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Titration curves for the binding of eight MAbs to 13 intact virions. The MAbs were tested at concentrations ranging from 0.0001 to 10.0 g/ml and virus was added to all wells at a constant concentration of 100 ng of p24/ml. The amounts of virus bound by MAbs are represented by the picograms of p24 per milliliter released by detergent treatment of bound virus. The binding curves for neutralizing MAbs (as defined in Table 2) are shown in red, those for nonneutralizing MAbs are blue, and those for the negative control MAb 1418 are green.

Titration curves for the binding of eight MAbs to 13 intact virions. The MAbs were tested at concentrations ranging from 0.0001 to 10.0 g/ml and virus was added to all wells at a constant concentration of 100 ng of p24/ml. The amounts of virus bound by MAbs are represented by the picograms of p24 per milliliter released by detergent treatment of bound virus. The binding curves for neutralizing MAbs (as defined in Table 2) are shown in red, those for nonneutralizing MAbs are blue, and those for the negative control MAb 1418 are green.

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Antibodies (Abs) against the V3 loop of the human immunodeficiency virus type 1 gp120 envelope glycoprotein were initially considered to mediate only type-specific neutralization of T-cell-line-adapted viruses. However, recent data show that cross-neutralizing V3 Abs also exist, and primary isolates can be efficiently neutralized with anti-V3 monoc...

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... coated with each of the MAbs at concentrations ranging from 0.0001 to 10 g/ml were used, and the quantity of virus bound was assessed by lysing the bound virus and measuring the levels of released p24, a technique previously used by us and others (2,4,34,38). Representative binding curves for the 90 anti-V3 MAb-virus combinations are shown in Fig. ...
Context 2
... gp120 binding indices had the highest values for the neutralization-sensitive viruses BaL, SF162, JR-CSF, US1, JR-FL, and 92US717, while the binding indices were lower for the other viruses, which are less sensitive to neutralization. A Table 2 and Fig. 4 and 5. The analyses show the correlation between percent neutralization and either the log of 50% maximal binding (A) or virus binding at saturation (using 10 g of MAb/ml) (B). Best-fit regression lines (solid lines) and 95% prediction intervals (dashed lines) are shown. regression analysis of the data revealed a significant ...
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... exposure differs with the virus and the conditions studied. Thus, by using saturating conditions in the virus binding assay, in which virions are captured on ELISA plates coated with 10 g of MAb/ml, we noted large differences in the capture of different viruses. For example, on average, much more BaL was captured by the anti-V3 MAbs than 92US717 (Fig. 4). Nonetheless, the V3 epitopes seemed to be comparably recognized when these same MAbs reacted with the solubilized gp120s from these two viruses (Table 4). These data lead to the conclusions that the relevant V3 epi- topes are present within the gp120s of these viruses but that the V3 loop is more accessible on the BaL virus than it is ...
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... and figures support the notion that V3 is differentially exposed by FIG. 7. Correlation between MAb binding to detergent-solubilized gp120 and capture of intact virions by MAbs under Ab-saturating conditions (A) and between MAb binding to gp120 and percent neutralization at 50 g of MAb/ml (B). The data analyzed were taken from Tables 2 and 4 and Fig. ...
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... eters contribute to the shape of the neutralization curve and the ultimate outcome of Ab-virus interactions. These multiple parameters, and most probably several others, contribute to the complex equation that determines neutralization sensitivity or resistance. Thus, viruses such as BaL, SF162, and JR-CSF, which according to the data shown in Fig. 4 have a very low threshold for resisting Ab-mediated neutralization, appear to have well-exposed V3 loops for which the anti-V3 MAbs have high affinities (Fig. 5); in contrast, viruses such as BR07, ADA, and aBL01 have higher thresholds as a consequence, at least in part, of a poorly exposed V3 loop and low-affinity interactions with ...
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... an Env oligomer reduced infectivity (46). Recently, Franti et al. also described a threshold effect when studying the interaction between JF-CSF and the anti- gp120 MAbs b12, 447-52D, and 2G12 (M. Franti, S. Frost, M. Guyader, K. Delgado, D. R. Burton, and P. Poignard, Abstr. AIDS Vaccine 2003Vaccine , abstr. 118, 2003. The data included in Fig. 4, however, indicate for the first time that the levels of these thresholds will vary with each individual strain of ...

