Naked DNA vaccines have a number of advantages over conventional vaccines, but induce only weak immune responses. We have here investigated if this inadequacy may be overcome by inducing muscle to secrete fusion proteins with the ability to target antigen-presenting cells (APC). The novel targeted vaccines are homodimers with (i) two identical single-chain fragment variable (scFv) targeting units specific for MHC class II molecules on mouse APC, (ii) a human Ig hinge and C(H)3 dimerization unit, and (iii) two identical scFv tumor antigenic units (idiotypes) from B cell cancers. After plasmid injection and electroporation of mouse muscle, secreted vaccine proteins (vaccibodies) delivered idiotypic tumor antigen to APC in draining lymph nodes for induction of T and B cell responses that protected mice against tumor challenges with a multiple myeloma (MOPC315) and a B cell lymphoma (A20). Targeting to APC was essential for these effects. The results show that immunogenicity of plasmid DNA vaccines can be increased by inducing muscle to secrete proteins that target antigen to APC.

New influenza A viruses with pandemic potential periodically emerge due to viral genomic reassortment. In the face of pandemic threats, production of conventional egg-based vaccines is time consuming and of limited capacity. We have developed in this study a novel DNA vaccine in which viral hemagglutinin (HA) is bivalently targeted to MHC class II (MHC II) molecules on APCs. Following DNA vaccination, transfected cells secreted vaccine proteins that bound MHC II on APCs and initiated adaptive immune responses. A single DNA immunization induced within 8 d protective levels of strain-specific Abs and also cross-reactive T cells. During the Mexican flu pandemic, a targeted DNA vaccine (HA from A/California/07/2009) was generated within 3 wk after the HA sequences were published online. These results suggest that MHC II–targeted DNA vaccines could play a role in situations of pandemic threats. The vaccine principle should be extendable to other infectious diseases.

IntroductionB lymphomas and multiple myelomas (MMs) produce monoclonal Ig, the V regions of which can serve as targets for tumor-specific immune responses. 1,2 The V region antigenic determinants (idiotype [Id]) arise as a consequence of clone-specific V(D)J rearrangements and somatic mutations. Id can be directly recognized in 3 ways: (1) by Ab, 3 (2) by CD4 ϩ , 4 and (3) by CD8 ϩ T cells. 5 Id-specific CD4 ϩ and CD8 ϩ T cells recognize V region-derived Id peptides presented on MHC class II and I molecules, respectively. While B-lymphoma cells display all 3 types of Id targets on their cell surface, MM cells often express little surface Ig and MHC class II. Nevertheless, Id-specific CD4 ϩ T cells can confer protection against MHC class II-negative MM cells because secreted myeloma protein is processed and presented by tumorinfiltrating antigen-presenting cells (APCs) to Id-specific CD4 ϩ T cells. [6][7][8] Immunization with myeloma protein in complete Freund adjuvant (CFA) induced an Id-specific resistance against tumor challenge in mice, 1 a finding that has been confirmed and extended in a number of experimental mouse studies. Id vaccination has entered clinical trials, with more promising results in B-cell lymphoma 9 than in MM 10 patients. However, Id is a weak antigen and a number of innovative strategies have been used to increase its immunogenicity. Id-pulsed dendritic cells (DCs) 11 are promising, but suffer from labor-intensive manufacture. Attractive alternatives are to fuse Ig, Fab, or single-chain fragment variable (scFv) to GM-CSF, 12 chemokines, [13][14][15] CD40Ligand, 16 tetanus toxin fragment C, and to deliver these as protein 12,13,[16][17][18] or DNA 13,17,18 vaccines. Such immunizations have generated tumorprotective responses by mechanisms that are not fully elucidated, but that most likely include targeting of Id to APCs, APC maturation, or both.Chemokines control migration of specific leukocyte populations during inflammatory responses, hematopoiesis, and routine immune surveillance. RANTES (regulated upon activation normal T-cell expressed, CCL5) and MIP-1␣ (macrophage inflammatory protein 1␣, CCL3) are inflammatory chemokines. They both have high affinity for CCR1 and CCR5 expressed on T cells, monocytes, natural killer cells, and DCs.MIP-1␣ and RANTES self-associate to form high-molecularmass aggregates. 19,20 However, the activation state of chemokine monomers versus oligomers has been a disputed field. [21][22][23] It has been shown that chemokine receptors initiate their ligand-induced signaling cascades by receptor dimerization. [24][25][26] Monomeric variants of RANTES and MIP-1␤ retain full activity in vitro, but are devoid of activity in vivo, suggesting that these chemokines require oligomerization to recruit cells in vivo. 27 It has been proposed that although chemokines are able to interact with receptors as monomers, 21 glycosaminoglycan-induced oligomerization of chemokines can achieve higher order oligomers in vivo, and they may interact with receptors differently as dime...

