Existing methods

Oncolytic viruses

Our project

Experiment

References

Existing methods

tumor treatment Method Advantages Disadvantages
Surgery Surgical removal of the tumor and surrounding tissue. Can be curative if the tumor is localized and completely removed. Invasive procedure with potential risks and complications. Not suitable for all types and stages of cancer.
Radiation Therapy High-energy radiation is used to kill or shrink cancer cells. Non-invasive treatment option. Can be used as a primary treatment or in combination with other methods. Potential side effects on healthy tissues near the tumor. Limited effectiveness in certain types of cancer.
Chemotherapy Drugs are used to kill or inhibit the growth of cancer cells throughout the body. Can be effective against cancers that have spread to multiple areas of the body. Can be used as a primary or adjuvant treatment. Systemic treatment that affects both cancerous and healthy cells, leading to various side effects. Development of drug resistance.
Targeted Therapy Drugs or other substances are used to target specific molecules or pathways involved in cancer growth. More precise targeting of cancer cells, potentially leading to fewer side effects. Can be effective against certain types of cancer with specific genetic mutations. Limited to specific types of cancer with identifiable targets. Development of resistance in some cases.
Immunotherapy Stimulating the immune system to recognize and attack cancer cells. Potential for long-lasting responses and effectiveness against various types of cancer. Can activate the immune system to target cancer cells. Response rates can vary, and not all patients benefit from immunotherapy. Potential for immune-related side effects.

Oncolytic viruses

Oncolytic viruses are a promising class of anticancer agents that are designed to selectively infect and kill cancer cells while sparing normal, healthy cells. They work by infecting cancer cells and replicating within them, leading to the destruction of the cancer cells. Additionally, oncolytic viruses can stimulate an immune response against the tumor, further aiding in tumor clearance[1].

Several oncolytic viruses have been studied and evaluated in clinical trials, including:

  1. T-Vec (talimogene laherparepvec): T-Vec is an oncolytic herpes simplex virus type 1 (HSV-1) modified to selectively replicate in and destroy cancer cells. It has been approved for the treatment of advanced melanoma.
  2. Rigvir: Rigvir is a non-engineered oncolytic virus derived from ECHO-7 virus that is used for the treatment of melanoma and other types of cancer. It is primarily used in Latvia and some other European countries.
  3. Pexa-Vec (JX-594): Pexa-Vec is an oncolytic vaccinia virus engineered to express granulocyte-macrophage colony-stimulating factor (GM-CSF). It has been evaluated in clinical trials for the treatment of advanced liver cancer.
  4. Reolysin (pelareorep): Reolysin is a wild-type reovirus that selectively infects and replicates in cancer cells with an activated Ras pathway. It has been investigated in clinical trials for various types of cancer.
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Fig 1. The induction of local and systemic anti-tumour immunity by oncolytic viruses[2]

Our project

Influenza virus, a well-known respiratory pathogen, has recently gained attention as a potential oncolytic agent for cancer therapy. The process of influenza virus invasion into host cells involves a series of coordinated steps, including entry, attachment, internalization, and replication. Notably, the interaction between the neuraminidase (NA) protein on the viral envelope and the transferrin receptor 1 (TfR1) on the cell surface plays a crucial role in the entry and internalization stages.

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Fig 2. TfR1 influence the Entry and Internalization (Unpublished work)

Given the observation that many tumor cells exhibit elevated expression levels of TfR1[3], it is hypothesized that influenza virus may possess an enhanced ability to infect and target tumor cells. This forms the primary focus of our research, where we aim to verify whether influenza virus demonstrates increased infectivity towards tumor cells compared to normal cells.

