In vivo and in vitro escape from neutralizing antibodies 2G12, 2F5, and 4E10

Recently, passive immunization of human immunodeficiency virus (HIV)-infected individuals with monoclonal antibodies (MAbs) 2G12, 2F5, and 4E10 provided evidence of the in vivo activity of 2G12 but raised concerns about the function of the two membrane-proximal external region (MPER)-specific MAbs (A. Trkola, H. Kuster, P. Rusert, B. Joos, M. Fischer, C. Leemann, A. Manrique, M. Huber, M. Rehr, A. Oxenius, R. Weber, G. Stiegler, B. Vcelar, H. Katinger, L. Aceto, and H. F. Gunthard, Nat. Med. 11:615-622, 2005). In the light of MPER-targeting vaccines under development, we performed an in-depth analysis of the emergence of mutations conferring resistance to these three MAbs to further elucidate their activity. Clonal analysis of the MPER of plasma virus samples derived during antibody treatment confirmed that no changes in this region had occurred in vivo. Sequence analysis of the 2G12 epitope relevant N-glycosylation sites of viruses derived from 13 patients during the trial supported the phenotypic evaluation, demonstrating that mutations in these sites are associated with resistance. In vitro selection experiments with isolates of four of these individuals corroborated the in vivo finding that virus strains rapidly escape 2G12 pressure. Notably, in vitro resistance mutations differed, in most cases, from those found in vivo. Importantly, in vitro selection with 2F5 and 4E10 demonstrated that resistance to these MAbs can be difficult to achieve and can lead to selection of variants with impaired infectivity. This remarkable vulnerability of the virus to interference within the MPER calls for a further evaluation of the safety and efficacy of MPER-targeting therapeutic and vaccination strategies.     

Neutralization profiles of newly transmitted human immunodeficiency virus type 1 by monoclonal antibodies 2G12, 2F5, and 4E10

As the AIDS epidemic continues unabated, the development of a human immunodeficiency virus (HIV) vaccine is critical. Ideally, an effective vaccine should elicit cell-mediated and neutralizing humoral immune responses. We have determined the in vitro susceptibility profile of sexually transmitted viruses from 91 patients with acute and early HIV-1 infection to three monoclonal antibodies, 2G12, 2F5, and 4E10. Using a recombinant virus assay to measure neutralization, we found all transmitted viruses were neutralized by 4E10, 80% were neutralized by 2F5, and only 37% were neutralized by 2G12. We propose that the induction of 4E10-like antibodies should be a priority in designing immunogens to prevent HIV-1 infection.     

Estimating the Probability of Polyreactive Antibodies 4E10 and 2F5 Disabling a gp41 Trimer after T Cell-HIV Adhesion

A few broadly neutralizing antibodies, isolated from HIV-1 infected individuals, recognize epitopes in the membrane proximal external region (MPER) of gp41 that are transiently exposed during viral entry. The best characterized, 4E10 and 2F5, are polyreactive, binding to the viral membrane and their epitopes in the MPER. We present a model to calculate, for any antibody concentration, the probability that during the pre-hairpin intermediate, the transient period when the epitopes are first exposed, a bound antibody will disable a trivalent gp41 before fusion is complete. When 4E10 or 2F5 bind to the MPER, a conformational change is induced that results in a stably bound complex. The model predicts that for these antibodies to be effective at neutralization, the time to disable an epitope must be shorter than the time the antibody remains bound in this conformation, about five minutes or less for 4E10 and 2F5. We investigate the role of avidity in neutralization and show that 2F5 IgG, but not 4E10, is much more effective at neutralization than its Fab fragment. We attribute this to 2F5 interacting more stably than 4E10 with the viral membrane. We use the model to elucidate the parameters that determine the ability of these antibodies to disable epitopes and propose an extension of the model to analyze neutralization data. The extended model predicts the dependencies of for neutralization on the rate constants that characterize antibody binding, the rate of fusion of gp41, and the number of gp41 bridging the virus and target cell at the start of the pre-hairpin intermediate. Analysis of neutralization experiments indicate that only a small number of gp41 bridges must be disabled to prevent fusion. However, the model cannot determine the exact number from neutralization experiments alone.     