Ligation of CD40 induces maturation of dendritic cells (DC) and could be a useful target for vaccines. In this study, we have constructed two types of Ab-based vaccine constructs that target mouse CD40. One type is a recombinant Ab with V regions specific for CD40 and has defined T cell epitopes inserted into its C region. The other type is a homodimer, each chain of which is composed of a targeting unit (single-chain fragment variable targeting CD40), a dimerization motif, and an antigenic unit. Such proteins bound CD40, stimulated maturation of DC, and enhanced primary and memory T cell responses. When delivered i.m. as naked DNA followed by electroporation, the vaccines induced T cell responses against MHC class II-restricted epitopes, Ab responses, and protection in two tumor models (myeloma and lymphoma). Two factors apparently contributed to these results: 1) agonistic ligation of CD40 and induction of DC maturation, and 2) delivery of Ag to APC and presentation on MHC class II molecules. These results highlight the importance of agonistic targeting of Ag to CD40 for induction of long-lasting and protective immune responses.

Different diseases require different immune responses for efficient protection. Thus, prophylactic vaccines should prime the immune system for the particular type of response needed for protection against a given infectious agent. We have here tested fusion DNA vaccines which encode proteins that bivalently target influenza hemagglutinins (HA) to different surface molecules on antigen presenting cells (APC). We demonstrate that targeting to MHC class II molecules predominantly induced an antibody/Th2 response, whereas targeting to CCR1/3/5 predominantly induced a CD8+/Th1 T cell response. With respect to antibodies, the polarizing effect was even more pronounced upon intramuscular (i.m) delivery as compared to intradermal (i.d.) vaccination. Despite these differences in induced immune responses, both vaccines protected against a viral challenge with influenza H1N1. Substitution of HA with ovalbumin (OVA) demonstrated that polarization of immune responses, as a consequence of APC targeting specificity, could be extended to other antigens. Taken together, the results demonstrate that vaccination can be tailor-made to induce a particular phenotype of adaptive immune responses by specifically targeting different surface molecules on APCs.

It has been difficult to translate promising results from DNA vaccination in mice to larger animals and humans. Previously, DNA vaccines encoding proteins that target Ag to MHC class II (MHC-II) molecules on APCs have been shown to induce rapid, enhanced, and long-lasting Ag-specific Ab titers in mice. In this study, we describe two novel DNA vaccines that as proteins target HLA class II (HLA-II) molecules. These vaccine proteins cross-react with MHC-II molecules in several species of larger mammals. When tested in ferrets and pigs, a single DNA delivery with low doses of the HLA-II–targeted vaccines resulted in rapid and increased Ab responses. Importantly, painless intradermal jet delivery of DNA was as effective as delivery by needle injection followed by electroporation. As an indication that the vaccines could also be useful for human application, HLA-II–targeted vaccine proteins were found to increase human CD4+ T cell responses by a factor of ×103 in vitro. Thus, targeting of Ag to MHC-II molecules may represent an attractive strategy for increasing efficacy of DNA vaccines in larger animals and humans.

Upon APC-targeted DNA vaccination, transfected cells secrete fusion proteins with targeting units specific for surface molecules on APC. In this study, we have tested several different targeting units for their ability to influence the magnitude and subclass of Ab responses to hemagglutinin from influenza A virus. The experiments employed bivalent homodimeric Ig-based molecules (vaccibodies). The overall efficiency in BALB/c mice depended on the targeting units in the following order: αMHC class II > αCD11c > αCD40 > Xcl-1 = MIP-1α > FliC > GM-CSF > Flt-3L > αDEC205. GM-CSF induced mainly IgG1, whereas Xcl1, MIP-1α, αCD40, and αDEC205 induced predominantly IgG2a. A more balanced mixture of IgG1 and IgG2a was observed with αCD11c, αMHC class II, Flt-3L, and FliC. Similar results of IgG subclass–skewing were obtained in Th1-prone C57BL/6 mice with a more limited panel of vaccines. IgG1 responses in BALB/c occurred early after immunization but declined relatively rapidly over time. IgG2a responses appeared later but lasted longer (>252 d) than IgG1 responses. The most efficient targeting units elicited short- and long-term protection against PR8 influenza (H1N1) virus in BALB/c mice. The results suggest that targeting of Xcr1+ conventional type 1 dendritic cells preferentially induces IgG2a responses, whereas simultaneous targeting of several dendritic cell subtypes also induces IgG1 responses. The induction of distinct subclass profiles by different surface molecules supports the APC–B cell synapse hypothesis. The results may contribute to generation of more potent DNA vaccines that elicit high levels of Abs with desired biologic effector functions.

Due to somatic recombination and hypermutation, Ig variable heavy (V H ) and light (V L ) regions contain unique immunogenic determinants, idiotopes (Id), which can stimulate T cells. To address the relevance of this in a human disease, monoclonal IgG (mAb)-secreting B cell clones were established from the cerebrospinal fluid (CSF) of two patients with multiple sclerosis (MS). HLA-DR-restricted CD4 + T cell lines and clones from CSF of both patients specifically recognized autologous CSF mAb. The CSF T cell clones produced IFN-c; some also produced TNF-a, IL-10 and IL-5. V H and V L on the monoclonal IgG derived from CSF B cells expressed amino acid replacements due to somatic mutations. A T cell epitope was mapped to a V H framework region, where an amino acid replacement was critical for the T cell recognition. The finding of Id-specific T cells and Id-bearing B cells in the CSF indicates that they coexist within the diseased organ, and provide a basis for the study of Id-driven T-B cell collaboration in a human autoimmune disease.