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... Additionally, results for 1F10 seemed viral strain dependent. This can be caused by antigenic variations of the V3 region and specific residues outside of V3, which can affect the presentation of the epitope hereby restricting access for antibodies (51)(52)(53). While V3 loop exposure has not been extensively studied, it is known that the V3 loop is shielded by several N-glycans and remains largely buried, and its accessibility is improved after conformational changes (54)(55)(56). ...
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The most effective treatment for HIV-1, antiretroviral therapy, suppresses viral replication and averts the disease from progression. Nonetheless, there is a need for alternative treatments as it requires daily administration with the possibility of side effects and occurrence of drug resistance. Broadly neutralizing antibodies or nanobodies targeting the HIV-1 envelope glycoprotein are explored as alternative treatment, since they mediate viral suppression and contribute to the elimination of virus-infected cells. Besides neutralization potency and breadth, Fc-mediated effector functions of bNAbs also contribute to the in vivo efficacy. In this study multivalent J3, 2E7 and 1F10 anti-HIV-1 broadly neutralizing nanobodies were generated to improve neutralization potency and IgG1 Fc fusion was utilized to gain Fc-mediated effector functions. Bivalent and trivalent nanobodies, coupled using long glycine-serine linkers, showed increased binding to the HIV-1 Env and enhanced neutralization potency compared to the monovalent variant. Fusion of an IgG1 Fc domain to J3 improved neutralization potency compared to the J3-bihead and restored Fc-mediated effector functions such as antibody-dependent cellular phagocytosis and trogocytosis, and natural killer cell activation. Due to their neutralization breadth and potency and their ability to induce effector functions these nanobody-IgG1 constructs may prove to be valuable towards alternative HIV-1 therapies.
... Conversely, after CD4 binding, the trimer transitions toward a low-energy, open state characterized by a dramatic displacement of the variable loop complex at the trimer apex, formed by the coalescence of the first two (V1V2) and third (V3) variable loops of gp120 (1,(19)(20)(21)(22). In this open conformation, the trimer is exposed to recognition by a wide variety of antibodies against sensitive regions such as the V3 tip and the so-called bridging sheet that provides the largest contact surface for coreceptor binding, which have otherwise little or no neutralizing activity because their target epitopes are largely concealed in the closed prefusion trimer (23)(24)(25). ...
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... This semiconserved and rather flexible loop of about 35 residues, which determines which co-receptor (CCR5 or CXCR4) is used by the virus to gain entry into cells, is recognized by many HIV-1 neutralizing antibodies present in infected inviduals. It is known that the V3 loop is able to induce anti HIV-1 neutralizing antibodies [33,83,84] but the precise V3 conformation that should be present in a V3 synthetic peptide vaccine has not been established. NMR studies of linear peptides complexed with Mabs specific for the conformation of the V3 loop present in intact virus and in gp120 protein showed that the conformation of the antibodybound V3 peptides was dictated by a process of induced fit to each Mab [85]. ...
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... For deeper interrogation of neutralization phenotype, a set of monoclonal antibodies that show a strong preference for tier 1 viruses was used. This set included V3-specific antibodies 2219 [66], 2557 [67], 3074 [68], 3869 [69], 447-52D [70] and 838-D [71], and the CD4bs antibodies 654-30D [72], 1008-30D [73], 1570D [73] and 729-30D [74] produced by Drs. Susan Zolla-Pazner and Miroslaw K. Gorny at New York University and the Veterans Affairs Medical Center, New York, New York. ...
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... Especially, mutation site 175 and 120 are closely neighboring the tip of the V3 loop. This tip section of the V3 loop, due to its exposed accessibility, was identified as the epitope of a variety of neutralizing and non-neutralizing antibodies which were elicited during HIV-1 infection in humans [121,141,142] . The mutation sites 120, 128 and 175 are referred to as the mutation cluster in the following. ...
