CpG Oligodeoxynucleotides for Avian Immune Stimulation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current CpG oligodeoxynucleotides (ODNs) for immunomodulation in non-mammalian species lack specificity and efficacy, with no clear correlation between in vitro and in vivo activities, and no high-throughput screening methods are available for selecting effective CpG motifs, especially for veterinary applications.
Innovation Solution
Development of novel immunostimulatory non-methylated oligodeoxynucleotides with specific sequences and modifications, such as phosphorothioate bonds, that stimulate NF-κB or IRF3 activation, using a secreted alkaline phosphatase-based detection system for sensitive and correlated in vitro and in vivo activity assessment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional CpG oligodeoxynucleotides are used for immunomodulation in non-mammalian species, then some immunostimulatory effect is achieved, but the efficacy is low and lacks specificity
Solution Approach 1:
The patent applies local quality by designing CpG motifs with specific local sequence characteristics (e.g., GpCpGpT patterns, flanking nucleotides) that are optimized for recognition by chicken TLR21. Rather than using generic CpG sequences, the invention introduces specific local sequence features including particular 5' and 3' flanking regions and spacing patterns that enhance binding affinity and immunostimulatory activity in avian species.
Solution Approach 2:
The patent employs parameter changes by systematically varying key parameters of the CpG oligodeoxynucleotide structure including the number of CpG repeats (n=2-100), the length of flanking regions (x=3-10, z=0-10), the specific nucleotide compositions at various positions, and the presence of phosphorothioate modifications. These parameter optimizations enable the design of highly specific and potent immunostimulatory sequences for non-mammalian species.
2Reliability
If high concentrations of CpG ODNs are used to achieve immunostimulation, then some immune activation is obtained, but the required doses are too high for practical application
Solution Approach 1:
The patent applies preliminary action by performing extensive in silico design and in vitro screening to pre-identify optimal CpG motifs with predicted high activity before in vivo application. The invention uses a systematic approach to pre-optimize sequence parameters, flanking regions, and modifications based on structural requirements for TLR21 recognition, thereby identifying motifs that achieve maximum immunostimulatory effect at minimal concentrations.
Solution Approach 2:
The patent replaces the trial-and-error empirical approach with a rational design system based on understanding TLR21 recognition mechanisms. By substituting mechanical testing of numerous sequences with a knowledge-based design framework that incorporates TLR binding requirements, structural features, and immunological principles, the invention efficiently identifies high-potency motifs without requiring extensive high-concentration testing.
3Reliability
If random CpG motif selection is used, then some immunostimulatory activity is achieved, but the selection process is inefficient and lacks correlation between in vitro and in vivo activity
Solution Approach 1:
The patent implements feedback by establishing a correlated in vitro assay system using chicken cells expressing TLR21 that accurately predicts in vivo immunostimulatory activity. The invention creates a feedback loop where in vitro screening results directly inform the selection of motifs for in vivo testing, and in vivo results refine the understanding of effective sequence features. This feedback mechanism ensures high correlation between preliminary screening and final efficacy.
Solution Approach 2:
The patent applies preliminary action by developing and validating an in vitro screening system before conducting in vivo experiments. The invention establishes a preliminary selection process using cell-based assays that measure NF-κB activation and cytokine production in response to candidate CpG motifs, allowing efficient pre-screening of numerous sequences to identify the most promising candidates for subsequent in vivo evaluation.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The novel CpG ODNs demonstrate high immunomodulatory effects at low doses, with some active in nanomolar or even picomolar concentrations, enabling effective immunostimulation in veterinary contexts and reducing the required antigen component amounts in vaccines.
Implementation Method 1
the cellular response in mice to CpG-ODNs is mediated by the toll-like receptor 9 (TLR9)... certain forms of pathogen deoxyribonucleic acid (DNA) are amongst these PAMPs... non-methylated CpG motifs in bacterial DNA trigger murine B-cell activation
Implementation Method 2
using a secreted alkaline phosphatase-based detection system for sensitive and correlated in vitro and in vivo activity assessment
Data Source
Figure 1
Figure 2
Figure 3
AI summary
The present invention relates to immunostimulatory oligodeoxynucleotides, vectors and vaccines comprising such oligodeoxynucleotides, to their use as a medicament, to their use in preventing or combating infectious disease, to methods for the detection of such oligodeoxynucleotides and to cells to be used in these method.