The neutralization properties of a HIV-specific antibody are markedly altered by glycosylation events outside the antigen-binding domain.
Neutralizing Abs constitute a pivotal mechanism of the adaptive immune response against HIV-1 infection. Yet, most of the Abs that appear in the circulation during HIV infection are nonneutralizing. In this study, we report a dramatic change of the neutralizing properties of a human Ab reactive with the nonneutralizing epitope termed cluster I on the HIV-1 transmembrane protein gp41 when the Ab was produced in Chinese hamster ovary (CHO)-K1 cells. Our laboratory has previously reported that the Ab F240, when produced in a hybridoma, is nonneutralizing as assessed by standard neutralization assays. The F240 IgG1 Ab expressed in CHO cells acquired a strong neutralization activity against a broad range of HIV isolates without a change in immunoreactivity. Sequencing of the F240 mRNAs produced in the parental hybridoma and CHO cells revealed identical sequences, suggesting that acquired neutralization resulted from cell-specific posttranslational modifications. We found that the Ab produced by CHO cells is glycosylated to a greater extent than the parental Ab produced by the hybridoma. Moreover, treatment with peptide N-glycosidase F abrogated F240 neutralization, in an isolate-specific manner, but not Ab b12 neutralization. Interestingly, the F240 isotype-switched variants IgG3 and IgG4, also expressed in CHO cells, exhibited identical immunoreactivity to IgG1 isotypes but had clear differences in viral neutralization. These results suggest that structural features of the Ig molecule other than the primary sequence of the variable regions play a more prominent role in HIV neutralization than anticipated.
Miranda
Luis R
LR
Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA.
Duval
Mark
M
Doherty
Heather
H
Seaman
Michael S
MS
Posner
Marshall R
MR
Cavacini
Lisa A
LA
D000818:Animals; D019380:Anti-HIV Agents / Q000737:chemistry / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D000918:Antibody Specificity; D001666:Binding Sites, Antibody / Q000235:genetics; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D015483:HIV Antibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015700:HIV Envelope Protein gp41 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D017578:Immunoglobulin Class Switching / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009500:Neutralization Tests; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D019380:Anti-HIV Agents; D000911:Antibodies, Monoclonal; D015483:HIV Antibodies; D015700:HIV Envelope Protein gp41; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins