Influenza is an acute respiratory infection caused by influenza virus that spreads worldwide. Influenza viruses are divided into four types: A, B, C, and D. Influenza A and B viruses can spread and cause seasonal epidemics. Influenza remains a major threat to global health, causing severe illness and death in high-risk populations.
It is reported that seasonal influenza can cause 3-5 million severe cases and 290,000-650,000 respiratory disease-related deaths worldwide each year. The most effective way to prevent influenza is to get vaccinated. Attenuated vaccines have become one of the important development directions because of their potential advantages in immune effects. Attenuated influenza vaccines can induce a broader immune response by retaining the natural structure of all or most of the antigens of the virus, including humoral immunity, respiratory mucosal immunity, cellular immunity, etc.
On July 4, 2022, Si Longlong's research group from the Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences published a research paper entitled: Generation of a live attenuated influenza A vaccine by proteolysis targeting in the journal Nature Biotechnology. Using influenza virus as a model virus, the research team established the technology of Proteolysis-Targeting Chimeric virus vaccine (PROTAC vaccine) as an attenuated vaccine, which provides a new idea for vaccine development.
The concept of PROTAC (Proteolysis-Targeting Chimeric) was first proposed in 2001. PROTAC technology is considered as a revolutionary technology in the field of biomedicine. PROTAC consists of three parts: target protein ligand, E3 ligase ligand, and a suitable linker in the middle. PROTAC recruits ubiquitin E3 ligase with protein degradation function to the surface of target protein, and utilizes the body's own natural protein processing system (ubiquitin-proteasome system) to selectively and effectively degrade and remove disease-causing proteins. In layman's terms, cells clean up waste proteins by "labeling" them. The special mechanism of PROTAC is expected to break the deadlock of "difficult to druggable" targets in the field of small molecule drugs.
Figure 1. Mode of action of PROTACs
PROTAC Attenuated Influenza Vaccine
PROTACs are bifunctional molecules that induce degradation of target proteins by targeting them to the ubiquitin-proteasome system. In this study, the research team constructed a PROTAC virus, which aims to attenuate the wild-type virus into a vaccine by manipulating the degradation of viral proteins to reduce the virus's ability to replicate. Specifically, the PROTAC virus combines a proteasome-targeting domain (PTD) with an influenza virus protein.
Given that viral replication depends on the proteins encoded by the virus, Si Longlong's group believes that manipulating the stability of viral proteins by using the host cell's protein-degradation mechanisms could represent a potential way to turn the viral life cycle on and off for vaccine development. In this study, the researchers designed a conditional knockout proteasome-targeting domain (PTD) on the influenza A virus matrix protein gene fragment. The PTD contains the ALAPYIP peptide, which can recognize the von Hippel–Lindau (VHL) tumor suppressor protein, and VHL is the substrate recognition component of CRL2VHL E3 ubiquitin ligase. The ubiquitous expression of VHL in most normal tissues and cell types may provide a critical basis for the safety of PROTAC vaccines.
The ubiquitin-proteasome system, a naturally occurring protein degradation machinery in host cells, provides a key biological basis for the design of the PROTAC virus vaccine. In recent years, the PROTAC technology based on ubiquitin-proteasome system has been successfully used to develop protein degradation agents based on chemical small molecules, and has become an international scientific research hotspot, that is, researchers designed a small molecule compound with two active ends, one of which can be combined with the target protein that needs to be degraded. The other active end can bind to a specific E3 ubiquitin ligase to induce ubiquitization of the target protein, which is then degraded by the proteasome.
In this study, the Si Longlong's team selected influenza virus as the model virus, used the protein degradation machinery naturally existing in host cells, designed components that could be conditioned to control the stability and degradation of viral proteins, and engineered the viral genome so that the corresponding viral proteins were recognized and degraded by the ubiquitin-proteasome system in normal cells, resulting in weakened virus replication ability, which becomes a potential vaccine. While in vaccine preparation cells, the inducible elements of viral protein degradation will be selectively removed, so that the viral protein can be retained. Therefore, PROTAC virus can be efficiently replicated and prepared in large quantities in vaccine preparation cells.
