Across Functional Boundaries: Making Nonneutralizing Antibodies To Neutralize HIV-1 and Mediate Fc-Mediated Effector Killing of Infected Cells

Highly conserved epitopes within the coreceptor binding site (CoRBS) and constant region 1 and 2 (C1-C2 or cluster A) are only available for antibody recognition after the HIV-1 Env trimer binds host cell CD4; therefore, they are not accessible on virions and infected cells, where the expression of CD4 is downregulated. Here, we have developed new antibody fusion molecules in which domains 1 and 2 of soluble human CD4 are linked with monoclonal antibodies of either the CoRBS or cluster A specificity.

Functional analysis of a monoclonal antibody reactive against the C1C2 of Env obtained from a patient infected with HIV-1 CRF02_AG

Background Recent data suggest the importance of non-neutralizing antibodies (nnAbs) in the development of vaccines against HIV-1 because two types of nnAbs that recognize the coreceptor binding site (CoRBS) and the C1C2 region mediate antibody-dependent cellular-cytotoxicity (ADCC) against HIV-1-infected cells. However, many studies have been conducted with nnAbs obtained from subtype B-infected individuals, with few studies in patients with non-subtype B infections. Results We isolated a monoclonal antibody 1E5 from a CRF02_AG-infected individual and constructed two forms of antibody with constant regions of IgG1 or IgG3. The epitope of 1E5 belongs to the C1C2 of gp120, and 1E5 binds to 27 out of 35 strains (77 %) across the subtypes. The 1E5 showed strong ADCC activity, especially in the form of IgG3 in the presence of small CD4-mimetic compounds (CD4mc) and 4E9C (anti-CoRBS antibody), but did not show any neutralizing activity even against the isolates with strong binding activities. The enhancement in the binding of A32, anti-C1C2 antibody isolated from a patient with subtype B infection, was observed in the presence of 1E5 and the combination of 1E5, A32 and 4E9C mediated a strong ADCC activity. Conclusions These results suggest that anti-C1C2 antibodies that are induced in patients with different HIV-1 subtype infections have common functional modality and may have unexpected interactions. These data may have implications for vaccine development against HIV-1. Graphical abstract

Elicitation of Cluster A and Co-Receptor Binding Site Antibodies Are Required to Eliminate HIV-1 Infected Cells

HIV-1-infected individuals raise a polyclonal antibody response targeting multiple envelope glycoprotein (Env) epitopes. Interestingly, two classes of non-neutralizing CD4-induced (CD4i) antibodies, present in the majority of HIV-1-infected individuals have been described to mediate antibody-dependent cellular cytotoxicity (ADCC) in the presence of small CD4 mimetic compounds (CD4mc). These antibodies recognize the coreceptor binding site (CoRBS) and the constant region one and two (C1C2 or inner domain cluster A) of the gp120. In combination with CD4mc they have been shown to stabilize an antibody-vulnerable Env conformation, known as State 2A. Here we evaluated the importance of these two families of Abs in ADCC responses by immunizing guinea pigs with gp120 immunogens that have been modified to elicit or not these types of antibodies. Underlying the importance of anti-CoRBS and anti-cluster A Abs in stabilizing State 2A, ADCC responses were only observed in the presence of these two types of CD4i antibodies. Altogether, our results suggest that these two families of CD4i antibodies must be taken into account when considering future strategies relying on the use of CD4mc to eliminate HIV-1-infected cells in vivo.

Exposing HIV-1 Env: Implications for therapeutic strategies

The human immunodeficiency virus (HIV-1) envelope glycoprotein trimer (Env) is exposed on the surfaces of both virions and infected cells. Thus, Env is the principal target for neutralizing antibodies and antibodies able to mediate antibodydependent cellular cytotoxicity (ADCC). The HIV-1 Env is a flexible molecule known to exist in at least three different conformational states: states 1, 2 and 3. Before interacting with the primary receptor, CD4, Env preferentially adopts a compact, “closed” conformation (state 1) that is largely antibody-resistant. The CD4 binding “opens” Env increasing the vulnerability of infected cells to ADCC mediated by non-neutralizing antibodies, as these easily-elicited antibodies preferentially recognize epitopes exposed in the open conformational states (states 2/3). These antibodies include the anti-coreceptor binding site and the anti-cluster A families of antibodies that, in combination with small CD4-mimetic compounds, stabilize a new asymmetric Env conformation (state 2A) that is vulnerable to ADCC. Approaches aimed at stabilizing this “open” conformation represent new interventional approaches to fight HIV-1 infection.

High-resolution cryo-EM structures of outbreak strain human norovirus shells reveal size variations

Noroviruses are a leading cause of foodborne illnesses worldwide. Although GII.4 strains have been responsible for most norovirus outbreaks, the assembled virus shell structures have been available in detail for only a single strain (GI.1). We present high-resolution (2.6- to 4.1-Å) cryoelectron microscopy (cryo-EM) structures of GII.4, GII.2, GI.7, and GI.1 human norovirus outbreak strain virus-like particles (VLPs). Although norovirus VLPs have been thought to exist in a single-sized assembly, our structures reveal polymorphism between and within genogroups, with small, medium, and large particle sizes observed. Using asymmetric reconstruction, we were able to resolve a Zn2+metal ion adjacent to the coreceptor binding site, which affected the structural stability of the shell. Our structures serve as valuable templates for facilitating vaccine formulations.

