A Therapeutic Antigen-Presenting Cell-Targeting DNA Vaccine VB10.16 in HPV16-Positive High-Grade Cervical Intraepithelial Neoplasia: Results from a Phase I/IIa Trial.

PURPOSE: To evaluate the safety, immunogenicity and efficacy of a therapeutic DNA vaccine VB10.16, using a unique modular vaccine technology that is based on linking antigens to CCL3L1 targeting module, in women with HPV16-positive high-grade cervical intraepithelial neoplasia (CIN). PATIENTS AND METHODS: We conducted a first-in-human, open-label, phase I/IIa clinical trial of VB10.16 in subjects with confirmed HPV16-positive CIN 2/3. The primary endpoint was the proportion of participants with adverse events, including dose-limiting toxicities. Secondary outcome measures included measuring the E6/E7-specific cellular immune response. In the Expansion cohort HPV16 clearance, regression of CIN lesion size and grading were assessed during a 12-month follow-up period. RESULTS: A total of 34 women were enrolled: 16 in two dose cohorts and 18 in the expansion cohort. No serious adverse events or dose-limiting toxicities were observed, and none of the subjects discontinued treatment with VB10.16 due to an adverse event. Mild to moderate injection site reactions were the most commonly reported adverse event (79%). HPV16-specific T-cell responses were observed after vaccination in the majority of the subjects. In the expansion cohort, HPV16 clearance was seen in 8 of 17 evaluable subjects (47%). Reductions in lesion size were seen in 16 subjects (94%) and 10 subjects (59%) had regression to CIN 0/1. Correlation between strong IFNγ T-cell responses and lesion size reduction was statistically significant (P < 0.001). CONCLUSIONS: The novel therapeutic DNA vaccine VB10.16 was well tolerated and showed promising evidence of efficacy and strong HPV16-specific T-cell responses in subjects with high-grade CIN.

The Magnitude and IgG Subclass of Antibodies Elicited by Targeted DNA Vaccines Are Influenced by Specificity for APC Surface Molecules.

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.

Increased generation of HIV-1 gp120-reactive CD8+ T cells by a DNA vaccine construct encoding the chemokine CCL3.

DNA vaccines based on subunits from pathogens have several advantages over other vaccine strategies. DNA vaccines can easily be modified, they show good safety profiles, are stable and inexpensive to produce, and the immune response can be focused to the antigen of interest. However, the immunogenicity of DNA vaccines which is generally quite low needs to be improved. Electroporation and co-delivery of genetically encoded immune adjuvants are two strategies aiming at increasing the efficacy of DNA vaccines. Here, we have examined whether targeting to antigen-presenting cells (APC) could increase the immune response to surface envelope glycoprotein (Env) gp120 from Human Immunodeficiency Virus type 1 (HIV-1). To target APC, we utilized a homodimeric vaccine format denoted vaccibody, which enables covalent fusion of gp120 to molecules that can target APC. Two molecules were tested for their efficiency as targeting units: the antibody-derived single chain Fragment variable (scFv) specific for the major histocompatibility complex (MHC) class II I-E molecules, and the CC chemokine ligand 3 (CCL3). The vaccines were delivered as DNA into muscle of mice with or without electroporation. Targeting of gp120 to MHC class II molecules induced antibodies that neutralized HIV-1 and that persisted for more than a year after one single immunization with electroporation. Targeting by CCL3 significantly increased the number of HIV-1 gp120-reactive CD8+ T cells compared to non-targeted vaccines and gp120 delivered alone in the absence of electroporation. The data suggest that chemokines are promising molecular adjuvants because small amounts can attract immune cells and promote immune responses without advanced equipment such as electroporation.

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.

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 (LD78ß 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 LD78ß outperformed full length LD78ß-vaccine proteins in vitro. LD78ß DNA fusion vaccines induced improved T cell (both CD4+ and CD8+) and antibody responses in mice following plasmid injection and skin electroporation. Finally, LD78ß-vaccine proteins bound Rhesus macaque CCR5, setting the stage for targeted DNA immunization in non-human primates.

Human receptors of innate immunity (CD14, TLR2) are promising targets for novel recombinant immunoglobulin-based vaccine candidates.

Experiments in mice have suggested that engagement of receptors of innate immunity has an adjuvant effect on adaptive immune responses. Such studies need to be extended to humans. We have here constructed recombinant scFv-based vaccine candidate proteins (vaccibodies) that target human TLR2 and CD14 for delivery of large antigens. Vaccibodies are homodimers, each chain consisting of scFv specific for surface molecules on antigen-presenting cells (APC), a homodimerization motif, and an antigenic unit. The TLR2- and CD14-specific vaccibodies bound their respective target receptors expressed on transfected CHO cells and PBMC. Large proteins such as paired mouse Ckappa-domains (229 aa) and fragment C of tetanus toxin (TetC, 451 aa) could be expressed as antigenic units with intact serological determinants detected by mAb or polyclonal antisera. In the presence of monocytes, TLR2- and CD14-specific vaccibodies having either Ckappa or TetC as antigenic unit were 100-10,000 more efficient at stimulating T cell clones in vitro compared to non-targeted vaccibodies expressing the same antigens. The results show that TLR2 and CD14 are efficient targets for delivery of antigen to APC for stimulation of HLA class II-restricted CD4(+) T cells. Thus, receptors of innate immunity should be further explored as targets for vaccines.

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.

Idiotype-specific immunotherapy in multiple myeloma: suggestions for future directions of research.

Multiple myeloma (MM) remains a difficult-to-cure cancer and less than 20% of patients achieve long-term survival irrespective of the treatment delivered, including high-dose chemotherapy. Thus, new treatment modalities are urgently needed. Myeloma cells produce a monoclonal immunoglobulin (Ig) which is a truly tumor-specific antigen. The tumor-specific antigenic determinants are localized in the variable regions of the monoclonal Ig and are termed idiotopes (Id). Id-vaccination, i.e., vaccination with the autologous monoclonal Ig, has been performed in MM patients in order to elicit tumor-specific immune responses and possibly elimination of myeloma cells. However, clinical trials have not given the promising results obtained in mice. This review focuses on tolerance mechanisms that might hinder Id-specific immune responses in MM patients. New strategies for Id vaccination in MM are discussed.

DNA vaccines increase immunogenicity of idiotypic tumor antigen by targeting novel fusion proteins to antigen-presenting cells.

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.

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 Ckappa, presented on human leukocyte antigen-DR4, were inserted into a loop connecting beta-strands in C(H)1 of human gamma3. In the presence of monocytes, CD14-specific Troybodies were >100 times as efficient as a nontargeting control antibody (Ab) at stimulating Ckappa(40-48)-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 Ckappa 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.