DIFFERENCE Bioscience Advances IND Filing for Next-Gen Intranasal Vaccines Powered by Proprietary Recombinant Viral Vector Platform and CRO Services

Two months ago, the nasal spray influenza virus-vectored COVID-19 vaccine, jointly developed by Xiamen University, the University of Hong Kong, and Wantai BioPharm, was approved for emergency use. This vaccine is the world’s first mucosal immune COVID-19 vaccine to be approved for clinical trials and to have its safety and broad-spectrum efficacy validated in Phase III clinical trials.This news has once again drawn attention to nasal spray vaccines, encouraging more research teams to accelerate their development efforts.

Nasal spray vaccines directly achieve mucosal blockade when viruses enter the human body through the nasal cavity, complementing the function of injectable vaccines and jointly establishing a triple immune defense comprising humoral, cellular, and mucosal immunity. In addition to offering greater convenience in administration, nasal spray vaccines not only block viral transmission but also provide a certain level of protection against various mutant strains, underscoring their significant importance.

Zhejiang Difu Runsi Biotechnology Co., Ltd. (“Difu Runsi”), which focuses on the development of mucosal immune vaccines, has entered the competition.Its intranasal Newcastle disease virus (NDV) vector-based multivalent COVID-19 vaccine, developed with independent intellectual property rights, has demonstrated excellent performance in animal studies.

Behind the robust nasal spray COVID-19 vaccine project lies its self-developed, world-class recombinant viral vector platform.The platform features the DIFF-Re-S-Omicron SARS-CoV-2 pseudovirus technology, a rarity in China, which can be utilized for serum neutralizing antibody detection, simulated infection experiments, and in vivo efficacy evaluation of vaccines and drugs. This technology fills a gap in the domestic COVID-19 vaccine industry and has attracted multiple listed companies and industry leaders to seek collaboration.

Meanwhile, the BSL-2 animal infection models established by Difu Runsi enable a series of non-clinical evaluations of safety, immunogenicity, and efficacy for products such as vaccines, antibodies, and drugs. These models demonstrate high sensitivity and accuracy, strong reproducibility, and the capacity for high-throughput screening, all while maintaining low costs. Benchmarking against Moderna’s mRNA technology platform, Difu Runsi aims to further open up its technology to develop more vaccine products with significant market value.

It is reported that Zhejiang Difu Runsi Biotechnology Co., Ltd. has completed a Series A financing round amounting to tens of millions of yuan. The funds will be used to file Investigational New Drug (IND) applications for its NDV vector-based multivalent COVID-19 nasal spray vaccine and another flagship product, a trivalent influenza nasal spray vaccine, with the aim of accelerating the entry of these two distinctive mucosal immune vaccines into clinical trials.

The three most advanced flagship projects of Difu Runsi are: the Newcastle disease virus (NDV) vector-based COVID-19 vaccine administered via nasal spray, the trivalent influenza vaccine administered via nasal spray, and the DIVA (Differentiating Infected from Vaccinated Animals) inactivated avian influenza vaccine.

The nasal spray COVID-19 vaccine currently has the most clearly defined market demand, and the core product with the fastest development progress at Zhejiang Difu Runsi Biotechnology Co., Ltd. is precisely this NDV viral vector-based COVID-19 vaccine.

Newcastle disease virus (NDV) vectors have been utilized in the development of vaccines for various infectious diseases in humans and animals (such as avian influenza, Ebola virus, and SARS-CoV) and in research on human cancer therapy. When employed for COVID-19 vaccines, NDV vectors offer numerous advantages: they readily induce mucosal immunity; exhibit a favorable safety profile in humans; involve minimal vector immune competition; are not interfered with by pre-existing antibodies; are suitable for repeated immunization; and feature low costs, making them amenable to industrial-scale production.

As early as the initial outbreak of the pandemic in 2020, Dr. Song Jiasheng, founder of Zhejiang Difu Runsi Biotechnology Co., Ltd., had already designed and planned the development of mucosal immune vaccines. In June 2020, it was officially reported that the world’s first recombinant NDV-vectored COVID-19 vaccine candidate strain had been constructed. At the time, this application faced considerable skepticism.

