ABSTRACT
Immunization against tumor-associated antigens is a promising approach to cancer therapy and prevention, but it faces several challenges and limitations, such as tolerance mechanisms associated with self-antigens expressed by the tumor cells. Costimulatory molecules B7.1 (CD80) and B7.2 (CD86) have improved the efficacy of gene-based and cell-based vaccines in animal models and are under investigation in clinical trials. However, their efficacy as vaccine adjuvants is likely limited by the fact that they mediate both stimulatory and inhibitory signals to T cells via CD28 and CTLA-4, respectively. To overcome these limitations, we have generated a B7.1-like, chimeric costimulatory molecule with preferential binding to CD28, named CD28-binding protein (CD28BP), which we combined with a modified, nonself tumor antigen variant of epithelial cell adhesion molecule (EpCAM), named TAg25. TAg25 induced a cross-reactive immune response against human wild-type EpCAM upon DNA vaccination in cynomolgus monkeys. However, TAg25 DNA immunization alone or in combination with human (h) B7.1 induced no detectable antigen-specific T cells in the peripheral blood of the animals. In contrast, TAg25 combined with CD28BP induced both CD4 and CD8 T cells specific for EpCAM. Moreover, TAg25 combined with CD28BP induced significantly higher levels of EpCAM-specific antibodies than TAg25 plus hB7.1. These improved adjuvant properties of CD28BP, when compared with hB7.1, illustrate the importance of CD28 costimulation in vaccine responses in nonhuman primates and warrant further studies on the potential of CD28BP in improving the efficacy of cancer vaccines.
Subject(s)
Antigens, Neoplasm/immunology , Cancer Vaccines , Carrier Proteins/immunology , Cell Adhesion Molecules/immunology , Recombinant Fusion Proteins/immunology , Recombinant Proteins/immunology , Animals , Antigens, Neoplasm/genetics , B7-1 Antigen/genetics , B7-1 Antigen/immunology , CD28 Antigens/immunology , Cancer Vaccines/genetics , Cancer Vaccines/immunology , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Carrier Proteins/pharmacology , Cell Adhesion Molecules/genetics , Cell Proliferation , Cross Reactions , Drug Synergism , Epithelial Cell Adhesion Molecule , Humans , Immunization, Secondary , Interferon-gamma/metabolism , Lymphocyte Activation/drug effects , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Macaca fascicularis , Neoplasms/therapy , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Peptide Fragments/immunology , Peptide Fragments/pharmacology , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/pharmacology , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Self Tolerance/drug effects , Self Tolerance/immunology , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
DNA shuffling and screening technologies were used to produce chimeric DNA constructs expressing antigens that shared epitopes from all four dengue serotypes. Three shuffled constructs (sA, sB and sC) were evaluated in the rhesus macaque model. Constructs sA and sC expressed pre-membrane and envelope genes, whereas construct sB expressed only the ectodomain of envelope protein. Five of six, and four of six animals vaccinated with sA and sC, respectively, developed antibodies that neutralized all 4 dengue serotypes in vitro. Four of six animals vaccinated with construct sB developed neutralizing antibodies against 3 serotypes (den-1, -2 and -3). When challenged with live dengue-1 or dengue-2 virus, partial protection against dengue-1 was observed. These results demonstrate the utility of DNA shuffling as an attractive tool to create tetravalent chimeric dengue DNA vaccine constructs, as well as a need to find ways to improve the immune responses elicited by DNA vaccines in general.
Subject(s)
Antibodies, Viral/biosynthesis , Dengue Virus/immunology , Dengue/prevention & control , Vaccines, DNA/immunology , Viral Vaccines/immunology , Animals , Antibodies, Viral/immunology , Antigens, Viral/genetics , Antigens, Viral/immunology , DNA Shuffling , Dengue/immunology , Dengue/virology , Dengue Virus/classification , Dengue Virus/drug effects , Dengue Virus/genetics , Directed Molecular Evolution , Epitopes , Humans , Macaca mulatta , Male , Neutralization Tests , Recombinant Fusion Proteins/immunology , Vaccines, DNA/administration & dosage , Vaccines, DNA/classificationABSTRACT
We employed DNA shuffling and screening technologies to develop a single recombinant dengue envelope (E) antigen capable of inducing neutralizing antibodies against all four antigenically distinct dengue serotypes. By DNA shuffling of codon-optimized dengue 1-4 E genes, we created a panel of novel chimeric clones expressing C-terminal truncated E antigens that combined epitopes from all four dengue serotypes. DNA vaccines encoding these novel chimeras induced multivalent T cell and neutralizing antibody responses against all four dengue serotypes in mice. By contrast, a mixture of four unshuffled, parental DNA vaccines failed to produce tetravalent neutralizing antibodies in mice. The neutralizing antibody titers for some of these antigens could be further improved by extending the sequences to express full-length pre-membrane and envelope proteins. The chimeric antigens also protected mice against a lethal dengue-2 virus challenge. These data demonstrate that DNA shuffling and associated screening can lead to the selection of multi-epitope antigens against closely related dengue virus serotypes and suggest a broad utility for these technologies in optimizing vaccine antigens.
