Biomedical subjects

Doris Apt

Publications and source records attributed to Doris Apt.

4 recordsLinked to original sources

PubMed2006

A chimeric tetravalent dengue DNA vaccine elicits neutralizing antibody to all four virus serotypes in rhesus macaques.

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.

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PubMed2005

Tetravalent neutralizing antibody response against four dengue serotypes by a single chimeric dengue envelope antigen.

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.

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PubMed2005

Diverse plasmid DNA vectors by directed molecular evolution of cytomegalovirus promoters.

Genetic vaccinations, gene therapy, and manufacturing of therapeutic proteins would benefit from promoter sequences that provide improved or prolonged expression levels. The cytomegalovirus (CMV) promoter is one of the most potent promoters known to date, and no previous examples of improved activity of this promoter by sequence mutagenesis have been reported. This study describes directed molecular evolution of CMV promoters derived from two human and two nonhuman primate strains of CMV by DNA shuffling and screening. Libraries of chimeric promoters were screened and analyzed for expression levels and immune responses, using plasmid DNA vectors encoding luciferase and beta-galactosidase. The results indicate that high functional diversity among CMV promoters can be generated, and the chimeric promoters selected after two rounds of DNA shuffling and particularly designed screening assays provided approximately 2-fold increased luciferase reporter gene expression and anti-beta-galactoside antibody response in vivo when compared with wild-type promoters. Sequence analysis of the shuffled promoters identified several mutations potentially contributing to the observed enhanced or reduced promoter activities and identified a 42-nucleotide region that appears obsolete for the functioning of the CMV promoter. Taken together, these data demonstrate the feasibility of generating diverse promoter sequences by DNA shuffling and screening methods, and provide novel structure- function information about CMV promoters. DNA shuffling and screening technologies provide a new approach to promoter optimization and development of optimal expression vectors for genetic vaccinations, gene therapy, and protein expression.

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PubMed2004

Overcoming antigenic diversity and improving vaccines using DNA shuffling and screening technologies.

Viral, bacterial and parasitic pathogens have evolved multiple strategies to evade the immune response, facilitate transmission and establish chronic infections. One of the underlying strategies that pathogens have evolved is antigenic variation of immune response targets that reduce the affinity of antigen binding to antibodies and major histocompatability complex class I and II receptors. Vaccine candidates generally target a limited number of these antigen variants or combine antigens from several variants to include in multivalent vaccine formulations. DNA shuffling and screening technologies, also known as MolecularBreeding (Maxygen, Inc.) directed molecular evolution, have been successfully used to identify and develop novel and chimaeric vaccine candidates capable of inducing immune responses that recognise and control multiple antigenic variants. DNA shuffling and screening strategies also select vaccine candidates with improved immunogenicity, increased expression as recombinant polypeptides and improved growth of whole viruses in cell culture. As DNA shuffling and screening strategies can be applied to many pathogens, there remain numerous applications of DNA shuffling to solve challenging problems in vaccine process development and manufacture.

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