Zika Virus Envelope Research Articles

Longitudinal analysis of the antibody repertoire of a Zika virus-infected patient revealed dynamic changes in antibody response

ABSTRACT The Zika virus (ZIKV) is a mosquito-borne flavivirus that causes neonatal abnormalities and other disorders. Antibodies to the ZIKV envelope (E) protein can block infection. In this study, next-generation sequencing (NGS) of immunoglobulin heavy chain (IgH) mRNA transcripts was combined with single-cell PCR cloning of E-binding monoclonal antibodies for analysing antibody response in a patient from the early stages of infection to more than one year after the clearance of the virus. The patient's IgH repertoire 14 and 64 days after symptom onset showed dramatic dominant clonal expansion but low clonal diversity. IgH repertoire 6 months after disease-free status had few dominant clones but increased diversity. E-binding antibodies appeared abundantly in the repertoire during the early stages of infection but quickly declined after clearance of the virus. Certain VH genes such as VH5-10-1 and VH4-39 appeared to be preferentially enlisted for a rapid antibody response to ZIKV infection. Most of these antibodies require relatively few somatic hypermutations to acquire the ability to bind to the E protein, pointing to a possible mechanism for rapid defence against ZIKV infection. This study provides a unique and holistic view of the dynamic changes and characteristics of the antibody response to ZIKV infection.

Optimization of Small-Scale Production of Zika Virus Envelope Glycoprotein by Transient Expression in HEK293 Cells for ELISA.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus, which has recently caused global epidemics with its association with congenital Zika syndrome such as severe microcephaly. The recombinant ZIKV envelope (Env) glycoprotein is useful for immunological applications such as serodiagnosis of ZIKV infection and for monitoring immune responses in preclinical and clinical ZIKV vaccine developments. In this chapter, we describe the optimization of production of Zika virus envelope glycoprotein in Human Embryonic Kidney (HEK 293T) cells by small-scale expression followed by large-scale protein production. Small-scale expression of HEK 293T cells allows screening of a large number of vectors simultaneously to select the vectors with best secretory profiles for scale-up in Expi293 mammalian system to maximize the protein yield followed by purification for research and clinical applications.

Glucose regulated protein 78 (GRP78) interacts with Zika virus envelope and is required for a productive infection

Zika virus (ZIKV) is a member of the Flaviviridae family and was until recently a relatively obscure tropical disease. Subsequently, ZIKV has been shown to be the causative agent of fetal abnormalities and Guillain-Barré syndrome in outbreaks across the Americas and so efforts towards delineating important factors in the viral lifecycle have increased. Combining protein pull-down with mass spectrometry, it was found that ZIKV envelope (Env) interacts with the endoplasmic reticulum (ER) resident chaperone, glucose regulated protein 78 (GRP78) in A549 cells. Flaviviruses such as Japanese encephalitis virus and dengue virus are known to co-opt ER resident proteins and members of the unfolded protein response, including GRP78, to enhance viral infectivity and propagation. The role these proteins play during the ZIKV lifecycle has yet to be elucidated. To determine the importance of this interaction during ZIKV infection, A549 cells were treated with GRP78-specific siRNAs prior to infection with a NanoLuc expressing reporter virus or a wild-type virus. Depletion of GRP78 significantly reduced both virus luciferase readings and viral titres, indicating that GRP78 is necessary for efficient infection of mammalian cell culture. In contrast, inhibition of GRP78 with small molecule inhibitors did not reduce ZIKV infection. Interestingly, immunofluorescence of ZIKV infected cells reveal that GRP78 re-localises following infection and co-localises with Env. Depletion of GRP78 abrogated localisation of viral replication factories. Further experiments have shown that GRP78 is important for infection post entry and replication, and that putative GRP78 interactions partners are also required during infection.