Moreover, a notable feature of influenza virus is the disproportionate abundance of the hemagglutinin (HA) protein relative to NA on its surface. To further enhance the selectivity of influenza virus towards tumor cells, we propose a novel approach. Specifically, we plan to swap the packaging signals of HA and NA, leading to the packaging of a higher quantity of NA on the viral surface. The rationale behind this strategy lies in the fact that tumor cells often overexpress TfR1, making them more susceptible to viral entry and internalization. By increasing the presence of NA on the viral envelope, we anticipate that the modified influenza viruses will exhibit enhanced affinity towards TfR1 on tumor cells, thereby increasing their selectivity and infectivity towards cancerous tissues.

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Fig 3. Design and rescue of PR8 virus with swapped HA and NA packaging signals[4]

In the next phase of our research, we plan to investigate the potential of extending the stalk domain of NA. The NA protein comprises a globular head domain and a stalk domain. Previous studies have shown that elongating the stalk domain of NA can enhance its immunogenicity. This effect may be attributed to the increased exposure of NA, facilitating its binding to ligands. Therefore, we aim to genetically engineer influenza viruses with elongated stalk domains of NA and evaluate their potential for increased infectivity towards tumor cells.

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Fig 4. The structure of NA and HA[5]

Finally, if both strategies prove successful in enhancing the specific infectivity of influenza virus towards tumor cells, we aim to combine these approaches. By exchanging the packaging signals of HA and NA to increase NA expression and extending the stalk domain of NA for enhanced recognition and binding to TfR1, we hope to generate an influenza virus strain with even stronger selectivity towards tumor cells. This constitutes the final phase of our project.

By elucidating the mechanisms underlying influenza virus infection and exploiting the specific interactions between viral surface proteins and cellular receptors, our research aims to contribute to the development of targeted oncolytic virotherapy, providing novel and effective treatment.

Influenza Virus Research: Experimental Design

Objective

To investigate the enhanced infectivity of influenza virus against tumor cells by validating the interaction between the surface neuraminidase (NA) protein of influenza virus and the transferrin receptor 1 (TfR1) on the cell surface, which influences the steps of entry and internalization.

Experimental Steps
  1. Comparative analysis of TfR1 expression:
    1. Collect different tumor cell lines and normal cell lines.
    2. Determine the expression levels of TfR1 in both tumor cells and normal cells using immunofluorescence staining, Western blotting, or quantitative PCR.
    3. Compare the expression levels of TfR1 between tumor cells and normal cells.
  2. Influenza virus infection assay:
    1. Infect tumor cell lines and normal cell lines with influenza virus.
    2. Monitor virus replication and cell destruction using methods such as viral titration, plaque assay, or cell viability assays.
    3. Compare the infectivity of influenza virus between tumor cells and normal cells.
  3. Interaction between influenza virus and TfR1:
    1. Perform co-immunoprecipitation assays to validate the interaction between NA of influenza virus and TfR1 on the surface of tumor cells.
    2. Use antibodies specific to NA and TfR1 to immunoprecipitate the protein complexes.
    3. Detect the interaction through Western blotting or mass spectrometry analysis.
  4. Data analysis and interpretation:
    1. Quantify the expression levels of TfR1 in tumor cells and normal cells.
    2. Compare the infectivity of influenza virus between tumor cells and normal cells based on virus replication and cell destruction data.
    3. Analyze the interaction between influenza virus NA and TfR1 in tumor cells.
Expected Outcome

If the expression of TfR1 is higher in tumor cells compared to normal cells and influenza virus demonstrates enhanced infectivity against tumor cells, it would support the hypothesis that the interaction between NA and TfR1 influences the entry and internalization of influenza virus, resulting in increased infectivity against tumor cells.

