Human Desmoglein-2 and Human CD46 Mediate Human Adenovirus Type 55 Infection, but Human Desmoglein-2 Plays the Major Roles.

Human adenovirus type 55 (HAdV55) represents an emerging respiratory pathogen and causes severe pneumonia with high fatality in humans. The cellular receptors, which are essential for understanding the infection and pathogenesis of HAdV55, remain unclear. In this study, we found that HAdV55 binding and infection were sharply reduced by disrupting the interaction of viral fiber protein with human desmoglein-2 (hDSG2) but only slightly reduced by disrupting the interaction of viral fiber protein with human CD46 (hCD46). Loss-of-function studies using soluble receptors, blocking antibodies, RNA interference, and gene knockout demonstrated that hDSG2 predominantly mediated HAdV55 infection. Nonpermissive rodent cells became susceptible to HAdV55 infection when hDSG2 or hCD46 was expressed, but hDSG2 mediated more efficient HAd55 infection than hCD46. We generated two transgenic mouse lines that constitutively express either hDSG2 or hCD46. Although nontransgenic mice were resistant to HAdV55 infection, infection with HAdV55 was significantly increased in hDSG2+/+ mice but was much less increased in hCD46+/+ mice. Our findings demonstrate that both hDSG2 and hCD46 are able to mediate HAdV55 infection but hDSG2 plays the major roles. The hDSG2 transgenic mouse can be used as a rodent model for evaluation of HAdV55 vaccine and therapeutics.IMPORTANCE Human adenovirus type 55 (HAdV55) has recently emerged as a highly virulent respiratory pathogen and has been linked to severe and even fatal pneumonia in immunocompetent adults. However, the cellular receptors mediating the entry of HAdV55 into host cells remain unclear, which hinders the establishment of HAdV55-infected animal models and the development of antiviral approaches. In this study, we demonstrated that human desmoglein-2 (hDSG2) plays the major roles during HAdV55 infection. Human CD46 (hCD46) could also mediate the infection of HAdV55, but the efficiency was much lower than for hDSG2. We generated two transgenic mouse lines that express either hDSG2 or hCD46, both of which enabled HAd55 infection in otherwise nontransgenic mice. hDSG2 transgenic mice enabled more efficient HAdV55 infection than hCD46 transgenic mice. Our study adds to our understanding of HAdV55 infection and provides an animal model for evaluating HAdV55 vaccines and therapeutics.

LncRNA PVT1 promotes gemcitabine resistance of pancreatic cancer via activating Wnt/ß-catenin and autophagy pathway through modulating the miR-619-5p/Pygo2 and miR-619-5p/ATG14 axes.

BACKGROUND: Pancreatic cancer is one of the most lethal malignancies and has an extremely poor diagnosis and prognosis. The development of resistance to gemcitabine is still a major challenge. The long noncoding RNA PVT1 was reported to be involved in carcinogenesis and chemoresistance; however, the mechanism by which PVT1 regulates the sensitivity of pancreatic cancer to gemcitabine remains poorly understood. METHODS: The viability of pancreatic cancer cells was assessed by MTT assay in vitro and xenograft tumor formation assay in vivo. The expression levels of PVT1 and miR-619-5p were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Western blotting analysis and qRT-PCR were performed to assess the protein and mRNA levels of Pygo2 and ATG14, respectively. Autophagy was explored via autophagic flux detection under confocal microscopy and autophagic vacuole investigation under transmission electron microscopy (TEM). The functional role and mechanism of PVT1 were further investigated by gain- and loss-of-function assays in vitro. RESULTS: In the present study, we demonstrated that PVT1 was up-regulated in gemcitabine-resistant pancreatic cancer cell lines. Gain- and loss-of-function assays revealed that PVT1 impaired sensitivity to gemcitabine in vitro and in vivo. We further found that PVT1 up-regulated the expression of both Pygo2 and ATG14 and thus regulated Wnt/ß-catenin signaling and autophagic activity to overcome gemcitabine resistance through sponging miR-619-5p. Moreover, we discovered three TCF/LEF binding elements (TBEs) in the promoter region of PVT1, and activation of Wnt/ß-catenin signaling mediated by the up-regulation of Pygo2 increased PVT1 expression by direct binding to the TBE region. Furthermore, PVT1 was discovered to interact with ATG14, thus promoting assembly of the autophagy specific complex I (PtdIns3K-C1) and ATG14-dependent class III PtdIns3K activity. CONCLUSIONS: These data indicate that PVT1 plays a critical role in the sensitivity of pancreatic cancer to gemcitabine and highlight its potential as a valuable target for pancreatic cancer therapy.