Dissecting the role of glycosylation in Zika virus envelope with biochemical methods

Dissecting the role of glycosylation in Zika virus envelope with biochemical methods

Assessment of Immunogenicity and Efficacy of a Zika Vaccine Using Modified Vaccinia Ankara Virus as Carriers.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that has spread to more than 70 countries worldwide since 2015. Despite active research, there are currently no licensed vaccines or therapeutics. We have previously reported the development of various adenoviral vectored vaccine candidates (ChAdOx1 ZIKV) with the ability to stimulate effective immunity in mice and provide protection upon a ZIKV challenge model, using a non-adjuvanted single vaccination approach. In this study, we constructed various modified vaccinia Ankara (MVA) viruses to express the ZIKV Envelope (E) with modifications on the precursor membrane (prM) or on the C-terminus envelope transmembrane domain (TM), similar to our ChAdOx1 vaccine candidates. MVA-ZIKV vaccine candidates were evaluated as a non-adjuvanted single vaccination regimen against a ZIKV Brazilian isolate, using viraemia as the correlate of protection. Here, we report the induction of a modest level of anti-ZIKV E antibodies by all MVA vectored vaccines and sub-optimal efficacy in a ZIKV challenge model. Our results indicate the requirement of additional strategies when using MVA-ZIKV vaccines to afford sterile protection upon a non-adjuvanted and single vaccination regime.

Effects of Adjuvants on the Immunogenicity and Efficacy of a Zika Virus Envelope Domain III Subunit Vaccine.

Zika virus (ZIKV), a mosquito-borne flavivirus, has attracted global attention due to its close association with congenital Zika syndrome and neurological diseases, and transmission through additional routes, such as sexual contact. Currently there are no vaccines approved for ZIKV, and thus, there is an urgent need to develop an effective and safe ZIKV vaccine. Domain III (DIII) of the ZIKV envelope (E) protein is an important vaccine target, and a vaccine developed using a mutant DIII of E (EDIII) protein protects adult and pregnant mice, and unborn offspring, against ZIKV infection. Here, we have used immunocompetent BALB/c mice treated with anti-interferon-α/β receptor 1 (Ifnar1) antibodies to investigate whether three adjuvants (aluminum (Alum), monophosphoryl lipid A (MPL), and MF59), either alone or in combination, could improve the efficacy of this EDIII subunit vaccine. Our data show that, although vaccine formulated with a single adjuvant induced a specific antibody and cellular immune response, and reduced viral load in mice challenged with ZIKV, the combination of Alum and MPL adjuvants led to a more robust and balanced immune response, stronger neutralizing activity against three recent ZIKV human strains, and greater protection against a high-dose ZIKV challenge. Particularly, the combination of Alum with MPL significantly reduced viral titers and viral RNA copy numbers in sera and tissues, including the male reproductive organs. Overall, this study has identified the combination of Alum and MPL as the most effective adjuvant for ZIKV EDIII subunit vaccines, and it has important implications for subunit vaccines against other enveloped viruses, including non-ZIKV flaviviruses.

Computationally Designed Peptides for Zika Virus Detection: An Incremental Construction Approach.

Herein, and in contrast to current production of anti-Zika virus antibodies, we propose a semi-combinatorial virtual strategy to select short peptides as biomimetic antibodies/binding agents for the detection of intact Zika virus (ZIKV) particles. The virtual approach was based on generating different docking cycles of tetra, penta, hexa, and heptapeptide libraries by maximizing the discrimination between the amino acid motif in the ZIKV and dengue virus (DENV) envelope protein glycosylation site. Eight peptides, two for each length (tetra, penta, hexa, and heptapeptide) were then synthesized and tested vs. intact ZIKV particles by using a direct enzyme linked immunosorbent assay (ELISA). As a reference, we employed a well-established anti-ZIKV antibody, the antibody 4G2. Three peptide-based assays had good detection limits with dynamic range starting from 105 copies/mL of intact ZIKV particles; this was one order magnitude lower than the other peptides or antibodies. These three peptides showed slight cross-reactivity against the three serotypes of DENV (DENV-1, -2, and -3) at a concentration of 106 copies/mL of intact virus particles, but the discrimination between the DENV and ZIKV was lost when the coating concentration was increased to 107 copies/mL of the virus. The sensitivity of the peptides was tested in the presence of two biological matrices, serum and urine diluted 1:10 and 1:1, respectively. The detection limits decreased about one order of magnitude for ZIKV detection in serum or urine, albeit still having for two of the three peptides tested a distinct analytical signal starting from 106 copies/mL, the concentration of ZIKV in acute infection.

Role of Zika Virus Envelope Protein Domain III as a Target of Human Neutralizing Antibodies.

