An Attenuated Zika Virus Encoding Non-Glycosylated Envelope (E) and Non-Structural Protein 1 (NS1) Confers Complete Protection against Lethal Challenge in a Mouse Model

Zika virus (ZIKV), a mosquito-transmitted flavivirus, emerged in the last decade causing serious human diseases, including congenital microcephaly in newborns and Guillain-Barré syndrome in adults. Although many vaccine platforms are at various stages of development, no licensed vaccines are currently available. Previously, we described a mutant MR766 ZIKV (m2MR) bearing an E protein mutation (N154A) that prevented its glycosylation, resulting in attenuation and defective neuroinvasion. To further attenuate m2MR for its potential use as a live viral vaccine, we incorporated additional mutations into m2MR by substituting the asparagine residues in the glycosylation sites (N130 and N207) of NS1 with alanine residues. Examination of pathogenic properties revealed that the virus (m5MR) carrying mutations in E (N154A) and NS1 (N130A and N207A) was fully attenuated with no disease signs in infected mice, inducing high levels of humoral and cell-mediated immune responses, and protecting mice from subsequent lethal virus challenge. Furthermore, passive transfer of sera from m5MR-infected mice into naïve animals resulted in complete protection from lethal challenge. The immune sera from m5MR-infected animals neutralized both African and Asian lineage viruses equally well, suggesting that m5MR virus could be developed as a potentially broad live virus vaccine candidate.

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Attenuated Vesicular Stomatitis Viruses as Vaccine Vectors

Journal of Virology ◽

10.1128/jvi.73.5.3723-3732.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 3723-3732 ◽

Cited By ~ 190

Author(s):

Anjeanette Roberts ◽

Linda Buonocore ◽

Ryan Price ◽

John Forman ◽

John K. Rose

Keyword(s):

Influenza Virus ◽

Vesicular Stomatitis Virus ◽

Neutralizing Antibodies ◽

Intranasal Administration ◽

Complete Protection ◽

Lethal Challenge ◽

Virus Challenge ◽

Influenza Virus Hemagglutinin ◽

Vesicular Stomatitis ◽

Ha Protein

ABSTRACT We showed previously that a single intranasal vaccination of mice with a recombinant vesicular stomatitis virus (VSV) expressing an influenza virus hemagglutinin (HA) protein provided complete protection from lethal challenge with influenza virus (A. Roberts, E. Kretzschmar, A. S. Perkins, J. Forman, R. Price, L. Buonocore, Y. Kawaoka, and J. K. Rose, J. Virol. 72:4704–4711, 1998). Because some pathogenesis was associated with the vector itself, in the present study we generated new VSV vectors expressing HA which are completely attenuated for pathogenesis in the mouse model. The first vector has a truncation of the cytoplasmic domain of the VSV G protein and expresses influenza virus HA (CT1-HA). This nonpathogenic vector provides complete protection from lethal influenza virus challenge after intranasal administration. A second vector with VSV G deleted and expressing HA (ΔG-HA) is also protective and nonpathogenic and has the advantage of not inducing neutralizing antibodies to the vector itself.

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Zika Virus Attenuation by Codon Pair Deoptimization Induces Sterilizing Immunity in Mouse Models

Journal of Virology ◽

10.1128/jvi.00701-18 ◽

2018 ◽

Vol 92 (17) ◽

Cited By ~ 18

Author(s):

Penghui Li ◽

Xianliang Ke ◽

Ting Wang ◽

Zhongyuan Tan ◽

Dan Luo ◽

...

Keyword(s):