Localization of the HIV-1 gp120 Conformational Epitope Recognized by Virus-Neutralizing Monoclonal Antibodies 2G12

A phage peptide library was used to select peptides interacting with virus-neutralizing monoclonal antibodies (mAb) 2G12 which recognize a discontinuous surface epitope of HIV-1 gp120. With the published X-ray data, gp120 regions involved in the antigenic determinant were predicted. Binding with mAb 2G12 was ascribed to Thr297, Phe383, Tyr384, Arg419, Ile240, Thr415, Leu416, Pro417, Lys421, and Trp112. Though distant in the gp120 sequence, these residues are close in space and form the 2G12 epitope on the gp120 surface.     

Broadly Neutralizing Monoclonal Antibodies 2F5 and 4E10 Directed against the Human Immunodeficiency Virus Type 1 gp41 Membrane-Proximal External Region Protect against Mucosal Challenge by Simian-Human Immunodeficiency Virus SHIVBa-L

The membrane-proximal external region (MPER) of HIV-1, located at the C terminus of the gp41 ectodomain, is conserved and crucial for viral fusion. Three broadly neutralizing monoclonal antibodies (bnMAbs), 2F5, 4E10, and Z13e1, are directed against linear epitopes mapped to the MPER, making this conserved region an important potential vaccine target. However, no MPER antibodies have been definitively shown to provide protection against HIV challenge. Here, we show that both MAbs 2F5 and 4E10 can provide complete protection against mucosal simian-human immunodeficiency virus (SHIV) challenge in macaques. MAb 2F5 or 4E10 was administered intravenously at 50 mg/kg to groups of six male Indian rhesus macaques 1 day prior to and again 1 day following intrarectal challenge with SHIV_Ba-L. In both groups, five out of six animals showed complete protection and sterilizing immunity, while for one animal in each group a low level of viral replication following challenge could not be ruled out. The study confirms the protective potential of 2F5 and 4E10 and supports emphasis on HIV immunogen design based on the MPER region of gp41.Eliciting broadly neutralizing antibodies is an important goal of HIV vaccine design efforts, and the study of broadly neutralizing monoclonal antibodies (bnMAbs) can assist in that goal. Human bnMAbs against both gp120 and gp41 of the HIV-1 envelope spike have been described. Three bnMAbs to gp41, 2F5, 4E10, and Z13e1, have been identified and shown to recognize neighboring linear epitopes on the membrane proximal external (MPER) region of gp41 (3, 24, 25, 37, 47). In a comprehensive cross-clade neutralization study by Binley et al., 2F5 neutralized 67% and 4E10 neutralized 100% of a diverse panel of 90 primary isolates (2). Similar broad neutralization was seen against sexually transmitted isolates cloned from acutely infected patients (22). More recently, a comprehensive study showed that 2F5 neutralized 97 isolates from a 162-virus panel (60%) and that 4E10 neutralized 159 isolates (98%) (41). Although less potent, the monoclonal antibody Z13, isolated from an antibody phage display library derived from a bone marrow donor whose serum was broadly neutralizing (47), has cross-clade neutralizing activity. Z13e1 is an affinity-enhanced variant of the earlier-characterized MAb Z13 that is directed against an access-restricted epitope between and overlapping the epitopes of 2F5 and 4E10. Both MAbs 2F5 and 4E10 were originally obtained as IgG3 antibodies in hybridomas derived from peripheral blood mononuclear blood lymphocytes (PBMCs) of HIV-1-seropositive nonsymptomatic patients and were later class switched to IgG1 to enable large-scale manufacturing and to prolong in vivo half-life (3, 6, 32).Despite the interest in the MPER as a vaccine target, there is limited information on the ability of MPER antibodies to act antivirally in vivo either in established infection or prophylactically. A study using the huPBL-SCID mouse model showed limited impact from 2F5 when the antibody was administered in established infection (31). Passive administration of 2G12, 2F5, and 4E10 to a cohort of acutely and chronically infected HIV-1 patients provided little direct evidence of 2F5 or 4E10 antiviral activity, whereas the emergence of escape variants indicated unequivocally the ability of 2G12 to act antivirally (18, 39). Indirect evidence did, however, suggest that the MPER MAbs may have affected virus replication, as indicated by viral rebound suppression in a patient known to have a 2G12-resistant virus prior to passive immunization (39). Another study of 10 individuals passively administered 2G12, 2F5, and 4E10 before and after cessation of combination antiretroviral therapy (ART) showed similarly that 2G12 treatment could delay viral rebound, but antiviral activity by 2F5 and 4E10 was not clearly demonstrated (21). In prophylaxis, an early 2F5 passive transfer study with chimpanzees suggested that the antibody could delay or lower the magnitude of primary viremia following HIV-1 challenge (7). A study using gene transfer of 2F5 in a humanized SCID mouse model suggested that continuous plasma levels of approximately 1 μg/ml of 2F5 may significantly reduce viral loads in LAI- and MN-challenged mice (34). Protection studies of rhesus macaques using simian-human immunodeficiency virus SHIV_89.6PD challenge did not provide definitive direct evidence for MPER antibody-mediated protection. One of three animals was protected against intravenous (i.v.) challenge when 2F5 was administered in a cocktail with HIVIG and 2G12 (19), but all three animals treated with 2F5 alone at high concentration became infected. In a vaginal challenge study with SHIV_89.6PD (20), four of five animals were protected with a cocktail of HIVIG, 2F5, and 2G12, but a 2F5/2G12 combination protected only two of five animals. Further protection studies have used MPER MAbs in combination with other MAbs, leaving the individual contributions of these antibodies uncertain (1, 8).In our previous studies, we successfully used the SHIV/macaque model to demonstrate neutralizing antibody protection against mucosal challenge, and we have begun to explore how that protection is achieved (12, 30). Here, we conducted a protection study with the two broadly neutralizing MPER-directed antibodies 2F5 and 4E10. We show that the antibodies can prevent viral infection and thereby support the MPER as a vaccine target.     

