FabULOUS™ in HIV Vaccine Research

January 26, 2023 | References |

The Fc N-glycosylation profile can be linked to non-neutralizing functions in a vaccine. In a recent study by Chen et al., FabULOUS digestion provided valuable insights in HIV vaccine research following N-glycosylation analysis of the Fc region in mouse antibodies.

Finding a successful vaccine against the global pathogen HIV is a demanding mission. Researchers believe that the virus needs to be attacked from multiple angles, including both extensive neutralizing antibody functions and non-neutralizing antibody functions.
A group of American researchers have been studying the effects of vaccination with HIV envelope antigen gp120 bound to nanofibers of peptide Q11 (gp120-Q11). An increased antibody response toward homologous and heterologous antigens have been observed in mice vaccinated with gp120-Q11 in comparison to soluble gp120. Recently, the same study was performed in rabbits. Comparable or improved binding capacity was observed toward all targets with gp120-Q11. However, with rabbit antibodies’ ability to bind to human Fc receptors, the IgG Fc N-glycosylation profile, which plays an important role in Fc-mediated non-neutralizing antibody functions, was analyzed as well. To investigate the reproducibility in other mammals, the same Fc N-glycosylation evaluation done in rabbits was also performed in mice.

FabULOUS was used to digest the gp120 specific serum mice antibodies into Fc and Fab after reaction with mouse HIV gp120 envelope protein on magnetic beads, enabling analysis of the Fc glycosylation profile. FabULOUS (SpeB) is a cysteine protease that digests in the hinge region of IgG from several different species and subclasses, generating a homogenous pool of Fab fragments. The serum was collected from the animals at a certain time after vaccinations. An adjuvant was given together with the immunization, with exception for some unadjuvanted mice.
The analysis of adjuvanted samples revealed both differences and similarities in the result from the mice and rabbits after vaccination with gp120-Q11 or gp120 in solution. Fucosylation and monogalactosylation increased after immunization with gp120-Q11 for both species, while the total glycosylation profile was similar for the two vaccination groups. However, while levels of sialylation, bisection, galactosylation, digalactosylation all increased with gp120-Q11 in rabbits, they decreased in mice. Agalactosylation on the other hand increased with gp120-Q11 in mice, while vaccination with gp120-Q11 or gp120 in solution gave similar results in rabbits. Interestingly, the unadjuvanted immunization with gp120-Q11 in mice showed an increase in sialylation and bisection, like the adjuvanted rabbits. In addition, gp120-Q11 or soluble gp120 unadjuvanted vaccinations in mice gave similar levels of total galactosylation, however, some offsetting differences were observed in the respective levels of a-, mono- and digalactosylated glycoforms.
Overall, the studies have shown effects of vaccination with gp120-Q11 both on neutralizing and non-neutralizing functions. In this process, FabULOUS served as a valuable tool to prove that modulation in N-glycosylation was not limited to rabbits but were also observed in mice.


Chen et al., 2022. Self-assembling peptide nanofiber HIV vaccine elicits robust vaccine-induced antibody functions and modulates Fc glycosylation Immunology.









FabULOUS™ – Above hinge digestion of IgG