Zika virus circumvents host innate immunity by targeting the adaptor proteins MAVS and MITA.

Recently, Zika virus (ZIKV) has generated extraordinary concern because of its severe neurotoxicity. Disturbingly, there is no vaccine or specific drug to prevent or treat the diseases caused by ZIKV infection. Thus, it is extremely urgent to characterize the pathogenesis of ZIKV. It has been documented that ZIKV can evade antiviral responses of host cells. Here, we demonstrate that ZIKV strain SZ-WIV01 down-regulates the production of type I IFN and IFN-stimulated genes along with the expression of mitochondrial antiviral signaling protein (MAVS) and mediator of IFN regulatory factor 3 activation (MITA). In the mechanism, ZIKV nonstructural (NS) 3 and NS2B3 negatively regulate IFN-related retinoic acid-inducible gene I-like receptor signaling pathway by targeting MAVS and MITA, respectively. Overexpression of ZIKV NS3 and NS2B3 dramatically inhibits expression of IFN-ß. ZIKV NS3 interacts with MAVS, and NS2B3 interacts with MITA, which catalyzes K48-linked polyubiquitination of MAVS and MITA for degradation. Further investigations suggest that ZIKV NS2B3 impairs polyinosinic:polycytidylic acid-triggered K63-linked polyubiquitination of MITA, thereby subverting the activation of downstream sensors. Our study reveals an undiscovered mechanism for ZIKV to escape the innate immune response, providing new insights into clinical study of vaccines or effective drugs.-Li, W., Li, N., Dai, S., Hou, G., Guo, K., Chen, X., Yi, C., Liu, W., Deng, F., Wu, Y., Cao, X. Zika virus circumvents host innate immunity by targeting the adaptor proteins MAVS and MITA.

Prognostic value of angiogenic T cells in hepatitis B-induced liver cirrhosis.

This study was performed to investigate the frequency of angiogenic T cells (CD4+ Tang cells) among CD4+ T cells in patients with hepatitis B-induced liver cirrhosis (HBV-LC) and to evaluate the predictive role of these cells in the clinical outcome. In total, 185 patients with HBV-LC were recruited to measure the frequency of CD4+ Tang cells and chemokine levels using flow cytometry. RESULTS: There was 11.4% of death after 3-momth follow-up. The AUC for the ability of the frequency of CD4+ Tang cell to predict death was 0.724 (higher than those for the MELD score, FIB-4 score, and Child-Pugh classification). Cox regression analysis revealed an association between the frequency of CD4+ Tang cells and a 3-month survival chance. CONCLUSIONS: The lower frequency of CD4+ T ang cells was correlated with the severity of HBV-LC and may serve as a prognostic predictor.

POSoligo software for in vitro gene synthesis.

Oligonucleotide synthesis is vital for molecular experiments. Bioinformatics has been employed to create various algorithmic tools for the in vitro synthesis of nucleotides. The main approach to synthesizing long-chain DNA molecules involves linking short-chain oligonucleotides through ligase chain reaction (LCR) and polymerase chain reaction (PCR). Short-chain DNA molecules have low mutation rates, while LCR requires complementary interfaces at both ends of the two nucleic acid molecules or may alter the conformation of the nucleotide chain, leading to termination of amplification. Therefore, molecular melting temperature, length, and specificity must be considered during experimental design. POSoligo is a specialized offline tool for nucleotide fragment synthesis. It optimizes the oligonucleotide length and specificity based on input single-stranded DNA, producing multiple contiguous long strands (COS) and short patch strands (POS) with complementary ends. This process ensures free 5'- and 3'-ends during oligonucleotide synthesis, preventing secondary structure formation and ensuring specific binding between COS and POS without relying on stabilizing the complementary strands based on Tm values. POSoligo was used to synthesize the linear RBD sequence of SARS-CoV-2 using only one DNA strand, several POSs for LCR ligation, and two pairs of primers for PCR amplification in a time- and cost-effective manner.

miR-214-3p promotes the pathogenesis of Parkinson's disease by inhibiting autophagy.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder characterized by dopaminergic neuron death in the substantia nigra, leading to motor dysfunction. Autophagy dysregulation has been implicated in PD pathogenesis. This study explores the role of miR-214-3p in PD, focusing on its impact on autophagy and dopaminergic neuron viability. Using in vitro and in vivo models, we demonstrate that miR-214-3p inhibits autophagy and promotes dopaminergic neuron apoptosis. Behavioral assessments and molecular analyses reveal exacerbation of PD symptoms upon miR-214-3p overexpression. Furthermore, mechanistic investigations identify ATG3 as a target, shedding light on miR-214-3p's regulatory role in autophagy. These findings enhance our understanding of PD pathogenesis and propose miR-214-3p as a potential biomarker and therapeutic target for modulating autophagy and neuronal survival in PD.