Antibody Titer ◽

Zika Virus ◽

Mammalian Cells ◽

Neutralizing Antibody ◽

Wild Type ◽

Complete Protection ◽

Lethal Challenge ◽

Vaccine Candidates ◽

Codon Pair ◽

Sterilizing Immunity

ABSTRACTZika virus (ZIKV) infection during the large epidemics in the Americas is related to congenital abnormities or fetal demise. To date, there is no vaccine, antiviral drug, or other modality available to prevent or treat Zika virus infection. Here we designed novel live attenuated ZIKV vaccine candidates using a codon pair deoptimization strategy. Three codon pair-deoptimized ZIKVs (Min E, Min NS1, and Min E+NS1) werede novosynthesized and recovered by reverse genetics and contained large amounts of underrepresented codon pairs in the E gene and/or NS1 gene. The amino acid sequence was 100% unchanged. The codon pair-deoptimized variants had decreased replication fitness in Vero cells (Min NS1 ≫ Min E > Min E+NS1), replicated more efficiently in insect cells than in mammalian cells, and demonstrated diminished virulence in a mouse model. In particular, Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer, and a single immunization achieved complete protection against lethal challenge and vertical ZIKV transmission during pregnancy. More importantly, due to the numerous synonymous substitutions in the codon pair-deoptimized strains, reversion to wild-type virulence through gradual nucleotide sequence mutations is unlikely. Our results collectively demonstrate that ZIKV can be effectively attenuated by codon pair deoptimization, highlighting the potential of Min E+NS1 as a safe vaccine candidate to prevent ZIKV infections.IMPORTANCEDue to unprecedented epidemics of Zika virus (ZIKV) across the Americas and the unexpected clinical symptoms, including Guillain-Barré syndrome, microcephaly, and other birth defects in humans, there is an urgent need for ZIKV vaccine development. Here we provided the first attenuated versions of ZIKV with two important genes (E and/or NS1) that were subjected to codon pair deoptimization. Compared to parental ZIKV, the codon pair-deoptimized ZIKVs were mammal attenuated and preferred insect to mammalian cells. Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer and achieved complete protection against lethal challenge and vertical virus transmission during pregnancy. More importantly, the massive synonymous mutational approach made it impossible for the variant to revert to wild-type virulence. Our results have proven the feasibility of codon pair deoptimization as a strategy to develop live attenuated vaccine candidates against flaviviruses such as ZIKV, Japanese encephalitis virus, and West Nile virus.

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A Single and Un-Adjuvanted Dose of a Chimpanzee Adenovirus-Vectored Vaccine against Chikungunya Virus Fully Protects Mice from Lethal Disease

Pathogens ◽

10.3390/pathogens8040231 ◽

2019 ◽

Vol 8 (4) ◽

pp. 231 ◽

Cited By ~ 4

Author(s):

Rafael Kroon Campos ◽

Lorena Preciado-Llanes ◽

Sasha R. Azar ◽

Cesar Lopez-Camacho ◽

Arturo Reyes-Sandoval ◽

...

Keyword(s):

Immune Responses ◽

Neutralizing Antibodies ◽

Chikungunya Virus ◽

Complete Protection ◽

Chikv Infection ◽

Lethal Challenge ◽

Virus Challenge ◽

Vectored Vaccine ◽

Global Health Problem

The mosquito-borne chikungunya virus (CHIKV) has become a major global health problem. Upon infection, chikungunya fever (CHIKF) can result in long-term joint pain and arthritis, and despite intense research, no licensed vaccine for CHIKV is available. We have developed two recombinant chimpanzee adenovirus-vectored vaccines (ChAdOx1) that induce swift and robust anti-CHIKV immune responses with a single dose, without the need for adjuvants or booster vaccines. Here, we report the vaccines’ protective efficacies against CHIKV infection in a lethal A129 mouse model. Our results indicate that a single, un-adjuvanted ChAdOx1 Chik or ChAdOx1 Chik ΔCap dose provided complete protection against a lethal virus challenge and prevented CHIKV-associated severe inflammation. These candidate vaccines supported survival equal to the attenuated 181/25 CHIKV reference vaccine but without the vaccine-related side effects, such as weight loss. Vaccination with either ChAdOx1 Chik or ChAdOx1 Chik ΔCap resulted in high titers of neutralizing antibodies that are associated with protection, indicating that the presence of the capsid within the vaccine construct may not be essential to afford protection under the conditions tested. We conclude that both replication-deficient ChAdOx1 Chik vaccines are safe even when used in A129 mice and afford complete protection from a lethal challenge.

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Development and Preclinical Evaluation of Virus Like Particle Vaccine Against COVID-19 Infection

10.22541/au.162615692.26217047/v2 ◽

2021 ◽

Author(s):

Ismail Yilmaz C ◽

Emre Ipekoglu ◽

Artun Bulbul ◽

Nilsu Turay ◽

Muzaffer Yildirim ◽

...