Zika virus (ZIKV) is a flavivirus that is structurally highly similar to the related viruses, dengue virus (DENV), West Nile virus, and yellow fever virus. ZIKV causes an acute infection that often results in mild symptoms but that can cause severe disease in rare instances. Following infection, individuals mount an adaptive immune response, composed of antibodies (Abs) that target the envelope (E) glycoprotein of ZIKV, which covers the surface of the virus. Groups have studied monoclonal antibodies and polyclonal immune sera isolated from individuals who recovered from natural ZIKV infections. Some of these antibodies bind to domain III of E (EDIII), but the functional importance of these antibodies is unknown. In this study, we aimed to determine if EDIII is a major target of the potent serum neutralizing antibodies present in people after ZIKV infection. By generating a chimeric virus containing ZIKV EDIII in a DENV4 virus backbone, our data show a minor role of EDIII-targeting antibodies in human polyclonal neutralization. These results reveal that while monoclonal antibody (MAb) studies are informative in identifying individual antibody epitopes, they can overestimate the importance of epitopes contained within EDIII as targets of serum neutralizing antibodies. Additionally, these results argue that the major target of human ZIKV neutralizing antibodies resides elsewhere in E; however, further studies are needed to assess the epitope specificity of the neutralizing response at the population level. Identification of the major epitopes on the envelope of ZIKV recognized by serum neutralizing antibodies is critical for understanding protective immunity following natural infection and for guiding the design and evaluation of vaccines.IMPORTANCE Zika virus is a flavivirus that was recently introduced to Latin America, where it caused a massive epidemic. Individuals infected with ZIKV generate an immune response composed of antibodies which bind to the envelope (E) protein. These anti-E antibodies are critical in protecting individuals from subsequent infection. Multiple groups have found that many ZIKV antibodies bind to domain III of E (EDIII), suggesting that this region is an important target of neutralizing antibodies. Here, we generated a chimeric virus containing ZIKV EDIII in a dengue virus backbone to measure ZIKV EDIII-specific antibody responses. We found that while polyclonal ZIKV immune serum contains antibodies targeting EDIII, they constitute only a small fraction of the total population of antibodies that neutralize ZIKV. Further studies are needed to define the main targets on the viral envelope recognized by human neutralizing antibodies, which is critical for guiding the development of ZIKV vaccines.

ZIKV Envelope Domain-Specific Antibodies: Production, Purification and Characterization.

Infection with Zika virus (ZIKV) came first to public attention after it was found to be associated with congenital microcephaly during the outbreak in Brazil (2015–2016). Diagnosis of ZIKV suffers from extensive cross-reactivity with other Flaviviruses, which are circulating in many ZIKV epidemic areas. Due to the fatal outcome of ZIKV infection during pregnancy, detailed knowledge about neutralizing and non-neutralizing epitopes is crucial for the development of robust detection systems of protective antibodies. Therefore, additional information about ZIKV immunogenicity and antibody response is required. In this project, we report the production, purification and characterization of six different polyclonal antibodies against ZIKV envelope (E) protein. The produced antibodies bind to isolated ZIKV E protein as well as to the surface of ZIKV particles, interestingly without being potently neutralizing. Surface plasmon resonance measurement showed that these antibodies bind with high affinity to ZIKV E protein. Epitope mapping revealed that the epitopes are distributed among the three ZIKV E domains with seven binding sites. These identified binding sites overlap only partially with the previously described epitopes recognized by neutralizing antibodies, which is in accordance with their lack of potent neutralizing activity. Additionally, these antibodies showed neither cross-reactivity nor potent neutralizing activity against West Nile virus, a related flavivirus. The gained set of data helps to extend our understanding about the distribution of neutralizing and non-/weak-neutralizing epitopes in ZIKV E protein, and provides a rationale for ZIKV vaccine design and development of robust detection assays for neutralizing antibodies.

Rational Design of Zika Virus Subunit Vaccine with Enhanced Efficacy.

