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BNT162b2, a lipid nanoparticle (LNP) formulated nucleoside-modified messenger RNA (mRNA) that encodes the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike glycoprotein (S) stabilized in the prefusion conformation, has demonstrated 95% efficacy in preventing coronavirus disease-19 (COVID-19)1. Here we extend our previous phase 1/2 trial report2 and present BNT162b2 prime/boost induced immune response data from a second phase 1/2 trial in healthy adults (18-55 years of age). BNT162b2 elicited strong antibody responses, with SARS-CoV-2 serum 50% neutralizing geometric mean titers up to 3.3-fold above those observed in COVID-19 human convalescent samples (HCS) one week post-boost. BNT162b2-elicited sera neutralized 22 pseudoviruses bearing SARS-CoV-2 S variants. Most participants had a strong IFNγ- or IL-2-positive CD8+ and CD4+ T helper type 1 (TH1) T cell response, detectable throughout the full observation period of nine weeks following the boost. pMHC multimer technology identified several BNT162b2-induced epitopes that were presented by frequent MHC alleles and conserved in mutant strains. One week post-boost, epitope-specific CD8+ T cells of the early differentiated effector-memory phenotype comprised 0.02-2.92% of total circulating CD8+ T cells and were detectable (0.01-0.28%) eight weeks later. In summary, BNT162b2 elicits an adaptive humoral and poly-specific cellular immune response against epitopes conserved in a broad range of variants at well tolerated doses.

Initial COVID-19 vaccine candidates were based on the original sequence of SARS-CoV-2. However, the virus has since accumulated mutations, among which the spike D614G is dominant in circulating virus, raising questions about potential virus escape from vaccine-elicited immunity. Here, we report that the D614G mutation modestly reduced (1.7–2.4-fold) SARS-CoV-2 neutralization by BNT162b2 vaccine-elicited mouse, rhesus, and human sera, concurring with the 95% vaccine efficacy observed in clinical trial.

A safe and effective vaccine against COVID-19 is urgently needed in quantities that are sufficient to immunize large populations. Here we report the preclinical development of two vaccine candidates (BNT162b1 and BNT162b2) that contain nucleoside-modified messenger RNA that encodes immunogens derived from the spike glycoprotein (S) of SARS-CoV-2, formulated in lipid nanoparticles. BNT162b1 encodes a soluble, secreted trimerized receptor-binding domain (known as the RBD–foldon). BNT162b2 encodes the full-length transmembrane S glycoprotein, locked in its prefusion conformation by the substitution of two residues with proline (S(K986P/V987P); hereafter, S(P2) (also known as P2 S)). The flexibly tethered RBDs of the RBD–foldon bind to human ACE2 with high avidity. Approximately 20% of the S(P2) trimers are in the two-RBD ‘down’, one-RBD ‘up’ state. In mice, one intramuscular dose of either candidate vaccine elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong T-helper-1 CD4+ and IFNγ+CD8+ T cell responses. Prime–boost vaccination of rhesus macaques (Macaca mulatta) with the BNT162b candidates elicits SARS-CoV-2-neutralizing geometric mean titres that are 8.2–18.2× that of a panel of SARS-CoV-2-convalescent human sera. The vaccine candidates protect macaques against challenge with SARS-CoV-2; in particular, BNT162b2 protects the lower respiratory tract against the presence of viral RNA and shows no evidence of disease enhancement. Both candidates are being evaluated in phase I trials in Germany and the USA1–3, and BNT162b2 is being evaluated in an ongoing global phase II/III trial (NCT04380701 and NCT04368728). BNT162b1 and BNT162b2 are two candidate mRNA vaccines against COVID-19 that elicit high virus-entry inhibition titres in mice, elicit high virus-neutralizing titres in rhesus macaques and protect macaques from SARS-CoV-2 challenge.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein substitution D614G became dominant during the coronavirus disease 2019 (COVID-19) pandemic1,2. However, the effect of this variant on viral spread and vaccine efficacy remains to be defined. Here we engineered the spike D614G substitution in the USA-WA1/2020 SARS-CoV-2 strain, and found that it enhances viral replication in human lung epithelial cells and primary human airway tissues by increasing the infectivity and stability of virions. Hamsters infected with SARS-CoV-2 expressing spike(D614G) (G614 virus) produced higher infectious titres in nasal washes and the trachea, but not in the lungs, supporting clinical evidence showing that the mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increase transmission. Sera from hamsters infected with D614 virus exhibit modestly higher neutralization titres against G614 virus than against D614 virus, suggesting that the mutation is unlikely to reduce the ability of vaccines in clinical trials to protect against COVID-19, and that therapeutic antibodies should be tested against the circulating G614 virus. Together with clinical findings, our work underscores the importance of this variant in viral spread and its implications for vaccine efficacy and antibody therapy.

