GP350 AS A NOVEL VACCINE PROTEIN CARRIER

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Purpose of the experiment: gp350 is an Epstein-Barr virus (EBV) protein that binds to CD21 on human and monkey, but not mouse, B cells. Previous studies have demonstrated that the co-crosslinking of CD21 and the B cell antigen receptor (BCR) on the surface of the B cell is highly synergistic for B cell activation and significantly lowers the amount of antigen required for optimal immunogenicity. CD21 is also expressed on follicular dendritic cells (FDC), present within germinal centers, where it is used to trap antigen (via bound complement) on the surface of the FDC and thus augment the germinal center reaction and subsequent immunity. A recent study demonstrated that linkage of C3d to a polysaccharide antigen leads to improved anti-polysaccharide responses in vivo. Thus, we propose that gp350 can be used as a novel carrier protein for polysaccharide conjugate vaccines in which gp350 not only serves to recruit T cell help (on the basis of it being a foreign protein) for the anti-polysaccharide response but provides an adjuvanting affect as well (on the basis of its ability to bind CD21), potentially allowing for the use of much lower doses of antigen in vivo. Further, we are constructing a dimer of gp350 that can be conjugated to peptides or proteins and used in a similar fashion to augment anti-peptide or anti-protein Ig responses. Preparation of conjugates: We have covalently linked gp350 to pneumococcal capsular polysaccharide, serotype 14 (PPS14). As a control we use the same chemistry and conjugation conditions to produce a similar conjugate of diphtheria toxoid (DT) covalently linked to PPS14. Further, we confirmed that the gp350-PPS14 conjugate retained its binding activity to CD21, using a CD21-expressing cell line. We also demonstrated that both conjugates are immunogenic in mice for both the anti-PPS14 and anti-protein response. Finally, we confirmed that the gp350 also specifically binds to peripheral blood B cells derived from either Cynomolgus or rhesus monkeys. Goals of current experiment: The goal of this initial experiment is a PROOF of PRINCIPLE, and are to directly compare the two conjugates using what historically should be optimal doses of antigen. This will give us baseline data that we can indeed elicit specific Ig responses in monkeys using these conjugates and to directly compare the quantitative responses between these two conjugates.