Zika virus (ZIKV) infection in pregnant women can lead to fetal deaths and malformations. We have previously reported that ZIKV envelope protein domain III (EDIII) is a subunit vaccine candidate with cross-neutralization activity; however, like many other subunit vaccines, its efficacy is limited. To improve the efficacy of this subunit vaccine, we identified a nonneutralizing epitope on ZIKV EDIII surrounding residue 375, which is buried in the full-length envelope protein but becomes exposed in recombinant EDIII. We then shielded this epitope with an engineered glycan probe. Compared to the wild-type EDIII, the mutant EDIII induced significantly stronger neutralizing antibodies in three mouse strains and also demonstrated significantly improved efficacy by fully protecting mice, particularly pregnant mice and their fetuses, against high-dose lethal ZIKV challenge. Moreover, the mutant EDIII immune sera significantly enhanced the passive protective efficacy by fully protecting mice against lethal ZIKV challenge; this passive protection was positively associated with neutralizing antibody titers. We further showed that the enhanced efficacy of the mutant EDIII was due to the shielding of the immunodominant nonneutralizing epitope surrounding residue 375, which led to immune refocusing on the neutralizing epitopes. Taken together, the results of this study reveal that an intrinsic limitation of subunit vaccines is their artificially exposed immunodominant nonneutralizing epitopes, which can be overcome through glycan shielding. Additionally, the mutant ZIKV protein generated in this study is a promising subunit vaccine candidate with high efficacy in preventing ZIKV infections in mice.IMPORTANCE Viral subunit vaccines generally show low efficacy. In this study, we revealed an intrinsic limitation of subunit vaccine designs: artificially exposed surfaces of subunit vaccines contain epitopes unfavorable for vaccine efficacy. More specifically, we identified an epitope on Zika virus (ZIKV) envelope protein domain III (EDIII) that is buried in the full-length envelope protein but becomes exposed in recombinant EDIII. We further shielded this epitope with a glycan, and the resulting mutant EDIII vaccine demonstrated significantly enhanced efficacy over the wild-type EDIII vaccine in protecting animal models from ZIKV infections. Therefore, the intrinsic limitation of subunit vaccines can be overcome through shielding these artificially exposed unfavorable epitopes. The engineered EDIII vaccine generated in this study is a promising vaccine candidate that can be further developed to battle ZIKV infections.

Rapid microsphere-assisted peptide screening (MAPS) of promiscuous MHCII-binding peptides in Zika virus envelope protein.

Despite promising developments in computational tools, peptide-class II MHC (MHCII) binding predictors continue to lag behind their peptide-class I MHC counterparts. Consequently, peptide-MHCII binding is often evaluated experimentally using competitive binding assays, which tend to sacrifice throughput for quantitative binding detail. Here, we developed a high-throughput semiquantitative peptide-MHCII screening strategy termed microsphere-assisted peptide screening (MAPS) that aims to balance the accuracy of competitive binding assays with the throughput of computational tools. Using MAPS, we screened a peptide library from Zika virus envelope (E) protein for binding to four common MHCII alleles (DR1, DR4, DR7, DR15). Interestingly, MAPS revealed a significant overlap between peptides that promiscuously bind multiple MHCII alleles and antibody neutralization sites. This overlap was also observed for rotavirus outer capsid glycoprotein VP7, suggesting a deeper relationship between B cell and CD4+ T cell specificity which can facilitate the design of broadly protective vaccines to Zika and other viruses.

Characterization of two engineered dimeric Zika virus envelope proteins as immunogens for neutralizing antibody selection and vaccine design

The envelope protein of Zika virus (ZIKV) exists as a dimer on the mature viral surface and is an attractive antiviral target because it mediates viral entry. However, recombinant soluble wild-type ZIKV envelope (wtZE) might preferentially exist as monomer (monZE). Recently, it has been shown that the A264C substitution could promote formation of dimeric ZIKV envelope protein (ZEA264C), requiring further characterization of purified ZEA264C for its potential applications in vaccine development. We also noted that ZEA264C, connected by disulfide bond, might be different from the noncovalent native envelope dimer on the virion surface. Because the antibody Fc fragment exists as dimer and is widely used for fusion protein construction, here we fused wtZE to human immunoglobulin G1 (IgG1) Fc fragment (ZE-Fc) for noncovalent wtZE dimerization. Using a multistep purification procedure, we separated dimeric ZEA264C and ZE-Fc, revealing that they both exhibit typical β-sheet-rich secondary structures and stabilities similar to those of monZE. The binding activities of monZE, ZEA264C, and ZE-Fc to neutralizing antibodies targeting different epitopes indicated that ZEA264C and ZE-Fc could better mimic the native dimeric status, especially in terms of the formation of tertiary and quaternary epitopes. Both ZEA264C and ZE-Fc recognize a ZIKV-sensitive cell line as does monZE, indicating that the two constructs are still functional. Furthermore, a murine immunization assay disclose that ZEA264C and ZE-Fc elicit more neutralizing antibody responses than monZE does. These results suggest that the two immunogen candidates ZEA264C and ZE-Fc have potential utility for neutralizing antibody selection and vaccine design against ZIKV.

