Kuan Rong Chan

Ligation of Fc gamma receptor IIB inhibits antibody-dependent enhancement of dengue virus infection

The interaction of antibodies, dengue virus (DENV), and monocytes can result in either immunity or enhanced virus infection. These opposing outcomes of dengue antibodies have hampered dengue vaccine development. Recent studies have shown that antibodies neutralize DENV by either preventing virus attachment to cellular receptors or inhibiting viral fusion intracellularly. However, whether the antibody blocks attachment or fusion, the resulting immune complexes are expected to be phagocytosed by Fc gamma receptor (FcγR)-bearing cells and cleared from circulation. This suggests that only antibodies that are able to block fusion intracellularly would be able to neutralize DENV upon FcγR-mediated uptake by monocytes whereas other antibodies would have resulted in enhancement of DENV replication. Using convalescent sera from dengue patients, we observed that neutralization of the homologous serotypes occurred despite FcγR-mediated uptake. However, FcγR-mediated uptake appeared to be inhibited when neutralized heterologous DENV serotypes were used instead. We demonstrate that this inhibition occurred through the formation of viral aggregates by antibodies in a concentration-dependent manner. Aggregation of viruses enabled antibodies to cross-link the inhibitory FcγRIIB, which is expressed at low levels but which inhibits FcγR-mediated phagocytosis and hence prevents antibody-dependent enhancement of DENV infection in monocytes.

Leukocyte immunoglobulin-like receptor B1 is critical for antibody-dependent dengue

Significance Dengue virus (DENV) infects almost 400 million people annually and some of these infections result in life threatening disease. An incomplete understanding of pathogenesis, particularly on how non- or subneutralizing levels of antibody augments DENV infection of cells expressing Fc-gamma receptors (FcγRs), has hampered vaccine development. Here, we show that, to overcome the activating FcγR-dependent expression of type-I interferon stimulated genes (ISGs), DENV binds and activates the inhibitory receptor, leukocyte immunoglobulin-like receptor-B1 (LILRB1). LILRB1 signals through its immunoreceptor tyrosine-based inhibition motif cytoplasmic tail to inhibit the expression of ISGs required for successful antibody-dependent DENV infection. Inhibition of DENV activation of LILRB1 could hence be a strategy for vaccine or therapeutic design. Viruses must evade the host innate defenses for replication and dengue is no exception. During secondary infection with a heterologous dengue virus (DENV) serotype, DENV is opsonized with sub- or nonneutralizing antibodies that enhance infection of monocytes, macrophages, and dendritic cells via the Fc-gamma receptor (FcγR), a process termed antibody-dependent enhancement of DENV infection. However, this enhancement of DENV infection is curious as cross-linking of activating FcγRs signals an early antiviral response by inducing the type-I IFN-stimulated genes (ISGs). Entry through activating FcγR would thus place DENV in an intracellular environment unfavorable for enhanced replication. Here we demonstrate that, to escape this antiviral response, antibody-opsonized DENV coligates leukocyte Ig-like receptor-B1 (LILRB1) to inhibit FcγR signaling for ISG expression. This immunoreceptor tyrosine-based inhibition motif-bearing receptor recruits Src homology phosphatase-1 to dephosphorylate spleen tyrosine kinase (Syk). As Syk is a key intermediate of FcγR signaling, LILRB1 coligation resulted in reduced ISG expression for enhanced DENV replication. Our findings suggest a unique mechanism for DENV to evade an early antiviral response for enhanced infection.