Stable Docking of Neutralizing Human Immunodeficiency Virus Type 1 gp41 Membrane-Proximal External Region Monoclonal Antibodies 2F5 and 4E10 Is Dependent on the Membrane Immersion Depth of Their Epitope Regions

The binding of neutralizing antibodies 2F5 and 4E10 to human immunodeficiency virus type 1 (HIV-1) gp41 involves both the viral membrane and gp41 membrane proximal external region (MPER) epitopes. In this study, we have used several biophysical tools to examine the secondary structure, orientation, and depth of immersion of gp41 MPER peptides in liposomes and to determine how the orientation of the MPER with lipids affects the binding kinetics of monoclonal antibodies (MAbs) 2F5 and 4E10. The binding of 2F5 and 4E10 both to their respective nominal epitopes and to a biepitope (includes 2F5 and 4E10 epitopes) MPER peptide-liposome conjugate was best described by a two-step encounter-docking model. Analysis of the binding kinetics and the effect of temperature on the binding stability of 2F5 and 4E10 to MPER peptide-liposome conjugates revealed that the docking of 4E10 was relatively slower and thermodynamically less favorable. The results of fluorescence-quenching and fluorescence resonance energy transfer experiments showed that the 2F5 epitope was more solvent exposed, whereas the 4E10 epitope was immersed in the polar-apolar interfacial region of the lipid bilayer. A circular dichroism spectroscopic study demonstrated that the nominal epitope and biepitope MPER peptides adopted ordered structures with differing helical contents when anchored to liposomes. Furthermore, anchoring of MPER peptides to the membrane via a hydrophobic anchor sequence was required for efficient MAb docking. These results support the model that the ability of 2F5 and 4E10 to bind to membrane lipid is required for stable docking to membrane-embedded MPER residues. These data have important implications for the design and use of peptide-liposome conjugates as immunogens for the induction of MPER-neutralizing antibodies.The two broadly neutralizing human monoclonal antibodies (MAbs) 2F5 and 4E10 target conserved core amino acid residues that lie in the membrane proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) gp41 (6, 9, 18, 25, 29). Structural studies of 2F5 and 4E10 in complex with their nominal epitope peptides led to the proposition that the long hydrophobic heavy chain CDR3 (CDR H3) loop might be involved in binding to the virion membrane due to the lack of direct contact of the tip of the CDR H3 loop with their bound epitopes (6, 25). MAbs 2F5 and 4E10 indeed were found to have enhanced binding to gp41 MPER in the presence of membrane (12, 25). Subsequent studies have revealed the lipid reactivity of both the 2F5 and 4E10 MAbs (2, 14, 23, 27), emphasizing the need to understand how MAbs 2F5 and 4E10 recognize their epitopes in the context of a membrane-gp41 MPER interface.It has been hypothesized that the ability of MAbs 2F5 and 4E10 to interact with membrane lipids is required for binding to the membrane-bound gp41 MPER region and subsequent HIV-1 neutralization (2, 14, 15). The binding of both the 2F5 and 4E10 MAbs to their epitope peptides presented on synthetic liposomes was remarkably different from that of epitope peptides alone and was best described by a two-step “encounter-docking” model (2). In this model, neutralizing MPER MAbs make an initial encounter complex, and such an interaction is associated with faster association and dissociation rates. The formation of the encounter complex induces the formation of the final “docked” complex, which is associated with slower dissociation rates and provides the stability of the overall interaction. A more recent study has also observed the same mode of interaction for MAb 4E10 when it binds to MPER peptide in liposomal form (31). The studies of Sun et al. revealed that critical residues of the 4E10 epitope may be buried in the viral membrane and that interaction of 4E10 with lipids is important in extracting the immersed residues from the lipid bilayer. Although 2F5 binding was not described in the study, the model shows that the N-terminal helix of the “L”-shaped MPER structure projects away from the membrane and that residues K₆₆₅ and W₆₆₆ of the core 2F5 epitope (residues DKW) are placed on the surface and in the interfacial region, respectively, of the membrane lipid (31). Thus, as for MAb 4E10, stable docking of 2F5 would also require some level of conformational rearrangement of MPER to release critical residues within the core epitope. This is consistent with binding kinetics data that showed that the final docking of MAbs 2F5 and 4E10 to MPER peptide-lipid conjugates might require conformational rearrangements (2). It is also likely that the CD4 and coreceptor-mediated triggering of HIV-1 Env (10, 28) that leads to the formation of the fusion intermediate conformation might also expose critical residues for MPER MAb binding. Both the 4E10 and 2F5 MAbs bound strongly to a recombinant trimeric gp41 intermediate design and either bound weakly or failed to bind, respectively, to the trimeric gp140 (11) and a putative prefusion-state trimeric MPER (22). However, the orientation of the MPER sequence in a viral-lipid-bound form is not known and, thus, it is possible that in the early stages of the triggered intermediate state, MPER residues may be lying in the plane of the membrane head groups and interaction of MPER MAbs with lipids and extraction of critical residues may be essential for stable docking (31).In order to gain further understanding of the binding mechanism involved in the interaction of MAbs 2F5 and 4E10 with their epitopes presented in the membrane environment, we have constructed three different novel gp41 MPER peptide-liposome conjugates, including a 2F5 nominal epitope peptide, a 4E10 nominal epitope peptide, and a peptide having sequences of epitopes for both the 2F5 and 4E10 MAbs. Unlike our previously designed constructs (2), the MPER peptides used in the current study were anchored to the liposomes by a hydrophobic sequence (YKRWIILGLNKIVRMYS), named GTH1, placed at their carboxyl termini. Using these second-generation peptide-liposome conjugates, we addressed the following questions. (i) How do MAbs 2F5 and 4E10 bind to the different peptide-liposome conjugates? (ii) How do the kinetics of MAb binding vary with temperature? (iii) How are the peptides oriented in the liposomal membrane in each construct? (iv) How does antibody binding correlate with differences in the membrane orientation of peptides? (v) Is there any difference in the secondary structures adopted by the peptides in the peptide-liposome complex?Our study of antibody interactions with their membrane-anchored epitope peptides indicates that both the 2F5 and 4E10 MAbs bind to their nominal epitope peptide-liposome conjugates with high affinity. The results of tryptophan fluorescence-quenching and fluorescence resonance energy transfer (FRET) experiments showed that the nominal 2F5 peptide is exposed on the surface of the membrane close to the polar head group, whereas the nominal 4E10 peptide is immersed in the interfacial region of the lipid bilayer. Circular dichroism (CD) spectroscopic studies revealed that the nominal epitope and biepitope peptides adopted ordered structures when anchored to the liposomal membrane. The membrane orientation data and secondary structural features of MPER peptides correlated well with antibody binding characteristics, thus suggesting that membrane-anchored MPER peptide conformations are a physiologic component of the native 2F5 and 4E10 binding epitopes in HIV-1 virions.     