Two Families of Env Antibodies Efficiently Engage Fc-Gamma Receptors and Eliminate HIV-1-Infected Cells

ABSTRACTHIV-1 conceals epitopes of its envelope glycoproteins (Env) recognized by antibody (Ab)-dependent cellular cytotoxicity (ADCC)-mediating antibodies. These Abs, including anti-coreceptor binding site (CoRBS) and anti-cluster A antibodies, preferentially recognize Env in its “open” conformation. The binding of anti-CoRBS Abs has been shown to induce conformational changes that further open Env, allowing interaction of anti-cluster A antibodies. We explored the possibility that CoRBS Abs synergize with anti-cluster A Abs to engage Fc-gamma receptors to mediate ADCC. We found that binding of anti-CoRBS and anti-cluster A Abs to the same gp120 is required for interaction with soluble dimeric FcγRIIIa in enzyme-linked immunosorbent assays (ELISAs). We also found that Fc regions of both Abs are required to optimally engage FcγRIIIa and mediate robust ADCC. Taken together, our results indicate that these two families of Abs act together in a sequential and synergistic fashion to promote FcγRIIIa engagement and ADCC.IMPORTANCEThe “open” CD4-bound conformation of HIV-1 envelope glycoproteins is the primary target of antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies present in HIV-positive (HIV+) sera, such as anti-coreceptor binding site and anti-cluster A antibodies. Here we report that the binding of these two families of antibodies is required to engage FcγRIIIa and mediate ADCC.

Structure of Simian Immunodeficiency Virus Envelope Spikes Bound with CD4 and Monoclonal Antibody 36D5

ABSTRACT The human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) envelope spike (Env) mediates viral entry into host cells. The V3 loop of the gp120 component of the Env trimer contributes to the coreceptor binding site and is a target for neutralizing antibodies. We used cryo-electron tomography to visualize the binding of CD4 and the V3 loop monoclonal antibody (MAb) 36D5 to gp120 of the SIV Env trimer. Our results show that 36D5 binds gp120 at the base of the V3 loop and suggest that the antibody exerts its neutralization effect by blocking the coreceptor binding site. The antibody does this without altering the dynamics of the spike motion between closed and open states when CD4 is bound. The interaction between 36D5 and SIV gp120 is similar to the interaction between some broadly neutralizing anti-V3 loop antibodies and HIV-1 gp120. Two conformations of gp120 bound with CD4 are revealed, suggesting an intrinsic dynamic nature of the liganded Env trimer. CD4 binding substantially increases the binding of 36D5 to gp120 in the intact Env trimer, consistent with CD4-induced changes in the conformation of gp120 and the antibody binding site. Binding by MAb 36D5 does not substantially alter the proportions of the two CD4-bound conformations. The position of MAb 36D5 at the V3 base changes little between conformations, indicating that the V3 base serves as a pivot point during the transition between these two states. IMPORTANCE Glycoprotein spikes on the surfaces of SIV and HIV are the sole targets available to the immune system for antibody neutralization. Spikes evade the immune system by a combination of a thick layer of polysaccharide on the surface (the glycan shield) and movement between spike domains that masks the epitope conformation. Using SIV virions whose spikes were “decorated” with the primary cellular receptor (CD4) and an antibody (36D5) at part of the coreceptor binding site, we visualized multiple conformations trapped by the rapid freezing step, which were separated using statistical analysis. Our results show that the CD4-induced conformational dynamics of the spike enhances binding of the antibody.

Cryo-EM structure of a CD4-bound open HIV-1 envelope trimer reveals structural rearrangements of the gp120 V1V2 loop

The HIV-1 envelope (Env) glycoprotein, a trimer of gp120–gp41 heterodimers, relies on conformational flexibility to function in fusing the viral and host membranes. Fusion is achieved after gp120 binds to CD4, the HIV-1 receptor, and a coreceptor, capturing an open conformational state in which the fusion machinery on gp41 gains access to the target cell membrane. In the well-characterized closed Env conformation, the gp120 V1V2 loops interact at the apex of the Env trimer. Less is known about the structure of the open CD4-bound state, in which the V1V2 loops must rearrange and separate to allow access to the coreceptor binding site. We identified two anti–HIV-1 antibodies, the coreceptor mimicking antibody 17b and the gp120–gp41 interface-spanning antibody 8ANC195, that can be added as Fabs to a soluble native-like Env trimer to stabilize it in a CD4-bound conformation. Here, we present an 8.9-Å cryo-electron microscopy structure of a BG505 Env–sCD4–17b–8ANC195 complex, which reveals large structural rearrangements in gp120, but small changes in gp41, compared with closed Env structures. The gp120 protomers are rotated and separated in the CD4-bound structure, and the three V1V2 loops are displaced by ∼40 Å from their positions at the trimer apex in closed Env to the sides of the trimer in positions adjacent to, and interacting with, the three bound CD4s. These results are relevant to understanding CD4-induced conformational changes leading to coreceptor binding and fusion, and HIV-1 Env conformational dynamics, and describe a target structure relevant to drug design and vaccine efforts.

Soluble Envelope Glycoprotein Trimers from a CD4-Independent HIV-1 Elicit Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies in Guinea Pigs

CD4-independent HIV-1 variants can infect coreceptor-expressing cells lacking CD4. The envelope (Env) glycoproteins on these HIV-1 variants expose a coreceptor binding site that overlaps some CD4-induced (CD4i) epitopes. Reports have demonstrated that CD4i antibodies mediate antibody-dependent cellular cytotoxicity (ADCC). Here we investigated the immunogenicity of soluble Env trimers (sgp140) from a CD4-independent HIV-1 in guinea pigs and found that the sgp140 elicited ADCC-mediating antibodies. Therefore, these sgp140 might be useful in vaccine regimens aimed at eliciting ADCC responses.