Hemagglutinin protein of measles virus induces apoptosis of HeLa cells via both extrinsic and intrinsic pathways.

In this study, we investigated the potential for different components of the measles virus (MV) to induce apoptosis of HeLa cells and explored the apoptotic molecular mechanisms. After testing the 2 envelope glycoproteins hemagglutinin (H) and fusion (F), we found that MV H alone was sufficient to induce the apoptosis of HeLa cells, whereas MV F did not. MV F also had no influence on MV-H-mediated apoptosis. MV H could induce cellular apoptosis in HeLa cells through its interaction with the cellular receptor CD46 via both the TRAIL-mediated extrinsic pathway and the mitochondria-controlled intrinsic pathway, and that cross talk between these 2 pathways occurred during the process. These findings extend the functions of MV envelope glycoproteins in the pathogenesis of MV infection and suggest that MV H may be a potential therapeutic in the treatment of some cancers.

Impact of vaccination on kinetics of neutralizing antibodies against SARS-CoV-2 by serum live neutralization test based on a prospective cohort.

How much the vaccine contributes to the induction and development of neutralizing antibodies (NAbs) of breakthrough cases relative to those unvaccinated-infected cases is not fully understood. We conducted a prospective cohort study and collected serum samples from 576 individuals who were diagnosed with SARS-CoV-2 Delta strain infection, including 245 breakthrough cases and 331 unvaccinated-infected cases. NAbs were analysed by live virus microneutralization test and transformation of NAb titre. NAbs titres against SARS-CoV-2 ancestral and Delta variant in breakthrough cases were 7.8-fold and 4.0-fold higher than in unvaccinated-infected cases, respectively. NAbs titres in breakthrough cases peaked at the second week after onset/infection. However, the NAbs titres in the unvaccinated-infected cases reached their highest levels during the third week. Compared to those with higher levels of NAbs, those with lower levels of NAbs had no difference in viral clearance duration time (P>0.05), did exhibit higher viral load at the beginning of infection/maximum viral load of infection. NAb levels were statistically higher in the moderate cases than in the mild cases (P<0.0001). Notably, in breakthrough cases, NAb levels were highest longer than 4 months after vaccination (Delta strain: 53,118.2 U/mL), and lowest in breakthrough cases shorter than 1 month (Delta strain: 7551.2 U/mL). Cross-neutralization against the ancestral strain and the current circulating isolate (Omicron BA.5) was significantly lower than against the Delta variant in both breakthrough cases and unvaccinated-infected cases. Our study demonstrated that vaccination could induce immune responses more rapidly and greater which could be effective in controlling SARS-CoV-2.

Clinical features and high-resolution chest computerized tomography findings of children infected by the B.1.617.2 variant of coronavirus disease 2019.

PURPOSE: To analyse the clinical symptoms, laboratory examinations and chest CT findings of children infected by the B.1.617.2 variant of COVID-19 and to compare the differences between clinical subtypes. METHODS: Fifty-three children (28 males, 25 females; age ranging from 4 months to 17 years) were included with B.1.617.2 variant infection in Nanjing, China, from July 21 to August 12 2021. Clinical data from patients were collected and analysed in groups of mild and common types. Imaging data were divided into three stages for evaluation: early, intermediate and late stages. RESULTS: In our study, fever (53%), cough (34%) and pharyngeal discomfort (28%) were the main symptoms. There were no differences in clinical symptoms between the mild and common type. The most common laboratory test items outside the normal range were decreased mean corpuscular volume (68%), lymphocyte percentage (64% elevated and 2% decreased) and decreased serum alkaline phosphatase concentration (66%). The differences in haemoglobin and monocyte percentages between the mild and common types were statistically significant (p = .037 and .033, respectively). No influencing factor was statistically significant in the regression analysis of both symptoms and clinical subtypes. The main CT findings were ground-glass opacity and consolidation located in the periphery and bilateral multilobed involvement. The mean CT score was 1.6. CT score correlated with packet cell volume, haemoglobin, mean erythrocyte volume, mean platelet volume and platelet distribution width. CONCLUSION: The pathogenetic condition of children with B.1.617.2 variant infection is mild. Although there were intergroup differences in some blood cell analyses, T-lymphocyte counts, and comprehensive biochemical indicators, no factors had a significant effect on clinical typing and the presence or absence of symptoms. CT findings and CT scores reflect disease stage and pathological changes and correlate moderately with laboratory tests, making them of good value for disease diagnosis and monitoring.Key MessagesPaediatric patients infected with B.1.617.2 variant have a milder clinical and imaging presentation than adults and are similar to the prototype infection.CT findings and scores which reflect disease stages and pathological changes.There is a correlation between chest CT and laboratory tests, which can be useful for the diagnosis and follow-up of the disease.

