Studies on the regulatory mechanisms of Fcγ receptor function

Receptors for immunoglobulins (Fc receptors) play a central role during an immune response, as they mediate the specific recognition of antigens of almost infinite diversity by leukocytes, thereby linking the innate with the adaptive branches of immunity. This thesis undertook a series of studies to further investigate the role of Fc receptors in innate immune responses, using in vitro approaches to examine functional activity and underlying regulatory mechanisms.FcγRIla (CD32a) is a member of the Fcγ receptor family and mediates binding of multivalent IgG. Although CD32a is expressed by a number of myeloid cell types, including neutrophils, macrophages, eosinophils and monocytes, we observed that ligand binding to this receptor was suppressed, as evidenced by the low levels of CD32a-mediated IgG binding to these cells. We therefore aimed to determine the mechanisms that account for the myeloid-specific suppression of CD32a ligand binding. A series of experiments were performed to examine the effects of proteases and sialylation of CD32a in the regulation of IgG binding. In addition, the role of association of CD32a with plasma membrane microdomains rich in cholesterol and sphingolipids in the regulation of IgG binding to CD32a was examined. These membrane microdomains, also termed lipid rafts, have previously been reported to be essential for efficient receptor signalling. Chemical disruption of lipid raft structure by depletion or sequestration of membrane cholesterol greatly inhibited CD32a-mediated IgG binding, strongly implicating lipid rafts in the control of CD32a function. To further investigate this suggestion, specific CD32a mutants were generated, which would be predicted to have altered association with lipid rafts. Both these mutants showed reduced association with lipid rafts (A224S and C241A) and displayed decreased levels of IgG binding compared with wild type CD32a. Additionally, we generated a chimaeric CD32a receptor containing a glycophosphatidylinositol (GPI] lipid anchor consensus sequence, which would constitutively associate with lipid rafts. GPI-anchored CD32a exhibited increased capacity for IgG binding compared with the full-length transmembrane CD32a. Our findings clearly suggest a major role for lipid rafts in the regulation of IgG binding and more specifically, that suppression of CD32a-mediated IgG binding in myeloid cells is achieved by receptor exclusion from lipid raft membrane microdomainsThis thesis also describes an investigation of the association of Fcγ receptor genetic variants with idiopathic pulmonary fibrosis (IPF) susceptibility and progression. In particular, two allelic variants of CD32a (H131/R131) and CD16 (NA1/NA2) were examined that confer altered IgG binding and may therefore contribute to disease pathogenesis. Copy number variation of the FCGR3B gene was also determined using a quantitative PCR-based approach. Susceptibility to IPF was found to be associated with the NA1 allele of FcγRIIIb and increased FCGR3B copy number. In addition, IPF disease severity at disease presentation and progression over a 1 2-month period following diagnosis was found to be linked to the FcγRIIa H131 variant. These results support the involvement of Fcγ-mediated interactions in IPF and reveal a novel role of Fcγ receptors in IPF disease pathogenesis and progression

A mouse model for HIV-1 entry

Passive transfer of neutralizing antibodies against HIV-1 can prevent infection in macaques and seems to delay HIV-1 rebound in humans. Anti-HIV antibodies are therefore of great interest for vaccine design. However, the basis for their in vivo activity has been difficult to evaluate systematically because of a paucity of small animal models for HIV infection. Here we report a genetically humanized mouse model that incorporates a luciferase reporter for rapid quantitation of HIV entry. An antibody’s ability to block viral entry in this in vivo model is a function of its bioavailability, direct neutralizing activity, and effector functions

Maternal Anti-Dengue IgG Fucosylation Predicts Susceptibility to Dengue Disease in Infants

Infant mortality from dengue disease is a devastating global health burden that could be minimized with the ability to identify susceptibility for severe disease prior to infection. Although most primary infant dengue infections are asymptomatic, maternally derived anti-dengue immunoglobulin G (IgGs) present during infection can trigger progression to severe disease through antibody-dependent enhancement mechanisms. Importantly, specific characteristics of maternal IgGs that herald progression to severe infant dengue are unknown. Here, we define \u3e /=10% afucosylation of maternal anti-dengue IgGs as a risk factor for susceptibility of infants to symptomatic dengue infections. Mechanistic experiments show that afucosylation of anti-dengue IgGs promotes FcgammaRIIIa signaling during infection, in turn enhancing dengue virus replication in FcgammaRIIIa(+) monocytes. These studies identify a post-translational modification of anti-dengue IgGs that correlates with risk for symptomatic infant dengue infections and define a mechanism by which afucosylated antibodies and FcgammaRIIIa enhance dengue infections

A Combination of Two Human Monoclonal Antibodies Limits Fetal Damage by Zika Virus in Macaques

