Novel Conjugates of Epitope Fusion Peptides with CpG ODN Display Enhanced Immunogenicity and HIV Recognition. (2004)

Abstract Vaccination strategies remain elusive that are effective against viral disease pathogens, yet remain gentle enough for widespread human use. We improved upon current strategies of utilizing CpG-ODN (CpG) in combination with peptide vaccines, by developing a model system that relies on the recognition of specific T cell epitopes from immunodominant antigens of HIV to explore single stranded CpG as an adjuvant. To take advantage of the specificity of epitope-based vaccines as well as the increased targeting capacity of the covalent addition of CpG to the peptide, we synthesized conjugates of CpG added to the amino terminus of our previously characterized epitope fusion peptides. We synthesized multiple forms of this CpG:peptide construct, and each possessed robust immunologic activity, in some cases exceeding 10-fold greater sensitivity than the equivalent mixture of peptide and CpG. Characterization of the immune recognition of DNA-peptide conjugates was carried out in a murine model of human HLA A2. Immunogenicity of DNA-peptide conjugates was superior in sensitivity to non-covalently linked mixtures of the same functional molecules as measured by peptide-mediated cytotoxicity and IFN-γ release, as well as protection against viral infection. Conjugate vaccines showed surprisingly robust activity against vaccinia virus (VV) expressing full-length HIV antigens in a challenge model, and efficiently lysed Jurkat T cells containing a functional HIV provirus. The minimal dose of 1.0 nmol of vaccine to accomplish this task is far less than the 50 nmol dose that was required using the free peptide and CpG combination. Using such a low level of vaccine translates well to future clinical use. However, we discovered that by including a booster, we could further reduce the dose by 10-fold (0.1 nmol) and still efficiently clear a VV infection. This experiment also highlighted that CpG or peptide alone was incapable of clearing the VV infection. The amount of vaccine (0.6 mg) is lower than comparable vaccines that have been published and demonstrated to be effective at clearing VV. It is likely that a successful HIV vaccine will require targeting multiple antigens to prevent escape by mutation of CTL epitopes. To address that concern, we synthesized a multiple epitope peptide, which encodes both the HIV-gag and -pol HLA A2-restricted CTL epitopes covalently linked to a T-help epitope as a model. Immunogenicity of both CTL epitopes was preserved as well as enhanced functional lytic recognition of HIV-infected Jurkat T cells. We have found that as many as four peptides can be easily attached to one ODN molecule with at least three epitopes assembled into each peptide, therefore, as many as 12 separate epitopes can be assembled onto the ODN. Receptor-mediated targeting of CpG may increase the effectiveness of peptide vaccines as antigens. This approach should be easily applicable to other infectious disease pathogens such as small pox, influenza virus, and SARS. Enhancement of sensitivity of immune recognition by covalent attachment of DNA to epitope peptides should be further evaluated as a novel prophylactic vaccine strategy for HIV infection and other infectious diseases.