Keyword(s):

High Titer ◽

Hek293 Cells ◽

Structural Proteins ◽

Lung Pathology ◽

Structural Protein ◽

Protein Antigens ◽

Live Virus ◽

Virus Challenge ◽

Humoral Immune ◽

Multimodal Chromatography

Background Vaccines that incorporate multiple SARS-CoV-2 antigens can further broaden the breadth of virus-specific cellular and humoral immunity. This study describes the development and immunogenicity of SARS-CoV-2 VLP vaccine that incorporates the 4 structural proteins of SARS-CoV-2. Methods VLPs were generated in transiently transfected HEK293 cells, purified by multimodal chromatography and characterized by tunable resistive pulse sensing, AFM, SEM, and TEM. Immunoblotting studies verified the protein identities of VLPs. Cellular and humoral immune responses of immunized animals demonstrated the immune potency of the formulated VLP vaccine. Results Transiently transfected HEK293 cells reproducibly generated vesicular VLPs that were similar in size to and expressing all four structural proteins of SARS-CoV-2. Alum adsorbed, K3-CpG ODN adjuvanted VLPs elicited high titer anti-S, anti-RBD, anti-N IgG, triggered multifunctional Th1 biased T cell responses, reduced virus load and prevented lung pathology upon live virus challenge in vaccinated animals. Conclusion These data suggest that VLPs expressing all four structural protein antigens of SARS-CoV-2 are immunogenic and can protect animals from developing COVID-19 infection following vaccination.

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Development and Preclinical Evaluation of Virus Like Particle Vaccine Against COVID-19 Infection

10.22541/au.162615692.26217047/v1 ◽

2021 ◽

Author(s):

Ismail Yilmaz C ◽

Emre Ipekoglu ◽

Muzaffer Yildirim ◽

Irem Evcili ◽

Naz Yilmaz ◽

...

Keyword(s):

High Titer ◽

Hek293 Cells ◽

Structural Proteins ◽

Lung Pathology ◽

Structural Protein ◽

Protein Antigens ◽

Live Virus ◽

Virus Challenge ◽

Humoral Immune ◽

Multimodal Chromatography

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A Label and Probe-Free Zika Virus Immunosensor Prussian Blue@carbon Nanotube-Based for Amperometric Detection of the NS2B Protein

Biosensors ◽

10.3390/bios11050157 ◽

2021 ◽

Vol 11 (5) ◽

pp. 157

Author(s):

Bárbara V. M. Silva ◽

Marli T. Cordeiro ◽

Marco A. B. Rodrigues ◽

Ernesto T. A. Marques ◽

Rosa F. Dutra

Keyword(s):

Carbon Nanotube ◽

Prussian Blue ◽

Zika Virus ◽

Point Of Care ◽

Amperometric Detection ◽

Cross Reactivity ◽

Structural Protein ◽

Serum Samples ◽

Polypyrrole Composite ◽

Congenital Disabilities

Zika virus (ZIKV) is a mosquito-borne infection, predominant in tropical and subtropical regions causing international concern due to the ZIKV disease having been associated with congenital disabilities, especially microcephaly and other congenital abnormalities in the fetus and newborns. Development of strategies that minimize the devastating impact by monitoring and preventing ZIKV transmission through sexual intercourse, especially in pregnant women, since no vaccine is yet available for the prevention or treatment, is critically important. ZIKV infection is generally asymptomatic and cross-reactivity with dengue virus (DENV) is a global concern. An innovative screen-printed electrode (SPE) was developed for amperometric detection of the non-structural protein (NS2B) of ZIKV by exploring the intrinsic redox catalytic activity of Prussian blue (PB), incorporated into a carbon nanotube–polypyrrole composite. Thus, this immunosensor has the advantage of electrochemical detection without adding any redox-probe solution (probe-less detection), allowing a point-of-care diagnosis. It was responsive to serum samples of only ZIKV positive patients and non-responsive to negative ZIKV patients, even if the sample was DENV positive, indicating a possible differential diagnosis between them by NS2B. All samples used here were confirmed by CDC protocols, and immunosensor responses were also checked in the supernatant of C6/36 and in Vero cell cultures infected with ZIKV.

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An epidemic Zika virus isolate suppresses antiviral immunity by disrupting antigen presentation pathways

Nature Communications ◽

10.1038/s41467-021-24340-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ryan D. Pardy ◽

Stefanie F. Valbon ◽

Brendan Cordeiro ◽

Connie M. Krawczyk ◽

Martin J. Richer

Keyword(s):

T Cell ◽

Zika Virus ◽

Cell Response ◽

Cd8 T Cell ◽

T Cell Response ◽

Cross Presentation ◽

Cell Immunity ◽

Asian Lineage ◽

Insight Into ◽

Host Tissues

AbstractZika virus (ZIKV) has emerged as an important global health threat, with the recently acquired capacity to cause severe neurological symptoms and to persist within host tissues. We previously demonstrated that an early Asian lineage ZIKV isolate induces a highly activated CD8 T cell response specific for an immunodominant epitope in the ZIKV envelope protein in wild-type mice. Here we show that a contemporary ZIKV isolate from the Brazilian outbreak severely limits CD8 T cell immunity in mice and blocks generation of the immunodominant CD8 T cell response. This is associated with a more sustained infection that is cleared between 7- and 14-days post-infection. Mechanistically, we demonstrate that infection with the Brazilian ZIKV isolate reduces the cross-presentation capacity of dendritic cells and fails to fully activate the immunoproteasome. Thus, our study provides an isolate-specific mechanism of host immune evasion by one Brazilian ZIKV isolate, which differs from the early Asian lineage isolate and provides potential insight into viral persistence associated with recent ZIKV outbreaks.

