The researchers used PROTAC technology to developed a PROTAC influenza virus vaccine called M1-PTD, which provides a new idea for vaccine development.

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PROTAC Technology: A New Application In
Influenza Vaccines
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.
PROTAC Technology
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

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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
(Source: Nature)
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

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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)

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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 [1])
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

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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
(Source: reference[2])

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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.
PROTAC Challenges
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.

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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.
Conclusion
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

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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.
References:
[1]. A new route to vaccines using PROTACs
[2]. Longlong Si. Generation of a live attenuated influenza A vaccine by proteolysis
targeting. Nature Biotechnology. 2022.

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Related articles:
[1]. Overview of New Targets And Technologies of PROTAC
[2]. Four Major Trends In The Development of PROTAC
[3]. PROTAC And Other Protein Degradation Technology
[4]. Peptide PROTAC in Drug Development