Abstract:

This final lecture of Virology Lectures 2025, delivered by Professor Vincent Racaniello, explores the field of therapeutic virology, focusing on how basic virology research enables the engineering of viruses to treat or prevent human diseases. The lecture covers four main applications: bacteriophage therapy against bacterial infections, viral vectors for gene therapy in monogenic diseases, viral vectors as vaccines, and viral oncotherapy for cancer treatment. Key technologies, such as the creation of infectious DNA copies of viral genomes for modification, are highlighted as foundational. Specific examples discussed include the use of lytic phages for antibiotic-resistant infections; Adenovirus, AAV, and Lentivirus vectors for delivering genes to correct conditions like X-linked SCID, sickle cell anemia, and inherited retinopathies (e.g., Luxturna), alongside challenges like immune responses and insertional mutagenesis. The lecture also reviews viral vector-based vaccines (e.g., Ervebo for Ebola, ChAdOx1 for COVID-19) and explores oncolytic viruses (e.g., T-VEC for melanoma), detailing strategies for tumor-specific replication, immune enhancement, the abscopal effect, and the role of viral vectors in CAR T-cell therapy. The overarching message emphasizes the critical importance of continued basic research in driving these therapeutic innovations.

Harnessing Viruses: A Journey into Therapeutic Virology

• 0:11 - Introduction to Therapeutic Viruses: This final lecture will focus on how viruses can be engineered and harnessed to benefit human health, covering phage therapy, gene therapy, viral vaccine vectors, and viral oncotherapy.
• 2:48 - Foundational Technology: Modifying Viral Genomes: The ability to create and manipulate infectious DNA copies of viral genomes (even RNA viruses like Poliovirus, first achieved for an animal virus by Racaniello in 1981) is crucial for developing all therapeutic viruses. This allows for gene insertion, deletion, and alteration.
• 5:19 - Bacteriophage (Phage) Therapy:
  • 5:19 - Historical Context: Discovered in 1915 by d'Herelle, phage therapy was explored before antibiotics but saw a resurgence due to antibiotic resistance.
  • 6:52 - Mechanism & Challenges: Uses lytic phages to kill specific bacteria. Challenges include identifying appropriate phages and FDA approval for personalized therapies.
  • 7:57 - Phage Lysins & Approved Uses: Purified phage lysins (e.g., AgriPhage for tomato disease, ListShield for food decontamination - GRAS status) are used for surface decontamination.
  • 9:53 - Human Case Studies: Examples include treating an aortic graft infection with Pseudomonas, engineered phages for Mycobacterium abscessus (from the Sea-Phages program), and a famous case of treating disseminated Acinetobacter baumannii in a patient (Stephanie Strathdee's husband).
  • 14:15 - Complication: Antibody responses against phages can interfere with treatment.
• 15:15 - Adenovirus Vectors for Gene Therapy:
  • 15:15 - Properties: dsDNA viruses efficient at infecting post-mitotic cells, episomal (non-integrating), with large capacity.
  • 16:17 - Generations:
    • First-generation: E1 (replication) and E3 (immunomodulatory) genes deleted.
    • Third-generation ("gutless"): All viral genes removed except ITRs and packaging signals, requiring a helper virus. A Cre-lox system is used to prevent helper virus packaging.
• 19:52 - Adeno-Associated Virus (AAV) Vectors:
  • 19:52 - Properties: ssDNA viruses. Vector construct deletes rep and cap genes, retaining ITRs.
  • 20:38 - Production & Advantages: Requires helper adenovirus functions (supplied on plasmids) for production. AAV vectors provide long-term gene expression without integrating into the host genome (they form persistent circular episomes).
• 22:54 - Retrovirus (Lentivirus) Vectors:
  • 22:54 - Production & Use: Typically uses a multi-plasmid system (gag-pol, envelope, transgene with packaging signal). Lentiviruses (like HIV-based vectors) can infect non-dividing cells.
  • 24:32 - Advantages & Risks: Provides long-term expression via integration into the host genome. Risk of insertional mutagenesis (e.g., activating oncogenes via LTRs), leading to development of safer vectors with modified LTRs. Can be pseudotyped (e.g., with VSV-G) for broader tropism.
