Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Minireviews
    • JVI Classic Spotlights
    • Archive
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JVI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Journal of Virology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Minireviews
    • JVI Classic Spotlights
    • Archive
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JVI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Pathogenesis and Immunity

Antibody Binding and Neutralization of Primary and T-Cell Line-Adapted Isolates of Human Immunodeficiency Virus Type 1

Joanne York, Kathryn E. Follis, Meg Trahey, Phillipe N. Nyambi, Susan Zolla-Pazner, Jack H. Nunberg
Joanne York
Montana Biotechnology Center, The University of Montana, Missoula, Montana 59812;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kathryn E. Follis
Montana Biotechnology Center, The University of Montana, Missoula, Montana 59812;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Meg Trahey
Montana Biotechnology Center, The University of Montana, Missoula, Montana 59812;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Phillipe N. Nyambi
New York University School of Medicine, New York, New York 10016; and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Susan Zolla-Pazner
New York University School of Medicine, New York, New York 10016; and
Veterans Affairs Medical Center, New York, New York 10010
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jack H. Nunberg
Montana Biotechnology Center, The University of Montana, Missoula, Montana 59812;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/JVI.75.6.2741-2752.2001
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

  • Tables
  • Fig. 1.
    • Open in new tab
    • Download powerpoint
    Fig. 1.

    HIVIG as a nondiscriminating measure of Env expression. COS-7 cells were transfected to express GFP and either 168P Env or 168C Env as described in Materials and Methods. (A) Flow cytometric analysis of HIVIG staining of GFP-positive cells expressing either 168P Env (dark line) or 168C Env (light line). (B) Transfected cultures were biotinylated on the cell surface with NHS-LC-biotin reagent (Pierce), and Env was immunoprecipitated with HIVIG and detected by Western blot analysis using avidin-horseradish peroxidase conjugate. Transfection efficiency was determined by immunochemical staining of fixed monolayers using HIVIG; the percentage of cells stained by HIVIG is indicated.

    • Open in new tab
    • Download powerpoint
    • Open in new tab
    • Download powerpoint
    • Open in new tab
    • Download powerpoint
    • Open in new tab
    • Download powerpoint
    Fig. 2.

    Flow cytometric analysis of MAb binding to 168P and 168C Envs. (A, C, E, and G) Neutralization sensitivity of 168P (dark line) and 168C (light line) viruses to the indicated MAbs was determined using U87-CD4-CXCR4 cells as described in Materials and Methods. Infected cells (foci) were determined by microscopic analysis of immunochemically stained monolayers, and the number of foci was compared to that obtained in the absence of MAb. (B, D, F, and H) COS-7 cells were transfected to express GFP and either 168P Env or 168C Env as described in Materials and Methods. Flow cytometric analysis of MAb binding to GFP-positive cells expressing either 168P Env (dark line) or 168C Env (light line) is illustrated. MAbs were used at the discriminating concentrations determined by neutralization as indicated in the legend to Fig. 3.

  • Fig. 3.
    • Open in new tab
    • Download powerpoint
    Fig. 3.

    Summary of MAb binding to 168P and 168C Envs. MAb binding was determined by flow cytometry as described in Materials and Methods and quantitated using MFC computation (CELLQuest software). The respective MFC values for binding to 168P and 168C Envs were normalized relative to HIVIG binding to account for modest differences in Env expression. The normalized values for MAb binding to 168P and 168C Envs are plotted. Among all experiments, MFC values for HIVIG binding to 168P and 168C Envs were 70 ± 35 and 67 ± 28, respectively. The ratio of HIVIG MFC values within any experiment (1.07 ± 0.39) was also consistent with the ratio of transfection efficiencies (1.01 ± 0.13). Representative MAb binding values compared within one experiment are shown. Purified MAbs were used at a concentration of 10 μg/ml (except for MAbs 58.2 and 59.1, which were used as a 1:100 dilution of ascites fluids), and sCD4 was used at a concentration of 50 μg/ml. At these concentrations, the relative amounts of neutralization of 168P and 168C, respectively, are shown in parentheses (as percentages); few if any of the antibody reagents significantly neutralized 168P. Symbols:Embedded Image, 50.1 (0, 95); ⊕, 58.2 (55, 99); Embedded Image, 59.1 (20, 70); ○, 257-D (50, 97);Embedded Image, 268-D (50, 95); ●, 447-52D (75, 99);Embedded Image, 559-64D (45, 95);Embedded Image, IgGb12 (25, 55); ⧫, F105 (30, 65); ▴, 17b (30, 80); ▵, sCD4 plus 17b (not applicable); ■, 2F5 (60, 75); □, F240 (0, 0);Embedded Image, sCD4 (80, 99 at 10 μg/ml).