Dengue Virus Neutralization in Cells Expressing Fc Gamma Receptors

Activating Fc gamma receptors (FcγRs) in hematopoietic cells serve to remove antibody-opsonized antigens, including dengue virus (DENV), from systemic circulation. While neutralizing antibody concentrations provide humoral immunity, cross-reactive or sub-neutralizing levels of antibody can result in antibody-dependent enhancement of DENV infection that increases overall viral burden. Recently, it has been suggested that the antibody levels needed for DENV neutralization differs when different FcγR is engaged. If this is true, the threshold titer used to infer immunity should be influenced by FcγR usage. Here, using cells that express both activating and inhibitory FcγRs, we show that the type of FcγR engaged during phagocytosis can influence the antibody concentration requirement for DENV neutralization. We demonstrate that phagocytosis through FcγRI requires significantly less antibody for complete DENV neutralization compared to FcγRIIA. Furthermore, when DENV is opsonized with sub-neutralizing levels of antibody, FcγRI-mediated phagocytosis resulted in significantly reduced DENV titers compared to FcγRIIA. However, while FcγRI may remove antibody-opsonized DENV more efficiently, this receptor is only preferentially engaged by clustering when neutralizing, but not sub-neutralizing antibody concentrations, were used. Collectively, our study demonstrates that activating FcγR usage may influence antibody titers needed for DENV neutralization.

Dengue E Protein Domain III-Based DNA Immunisation Induces Strong Antibody Responses to All Four Viral Serotypes.

Dengue virus (DENV) infection is a major emerging disease widely distributed throughout the tropical and subtropical regions of the world affecting several millions of people. Despite constants efforts, no specific treatment or effective vaccine is yet available. Here we show a novel design of a DNA immunisation strategy that resulted in the induction of strong antibody responses with high neutralisation titres in mice against all four viral serotypes. The immunogenic molecule is an engineered version of the domain III (DIII) of the virus E protein fused to the dimerising CH3 domain of the IgG immunoglobulin H chain. The DIII sequences were also codon-optimised for expression in mammalian cells. While DIII alone is very poorly secreted, the codon-optimised fusion protein is rightly expressed, folded and secreted at high levels, thus inducing strong antibody responses. Mice were immunised using gene-gun technology, an efficient way of intradermal delivery of the plasmid DNA, and the vaccine was able to induce neutralising titres against all serotypes. Additionally, all sera showed reactivity to a recombinant DIII version and the recombinant E protein produced and secreted from mammalian cells in a mono-biotinylated form when tested in a conformational ELISA. Sera were also highly reactive to infective viral particles in a virus-capture ELISA and specific for each serotype as revealed by the low cross-reactive and cross-neutralising activities. The serotype specific sera did not induce antibody dependent enhancement of infection (ADE) in non-homologous virus serotypes. A tetravalent immunisation protocol in mice showed induction of neutralising antibodies against all four dengue serotypes as well.

Cross-reactive antibodies enhance live attenuated virus infection for increased immunogenicity.

Vaccination has achieved remarkable successes in the control of childhood viral diseases. To control emerging infections, however, vaccines will need to be delivered to older individuals who, unlike infants, probably have had prior infection or vaccination with related viruses and thus have cross-reactive antibodies against the vaccines. Whether and how these cross-reactive antibodies impact live attenuated vaccination efficacy is unclear. Using an open-label randomized trial design, we show that subjects with a specific range of cross-reactive antibody titres from a prior inactivated Japanese encephalitis vaccination enhanced yellow fever (YF) immunogenicity upon YF vaccination. Enhancing titres of cross-reactive antibodies prolonged YF vaccine viraemia, provoked greater pro-inflammatory responses, and induced adhesion molecules intrinsic to the activating Fc-receptor signalling pathway, namely immune semaphorins, facilitating immune cell interactions and trafficking. Our findings clinically demonstrate antibody-enhanced infection and suggest that vaccine efficacy could be improved by exploiting cross-reactive antibodies.

Fc receptors and their influence on efficacy of therapeutic antibodies for treatment of viral diseases

The lack of vaccines against several important viral diseases necessitates the development of therapeutics to save lives and control epidemics. In recent years, therapeutic antibodies have received considerable attention due to their good safety profiles and clinical success when used against viruses such as respiratory syncytial virus, Ebola virus and Hendra virus. The binding affinity of these antibodies can directly impact their therapeutic efficacy. However, we and others have also demonstrated that the subtype of Fc-gamma receptors (FcγRs) engaged influences the stoichiometric requirement for virus neutralization. Hence, the development of therapeutic antibodies against infectious diseases should consider the FcγRs engaged and Fc-effector functions involved. This review highlights the current state of knowledge about FcγRs and FcγR effector functions involved in virus neutralization, with emphasis on factors that can affect FcγR engagement. A better understanding of Fc-FcγR interactions during virus neutralization will allow development of therapeutic antibodies that are efficacious and can be administered with minimal side effects.

