Improving the immunogenicity of native-like HIV-1 envelope trimers by hyperstabilization

The production of native-like recombinant versions of the HIV-1 envelope glycoprotein (Env) trimer requires overcoming the natural flexibility and instability of the complex. The engineered BG505 SOSIP.664 trimer mimics the structure and antigenicity of native Env. Here, we describe how the introduction of new disulfide bonds between the glycoprotein (gp)120 and gp41 subunits of SOSIP trimers of the BG505 and other genotypes improves their stability and antigenicity, reduces their conformational flexibility, and helps maintain them in the unliganded conformation. The resulting next-generation SOSIP.v5 trimers induce strong autologous tier-2 neutralizing antibody (NAb) responses in rabbits. In addition, the BG505 SOSIP.v6 trimers induced weak heterologous NAb responses against a subset of tier-2 viruses that were not elicited by the prototype BG505 SOSIP.664. These stabilization methods can be applied to trimers from multiple genotypes as components of multivalent vaccines aimed at inducing broadly NAbs (bNAbs)

Immunoassay for quantification of antigen-specific IgG fucosylation

BACKGROUND: Immunoglobulin G (IgG) antibodies serve a crucial immuno-protective function mediated by IgG Fc receptors (FcγR). Absence of fucose on the highly conserved N-linked glycan in the IgG Fc domain strongly enhances IgG binding and activation of myeloid and natural killer (NK) cell FcγRs. Although afucosylated IgG can provide increased protection (malaria and HIV), it also boosts immunopathologies in alloimmune diseases, COVID-19 and dengue fever. Quantifying IgG fucosylation currently requires sophisticated methods such as liquid chromatography-mass spectrometry (LC-MS) and extensive analytical skills reserved to highly specialized laboratories. METHODS: Here, we introduce the Fucose-sensitive Enzyme-linked immunosorbent assay (ELISA) for Antigen-Specific IgG (FEASI), an immunoassay capable of simultaneously quantitating and qualitatively determining IgG responses. FEASI is a two-tier immunoassay; the first assay is used to quantify antigen-specific IgG (IgG ELISA), while the second gives FcγRIIIa binding-dependent readout which is highly sensitive to both the IgG quantity and the IgG Fc fucosylation (FcγR-IgG ELISA). FINDINGS: IgG Fc fucosylation levels, independently determined by LC-MS and FEASI, in COVID-19 responses to the spike (S) antigen, correlated very strongly by simple linear regression (R2=0.93, p < 0.0001). The FEASI method was then used to quantify IgG levels and fucosylation in COVID-19 convalescent plasma which was independently validated by LC-MS. INTERPRETATION: FEASI can be reliably implemented to measure relative and absolute IgG Fc fucosylation and quantify binding of antigen-specific IgG to FcγR in a high-throughput manner accessible to all diagnostic and research laboratories. FUNDING: This work was funded by the Stichting Sanquin Bloedvoorziening (PPOC 19-08 and SQI00041) and ZonMW 10430 01 201 0021

Antibody Conjugates for Targeted Therapy Against HIV-1 as an Emerging Tool for HIV-1 Cure

Although advances in antiretroviral therapy (ART) have significantly improved the life expectancy of people living with HIV-1 (PLWH) by suppressing HIV-1 replication, a cure for HIV/AIDS remains elusive. Recent findings of the emergence of drug resistance against various ART have resulted in an increased number of treatment failures, thus the development of novel strategies for HIV-1 cure is of immediate need. Antibody-based therapy is a well-established tool in the treatment of various diseases and the engineering of new antibody derivatives is expanding the realms of its application. An antibody-based carrier of anti-HIV-1 molecules, or antibody conjugates (ACs), could address the limitations of current HIV-1 ART by decreasing possible off-target effects, reduce toxicity, increasing the therapeutic index, and lowering production costs. Broadly neutralizing antibodies (bNAbs) with exceptional breadth and potency against HIV-1 are currently being explored to prevent or treat HIV-1 infection in the clinic. Moreover, bNAbs can be engineered to deliver cytotoxic or immune regulating molecules as ACs, further increasing its therapeutic potential for HIV-1 cure. ACs are currently an important component of anticancer treatment with several FDA-approved constructs, however, to date, no ACs are approved to treat viral infections. This review aims to outline the development of AC for HIV-1 cure, examine the variety of carriers and payloads used, and discuss the potential of ACs in the current HIV-1 cure landscape

