SOSIP trimer-specific antibodies isolated from a SHIV-infected monkey with versus without a pre-blocking step with gp41

2021 ◽  
Author(s):  
Natasha N. Duggan ◽  
Kim L. Weisgrau ◽  
Diogo M. Magnani ◽  
Eva G. Rakasz ◽  
Ronald C. Desrosiers ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Mononuclear Cells ◽  
Broadly Neutralizing Antibodies ◽  
Peripheral Blood Mononuclear ◽  
Specific Antibodies ◽  
Neutralizing Activity ◽  
Infected Monkey ◽  
External Domain ◽  
Hiv 1

BG505 SOSIP.664 (hereafter referred to as SOSIP), a stabilized trimeric mimic of the HIV-1 envelope spike resembling the native viral spike, is a useful tool for isolating anti-HIV-1 neutralizing antibodies. We screened long-term SHIV-AD8 infected rhesus monkeys for potency and breadth of serum neutralizing activity against autologous and heterologous viruses: SHIV-AD8, HIV-1 YU2, HIV-1 JR-CSF, and HIV-1 NL4-3. Monkey rh2436 neutralized all viruses tested and showed strong reactivity to the SOSIP trimer, suggesting this was a promising candidate for attempts at monoclonal antibody (mAb) isolation. Monoclonal antibodies were isolated by performing single B-cell sorts from peripheral blood mononuclear cells (PBMC) by FACS using the SOSIP trimer as a probe. An initial round of sorted cells revealed the majority of isolated mAbs were directed to the gp41 external domain portion of the SOSIP trimer and were mostly non-neutralizing against tested isolates. A second sort was performed, introducing a gp41 blocking step prior to PBMC staining and FACS sorting. These isolated mAbs bound SOSIP trimer but were no longer directed to the gp41 external domain portion. A significantly higher proportion of mAbs with neutralizing activity were obtained with this strategy. Our data show this pre-blocking step with gp41 greatly increases the yield of non-gp41 reactive, SOSIP- specific mAbs and increases the likelihood of isolating mAbs with neutralizing activity. Importance Recent advancements in the field have focused on the isolation and use of broadly neutralizing antibodies for both prophylaxis and therapy. Finding a useful probe to isolate broad potent neutralizing antibodies while avoiding non-neutralizing antibodies is important. The SOSIP trimer has been shown to be a great tool for this purpose because it binds known broadly neutralizing antibodies. However, the SOSIP trimer can isolate non-neutralizing antibodies as well, including gp41-specific mAbs. Introducing a pre-blocking step with gp41 recombinant protein decreased the percent of gp41-specific antibodies isolated with SOSIP probe, as well as increased the number of neutralizing antibodies isolated. This method could be used as a tool to increase the chances of isolating neutralizing antibodies.

scholarly journals A glycoside analog of mammalian oligomannose formulated with a TLR4-stimulating adjuvant elicits HIV-1 cross-reactive antibodies

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jean-François Bruxelle ◽  
Tess Kirilenko ◽  
Nino Trattnig ◽  
Yiqiu Yang ◽  
Matteo Cattin ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Envelope Protein ◽  
Protein Sequence ◽  
Neutralizing Antibodies ◽  
Human Antibody ◽  
Broadly Neutralizing Antibodies ◽  
Specific Antibodies ◽  
Strong Binding ◽  
Hiv Envelope ◽  
Hiv 1

AbstractThe occurrence of oligomannose-specific broadly neutralizing antibodies (bnAbs) has spurred efforts to develop immunogens that can elicit similar antibodies. Here, we report on the antigenicity and immunogenicity of a CRM197-conjugate of a previously reported oligomannose mimetic. Oligomannose-specific bnAbs that are less dependent on interactions with the HIV envelope protein sequence showed strong binding to the glycoconjugates, with affinities approximating those reported for their cognate epitope. The glycoconjugate is also recognized by inferred germline precursors of oligomannose-specific bnAbs, albeit with the expected low avidity, supporting its potential as an immunogen. Immunization of human-antibody transgenic mice revealed that only a TLR4-stimulating adjuvant formulation resulted in antibodies able to bind a panel of recombinant HIV trimers. These antibodies bound at relatively modest levels, possibly explaining their inability to neutralize HIV infectivity. Nevertheless, these findings contribute further to understanding conditions for eliciting HIV-cross-reactive oligomannose-specific antibodies and inform on next steps for improving on the elicited response.

scholarly journals Transcriptome Sequencing of Peripheral Blood Mononuclear Cells from Elite Controller-Long Term Non Progressors

