Heterodimeric Barnase-Barstar Vaccine Molecules: Influence of One versus Two Targeting Units Specific for Antigen Presenting Cells

<div><p>It is known that targeting of antigen to antigen presenting cells (APC) increases immune responses. However, it is unclear if more than one APC-specific targeting unit in the antigenic molecule will increase responses. To address this issue, we have here made heterodimeric vaccine molecules that each express four different fusion subunits. The bacterial ribonuclease barnase and its inhibitor barstar interact with high affinity, and the barnase-barstar complex was therefore used as a dimerization unit. Barnase and barstar were fused N-terminally with single chain fragment variable (scFv)s targeting units specific for either MHC class II molecules on APC or the hapten 5-iodo-4-hydroxy-3-nitrophenylacetyl (NIP). C-terminal antigenic fusions were either the fluorescent protein mCherry or scFv³¹⁵ derived from myeloma protein M315. The heterodimeric vaccine molecules were formed both <em>in vitro</em> and <em>in vivo</em>. Moreover, the four different fused moieties appeared to fold correctly since they retained their specificity and function. DNA vaccination with MHC class II-targeted vaccine induced higher mCherry-specific IgG1 responses compared to non-targeted control. Since mCherry and MHC class II are in trans in this heterodimer, this suggests that heterodimeric proteins are formed <em>in vivo</em> without prior protein purification. Surprisingly, one targeting moiety was sufficient for the increased IgG1 response, and addition of a second targeting moiety did not increase responses. Similar results were found in <em>in vitro</em> T cell assays; vaccine molecules with one targeting unit were as potent as those with two. In combination with the easy cloning strategy, the heterodimeric barnase-barstar vaccine molecule could provide a flexible platform for development of novel DNA vaccines with increased potency.</p> </div

<i>In vitro</i> characterization of the heterodimeric vaccine proteins.

<p>(A) Supernatants of transiently transfected HEK293 cells were analyzed by ELISA for secretion of heterodimeric vaccine proteins. Ab2.1-4 mAb was used as coat (binds scFv³¹⁵), and bound heterodimers were detected with biotinylated anti-mCherry mAb. (B) Western blot of DNP-purified vaccine proteins probed with biotinylated α6xHis under non-reducing (left) and reducing (right) conditions. (C) MHC class II specificity of proteins measured in flow cytometry. Bound proteins were detected with biotinylated anti-mCherry mAb and Streptavidin-PE. (D) Titration curves of affinity-purified vaccine proteins normalized by Western were analyzed in a NIP-specific ELISA. NIP-BSA was used as coat and bound proteins were detected with biotinylated anti-mCherry mAb. (E) Fluorescence microscopic analysis of transiently transfected HEK293 cells. 1. αMHCII/αMHCII, 2. αNIP/αNIP, 3. αMHCII/αNIP, and 4. Mock. ELISA results are shown as mean ± SD.</p

Heterodimer plasmid pairs used <i>f</i>or DNA immunization.

a<p>scFv from mAb αI-E^d, αNIP and myeloma protein M315 are denoted Fv^I-E, Fv^NIP and Fv³¹⁵, respectively. Barnase, Barstar and mCherry are denoted Bn, Bs and Ch, respectively.</p

Targeted heterodimers induced high levels of anti-mCherry antibodies.

<p>Mice were immunized by intradermal injection of DNA immediately followed by electroporation. Sera obtained at different time-points were analyzed for antigen-specific antibodies by ELISA. Levels of mCherry-specific Ig were measured in sera for 140 days. (A) Total κ⁺Ig anti-mCherry antibodies, (B) IgG1anti-mCherry antibodies, and (C) IgG2a anti-mCherry antibodies. ELISA results are shown as mean ± SEM.</p

The carboxyl-terminal domains of IgA and IgM direct isotype-specific polymerization and interaction with the polymeric immunoglobulin receptor

Mucosal surfaces are protected by polymeric immunoglobulins that are transported across the epithelium by the polymeric immunoglobulin receptor (pIgR). Only polymeric IgA and IgM containing a small polypeptide called the "joining" (J) chain can bind to the pIgR. J chain-positive IgA consists of dimers, and some larger polymers, whereas only IgM pentamers incorporate the J chain. We made domain-swap chimeras between human IgA1 and IgM and found that the COOH-terminal domains of the heavy chains (Ca3 and Cm4, respectively) dictated the size of the polymers formed and also which polymers incorporated the J chain. We also showed that chimeric IgM molecules engineered to contain Ca3 were able to bind the rabbit pIgR. Since the rabbit pIgR normally does not bind IgM, these results suggest that the COOH-terminal domain of the polymeric immunoglobulins is primarily responsible for interaction with the pIgR. Finally, we made a novel chimeric IgA immunoglobulin, containing the terminal domain from IgM. This recombinant molecule formed J chain-containing pentamers that could, like IgA, efficiently form covalent complexes with the human pIgR ectodomain, known as secretory component. This research was originally published in: Journal of Biological Chemistry. © the American Society for Biochemistry and Molecular Biology

Plasmid vectors expressing vaccine polypeptides.

a<p>scFv from mAb αI-E^d, αNIP and myeloma protein M315 are denoted Fv^I-E, Fv^NIP and Fv³¹⁵, respectively. Barnase, Barstar and mCherry are denoted Bn, Bs and Ch, respectively.</p