Antibodies Elicited by an NS1-Based Vaccine Protect Mice against Zika Virus

ABSTRACTZika virus is a mosquito-borne flavivirus which can cause severe disease in humans, including microcephaly and other congenital malformations in newborns and Guillain-Barré syndrome in adults. There are currently no approved prophylactics or therapeutics for Zika virus; the development of a safe and effective vaccine is an urgent priority. Preclinical studies suggest that the envelope glycoprotein can elicit potently neutralizing antibodies. However, such antibodies are implicated in the phenomenon of antibody-dependent enhancement of disease. We have previously shown that monoclonal antibodies targeting the Zika virus nonstructural NS1 protein are protective without inducing antibody-dependent enhancement of disease. Here, we investigated whether the NS1 protein itself is a viable vaccine target. Wild-type mice were vaccinated with an NS1-expressing DNA plasmid followed by two adjuvanted protein boosters, which elicited high antibody titers. Passive transfer of the immune sera was able to significantly protect STAT2 knockout mice against lethal challenge by Zika virus. In addition, long-lasting NS1-specific IgG responses were detected in serum samples from patients in either the acute or the convalescent phase of Zika virus infection. These NS1-specific antibodies were able to functionally engage Fcγ receptors. In contrast, envelope-specific antibodies did not activate Fc-mediated effector functions on infected cells. Our data suggest that the Zika virus NS1 protein, which is expressed on infected cells, is critical for Fc-dependent cell-mediated immunity. The present study demonstrates that the Zika virus NS1 protein is highly immunogenic and can elicit protective antibodies, underscoring its potential for an effective Zika virus vaccine.IMPORTANCEZika virus is a global public health threat that causes microcephaly and congenital malformations in newborns and Guillain-Barré syndrome in adults. Currently, no vaccines or treatments are available. While antibodies targeting the envelope glycoprotein can neutralize virus, they carry the risk of antibody-dependent enhancement of disease (ADE). In contrast, antibodies generated against the NS1 protein can be protective without eliciting ADE. The present study demonstrates the effectiveness of an NS1-based vaccine in eliciting high titers of protective antibodies against Zika virus disease in a mouse model. Sera generated by this vaccine can elicit Fc-mediated effector functions against Zika virus-infected cells. Lastly, we provide human data suggesting that the antibody response against the Zika virus NS1 protein is long-lasting and functionally active. Overall, our work will inform the development of a safe and effective Zika virus vaccine.

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Mass Spectrometry versus Conventional Techniques of Protein Detection: Zika Virus NS3 Protease Activity towards Cellular Proteins

Molecules ◽

10.3390/molecules26123732 ◽

2021 ◽

Vol 26 (12) ◽

pp. 3732

Author(s):

Agnieszka Dabrowska ◽

Aleksandra Milewska ◽

Joanna Ner-Kluza ◽

Piotr Suder ◽

Krzysztof Pyrc

Keyword(s):

Mass Spectrometry ◽

Zika Virus ◽

Viral Infections ◽

Protein Detection ◽

Extensive Discussion ◽

Protein Targets ◽

Highly Sensitive ◽

Infected Cells ◽

Ns3 Protease ◽

To Receive

Mass spectrometry (MS) used in proteomic approaches is able to detect hundreds of proteins in a single assay. Although undeniable high analytical power of MS, data acquired sometimes lead to confusing results, especially during a search of very selective, unique interactions in complex biological matrices. Here, we would like to show an example of such confusing data, providing an extensive discussion on the observed phenomenon. Our investigations focus on the interaction between the Zika virus NS3 protease, which is essential for virus replication. This enzyme is known for helping to remodel the microenvironment of the infected cells. Several reports show that this protease can process cellular substrates and thereby modify cellular pathways that are important for the virus. Herein, we explored some of the targets of NS3, clearly shown by proteomic techniques, as processed during infection. Unfortunately, we could not confirm the biological relevance of protein targets for viral infections detected by MS. Thus, although mass spectrometry is highly sensitive and useful in many instances, also being able to show directions where cell/virus interaction occurs, we believe that deep recognition of their biological role is essential to receive complete insight into the investigated process.

