Characterization of a Large Panel of Rabbit Monoclonal Antibodies against HIV-1 gp120 and Isolation of Novel Neutralizing Antibodies against the V3 Loop.
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ABSTRACT:
SUBMITTER: Qin Y
PROVIDER: S-EPMC4454676 | biostudies-literature | 2015
REPOSITORIES: biostudies-literature
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Project description:Monoclonal antibodies (MAbs) were obtained by immunizing mice with synthetic peptides corresponding to the third variable (V3) or the third conserved (C3) domain of the external envelope protein (gp120) of human immunodeficiency virus type 2 (HIV-2ROD). One MAb, designated B2C, which was raised against V3 peptide NKI26, bound to the surface of HIV-2-infected cells but not to their uninfected counterparts. B2C was capable of neutralizing cell-free and cell-associated virus infection in an isolate-specific fashion. The antibody-binding epitope was mapped to a 6-amino-acid peptide in the V3 variable domain which had the core sequence His-Tyr-Gln. Two MAbs, 2H1B and 2F19C, which were raised against the C3 peptide TND27 reacted with gp120 of HIV-2ROD in a Western immunoblot assay. The C3 epitopes recognized by these two MAbs appeared inaccessible because of their poor reactivity in a surface immunofluorescence assay. Although partial inhibition of syncytium formation was observed in the presence of the anti-C3 MAbs, their neutralizing activity appeared weak. Finally, the effects of these MAbs against CD4-gp120 binding were assessed. Partial inhibition of CD4-gp120 binding was observed in the presence of high concentrations of B2C. On the other hand, no inhibition of CD4-gp120 binding was observed in the presence of anti-C3 MAbs. Since complete neutralization could be achieved at a concentration corresponding to that of partial binding inhibition by B2C, some different mechanisms may be involved in the B2C-mediated neutralization. These results, taken together, indicated that analogous to the function of the V3 region of HIV-1, the V3 region of HIV-2ROD contained at least a type-specific fusion-inhibiting neutralizing epitope. In this respect, the V3 sequence of HIV-2 may be a useful target in an animal model for HIV vaccine development.
| S-EPMC189045 | biostudies-literature
Project description:The V3 loop of HIV-1 gp120 is considered occluded on many primary viruses. However, virus sensitivity to neutralization by different V3 mAbs often varies, indicating that access to V3 is not restricted equally for all antibodies. Here, we have sought to gain a better understanding of these restrictions by determining the neutralizing activities of 7 V3 mAbs (19b, 39F, CO11, F2A3, F530, LA21, and LE311) against 15 subtype B primary isolates and relating these activities to the fine specificity of the mAbs. Not surprisingly, we found that most mAbs neutralized the same 2-3 viruses, with only mAb F530 able to neutralize 2 additional viruses not neutralized by the other mAbs. Epitope mapping revealed that positively-charged residues in or near the V3 stem are important for the binding of all the mAbs and that most mAbs seem to require the Pro residue that forms the GPGR beta hairpin turn in the V3 tip for binding. Based on the mapping, we determined that V3 sequence variation accounted for neutralization resistance of approximately half the viruses tested. Comparison of these results to those of select V3 mAbs with overall better neutralizing activities in the light of structural information illustrates how an antibody's mode of interaction with V3, driven by contact residue requirements, may restrict the antibody from accessing its epitope on different viruses. Based on the data we propose an angle of interaction with V3 that is less stringent on access for antibodies with cross-neutralizing activity compared to antibodies that neutralize relatively fewer viruses.
