virology exam 4

adenoviruses

• non-enveloped, dsDNA viruses

• belong to the adenoviridae family

• moderately sized (around 100 nm)

• have fibers that protrude from the five-fold axis of symmetry and play a role in viral binding to the host cell receptors

genome structure of adenoviruses

• linear, dsDNA genome

• coordinate expression of genes in similar fashion to what is done by herpes and pox viruses

• genes on the left side of the genome are transcribed first, and then these transcripts and resulting proteins are used for expression of genes further down the genome

• the first viral gene to be transcribed, and one of the first to be used in replication is the E1A gene’ it’s protein product has two functions

two functions of E1A protein product

• promotes transcription of other viral genes

  • similar to herpes and pox viruses

• bind to specific host cell proteins that regulate the growth cycle of that cell

  • the results of interacting with this protein is that it essentially locks the cell in a DNA synthesis phase, which is an advantage for a dsDNA virus

• the specific role this gene plays in viral replication makes it critical for controlling replication, and so it is one of the genes that is modified in adenoviruses that are being used as gene therapy vectors

E3 gene

• another gene that is often modified and actually deleted in adenovirus gene therapy vectors

• expressed very late in replication, and doesn’t play a critical role in the early stages of infection like the E1A gene

• the non-critical nature of the gene is useful when generating viruses to be used in gene therapy

adenovirus attachment and entry

• infectious cycle begins with binding of characteristic protruding fibers on the surface of the virus to its host cell

  • these viruses bind the same receptor as coxsackie viruses, and the receptor is named coxsackie-adenovirus receptor (CAR)

• after binding, the virus moves along the surface of the cell until it comes in contact with a clathrin-coated pit, and enters through clathrin-mediated endocytosis

• once inside the vesicle, the protrusions are no longer present, and the base of the fiber (the penton) interacts with the cellular protein on the inside of that endoscopic vesicle (the integrin)

• this interaction begins the disassembly process for the viral capsud

adenovirus replication

• the virus continues to uncoat as it moves through the cytoplasm, and ultimately the genome is imported into the nucleus

• at this point, the host cell pols being transcribing the immediate early gene E1A

• these mRNAs are spliced and transported to the cytoplasm, where the E1A protein gets made

• these proteins then migrate back into the nucleus, where they regulate both cellular and viral genes

• the E1A protein stimulates transcription of the early genes, and they go on to interact with host proteins to lock the cell in a DNA synthesis phase

• the host cell pols also begin transcription of VA genes during this early phase of infection

• after the early proteins are made, they are going to migrate into the nucleus, and along with the cellular proteins, they will be involved in copying of the viral genome

  • this new genome then serves as a template for either more DNA replication or the transcription of late genes

• once these late proteins are made, which are going to be largely encoded for structural proteins, they are also going to go back into the nucleus, where new viral particles are formed

• these first particles formed are actually non-infectious, immature viral particles

  • like other viruses, viral proteins are packaged within the viral capsid, and once they are enclosed, they begin to cleave viral proteins that allow for maturation of the virus

  • however, unlike HIV, maturation of adenoviruses occurs within the nucleus

• after this maturation process, the virus typically lysis in the cell and then move on to infect neighboring cells

how the genome is manipulated for gene therapy

• when using viruses in gene therapy, the ability of the virus to infect and deliver its genetic material has to be balanced with the ability of the virus to induce inflammation or cause disease

  • for most gene therapy treatments, what is desired is that the virus will infect a cell, deliver its genome, but then not actually replicate

  • don’t want the virus reproducing, potentially mutating, and then going on to infect other cells of the body; also don’t want it to replicate and initiate a strong immune response

• in the case of adenoviruses, scientists have discovered that by deleting the E1A gene from the chromosome, they can accomplish both of these goals

• the gene is not at all involved in attachment, entry, or delivery of the DNA to the nucleus, but it is heavily involved in replication

  • so by deleting the gene, we can use the modified viruses to deliver new genes to targeted cells and mitigate/reduce the impact of the virus on the patient

how do scientists make the virus used for gene therapy

• since the virus can’t replicate, they supply the gene from an outside source

• the way all viruses are generated is by growing them in permissive cells

• scientists have engineered cell lines that will express the E1A gene and the E1A protein

• they make a virus that has the E1A gene deleted, meaning that it can attach, enter, and deliver its genome, but it can’t progress past that point

• but if scientists make a cell that produces this E1A protein, then while the virus doesn’t make that protein itself, it can use the protein that’s made by the host cell to complete its replication process

  • this results in the formation of a mutated virus that can only replicate when E1A is supplied from an outside source