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Francisco Díez-Fuertes ◽  
Humberto Erick De La Torre-Tarazona ◽  
Esther Calonge ◽  
Maria Pernas ◽  
María del Mar Alonso-Socas ◽  
...  
Keyword(s):  
Disease Progression ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Elite Controller ◽  
Type I ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Hiv 1

Abstract The elite controller (EC)-long term non-progressor (LTNP) phenotype represent a spontaneous and advantageous model of HIV-1 control in the absence of therapy. The transcriptome of peripheral blood mononuclear cells (PBMCs) collected from EC-LTNPs was sequenced by RNA-Seq and compared with the transcriptomes from other phenotypes of disease progression. The transcript abundance estimation combined with the use of supervised classification algorithms allowed the selection of 20 genes and pseudogenes, mainly involved in interferon-regulated antiviral mechanisms and cell machineries of transcription and translation, as the best predictive genes of disease progression. Differential expression analyses between phenotypes showed an altered calcium homeostasis in EC-LTNPs evidenced by the upregulation of several membrane receptors implicated in calcium-signaling cascades and intracellular calcium-mobilization and by the overrepresentation of NFAT1/Elk-1-binding sites in the promoters of the genes differentially expressed in these individuals. A coordinated upregulation of host genes associated with HIV-1 reverse transcription and viral transcription was also observed in EC-LTNPs –i.e. p21/CDKN1A, TNF, IER3 and GADD45B. We also found an upregulation of ANKRD54 in EC-LTNPs and viremic LTNPs in comparison with typical progressors and a clear alteration of type-I interferon signaling as a consequence of viremia in typical progressors before and after receiving antiretroviral therapy.

scholarly journals A glycoside analog of mammalian oligomannose formulated with a TLR4-stimulating adjuvant elicits HIV-1 cross-reactive antibodies

2021 ◽  
Author(s):  
Jean-Francois Bruxelle ◽  
Tess Kirilenko ◽  
Nino Trattnig ◽  
Yiqiu Yang ◽  
Matteo Cattin ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Envelope Protein ◽  
Protein Sequence ◽  
Neutralizing Antibodies ◽  
Human Antibody ◽  
Broadly Neutralizing Antibodies ◽  
Specific Antibodies ◽  
Strong Binding ◽  
Hiv Envelope ◽  
Hiv 1

Abstract The occurrence of oligomannose-specific broadly neutralizing antibodies (bnAbs) has spurred efforts to develop immunogens that can elicit similar antibodies. Here, we report on the antigenicity and immunogenicity of a CRM197-conjugate of a previously reported oligomannose mimetic. Oligomannose-specific bnAbs that are less dependent on interactions with the HIV envelope protein sequence showed strong binding to the glycoconjugates, with affinities approximating those reported for their cognate epitope. The glycoconjugate is also recognized by germline precursors of oligomannose-specific bnAbs, albeit with the expected low avidity, supporting its potential as an immunogen. Immunization of human-antibody transgenic mice revealed that only a TLR4-stimulating adjuvant formulation evoked antibodies able to bind a panel of recombinant HIV trimers. These antibodies bound at relatively modest levels, likely explaining their inability to neutralize HIV infectivity. Nevertheless, these findings contribute further to understanding conditions for eliciting HIV-cross-reactive oligomannose-specific antibodies and inform on next steps for improving on the elicited response.

scholarly journals Insight in miRNome of Long-Term Non-Progressors and Elite Controllers Exposes Potential RNAi Role in Restraining HIV-1 Infection

2020 ◽  
Vol 9 (8) ◽  
pp. 2452
Author(s):  
Rubén Ayala-Suárez ◽  
Francisco Díez-Fuertes ◽  
Esther Calonge ◽  
Humberto Erick De La Torre Tarazona ◽  
María Gracia-Ruíz de Alda ◽  
...  
Keyword(s):  
Immune Response ◽  
Mononuclear Cells ◽  
Elite Controllers ◽  
Peripheral Blood Mononuclear ◽  
Extreme Phenotypes ◽  
Blood Mononuclear Cells ◽  
Before And After ◽  
Anti Hiv ◽  
Hiv 1

Long-term non-progressors (LTNP) and elite controllers (EC) represent spontaneous natural models of efficient HIV-1 response in the absence of treatment. The main purposes of this work are to describe the miRNome of HIV-1 infected patients with different extreme phenotypes and identify potentially altered pathways regulated by differentially expressed (DE) miRNAs. The miRNomes from peripheral blood mononuclear cells (PBMCs) of dual phenotype EC-LTNP or LTNP with detectable viremia and HIV-infected patients with typical progression before and after treatment, were obtained through miRNA-Seq and compared among them. The administration of treatment produces 18 DE miRNAs in typical progressors. LTNP condition shows 14 DE miRNA when compared to typical progressors, allowing LTNP phenotype differentiation. A set of four miRNAs: miR-144-3p, miR-18a-5p, miR-451a, and miR-324 is strongly downregulated in LTNP and related to protein regulation as AKT, mTOR, ERK or IKK, involved in immune response pathways. Deregulation of 28 miRNA is observed between EC-LTNP and viremic-LTNP, including previously described anti-HIV miRNAs: miR-29a, associated with LTNP phenotype, and miR-155, targeting different pre-integration complexes such as ADAM10 and TNPO3. A holistic perspective of the changes observed in the miRNome of patients with different phenotypes of HIV-control and non-progression is provided.

scholarly journals Neutralizing Antibody Responses following Long-Term Vaccination with HIV-1 Env gp140 in Guinea Pigs

2018 ◽  
Vol 92 (13) ◽  
pp. e00369-18 ◽  
Author(s):  
Christine A. Bricault ◽  
James M. Kovacs ◽  
Alexander Badamchi-Zadeh ◽  
Krisha McKee ◽  
Jennifer L. Shields ◽  
...  
Keyword(s):  
Guinea Pigs ◽  
Neutralizing Antibodies ◽  
Antibody Responses ◽  
Tier 2 ◽  
Broadly Neutralizing Antibodies ◽  
Vaccination Regimen ◽  
Tier 1 ◽  
Clade C ◽  
Hiv 1

ABSTRACTA vaccination regimen capable of eliciting potent and broadly neutralizing antibodies (bNAbs) remains an unachieved goal of the HIV-1 vaccine field. Here, we report the immunogenicity of longitudinal prime/boost vaccination regimens with a panel of HIV-1 envelope (Env) gp140 protein immunogens over a period of 200 weeks in guinea pigs. We assessed vaccine regimens that included a monovalent clade C gp140 (C97ZA012 [C97]), a tetravalent regimen consisting of four clade C gp140s (C97ZA012, 459C, 405C, and 939C [4C]), and a tetravalent regimen consisting of clade A, B, C, and mosaic gp140s (92UG037, PVO.4, C97ZA012, and Mosaic 3.1, respectively [ABCM]). We found that the 4C and ABCM prime/boost regimens were capable of eliciting greater magnitude and breadth of binding antibody responses targeting variable loop 2 (V2) over time than the monovalent C97-only regimen. The longitudinal boosting regimen conducted over more than 2 years increased the magnitude of certain tier 1 NAb responses but did not increase the magnitude or breadth of heterologous tier 2 NAb responses. These data suggest that additional immunogen design strategies are needed to induce broad, high-titer tier 2 NAb responses.IMPORTANCEThe elicitation of potent, broadly neutralizing antibodies (bNAbs) remains an elusive goal for the HIV-1 vaccine field. In this study, we explored the use of a long-term vaccination regimen with different immunogens to determine if we could elicit bNAbs in guinea pigs. We found that longitudinal boosting over more than 2 years increased tier 1 NAb responses but did not increase the magnitude and breadth of tier 2 NAb responses. These data suggest that additional immunogen designs and vaccination strategies will be necessary to induce broad tier 2 NAb responses.

scholarly journals PCR-Based Assay To Quantify Human Immunodeficiency Virus Type 1 DNA in Peripheral Blood Mononuclear Cells

2000 ◽  
Vol 38 (2) ◽  
pp. 630-634 ◽  
Author(s):  
Cindy Christopherson ◽  
Yorda Kidane ◽  
Brian Conway ◽  
John Krowka ◽  
Haynes Sheppard ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells ◽  
The Mean ◽  
Long Term Survivors ◽  
Hiv 1

An assay that quantifies the amount of human immunodeficiency virus type 1 (HIV-1) DNA in peripheral blood mononuclear cells has been developed. PCR amplification of the HIV-1 DNA is performed in the presence of an internal quantitation standard, and colorimetric detection of the amplified product is performed with microwell plates. The copies of HIV-1 DNA are normalized to total genomic DNA input. The assay has an analytical sensitivity of 10 input copies per amplification reaction and a three-log detection range. In an analysis of sequential samples from patients on combination therapy, HIV-1 DNA was quantifiable for all individuals tested, including those with undetectable plasma HIV-1 RNA. In a separate study, a comparison of HIV-1 DNA levels was made with a group of long-term survivors and progressors. The mean HIV-1 DNA levels were lower in the long-term survivors than in the progressors (P, 0.04). The mean HIV-1 RNA levels were also lower, but the difference was not statistically significant (P, 0.164). A quantitative DNA assay will provide an additional tool to gain insight into the natural history of infection and the continued efficacy of potent antiretroviral therapies.