Figure 2. Principle of PROTAC virus vaccine. VP, viral protein; Ub, ubiquitin; PTD, proteolysis-targeting domain
(Source: Reference 2)
Based on the above design principles, the research team first developed a PROTAC influenza virus vaccine, named M1-PTD. PROTAC viruses are attenuated by proteasome-mediated targeted degradation of ubiquitinated viral proteins (M1-PTD), resulting in insufficient protein synthesis and weakened viral replication. The viral nucleoprotein (NP) not fused to PTD will not be degraded. Viral replication should not be attenuated during vaccine production, so the PTD also contains the tobacco etch virus cleavage site (TEVcs) linker, ENLYFQG, which can be selectively cleaved by the tobacco etch virus protease (TEVp), thereby avoiding viral proteins in stable expression of TEV Cellular degradation by proteases.
Figure 3. Overview of the PROTAC virus production system
(Source: Reference )
The safety of PROTAC viruses as potential vaccines depends largely on the degree to which they are attenuated in conventional cells. M1-PTD can only be prepared by efficient replication in PROTAC virus preparation cells, while its replication ability is significantly reduced and safe in normal cells. In addition, immunofluorescence assay results showed that M1-PTD virus protein was degraded in normal cells. The results of plaque assay showed that M1-PTD could form plaque only in PROTAC virus preparation cells, but not in normal cells. Cytopathic test results showed that M1-PTD did not cause obvious lesions in normal cells. Therefore, M1-PTD successfully attenuated wild-type virus into a safe influenza vaccine.
In addition, the attenuated effects of PROTAC virus were evaluated in animal models of mice and ferrets. The results showed that the median lethal dose (LD50) of WT virus in mice was 104PFU, and 10×LD50 WT virus resulted in death in all mice with significant weight loss. In contrast, no death, weight loss, or other indicative clinical symptoms were observed in M1-PTD infected mice at the same dose (Figure 4a, b). In ferret models, the researchers further confirmed the safety of M1-PTD. On day 3 after inoculation, the titers of M1-PTD in the nasal rinse solution, trachea and lung of ferrets showed approximately -log2.0, approximately -log2.1, and approximately -log2.9, respectively, compared with WT virus (Figure 4c, d). All data from animal models indicate that influenza viruses are greatly attenuated in vivo by using host protein degradation machinery.
Figure 4. In vivo safety evaluation of PROTAC virus M1-PTD in mice and ferrets
At the same time, there are also data showing that PROTAC vaccine can induce a strong and widespread immune response in mice and ferrets. Immunological evaluation tests on M1-PTD influenza vaccine in mouse and ferret animal models showed that M1-PTD activated humoral, mucosal and cellular immune responses at the same time, and the body can provide strong protection against homologous and heterologous virus attacks.
The design of PROTAC technology in virus vaccines provides a new idea for the development of virus vaccines, enriches the vaccine technology arsenal for humans to resist viruses, and also helps to promote the basic biological research of cellular protein degradation machines and the depth of medical transformation of vaccine research and development cross fusion.
In recent years, PROTAC has developed rapidly, but compared with ADC, monoclonal antibody and other technologies, PROTAC technology is not mature, and it still faces many problems and challenges.
The relative molecular weight of PROTAC is too large, and the cell permeability is poor: at present, the general molecular weight of PROTAC drugs is mostly above 700 Dalton, which breaks the "5 principle of class drugs" that the molecular weight of small and medium-sized drugs is less than 500 Dalton. The relatively large molecular weight means that oral absorption and membrane permeability are relatively poor.