• 25:35 - Poxvirus Vectors (e.g., MVA):
  • 25:35 - Modified Vaccinia Ankara (MVA): Highly attenuated, replication-deficient in mammalian cells (BSL1). Large capacity. Recombinants made via homologous recombination.
• 27:26 - Other Viral Vectors:
  • 27:26 - VSV: Negative-strand RNA virus; transgene can replace glycoprotein gene.
  • 28:20 - Flaviviruses (e.g., Yellow Fever Virus vaccine strain): Used as a backbone to create chimeric vaccines (e.g., Dengvaxia by swapping structural proteins).
  • 29:33 - Newcastle Disease Virus (NDV): Avian virus, used as a vaccine vector for poultry (e.g., expressing H5 hemagglutinin for avian influenza).
• 30:20 - Licensed Vaccines Using Viral Vectors: Examples include Ervebo (VSV for Ebola), Dengvaxia (YFV backbone for Dengue), ChAdOx1 (chimpanzee adenovirus for COVID-19), and Sputnik V (Ad5/Ad26 for COVID-19).
• 32:32 - Self-Amplifying mRNA Vaccines: Based on alphavirus or flavivirus replicons, these mRNAs encode their own polymerase to amplify the antigen-encoding RNA, potentially increasing protein expression.
• 34:18 - Gene Therapy for Monogenic Diseases:
  • 34:18 - Overview: Over 6,000 diseases caused by single-gene mutations (1 in 200 live births).
  • 37:32 - Early Trials & Setbacks:
    • Cystic Fibrosis (CFTR): Adenovirus vector showed transient expression, limited by immune response.
    • Jesse Gelsinger (1999): Died after Ad-vector treatment for ornithine transcarbamylase deficiency due to a massive immune response, halting many trials.
    • X-linked SCID (IL2RG): Retroviral vector cured infants but later caused leukemia in some due to insertional mutagenesis near oncogenes (e.g., LMO2). Led to safer vector designs (e.g., self-inactivating LTRs).
  • 43:43 - Success Stories & High Costs:
    • Sickle Cell Disease: Corrected using lentiviral vectors or CRISPR-Cas9 (e.g., Lyfgenia, Casgevy).
    • Inherited Retinopathies (e.g., Leber Congenital Amaurosis - LCA): Luxturna (AAV vector for RPE65 gene) approved, costing ~$895,000 for two eyes.
    • Other successes: Spinal Muscular Atrophy (Zolgensma, AAV9, ~$2.25M), Hemophilia A/B (AAV, ~$3M).
• 50:13 - Viral Oncotherapy (Using Viruses to Kill Tumors):
  • 51:03 - Historical Attempts: Early, often unethical trials in the 1940s-50s hinted at viruses' potential to cause tumor regression, despite severe side effects.
  • 53:21 - Tumor Selectivity Strategy 1: Exploiting Interferon Defects: Many cancer cells have defective interferon pathways. Viruses can be engineered (e.g., Ad E1B deletion, HSV ICP34.5 deletion) to be unable to counter interferon, thus replicating only in tumor cells.
  • 55:08 - Tumor Selectivity Strategy 2: Receptor Targeting & p53/RB Defects:
    • Measles vaccine targets CD46, often upregulated on tumors.
    • Adenovirus can be modified (e.g., Onyx-015 lacking E1B) to only replicate in cells with defective p53/RB pathways (common in tumors).
  • 58:28 - Post-Entry Targeting: Using tumor-specific promoters or exploiting differential microRNA expression between normal and tumor cells.
  • 59:44 - Arming Oncolytic Viruses:
    • Expressing pro-drug converting enzymes (e.g., HSV TK for Ganciclovir, cytosine deaminase for 5-Fluorocytosine into 5-FU like in Toca 511).
    • Expressing immunostimulatory molecules (e.g., GM-CSF).
  • 1:03:20 - Licensed Oncolytic Virus: T-VEC (Imlygic): A modified Herpes Simplex Virus for melanoma. Expresses GM-CSF, deleted for ICP34.5 (tumor specificity) and ICP47 (enhances antigen presentation).
  • 1:04:43 - Other Oncolytic Candidates: Myxoma virus (rabbit virus) and Measles virus vaccine strain (can be engineered with sodium-iodide symporter for imaging/radiotherapy).
  • 1:06:10 - Mechanism: Direct Lysis & Abscopal Effect: Viruses directly kill tumor cells and release tumor antigens, triggering a systemic anti-tumor immune response that can affect distant, untreated tumors (abscopal effect).
• 1:08:12 - CAR T-Cell Therapy: Viral vectors (typically lentiviruses) are used to engineer a patient's T-cells to express a Chimeric Antigen Receptor (CAR) that targets cancer cells. Approved for certain blood cancers.
• 1:09:17 - Conclusion: Importance of Basic Research: All therapeutic virology advancements rely heavily on fundamental discoveries in virology, molecular biology, and immunology. Continued support for basic research is essential.