Development of envelope protein antigens to serologically differentiate zika virus infection from dengue virus infection

Zika virus (ZIKV) is an emerging flavivirus that can cause birth defects and neurologic complications. Molecular tests are effective for diagnosing acute ZIKV infection, although the majority of infections produce no symptoms at all or present after the narrow window in which molecular diagnostics are dependable. Serology is a reliable method for detecting infections after the viremic period; however, most serological assays have limited specificity due to cross-reactive antibodies elicited by flavivirus infections. Since ZIKV and dengue virus (DENV) widely cocirculate, distinguishing ZIKV infection from DENV infection is particularly important for diagnosing individual cases or for surveillance to coordinate public health responses. Flaviviruses also elicit type-specific antibodies directed to non-cross-reactive epitopes of the infecting virus; such epitopes are attractive targets for the design of antigens for development of serological tests with greater specificity. Guided by comparative epitope modeling of the ZIKV envelope protein, we designed two recombinant antigens displaying unique antigenic regions on domain I (Z-EDI) and domain III (Z-EDIII) of the ZIKV envelope protein. Both the Z-EDI and Z-EDIII antigens consistently detected ZIKV-specific IgG in ZIKV-immune sera but not cross-reactive IgG in DENV-immune sera in late convalescence (12 weeks postinfection). In contrast, during early convalescence (2 to 12 weeks postinfection), secondary DENV-immune sera and some primary DENV-immune sera cross-reacted with the Z-EDI and Z-EDIII antigens. Analysis of sequential samples from DENV-immune individuals demonstrated that Z-EDIII cross-reactivity peaked in early convalescence and declined steeply over time. The Z-EDIII antigen has much potential as a diagnostic antigen for population-level surveillance and for detecting past infections in patients

Is there a silver lining to the Zika virus epidemic in the Americas?

It is hard to find anything positive to say about an epidemic of an emerging virus that infects pregnant women, targets developing fetuses’ neural progenitor cells, and disrupts the sequence of neural development to cause a devastating syndrome resulting in lifelong disabilities. Infants with microcephaly became the faces of the Zika virus epidemic in the Americas, which affected almost all countries in the western hemisphere in 2014–17. By the end of 2017, Zika virus has nearly disappeared from the Americas as quickly as it emerged. Now that the dust is settling, what have the scientific and public health communities learned? Is there a silver lining to Zika’s devastation

Antibody Cross-Reactivity Between Zika Virus, Kedougou Virus, and Spondweni Virus

Zika virus (ZIKV), dengue virus (DENV), Kedougou virus (KEDV), and Spondweni virus (SPOV) are closely-related, mosquito-borne flaviviruses. It is well-established that ZIKV and DENV both cause fever, rash, and joint pain, in addition to more severe disease states such as microcephaly and hemorrhagic fever, respectively. However, little is known about KEDV or SPOV infections because they have not yet caused large human outbreaks. Given that cross- reactive antibodies between ZIKV and DENV complicate serodiagnostic tests in regions where both viruses circulate, it is crucial to understand and develop serological applications for other flaviviruses, such as KEDV and SPOV, which are even more closely related to ZIKV than DENV is. In addition, identifying a molecular basis for the cross-neutralization of ZIKV, KEDV, and SPOV can help inform the development of neutralizing antibodies and broadly protective vaccines. The objective of this project was to assess the neutralization of ZIKV (strain H/PF/ 2013), KEDV (D14701), and SPOV (SA Ar 94) by mouse polyclonal immune sera (anti-ZIKV, anti-KEDV, and anti-SPOV) and human convalescent sera (ZIKV-immune and DENV-immune). Another goal was to determine whether a panel of monoclonal antibodies (mAbs), specific to particular epitopes on the flavivirus E protein (domain I, domain II, domain III, fusion loop), broadly neutralized ZIKV, KEDV, and SPOV. Here, we found that anti-ZIKV mouse immune sera cross-neutralized KEDV and SPOV, though anti-KEDV and anti-SPOV sera did not cross- neutralize ZIKV (FRNT≤25). We observed that several mouse and human mAbs targeting the fusion loop bound ZIKV, KEDV, and SPOV. However, these fusion loop mAbs neutralized SPOV but not ZIKV, suggesting that virion maturation state could impact cross-neutralization. In addition, we identified two mAbs—G9E and EDEC8—that cross-neutralized both ZIKV and SPOV by targeting the E protein cross-dimer interface, including domains I and II. These findings allow for subsequent studies on the cross-neutralization of ZIKV, KEDV, and SPOV by an expanded panel of ZIKV-immune and DENV-immune human convalescent sera samples, as well as other mAbs that cross-neutralize these viruses. This would facilitate both vaccine design and serodiagnostics in the event that a new flavivirus emerges in ZIKV/DENV co-endemic areas.Bachelor of Scienc

Human antibody response to Zika targets type-specific quaternary structure epitopes