Effectiveness of inactivated COVID-19 vaccines against severe illness in B.1.617.2 (Delta) variant-infected patients in Jiangsu, China.

BACKGROUND: The SARS-CoV-2 B.1.617.2 (Delta) variant has caused a new surge in the number of COVID-19 cases. The effectiveness of inactivated vaccines against this variant is not fully understood. METHODS: Using data from a recent large-scale outbreak of B.1.617.2 SARS-CoV-2 infection in Jiangsu, China, we conducted a real-world study to explore the effect of inactivated vaccine immunization on the course of disease in patients infected with the Delta variant. RESULTS: Of 476 patients with B.1.617.2 infection, 184 were unvaccinated, 105 were partially vaccinated, and 187 were fully vaccinated. A total of 42 (8.8%) patients developed severe illness, of whom, 27 (14.7%), 13 (12.4%), and 2 (1.1%) were unvaccinated, partially vaccinated, and fully vaccinated, respectively (P <0.001). All 15 (3.2%) patients who required mechanical ventilation were unvaccinated. After adjusting for age, sex, and comorbidities, fully vaccinated patients had an 88% reduced risk of progressing to severe illness (ORadjusted: 0.12, 95% CI: 0.02-0.45). However, this protective effect was not observed in partially vaccinated patients (ORadjusted: 1.11, 95% CI: 0.51-2.36). Full immunization offered 100% protection from severe illness among women. The effect of the vaccine was potentially affected by underlying medical conditions (ORadjusted: 0.26, 95% CI: 0.03-1.23). CONCLUSION: Full vaccination with inactivated vaccines is highly effective in preventing severe illness in Delta variant-infected patients. However, partial vaccination does not offer clinically meaningful protection against severe disease.

Nogo-B mediates HeLa cell adhesion and motility through binding of Fibulin-5.

Nogo-B is a known regulator of neural functions and plays an important role in cell adhesion and migration. To our knowledge, the molecular mechanism behind its regulation of cell motility is still unknown. Here, we identified Fibulin-5, a secreted extracellular matrix protein, as a binding partner of Nogo-B. Using HeLa cells as a model, we found that Nogo-B and Fibulin-5 co-localize in the cytoplasm and plasma membrane. Furthermore, in HeLa cells that overexpress Nogo-B, cell migration and invasion was promoted by the elevated secretion of Fibulin-5. Thus, identification of the Nogo-B binding protein Fibulin-5 may contribute to uncover the pathway in which Nogo-B regulates tumor cell movement.

An unusual COVID-19 case with over four months of viral shedding in the presence of low neutralizing antibodies: a case report.

The ongoing coronavirus disease 2019 (COVID-19) pandemic is a global public health crisis, causing social and economic disasters in many countries. In China, two-consecutive negative results of nucleic acid tests for SARS-CoV-2 from the respiratory samples are required to end the quarantine of COVID-19 patients. However, clinicians face a dilemma in case of patients with long-term viral shedding. This report described an unusual COVID-19 case who had persistent viral RNA positivity for more than 4 months after initial illness in the presence of low neutralizing antibodies, but without prolonged clinical symptoms. Multiple anti-viral drug treatments had no impact and there was no evidence of re-infection. When the patient was self-quarantined at home, no infection occurred to the three family members living with her for 15 to 19 days. Sputum viral culture in BSL-3 laboratory on the 102 nd day after symptom onset was negative. From the 129 th day on, 8 continuous nucleic acid tests of sputum samples showed negative results. The patient was discharged on 137 th days since symptom onset. In conclusion, viral RNA shedding in the sputum of the COVID-19 patient may last over 4 months. As no evidence shows the existence of infectious virus, two-consecutive negative nucleic acid tests may not be the prerequisite for ending quarantine of COVID-19 patients with prolonged viral shedding.