| S-EPMC2613967 | biostudies-literature
Project description:The third variable (V3) loop of HIV-1 gp120 is an immunodominant region targeted by neutralizing antibodies (nAbs). Despite limited breadth, better characterization of the structural details of the interactions between these nAbs and their target epitopes would enhance our understanding of the mechanism of neutralization and facilitate designing better immunogens to induce nAbs with greater breadth. Recently, we isolated two anti-V3 neutralizing monoclonal antibodies (MAbs), 10A3 and 10A37, from a rabbit immunized with gp120 of the M group consensus sequence. In this study, crystal structures of these MAbs bound to target epitopes were determined. 10A3 binds to the V3 crown (303TRKSIHIGPGRAF317) using the cradle binding mode, similar to human V3 MAbs encoded by IGHV5-51 germ line genes, and its epitope structure resembles that bound to the human antibodies. In contrast, 10A37, which exhibits greater breadth and potency than 10A3, binds the V3 crown and the succeeding stem region (308HIGPGRAFYTTGEI323). Unexpectedly, the 315RAFYTT320 portion of the epitope existed as helical turns, a V3 structure that has not been observed previously. Its main chain-dominated antigen-antibody interactions not only explain the broad neutralization of 10A37 but also show that its epitope is a potential vaccine target to be further evaluated. In conclusion, our study provides novel insights about neutralization-susceptible epitope structures of the V3 loop of HIV-1 gp120 and demonstrates that, despite low amino acid sequence similarity to human antibody germ line genes, rabbits can serve as a useful animal model to evaluate human vaccine candidates.IMPORTANCE The apex crown of V3 of HIV-1 gp120 is the most immunogenic region of the surface glycoprotein, and many MAbs targeting this region have been developed. Structural understanding of V3 crown MAbs not only can help understand how antibody responses target this unique region but also contribute to immunogen design for vaccine development. We present here crystal structures of two neutralizing V3 MAbs, 10A3 and 10A37, developed from a rabbit immunized with gp120. Our analysis of 10A3 in complex with V3 provided a detailed example of how epitope complexity can evolve with affinity maturation, while that of 10A37 revealed a novel V3 binding mode targeting the C-terminal side of the V3 crown and showed that this region can form a helical structure. Our study provides novel insights about neutralization-susceptible V3 epitope structures and demonstrates that rabbits can serve as a useful animal model to evaluate human vaccine candidates.
| S-EPMC5972897 | biostudies-literature
Project description:IntroductionImprinting broadly neutralizing antibody (bNAb) paratopes by shape complementary protein mimotopes represents a potential alternative for developing vaccine immunogens. This approach, designated as a Non-Cognate Ligand Strategy (NCLS), has recently been used for the identification of protein variants mimicking CD4 binding region epitope or membrane proximal external region (MPER) epitope of HIV-1 envelope (Env) glycoprotein. However, the potential of small binding proteins to mimic viral glycan-containing epitopes has not yet been verified.MethodsIn this work, we employed a highly complex combinatorial Myomedin scaffold library to identify variants recognizing paratopes of super candidate bNAbs, PGT121 and PGT126, specific for HIV-1 V3 loop epitopes.ResultsIn the collection of Myomedins called MLD variants targeted to PGT121, three candidates competed with gp120 for binding to this bNAb in ELISA, thus suggesting an overlapping binding site and epitope-mimicking potential. Myomedins targeted to PGT126 designated MLB also provided variants that competed with gp120. Immunization of mice with MLB or MLD binders resulted in the production of anti-gp120 and -Env serum antibodies. Mouse hyper-immune sera elicited with MLB036, MLB041, MLB049, and MLD108 moderately neutralized 8-to-10 of 22 tested HIV-1-pseudotyped viruses of A, B, and C clades in vitro.DiscussionOur data demonstrate that Myomedin-derived variants can mimic particular V3 glycan epitopes of prominent anti-HIV-1 bNAbs, ascertain the potential of particular glycans controlling neutralizing sensitivity of individual HIV-1 pseudoviruses, and represent promising prophylactic candidates for HIV-1 vaccine development.
| S-EPMC9772448 | biostudies-literature
Project description:The pandemic of Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome 2 coronavirus (SARS-CoV-2) is a continuing worldwide threat to human health and social economy. Historically, SARS-CoV-2 follows SARS and MERS as the third coronavirus spreading across borders and continents, but far more dangerous with long-lasting symptomatic consequences. The current situation is strong evidence that coronaviruses will continue to be pathogens of consequence in the future, thus calling for the development of neutralizing antibody-based prophylactics and therapeutics for prevention and treatment of COVID-19 and other human coronavirus diseases. This review summarized the progresses of developing neutralizing monoclonal antibodies against infection of SARS-CoV-2, SARS-CoV, and MERS-CoV, and discussed their potential applications in prevention and treatment of COVID-19 and other human coronavirus diseases.
| S-EPMC8716168 | biostudies-literature
Project description:Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic tick-borne virus, prevalent in more than 30 countries worldwide. Human infection by this virus leads to severe illness, with an average case fatality of 40%. There is currently no approved vaccine or drug to treat the disease. Neutralizing antibodies are a promising approach to treat virus infectious diseases. This study generated 37 mouse-derived specific monoclonal antibodies against CCHFV Gc subunit. Neutralization assays using pseudotyped virus and authentic CCHFV identified Gc8, Gc13, and Gc35 as neutralizing antibodies. Among them, Gc13 had the highest neutralizing activity and binding affinity with CCHFV Gc. Consistently, Gc13, but not Gc8 or Gc35, showed in vivo protective efficacy (62.5% survival rate) against CCHFV infection in a lethal mouse infection model. Further characterization studies suggested that Gc8 and Gc13 may recognize a similar, linear epitope in domain II of CCHFV Gc, while Gc35 may recognize a different epitope in Gc. Cryo-electron microscopy of Gc-Fab complexes indicated that both Gc8 and Gc13 bind to the conserved fusion loop region and Gc13 had stronger interactions with sGc-trimers. This was supported by the ability of Gc13 to block CCHFV GP-mediated membrane fusion. Overall, this study provides new therapeutic strategies to treat CCHF and new insights into the interaction between antibodies with CCHFV Gc proteins.
