Advanced next-generation vaccines for dengue and other flaviviral infections.

Notifiable flaviviral pathogens, particularly dengue, continue to pose significant health and economic challenges worldwide, especially in tropical regions where these infections remain endemic. In the Philippines, the risk remains high following the 2019 withdrawal of the Dengvaxia vaccine, with no alternative immunization currently available locally. Recent interest in nucleic acid-based immunogens highlights the need for scalable, non-infectious vaccine production platforms; however, generating high-dose DNA constructs capable of inducing effective immune responses without live-virus propagation remains challenging. Our work from 2018 to 2024 addressed this need through the development of computationally designed, cell-free, PCR-amplifiable DNA constructs using algorithmic overlap extension PCR (OE-PCR) and an improved technique, asymmetric extension supported by a simulator for oligonucleotide extension (AESOE). Subsequent integration of machine learning, including recurrent neural network-based amplification prediction and LAMP primer design, further improved synthesis efficiency and expanded applications for surveillance and diagnostics. The AESOE-guided OE-PCR platform enabled the assembly of synthetic flaviviral DNA fragments of at least 1 kilobase in length from overlapping oligonucleotides, supporting scalable production of synthetic immunogens without the need for infectious viral materials. These live-virus-free strategies may enhance biosafety, reduce development timelines, and improve accessibility for laboratories in resource-limited settings, potentially supporting next-generation vaccine development for dengue and other flaviviral diseases.