Arboviruses maintain high mutation rates due to lack of proofreading ability of their viral polymerases, in some cases facilitating adaptive evolution and emergence. Here we show that, just before its 2013 spread to the Americas, Zika virus (ZIKV) underwent an envelope protein V473M substitution (E-V473M) that increased neurovirulence, maternal-to-fetal transmission, and viremia to facilitate urban transmission. A preepidemic Asian ZIKV strain (FSS13025 isolated in Cambodia in 2010) engineered with the V473M substitution significantly increased neurovirulence in neonatal mice and produced higher viral loads in the placenta and fetal heads in pregnant mice. Conversely, an epidemic ZIKV strain (PRVABC59 isolated in Puerto Rico in 2015) engineered with the inverse M473V substitution reversed the pathogenic phenotypes. Although E-V473M did not affect oral infection of Aedes aegypti mosquitoes, competition experiments in cynomolgus macaques showed that this mutation increased its fitness for viremia generation, suggesting adaptive evolution for human viremia and hence transmission. Mechanistically, the V473M mutation, located at the second transmembrane helix of the E protein, enhances virion morphogenesis. Overall, our study revealed E-V473M as a critical determinant for enhanced ZIKV virulence, intrauterine transmission during pregnancy, and viremia to facilitate urban transmission.

BNT162b2 Vaccine and Emerging SARS-CoV-2 Variants A total of 20 serum samples from 15 persons who received the BNT162b2 vaccine showed strong neutralization activity against recombinant viruses eng...

This work was supported by Amon G. Carter Foundation; Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico: [Grant Number 303999/2016-0,440405/2016-5]; Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior: [Grant Number Zika fast track project]; John S. Dunn Foundation; Gilson Longenbaugh Foundation; NIH: [Grant Number AI127744,AI136126,AI142759];PAHO: [Grant Number SCON2016-01353]; Summerfield Robert Foundation; Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation; Sealy & Smith Foundation. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil / Department of Biochemistry & Molecular Biology. Galveston, TX, USA / Health Sciences Institute. Belem, PA, Brazil. Department of Biochemistry & Molecular Biology. Galveston, TX, USA. Department of Biochemistry & Molecular Biology. Galveston, TX, USA. Department of Biochemistry & Molecular Biology. Galveston, TX, USA / Department of Microbiology & Immunology. Galveston, TX, USA. Department of Biochemistry & Molecular Biology. Galveston, TX, USA / Health Sciences Institute. Belem, PA, Brazil. Health Sciences Institute. Belem, PA, Brazil / Federal University of Par?. Biological Sciences Institute. Belem, PA, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil / Par? State University. Department of Pathology. Bel?m, PA, Brazil. Department of Biochemistry & Molecular Biology. Galveston, TX, USA / Institute for Human Infections & Immunity. Galveston, TX, USA / Institute for Translational Science. Galveston, TX, USA / Sealy Institute of Vaccine Sciences. Galveston, TX, USA / Texas Medical Branch. Sealy Center for Structural Biology & Molecular Biophysics. Galveston, TX, USA. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil / Department of Biochemistry & Molecular Biology. Galveston, TX, USA / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Programa de P?s-Gradua??o em Virologia. Ananindeua, PA, Brasil / Health Sciences Institute. Bel?m, PA, Brazil. The Asian lineage of Zika virus (ZIKV) is responsible for the recent epidemics in the Americas and severe disease, whereas the African lineage of ZIKV has not been reported to cause epidemics or severe disease. We constructed a cDNA infectious clone (IC) of an African ZIKV strain, which, together with our previously developed Asian ZIKV strain IC, allowed us to engineer chimeric viruses by swapping the structural and non-structural genes between the two lineages. Recombinant parental and chimeric viruses were analyzed in A129 and newborn CD1 mouse models. In the A129 mice, the African strain developed higher viremia, organ viral loading, and mortality rate. In CD1 mice, the African strain exhibited a higher neurovirulence than the Asian strain. A chimeric virus containing the structural genes from the African strain is more virulent than the Asian strain, whereas a chimeric virus containing the non-structural genes from the African strain exhibited a virulence comparable to the Asian strain. These results suggest that (i) African strain is more virulent than Asian strain and (ii) viral structural genes primarily determine the virulence difference between the two lineages in mouse models. Other factors may contribute to the discrepancy between the mouse and epidemic results.