Experimental Expansion

To enhance the selectivity of influenza virus against tumor cells, considering the higher abundance of the hemagglutinin (HA) protein compared to the neuraminidase (NA) protein on the surface of influenza virus (data can be supplemented), we propose a strategy to swap the packaging signals of HA and NA proteins. By doing so, we aim to generate recombinant influenza viruses with an increased presence of NA on their viral surface. This alteration inthe viral surface composition will be investigated for its potential to enhance tumor cell selectivity. Experimental Details

  1. Construction of Recombinant Influenza Viruses:
    1. Generate plasmids containing the HA and NA genes with their packaging signals swapped.
    2. Use molecular cloning techniques, such as PCR, restriction enzyme digestion, and ligation, to introduce the packaging signal swap.
    3. Verify the successful generation of recombinant plasmids through DNA sequencing.
  2. Generation of Recombinant Influenza Viruses:
    1. Transfect appropriate host cells, such as embryonated chicken eggs or mammalian cell lines, with the recombinant plasmids.
    2. Allow the recombinant plasmids to undergo recombination and produce recombinant influenza viruses.
    3. Harvest the viral supernatant or allantoic fluid from infected host cells.
  3. Quantification of HA and NA Proteins:
    1. Determine the levels of HA and NA proteins on the viral surface using techniques like enzyme-linked immunosorbent assay (ELISA) or flow cytometry.
    2. Compare the relative abundance of NA between the recombinant viruses and wild-type influenza viruses.
  4. Assessment of Tumor Cell Selectivity:
    1. Infect both tumor cell lines and normal cell lines with the recombinant viruses and wild-type influenza viruses.
    2. Monitor virus entry, internalization, and replication in the infected cells.
    3. Assess virus replication and cytopathic effects specifically in tumor cells using methods such as viral titration, plaque assay, or qRT-PCR.
    4. Compare the selectivity of recombinant viruses and wild-type viruses in terms of their ability to infect and replicate within tumor cells.
  5. Analysis of Virus-Cell Interactions:
    1. Investigate the interaction between recombinant viruses and tumor cells by employing techniques such as immunofluorescence staining, confocal microscopy, or electron microscopy.
    2. Examine the binding and internalization of recombinant viruses in tumor cells compared to wild-type viruses.
  6. Data Analysis and Interpretation:
    1. Quantify the levels of NA on the viral surface of recombinant viruses compared to wild-type viruses.
    2. Compare the infectivity and replication efficiency of recombinant viruses and wild-type viruses specifically in tumor cells.
    3. Analyze the significance of the altered HA-NA packaging signals in enhancing the tumor cell selectivity of influenza viruses.

The results from this experiment will shed light on the potential of manipulating the viral surface composition through HA-NA packaging signal swap to improve the selectivity of influenza viruses towards tumor cells.

Reference

[1]Lan, Q., Xia, S., Wang, Q. et al. Development of oncolytic virotherapy: from genetic modification to combination therapy. Front. Med. 14, 160-184 (2020).https://doi.org/10.1007/s11684-020-0750-4

[2]Kaufman, H., Kohlhapp, F. & Zloza, A. Oncolytic viruses: a new class of immunotherapy drugs. Nat Rev Drug Discov 14, 642-662 (2015).https://doi.org/10.1038/nrd4663

[3]Candelaria PV, Leoh LS, Penichet ML, Daniels-Wells TR. Antibodies Targeting the Transferrin Receptor 1 (TfR1) as Direct Anti-cancer Agents. Front Immunol. 2021;12:607692. Published 2021 Mar 17. https://doi.org/10.3389/fimmu.2021.607692

[4]Zheng, A., Sun, W., Xiong, X., Freyn, A. W., Peukes, J., Strohmeier, S., Nachbagauer, R., Briggs, J. A. G., Krammer, F., & Palese, P. (2020). Enhancing Neuraminidase Immunogenicity of Influenza A Viruses by Rewiring RNA Packaging Signals. Journal of virology, 94(16), e00742-20.https://doi.org/10.1128/JVI.00742-20

[5]Broecker, F., Zheng, A., Suntronwong, N., Sun, W., Bailey, M. J., Krammer, F., & Palese, P. (2019). Extending the Stalk Enhances Immunogenicity of the Influenza Virus Neuraminidase. Journal of virology, 93(18), e00840-19.https://doi.org/10.1128/JVI.00840-19