| S-EPMC10863865 | biostudies-literature
Project description:To provide protective immunity against circulating primary HIV-1 strains, a vaccine most likely has to induce broadly neutralizing antibodies to the HIV-1 envelope glycoprotein (Env) spike. Recombinant Env trimers such as the prototype BG505 SOSIP.664 that closely mimic the native Env spike can induce autologous neutralizing antibodies (NAbs) against relatively resistant (tier 2) primary viruses. Ideally, Env immunogens should present broadly neutralizing antibody epitopes but limit the presentation of immunodominant non-NAb epitopes that might induce off-target and potentially interfering responses. The V3 loop in gp120 is such a non-NAb epitope that can effectively elicit non-NAbs when animals are immunized with SOSIP.664 trimers. V3 immunogenicity can be diminished, but not abolished, by reducing the conformational flexibility of trimers via targeted sequence changes, including an A316W substitution in V3, that create the SOSIP.v4.1 and SOSIP.v5.2 variants. Here, we further modified these trimer designs by introducing leucine residues at V3 positions 306 and 308 to create hydrophobic interactions with the tryptophan residue at position 316 and with other topologically proximal sites in the V1V2 domain. Together, these modifications further stabilized the resulting SOSIP.v5.2 S306L/R308L trimers in the prefusion state in which V3 is sequestered. When we tested these trimers as immunogens in rabbits, the induction of V3 non-NAbs was significantly reduced compared with the SOSIP.v5.2 trimers and even more so compared with the SOSIP.664 prototype, without affecting the autologous NAb response. Hence, these additional trimer sequence modifications may be beneficial for immunization strategies that seek to minimize off-target non-NAb responses.
| S-EPMC5798299 | biostudies-literature
Project description:There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs: 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infection yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at subneutralizing concentrations. The neutralization activities of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies.
| S-EPMC3536401 | biostudies-literature
Project description:Ebola virus infections cause a deadly hemorrhagic disease for which no vaccines or therapeutics has received regulatory approval. Here we show isolation of three (Q206, Q314 and Q411) neutralizing monoclonal antibodies (mAbs) against the surface glycoprotein (GP) of Ebola virus identified in West Africa in 2014 through sequential immunization of Chinese rhesus macaques and antigen-specific single B cell sorting. These mAbs demonstrated potent neutralizing activities against both pseudo and live Ebola virus independent of complement. Biochemical, single particle EM, and mutagenesis analysis suggested Q206 and Q411 recognized novel epitopes in the head while Q314 targeted the glycan cap in the GP1 subunit. Q206 and Q411 appeared to influence GP binding to its receptor NPC1. Treatment with these mAbs provided partial but significant protection against disease in a mouse model of Ebola virus infection. These novel mAbs could serve as promising candidates for prophylactic and therapeutic interventions against Ebola virus infection.
| S-EPMC4867612 | biostudies-literature
Project description:Tetanus is a fatal disease caused by tetanus neurotoxin (TeNT). TeNT is composed of a light chain (Lc) and a heavy chain, the latter of which is classified into two domains, N-terminus Hn and C-terminus Hc. Several TeNT-neutralizing antibodies have been reported, but it remains unclear which TeNT domains are involved in neutralization. To further understand the mechanism of these antibodies, we isolated TeNT-reactive human antibody clones from peripheral blood mononuclear cells. We then analyzed the reactivity of the isolated antibody clones to each protein domain and their inhibition of Hc-ganglioside GT1b binding, which is critical for TeNT toxicity. We also investigated the TeNT-neutralizing ability of isolated antibody clones and showed that an Hn-reactive clone protected strongly against TeNT toxicity in mice. Furthermore, combination treatment of Hn-reactive antibody clones with both Hc-reactive and TeNT mix (the mixture of Hc, Hn, and Lc proteins)-reactive antibody clones enhanced the neutralizing effect. These results indicated that antibody clones targeting Hn effectively neutralized TeNT. In addition, the use of a cocktail composed of Hc-, Hn-, and TeNT mix-reactive antibodies provided enhanced protection compared to the use of each antibody alone.
| S-EPMC8190289 | biostudies-literature