The original version of this Article contained an error in the Acknowledgements, which incorrectly listed the NIH grant ‘AI142759’. This has been corrected in both the PDF and HTML versions of the Article. © The Author(s) 2021.

Variant SARS-CoV-2 and BNT162b2 Vaccine How well do serum samples obtained from persons injected with the BNT162b2 vaccine neutralize the P.1, B.1.1.7, and B.1.135 lineages of SARS-CoV-2, first ide...

Although live attenuated vaccines (LAVs) have been effective in the control of flavivirus infections, to date they have been excluded from Zika virus (ZIKV) vaccine trials due to safety concerns. We have previously reported two ZIKV mutants, each of which has a single substitution in either envelope (E) glycosylation or nonstructural (NS) 4B P36 and displays a modest reduction in mouse neurovirulence and neuroinvasiveness, respectively. Here, we generated a ZIKV mutant, ZE4B-36, which combines mutations in both E glycosylation and NS4B P36. The ZE4B-36 mutant is stable and attenuated in viral replication. Next-generation sequence analysis showed that the attenuating mutations in the E and NS4B proteins are retained during serial cell culture passages. The mutant exhibits a significant reduction in neuroinvasiveness and neurovirulence and low infectivity in mosquitoes. It induces robust ZIKV-specific memory B cell, antibody, and T cell-mediated immune responses in type I interferon receptor (IFNR) deficient mice. ZIKV-specific T cell immunity remains strong months post-vaccination in wild-type C57BL/6 (B6) mice. Vaccination with ZE4B-36 protects mice from ZIKV-induced diseases and vertical transmission. Our results suggest that combination mutations in E glycosylation and NS4B P36 contribute to a candidate LAV with significantly increased safety but retain strong immunogenicity for prevention and control of ZIKV infection.

We engineered three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses containing key spike mutations from the newly emerged United Kingdom (UK) and South African (SA) variants: N501Y from UK and SA; 69/70-deletion + N501Y + D614G from UK; and E484K + N501Y + D614G from SA. Neutralization geometric mean titers (GMTs) of 20 BTN162b2 vaccine-elicited human sera against the three mutant viruses were 0.81- to 1.46-fold of the GMTs against parental virus, indicating small effects of these mutations on neutralization by sera elicited by two BNT162b2 doses. Human sera from recipients of the BNT162b2 vaccine can neutralize SARS-CoV-2 viruses containing spike mutations present in globally circulating variants of concern.

Rapidly spreading variants of SARS-CoV-2 that have arisen in the United Kingdom and South Africa share the spike N501Y substitution, which is of particular concern because it is located in the viral receptor binding site for cell entry and increases binding to the receptor (angiotensin converting enzyme 2). We generated isogenic N501 and Y501 SARS-CoV-2. Sera of 20 participants in a previously reported trial of the mRNA-based COVID-19 vaccine BNT162b2 had equivalent neutralizing titers to the N501 and Y501 viruses.