SARS-CoV-2 neutralizing antibody levels are correlated with severity of COVID-19 pneumonia.

The emerging coronavirus disease 2019 (COVID-19) has become a serious global public health threat. With more and more recovered patients, it is urgently needed for evaluation of the neutralizing antibody (NAb) in these patients. In this study, we collected blood samples from 49 patients recently recovered from COVID-19. Serum NAbs were measured using a novel surrogate virus neutralization test (sVNT). Factors associated with NAb titers were analyzed using Ordinary Least Squares regression model. The median age of the study participants was 37 years (IQR, 30.0-54.5) and 55.1 % (27/49) of which were male. The median time to blood collection (for NAb analysis) from illness onset, viral clearance and discharge were 43.0 days (IQR, 36.0-50.0), 27.0 days (IQR, 20.5-37) and 17.0 days (IQR, 15.0-33.0), respectively. Patients had a median NAb titer of 1: 40 (IQR, 1:15-1:120). NAbs were not detected in two asymptomatic children who quickly cleared the virus. NAb titers were higher in patients with older age (p = 0.020), symptomatic infection (p = 0.044), more profound lung involvement (p＜0.001), abnormal C-reactive protein level (p＜0.01) and elevated lactate dehydrogenase (p = 0.019). Multivariable analysis revealed that severity of pneumonia and having comorbidity positively correlated with NAb titers in recovered patients (p = 0.02), while use of corticosteroids negatively impacted NAb titers (p = 0.01). Our study suggests that some COVID-19 patients may not have detectable NAb after recovery. SARS-CoV-2 NAb titers are positively correlated with severity of COVID-19 pneumonia.

Chloroquine inhibits endosomal viral RNA release and autophagy-dependent viral replication and effectively prevents maternal to fetal transmission of Zika virus.

Zika virus (ZIKV) infection can cause neonatal microcephaly and neurological disorders. Currently, there is no designated drug for treating ZIKV infection and preventing neonatal microcephaly. In this study, we evaluated the effect of chloroquine, an anti-malaria drug, in ZIKV infected cells and mouse models. Chloroquine significantly inhibited ZIKV infection in multiple mammalian cell lines. Chloroquine treatment significantly improved the survival of ZIKV-infected 1-day old suckling SCID Beige mice and reduced viremia in adult SCID Beige mice. Importantly, chloroquine protected the fetus from maternal infection by reducing placenta to fetus viral transmission. We found that chloroquine exerts at least two mechanisms in protecting against ZIKV infection: 1) inhibiting endosomal disassembly of the internalized virus and thus reducing the release of viral RNA to the cytoplasm for replication; 2) inhibiting ZIKV RNA replication through blocking ZIKV induced autophagy. Our study suggests that chloroquine treatment warrants to be considered as a mitigation strategy for treating ZIKV infection and preventing ZIKV-associated microcephaly in pregnant women.

Convalescent patient-derived monoclonal antibodies targeting different epitopes of E protein confer protection against Zika virus in a neonatal mouse model.

The Zika virus (ZIKV) outbreak and its link to microcephaly triggered a public health concern. To examine antibody response in a patient infected with ZIKV, we used single-cell PCR to clone 31 heavy and light chain-paired monoclonal antibodies (mAbs) that bind to ZIKV envelope (E) proteins isolated from memory B cells of a ZIKV-infected patient. Three mAbs (7B3, 1C11, and 6A6) that showed the most potent and broad neutralization activities against the African, Asian, and American strains were selected for further analysis. mAb 7B3 showed an IC50 value of 11.6 ng/mL against the circulating American strain GZ02. Epitope mapping revealed that mAbs 7B3 and 1C11 targeted residue K394 of the lateral ridge (LR) epitope of the EDIII domain, but 7B3 has a broader LR epitope footprint and recognizes residues T335, G337, E370, and N371 as well. mAb 6A6 recognized residues D67, K118, and K251 of the EDII domain. Interestingly, although the patient was seronegative for DENV infection, mAb 1C11, originating from the VH3-23 and VK1-5 germline pair, neutralized both ZIKV and DENV1. Administration of the mAbs 7B3, 1C11, and 6A6 protected neonatal SCID mice infected with a lethal dose of ZIKV. This study provides potential therapeutic antibody candidates and insights into the antibody response after ZIKV infection.

