Science Spotlight

Antibodies are not only for neutralization

Vaccine and Infectious Disease Division and Public Health Sciences Division

Antibodies can protect against viral infection by many mechanisms, some of which include neutralization of the virus, agglutination, phagocytosis, and lysis of infected cells. Many of these mechanisms are difficult to test with multiple assays that can be performed for each. Most commonly, neutralization of the virus is measured, looking at antibody blocking of viral entry into a cell. Neutralization is thought to be especially important in protection against viruses like HIV. However, recent vaccine trials have shown contribution of non-neutralizing antibodies in protection. It is thought that a Fc receptor-medicated antibody effect is contributing. Antibodies from patients with early HIV infection and HIV nonprogressors have been found to induce antibody-dependent cellular toxicity (ADCC). These antibodies are also associated with better outcomes in mother to child transmission and overall HIV control. Because FcR-mediated activities can be produced by varying mechanisms there are multiple effector functional assays currently available. These include: ADCC, cell-mediated viral inhibition (ADCVI) and antibody- dependent cellular phagocytosis (ADCP). In a paper recently published in Journal of Immunology Dr. Huang (Vaccine and Infectious Disease Division, Fred Hutch) and colleges sought to define assays that best captured the entire Fc-mediated antibody effect, with the goal being to facilitate better understanding on the effects of non-neutralizing antibodies in protection.

In order to understand what potential Fc-mediated functional assays would be best, the authors isolated antibodies from HIV infected, uninfected, uninfected vaccinated (gp120 vaccination) participants and subjected them to seven functional assays using a known HIV antibody, b12 as a positive control. Of the seven assays four tested ADCC, two ADCP, and one of ADCVI. The assays are explained in greater detail in table 1. All seven assays demonstrated strong separation between positive and negative controls with high reproducibility. The authors compared antibodies from HIV-infected subjects against antibodies from uninfected vaccinated subjects using the seven assays. Small differences were found between groups in the BVADCC, GTL ADCC, and RFADCC assays with more activity from infected samples. The other tests showed no differences. When comparing antibodies from the placebo group to the vaccinated group the outcome was the same, showing a small difference in the same assays. Results from these tests led the researchers to conclude that the different assays captured relatively distinct functional activities. To compare directly across the assays, the researchers used all 90 HIV infected samples to compare each individual assay against the other six assays. This created a tiered clustering of ADCC assays (BVADCC, RFADCC, and GTL ADCC) together in a subclade and the other assays were in separate individual subclades. This further confirms the authors hypothesis that various functional assays are needed to fully understand an antibodies functional mechanism.

Table 1. Name and description of functional assays.

Assay Description


inhibition of infection of target cells at the bound virion stage


induction of granzyme B and uptake by target cells


killing of sensitized target cells labeled with dye


reduction of viral LUC activity in infected target cells


inhibition of virus production by target cells

Virion phagocytosis

phagocytosis of virus by effector cells


phagocytosis of antigen coated beads by effector cells

The antibodies collected from the HIV infected group were further broken down to allow for intragroup comparisons. The samples collected were from participants with three distinct histories of disease: HIV chronically infected with treatment, chronically infected without treatment, and elite controllers (people infected with HIV who control the virus). This allowed the team to compare the functional assays by HIV history. The results of this comparison indicated that the elite controller group had the highest functional correlation compared to all other groups. A principal component analysis (PCA) was conducted and the analysis confirmed similarities between the ADCC assays and discordance between the other assays. Lastly, the authors modified the b12 antibody by making point mutations in order to increase or decrease Fc receptor-mediated antibody function. The majority of the seven assays followed activity predicted by the mutations, however not all did. The authors attribute this discrepancy to the fact that this is a complex activity and that multiple interactions may be needed and that their mutations may not account for this.  

This paper is the first to use a common set of samples against multiple assays of Fc-mediated functional activity in a blinded study. With more and more vaccine studies showing the added benefit of non-neutralizing antibody mediated protection a study of this nature is of importance. This study identified individual assays that captured diverse activity, suggesting that multiple assays may be needed to fully elucidate an antibodies functional significance.   

Huang Y, Ferrari G, Alter G, Forthal DN, Kappes JC, Lewis GK, Love JC, Borate B, Harris L, Greene K, Gao H, Phan TB, Landucci G, Goods BA, Dowell KG, Cheng HD, Bailey-Kellogg C, Montefiori DC, Ackerman ME. 2016. Diversity of Antiviral IgG Effector Activities Observed in HIV-Infected and Vaccinated Subjects. J Immunol, 197(12), 4603-4612. PMCID: PMC5137799.

This work was supported by The Collaboration for AIDS Vaccine Discovery and the National Institutes of Health.