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and the resulting disease, coronavirus disease 2019 (Covid-19), have spread to millions of persons worldwide. Multiple vaccine candidates are under development, but no vaccine is currently available. Interim safety and immunogenicity data about the vaccine candidate BNT162b1 in younger adults have been reported previously from trials in Germany and the United States. Methods In an ongoing, placebo-controlled, observer-blinded, dose-escalation, phase 1 trial conducted in the United States, we randomly assigned healthy adults 18 to 55 years of age and those 65 to 85 years of age to receive either placebo or one of two lipid nanoparticle–formulated, nucleoside-modified RNA vaccine candidates: BNT162b1, which encodes a secreted trimerized SARS-CoV-2 receptor–binding domain; or BNT162b2, which encodes a membrane-anchored SARS-CoV-2 full-length spike, stabilized in the prefusion conformation. The primary outcome was safety (e.g., local and systemic reactions and adverse events); immunogenicity was a secondary outcome. Trial groups were defined according to vaccine candidate, age of the participants, and vaccine dose level (10 μg, 20 μg, 30 μg, and 100 μg). In all groups but one, participants received two doses, with a 21-day interval between doses; in one group (100 μg of BNT162b1), participants received one dose. Results A total of 195 participants underwent randomization. In each of 13 groups of 15 participants, 12 participants received vaccine and 3 received placebo. BNT162b2 was associated with a lower incidence and severity of systemic reactions than BNT162b1, particularly in older adults. In both younger and older adults, the two vaccine candidates elicited similar dose-dependent SARS-CoV-2–neutralizing geometric mean titers, which were similar to or higher than the geometric mean titer of a panel of SARS-CoV-2 convalescent serum samples. Conclusions The safety and immunogenicity data from this U.S. phase 1 trial of two vaccine candidates in younger and older adults, added to earlier interim safety and immunogenicity data regarding BNT162b1 in younger adults from trials in Germany and the United States, support the selection of BNT162b2 for advancement to a pivotal phase 2–3 safety and efficacy evaluation. (Funded by BioNTech and Pfizer; ClinicalTrials.gov number, NCT04368728.)

A safe and effective vaccine against COVID-19 is urgently needed in quantities sufficient to immunise large populations. We report the preclinical development of two BNT162b vaccine candidates, which contain lipid-nanoparticle (LNP) formulated nucleoside-modified mRNA encoding SARS-CoV-2 spike glycoprotein-derived immunogens. BNT162b1 encodes a soluble, secreted, trimerised receptor-binding domain (RBD-foldon). BNT162b2 encodes the full-length transmembrane spike glycoprotein, locked in its prefusion conformation (P2 S). The flexibly tethered RBDs of the RBD-foldon bind ACE2 with high avidity. Approximately 20% of the P 2S trimers are in the two-RBD ‘down,’ one-RBD ‘up’ state. In mice, one intramuscular dose of either candidate elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong TH1 CD4+ and IFNγ+ CD8+ T-cell responses. Prime/boost vaccination of rhesus macaques with BNT162b candidates elicits SARS-CoV-2 neutralising geometric mean titres 8.2 to 18.2 times that of a SARS-CoV-2 convalescent human serum panel. The vaccine candidates protect macaques from SARS-CoV-2 challenge, with BNT162b2 protecting the lower respiratory tract from the presence of viral RNA and with no evidence of disease enhancement. Both candidates are being evaluated in phase 1 trials in Germany and the United States. BNT162b2 is being evaluated in an ongoing global, pivotal Phase 2/3 trial (NCT04380701, NCT04368728).

To contain the coronavirus disease 2019 (COVID-19) pandemic, a safe and effective vaccine against the new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is urgently needed in quantities sufficient to immunise large populations. In this study, we report the design, preclinical development, immunogenicity and anti-viral protective effect in rhesus macaques of the BNT162b2 vaccine candidate. BNT162b2 contains an LNP-formulated nucleoside-modified mRNA that encodes the spike glycoprotein captured in its prefusion conformation. After expression of the BNT162b2 coding sequence in cells, approximately 20% of the spike molecules are in the one-RBD ‘up’, two-RBD ‘down’ state. Immunisation of mice with a single dose of BNT162b2 induced dose level-dependent increases in pseudovirus neutralisation titers. Prime-boost vaccination of rhesus macaques elicited authentic SARS-CoV-2 neutralising geometric mean titers 10.2 to 18.0 times that of a SARS-CoV-2 convalescent human serum panel. BNT162b2 generated strong TH1 type CD4+ and IFNγ+ CD8+ T-cell responses in mice and rhesus macaques. The BNT162b2 vaccine candidate fully protected the lungs of immunised rhesus macaques from infectious SARS-CoV-2 challenge. BNT162b2 is currently being evaluated in a global, pivotal Phase 2/3 trial (NCT04368728).