  • Fig. 4.
    • Open in new tab
    • Download powerpoint
    Fig. 4.

    Summary of MAb binding to 320SI and 320SI-C3.3 Envs. Methods and MAb concentrations are as described in the legend to Fig.3. At these concentrations, the relative amounts of neutralization of 320SI and 320SI-C3.3, respectively, are shown in parentheses (as percentages); none of the antibody reagents significantly neutralized 320SI. Symbols:Embedded Image, 50.1 (0, 45); ⊕, 58.2 (10, 70); Embedded Image, 59.1 (15, 70); ⊚, 257-D (0, 60);Embedded Image, 268-D (0, 0); ●, 447-52D (2, 95);Embedded Image, 559-64D (20, 30); ◊, lgGb12 (25, 99); ⧫, F105 (10, 70); ▴, 17b (10, 60); ▵, sCD4 plus 17b (not applicable); ■, 2F5 (40, 99); □, F240 (0, 0);Embedded Image, sCD4 (0, 99 at 10 μg/ml). The mean ratio of HIVIG MFC values for 320SI and 320SI-C3.3 Envs was 0.87 ± 0.21, and these values varied among experiments similarly to those of 168P and 168C.

  • Fig. 5.
    • Open in new tab
    • Download powerpoint
    Fig. 5.

    Relative capture of PI and TCLA virions. Virus stocks were prepared from supernatants of acutely infected peripheral blood mononuclear cells (PI virus) or H9 cells (TCLA virus). The virion capture ELISA was performed as described by Nyambi and colleagues (42). V3-loop-directed MAbs 58.2, 268-D, 257-D, and 447-52D were bound to microtiter wells in 100 μl at a concentration of 10 μg/ml; HIVIG was bound at 50 μg/ml. Virions were subsequently captured from 100 μl containing 100 ng of p24 per ml. The amount of virion captured by the specific MAbs is expressed as a percentage of that captured by HIVIG, in order to normalize results across virus stocks. For clarity, we show the relative capture of TCLA virion versus PI virion as the ratio of the HIVIG-normalized percentages. In the experiments shown, the underlying amounts of virion p24 captured by HIVIG were as follows: 168P, 902 pg/ml; 168C, 927 pg/ml; 320SI, 370 pg/ml; and 320SI-C3.3, 257 pg/ml. The amounts of 168P and 320SI PI virus captured by the MAbs were as follows, respectively: 58.2, 299 and 384 pg/ml; 268-D, 439 and 116 pg/ml; 257-D, 665 and 196 pg/ml; and 447-52D, 864 and 180 pg/ml. The relative capture of 168C and 168P is shown in dark bars, and 320SI-C3.3 and 320SI in light bars. As calculable, correspondingly less TCLA virion was captured than PI virion. ∗, 320SI-C3.3 virions whose V3 loop sequence contains an alteration of the nominal MAb epitope.

  • Fig. 6.
    • Open in new tab
    • Download powerpoint
    Fig. 6.

    MAb capture of infectious PI and TCLA virions. Virions were captured from 168P and 168C virus stocks (dark and light symbols, respectively) using streptavidin-coated M-280 Dynabeads to which biotinylated MAb 50.1 had been bound. Infectivity retained by the MAb was assessed by culturing U87-CD4-CXCR4 cells with the extensively washed magnetic beads, and the numbers of infected cell foci were determined as described in Materials and Methods. Incubations of virions with MAb-coated beads were at 4 or 37°C (circle and square symbols), and retained infectivity is compared to that in the initial virus stock used for capture (triangle symbols). Streptavidin-coated beads that were incubated with nonbiotinylated MAb 50.1 (mock) served as specificity controls (data not plotted; all, ≤5 foci/well).