A non-coated enzyme-linked immunosorbent assay for screening zika virus envelope protein

To establish a non-coated enzyme-linked immunosorbent assay (ELISA) based on zika virus envelope (E) protein for detecting the expression of E protein in infected cells. Adherent Vero-143 cells infected with zika virus in a 96-well plate were fixed, and the antibodies against zika virus E protein were added at an optimized concentration to establish the non-coated ELISA method for E protein. The antiviral activities of lignans compound C1 was evaluated using this method. The accuracy of this non-coated ELISA was verified by RT-PCR, and the cross reaction with dengue virus was assessed. After optimization, the background absorbance at 450 nm of uninfected cells was reduced to about 0.20. The antiviral activities of lignans compound C1 detected by this method were basically consistent with the results of RT-PCR. No cross reaction with dengue virus was found in this assay. A non- coated ELISA method based on zika virus E protein was established, which can be used for screening antiviral agents against zika virus.

A common conserved peptide harboring predicted T and B cell epitopes in domain III of envelope protein of Japanese Encephalitis Virus and West Nile Virus for potential use in epitope based vaccines

Japanese encephalitis virus (JEV) and West Nile virus (WNV) are two major mosquito borne flaviviruses belonging to same serocomplex. JEV is transmitted by Culex mosquitoes and the reservoir host for the virus is pigs and/or water birds. WNV is also transmitted by Culex mosquitoes and reservoir host in this case is birds. It can also be transmitted through contact with other infected animals, their blood, or other tissues. The envelope protein of these viruses is the major source of epitopes and provides protective immunity. Bioinformatics tools were used to identify conserved epitopes in the envelope protein of these viruses. A conserved peptide "TPVGRLVTVNPFV" present in both the viruses containing predicted T and B cell epitopes was found. The model of one of the predicted epitope was generated and upon docking it bound in the groove of HLA-A0201 Class I MHC molecule. Further, it was amenable to proteasomal cleavage enhancing its chances of processing by cytosolic pathway. The peptide was found to be non toxic, non allergenic and stable in mammalian cells based on database search. The population coverage was pan world and nearly 70% identity of the peptide was found in the Zika virus envelope protein. The peptide was located in the domain III of envelope protein which is the exposed domain therefore B cell receptors may recognize this peptide easily. The conserved peptide containing T and B cell epitopes can have future application for designing epitope based vaccines for both JEV and WNV.

ZIKV-Specific NS1 Epitopes as Serological Markers of Acute Zika Virus Infection

Zika virus (ZIKV) infections have reemerged as a global health issue due to serious clinical complications. Development of specific serological assays to detect and differentiate ZIKV from other cocirculating flaviviruses for accurate diagnosis remains a challenge. We investigated antibody responses in 51 acute ZIKV-infected adult patients from Campinas, Brazil, including 7 pregnant women who later delivered during the study. Using enzyme-linked immunosorbent assays, levels of antibody response were measured and specific epitopes identified. Several antibody-binding hot spots were identified in ZIKV immunogenic antigens, including membrane, envelope (E) and nonstructural protein 1 (NS1). Interestingly, specific epitopes (2 from E and 2 from NS1) strongly recognized by ZIKV-infected patients' antibodies were identified and were not cross-recognized by dengue virus (DENV)-infected patients' antibodies. Corresponding DENV peptides were not strongly recognized by ZIKV-infected patients' antibodies. Notably, ZIKV-infected pregnant women had specific epitope recognition for ZIKV NS1 (amino acid residues 17-34), which could be a potential serological marker for early ZIKV detection. This study identified 6 linear ZIKV-specific epitopes for early detection of ZIKV infections. We observed differential epitope recognition between ZIKV-infected and DENV-infected patients. This information will be useful for developing diagnostic methods that differentiate between closely related flaviviruses.

Pichia pastoris-expressed Zika virus envelope domain III on a virus-like particle platform: design, production and immunological evaluation.