• this process of using engineered cell lines to generate large quantities of this virus that are missing that E1A gene is how we make adenovirus vectors for gene therapy

issue in generating viruses for gene therapy

• the size of the viral genome compared to the size of human genes is an issue

• if doctors are trying to replace a dysfunctional gene, then that whole gene needs to be encoded in the viral genome so it can function properly once inside the host cell

• however, all viruses are sensitive to the size of their genomes, and changing the length of the genome too much will impact replication

  • in the case of adenoviruses, more than a 5% change in genome size in either direction (shorter or longer) will cause packaging of the virus to fail

• scientists have gotten around this issue through deletion of other non-essential genes or genes that aren’t essential for the early phases of infection

  • ex. E3 gene; involved in the final stages of forming new viral particles, but since it isn’t necessary in delivery of genetic material

gene therapy

the taking of a gene and inserting it into a cell to replace the function of a missing or non-functional gene in order to correct some type of genetic disorder

genetic diseases

• the discovery of heritable genetic disorders was made in the early 1900s, and this was before we had any understanding of what DNA was or that it was the genetic material

• using mendelian genetics, scientists were able to determine that there were certain diseases that could be transmitted from parent to child through a family line

• over several decades, the understanding of genetics, and how genes and chromosomes function, has increased

beginning of gene therapy

• in the early 1980s, scientists were able to begin doing gene-therapy experiments

• done using tissue culture systems, and scientists were able to demonstrate taking a functional gene, insert it into a cell, and actually correct or fix the genetic disorder

• wasn’t until 1990, that the first gene-therapy clinical trial was actually performed; it was a small clinical trial that was only done in two patients, but it was successful, and able to correct the disorder that those patients were suffering from through multiple treatments using gene therapy

  • broke open the field of gene therapy and led to the advancement of gene therapy and gene-delivery mechanisms

delivery vehicles used for gene therapy treatments

• almost all viruses

• there are a couple chemical treatments or injection of DNA directly, but viruses are the primary vector vehicle used for the delivery of functional genes

• three most important ones:

  • adenovirus (20.5%) - make up the largest percentage of viral vector use in gene therapy techniques partly bc it was the first virus that was ever used for gene therapy treatments, so it makes up a lot of historical clinical trials; however, its use is decreasing, bc other types of viral vectors are being used

  • adeno-associated virus (AAV) (7.6%) - only makes up a small portion of the type of vector delivery methods that are used, but is a very versatile virus; increasingly being used for a variety of different gene therapy techniques

  • retroviruses (17.9) - need to be very highly-genetically manipulated in order to be used as a vector for gene therapy, but their rise has been very successful

good characteristics for viral vectors

1. the most important is the genome capacity of the virus, which refers to how much genetic space is in that virus

   • larges viruses have a lot of genetic space, so you have a lot of room to incorporate a human gene; human genes are very large, especially in comparison to most viral genes

2. also cellular tropism, meaning the type of cell the virus infects

   • if you’re targeting a liver disorder, then you will want to choose a virus that infects hepatocytes, not epithelial cells

3. stability of gene expression, like how long will the gene be expressed in that cell after the virus has delivered it

   • this is a fundamental characteristic of the virus, and how that particular virus goes about gene expression after infection

   • some viruses express genes for an extended length of time, which is ideal for gene therapy, but some don’t

4. want viral vectors that infect cells efficiently

   • if it takes a virus a long time to get inside of a cell and release its genetic content, that gives the immune response a greater amount of time to recognize that virus and develop an immune response which would clear the virus from the cell

5. don’t want a virus that is going to be pathogenic; don’t want to give a patient another disease

   • most of the viruses chosen for gene delivery have low pathogenicity

6. immunogenicity is also important; want a virus that is able to avoid the immune response bc you don’t want the immune system to immediately recognize the virus, and then clear it before it has time to reach the cell

   • deal with this by using a variety of different viral serotype

7. whether or not the virus can infect a resting cell

   • if you’re looking to correct a gene in cells that don’t replicate often, then you need a virus that’s going to be able to infect and replicate in cells that aren’t actively dividing

8. how easily a virus is propagated in cell culture; functionality and logistics

   • if you can’t grow the virus in cell culture, then you’re not going to be able to make enough of the virus to use as a treatment

serotypes

• refers to the immune response that the body produces against a particular type of virus; somewhat analogous to viral strain, but not exactly the same

• the way that serotypes work is that you can have the same virus, and the immune system will recognize this virus, but it won’t recognize it the same way that it will recognize a virus that has a different serotype

• so if a person is first infected with serotype 1 virus, then later is infected with a virus that has serotype 2, the immune system won’t recognize the second virus in the same way and won’t respond as quickly