Lipid binding properties of 4E10, 2F5, and WR304 monoclonal antibodies that neutralize HIV-1

Two human mAbs (2F5 and 4E10), originally derived from HIV-1-infected patients, are important, but rare, mAbs that exhibit broad cross-clade neutralizing activities against HIV-1. In addition to peptide sequences on the gp41 envelope protein, both antibodies reportedly also bound specifically to several phospholipid antigens. However, the phospholipid binding property of 2F5 has been disputed and, because of uncertainly regarding phospholipid binding, the modeling of neutralizing mechanisms has been difficult. To explore this issue, we examined the binding of 4E10 and 2F5 to a broad range of lipid antigens by ELISA. 4E10 and 2F5 both bound to a variety of purified phospholipids, and 4E10 bound, but 2F5 did not bind, to cardiolipin. Both mAbs also bound to a sulfated glycolipid, sulfogalactosyl ceramide (sulfatide), and to two neutral glycolipids, galactosyl ceramide and glucosyl ceramide, but not to other galactosyl glycolipids. 4E10, but not 2F5, also bound to cholesterol, although both mAbs bound to squalene. Interestingly, 4E10, but not 2F5, exhibited striking binding to lipid A, the lipid moiety of Gram-negative bacterial lipopolysaccharide. The binding properties of 4E10 to phospholipids, sulfatide, cholesterol, squalene, and lipid A were similar to those of a neutralizing murine mAb (WR304) induced by liposomes containing phosphatidylinositol phosphate and lipid A, although WR304 did not bind to neutral glycolipids. The discovery of a binding specificity of 4E10 for lipid A, a widely used vaccine adjuvant, suggests that innate immunity stimulated by lipid A could have played a role for induction of multispecific antibodies that simultaneously recognize both HIV-1 protein and lipid antigens.     

Steric Accessibility of the Cleavage Sites Dictates the Proteolytic Vulnerability of the Anti‐HIV‐1 Antibodies 2F5, 2G12, and PG9 in Plants

Broadly neutralizing antibodies (bNAbs) to human immunodeficiency virus type 1 (HIV‐1) hold great promise for immunoprophylaxis and the suppression of viremia in HIV‐positive individuals. Several studies have demonstrated that plants as Nicotiana benthamiana are suitable hosts for the generation of protective anti‐HIV‐1 antibodies. However, the production of the anti‐HIV‐1 bNAbs 2F5 and PG9 in N. benthamiana is associated with their processing by apoplastic proteases in the complementarity‐determining‐region (CDR) H3 loops of the heavy chains. Here, it is shown that apoplastic proteases can also cleave the CDR H3 loop of the bNAb 2G12 when the unusual domain exchange between its heavy chains is prevented by the replacement of Ile¹⁹ with Arg. It is demonstrated that CDR H3 proteolysis leads to a strong reduction of the antigen‐binding potencies of 2F5, PG9, and 2G12‐I19R. Inhibitor profiling experiments indicate that different subtilisin‐like serine proteases account for bNAb fragmentation in the apoplast. Differential scanning calorimetry experiments corroborate that the antigen‐binding domains of wild‐type 2G12 and 4E10 are more compact than those of proteolysis‐sensitive antibodies, thus shielding their CDR H3 regions from proteolytic attack. This suggests that the extent of proteolytic inactivation of bNAbs in plants is primarily dictated by the steric accessibility of their CDR H3 loops.     

Epitope Mapping of Ibalizumab,a Humanized Anti-CD4 Monoclonal Antibody with Anti-HIV-1 Activity in Infected Patients