A spike protein mutation D614G became dominant in SARS-CoV-2 during the COVID-19 pandemic. However, the mutational impact on viral spread and vaccine efficacy remains to be defined. Here we engineer the D614G mutation in the SARS-CoV-2 USA-WA1/2020 strain and characterize its effect on viral replication, pathogenesis, and antibody neutralization. The D614G mutation significantly enhances SARS-CoV-2 replication on human lung epithelial cells and primary human airway tissues, through an improved infectivity of virions with the spike receptor-binding domain in an “up” conformation for binding to ACE2 receptor. Hamsters infected with D614 or G614 variants developed similar levels of weight loss. However, the G614 virus produced higher infectious titers in the nasal washes and trachea, but not lungs, than the D614 virus. The hamster results confirm clinical evidence that the D614G mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increases transmission. For antibody neutralization, sera from D614 virus-infected hamsters consistently exhibit higher neutralization titers against G614 virus than those against D614 virus, indicating that (i) the mutation may not reduce the ability of vaccines in clinical trials to protect against COVID-19 and (ii) therapeutic antibodies should be tested against the circulating G614 virus before clinical development.ImportanceUnderstanding the evolution of SARS-CoV-2 during the COVID-19 pandemic is essential for disease control and prevention. A spike protein mutation D614G emerged and became dominant soon after the pandemic started. By engineering the D614G mutation into an authentic wild-type SARS-CoV-2 strain, we demonstrate the importance of this mutation to (i) enhanced viral replication on human lung epithelial cells and primary human airway tissues, (ii) improved viral fitness in the upper airway of infected hamsters, and (iii) increased susceptibility to neutralization. Together with clinical findings, our work underscores the importance of this mutation in viral spread, vaccine efficacy, and antibody therapy.

Virus neutralization remains the gold standard for determining antibody efficacy. Therefore, a high-throughput assay to measure SARS-CoV-2 neutralizing antibodies is urgently needed for COVID-19 serodiagnosis, convalescent plasma therapy, and vaccine development. Here, we report on a fluorescence-based SARS-CoV-2 neutralization assay that detects SARS-CoV-2 neutralizing antibodies in COVID-19 patient specimens and yields comparable results to plaque reduction neutralizing assay, the gold standard of serological testing. The fluorescence-based neutralization assay is specific to measure COVID-19 neutralizing antibodies without cross reacting with patient specimens with other viral, bacterial, or parasitic infections. Collectively, our approach offers a rapid platform that can be scaled to screen people for antibody protection from COVID-19, a key parameter necessary to safely reopen local communities., Neutralizing antibody titers in SARS-CoV-2 infected or vaccinated people are an important measure for vaccine development and public health decision-making. Here, the authors develop a fluorescence based SARS-CoV-2 assay to determine neutralizing antibody titers in COVID-19 patient sera in a high throughput set-up.

A high-throughput platform would greatly facilitate coronavirus disease 2019 (COVID-19) serological testing and antiviral screening. Here we present a high-throughput nanoluciferase severe respiratory syndrome coronavirus 2 (SARS-CoV-2-Nluc) that is genetically stable and replicates similarly to the wild-type virus in cell culture. SARS-CoV-2-Nluc can be used to measure neutralizing antibody activity in patient sera within 5 hours, and it produces results in concordance with a plaque reduction neutralization test (PRNT). Additionally, using SARS-CoV-2-Nluc infection of A549 cells expressing human ACE2 receptor (A549-hACE2), we show that the assay can be used for antiviral screening. Using the optimized SARS-CoV-2-Nluc assay, we evaluate a panel of antivirals and other anti-infective drugs, and we identify nelfinavir, rupintrivir, and cobicistat as the most selective inhibitors of SARS-CoV-2-Nluc (EC50 0.77 to 2.74 µM). In contrast, most of the clinically approved antivirals, including tenofovir alafenamide, emtricitabine, sofosbuvir, ledipasvir, and velpatasvir were inactive at concentrations up to 10 µM. Collectively, this high-throughput platform represents a reliable tool for rapid neutralization testing and antiviral screening for SARS-CoV-2., A high-throughput platform would greatly facilitate coronavirus disease 2019 (COVID-19) serological testing and antiviral screening. To address this, Shi and colleagues present a high-throughput nanoluciferase severe respiratory syndrome coronavirus 2 (SARS-CoV2-Nluc), and show that it has potential for large-scale vaccine evaluation and neutralizing antibody testing.