Hexon and fiber of adenovirus type 14 and 55 are major targets of neutralizing antibody but only fiber-specific antibody contributes to cross-neutralizing activity.

Re-emerging human adenoviruses type 14 (HAdV14) and 55 (HAdV55) represent two highly virulent adenoviruses. The neutralizing antibody (nAb) responses elicited by infection or immunization remain largely unknown. Herein, we generated hexon-chimeric HAdV14 viruses harboring each single or entire hexon hyper-variable-regions (HVR) from HAdV55, and determined the neutralizing epitopes of human and mouse nAbs. In human sera, hexon-targeting nAbs are type-specific and mainly recognize HVR2, 5, and 7. Fiber-targeting nAbs are only detectable in sera cross-neutralizing HAdV14 and HAdV55 and contribute substantially to cross-neutralization. Penton-binding antibodies, however, show no significant neutralizing activities. In mice immunized with HAdV14 or HAdV55, a single immunization mainly elicited hexon-specific nAbs, which recognized HAdV14 HVR1, 2, and 7 and HAdV55 HVR1 and 2, respectively. After a booster immunization, cross-neutralizing fiber-specific nAbs became detectable. These results indicated that hexon elicits type-specific nAbs whereas fiber induces cross-neutralizing nAbs to HAdV14 and HAdV55, which are of significance in vaccine development.

An adenovirus serotype 2-vectored ebolavirus vaccine generates robust antibody and cell-mediated immune responses in mice and rhesus macaques.

Ebolavirus vaccines based on several adenoviral vectors have been investigated in preclinical studies and clinical trials. The use of adenovirus serotype 2 as a vector for ebolavirus vaccine has not been reported. Herein, we generated rAd2-ZGP, a recombinant replication-incompetent adenovirus serotype 2 expressing codon-optimized Zaire ebolavirus glycoprotein, and evaluated its immunogenicity in mice and rhesus macaques. rAd2-ZGP induced significant antibody and cell-mediated immune responses at 2 weeks after a single immunization. The glycoprotein (GP)-specific immune responses could be further enhanced with a booster immunization. Compared to protein antigens, Zaire ebolavirus GP and Zaire ebolavirus-like particles, rAd2-ZGP could induce stronger cross-reactive antibody and cell-mediated immune responses to heterologous Sudan ebolavirus in mice and rhesus macaques. In rAd2-ZGP-immunized macaques, GP-specific CD8+ T cells could secret IFN-γ and IL-2, indicating a Th1-biased response. In adenovirus serotype 5 seropositive macaques, rAd2-ZGP could induce robust antibody and cell-mediated immune responses, suggesting that the efficacy of rAd2-ZGP is not affected by pre-existing immunity to adenovirus serotype 5. These results demonstrated that rAd2-ZGP can be considered an alternative ebolavirus vaccine for use in adenovirus serotype 5 seropositive subjects or as a sequential booster vaccine after the subjects have been immunized with a recombinant adenovirus serotype 5-based vaccine.

Incorporation of NS1 and prM/M are important to confer effective protection of adenovirus-vectored Zika virus vaccine carrying E protein.

Current design of Zika virus (ZIKV) vaccine mainly considered envelope (E) as the major target antigen. Non-structural protein NS1 was seldom considered. Herein, we generated three adenovirus-vectored vaccines carrying E (Ad2-E), or premembrane/membrane (prM/M) with E (Ad2-prME), or NS1 in addition to prM/M with E (Ad2-prME-NS1). Ad2-prME induced higher neutralizing antibody response to ZIKV than Ad2-E, suggesting prM/M is important for the folding of immunogenic E. Most intriguingly, Ad2-prME-NS1 elicited the best viral inhibition when the immune sera were added to ZIKV-infected cells. In ZIKV-challenged neonatal mice born to maternally immunized dams, Ad2-prME-NS1 conferred the best protection in preventing weight loss, neurological disorders, and viral replication. Ad2-prME also conferred significant protection but was less effective than Ad2-prME-NS1, whereas Ad2-E only alleviated neurological symptoms but did not inhibit viral replication. Our study suggested that NS1 should be considered in the design of ZIKV vaccine in addition to prM/M and E.