lipid peroxidation, regulated in part through sphingosine kinase-2, severely restricts HCV replication in Huh-7 cells and primary human hepatoblasts. Endogenous oxidative membrane damage lowers the 50% effective concentration of direct-acting antivirals in vitro, suggesting critical regulation of the conformation of the NS3-4A protease and the NS5B polymerase, membrane-bound HCV replicase components. Resistance to lipid peroxidation maps genetically to transmembrane and membrane-proximal residues within these proteins and is essential for robust replication in cell culture, as exemplified by the atypical JFH1 strain of HCV. Thus, the typical, wild-type HCV replicase is uniquely regulated by lipid peroxidation, providing a mechanism for attenuating replication in stressed tissue and possibly facilitating long-term viral persistence.

Mutational analysis of the West Nile virus NS4B protein.

West Nile virus NS4B is a small hydrophobic nonstructural protein approximately 27 kDa in size whose function is poorly understood. Amino acid substitutions were introduced into the NS4B protein primarily targeting two distinct regions; the N-terminal domain (residues 35 through 60) and the central hydrophobic domain (residues 95 through 120). Only the NS4B P38G substitution was associated with both temperature-sensitive and small-plaque phenotypes. Importantly, this mutation was found to attenuate neuroinvasiveness greater than 10,000,000-fold and lower viremia titers compared to the wild-type NY99 virus in a mouse model. Full genome sequencing of the NS4B P38G mutant virus revealed two unexpected mutations at NS4B T116I and NS3 N480H (P38G/T116I/N480H), however, neither mutation alone was temperature sensitive or attenuated in mice. Following incubation of P38G/T116I/N480H at 41°C, five mutants encoding compensatory substitutions in the NS4B protein exhibited a reduction in the temperature-sensitive phenotype and reversion to a virulent phenotype in the mouse model.

Vector competence of Australian mosquitoes for yellow fever virus.

The vector competence of Australian mosquitoes for yellow fever virus (YFV) was evaluated. Infection and transmission rates in Cairns and Townsville populations of Aedes aegypti and a Brisbane strain of Ae. notoscriptus were not significantly different from a well-characterized YFV-susceptible strain of Ae. aegypti. After exposure to 107·² tissue culture infectious dose (TCID50)/mL of an African strain of YFV, > 70% of Ae. aegypti and Ae. notoscriptus became infected, and > 50% transmitted the virus. When exposed to 106·7) TCID50/mL of a South American strain of YFV, the highest infection (64%) and transmission (56%) rates were observed in Ae. notoscriptus. The infection and transmission rates in the Cairns Ae. aegypti were both 24%, and they were 36% and 28%, respectively, for the Townsville population. Because competent vectors are present, the limited number of travelers from endemic areas and strict vaccination requirements will influence whether YFV transmission occurs in Australia.

Stability of yellow fever virus under recombinatory pressure as compared with chikungunya virus.

Recombination is a mechanism whereby positive sense single stranded RNA viruses exchange segments of genetic information. Recent phylogenetic analyses of naturally occurring recombinant flaviviruses have raised concerns regarding the potential for the emergence of virulent recombinants either post-vaccination or following co-infection with two distinct wild-type viruses. To characterize the conditions and sequences that favor RNA arthropod-borne virus recombination we constructed yellow fever virus (YFV) 17D recombinant crosses containing complementary deletions in the envelope protein coding sequence. These constructs were designed to strongly favor recombination, and the detection conditions were optimized to achieve high sensitivity recovery of putative recombinants. Full length recombinant YFV 17D virus was never detected under any of the experimental conditions examined, despite achieving estimated YFV replicon co-infection levels of ∼2.4 x 106 in BHK-21 (vertebrate) cells and ∼1.05 x 105 in C710 (arthropod) cells. Additionally YFV 17D superinfection resistance was observed in vertebrate and arthropod cells harboring a primary infection with wild-type YFV Asibi strain. Furthermore recombination potential was also evaluated using similarly designed chikungunya virus (CHIKV) replicons towards validation of this strategy for recombination detection. Non-homologus recombination was observed for CHIKV within the structural gene coding sequence resulting in an in-frame duplication of capsid and E3 gene. Based on these data, it is concluded that even in the unlikely event of a high level acute co-infection of two distinct YFV genomes in an arthropod or vertebrate host, the generation of viable flavivirus recombinants is extremely unlikely.

Chikungunya virus adaptation to Aedes albopictus mosquitoes does not correlate with acquisition of cholesterol dependence or decreased pH threshold for fusion reaction.

BACKGROUND: Chikungunya virus (CHIKV) is a mosquito transmitted alphavirus that recently caused several large scale outbreaks/epidemics of arthritic disease in tropics of Africa, Indian Ocean basin and South-East Asia. This re-emergence event was facilitated by genetic adaptation (E1-A226V substitution) of CHIKV to a newly significant mosquito vector for this virus; Aedes albopictus. However, the molecular mechanism explaining the positive effect of the E1-A226V mutation on CHIKV fitness in this vector remains largely unknown. Previously we demonstrated that the E1-A226V substitution is also associated with attenuated CHIKV growth in cells depleted by cholesterol. METHODS: In this study, using a panel of CHIKV clones that varies in sensitivity to cholesterol, we investigated the possible relationship between cholesterol dependence and Ae. albopictus infectivity. RESULTS: We demonstrated that there is no clear mechanistic correlation between these two phenotypes. We also showed that the E1-A226V mutation increases the pH dependence of the CHIKV fusion reaction; however, subsequent genetic analysis failed to support an association between CHIKV dependency on lower pH, and mosquito infectivity phenotypes. CONCLUSION: the E1-A226V mutation probably acts at different steps of the CHIKV life cycle, affecting multiple functions of the virus.