The Ligands for Human IgG and Their Effector Functions

Activation of the humoral immune system is initiated when antibodies recognize an antigen and trigger effector functions through the interaction with Fc engaging molecules. The most abundant immunoglobulin isotype in serum is Immunoglobulin G (IgG), which is involved in many humoral immune responses, strongly interacting with effector molecules. The IgG subclass, allotype, and glycosylation pattern, among other factors, determine the interaction strength of the IgG-Fc domain with these Fc engaging molecules, and thereby the potential strength of their effector potential. The molecules responsible for the effector phase include the classical IgG-Fc receptors (Fc&#947;R), the neonatal Fc-receptor (FcRn), the Tripartite motif-containing protein 21 (TRIM21), the first component of the classical complement cascade (C1), and possibly, the Fc-receptor-like receptors (FcRL4/5). Here we provide an overview of the interactions of IgG with effector molecules and discuss how natural variation on the antibody and effector molecule side shapes the biological activities of antibodies. The increasing knowledge on the Fc-mediated effector functions of antibodies drives the development of better therapeutic antibodies for cancer immunotherapy or treatment of autoimmune diseases

The Ligands for Human IgG and Their Effector Functions

Activation of the humoral immune system is initiated when antibodies recognize an antigen and trigger effector functions through the interaction with Fc engaging molecules. The most abundant immunoglobulin isotype in serum is Immunoglobulin G (IgG), which is involved in many humoral immune responses, strongly interacting with effector molecules. The IgG subclass, allotype, and glycosylation pattern, among other factors, determine the interaction strength of the IgG-Fc domain with these Fc engaging molecules, and thereby the potential strength of their effector potential. The molecules responsible for the effector phase include the classical IgG-Fc receptors (FcγR), the neonatal Fc-receptor (FcRn), the Tripartite motif-containing protein 21 (TRIM21), the first component of the classical complement cascade (C1), and possibly, the Fc-receptor-like receptors (FcRL4/5). Here we provide an overview of the interactions of IgG with effector molecules and discuss how natural variation on the antibody and effector molecule side shapes the biological activities of antibodies. The increasing knowledge on the Fc-mediated effector functions of antibodies drives the development of better therapeutic antibodies for cancer immunotherapy or treatment of autoimmune diseases

Variable Domain N-Linked Glycans Acquired During Antigen-Specific Immune Responses Can Contribute to Immunoglobulin G Antibody Stability

Immunoglobulin G (IgG) can contain N-linked glycans in the variable domains, the so-called Fab glycans, in addition to the Fc glycans in the CH2 domains. These Fab glycans are acquired following introduction of N-glycosylation sites during somatic hypermutation and contribute to antibody diversification. We investigated whether Fab glycans may—in addition to affecting antigen binding—contribute to antibody stability. By analyzing thermal unfolding profiles of antibodies with or without Fab glycans, we demonstrate that introduction of Fab glycans can improve antibody stability. Strikingly, removal of Fab glycans naturally acquired during antigen-specific immune responses can deteriorate antibody stability, suggesting in vivo selection of stable, glycosylated antibodies. Collectively, our data show that variable domain N-linked glycans acquired during somatic hypermutation can contribute to IgG antibody stability. These findings indicate that introducing Fab glycans may represent a mechanism to improve therapeutic/diagnostic antibody stability