In 2015-2016, the little known Zika virus (ZIKV) caused an epidemic, in which it became recognized as a unique human pathogen associated with a range of devastating congenital abnormalities collectively categorized as congenital Zika syndrome (CZS). In adults, the virus can trigger the autoimmune disorder Guillain-Barre syndrome (GBS), characterized by ascending paralysis. In February 2016, the World Health Organization (WHO) declared ZIKV to be a Public Health Emergency of International Concern. The global public health problem prompted academia, industry, and governments worldwide to initiate development of an effective vaccine to prevent another ZIKV epidemic that would put millions at risk. The development of reverse genetic systems for the study and manipulation of RNA viral genomes has revolutionized the field of virology, providing platforms for vaccine and antiviral development. In this review, we discuss the impact of reverse genetic systems on the rapid progress of ZIKV vaccines and antiviral therapeutics.

University of Texas Medical Branch. Department of Biochemistry & Molecular Biology. Galveston, TX, USA. University of Texas Medical Branch. Department of Biochemistry & Molecular Biology. Galveston, TX, USA. University of Texas Medical Branch. Department of Microbiology & Immunology. Galveston, TX, USA. University of Texas Medical Branch. Institute for Human Infections & Immunity. Galveston, TX, USA / University of Texas Medical Branch. Institute for Translational Science. Galveston, TX, USA. University of Texas Medical Branch. Department of Microbiology & Immunology. Galveston, TX, USA / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. University of Texas Medical Branch. Department of Microbiology & Immunology. Galveston, TX, USA . University of Texas Medical Branch. Department of Biochemistry & Molecular Biology. Galveston, TX, USA. University of Texas Medical Branch. Department of Biochemistry & Molecular Biology. Galveston, TX, USA. University of Texas Medical Branch. Institute for Human Infections & Immunity. Galveston, TX, USA / University of Texas Medical Branch. Institute for Translational Science. Galveston, TX, USA. University of Texas Medical Branch. Department of Biochemistry & Molecular Biology. Galveston, TX, USA. University of Texas Medical Branch. Institute for Human Infections & Immunity. Galveston, TX, USA / University of Texas Medical Branch. Department of Pathology and Center for Biodefense & Emerging Infectious Diseases. Galveston, TX, USA. University of Texas Medical Branch. Department of Microbiology & Immunology. Galveston, TX, USA / University of Texas Medical Branch. Department of Pathology and Center for Biodefense & Emerging Infectious Diseases. Galveston, TX, USA / University of Texas Medical Branch. Sealy Center for Vaccine Development. Galveston, TX, USA. University of Texas Medical Branch. Institute for Human Infections & Immunity. Galveston, TX, USA / University of Texas Medical Branch. Institute for Translational Science. Galveston, TX, USA / University of Texas Medical Branch. Department of Microbiology & Immunology. Galveston, TX, USA / University of Texas Medical Branch. Sealy Center for Vaccine Development. Galveston, TX, USA / University of Texas Medical Branch. Sealy Center for Structural Biology & Molecular Biophysics. Galveston, TX, USA. University of Texas Medical Branch. Department of Biochemistry & Molecular Biology. Galveston, TX, USA / University of Texas Medical Branch. Sealy Center for Vaccine Development. Galveston, TX, USA / University of Texas Medical Branch. Department of Pharmacology & Toxicology. Galveston, TX, USA / University of Texas Medical Branch. Sealy Center for Structural Biology & Molecular Biophysics. Galveston, TX, USA. Zika virus (ZIKV) infection causes devastating congenital abnormities and Guillain-Barre? syndrome. The ZIKV envelope (E) protein is responsible for viral entry and represents a major determinant for viral pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154. To study the function of E glycosylation, we generated a recombinant N154Q ZIKV that lacks the E glycosylation and analyzed the mutant virus in mammalian and mosquito hosts. In mouse models, the mutant was attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; however, knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts.