However, more than a month later, the team led by U.S. National Academy of Sciences member Peter Palese also officially reported the development of an intranasal NDV-vector COVID-19 vaccine. Currently, this NDV-based COVID-19 vaccine, NDV-HXP-S, has successfully entered Phase II clinical trials, with data confirming the safety and efficacy of the NDV vector for COVID-19 vaccination. Regrettably, although Dr. Song Jiasheng recognized the application prospects of Newcastle disease virus vectors in vaccines for human respiratory diseases at an early stage, the team at Zhejiang Difu Runsi Biotechnology Co., Ltd. lagged behind their foreign counterparts in the industrialization of NDV-vector COVID-19 vaccines due to limitations in funding and resources.

Over the next two-plus years, the R&D team at Zhejiang Difu Runsi Biotechnology Co., Ltd. constructed and tested hundreds of candidate strains, completing three iterations of their NDV-vector COVID-19 vaccine technology. In fact, when the second-generation vaccine was developed, many people advised Dr. Song Jiasheng to proceed to clinical trials.

But Dr. Song Jiasheng insisted on completing three iterations and achieved breakthrough results before filing the IND, ““We do not want to create products with identical efficacy; latecomers must strive to do better.” This is also the aspect that investors value most about Zhejiang Difu Runsi Biotechnology Co., Ltd.: a pragmatic team that has long maintained a steady development model, aligning with the current era’s emphasis on high-quality growth.

The Difu team has conducted extensive work on recombinant antigens and multi-target optimization for third-generation vaccines, successfully developing them into multivalent vaccines. In humanized hACE2 mouse models, these vaccines achieved nearly 100% protection, with neutralizing antibody titers reaching 1:1600 and demonstrating superior clearance of live virus. Their protective efficacy was better than the experimental data reported for the vaccine developed by Peter Palese’s team. In light of this,Difu’s third-generation NDV vector vaccine technology is poised to become a global leader in this technical route, achieving overtaking on the bend.

Currently, this nasal spray COVID-19 vaccine is in the pilot-scale production phase, with clinical trials expected to begin in 5–7 months. Meanwhile, the R&D team at Zhejiang Difu Runsi Biotechnology Co., Ltd. continues to advance the fourth-generation innovation of this vaccine.

Another human mucosal immune vaccine product from Difu Runsi is a broad-spectrum, trivalent nasal spray influenza vaccine. In 2022, the R&D team discovered for the first time a novel attenuation method based on M2 gene modification to restrict the replication of influenza A virus, and developed this vaccine using the technology.

This technological approach offers significant application value. The trivalent nasal spray influenza vaccine developed using this technology has achieved breakthroughs in both safety and protective efficacy, while also demonstrating broad-spectrum activity, holding promise for overcoming the challenges posed by continuously emerging variant strains to existing vaccines. Challenge studies have shown that immunization with antigens derived from 2021 circulating strains can provide protection against heterologous strains that circulated as early as 50 years ago or even longer (such as the H1N1 strain prevalent in 1934 and the H3N2 strain prevalent in 1968). This product is benchmarked against Flumist, a marketed vaccine from MedImmune, a leading U.S. vaccine company, and is expected to achieve new breakthroughs in broad-spectrum coverage and safety, making it more suitable for vulnerable populations such as the elderly and children.

In addition to its two human vaccines, Difu Runsi continues to advance a project initiated at its inception—the development of a DIVA (Differentiating Infected from Vaccinated Animals) inactivated avian influenza vaccine for animals, which is also scheduled to enter clinical trials this year.This vaccine primarily aims to achieve source-level eradication of avian influenza in poultry farms, preventing virus-contaminated food products from entering the distribution chain, and holds promise for eliminating the significant threats to food safety and public health posed by avian influenza strains such as H7N9 and H5N1.

Furthermore, multiple pipelines are advancing, including intranasal respiratory syncytial virus (RSV) vaccines, viral vector-based mpox vaccines, and oncolytic virus anticancer therapeutics. Dr. Song Jiasheng plans to bring two human intranasal vaccines for COVID-19 and influenza to market within two to three years, and to advance one or two products in the fields of oncolytic viruses and gene therapy into clinical stages.