Complement evasion by Borrelia burgdorferi: it takes three to tango

The complement system is one of the major innate defense mechanisms Borrelia burgdorferi sensu lato has to overcome to establish an infection of mammalian hosts and to cause Lyme borreliosis in humans. Borrelia prevents complement-mediated killing during host colonization through (i) recruitment of host complement regulators by Borrelia, (ii) evasion mechanisms by Borrelia itself, and (iii) exploitation of tick proteins by Borrelia. These interactions with complement can be host species-specific. This review provides an overview of interactions between Borrelia, tick, and host leading to evasion of complement-mediated killin

HIV-1 anchor inhibitors and membrane fusion inhibitors target distinct but overlapping steps in virus entry

HIV-1 entry into cells is mediated by the envelope glycoprotein (Env) and represents an attractive target for therapeutic intervention. Two drugs that inhibit HIV entry are approved for clinical use: the membrane fusion-inhibitor T20 (Fuzeon, enfuvirtide) and the C-C chemokine receptor type 5 (CCR5) blocker maraviroc (Selzentry). Another class of entry inhibitors supposedly target the fusion peptide (FP) and are termed anchor inhibitors. These include the VIRIP peptide and VIRIP derivatives such as VIR165, VIR353, and VIR576. Here, we investigated the mechanism of inhibition by VIR165. We show that substitutions within the FP modulate sensitivity to VIR165, consistent with the FP being the drug target. Our results also revealed that VIR165 acts during an intermediate post-CD4 – binding entry step that is overlapping but not identical to the step inhibited by fusion inhibitors such as T20. We found that some but not all resistance mutations to heptad repeat 2 (HR2)-targeting fusion inhibitors can provide cross-resistance to VIR165. In contrast, resistance mutations in the HR1-binding site for the fusion inhibitors did not cause cross-resistance to VIR165. However, Env with mutations located outside this binding site and thought to affect fusion kinetics, exhibited decreased sensitivity to VIR165. Although we found a strong correlation between Env stability and resistance to HR2-based fusion inhibitors, such correlation was not observed for Env stability and VIR165 resistance. We conclude that VIRIP analogs target the FP during an intermediate, post-CD4 – binding entry step that overlaps with but is distinct from the step(s) inhibited by HR2-based fusion inhibitors

HIV-1 Escape from a Peptidic Anchor Inhibitor through Stabilization of the Envelope Glycoprotein Spike

The trimeric HIV-1 envelope glycoprotein spike (Env) mediates viral entry into cells by using a spring-loaded mechanism that allows for the controlled insertion of the Env fusion peptide into the target membrane, followed by membrane fusion. Env is the focus of vaccine research aimed at inducing protective immunity by antibodies as well as efforts to develop drugs that inhibit the viral entry process. The molecular factors contributing to Env stability and decay need to be understood better in order to optimally design vaccines and therapeutics. We generated viruses with resistance to VIR165, a peptidic inhibitor that binds the fusion peptide of the gp41 subunit and prevents its insertion into the target membrane. Interestingly, a number of escape viruses acquired substitutions in the C1 domain of the gp120 subunit (A60E, E64K, and H66R) that rendered these viruses dependent on the inhibitor. These viruses could infect target cells only when VIR165 was present after CD4 binding. Furthermore, the VIR165-dependent viruses were resistant to soluble CD4-induced Env destabilization and decay. These data suggest that VIR165-dependent Env proteins are kinetically trapped in the unliganded state and require the drug to negotiate CD4-induced conformational changes. These studies provide mechanistic insight into the action of the gp41 fusion peptide and its inhibitors and provide new ways to stabilize Env trimer vaccines. Because of the rapid development of HIV-1 drug resistance, new drug targets need to be explored continuously. The fusion peptide of the envelope glycoprotein can be targeted by anchor inhibitors. Here we describe virus escape from the anchor inhibitor VIR165. Interestingly, some escape viruses became dependent on the inhibitor for cell entry. We show that the identified escape mutations stabilize the ground state of the envelope glycoprotein and should thus be useful in the design of stabilized envelope-based HIV vaccine