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., Zika virus vaccination: identifying NS protein mutations for protection Live attenuated vaccines are used to protect against some flavivirus infections; however, no Zika virus (ZIKV) vaccine has yet been approved for human use. Here, Tian Wang and colleagues use nonstructural protein 4B (NS4B), which is involved in virus replication and evasion of host immunity, as a basis for a potential ZIKV vaccine. Using site-directed mutagenesis, they generated mutations in the conserved N-terminal and central hydrophobic regions of this protein, and identify three single-site mutations that caused the virus to become attenuated. One mutation in particular was found to be more attenuated in an immunocompromised mouse model of ZIKV—the NS4B-C100S mutant. When compared with the wild-type virus strain and a previously mutated ZIKV strain, it induced stronger antiviral immune responses in this mouse model, including increased type I IFN and IL-6 production, and increased ZIKV-specific CD4+ and CD8+ T-cell responses, yet similar neutralizing antibody titerss. Both active and passive immunization with this mutant provided protection when mice were challenged with wild-type ZIKV, suggesting that this mutant strain may warrant further studies as a potential vaccine.

The original version of this Article contained an error in the Acknowledgements, which incorrectly listed the NIH grant ‘AI142759’. This has been corrected in both the PDF and HTML versions of the Article.

The emergence of Zika virus (ZIKV) and its association with congenital malformations has prompted the rapid development of vaccines. Although efficacy with nucleic acid or inactivated viral vaccine platforms has been established in animals, no study has addressed protection during pregnancy. We tested in mice two vaccine platforms, a lipid nanoparticle-encapsulated modified mRNA vaccine encoding ZIKV prM and E genes and a live-attenuated ZIKV strain encoding an NS1 protein without glycosylation, for their ability to protect against transmission to the fetus. Vaccinated dams challenged with a heterologous ZIKV strain at embryo day 6 (E6) and evaluated at E13 showed markedly diminished levels of viral RNA in maternal, placental, and fetal tissues, which resulted in protection against placental damage and fetal demise. As modified mRNA and live-attenuated vaccine platforms can restrict in utero transmission of ZIKV in mice, their further development in humans to prevent congenital ZIKV syndrome is warranted.

Zika virus (ZIKV) has drawn global attention as the etiologic agent of Zika Congenital Syndrome in babies born to infected pregnant women. To prevent future ZIKV outbreaks and protect persons at risk for severe disease, we developed two live-attenuated vaccine (LAV) candidates containing 10- or 20-nucleotide deletions in the 3′UTR of the viral genome (Δ10 and Δ20). After a single-dose immunization, both Δ10 and Δ20 LAVs protected mice and non-human primates against ZIKV infection. Here, we characterized the stability, safety, and efficacy of the LAVs after continuously culturing them on manufacture Vero cells for ten rounds. Whole genome sequencing showed that passage 10 (P10) LAVs retained the engineered Δ10 and Δ20 deletions; one to four additional mutations emerged at different regions of the genome. In A129 mice, the P10 LAVs exhibited viremia higher than the un-passaged LAVs, but lower than wild-type ZIKV; unlike wild-type ZIKV-infected mice, none of the P10 LAV-infected mice developed disease or death, demonstrating that the P10 LAVs remained attenuated. Mice immunized with a single dose of the P10 LAVs developed robust neutralizing antibody titers (1/1,000 to 1/10,000) and were protected against epidemic ZIKV challenge. The P10 LAVs did not exhibit increased neurovirulence. Intracranial inoculation of one-day-old CD1 pups with 103 focus-forming units of the P10 Δ10 and Δ20 LAVs resulted in 100% and ≥80% survival, respectively. Furthermore, the P10 LAVs remained incompetent in infecting Aedes aegypti mosquitoes after intrathoracic microinjection. Our results support the phenotypic stability and further development of these promising LAVs for ZIKV.