Therapeutic antibodies as a treatment option for dengue fever

Dengue fever is the most prevalent mosquito-borne viral disease globally with about 100 million cases of acute dengue annually. Severe dengue infection can result in a life-threatening illness. In the absence of either a licensed vaccine or antiviral drug against dengue, therapeutic antibodies that neutralize dengue virus (DENV) may serve as an effective medical countermeasure against severe dengue. However, therapeutic antibodies would need to effectively neutralize all four DENV serotypes. It must not induce antibody-dependent enhancement of DENV infection in monocytes/macrophages through Fc gamma receptor (FcγR)-mediated phagocytosis, which is hypothesized to increase the risk of severe dengue. Here, we review the strategies and technologies that can be adopted to develop antibodies for therapeutic applications. We also discuss the mechanism of antibody neutralization in the cells targeted by DENV that express Fc gamma receptor. These studies have provided significant insight toward the use of therapeutic antibodies as a potentially promising bulwark against dengue.

Neutralization of dengue virus in the presence of Fc receptor-mediated phagocytosis distinguishes serotype-specific from cross-neutralizing antibodies.

Although several vaccine candidates are presently in various phases of clinical trials, the field still lacks an effective tool to determine protective immunity. The presence of cross-neutralizing antibodies limits a serological approach to identify the etiology and distinguish lifelong from short-lived humoral protection. A recent study indicated that cross-reactive but not serotype-specific antibodies require high antibody concentration to co-ligate FcγRIIB and inhibit infection. Here, we tested if these differences could allow us to distinguish serotype-specific from cross-neutralizing antibodies. Using 30 blinded early convalescent serum samples from patients with virologically confirmed dengue, we demonstrate that neutralization in the presence of FcγR-mediated phagocytosis in THP-1 correctly identifies the DENV serotype of the infection in 93.3% of the cases compared to 76.7% with plaque reduction neutralization test. Our findings could provide a new approach for evaluating DENV neutralization and suggest that in addition to blocking specific ligand-receptor interactions for viral entry, antibodies must prevent viral uncoating during FcγR-mediated phagocytosis for complete humoral protection.

A systematic approach to the development of a safe live attenuated Zika vaccine

Zika virus (ZIKV) is a flavivirus that can cause congenital disease and requires development of an effective long-term preventative strategy. A replicative ZIKV vaccine with properties similar to the yellow fever 17D (YF17D) live-attenuated vaccine (LAV) would be advantageous, as a single dose of YF17D produces lifelong immunity. However, a replicative ZIKV vaccine must also be safe from causing persistent organ infections. Here we report an approach to ZIKV LAV development. We identify a ZIKV variant that produces small plaques due to interferon (IFN)-restricted viral propagation and displays attenuated infection of endothelial cells. We show that these properties collectively reduce the risk of organ infections and vertical transmission in a mouse model but remain sufficiently immunogenic to prevent wild-type ZIKV infection. Our findings suggest a strategy for the development of a safe but efficacious ZIKV LAV.A Zika virus (ZIKV) vaccine should provide long-lasting immunity, which may be achieved with a live-attenuated vaccine. Here, Kwek et al. select an interferon-restricted, attenuated ZIKV variant and evaluate replication and immunogenicity in mouse and mosquito models.

Viral Manipulation of Host Inhibitory Receptor Signaling for Immune Evasion.

The immune system has evolved pairs of activating and inhibitory receptors that modulate the magnitude of immune responses, enabling the maintenance of immune homeostasis. Inhibitory signaling dampens the immune response, which prevents inflammatory damage to the host. It has now become increasingly clear that viruses have evolved means of exploiting the inhibitory signaling pathways of the immune system in order to blunt the responses that would otherwise abrogate infection. Recent evidence demonstrates how viruses exploit inhibitory receptors both for host cell entry and to down-regulate antiviral responses for enhanced viral pathogenesis. Both acute and chronic viral infections also induce expression of intermediates of inhibitory signaling for improved odds of survival within the intracellular environment. This review highlights and synthesizes from recent findings how medically important viruses exploit the inhibitory pathways that maintain immune homeostasis for successful human infection.