Ibalizumab is a humanized monoclonal antibody that binds human CD4, the primary receptor for human immunodeficiency virus type 1 (HIV-1). With its unique specificity for domain 2 of CD4, this antibody potently and broadly blocks HIV-1 infection in vitro by inhibiting a postbinding step required for viral entry but without interfering with major histocompatibility complex class II (MHC-II)-mediated immune function. In clinical trials, ibalizumab has demonstrated anti-HIV-1 activity in patients without causing immunosuppression. Thus, a characterization of the ibalizumab epitope was conducted in an attempt to gain insight into the underlying mechanism of its antiviral activity as well as its safety profile. By studying mouse/human chimeric CD4 molecules and site-directed point mutants of CD4, amino acids L96, P121, P122, and Q163 in domain 2 were found to be important for ibalizumab binding, with E77 and S79 in domain 1 also contributing. All these residues appear to cluster on the interface between domains 1 and 2 of human CD4 on a surface opposite the site where gp120 and the MHC-II molecule bind on domain 1. Separately, the epitope of M-T441, a weakly neutralizing mouse monoclonal antibody that competes with ibalizumab, was localized entirely within domain 2 on residues 123 to 125 and 138 to 140. The results reported herein not only provide an appreciation for why ibalizumab has not had significant adverse immunological consequences in infected patients to date but also raise possible steric hindrance mechanisms by which this antibody blocks HIV-1 entry into a CD4-positive cell.The human immunodeficiency virus type 1 (HIV-1) epidemic continues to spread at the alarming rate of approximately 2.5 million new cases per year, despite intensive efforts from the scientific community. A safe and effective HIV-1 vaccine would be a key weapon to fight this epidemic; however, vaccine development has not yet proven successful. The extraordinary diversity of the virus, its capacity to evade adaptive immune responses, and the inability to induce broadly neutralizing antibodies against HIV-1 represent unprecedented challenges for vaccine development (3). Alternatively, the strategy of preexposure prophylaxis (PrEP) with antiretroviral drugs or even virus-specific immunoglobulins (Igs) (11) is gaining traction. Protection of rhesus macaques from challenge with simian immunodeficiency virus (SIV) has been observed after passive administration of anti-gp120 or anti-gp41 monoclonal antibodies, such as b12, 2G12, 2F5, and 4E10 (2, 20). However, the application of these antibodies as PrEP has been hindered due to their lack of potency or breadth or both. To this end, PrEP strategies could also consider antibodies to CCR5 (13) or CD4 (8, 12, 14), which have potent and broad inhibitory activities against HIV-1 without unwanted side effects.The CD4 molecule, a cell surface glycoprotein found primarily on T lymphocytes, is the primary receptor for the HIV-1 envelope gp160 glycoprotein (7, 18). A member of the immunoglobulin superfamily (19), CD4 consists of an extracellular segment composed of four tandem immunoglobulin-like domains (D1, D2, D3, and D4), a single transmembrane span, and a short C-terminal cytoplasmic tail (15, 24). It is worth noting that both human major histocompatibility complex (MHC) class II (26) and HIV-1 gp120 (16, 24) bind to the same surface on the first domain (D1) of the CD4 molecule.Ibalizumab (formerly known as TNX-355) is a humanized IgG4 monoclonal antibody that blocks HIV-1 entry by binding to human CD4 (8, 12, 14, 33). It was engineered from its mouse progenitor (5A8) by grafting the mouse complementary-determining region (CDR) onto a human IgG4 construct (4, 5). The IgG4 isotype was chosen to minimize the chances for CD4⁺ T-cell depletion by antibody- and complement-dependent cytotoxicity mediated by binding to Fc receptors. Ibalizumab or 5A8 blocks CD4-dependent virus entry and inhibits a broad spectrum of both laboratory-adapted and clinical HIV-1 isolates, including CCR5-tropic and CXCR4-tropic strains from multiple subtypes, with 50% inhibitory concentrations (IC₅₀s) of 0.0004 to 0.152 μg/ml (4, 5). In vivo, treatment with ibalizumab prominently reduced plasma viremia in rhesus macaques infected with SIV, because this monoclonal antibody has equal affinity for rhesus CD4 (22, 23). In HIV-1 patients, single as well as multiple doses of ibalizumab resulted in substantial reductions (∼10-fold) in viral loads and increases in CD4⁺ T-cell counts without evidence of serious adverse effects or immunologic impairments (12, 14).Efforts were made years ago to delineate the antibody binding site of 5A8 on human CD4 (hCD4) (5). Two stretches, amino acids (aa) 121 to 124 and aa 127 to 134, in domain 2 (D2) were found to be critical for binding. Since then, however, little work has been done to fine-map this epitope, and whether other parts of hCD4 are involved in the binding of this antibody has remained unexplored. The fact that an anti-hCD4-D2 antibody can noncompetitively, yet potently, block HIV-1 entry is intriguing, as viral gp120 binds to D1 of hCD4 (16, 24). Therefore, to gain a better understanding of the mechanism by which ibalizumab inhibits HIV-1 infection while avoiding undesired side effects, we sought to fine-map the epitope of this unique monoclonal antibody.     