The recent Zika virus (ZIKV) epidemic in the Americas has revealed rare but serious manifestations of infection. ZIKV has emerged in regions endemic for dengue virus (DENV), a closely related mosquito-borne flavivirus. Cross-reactive antibodies confound studies of ZIKV epidemiology and pathogenesis. The immune responses to ZIKV may be different in people, depending on their DENV immune status. Here, we focus on the human B cell and antibody response to ZIKV as a primary flavivirus infection to define the properties of neutralizing and protective antibodies generated in the absence of preexisting immunity to DENV. The plasma antibody and memory B cell response is highly ZIKV type–specific, and ZIKV-neutralizing antibodies mainly target quaternary structure epitopes on the viral envelope. To map viral epitopes targeted by protective antibodies, we isolated 2 type-specific monoclonal antibodies (mAbs) from a ZIKV case. Both mAbs were strongly neutralizing in vitro and protective in vivo. The mAbs recognize distinct epitopes centered on domains I and II of the envelope protein. We also demonstrate that the epitopes of these mAbs define antigenic regions commonly targeted by plasma antibodies in individuals from endemic and nonendemic regions who have recovered from ZIKV infections

Complete absence of linear immunodominant epitope regions recognized by IgG after flavivirus infection and vaccination in whole proteome analyses

The flavivirus genera comprises more than 70 viruses and has an important impact on public health in their endemic and epidemic regions all over the world. Serodiagnosis of specific flavivirus infections is difficult or even impossible, due to the high degree of antibody cross-reactivity. It is however conceivable that serodiagnosis based on one or more individual flavivirus species derived peptides allows to differentiate with high specificity between different viral infections. The primary objective of my doctoral thesis therefore was to map specific immunoglobulin G (IgG) antibody responses after natural flavivirus infections and yellow fever vaccination to identify type-specific signatures of different flaviviruses as well as to study dynamics of cross-reactivity and crossrecognition between them. Therefore, a panel of sera from flavivirus-infected study subjects as well as sera before (D0) and 28 days after (D28) YFV vaccination were tested with the RepliTope™pan-Flavivirus peptide array by the company JPT Peptide Technologies displaying 6253 peptides of various flaviviruses. In general, peptide array technique allows high-resolution, high-throughput, ultra-high-density mapping of linear antibody epitopes. Serum samples prior yellow fever vaccination (timepoint D0) served as negative controls. Data analyses were performed using R-script as well as GraphPad Prism software to further dissect the IgG immune response and to identify potential serodiagnostic peptides. The analysis was focused on detection of potential targets of IgG immune responses located in prM, E, NS1 and NS5 proteins. To summarize the data obtained by heat maps and graphs, our data has shown a high level of individual variation in antibody specificities as well as no flavivirus type-specific IgG peptide recognition signature between different flaviviruses across flavivirus-infected and YFV vaccinated study subjects. This study points out the difficulties of detecting a flavivirusspecific immune response but gives further insight into a more detailed epitope mapping and understanding of antibody responses in flavivirus infections.Die Gattung der Flaviviren umfasst über 70 Viren und hat einen wichtigen Einfluss auf das öffentliche Gesundheitswesen in endemischen sowie epidemischen Regionen weltweit. Die Serodiagnostik von speziellen Flavivirus Infektionen stellt sich aufgrund eines hohen Grades an Kreuzreaktivität der Antikörper als schwierig dar. Es ist jedoch vorstellbar, dass Serodiagnostik basierend auf einem oder mehreren individuellen Flavivirus-Peptiden die Differenzierung zwischen verschiedenen Flavivirus Infektionen mit hoher Spezifität möglich macht. Das vorrangige Ziel dieser Doktorarbeit ist die Kartierung spezifischer Immunglobulin G (IgG) Reaktionen nach natürlichen Flavivirus- Infektionen und nach Gelbfieberimpfung, um spezifische Signaturen verschiedener Flaviviren zu identifizieren sowie die Dynamik der Kreuzreaktivität und Kreuzerkennung zwischen diesen zu untersuchen. Daher wurde eine Auswahl an Seren von Flavivirus-infizierten Studienteilnehmer*innen sowie Seren von gesunden Proband*innen vor (Zeitpunkt D0) und 28 Tage nach Gelbfieber Impfung (Zeitpunkt D28) mit dem RepliTope™pan-Flavivirus Peptidarray der Firma JPT Peptide Technologies getestet, welches 6253 Peptide verschiedener Flaviviren abbildet. Im Allgemeinen ermöglicht die Peptid-Array-Technik eine hochauflösende Abbildung von linearen Antikörper-Epitopen. Serumproben vor der Gelbfieberimpfung (Zeitpunkt D0) dienten als Negativkontrollen. Die Analyse der mit den Peptidarrays gewonnenen Daten umfasste Auswertungs-methoden unter Verwendung von R-Skripten sowie der GraphPad-Prism-Software zur weiteren Aufschlüsselung der IgG-Immunantwort und zur Identifizierung serodiagnostischer Peptide. Die Analyse konzentrierte sich auf den Nachweis potenzieller, spezifischer Erkennungsstrukturen der IgG-Immunantwort, die in prM-, E, NS1- und NS5-Proteinen lokalisiert sind. Unsere Daten zeigten ein hohes Maß an individueller Variation der Antikörperspezifitäten sowie eine fehlende Flavivirus typ-spezifische IgG-Peptid-Signatur bei Flavivirus infizierten und Gelbfieber geimpften Studienteilnehmer*innen bzw. Proband*innen. Diese Studie weist auf die Schwierigkeiten des Nachweises einer Flavivirus-spezifischen Immunantwort hin, gibt jedoch Einblicke in eine detailliertere Epitop Kartierung und ein besseres Verständnis der Antikörper-Antworten bei Flavivirus-Infektionen