Human infection by Zika virus (ZIKV) during pregnancy can lead to vertical transmission and fetal aberrations, including microcephaly. Prophylactic administration of antibodies can diminish or prevent ZIKV infection in animal models, but whether passive immunization can protect nonhuman primates and their fetuses during pregnancy has not been determined. Z004 and Z021 are neutralizing monoclonal antibodies to domain III of the envelope (EDIII) of ZIKV. Together the two antibodies protect nonpregnant macaques against infection even after Fc modifications to prevent antibody-dependent enhancement in vitro (ADE) and extend their half-lives. Here we report on prophylactic co-administration of the Fc-modified antibodies to pregnant rhesus macaques challenged 3 times with ZIKV during first and second trimester. The two antibodies did not entirely eliminate maternal viremia but limited vertical transmission protecting the fetus from neurologic damage. Thus, maternal passive immunization with two antibodies to EDIII can shield primate fetuses from the harmful effects of ZIKV

HIV therapy by a combination of broadly neutralizing antibodies in humanized mice

Human antibodies to human immunodeficiency virus-1 (HIV-1) can neutralize a broad range of viral isolates in vitro and protect non-human primates against infection. Previous work showed that antibodies exert selective pressure on the virus but escape variants emerge within a short period of time. However, these experiments were performed before the recent discovery of more potent anti-HIV-1 antibodies and their improvement by structure-based design. Here we re-examine passive antibody transfer as a therapeutic modality in HIV-1-infected humanized mice. Although HIV-1 can escape from antibody monotherapy, combinations of broadly neutralizing antibodies can effectively control HIV-1 infection and suppress viral load to levels below detection. Moreover, in contrast to antiretroviral therapy the longer half-life of antibodies led to control of viraemia for an average of 60 days after cessation of therapy. Thus, combinations of potent monoclonal antibodies can effectively control HIV-1 replication in humanized mice, and should be re-examined as a therapeutic modality in HIV-1-infected individuals

A Combination of Two Human Monoclonal Antibodies Limits Fetal Damage by Zika Virus in Macaques

Human infection by Zika virus (ZIKV) during pregnancy can lead to vertical transmission and fetal aberrations, including microcephaly. Prophylactic administration of antibodies can diminish or prevent ZIKV infection in animal models, but whether passive immunization can protect nonhuman primates and their fetuses during pregnancy has not been determined. Z004 and Z021 are neutralizing monoclonal antibodies to domain III of the envelope (EDIII) of ZIKV. Together the two antibodies protect nonpregnant macaques against infection even after Fc modifications to prevent antibody-dependent enhancement in vitro (ADE) and extend their half-lives. Here we report on prophylactic co-administration of the Fc-modified antibodies to pregnant rhesus macaques challenged 3 times with ZIKV during first and second trimester. The two antibodies did not entirely eliminate maternal viremia but limited vertical transmission protecting the fetus from neurologic damage. Thus, maternal passive immunization with two antibodies to EDIII can shield primate fetuses from the harmful effects of ZIKV

Association of FcγRIIa R131H polymorphism with idiopathic pulmonary fibrosis severity and progression

<p>Abstract</p> <p>Background</p> <p>A significant genetic component has been described for idiopathic pulmonary fibrosis (IPF). The R131H (rs1801274) polymorphism of the IgG receptor FcγRIIa determines receptor affinity for IgG subclasses and is associated with several chronic inflammatory diseases. We investigated whether this polymorphism is associated with IPF susceptibility or progression.</p> <p>Methods</p> <p>In a case-control study, we compared the distribution of FcγRIIa R131H genotypes in 142 patients with IPF and in 218 controls using allele-specific PCR amplification.</p> <p>Results</p> <p>No differences in the frequency of FcγRIIa genotypes were evident between IPF patients and control subjects. However, significantly impaired pulmonary function at diagnosis was observed in HH compared to RR homozygotes, with evidence of more severe restriction (reduced forced vital capacity (FVC)) and lower diffusing capacity for carbon monoxide (D<smcaps>L</smcaps>_CO). Similarly, increased frequency of the H131 allele was observed in patients with severe disease (D<smcaps>L</smcaps>_CO< 40% predicted) (0.53 vs. 0.38; p = 0.03). Furthermore, the H131 allele was associated with progressive pulmonary fibrosis as determined by > 10% drop in FVC and/or > 15% fall in D<smcaps>L</smcaps>_COat 12 months after baseline (0.48 vs. 0.33; p = 0.023).</p> <p>Conclusions</p> <p>These findings support an association between the FcγRIIa R131H polymorphism and IPF severity and progression, supporting the involvement of immunological mechanisms in IPF pathogenesis.</p