G1 cyclin/cyclin-dependent kinase-coordinated phosphorylation of endogenous pocket proteins differentially regulates their interactions with E2F4 and E2F1 and gene expression

Mitogenic stimulation leads to activation of G(1) cyclin-dependent kinases (CDKs), which phosphorylate pocket proteins and trigger progression through the G(0)/G(1) and G(1)/S transitions of the cell cycle. However, the individual role of G(1) cyclin-CDK complexes in the coordinated regulation of pocket proteins and their interaction with E2F family members is not fully understood. Here we report that individually or in concert cyclin D1-CDK and cyclin E-CDK complexes induce distinct and coordinated phosphorylation of endogenous pocket proteins, which also has distinct consequences in the regulation of pocket protein interactions with E2F4 and the expression of p107 and E2F1, both E2F-regulated genes. The up-regulation of these two proteins and the release of p130 and pRB from E2F4 complexes allows formation of E2F1 complexes not only with pRB but also with p130 and p107 as well as the formation of p107-E2F4 complexes. The formation of these complexes occurs in the presence of active cyclin D1-CDK and cyclin E-CDK complexes, indicating that whereas phosphorylation plays a role in the abrogation of certain pocket protein/E2F interactions, these same activities induce the formation of other complexes in the context of a cell expressing endogenous levels of pocket and E2F proteins. Of note, phosphorylated p130 "form 3," which does not interact with E2F4, readily interacts with E2F1. Our data also demonstrate that ectopic overexpression of either cyclin is sufficient to induce mitogen-independent growth in human T98G and Rat-1 cells, although the effects of cyclin D1 require downstream activation of cyclin E-CDK2 activity. Interestingly, in T98G cells, cyclin D1 induces cell cycle progression more potently than cyclin E. This suggests that cyclin D1 activates pathways independently of cyclin E that ensure timely progression through the cell cycle.     

Monoclonal Antibodies 1G12 and 6A12 to the N-domain of human angiotensin-converting enzyme: fine epitope mapping and antibody-based detection of ACE inhibitors in human blood

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N- and C-), each bearing a Zn-dependent active site. ACE inhibitors are among the most prescribed drugs in the treatment of hypertension and cardiac failure. Fine epitope mapping of two monoclonal antibodies (mAb), 1G12 and 6A12, against the N-domain of human ACE, was developed using the N-domain 3D-structure and 21 single and double N-domain mutants. The binding of both mAbs to their epitopes on the N-domain of ACE is significantly diminished by the presence of the C-domain in the two-domain somatic tissue ACE and further diminished by the presence of sialic acid residues on the surface of blood ACE. The binding of these mAbs to blood ACE, however, increased dramatically (5-10-fold) in the presence of ACE inhibitors or EDTA, whereas the effect of these compounds on the binding of the mAbs to somatic tissue ACE was less pronounced and even less for truncated N-domain. This implies that the binding of ACE inhibitors or removal of Zn2+ from ACE active centers causes conformational adjustments in the mutual arrangement of N- and C-domains in the two-domain ACE molecule. As a result, the regions of the epitopes for mAb 1G12 and 6A12 on the N-domain, shielded in somatic ACE by the C-domain globule and additionally shielded in blood ACE by sialic acid residues in the oligosaccharide chains localized on Asn289 and Asn416, become unmasked. Therefore, we demonstrated a possibility to employ these mAbs (1G12 or 6A12) for detection and quantification of the presence of ACE inhibitors in human blood. This method should find wide application in monitoring clinical trials with ACE inhibitors as well as in the development of the approach for personalized medicine by these effective drugs.     

Specificity of E2F1, E2F2, and E2F3 in mediating phenotypes induced by loss of Rb.

The Rb/E2F pathway plays a critical role in the control ofcellular proliferation. Here, we report that E2F1, E2F2, and E2F3 make major individual contributions toward the in vivo phenotypic consequences of Rb deficiency. In the developing lens of Rb(-/-) embryos, loss of E2F1, E2F2, or E2F3 reduces the unscheduled proliferation of fiber cells, with the loss of E2F3 having the most pronounced effect. In Rb-deficient retinas, all three E2Fs contribute equally to the ectopic proliferation of postmitotic neuronal cells. In contrast, E2F1 is unique in mediating apoptosis in both Rb(-/-) lenses and retinas. In the central nervous system, loss of E2F1 or E2F3 can almost completely eliminate the ectopic DNA replication and apoptosis observed in Rb(-/-) embryos, and loss of E2F2 partially reduces the unscheduled DNA replication and has no effect on apoptosis. These results provide clear evidence for functional specificity among E2Fs in the control of Rb-dependent proliferation and apoptosis in a tissue-specific manner.