Determinants Of Antibody Immunity In Flavivirus Infections And Immunization

The most significant arthropod-borne viral infections in modern history are caused by species of the Flavivirus (FV) genus due to their global transmission. While immunization is effective at decreasing the burden imposed by some FV, vaccines for other FV such as Dengue virus (DENV) and Zika virus (ZIKV) are still in development or not widely available. A better understanding of the protective immune response, specifically the antibody response, to DENV and ZIKV will advance the vaccine development endeavors for these viruses. Utilizing longitudinal samples from controlled human immunization/challenge studies and natural infections, we investigated the antibody response to these viruses in both the cellular and serological compartments. Primary DENV or ZIKV infection results in an antibody response specific to the infecting virus with little cross-reactivity to other related FV. This specificity profile is observed in the immune serum, and both the acute plasmablast and convalescent memory B cell sub-populations. Neutralizing antibodies were identified in these compartments of the antibody response, suggesting their functional association with homologous immunity to the infecting virus. In contrast to primary infection, the antibody response induced by immunization with the tetravalent live attenuated vaccine TV003 is cross-reactive in both the serological and cellular compartments. Interpretation of these findings reveals an intimate and complex relationship between B cell sub-populations and serum antibodies, and contributes to our knowledge of various differentiation routes amongst these B cells. Further analysis using monoclonal antibodies reveals structural vulnerabilities of FV. We identified domain III of the viral envelope glycoprotein to be one of the immunodominant and neutralizing targets for DENV- and ZIKV-specific antibodies. Further studies will continue to elucidate the connections between different B cell sub-populations engaged in FV infections, and identify specific subsets that either confer protection or pose a disease risk in the secondary antibody response invoked by multiple infections. Characterization of antibodies produced by these B cell subsets will further define the determinants of antibody-mediated protection. Application of these findings will benefit the endeavor to combat the burden of FV. Better understanding of the structural-functional relationship between viral epitopes and neutralizing antibodies will create avenues for antibody-based therapies as well as vaccine design. Leveraging virus-specific antibodies induced by primary exposures will improve the specificity of DENV or ZIKV diagnosis to differentiate infection by each of these viral species or serotypes. Accurate diagnoses will lead to proper treatments to improve disease outcomes. Furthermore, accurate diagnoses will aid in sero-surveillance efforts to ultimately inform vaccine strategies for endemic populations

ZIKA VIRUS PATHOGENESIS AT SITES OF VECTOR-INDEPENDENT TRANSMISSION AND A FORAY INTO APPLIED SEROPREVALENCE

Zika virus (ZIKV) recently emerged in 2016 spread by mosquito-borne, sexual, and congenital transmission. During the course of this work, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also emerged and has been one of the most significant causes of mortality in 2020 as a result of the disease it causes, coronavirus disease 2019 (COVID-19). Because ZIKV causes placental damage, I set out to establish in collaboration with Tulika Singh in Dr. Sallie Permar’s laboratory whether antibody transfer is preserved during gestational ZIKV infection. I found that approximately half of our small 20-person cohort had serologic evidence of ZIKV infection during gestation and that ZIKV infection does not impair transfer of ZIKV and DENV-neutralizing antibodies. One individual had prolonged infection during gestation, and I showed that an IgM isolated from this individual potently neutralizes ZIKV and can protect mice from severe disease. I also investigated ZIKV replication in the vagina of wild-type (WT) mice. Typically, ZIKV infections in the laboratory are performed in mice deficient in IFN-αβ signaling because these mice are permissive to disseminated infection following inoculation by footpad. I found that ZIKV infection in the vagina is regulated by progesterone but not IFN-αβ signaling. I also found that progesterone-induced susceptibility to ZIKV is unlikely to be due to decreased epithelial integrity, changes in leukocyte numbers in vaginal tissue, or dampened antiviral signaling in the vagina. Finally, I coordinated a multi-site effort to measure SARS-CoV-2 seroprevalence over a 6-month time frame. We found that Latinx and underinsured individuals were at greatest risk of infection. Also, SARS-CoV-2 neutralizing antibody titers for individuals without respiratory symptoms were lower than for those with symptoms or COVID-19 diagnosis, supporting that asymptomatic infections may result in lower neutralizing titers.My results support that transplacental antibody transfer is not solely a result of placental damage. They also support that progesterone regulates some aspect of viral susceptibility independent of the interferon response. My results also emphasize that those at risk of systemic racism also may be at greatest risk of infection of emerging viruses that are driven by transmission in close quarters.Doctor of Philosoph