Thesis
Peptides are versatile tools to investigate protein-protein interaction on the amino acid sequence level. The entry to the host cell of HIV-1 is a crucial step of the viral replication cycle. The investigation of inhibitory peptides was a promising starting point to gain further insight into the molecular mechanism of viral host cell entry. Moreover, the characterization of the structural activity of peptides as HIV-1 entry inhibitors contributed to a better understanding of the complex process of HIV-1 entry and its inhibition respectively. During this work two different approaches were pursued. The first approach used a known inhibitory protein-derived peptide (PEGI) as a starting point. The PEGI peptide is derived from the E2 protein of HPgV-1, a non-pathogen human virus, which is a commonly occurring co-infection of HIV-1 infected individuals. 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In addition, reduced cysteines instead of a connection via inter- or intramolecular disulfide or lactam bridges were found to be beneficial for HIV-1 inhibitory activity and thus, it can be speculated that redox-active cysteines are involved in the HIV 1 inhibition by PEGI. Moreover, truncation variants revealed a five fold more potent HIV-1 peptide inhibitor (PEGI-D35), which is a promising candidate for follow up studies (Ac LCDCPNGPWVWVPAFCQD-NH2). The PEGI sequence represents a highly conserved motif within the E2 protein with only three flexible positions among the circulating HPgV-1 isolates. The relevance of the PEGI sequence for HPgV-1 isolates was proven by testing a panel of mainly occurring variants of these E2 section and HPgV 1 strain variants, which were inhibitory active. The breadth of HIV-1 inhibition was determined with a HIV-1 strain panel. 4 of 20 tested HIV 1 isolates were resistant against inhibition by PEGI. 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The successful cultivation of a PEGI resistant HIV 1NL4 3 virus and subsequent sequencing of the viral genes discovered five single mutations within gp120, partly located in conserved sections of the protein. Especially the mutation of conserved positions suggested that an important binding site of PEGI is located within gp120. Binding experiments were performed with a set of PEGI peptides to further support this hypothesis. The results outstandingly correlated with the results in HIV-1 neutralization assay. Based on this correlation, gp120 binding of PEGI is a plausible mechanism enabling HIV-1 inhibition. Moreover, it could be shown that PEGI inhibits the binding of gp120 targeting antibodies to their epitope. It was an especially potent inhibitor of the V3 loop antibody F425 B4e8 -gp120 interaction. The inhibition could be narrowed down to the epitope level in binding and HIV-1 neutralization experiments. After a profound analysis of all available results it could be concluded that the PEGIs HIV-1 inhibitory activity is based on binding to gp120, especially the V3 loop, and thereby either directly or indirectly destabilizing crucial gp120 conformation for a successful HIV-1 entry process. The HPgV-1 protein derived peptide PEGI, including its variants, analysis of mode of action and neutralization breadth provided new important insights into to understand the viral interference of HIV-1 and HPgV-1 and HIV 1 inhibition on the protein level. To further support the hypothesized molecular mechanism of HIV-1 inhibition by PEGI, a follow up on the peptide level can be recommended which uses V3 loop sequences of resistant and susceptible HIV-1 isolates. The second approach was the screening of synthetic combinatorial libraries in HIV 1 neutralizing antibody assays. The aim was to identify new peptide sequences that inhibit a designated antibody-protein interaction. 18 scaffold ranking samples of peptide libraries were tested in antibody-protein competitive assays. The following HIV-1 neutralizing antibodies were selected for screening: b12, VRC01, VRC03, three gp120 targeting antibodies, and F240, a gp41 targeting antibody. The screening results proved the suitability of the scaffold ranking to detect active peptide libraries in the field of HIV-1 neutralizing antibodies. A linear D-AA hexapeptide scaffold was selected as a promising candidate to further identify individual peptides which could inhibit the VRC01-gp120HXBc2 interaction. Following the principles for a successful deconvolution, positional scanning samples of the D-AA hexapeptide library were tested. The evaluation of the positional scanning profiles was based on the newly introduced evaluation parameter called fold over X (FOX). FOX enabled the deconvolution of profiles with a moderate level of differentiation. The 24 peptides, selected during the deconvolution process, were synthesized and their ability to inhibit VRC01-gp120HXBc2 interaction was tested. Two active hexapeptides were identified with a high sequence homology of five positions. Both peptides, VRC01-ReH12 (Ac-efvwvy-NH2) and VRC01-ReH11 (Ac-efvwvl-NH2), inhibited the VRC01-gp120HXBc2 binding with moderate inhibitory activity (IC30 = 2 respectively 12 µM), whereas L-AA variants of the active peptides did not show inhibitory activity. The activity was not limited to the VRC01 gp120HXBc2 interaction because both peptides also inhibited the b12- gp120HXBc2 interaction. The identified peptides were the first reported D AA hexapeptide competitors of HIV-1 neutralizing antibodies targeting the CD4 binding site. 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... Monoclonal Abs 1357 [28], 1361 [28], 2158 [29], and 830A [26] are specific for the V2i epitope of gp120 as defined in Spurrier et al. [30]. Monoclonal Abs 2219 [28], 2557 [31], and 3792 [32] are specific for the V3 loop of gp120. Recombinant mAbs were produced by conventional molecular techniques as previously described [33] in 293T cells. ...