• so if you have a that has a lot of available serotypes, then if you need to give somebody multiple treatments for your gene therapy, you can administer that therapy using a virus of one serotype the first time, another the second time, and use this method to prevent rapid immune responses from developing

advantages of adenovirus vectors

• they have a very large dsDNA genome, meaning that they have a high-genome capacity

• the first generation of adenovirus gene therapy vectors were only minimally modified

  • they had only a few genes removed, and then the functional genes inserted before they were used in early trials

• since, there has been a few generations of adenovirus vectors, and the viruses that are used today, have been cut down to only the very few genes that are absolutely essential for infection and delivery of the therapeutic gene

  • these modifications have provided a lot of genetic room, so these viruses can include not just one gene, but sometimes multiple genes, which has made them useful for correcting some genetic disorders

• these viruses also efficiently infect non-dividing cells, and they rapidly express their genes, so gene therapy treatments using adenoviruses can show results very quickly

• they also don’t integrate into the chromosome, which can be an advantage or disadvantage depending on what you’re trying to accomplish with the therapy

  • advantage is that you don’t have a risk of genetic mutation within those cells bc they integrate randomly, which could possibly disrupt cellular function and growth, leading to transformation and tumor formation

  • disadvantage is that if the cell being treated divides rapidly when cell division occurs, the viral genome isn’t going to be propagated into the daughter cells, and the virus will eventually be cleared from the body

• can be highly purified and concentrated in large quantities, which is one of the reasons it was initially chosen as a gene-therapy vector

• also a lot of serotypes available, so if a patient requires multiple doses, then there are a lot of different adenoviruses to choose form, and they can be given multiple doses with different serotypes

• have a broad cellular tropism, so you can use them to target hepatocytes or epithelial cells; can be engineered to target a lot of different cell types

disadvantages of adenovirus vectors

• the can be mildly pathogenic; originally discovered in the adenoids, and can infect inside the back of the throat

• also elicit an immune response, which is why having many serotypes is useful

  • of someone is given adenovirus as a gene therapy, they will mount an immune response against that serotype, so you need to have another available

AAV structure

• has a linear, non-enveloped ssRNA genome that does not encode polymerase, and relies on cellular pols for replication; entire genome only encodes the viral replication and capsid genes

• these two genes encode all nonstructural and structural proteins from replication regulation and capsid structure

• capsid proteins assemble into a near spherical protein shell of 60 subunits

advantages of AAV vectors

• has helped move science and research forward due to their excellence as a gene delivery system; revolutionized the usage of viral vectors for gene therapy and transgenic expression

• has very low pathogenicity, which can be attributed to its inability to replicate on its own; requires a cofactor to replicate and cause a productive infection in the body

  • even at worst, AAV causes a very mild immune response making it a great candidate for gene therapy due to the low damage it can potentially cause

• unique from other viral vectors due to its natural tropism towards specific cell types determined by the serotype; has many serotypes

  • lots of research going into modifying these various serotypes and genetically engineering the capsid, so it can target different cell types

• infects both dividing and quiescent cells, allowing the genetic material to be delivered to highly diverse range of cell types

• AAV can persist in an extra-chromosomal form, and can cause long-term expression in non-dividing cells, as it is not diluted until a host cell divides and AAV has a negligible pathogenicity and induces a very mild immune response

  • can integrate into the host chromosome; incorporate into specific parts (not random), so the risk for developing tumors is extremely low

• have been used as viral vectors in over 117 clinical trials worldwide, with promising results in trials for leber’s congenital amaurosis, hemophilia, congestive heart failure, lipoprotein lipase deficiency, and parkinson’s disease

how AAV works

• when AAV infects a human cell alone, its gene-expression program is auto-repressed in latencies ensued by integration of the virus into a two-kb region of chromosome 19, called AAVS1, allowing for the virus to sustain a lysogenic state in the cell/infected tissue until a helper virus is brought into the infected cells

• once introduced, AAV can enter a lytic cycle and replicate along with the adenovirus

• E1a, E1b55k, E2a, E4orf6, and viral associated genes from adenovirus are the known helper genes required for AAV replication

other AAV serotypes

• serotype 2 is the most studied, and presents a natural tropism towards skeletal muscles, neurons, vascular smooth-muscle cells, and hepatocytes

• AAV 1 has been found to excel in gene delivery to vascular endothelial cells, as well as retina, heart, and lung

• AAV 5 exhibits tropism towards vascular endothelial cells, and is efficient in reducing astrocytes

• AAV 6 has been found to be excellent in reducing airway epithelial cells, as well as hepatocytes

• AAV 7 is good at introducing murine smooth muscle cells, like AAV 1 and 5

• both AAV 8 and 3 show a natural tropism towards hepatocytes and are excellent at transducing them

• AAV 4 has tropism towards both kidney and heart cells

disadvantages of AAV viral vectors

• major drawback is its small size; about 5 to 10 times smaller than adenoviruses

