Construction of Engineered Nanoparticles with Tailored Densities of Self-Antigen for the Study of B-cell Activation

Abstract

Epitope density appears to be a critical factor in pathogens that can elicit effective immune responses. All the viruses that have efficacious FDA-approved target vaccines have high surface densities of antigens. HIV, on the other hand, has a limited number of envelope glycoproteins present on individual virions on average, a characteristic with which HIV can evade from human immunity. To investigate how immune cells, particularly B cells, recognize and differentiate surface antigen density, we aim to build nanoparticles with different densities of antigens on the surface. Liposomes are chosen as antigen carriers for their clinical safety and engineering versatility. Both ensemble biochemistry assay and single-molecule biophysical techniques are developed to determine the spatial density of two model proteins on liposomal surface. Our results showed the initial density of protein conjugated on Ni-chelating liposomes could be finely controlled but decreased overtime. In comparison, maleimide-liposomes performed well in both protein density control and conjugation stability. Through maleimide-cysteine reaction, we successfully constructed liposomes with varied surface density levels of TNF-α peptide, a model self-antigen that is critical in the pathogenesis of rheumatoid arthritis. Immunization of mice using liposomes that display TNF-α peptide at high density consistently elicited both IgM and class-switched IgG antibodies that are reactive towards self-antigen TNF-α protein. In transgenic mice lacking either functional T-cell receptors or MHC II on B cells, the liposomal particles elicited similar levels of IgM and IgG responses, implying that this is a T-independent process. Addition of CpG or T-cell epitope tetanus toxin peptide improved antibody responses elicited by liposomal particles but not by soluble antigen peptide. Furthermore, the Ab titer elicited can be increased by 1000-fold upon replacement of liposomes by bacteriophage Qβ virus-like particles of similar epitope densities. Remarkably, this enhancement of Ab titer is almost lost entirely in transgenic mice lacking TCR or MHC II, which uncovers T cell help as the dominant mechanism behind this enhancement. In conclusion, high epitope density alone can trigger antibody class switching in B cells, and the cognate T-cell help recruited by components in VLPs can further boost antibody response by promoting affinity maturation. As an exploratory work to build on this mechanism of immunogenicity, we attempted next to build HIV VLP using the recombinant HIV Gag protein, which is known to contain many well-defined human T cell epitopes. If successful, the display of HIV envelope glycoproteins on the surface of this VLP at high density may afford an attractive lead for HIV vaccine development. HIV Gag protein can self-assemble in vitro to HIV virus-like particles, however, the quantitative aspects and in particular, the yield of this process are poorly characterized. Our results revealed that purified Gag proteins assembled into aggregates as large as micron-sized particles that are visible under light microscope. This self-assembly is induced by addition of nucleic acids and interestingly, it is reversible with the addition of excess nucleic acids. Thus, further engineering of this process is required in order to make it useful for production of HIV VLPs. In summary, our study overall has answered a fundamental question in immunology regarding B-cell activation by particulate antigens, offering insights into the process of pathogen recognition. These findings will help guide future design of therapeutic vaccines for chronic conditions including cancer and Alzheimer’s disease, as well as prophylactic vaccine for infectious agents like HIV.