Difu Runsi boasts world-class platforms for the construction and modification of diverse RNA and DNA viral vectors, which constitute its core competitiveness.This platform enables a range of operations, including pseudovirus construction, in vivo viral tracing, high-throughput screening, and reverse genetics. It can be utilized for the development of vaccines against major infectious diseases and tumors, as well as for various therapies such as gene therapy and oncolytic virotherapy, offering broad market prospects.

Leveraging this recombinant viral vector platform, Difu Runsi currently adopts two collaboration models: CRO services and co-development. The company’s CRO services have established partnerships with numerous publicly listed companies and industry leaders in the fields of animal vaccines and human pharmaceuticals. This business segment also generates tens of millions in stable annual cash flow for the company.

Why do large pharmaceutical companies choose Difu Runsi’s platform for R&D? Dr. Song Jiasheng stated that it is because the recombinant viral vector platform can accomplish “things that other companies cannot.”

Zhejiang Difu Runsi Biotechnology Co., Ltd.’s recombinant viral vector platform-based pseudovirus technology holds significant application value. Pseudoviruses are surrogate agents prepared based on publicly available sequences, possessing the fundamental characteristics and immunogenicity of native viruses while lacking pathogenicity, thereby facilitating large-scale laboratory use. Due to their low biosafety risk (requiring only Biosafety Level 2 [BSL-2] containment), controllable quality control methods, and capacity for long-term stable production and supply, pseudoviruses have been widely employed in research on infection mechanisms, receptor recognition, and viral inhibition, as well as in the development and evaluation of antibodies and vaccines, owing to their authenticity, stability, and safety.

DIFF-Re-S-Omicron SARS-CoV-2 Pseudovirus is a novel SARS-CoV-2 pseudovirus developed by the R&D team at Difu Runsi using the vesicular stomatitis virus (VSV) vector system. VSV, an RNA-enveloped virus, shares similarities with SARS-CoV-2. By replacing the VSV G protein with the SARS-CoV-2 S protein and inserting reporter genes such as eGFP or Luciferase, the researchers constructed the DIFF-Re-S-Omicron SARS-CoV-2 pseudovirus.

Research data indicate that DIFF-Re-S-Omicron can successfully enter target cells and infect hACE2-transgenic mice. This pseudovirus exhibits a high safety profile, allowing for safe handling in Biosafety Level 2 (BSL-2) laboratories. Furthermore, its surface protein conformation closely resembles that of the native viral protein, enabling it to mimic the mechanisms of viral entry and cellular infection. Consequently, it serves as a high-quality model for replacing wild-type SARS-CoV-2 in applications such as vaccine development and drug efficacy evaluation. In similar international studies, pseudoviruses with comparable structures have been utilized in the development of human COVID-19 vaccines and have successfully passed Phase I clinical safety assessments.

The novel DIFF-Re-S-Omicron SARS-CoV-2 pseudovirus technology and BSL-2 animal infection models developed by the Difu Runsi team offer broad applicability, high sensitivity, accuracy, and reproducibility, enabling high-throughput screening while maintaining low costs. “We have engineered a safer mimic of the SARS-CoV-2 virus, which can be used to rapidly screen vaccine candidates or evaluate drugs, vaccines, and other therapeutic interventions in animal models,” stated Dr. Song Jiasheng, noting that this technology remains relatively scarce both domestically and internationally.

Currently, the company operates professional laboratories with Biosafety Level 2 (BSL-2) containment and has obtained dual certifications: the Laboratory Animal Use License and ABSL-2 registration. Meanwhile, its technological achievements in vaccine-related fields have resulted in more than 30 domestic and international invention patents, including over 10 PCT international patents.

Targeting the global markets for COVID-19, influenza, and avian influenza vaccines, as well as recombinant viral vector platforms, Difu Runsi will commence its strategic expansion in Singapore starting in 2023 (having already established its wholly-owned subsidiary, DIFFERENCE BIOTECH PTE. LTD.), thereby further expanding profit margins through technology licensing. The company plans to list on the STAR Market in 2025, promote the commercial launch of its three flagship vaccines, and establish a competitive animal health business segment.