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The only clinical HIV vaccine trial to demonstrate efficacy, RV144, correlated protection with the antibodies (Abs) mediating function via the "constant" immunoglobulin region, the crystallizable fragment (Fc). These data have supported a focus on the induction of Abs by vaccines that trigger antiviral activities by relevant leukocytes via Fc receptors (FcRs). Neutrophils are phagocytes that comprise > 50% of leukocytes and display unique FcRs. We sought to compare the Ab-dependent cellular phagocytosis (ADCP) activity of human neutrophils to the commonly assayed THP-1 cell line. HIV-specific Abs were employed to elicit ADCP of beads coated with HIV envelope protein. Overall, trends were noted among neutrophil donors and the ADCP profile was different from that of THP-1 cells. mAb ELISA titers correlated with ADCP by THP-1 cells but not neutrophils. Monoclonal (m)Abs were also tested with primary monocytes. Donor-to-donor variation was high, and hindered the analysis of this dataset, but it was, in itself, an important finding. This study illustrates the concept that the assessment of FcR-mediated Ab activity with a frequently used cell line such as THP-1 is not necessarily indicative of relevant Ab functionality in vivo, and this calls for in-depth study of the properties of the HIV antibodies best-suited to eliciting antiviral activities by primary cells.
... loop is an immunodominant regions, and is the target of a large proportion of antibodies, especially early in infection [238, 254,255]. Some studies have shown that V3 loop antibodies have promise, with the V3 mAbs 2219, 2557 and 3074 neutralizing around 30% of pseudoviruses they were tested against from both tier 1 viruses, which are sensitive to antibody neutralization, and tier 2 viruses, which are more resistant to antibody neutralization, including representatives from clades A, B and C [256]. ...
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... [2,3,210,275]. The V3 loop is also highly 72 immunogenic, and is an important target of antibodies both in natural infection and in vaccination studies [255,306,[313][314][315]. Because of this, recent efforts to use the SOSIP trimer as a vaccine immunogen have shown that non-cross neutralizing V3 antibodies are generated in high numbers [220,257,272,277]. ...
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Human Immunodeficiency Virus (HIV) is the etiological agent of AIDS and is responsible for the AIDS pandemic worldwide. According to UNAIDS, as of 2016, there were approximately 36.7 million people living with HIV globally, and 1.8 million new infections that year. While antiretroviral therapies and education continue to reduce these numbers, a preventative vaccine is still required to curb this epidemic. The envelope glycoprotein trimer of HIV, which is the sole protein on the surface of the virus and facilitates entry of the virus into host cells, is of keen interest to the HIV vaccine and drug-development field. Recently, the structure of this trimer was solved which has opened the door for the design of new immunogens and drugs. These solved envelope protein trimer structures demonstrate that the major variable loops of the env protein are positioned at the apex of the trimer where they play a role in trimer stability. Data from our lab has shown previously that both the V2 and V3 loop contain elements that are critical for envelope trimer stability. The work presented in this dissertation expands on these initial studies and confirms that the V2 and V3 loops contribute to envelope trimer stability and functionality in HIV-1, HIV-2 and in Simian Immunodeficiency Virus (SIV) species. We have demonstrated that the hydrophobic patch near the tip of the V3 loop, which is present in all primate lentiviruses, is critical for trimer stability in subtype C HIV-1, HIV-2 and in SIV species. Additionally, we further explored the role of the twin-cysteine motif found in the V2 loop of HIV-2 and SIV species, but not in HIV-1. We have shown that this motif is critical for the stability of the envelope protein of these viruses. Both observations are supported by our experimental data and by molecular modeling. This work supports the released trimer structures and provides a further understanding of the forces that stabilize the envelope trimer of primate lentiviruses, and will aid in the development of stabilized envelope trimer based immunogens in the effort to develop an effective HIV vaccine. Advisor: Shi-Hua Xiang
... The V3-specific mAb 2219 (recognizing an epitope in beta-hairpin of the V3 crown), [77,78] was shown to bind both the A244.AE and 6240.B gp120 proteins with KD of 90 nM and 243 nM, respectively (Table 1 and S4 Fig). Higher affinities were observed with the V3 crown mAb 2557 [79] for both A244.AE (32 nM) and 6240.B (4.01 nM) proteins. In contrast to the V2i mAbs, the V3 mAbs showed relatively slow on and fast off rates. ...
