Jawaharlal Nehru Center for Advanced Scientific ResearchFigure 1. The wild type Tat amino acid sequenceCodonFigure 2. Codon optimization at three different places in the Tat amino acid sequenceA Pattern Matching Algorithm for Codon Optimization and CpG Motif-Engineering in DNA Expression Vectors Ravi Vijaya Satya and Amar Mukherjee School of Engineering and Computer Science University of Central Florida Orlando, FL 32816 rvijaya, [email protected] Udaykumar Ranga Jawaharlal Nehru Center for Advanced Scientific Research Jakkur, Bangalore, India [email protected] Abstract Codon optimization enhances the efficiency of DNA expression vectors used in DNA vaccination and gene therapy by increasing protein expression. Additionally, certain nucleotide motifs have experimentally been shown to be immuno-stimulatory while certain others immuno-suppressive. In this paper, we present algorithms to locate a given set of immuno-modulatory motifs in the DNA expression vectors corresponding to a given amino acid sequence and maximize or minimize the number and the context of the immuno-modulatory motifs in the DNA expression vectors. The main contribution is to use multiple pattern matching algorithms to synthesize a DNA sequence for a given amino acid sequence and a graph theoretic approach for finding the longest weighted path in a directed graph that will maximize or minimize certain motifs. This is achieved using O(n2) time, where n is the length of the amino acid sequence. Based on this, we develop a software tool. Key Words: Codon optimization, immuno-modulatory motifs, multiple pattern matching, longest weighted path. 1. Introduction DNA vaccines have revolutionized the field of vaccine technology by demonstrating the ability to induce humoral and cellular immune responses in experimental animals and humans [9]. Immunization of animals with plasmid DNA encoding a protein antigen was an accidental observation that eventually led the way to a novel strategy of immunization. DNA vaccines, also known as "naked DNA" or "nucleic acid" vaccines, encode the antigens of pathogenic organisms including viruses, bacteria, fungi and parasites [40]. The protein antigen is processed within the cell and presented by the MHC-I and –II pathways thereby eliciting specific immune responses essential for controlling pathogenic infections [2]. Although DNA vaccines have been successful in generating strong immune responses in smaller animal model such as mouse, they have not been as efficient in larger species such as primates and humans [23, 3, 25, 4]. Stimulating both the arms of the immune system is often desirable for efficient control of infectious diseases especially in the larger animals. In the case of recombinant protein vaccines, immune-enhancers technically known as adjuvants, such as Freund’s adjuvants and Alum, are in use to enhance antigen specific immune responses. However, no such adjuvants are available for use in the context of DNA vaccines. The lack of suitable adjuvants for DNA vaccines is one important reason for the poor performance of the DNA vaccines in larger animals. Nucleotide sequence encoding a foreign protein is directly placed under the control of a mammalian promoter to construct a DNA vaccine. Several amino acids are encoded by more than one triplet codon and different organisms have variable requirement for codon preference [13]. Cloning of a wild type gene from a parasite into a DNA vaccine often leads to insufficient levels of protein synthesis in the host cell as a result of codon bias between the species. Successful immunization with DNA vaccines requires high expression of cloned genes to synthesize large quantities of the foreign protein. For instance, the overall genetic content of Human Immunodeficiency Virus-1 is AT-rich, while that of the human beings is CG-rich. The codon frequency of the pathogenic DNA embedded into the mammalian expression vector may not be optimal for adequate protein expression in the host resulting in low level protein expression. A potential solution for the codon bias is to optimize the codon sequences of a gene to suit the requirements of the host without altering the original amino acid sequence of the protein [41, 16]. This approach has been successful in eliciting strong immune responses in several species of experimental animals [8,28]. While immunization with synthetic genes, codon-optimized for mammalian expression stimulated strong immune response, immunization performed in parallel with wild type genes generated low or moderate levels of immune response [34, 36]. In addition to codon optimization of the synthetic genes, a range of molecular approaches is being evaluated to up-regulate immune responses generated by DNA vaccines. Co-expression of cytokine genes [20], co-stimulatory receptors [12, 35] or other immunemodulators [33], synthetic assembly of T-helper or CTL epitopes [6] and formulation with a variety of chemical adjuvants [11, 24, 33, 37, 39] have been some of the approaches reported. However, most of these approaches may not be suitable for human application due to toxic manifestations of the adjuvants. An ideal agent used as an adjuvant for DNA vaccine must enhance the immunogenicity without apparent cytotoxicity to the host. Engineering CpG islands into DNA vaccines has been one promising approach that showed enhanced immune responses [19]. The well-established immune-enhancing property of bacterial DNA has been mapped to sequence motifs consisting of un-methylated CpG dinucleotides flanked by base pairs in a specific context [18]. Two important differences between the bacterial and mammalian DNA enable the mammalian innate immune system to recognize the former as a foreign component. CpG motifs in bacteria are found at the expected 1:16 frequency; however, their frequency in mammals is 4 times less than expected. Bacterial CpG are non-methylated while those of mammals are mostly methylated. The mammalian immune system takes advantage of these two chemical differences between the bacterial and mammalian DNA to identify a bacterial infection and wage strong and rapid anti-bacterial immune responses [29]. CpG-mediated activation of the mammalian innate immune system has been extensively exploited in the vaccination technology. Co-injection of CpG containing oligonucleotides or empty vectors with protein antigens elicited potent immune response to the antigen. Methylation of the CpG motifs, on the