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Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins

Scientific Reports ◽

10.1038/s41598-021-84682-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Alexander Pralow ◽

Alexander Nikolay ◽

Arnaud Leon ◽

Yvonne Genzel ◽

Erdmann Rapp ◽

...

Keyword(s):

Zika Virus ◽

Animal Cell ◽

Gradient Centrifugation ◽

Viral Envelope ◽

Protein Glycosylation ◽

High Yield ◽

Structural Protein ◽

Site Specific ◽

E Protein ◽

The Impact

AbstractHere, we present for the first time, a site-specific N-glycosylation analysis of proteins from a Brazilian Zika virus (ZIKV) strain. The virus was propagated with high yield in an embryo-derived stem cell line (EB66, Valneva SE), and concentrated by g-force step-gradient centrifugation. Subsequently, the sample was proteolytically digested with different enzymes, measured via a LC–MS/MS-based workflow, and analyzed in a semi-automated way using the in-house developed glyXtoolMS software. The viral non-structural protein 1 (NS1) was glycosylated exclusively with high-mannose structures on both potential N-glycosylation sites. In case of the viral envelope (E) protein, no specific N-glycans could be identified with this method. Nevertheless, N-glycosylation could be proved by enzymatic de-N-glycosylation with PNGase F, resulting in a strong MS-signal of the former glycopeptide with deamidated asparagine at the potential N-glycosylation site N444. This confirmed that this site of the ZIKV E protein is highly N-glycosylated but with very high micro-heterogeneity. Our study clearly demonstrates the progress made towards site-specific N-glycosylation analysis of viral proteins, i.e. for Brazilian ZIKV. It allows to better characterize viral isolates, and to monitor glycosylation of major antigens. The method established can be applied for detailed studies regarding the impact of protein glycosylation on antigenicity and human pathogenicity of many viruses including influenza virus, HIV and corona virus.

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Complications and Sequelae in Patients With Congenital Microcephaly Associated With Zika Virus Infection: Two-Year Follow-Up

Journal of Child Neurology ◽

10.1177/0883073820983163 ◽

2021 ◽

pp. 088307382098316

Author(s):

Luane A. Gouvea ◽

Marlos Martins ◽

Daniela Vivacqua ◽

Julia Rosseto ◽

Giulia Lima ◽

...

Keyword(s):

Developmental Delay ◽

Zika Virus ◽

Median Number ◽

Global Developmental Delay ◽

Spastic Diplegia ◽

Long Term Follow Up ◽

Congenital Microcephaly ◽

Auditory Impairment ◽

Worse Prognosis

Background: We aim to describe the long term follow-up of a cohort of children exposed in utero to the Zika virus. Methods: Descriptive study of a cohort of microcephalic children due to Zika virus. Logistic regression was used to evaluate variables associated with worse prognosis epilepsy. Results: We followed 28 children (15 females), with a median follow-up of 24 months (IQR = 12-28). During the follow-up, 1 infant died. The median head circumference at birth was 29 cm (IQR = 27-31). All presented a global developmental delay. The most frequent central nervous system abnormalities were on cortical development in 22 participants; dysgenesis of corpus callosum in 13; ventriculomegaly in 25; and calcifications in 24. A total of 9 presented ocular abnormalities, 4 auditory impairment. During follow-up, 12 presented with sleep disorders, 10 with irritability, and 23 with epilepsy (2 with generalized tonic-clonic, 3 with generalized tonic-clonic and spasms, 12 with spasms, 3 tonic and spasms, and 3 motor focal and spasms). The median age at the begin of the epilepsy was 4 months (IQR = 2-10), the median number of drugs used to control the epilepsy was 2 (IQR = 2-3). Maternal illicit drug use during pregnancy was associated with worse prognosis epilepsy (Lennox-Gastaut syndrome, West syndrome, or status epilepticus). A total of 19 presented with dysphagia, 10 children required gastrostomy. Conclusion: Children with microcephaly due to Zika virus presented with several complications during follow-up, as epilepsy, spastic diplegia, and global developmental delay.

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