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... H IV-1 Env proteins contain highly immunogenic epitopes that readily induce antibody responses upon either infection or vaccination (reviewed in reference 1). These responses are represented by large numbers of monoclonal antibodies (MAbs) isolated from vaccinated or infected subjects that are directed against standard nonglycosylated epitopes in the V3 region (2)(3)(4)(5)(6)(7)(8)(9), the V1/V2 domain (10)(11)(12)(13)(14), the CD4binding domain (15)(16)(17)(18), and CD4-induced epitopes (16,(19)(20)(21)(22). Such so-called conventional antibodies possess a narrow neutralization breadth, limited mostly to a small class of neutralization-sensitive tier 1a and tier 1b Envs, with little, if any, neutralization activity against typical tier 2/3 primary isolates (8,(23)(24)(25). ...
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The subtype C HIV-1 isolate MW965.26 is a highly neutralization-sensitive tier 1a primary isolate that is widely used in vaccine studies, but the basis for the sensitive neutralization phenotype of this isolate is not known. Substituting the MW965.26 V1/V2 domain into a neutralization-sensitive SF162 Env clone resulted in high resistance to standard anti-V3 monoclonal antibodies, demonstrating that this region possesses strong masking activity in a standard Env backbone and indicating that determinants elsewhere in MW965.26 Env are responsible for its unusual neutralization sensitivity. Key determinants for this phenotype were mapped by generating chimeric Envs between MW965.26 Env and a typical resistant Env clone, the consensus C (ConC) clone, and localized to two residues, Cys384 in the C3 domain and Asn502 in the C5 domain. Substituting the sensitizing mutations Y384C and K502N at these positions into several resistant primary Envs resulted in conversion to neutralization-sensitive phenotypes, demonstrating the generalizability of this effect. In contrast to the sensitizing effects of these substitutions on normally masked epitopes, these mutations reduced the sensitivity of VRC01-like epitopes overlapping the CD4-binding domain, while they had no effect on several other classes of broadly neutralizing epitopes, including members of several lineages of V2-dependent quaternary epitopes and representatives of N332 glycan-dependent epitopes (PGT121) and quaternary, cleavage-dependent epitopes centered at the gp41-gp120 interface on intact HIV-1 Env trimers (PGT151). These results identify novel substitutions in gp120 that regulate the expression of alternative conformations of Env and differentially affect the exposure of different classes of epitopes, thereby influencing the neutralization phenotype of primary HIV-1 isolates. IMPORTANCE A better understanding of the mechanisms that determine the wide range of neutralization sensitivity of circulating primary HIV-1 isolates would provide important information about the natural structural and conformational diversity of HIV-1 Env and how this affects the neutralization phenotype. A useful way of studying this is to determine the molecular basis for the unusually high neutralization sensitivities of the limited number of available tier 1a viruses. This study localized the neutralization sensitivity of MW965.26, an extremely sensitive subtype C-derived primary isolate, to two rare substitutions in the C3 and C5 domains and demonstrated that the sequences at these positions differentially affect the presentation of epitopes recognized by different classes of standard and conformation-dependent broadly neutralizing antibodies. These results provide novel insight into how these regions regulate the neutralization phenotype and provide tools for controlling the Env conformation that could have applications both for structural studies and in vaccine design.
... The V3 loop of gp120 serves as a component of the coreceptor binding site and is an important target for neutralizing antibodies (49)(50)(51)(52). V3-mediated neutralization sensitivity varies widely among HIV-1 strains (53,54). ...
Article
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.