21 - Viruses in Biomedicine and Biotechnology

Viruses in science

• small genomes

• ‘simple’ systems

• manipulation of cells

• pathogens

  • humans

  • animals

  • insects

  • plants

  • bacteria

• infections

• genetics

• immunology

• cancer

• biotechnology

• medicine

• molecular biology

Application of viruses in medicine and science

• genetic engineering → cloning and expression vectors

• biological weapons

• vaccines

• cancer therapy

• nanotechnology → viruses as carrier

• antibiotics → bacteriophages

• agriculture → transgenic animals and plants

• gene therapy

viral elements in cloning and expression vectors

• promoter → CMV promoter (human cytomegalovirus major): major TF bind = works in a variety of cells

• Enhancer: boosting protein transcription

• origin of replication

• Internal ribosomal entry site: secondary structure of RNA, interact with 40s subunit

nanotechnology - viruses as carrier

Virus like particles (VPLs)

• only capsid/envelope

• without genetic material

→ Viruses tolerate a wide range of chemical modifications:

• antimicrobial agent

• detection

• imaging

• photosensitive material

• drug delivery

• biosensor

• batteries

• tissue regeneration

→ viruses chemically augmented with capabilities limited only by imagination.

example: tobacco mosaic virus TMV

• structural feature: helical symmetry → form long and very stable tubes

• metal deposition of TMV or its VLPs → metallized TMV or its VLPs used for

  • nanoelectronics

  • batteries

  • catalysts

  • (bio)sensors

bacteriophages

• huge variety, many shapes

  

  • ds DNA, SS DNA, RNA

• life cycle: lytic or lysogenic

  

• high host specificity → good for antibiotics = less side effects

Factors affecting the effectiveness of phage use against pathogenic bacteria

8

phage administration

• phage treatment by: oral, topical, intraperitoneal, intravenous and intranasal administration depending on site of infection

• some cases: iv treatment was faster than the intramammary one

phage concentration (MOI)

• MOI (multiplicity of infection) = ratio of phage/bacteria

• for in vivo and in vitro experiments, MOI varied from 0.01 to 100

dose and moment of treatment

• application of phage was most useful when the treatment was early

• if treated early, multiple doses are better than a single one

environment conditions

• phages survival and persistence affected by physicochemical factors (pH, T)

• eg: proliferation of several phages is limited when pH < 4.5

neutralization

• by AB or other compounds

• need to

  • repeat the administration

  • increase the dose

  • administration of different phages more resistant

  • protection of phages by encapsulation

accessibility to target bacteria

• pathogens develop in tissue or organ compartment inaccessible to phages

• phages diffusion limited in solid matrices

• immune factors in raw milk could protect bacteria from phages

resistance to phage

• bacteria may become resistant → use cocktail of phage or isolate the new phages

specificity

• phages must be lytic and able to infect the target bacteria

• spectrum of phage activity may be increased by use of a cocktail of phage

gene therapy

basic principles:

• gene augmentation therapy

  • insert functioning gene in a cell with non-functioning gene

  • use: monogenic diseases (sickle cell anemia, certain muscle dystrophies, cystic fibrosis,…)

• gene inhibition therapy

  • insert blocking gene in a cell containing faulty gene

  • use: certain cancers (oncogenes)

• killing of specific cells

  • insert suicide gene in a diseased cell → produce toxic product → cell death

  • insert marker gene → marker prot on cell surface recognized by IS → cell death

  • use: cancer/infectionss

Challenges in gene therapy

• delivery (right cell, right place, sufficient number of cells targeted, …)

• required expression level

• duration of desired effect

• avoiding immune responses

• safety (not the right cell, not the right place, …)

• manufacturing and costs

• ethical issues

monogenic disease vector treatment vs treat viral infection

monogenic disease vector treatment advantages:

• no need to target 100% of cells

  • replacing only a few is efficient

  • not the case with viral infections

treat viral infection advantages

• no need for lifelong expression

Examples of viral vectors in gene therapy

most used:

1. adenovirus 20.5%

2. retrovirus 17.9%

3. naked/plasmid DNA 16.6%

4. adeno-associated virus 7.6%

5. lentivirus 7.3%

cancer therapy - Oncolytic viruses

• mode of action

modes of action

1. OV colonizing the tumor

2. direct OV-mediated tumor lysis

3. recruitment of immune cells to the inflamed tumor

4. engulfment of OV-infected tumor cells by DC

5. AG cross-presentation to specific CTLs

6. migration of effector CTLs to the tumor site

7. tumor cell lysis by TAA-specific CTLs

cancer therapy - Oncolytic viruses

• OV in the TME

OV in the TME

• cancer cell lysis

• CAF attack

• vascular endothelial cell attack

• overcome immune suppression

• inflammatory cytokines

• antitumor-adaptive immunity

• PRR activation

• activation of innate IR

• immunogenic cell death

• combination with other anticancer therapies

cancer therapy - Oncolytic viruses

• Barriers to effective OV therapy

→ factors affecting systemic OV delivery

1. anti-viral serum factors

2. sequestration by the mononuclear phagocyte system

3. non-specific binding to red blood cells

4. high interstitial fluid pressure → inadequate extravasation

→ factors affecting intratumoral OV spread

5. dense network of ECM

6. resistance to infection/direct cytotoxicity

7. infiltrating immune cells

8. innate anti-viral response (IFN-α/β)

→ factors affecting the generation of anti-tumor immunity

9. regulatory immune cell infiltration

10. immune checkpoints (PD-1)

11. tumor cell downregulation of MHC/expression of immune-inhibitory proteins

12. aberrant chemokine/cytokine milieu