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Development of Bacteriophage Virus-Like Particle Vaccines Displaying Conserved Epitopes of Dengue Virus Non-Structural Protein 1

Vaccines ◽

10.3390/vaccines9070726 ◽

2021 ◽

Vol 9 (7) ◽

pp. 726

Author(s):

Nikole L. Warner ◽

Kathryn M. Frietze

Keyword(s):

Dengue Virus ◽

Population Based ◽

Ns1 Protein ◽

Structural Protein ◽

Denv Serotypes ◽

Conserved Regions ◽

Virus Like Particle ◽

Dependent Cell ◽

Infected Cells ◽

Plasma Leakage

Dengue virus (DENV) is a major global health problem, with over half of the world’s population at risk of infection. Despite over 60 years of efforts, no licensed vaccine suitable for population-based immunization against DENV is available. Here, we describe efforts to engineer epitope-based vaccines against DENV non-structural protein 1 (NS1). NS1 is present in DENV-infected cells as well as secreted into the blood of infected individuals. NS1 causes disruption of endothelial cell barriers, resulting in plasma leakage and hemorrhage. Immunizing against NS1 could elicit antibodies that block NS1 function and also target NS1-infected cells for antibody-dependent cell cytotoxicity. We identified highly conserved regions of NS1 from all four DENV serotypes. We generated synthetic peptides to these regions and chemically conjugated them to bacteriophage Qβ virus-like particles (VLPs). Mice were immunized two times with the candidate vaccines and sera were tested for the presence of antibodies that bound to the cognate peptide, recombinant NS1 from all four DENV serotypes, and DENV-2-infected cells. We found that two of the candidate vaccines elicited antibodies that bound to recombinant NS1, and one candidate vaccine elicited antibodies that bound to DENV-infected cells. These results show that an epitope-specific vaccine against conserved regions of NS1 could be a promising approach for DENV vaccines or therapeutics to bind circulating NS1 protein.

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Palmitoleate Protects against Zika Virus-Induced Placental Trophoblast Apoptosis

Biomedicines ◽

10.3390/biomedicines9060643 ◽

2021 ◽

Vol 9 (6) ◽

pp. 643

Author(s):

Philma Glora Muthuraj ◽

Aryamav Pattnaik ◽

Prakash K. Sahoo ◽

Md Torikul Islam ◽

Asit K. Pattnaik ◽

...

Keyword(s):

Fatty Acid ◽

Er Stress ◽

Zika Virus ◽

Intrauterine Growth Restriction ◽

Maternal Circulation ◽

Zikv Infection ◽

Human Trophoblast ◽

Homologous Protein ◽

Stress Markers ◽

Infected Cells

Zika virus (ZIKV) infection in pregnancy is associated with the development of microcephaly, intrauterine growth restriction, and ocular damage in the fetus. ZIKV infection of the placenta plays a crucial role in the vertical transmission from the maternal circulation to the fetus. Our previous study suggested that ZIKV induces endoplasmic reticulum (ER) stress and apoptosis of placental trophoblasts. Here, we showed that palmitoleate, an omega-7 monounsaturated fatty acid, prevents ZIKV-induced ER stress and apoptosis in placental trophoblasts. Human trophoblast cell lines (JEG-3 and JAR) and normal immortalized trophoblasts (HTR-8) were used. We observed that ZIKV infection of the trophoblasts resulted in apoptosis and treatment of palmitoleate to ZIKV-infected cells significantly prevented apoptosis. However, palmitate (saturated fatty acid) did not offer protection from ZIKV-induced ER stress and apoptosis. We also observed that the Zika viral RNA copies were decreased, and the cell viability improved in ZIKV-infected cells treated with palmitoleate as compared to the infected cells without palmitoleate treatment. Further, palmitoleate was shown to protect against ZIKV-induced upregulation of ER stress markers, C/EBP homologous protein and X-box binding protein-1 splicing in placental trophoblasts. In conclusion, our studies suggest that palmitoleate protects placental trophoblasts against ZIKV-induced ER stress and apoptosis.

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Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses

Journal of Virology ◽

10.1128/jvi.01455-17 ◽

2017 ◽

Vol 91 (23) ◽

Cited By ~ 13

Author(s):

Nicholas S. Eyre ◽

Stephen M. Johnson ◽

Auda A. Eltahla ◽

Maria Aloi ◽

Amanda L. Aloia ◽

...