Off-target toxicity of PROTAC: PROTAC can completely degrade the target protein, thereby inhibiting all functions of the target protein, but it may accidentally injure normal proteins. Off-target effects and toxicity are the biggest concerns about the safety of PROTAC technology.
Molecular stability of PROTAC: Compared with traditional small molecule drugs, the more complex structure of PROTAC molecules leads to more potential metabolic sites, which in turn has the disadvantage of poor metabolic stability.
In terms of details,, PROTAC's chain design and selection, as well as the E3 ligand, are equally critical. The former is also one of the difficulties in PROTAC drug development. Its length and chemical composition will affect the structural rigidity, hydrophobicity and solubility of PROTAC. The latter is the development of the existing species is not much, there are still limitations.
Key Features of PROTAC Virus Vaccines
In addition to clinically used methods for producing inactivated vaccines (IIV) and attenuated vaccines (CAIV), several other strategies have been developed to attenuate viruses in preclinical studies. Compared with these existing approaches, the PROTAC virus vaccine technology employs a unique vaccine design principle—that is, to conditionally target viral proteins to the host's protein degradation system to generate proteolytically targeted viruses as vaccines. The method has five key features:
1. PROTAC virus vaccines can highly attenuate the virus to a low level of replication ability, which may be safer than existing methods.
2. PROTAC virus vaccines may be able to attenuate multiple seasonal or pandemic virus strains, providing a sufficient antigenic match between the vaccine and the target virus to enhance efficacy.
3. Because more than 600 E3 ligases are found in the human ubiquitin-proteasome system, theoretically many PTDs can be used to generate PROTAC viruses.
4. PROTAC virus vaccines are a simple and general approach that may be applicable to many other viruses and are available in most laboratories.
5. By using TeVp-expressing cells or viral proteins expressing interest, it can achieve cost-effective vaccine production within weeks under normal cell culture conditions.
An ideal vaccine should be sufficiently attenuated in the host to ensure safety, while maintaining strong immunogenicity to ensure efficacy, and be able to be efficiently produced in a tissue culture platform suitable for manufacturing. Si Longlong's research group successfully developed a PROTAC virus vaccine by using the host's protein degradation mechanism to control the stability of viral proteins. PROTAC virus can be highly attenuated in conventional cells, but can retain its ability to replicate efficiently in the engineered TEVp-expressing MDCK cell line that have been approved by the US FDA for human vaccine production. PROTAC viruses can be sufficiently attenuated in vivo, but still induce strong and diverse humoral, mucosal, and cellular immunity, thus providing extensive protection against homologous and heterologous virus attacks.
Finally, the potential safety issues of the PROTAC vaccine require further study. The results also raise concerns about the use of VHL as a PROTAC target, as VHL is a tumor suppressor protein and was found to be lost in clear cell kidney cancer. As Si Longlong and others have pointed out, the PROTAC method or specific PROTAC viruses may not be appropriate for some people, such as those who are treated with proteasome inhibitors or who have defective expression of specific E3 ligases. However, many different PTD-E3 ligase pairs and PTD-viral protein junctions can also be studied. Although personalizing selection from a suite of E3 ligases is not feasible for population-based vaccine production, there is still room to develop safe, effective and cost-effective candidates for improved PROTAC vaccines. As the method expands to other viral pathogens, selecting the best viral proteins to target and ensuring efficient cleavage is one of the details that needs to be studied.
The commonly used linkers in the development of PROTACs are PEGs. As a leading PEG derivatives supplier, Biopharma PEG provides high-purity PROTAC PEG linkers with various active groups to assist with your PROTAC-related projects.
. A new route to vaccines using PROTACs
. Longlong Si. Generation of a live attenuated influenza A vaccine by proteolysis targeting. Nature Biotechnology. 2022.
. Overview of New Targets And Technologies of PROTAC
. Four Major Trends In The Development of PROTAC
. PROTAC And Other Protein Degradation Technology
. Peptide PROTAC in Drug Development