A single-dose plasmid-launched live-attenuated Zika vaccine induces protective immunityResearch in context

Background: Vaccines are the most effective means to fight and eradicate infectious diseases. Live-attenuated vaccines (LAV) usually have the advantages of single dose, rapid onset of immunity, and durable protection. DNA vaccines have the advantages of chemical stability, ease of production, and no cold chain requirement. The ability to combine the strengths of LAV and DNA vaccines may transform future vaccine development by eliminating cold chain and cell culture with the potential for adventitious agents. Methods: A DNA-launched LAV was developed for ZIKV virus (ZIKV), a pathogen that recently caused a global public health emergency. The cDNA copy of a ZIKV LAV genome was engineered into a DNA plasmid. The DNA-LAV plasmid was delivered into mice using a clinically proven device TriGrid™ to launch the replication of LAV. Findings: A single-dose immunization as low as 0.5 μg of DNA-LAV plasmid conferred 100% seroconversion in A129 mice. All seroconverted mice developed sterilizing immunity, as indicated by no detectable infectious viruses and no increase of neutralizing antibody titers after ZIKV challenge. The immunization also elicited robust T cell responses. In pregnant mice, the DNA-LAV vaccination fully protected against ZIKV-induced disease and maternal-to-fetal transmission. High levels of neutralizing activities were detected in fetal serum, indicating maternal-to-fetal humoral transfer. In male mice, a single-dose vaccination completely prevented testis infection, injury, and oligospermia. Interpretation: The remarkable simplicity and potency of ZIKV DNA-LAV warrant further development of this vaccine candidate. The DNA-LAV approach may serve as a universal vaccine platform for other plus-sense RNA viruses. Fund: National Institute of Health, Kleberg Foundation, Centers for Disease Control and Prevention, University of Texas Medical Branch. Keywords: Zika virus, DNA vaccine, live-attenuated vaccine, flaviviru

A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation

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 cDNA Clone-Launched Platform for High-Yield Production of Inactivated Zika Vaccine

A purified inactivated vaccine (PIV) using the Zika virus (ZIKV) Puerto Rico strain PRVABC59 showed efficacy in monkeys, and is currently in a phase I clinical trial. High-yield manufacture of this PIV is essential for its development and vaccine access. Here we report an infectious cDNA clone-launched platform to maximize its yield. A single NS1 protein substitution (K265E) was identified to increase ZIKV replication on Vero cells (a cell line approved for vaccine production) for both Cambodian FSS13025 and Puerto Rico PRVABC59 strains. The NS1 mutation did not affect viral RNA synthesis, but significantly increased virion assembly through an increased interaction between NS1 and NS2A (a known regulator of flavivirus assembly). The NS1 mutant virus retained wild-type virulence in the A129 mouse model, but decreased its competence to infect Aedes aegypti mosquitoes. To further increase virus yield, we constructed an infectious cDNA clone of the clinical trial PIV strain PRVABC59 containing three viral replication-enhancing mutations (NS1 K265E, prM H83R, and NS3 S356F). The mutant cDNA clone produced >25-fold more ZIKV than the wild-type parent on Vero cells. This cDNA clone-launched manufacture platform has the advantages of higher virus yield, shortened manufacture time, and minimized chance of contamination

Understanding Zika Virus Stability and Developing a Chimeric Vaccine through Functional Analysis

Compared with other flaviviruses, Zika virus (ZIKV) is uniquely associated with congenital diseases in pregnant women. One recent study reported that (i) ZIKV has higher thermostability than dengue virus (DENV [a flavivirus closely related to ZIKV]), which might contribute to the disease outcome; (ii) the higher thermostability of ZIKV could arise from an extended loop structure in domain III of the viral envelope (E) protein and an extra hydrogen-bond interaction between E molecules (V. A. Kostyuchenko, E. X. Y. Lim, S. Zhang, G. Fibriansah, T.-S. Ng, J. S. G. Ooi, J. Shi, and S.-M. Lok, Nature 533:425–428, 2016, https://doi.org/10.1038/nature17994). Here we report the functional analysis of the structural information in the context of complete ZIKV and DENV-2 virions. Swapping the prM-E genes between ZIKV and DENV-2 switched the thermostability of the chimeric viruses, identifying the prM-E proteins as the major determinants for virion thermostability. Shortening the extended loop of the E protein by 1 amino acid was lethal for ZIKV assembly/release. Mutations (Q350I and T351V) that abolished the extra hydrogen-bond interaction between the E proteins did not reduce ZIKV thermostability, indicating that the extra interaction does not increase the thermostability. Interestingly, mutant T351V was attenuated in A129 mice defective in type I interferon receptors, even though the virus retained the wild-type thermostability. Furthermore, we found that a chimeric ZIKV with the DENV-2 prM-E and a chimeric DENV-2 with the ZIKV prM-E were highly attenuated in A129 mice; these chimeric viruses were highly immunogenic and protective against DENV-2 and ZIKV challenge, respectively. These results indicate the potential of these chimeric viruses for vaccine development

