Covalently linked dengue virus envelope glycoprotein dimers reduce exposure of the immunodominant fusion loop epitope

A problem in the search for an efficient vaccine against dengue virus is the immunodominance of the fusion loop epitope (FLE), a segment of the envelope protein E that is buried at the interface of the E dimers coating mature viral particles. Anti-FLE antibodies are broadly cross-reactive but poorly neutralizing, displaying a strong infection enhancing potential. FLE exposure takes place via dynamic “breathing” of E dimers at the virion surface. In contrast, antibodies targeting the E dimer epitope (EDE), readily exposed at the E dimer interface over the region of the conserved fusion loop, are very potent and broadly neutralizing. We have engineered E dimers locked by inter-subunit disulphide bonds, and show here by X-ray crystallography and by binding to a panel of human antibodies that these engineered dimers do not expose the FLE while retaining the EDE exposure. These locked dimers are strong immunogen candidates for a next-generation vaccin

Structural analysis of a dengue cross-reactive antibody complexed with envelope domain III reveals the molecular basis of cross-reactivity.

Dengue virus infections are still increasing at an alarming rate in tropical and subtropical countries, underlying the need for a dengue vaccine. Although it is relatively easy to generate Ab responses to dengue virus, low avidity or low concentrations of Ab may enhance infection of FcR-bearing cells with clinical impact, posing a challenge to vaccine production. In this article, we report the characterization of a mAb, 2H12, which is cross-reactive to all four serotypes in the dengue virus group. Crystal structures of 2H12-Fab in complex with domain III of the envelope protein from three dengue serotypes have been determined. 2H12 binds to the highly conserved AB loop of domain III of the envelope protein that is poorly accessible in the mature virion. 2H12 neutralization varied between dengue serotypes and strains; in particular, dengue serotype 2 was not neutralized. Because the 2H12-binding epitope was conserved, this variation in neutralization highlights differences between dengue serotypes and suggests that significant conformational changes in the virus must take place for Ab binding. Surprisingly, 2H12 facilitated little or no enhancement of infection. These data provide a structural basis for understanding Ab neutralization and enhancement of infection, which is crucial for the development of future dengue vaccines

A protective Zika virus E-dimer-based subunit vaccine engineered to abrogate antibody-dependent enhancement of dengue infection

Infections with dengue (DENV) and Zika (ZIKV) viruses can induce cross-reactive antibody responses. Two immunodominant epitopes, to precursor membrane protein (prM) or the fusion loop epitope (FLE) on envelope (E) protein are recognized by cross-reactive antibodies1, 2, 3 that are not only poorly neutralizing, but can also promote increased viral replication and disease seerity via Fc-gamma receptor mediated infection of myeloid cells, a process termed antibody-dependent enhancement (ADE)1, 4, 5 . ADE is a significant concern for both ZIKV and DENV vaccines as the induction of poorly-neutralizing cross-reactive antibodies may prime an individual for ADE upon natural infection. In this report, we describe the design and production of covalently-stabilized ZIKV E-dimers, which lack prM and do not expose the immunodominant FLE. Immunization of mice with ZIKV E-dimers induces dimer-specific antibodies, which protected against ZIKV challenge during pregnancy. Importantly, the ZIKV E-dimer-induced response does not cross-react with DENV or induce ADE of DENV infection

Human antibodies to the dengue virus E-dimer epitope have therapeutic activity against Zika virus infection

The Zika virus (ZIKV) epidemic has resulted in congenital abnormalities in fetuses and neonates. Although some cross-reactive dengue virus (DENV)-specific antibodies can enhance ZIKV infection in mice, those recognizing the DENV E-dimer epitope (EDE) can neutralize ZIKV infection in cell culture. We evaluated the therapeutic activity of human monoclonal antibodies to DENV EDE for their ability to control ZIKV infection in the brains, testes, placentas, and fetuses of mice. A single dose of the EDE1-B10 antibody given 3 d after ZIKV infection protected against lethality, reduced ZIKV levels in brains and testes, and preserved sperm counts. In pregnant mice, wild-type or engineered LALA variants of EDE1-B10, which cannot engage Fcg receptors, diminished ZIKV burden in maternal and fetal tissues, and protected against fetal demise. Because neutralizing antibodies to EDE have therapeutic potential against ZIKV, in addition to their established inhibitory effects against DENV, it may be possible to develop therapies that control disease caused by both viruses

Characterization of a potent and highly unusual minimally enhancing antibody directed against dengue virus

