Agnete Brunsvik Fredriksen

Delivery of Antigen to CD40 Induces Protective Immune Responses against Tumors

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.

Human chemokine MIP1α increases efficiency of targeted DNA fusion vaccines

Efficacy of DNA vaccination has been improved in mice by fusion vaccines targeting antigen to antigen-presenting cells (APC) via chemokine receptors. Here, we aimed at extending this strategy to large animals and humans. Fusion proteins equipped with human MIP1α (LD78β isoform) retained functional activity and conformational correctness of targeting and antigenic units, respectively. Fusion proteins improved responses of cloned human CD4+ T cells, and a two amino acid NH(2)-truncated version of LD78β outperformed full length LD78β-vaccine proteins in vitro. LD78β DNA fusion vaccines induced improved T cell (both CD4+ and CD8+) and antibody responses in mice following plasmid injection and skin electroporation. Finally, LD78β-vaccine proteins bound Rhesus macaque CCR5, setting the stage for targeted DNA immunization in non-human primates.

DNA Vaccine that Targets Hemagglutinin to MHC Class II Molecules Rapidly Induces Antibody-Mediated Protection against Influenza

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.

Cerebrospinal fluid T cell clones from patients with multiple sclerosis : recognition of idiotopes on monoclonal IgG secreted by autologous cerebrospinal fluid B cells

Due to somatic recombination and hypermutation, Ig variable heavy (VH) and light (VL) 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‐γ; some also produced TNF‐α, IL‐10 and IL‐5. VH and VL 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 VH 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.

The Specificity of Targeted Vaccines for APC Surface Molecules Influences the Immune Response Phenotype

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.

Targeted DNA vaccines for enhanced induction of idiotype-specific B and T cells

Background: Idiotypes (Id) are antigenic determinants localized in variable (V) regions of Ig. Id-specific T and B cells (antibodies) play a role in immunotherapy of Id+ tumors. However, vaccine strategies that enhance Id-specific responses are needed. Methods: Id+ single-chain fragment variable (scFv) from multiple myelomas and B cell lymphomas were prepared in a fusion format that bivalently target surface molecules on antigen-presenting cells (APC). APC-specific targeting units were either scFv from APC-specific mAb (anti-MHC II, anti-CD40) or chemokines (MIP-1α, RANTES). Homodimeric Id-vaccines were injected intramuscularly or intradermally as plasmids in mice, combined with electroporation. Results: (i) Transfected cells secreted plasmid-encoded Id+ fusion proteins to extracellular fluid followed by binding of vaccine molecules to APC. (ii) Targeted vaccine molecules increased Id-specific B and T cell responses. (iii) Bivalency and xenogeneic sequences both contributed to enhanced responses. (iv) Targeted Id DNA vaccines induced tumor resistance against challenges with Id+ tumors. (v) Human MIP-1α targeting units enhanced Id-specific responses in mice, due to a cross reaction with murine chemokine receptors. Thus, targeted vaccines designed for humans can be quality tested in mice. (vi) Human Id+ scFv from four multiple myeloma patients were inserted into the vaccine format and were successfully tested in mice. (vii) Human MIP-1α vaccine proteins enhanced human T cell responses in vitro. (viii) A hypothetical model for how the APC-targeted vaccine molecules enhance Id-specific T and B cells is presented. Conclusion: Targeted DNA Id-vaccines show promising results in preclinical studies, paving the way for testing in patients.

Targeted idiotype‐fusion DNA vaccines for human multiple myeloma: preclinical testing

Objectives:  A homodimeric fusion DNA vaccine targeting idiotype (Id) to antigen‐presenting cells (APC) induced robust tumor protection in a mouse model of multiple myeloma (MM). Similar Id vaccine molecules were generated for four patients with MM with three main objectives: (i) do the vaccine molecules induce bona fide anti‐Id immune responses in mice? (ii) does targeting of the vaccine molecules to APC enhance immune responses? (iii) can anti‐Id antibodies, generated as by‐product in vaccinated mice, be used to establish sensitive assays for complete remission (CR) prior to patient vaccination?

T cells from multiple sclerosis patients recognize multiple epitopes on Self-IgG.

Abstract The highly diversified variable regions of immunoglobulin (Ig) molecules contain immunogenic determinants denoted idiotopes. We have previously reported that T cells from multiple sclerosis (MS) patients recognize IgG from autologous cerebrospinal fluid (CSF), and mapped a T‐cell epitope to an IgG idiotope. To test the ability of CSF IgG molecules to elicit a broad polyclonal T‐cell response in MS, we have analysed T‐cell responses in the blood and CSF against idiotope peptides spanning complementarity determining region (CDR) 3 and somatic mutations within the variable regions of monoclonal CSF IgG. Consistent with a diversified idiotope‐specific T‐cell repertoire, CD4+ T cells from both patients recognized several idiotope peptides presented by HLA‐DR molecules. Mutations were critical for T‐cell recognition, as T cells specific for a mutated CDR1 peptide did not recognize corresponding germline‐encoded peptides. One T‐cell clone recognized both an idiotope peptide and the B‐cell clone expressing this idiotope, compatible with endogenous processing and presentation of this idiotope by B cells. These results suggest that mutated CSF IgG from MS patients carry several T‐cell epitopes, which could mediate intrathecal IgG production and inflammation in MS through idiotope‐driven T–B‐cell collaboration.

Antigen Targeting to Human HLA Class II Molecules Increases Efficacy of DNA Vaccination

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.

Human CD14 is an efficient target for recombinant immunoglobulin vaccine constructs that deliver T cell epitopes.

It has been shown in the mouse that recombinant immunoglobulin (Ig) molecules with T cell epitopes inserted into the constant domain (Troybodies) can target antigen‐presenting cells (APC) for efficient delivery of T cell epitopes. Here, we have extended the Troybody concept to human applications. Moreover, we show that a receptor of innate immunity, CD14, which is a part of the lipopolysaccharide receptor complex on monocyte APC, is an efficient target. For construction of CD14‐specific Troybodies, we used rearranged variable(diversity)joining regions cloned from the 3C10 mouse B cell hybridoma. As a model T cell epitope, amino acids 40–48 of mouse Cκ, presented on human leukocyte antigen‐DR4, were inserted into a loop connecting β‐strands in CH1 of human γ3. In the presence of monocytes, CD14‐specific Troybodies were >100 times as efficient as a nontargeting control antibody (Ab) at stimulating Cκ40–48‐specific/DR4‐restricted T cells. Presentation was dependent on the conventional processing pathway for presentation on major histocompatibility complex (MHC) class II molecules. Enhanced presentation of the Cκ epitope was most likely a result of increased loading of MHC class II molecules, as the CD14‐specific monoclonal Ab 3C10 did not induce maturation of the APC. The results show that CD14, a receptor of innate immunity, may be a promising target of recombinant Ig‐based vaccines for elicitation of T cell responses in humans.