Functional Analysis of Glycosylation of Zika Virus Envelope Protein

Summary: Zika virus (ZIKV) infection causes devastating congenital abnormities and Guillain-Barré 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. : Zika virus (ZIKV) causes devastating congenital abnormities and Guillain-Barré syndrome. Fontes-Garfias et al. showed that the glycosylation of ZIKV envelope protein plays an important role in infecting mosquito vectors and pathogenesis in mouse. Keywords: Zika virus, glycosylation, flavivirus entry, mosquito transmission, vaccin

Zika structural genes determine the virulence of African and Asian lineages

This work was supported by Amon G. Carter Foundation; Conselho Nacional de Desenvolvimento Científico e Tecnológico: [Grant Number 303999/2016-0,440405/2016-5]; Coordenação de Aperfeiçoamento de Pessoal de Nível 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.Ministério da Saúde. Secretaria de Vigilância em Saúde. 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 Pará. Biological Sciences Institute. Belem, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil / Pará State University. Department of Pathology. Belém, 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.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil / Department of Biochemistry & Molecular Biology. Galveston, TX, USA / Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Programa de Pós-Graduação em Virologia. Ananindeua, PA, Brasil / Health Sciences Institute. Belém, 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

An evolutionary NS1 mutation enhances Zika virus evasion of host interferon induction

The Asian lineage of Zika virus (ZIKV) has acquired a mutation in NS1 that enhances mosquito infection. Here, Xia et al. show that the same mutation interferes with interferon production through interaction with TBK1 and affects ZIKV replication in mice

A nanoluciferase SARS-CoV-2 for rapid neutralization testing and screening of anti-infective drugs for COVID-19

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

A Single-Dose Live-Attenuated Zika Virus Vaccine with Controlled Infection Rounds that Protects against Vertical Transmission

Zika virus (ZIKV) infection of the mother during pregnancy causes devastating Zika congenital syndrome in the offspring. A ZIKV vaccine with optimal safety and immunogenicity for use in pregnant women is critically needed. Toward this goal, we have developed a single-dose live-attenuated vaccine candidate that infects cells with controlled, limited infection rounds. The vaccine contains a 9-amino-acid deletion in the viral capsid protein and replicates to titers of > 106 focus-forming units (FFU)/mL in cells expressing the full-length capsid protein. Immunization of A129 mice with one dose (105 FFU) did not produce viremia, but elicited protective immunity that completely prevented viremia, morbidity, and mortality after challenge with an epidemic ZIKV strain (106 PFU). A single-dose vaccination also fully prevented infection of pregnant mice and maternal-to-fetal transmission. Intracranial injection of the vaccine (104 FFU) to 1-day-old mice did not cause any disease or death, underscoring the safety of this vaccine candidate.status: publishe

A live-attenuated Zika virus vaccine candidate induces sterilizing immunity in mouse models

P.-Y.S. was supported by a University of Texas Medical Branch (UTMB) start-up award, UTMB Innovation and Commercialization award, University of Texas STARs Award, CDC grant for the Western Gulf Center of Excellence for Vector-Borne Diseases, Pan American Health Organization grant SCON2016- 01353, and UTMB CTSA UL1TR-001439. This research was also partially supported by a US National Institutes of Health grant AI120942 to S.C.W.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 Sciences. Galveston, TX, USA.University of Texas Medical Branch. Department of Biochemistry & Molecular Biology. Galveston, TX, USA / Ministério da Saúde. Secretaria de Vigilância em Saúde. 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 / Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.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. 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. Sealy Center for Vaccine Development. 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 Sciences. 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 Vaccine Development. Galveston, TX, USA.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil / Pará State University. Department of Pathology. Belém, PA, Brasil.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 Biochemistry & Molecular Biology. 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 Phamarcology & Toxicology. Galveston, TX, USA.Zika virus (ZIKV) infection of pregnant women can cause a wide range of congenital abnormalities, including microcephaly, in the infant, a condition now collectively known as congenital ZIKV syndrome. A vaccine to prevent or significantly attenuate viremia in pregnant women who are residents of or travelers to epidemic or endemic regions is needed to avert congenital ZIKV syndrome, and might also help to suppress epidemic transmission. Here we report on a live-attenuated vaccine candidate that contains a 10-nucleotide deletion in the 3' untranslated region of the ZIKV genome (10-del ZIKV). The 10-del ZIKV is highly attenuated, immunogenic, and protective in type 1 interferon receptor-deficient A129 mice. Crucially, a single dose of 10-del ZIKV induced sterilizing immunity with a saturated neutralizing antibody titer, which no longer increased after challenge with an epidemic ZIKV, and completely prevented viremia. The immunized mice also developed a robust T cell response. Intracranial inoculation of 1-d-old immunocompetent CD-1 mice with 1 × 104 infectious focus units (IFU) of 10-del ZIKV caused no mortality, whereas infections with 10 IFU of wild-type ZIKV were lethal. Mechanistically, the attenuated virulence of 10-del ZIKV may be due to decreased viral RNA synthesis and increased sensitivity to type-1-interferon inhibition. The attenuated 10-del ZIKV was incapable of infecting mosquitoes after oral feeding of spiked-blood meals, representing an additional safety feature. Collectively, the safety and efficacy results suggest that further development of this promising, live-attenuated ZIKV vaccine candidate is warranted