A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples

Ackerman, Margaret E.; Moldt, Brian; Wyatt, Richard T.; Dugast, Anne‐Sophie; McAndrew, Elizabeth; Tsoukas, Stephen; Jost, Stéphanie; Berger, Christoph T.; Sciaranghella, Gaia; Liu, Qingquan; Irvine, Darrell J.; Burton, Dennis R.; Alter, Galit

doi:10.1016/j.jim.2010.12.016

Cited by 402 publications

(442 citation statements)

References 47 publications

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“…To reduce inhibition due to NK cells alone, only KIR3DS1 -donor cells were used (31). A subset of antibody samples were treated with PNGaseF (New England Biolabs) to remove N-linked glycans, as previously described (32). The presence of inactivated PNGaseF did not affect the ADCVI assay results.…”

Section: Methodsmentioning

confidence: 99%

Natural variation in Fc glycosylation of HIV-specific antibodies impacts antiviral activity

Ackerman

Crispin²,

Yu³

et al. 2013

J. Clin. Invest.

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313

349

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While the induction of a neutralizing antibody response against HIV remains a daunting goal, data from both natural infection and vaccine-induced immune responses suggest that it may be possible to induce antibodies with enhanced Fc effector activity and improved antiviral control via vaccination. However, the specific features of naturally induced HIV-specific antibodies that allow for the potent recruitment of antiviral activity and the means by which these functions are regulated are poorly defined. Because antibody effector functions are critically dependent on antibody Fc domain glycosylation, we aimed to define the natural glycoforms associated with robust Fc-mediated antiviral activity. We demonstrate that spontaneous control of HIV and improved antiviral activity are associated with a dramatic shift in the global antibody-glycosylation profile toward agalactosylated glycoforms. HIV-specific antibodies exhibited an even greater frequency of agalactosylated, afucosylated, and asialylated glycans. These glycoforms were associated with enhanced Fc-mediated reduction of viral replication and enhanced Fc receptor binding and were consistent with transcriptional profiling of glycosyltransferases in peripheral B cells. These data suggest that B cell programs tune antibody glycosylation actively in an antigen-specific manner, potentially contributing to antiviral control during HIV infection.

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Section: Methodsmentioning

confidence: 99%

Natural variation in Fc glycosylation of HIV-specific antibodies impacts antiviral activity

Ackerman

Crispin²,

Yu³

et al. 2013

J. Clin. Invest.

Self Cite

313

349

View full text Add to dashboard Cite

show abstract

“…ADCP activity was determined using a flow cytometric assay that measures the uptake of fluorescent gp120-coated beads by monocytic THP-1 cells in the presence of subject antibody. The antibodydependent phagocytosis assay was performed as previously described, with subject antibodies tested at a concentration of 6.25 g/ml (29,30). ADCC activity was characterized using the rapid fluorometric ADCC (RFADCC) assay with NK cells as effectors, as previously described (31).…”

Section: Methodsmentioning

confidence: 99%

Divergent Antibody Subclass and Specificity Profiles but Not Protective HLA-B Alleles Are Associated with Variable Antibody Effector Function among HIV-1 Controllers

Lai

Licht

Dugast

et al. 2014

J Virol

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Understanding the coordination between humoral and cellular immune responses may be the key to developing protective vaccines, and because genetic studies of long-term HIV-1 nonprogressors have associated specific HLA-B alleles with spontaneous control of viral replication, this subject group presents an opportunity to investigate relationships between arms of the adaptive immune system. Given evidence suggesting that cellular immunity may play a role in viral suppression, we sought to determine whether and how the humoral immune response might vary among controllers. Significantly, Fc-mediated antibody effector functions have likewise been associated with durable viral control. In this study, we compared the effector function and biophysical features of HIV-specific antibodies in a cohort of controllers with and without protective HLA-B alleles in order to investigate whether there was evidence for multiple paths to HIV-1 control, or whether cellular and humoral arms of immunity might exhibit coordinated profiles. However, with the exception of IgG2 antibodies to gp41, HLA status was not associated with divergent humoral responses. This finding did not result from uniform antibody responses across subjects, as controllers could be regrouped according to strong differences in their HIV-specific antibody subclass specificity profiles. These divergent antibody profiles were further associated with significant differences in nonneutralizing antibody effector function, with levels of HIVspecific IgG1 acting as the major distinguishing factor. Thus, while HLA background among controllers was associated with minimal differences in humoral function, antibody subclass and specificity profiles were associated with divergent effector function, suggesting that these features could be used to make functional predictions. Because these nonneutralizing antibody activities have been associated with spontaneous viral control, reduced viral load, and nonprogression in infected subjects and protection in vaccinated subjects, understanding the specific features of IgGs with potentiated effector function may be critical to vaccine and therapeutic antibody development. IMPORTANCEIn this study, we investigated whether the humoral and cellular arms of adaptive immunity exhibit coordinated or compensatory activity by studying the antibody response among HIV-1 controllers with different genetic backgrounds.