  • Fig. 7.
    • Open in new tab
    • Download powerpoint
    Fig. 7.

    Flow cytometric determination of the binding affinity of MAbs 50.1 and 447-52D to 168P and 168C Envs. In this experiment, cotransfected COS-7 cells expressing GFP and either 168P Env (dark lines) or 168C Env (light lines) were stained using the indicated concentrations of MAb 50.1 (A) or 447-52D (B). MFC values for MAb binding to GFP-positive cells were determined as described for Fig. 1and 2. The percentage of maximal binding was determined for each MAb dilution as the ratio of MFC values.

Tables

  • Figures
  • Table 1.

    Pedigreed PI and TCLA virusesa

    VirusIsolate typeCoreceptor(s) usedCentral V3 loopAdaptation-associated aa changesReference(s)
    168PPICCR5 and CXCR4 NIRKRIHIGPGRAFYTTG 28, 65
    168CTCLACXCR4 NIRKRIHIGPGRAFYTTR V2 I166R, C2 I282N, and V3 G318R
    320SIPICCR5 and CXCR4 NTRKGIHIGPGRAFYAAR 16
    320SI-C3.3TCLACCR5 and CXCR4 NTRKGIRIGPGRAFYAAR V2 I166K and V3 H317R
    • ↵a Amino acid (aa) changes in the central V3 loop of the TCLA virus are underlined.

  • Table 2.

    Env-directed MAbsa

    AntibodyTargetNominal epitopebSourceReference
    50.1V3 loop ....RIHIG......... Repligen Corp.63
    58.2V3 loop .......IGPGRAF.... Repligen Corp.63
    59.1V3 loop ........GPGRAF.... Repligen Corp.63
    257-DV3 loop ...KRIHI.......... S. Zolla-Pazner19
    268-DV3 loop ......HIGPGR...... S. Zolla-Pazner19
    447-52DV3 loop ........GPGR...... S. Zolla-Pazner19
    IgG1b12CD4-bsD. Burton, from ARRRP5 
    559-64DCD4-bsS. Zolla-Pazner25
    F105CD4-bsM. Posner50
    17bCD4-iSee reference 27 J. Robinson60
    2F5gp41gp41...ELDKWAH. Katinger40
    F240gp41gp41...aa 592–606M. Posner7 
    • ↵a CD4-bs, CD4-binding site; CD4-i, CD4 induced; aa, amino acids; ARRRP, NIH AIDS Research and Reference Reagent Program.

    • ↵b Nominal MAb epitopes are reported in the associated references. Central V3 loop epitopes are arrayed as in Table1.

PreviousNext
Back to top
Download PDF
Citation Tools
Antibody Binding and Neutralization of Primary and T-Cell Line-Adapted Isolates of Human Immunodeficiency Virus Type 1
Joanne York, Kathryn E. Follis, Meg Trahey, Phillipe N. Nyambi, Susan Zolla-Pazner, Jack H. Nunberg
Journal of Virology Mar 2001, 75 (6) 2741-2752; DOI: 10.1128/JVI.75.6.2741-2752.2001

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Journal of Virology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Antibody Binding and Neutralization of Primary and T-Cell Line-Adapted Isolates of Human Immunodeficiency Virus Type 1
(Your Name) has forwarded a page to you from Journal of Virology
(Your Name) thought you would be interested in this article in Journal of Virology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Antibody Binding and Neutralization of Primary and T-Cell Line-Adapted Isolates of Human Immunodeficiency Virus Type 1
Joanne York, Kathryn E. Follis, Meg Trahey, Phillipe N. Nyambi, Susan Zolla-Pazner, Jack H. Nunberg
Journal of Virology Mar 2001, 75 (6) 2741-2752; DOI: 10.1128/JVI.75.6.2741-2752.2001
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

Antibodies, Monoclonal
HIV Antibodies
HIV Infections
HIV-1

Related Articles

Cited By...

About

  • About JVI
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #Jvirology

@ASMicrobiology

       

 

JVI in collaboration with

American Society for Virology

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0022-538X; Online ISSN: 1098-5514