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Insertional Mutagenesis ◽

High Resolution Imaging ◽

Ns1 Protein ◽

Reporter Virus ◽

Genome Wide ◽

Infected Cells ◽

Resolution Imaging ◽

Virus Replication Cycle

ABSTRACT Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3′ untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector, and β-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies. IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in the generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here, we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the viral NS1 protein displayed remarkable tolerance of small insertions. This genetic flexibility enabled generation of several novel NS1-tagged reporter viruses, including an APEX2-tagged virus that we used in high-resolution imaging of NS1 localization in infected cells by electron microscopy. For the first time, this analysis revealed the localization of NS1 within viral replication factories known as “vesicle packets” (VPs), in addition to its acknowledged localization to the luminal surface of these VPs. Together, this genetic profile of DENV may be further refined and exploited in the identification of antiviral targets and the generation of reporter virus tools.

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A Recombinant Subunit Based Zika Virus Vaccine Is Efficacious in Non-human Primates

Frontiers in Immunology ◽

10.3389/fimmu.2018.02464 ◽

2018 ◽

Vol 9 ◽

Cited By ~ 18

Author(s):

Liana O. Medina ◽

Albert To ◽

Michael M. Lieberman ◽

Teri Ann S. Wong ◽

Madhuri Namekar ◽

...

Keyword(s):

Virus Vaccine ◽

Zika Virus

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Fast Tracks and Roadblocks for Zika Vaccines

Vaccines ◽

10.3390/vaccines6040077 ◽

2018 ◽

Vol 6 (4) ◽

pp. 77 ◽

Cited By ~ 4

Author(s):

Khairunnisa Ghaffar ◽

Lisa Ng ◽

Laurent Renia

Keyword(s):

Clinical Trials ◽

Zika Virus ◽

Congenital Malformations ◽

Immunological Factors

In early 2014, a relatively obscure virus, the Zika virus, made headlines worldwide following an increase in the number of congenital malformations. Since then, research on Zika virus, treatment and vaccines have progressed swiftly with various drugs being repurposed and vaccines heading into clinical trials. Nonetheless, the need for a vaccine is crucial in order to eradicate this re-emerging arthropod-borne virus which remained silent since its first discovery in 1947. In this review, we focused on how the inconspicuous virus managed to spread, the key immunological factors required for a vaccine and the various vaccine platforms that are currently being studied.

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A GFP Reporter MR766-Based Flow Cytometry Neutralization Test for Rapid Detection of Zika Virus-Neutralizing Antibodies in Serum Specimens

Vaccines ◽

10.3390/vaccines7030066 ◽

2019 ◽

Vol 7 (3) ◽

pp. 66 ◽

Cited By ~ 5

Author(s):

Frumence ◽

Viranaicken ◽

Gadea ◽

Desprès

Keyword(s):

Flow Cytometry ◽

Zika Virus ◽

Neutralizing Antibodies ◽

Neutralization Test ◽

Health Concern ◽

Structural Protein ◽

Adult Mice ◽

Gfp Reporter ◽

Infected Cells ◽

High Level

Zika virus (ZIKV) is an emerging arthropod-borne virus of major public health concern. ZIKV infection is responsible for congenital Zika disease and other neurological defects. Antibody-mediated virus neutralization is an essential component of protective antiviral immunity against ZIKV. In the present study, we assessed whether our GFP reporter ZIKV derived from African viral strain MR766 could be useful for the development of a flow cytometry neutralization test (FNT), as an alternative to the conventional plaque-reduction neutralization test (PRNT). To improve the efficacy of GFP-expressing MR766, we selected virus variant MR766GFP showing a high level of GFP signal in infected cells. A MR766GFP-based FNT was assayed with immune sera from adult mice that received ZIKBeHMR-2. The chimeric ZIKV clone ZIKBeHMR-2 comprises the structural protein region of epidemic strain BeH819015 into MR766 backbone. We reported that adult mice inoculated with ZIKBeHMR-2 developed high levels of neutralizing anti-ZIKV antibodies. Comparative analysis between MR766GFP-based FNT and conventional PRNT was performed using mouse anti-ZIKBeHMR-2 immune sera. Indistinguishable neutralization patterns were observed when compared with PRNT50 and FNT50. We consider that the newly developed MR766GFP-based FNT is a valid format for measuring ZIKV-neutralizing antibodies in serum specimens.