Dengue virus is a major pathogen and severe infections can lead to life threatening dengue hemorrhagic fever (DHF). Dengue exists as four serotypes and DHF is often associated with secondary heterologous infections. Antibody dependent enhancement (ADE) may drive higher virus loads in these secondary infections, and is purported to result from antibodies that recognize dengue but fail to fully neutralize. We have characterized two antibodies, 2C8 and 3H5, which bind to the envelope protein. 3H5 is highly unusual as it is both potently neutralizing, but promotes little if any ADE, whereas 2C8 has strong capacity to promote ADE. We show that 3H5 shows resilient binding in endosomal pH conditions and neutralizes at low occupancy. Immune complexes of 3H5 and dengue virus do not efficiently interact with Fcγ receptors, which we propose is due to the binding mode of 3H5 and which constitutes the primary mechanism of how ADE is avoided

Characterization of a potent and highly unusual minimally enhancing antibody directed against dengue virus

Dengue virus is a major pathogen and severe infections can lead to life threatening dengue hemorrhagic fever (DHF). Dengue exists as four serotypes and DHF is often associated with secondary heterologous infections. Antibody dependent enhancement (ADE) may drive higher virus loads in these secondary infections, and is purported to result from antibodies that recognize dengue but fail to fully neutralize. We have characterized two antibodies, 2C8 and 3H5, which bind to the envelope protein. 3H5 is highly unusual as it is both potently neutralizing, but promotes little if any ADE, whereas 2C8 has strong capacity to promote ADE. We show that 3H5 shows resilient binding in endosomal pH conditions and neutralizes at low occupancy. Immune complexes of 3H5 and dengue virus do not efficiently interact with Fcγ receptors, which we propose is due to the binding mode of 3H5 and which constitutes the primary mechanism of how ADE is avoided

The epitope arrangement on flavivirus particles contributes to Mab C10’s extraordinary neutralization breadth across Zika and dengue viruses

The human monoclonal antibody C10 exhibits extraordinary cross-reactivity, potently neutralizing Zika virus (ZIKV) and the four serotypes of dengue virus (DENV1–DENV4). Here we describe a comparative structure-function analysis of C10 bound to the envelope (E) protein dimers of the five viruses it neutralizes. We demonstrate that the C10 Fab has high affinity for ZIKV and DENV1 but not for DENV2, DENV3, and DENV4. We further show that the C10 interaction with the latter viruses requires an E protein conformational landscape that limits binding to only one of the three independent epitopes per virion. This limited affinity is nevertheless counterbalanced by the particle’s icosahedral organization, which allows two different dimers to be reached by both Fab arms of a C10 immunoglobulin. The epitopes’ geometric distribution thus confers C10 its exceptional neutralization breadth. Our results highlight the importance not only of paratope/epitope complementarity but also the topological distribution for epitope-focused vaccine design

Epstein-Barr virus genome deletions in Epstein-Barr virus-positive T/NK cell lymphoproliferative diseases

The main target cells for Epstein-Barr virus (EBV) infection and persistence are B lymphocytes, although T and NK cells can also become infected. In this paper we characterise the EBV present in 21 pediatric and adult patients treated in France for a range of diseases that involve infection of T or NK cells. Of these 21 cases, 5 pediatric patients (21%) and 11 adult patients (52%) are of Caucasian origin. In about 30% of the cases, some of the EBV genomes contain a large deletion. The deletions are different in every patient but tend to cluster near the BART region of the viral genome. Detailed investigation of a family, in which several members have persistent T or NK cell infection by EBV, indicates that the virus genome deletions arise or are selected independently in each individual patient. Genome sequence polymorphisms in the EBV in these T or NK cell diseases reflect the geographic origin of the patient, not a distinct type of EBV (the 21 cases studied included examples of both type 1 and type 2 EBV infection). Using virus produced from type 1 or type 2 EBV genomes cloned in bacterial artificial chromosome (BAC) vectors, we demonstrate infection of T cells in cord blood from healthy donors. Our results are consistent with transient infection of some T cells being part of normal asymptomatic infection by EBV in young children

A new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus

Dengue is a rapidly emerging, mosquito-borne viral infection, with an estimated 400 million infections occurring annually. To gain insight into dengue immunity, we characterized 145 human monoclonal antibodies (mAbs) and identified a previously unknown epitope, the envelope dimer epitope (EDE), that bridges two envelope protein subunits that make up the 90 repeating dimers on the mature virion. The mAbs to EDE were broadly reactive across the dengue serocomplex and fully neutralized virus produced in either insect cells or primary human cells, with 50% neutralization in the low picomolar range. Our results provide a path to a subunit vaccine against dengue virus and have implications for the design and monitoring of future vaccine trials in which the induction of antibody to the EDE should be prioritized.</p