scholarly journals Primary HIV-1 and Infectious Molecular Clones Are Differentially Susceptible to Broadly Neutralizing Antibodies

Vaccines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 782
Author(s):  
Jiae Kim ◽  
Venigalla B. Rao ◽  
Mangala Rao
Keyword(s):  
Neutralizing Antibodies ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Virus Transmission ◽  
Early Time ◽  
Design Strategies ◽  
Peripheral Blood Mononuclear ◽  
Infectious Molecular Clones ◽  
Hiv 1

To prevent the spread of HIV-1, a vaccine should elicit antibodies that block viral entry for all cell types. Recently, we have developed a virus capture assay to quantitatively examine early time points of infection. Here we present data on the ability of bNAbs to inhibit capture (1 h) or replication (48 h) of purified primary acute or chronic HIV or infectious molecular clones (IMCs) in human peripheral blood mononuclear cells (PBMCs) as quantified by qRT-PCR. Although bNAbs significantly inhibited HIV-1 replication in PBMCs in a virus subtype and in a PBMC-donor specific manner, they did not inhibit virus capture of primary viruses. In contrast, IMC capture and replication in PBMCs and purified CD4+ T cells were significantly inhibited by bNAbs, thus indicating that unlike IMCs, primary HIV-1 may initially bind to other cell surface molecules, which leads to virus capture even in the presence of bNAbs. Our results demonstrate that the initial interactions and some aspects of infectivity of primary HIV-1 and IMCs are inherently different, which underscores the importance of studying virus transmission using primary viruses in in vitro studies, an issue that could impact HIV-1 vaccine design strategies.

scholarly journals Neutralization Breadth and Potency of Single-Chain Variable Fragments Derived from Broadly Neutralizing Antibodies Targeting Multiple Epitopes on the HIV-1 Envelope

2019 ◽  
Vol 94 (2) ◽  
Author(s):  
Rebecca T. van Dorsten ◽  
Bronwen E. Lambson ◽  
Constantinos Kurt Wibmer ◽  
Marc S. Weinberg ◽  
Penny L. Moore ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Passive Immunization ◽  
Loss Of Function ◽  
Single Chain ◽  
Broadly Neutralizing Antibodies ◽  
Variable Regions ◽  
Neutralizing Activity ◽  
Single Chain Variable Fragments ◽  
Key Residues ◽  
Hiv 1

ABSTRACT Passive administration of HIV-directed broadly neutralizing antibodies (bNAbs) can prevent infection in animal models, and human efficacy trials are under way. Single-chain variable fragments (scFv), comprised of only the variable regions of antibody heavy and light chains, are smaller molecules that may offer advantages over full-length IgG. We designed and expressed scFv of HIV bNAbs prioritized for clinical testing that target the V2-apex (CAP256-VRC26.25), V3-glycan supersite (PGT121), CD4 binding site (3BNC117), and MPER (10E8v4). The use of either a 15- or 18-amino-acid glycine-serine linker between the heavy- and light-chain fragments provided adequate levels of scFv expression. When tested against a 45-multisubtype virus panel, all four scFv retained good neutralizing activity, although there was variable loss of function compared to the parental IgG antibodies. For CAP256-VRC26.25, there was a significant 138-fold loss of potency that was in part related to differential interaction with charged amino acids at positions 169 and 170 in the V2 epitope. Potency was reduced for the 3BNC117 (13-fold) and PGT121 (4-fold) scFv among viruses lacking the N276 and N332 glycans, respectively, and in viruses with a longer V1 loop for PGT121. This suggested that scFv interacted with their epitopes in subtly different ways, with variation at key residues affecting scFv neutralization more than the matched IgGs. Remarkably, the scFv of 10E8v4 maintained breadth of 100% with only a minor reduction in potency. Overall, scFv of clinically relevant bNAbs had significant neutralizing activity, indicating that they are suitable for passive immunization to prevent HIV-1 infection. IMPORTANCE Monoclonal antibodies have been isolated against conserved epitopes on the HIV trimer and are being investigated for passive immunization. Some of the challenges associated with full-sized antibody proteins may be overcome by using single-chain variable fragments (scFv). These smaller forms of antibodies can be produced more efficiently, may show fewer off-target effects with increased tissue penetration, and are more adaptable to vectored-mediated expression than IgG. Here, we demonstrate that scFv of four HIV-directed bNAbs (CAP256-VRC26.25, PGT121, 3BNC117, and 10E8v4) had significant neutralizing activity against diverse global strains of HIV. Loss of potency and/or breadth was shown to be due to increased dependence of the scFv on key residues within the epitope. These smaller antibody molecules with functional activity in the therapeutic range may be suitable for further development as passive immunity for HIV prevention.

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