In vivo imaging of chikungunya virus in mice and Aedes mosquitoes using a Renilla luciferase clone.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that in humans causes an acute febrile illness characterized by fever, arthralgia, and rash. It is currently associated with large outbreaks in Asia, Africa, and islands of the Indian Ocean and has been introduced from these tropical regions into Europe, where local transmission has been recorded on two occasions. The underlying basis of the pathogenesis of CHIKV and related alphaviruses that produce similar symptoms remains unclear. By applying new techniques, for example, in vivo imaging in live animals and arthropods, we may improve our understanding of viral pathogenesis in vertebrates and viral replication in mosquitoes. This technical report describes the evaluation of a CHIKV?luciferase clone to visualize infection and dissemination in both Aedes aegypti and Aedes albopictus mosquitoes and mice. In mosquitoes, luciferase activity was seen at 3 and 7 days post-infection in both head and abdomens. In vivo imaging of CHIKV-luciferase was detected in mice for up to 5 days post-infection at the site of inoculation with limited dissemination to the skeletal muscle.

Aedes aegypti saliva alters leukocyte recruitment and cytokine signaling by antigen-presenting cells during West Nile virus infection.

West Nile virus (WNV) is transmitted during mosquito bloodfeeding. Consequently, the first vertebrate cells to contact WNV are cells in the skin, followed by those in the draining lymph node. Macrophages and dendritic cells are critical early responders in host defense against WNV infection, not just because of their role in orchestrating the immune response, but also because of their importance as sites of early peripheral viral replication. Antigen-presenting cell (APC) signals have a profound effect on host antiviral responses and disease severity. During transmission, WNV is intimately associated with mosquito saliva. Due to the ability of mosquito saliva to affect inflammation and immune responses, and the importance of understanding early events in WNV infection, we investigated whether mosquito saliva alters APC signaling during arbovirus infection, and if alterations in cell recruitment occur when WNV infection is initiated with mosquito saliva. Accordingly, experiments were performed with cultured dendritic cells and macrophages, flow cytometry was used to characterize infiltrating cell types in the skin and lymph nodes during early infection, and real-time RT-PCR was employed to evaluate virus and cytokine levels. Our in vitro results suggest that mosquito saliva significantly decreases the expression of interferon-beta and inducible nitric oxide synthase in macrophages (by as much as 50 and 70%, respectively), whilst transiently enhancing interleukin-10 (IL-10) expression. In vivo results indicate that the predominate effect of mosquito feeding is to significantly reduce the recruitment of T cells, leading the inoculation site of mice exposed to WNV alone to have up to 2.8 fold more t cells as mice infected in the presence of mosquito saliva. These shifts in cell population are associated with significantly elevated IL-10 and WNV (up to 4.0 and 10 fold, respectively) in the skin and draining lymph nodes. These results suggest that mosquito saliva dysregulates APC antiviral signaling, and reveal a possible mechanism for the observed enhancement of WNV disease mediated by mosquito saliva via a reduction of T lymphocyte and antiviral activity at the inoculation site, an elevated abundance of susceptible cell types, and a concomitant increase in immunoregulatory activity of IL-10.

Transcriptome changes in Culex quinquefasciatus (Diptera: Culicidae) salivary glands during West Nile virus infection.

Persistent West Nile virus (WNV) infection in the mosquito Culex quinquefasciatus Say (Diptera: Culicidae) is associated with pathological changes in the salivary glands, including apoptotic cell death and a corresponding reduction in virus transmission over time. The vector host response to WNV infection and the molecular basis of WNV pathogenesis in Cx. quinquefasciatus was investigated using oligonucleotide microarrays designed to detect differences in the salivary gland transcriptome between WNV-infected mosquitoes and uninfected controls. Transcripts with increased abundance in infected salivary glands included those related to immunity, transcription, protein transport and degradation, amino acid and nucleotide metabolism, signal transduction, and cellular detoxification. Microarray-based analysis detected a decrease in transcript levels of a Culex inhibitor of apoptosis gene (IAP-1) and a decrease in abundance of 11 transcripts encoding salivary gland proteins. Transcript levels for an endonuclease, a proline-rich mucin, and several D7 protein family members also decreased. Transcripts with the greatest change in abundance during infection had either no similarity to sequences found in GenBank, VectorBase, and FlyBase, or were similar to sequences with uncharacterized protein products. These transcripts represent exciting targets for future analysis. Results from this study suggest that WNV infection influences transcriptional changes in an invertebrate host target tissue that may confer an advantage to the replicating virus, induce a host defense response, and alter the composition of vector saliva. The ramifications of these changes are discussed in terms of mosquito vector competence and WNV pathogenesis.

Infection, dissemination, and transmission of a West Nile virus green fluorescent protein infectious clone by Culex pipiens quinquefasciatus mosquitoes.

We report the construction and comparative characterization of a full-length West Nile virus (WNV) cDNA infectious clone (ic) that contains a green fluorescent protein (GFP) expression cassette fused within the viral open reading frame. Virus derived from WNV-GFP ic stably infected Culex pipiens quinquefasciatus mosquitoes at comparable rates to virus derived from the parental (non-GFP) ic. However, insertion of this GFP cassette resulted in a temporal delay in in vivo replication kinetics and significantly decreased dissemination to head tissue. Consistent with previous reports of WNV-infected mosquito midguts, focal GFP expression was observed at 3 days post-infection (dpi), with the majority of posterior midgut epithelial cells being positive by 7 dpi. GFP foci were observed in one pair of salivary glands (1/15) dissected 14 dpi. Mice exposed to WNV-GFP-infected mosquitoes developed viremia, and GFP was detected in lymph node homogenates. These data demonstrate the effectiveness of our strategy to generate a replication competent construct with increased reporter gene stability that may be used to study early events in infection.