Zika virus (ZIKV) is an arbovirus which shares antigenic similarity and the mosquito vector with dengue viruses (DENVs). ZIKV is a neurotropic virus capable of causing congenital neurodevelopmental birth defects. As ZIKV antibodies (Abs) can potentially enhance infection by DENVs, a preventive ZIKV vaccine must be designed to eliminate antibody dependent enhancement of infection. We developed a Zika Subunit Vaccine (ZSV) consisting of two proteins, ZS and S, in a genetically pre-determined ratio of 1:4, using the methylotrophic yeast Pichia pastoris. ZS is an in-frame fusion of ZIKV envelope domain III with the Hepatitis B virus (HBV) surface antigen, and S is the un-fused HBV surface antigen. Using specific monoclonal Abs we showed the presence of ZS and S in the co-purified material which were found to co-assemble into virus-like particles (VLPs), based on dynamic light scattering and electron microscopic analyses. These VLPs were immunogenic in BALB/c mice, eliciting Abs capable of neutralizing ZIKV reporter virus particles. Further, the VLP-induced Abs did not enhance a sub-lethal DENV-2 challenge in AG129 mice. This important safety feature, coupled to the well-documented advantage of P. pastoris expression system, warrants further exploration of ZSV VLP as a possible vaccine candidate.

Quantum dots-based fluoroimmunoassay for anti-Zika virus IgG antibodies detection

Zika virus (ZIKV) has been declared a public health emergency of international concern. ZIKV has been associated with some neurological disorders, and their long-term effects are not completely understood. The majority of the methods for ZIKV diagnosis are based on the detection of IgM antibodies, which are the first signs of immunological response. However, the detection of IgG antibodies can be an important approach for ZIKV past infection diagnosis, especially for pregnant women, helping the comprehension/treatment of this disease. There has been a growing interest in applying nanoparticles for efficient ZIKV or antibodies detection. Quantum dots (QD) are unique fluorescent semiconductor nanoparticles, highly versatile for biological applications. In the present study, we explored the special QD optical properties to develop an immunofluorescence assay for anti-ZIKV IgG antibodies detection. Anti-IgG antibodies were successfully conjugated with QDs and applied in a fluorescence sensing nanoplatform. After optimization using IgG antibodies, the conjugates were employed to detect anti-ZIKV IgG antibodies in polystyrene microplates sensitized with ZIKV envelope E protein. The nanoplatform was able to detect anti-ZIKV IgG antibodies in a concentration at least 100-fold lower than the amount expected for protein E immune response. Moreover, conjugates were able to detect the antibodies for at least 4 months. Thus, our results showed that this QDs-based fluoroimmunoplatform can be considered practical, simple and promising to detect Zika past infections and/or monitoring immune response in vaccine trials.

Identification of small molecule inhibitors targeting the Zika virus envelope protein

The recent emergence of Zika virus, a mosquito-borne flavivirus, in the Americas has shed light on the severe neurological diseases associated with infection, notably congenital microcephaly in newborns and Guillain-Barré syndrome in adults. Despite the recent focus on Zika virus, there are currently no approved vaccines or antiviral therapies available to treat or prevent infection. In this study we established a competitive amplified luminescent proximity homogeneous assay (ALPHAscreen) to identify small molecule inhibitors targeting the envelope protein of Zika virus (Zika E). We utilized this assay to screen two libraries of nearly 27,000 compounds and identified seven novel inhibitors of Zika E. Characterization of these primary screening leads demonstrated that inhibition of Zika virus occurs at non-cytotoxic concentrations for all seven lead compounds. In addition, we found that all seven lead compounds have potent activity against the closely related dengue virus 2 but not vesicular stomatitis virus, an unrelated enveloped virus. Biochemical experiments indicate that these compounds act by preventing E-mediated membrane fusion. This work highlights a new method for the discovery and optimization of direct-acting antivirals targeting the E protein of Zika and other flaviviruses.

An ultra-sensitive capacitive microwire sensor for pathogen-specific serum antibody responses

Detection of viral infection is commonly performed using serological techniques like the enzyme-linked immunosorbent assay (ELISA) to detect antibody responses. Such assays may also be used to determine the infection phase based on isotype prevalence. However, ELISAs demonstrate limited sensitivity and are difficult to perform at the point of care. Here, we present a novel technique for label-free, rapid detection of ultra-low concentrations of virus specific antibodies. We have developed a simple, robust capacitive biosensor using microwires coated with Zika or Chikungunya virus envelope antigen. With little discernable nonspecific binding, the sensor can detect as few as 10 antibody molecules in a small volume (10 molecules/30 µL) within minutes. It can also be used to rapidly, specifically, and accurately determine the isotype of antigen-specific antibodies. Finally, we demonstrate that anti-Zika virus antibody can be sensitively and specifically detected in dilute mouse serum and can be isotyped using the sensor. Overall, our findings suggest that our microwire sensor platform has the potential to be used as a reliable, sensitive, and inexpensive diagnostic tool to detect immune responses at the point of care.

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

ABSTRACT 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.