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Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner

mBio ◽

10.1128/mbio.01624-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 64

Author(s):

Caitlin E. Mullarkey ◽

Mark J. Bailey ◽

Diana A. Golubeva ◽

Gene S. Tan ◽

Raffael Nachbagauer ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Fc Receptor ◽

Igg Antibodies ◽

Specific Antibodies ◽

Effector Functions ◽

Iga Antibodies ◽

Specific Igg ◽

Optimal Protection ◽

Specific Igg Antibodies

ABSTRACTBroadly neutralizing antibodies that recognize the conserved hemagglutinin (HA) stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR) interactions for optimal protectionin vivo. Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using anin vitroassay to detect the production of reactive oxygen species (ROS), we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR) engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection.IMPORTANCEThe present study provides evidence that broadly neutralizing HA stalk-specific antibodies induce downstream Fc-mediated neutrophil effector functions. In addition to their ability to neutralize, this class of antibodies has been shown to rely on Fc-Fc receptor interactions for optimal protectionin vivo. Curiously, neutralizing antibodies that bind the HA head domain do not require such interactions. Our findings build on these previous observations and provide a more complete picture of the relationship between stalk-specific antibodies and cells of the innate immune compartment. Furthermore, our data suggest that the ability of HA stalk-specific antibodies to mediate Fc-Fc receptor engagement is epitope dependent. Overall, this work will inform the rational design of improved influenza virus vaccines and therapeutics.

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T Cell Immunity and Zika Virus Vaccine Development

Trends in Immunology ◽

10.1016/j.it.2017.05.004 ◽

2017 ◽

Vol 38 (8) ◽

pp. 594-605 ◽

Cited By ~ 20

Author(s):

Noemia S. Lima ◽

Morgane Rolland ◽

Kayvon Modjarrad ◽

Lydie Trautmann

Keyword(s):

T Cell ◽

Virus Vaccine ◽

Zika Virus ◽

Vaccine Development ◽

T Cell Immunity ◽

Cell Immunity

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Infection of HLA-DR1 Transgenic Mice with a Human Isolate of Influenza A Virus (H1N1) Primes a Diverse CD4 T-Cell Repertoire That Includes CD4 T Cells with Heterosubtypic Cross-Reactivity to Avian (H5N1) Influenza Virus

Journal of Virology ◽

10.1128/jvi.00302-09 ◽

2009 ◽

Vol 83 (13) ◽

pp. 6566-6577 ◽

Cited By ~ 52

Author(s):

Katherine A. Richards ◽

Francisco A. Chaves ◽

Andrea J. Sant

Keyword(s):

T Cells ◽

Influenza Virus ◽

T Cell ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Cd4 T Cells ◽

Cross Reactivity ◽

Cd4 T Cell ◽

Ns1 Protein ◽

Infected Cells

ABSTRACT The specificity of the CD4 T-cell immune response to influenza virus is influenced by the genetic complexity of the virus and periodic encounters with variant subtypes and strains. In order to understand what controls CD4 T-cell reactivity to influenza virus proteins and how the influenza virus-specific memory compartment is shaped over time, it is first necessary to understand the diversity of the primary CD4 T-cell response. In the study reported here, we have used an unbiased approach to evaluate the peptide specificity of CD4 T cells elicited after live influenza virus infection. We have focused on four viral proteins that have distinct intracellular distributions in infected cells, hemagglutinin (HA), neuraminidase (NA), nucleoprotein, and the NS1 protein, which is expressed in infected cells but excluded from virion particles. Our studies revealed an extensive diversity of influenza virus-specific CD4 T cells that includes T cells for each viral protein and for the unexpected immunogenicity of the NS1 protein. Due to the recent concern about pandemic avian influenza virus and because CD4 T cells specific for HA and NA may be particularly useful for promoting the production of neutralizing antibody to influenza virus, we have also evaluated the ability of HA- and NA-specific CD4 T cells elicited by a circulating H1N1 strain to cross-react with related sequences found in an avian H5N1 virus and find substantial cross-reactivity, suggesting that seasonal vaccines may help promote protection against avian influenza virus.

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