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“…Cells containing small concentrated fluorescent spots have positive scores, whereas cells showing little and diffuse fluorescence have negative scores. For the analysis of the proteins' nuclear internalization, we used an IDEAS software feature, the similarity score (SS) (22), to compare the similarity of the nuclear (DRAQ5/CyTRAK) and labeled-protein staining patterns. All events showing a positive SS were considered with high similarity between proteins and DRAQ5/CyTRAK, thus indicating a nuclear localization of the proteins.…”

Section: Methodsmentioning

confidence: 99%

Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 1 Matrix Proteins Specify Different Capabilities To Modulate B Cell Growth

Caccuri

Giagulli

Reichelt

et al. 2014

J Virol

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Exogenous HIV-1 matrix protein p17 (p17) deregulates the function of different cells after its N-terminal loop (AT20) binding to the chemokine receptors CXCR1 and CXCR2. One site within AT20 has been recently found to be the major determinant of viral fitness following transmission of simian immunodeficiency virus (SIV) to the human host. Therefore, we sought to determine whether SIV matrix protein (MA) was already capable of interacting with CXCR1 and CXCR2 and mimic p17 biological activities rather than this being a newly acquired function during host adaptation. We show here that SIV MA binds with the same affinity of p17 to CXCR1 and CXCR2 and displays both p17 proangiogenic on human primary endothelial cells and chemotactic activity on human primary monocytes and B cells. However, SIV MA exhibited a higher degree of plasticity than p17 in the C terminus, a region known to play a role in modulating B cell growth. Indeed, in contrast to p17, SIV MA was found to activate the phosphatidylinositol 3-kinase/Akt signaling pathway and strongly promote B cell proliferation and clonogenic activity. Interestingly, we have recently highlighted the existence of a Ugandan HIV-1 strain-derived p17 variant (S75X) with the same B cell growth-promoting activity of SIV MA. Computational modeling allowed us to hypothesize an altered C terminus/core region interaction behind SIV MA and S75X activity. Our findings suggest the appearance of a structural constraint in the p17 C terminus that controls B cell growth, which may help to elucidate the evolutionary trajectory of HIV-1. IMPORTANCEThe HIV-1 matrix protein p17 (p17) deregulates the biological activities of different cells after binding to the chemokine receptors CXCR1 and CXCR2. The p17 functional domain responsible for receptors interaction includes an amino acid which is considered the major determinant of SIV replication in humans. Therefore, we sought to determine whether SIV matrix protein (SIV MA) already had the ability to bind to both chemokine receptors rather than being a function newly acquired during host adaptation. We show here that SIV MA binds to CXCR1 and CXCR2 and fully mimics the p17 proangiogenic and chemokine activity. However, it differs from p17 in its ability to signal into B cells and promote B cell growth and clonogenicity. Computational analysis suggests that the accumulation of mutations in the C-terminal region may have led to a further SIV MA adaptation to the human host. This finding in turn sheds light on the evolutionary trajectory of HIV-1.

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A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples

Cited by 402 publications

References 47 publications

Natural variation in Fc glycosylation of HIV-specific antibodies impacts antiviral activity

Natural variation in Fc glycosylation of HIV-specific antibodies impacts antiviral activity

Divergent Antibody Subclass and Specificity Profiles but Not Protective HLA-B Alleles Are Associated with Variable Antibody Effector Function among HIV-1 Controllers

Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 1 Matrix Proteins Specify Different Capabilities To Modulate B Cell Growth

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