• greatly restricts the size of the gene that can be inserted into the AAV viral vector, as the transgene itself can only be less than 4.5 kb in length

• can be difficult to produce and culture; don’t want to impure prep to be injected into somebody bc that could cause adverse side effects

• don’t want to give in low doses; usually want high doses, so we cna be sure that the virus reaches the target in a quantity that is necessary and needed for it to infect

advantages of retroviruses as viral vectors

• have a fairly large genome, not as big as adenoviruses, but we now have the genetic tools available to manipulate viruses; can cut out anything that isn’t absolutely required and put in as much of our human genes as needed

• infects with very high efficiency, and also integrate into the chromosome, allowing for long-term expression

  • however, also a disadvantage bc they could cause tumors if they insert into the genome in the wrong place

  • but since we have the ability to modify viral genomes, some of the research being down in retroviruses is to see if we can direct what sites of the chromosome the virus will integrate into

• there are a lot of different pseudo-types available (pseudo-type is mostly analogous to serotype, difference is that it’s not really naturally occurring)

• genetically modified to express different proteins on their cell surface, which alters both the immunogenicity, as well as the cellular tropism; can heavily modify retroviruses to target almost any specific cell type we want

• have a fairly low inflammatory response, which is natural to the virus

  • don’t tend to elicit really rapid and profound immune responses, which is why they’re able to cause long-term disease upon infection

disadvantages of retroviruses as viral vectors

• can have low capsid stability; possible, once they’re purified, that the capsids degrade if the vector isn’t handled properly

  • could also degrade after they’ve been infected into an individual before the virus actually reaches the target tissue that you’re really trying to get to the cell in order to correct that genetic disorder

• typically only infect dividing cells, but we can go through genetic manipulations and make it so that this virus is able to infect both dividing and non-dividing cells

• mildly pathogenic, low concern, but could always somehow revert and cause potential problems and actually integrate into a chromosome and cause long-term disease in the absence of gene therapy

two viral vector delivery methods

• direct delivery

• cell-based delivery

direct delivery

• first, the therapeutic gene is inserted inside the viral genome, and then the viruses must be generated in culture

• the viruses are collected into highly-concentrated stocks, and then they’re purified and diluted to an appropriate concentration

• this virus prep is the directly delivered into the affected individual; can be injected directly into the liver or into the spleen

  • there are a lot of different organs that have been tested with the delivery method, and have been successful

cell-based delivery

• but not every cell type that needs to be infected can be directly injected as they are not as easily accessible

  • ex. trying to correct a disorder in stem cells or cells that are found in the bone marrow

• process starts about the same as direct-delivery, therapeutic genes are put into the viral genome, and then the virus is made in culture

• then, go into the sick individual and remove the cell type that you are trying to correct

• culture these cells in the lab, and while they are growing you infect them with the virus that you’ve made

• these cells are allowed to grow for a short period of time, and once they have gotten to the point where all the cells in the culture have the corrected gene inside them and are functioning properly, they are injected back into the patient

• works bc our cells (particularly stem cells) have homing properties, meaning that after being injected, they will go to the tissue where they are supposed to be

viruses fighting disease

• understanding viruses has helped to fight chronic and deadly disorders

• the special nature of viruses can be exploited for treating a variety of diseases, including genetic disorders and cancers

• understanding cells and viruses can help to come up with new and creative ways to help the population

• our understanding of how viruses function allows us to exploit them for our use in treating disease

using viruses to target diseases

• alter virus to only infect cancer cells

• deliver tumor killing drugs to cell

• insert genes to alter environment outside of the cancer cell

• stimulate the immune response to recognize cancer cells

• alter virus to only replicate in cancer cells

• deliver genes for long term protein expression

characteristics that are exploited

• the basic nature of viruses are ideal for killing cells, and it doesn’t require manipulation, so many of the innate characteristics of viruses can be exploited to treat disease

• extreme host and sometimes even cell type specificity; can take known information, like surface proteins what molecules are used to attach to specific cells, and engineer viruses that are specifically designed to only target certain cell types

• can also modify viruses to selectively replicate in certain cell types; cancer cells are immortal and produce high levels of proteins and enzymes that aren’t normally found in typical cells, so we develop viruses that only replicate in cells that have high concentrations of certain enzymes

• using viruses as a delivery system for drugs or therapeutic genes

• viruses can also be used to alter the extracellular environment

• can be used to module the immune response, called immunotherapy

oncolytic viruses

• in cancer, we can develop a virus that will only bind to or infect a certain cancer cell type, and once the virus starts replicating and producing viral particles, the cell will be lyses or destroyed

• outcome of this infection is that the target cancer cell is destroyed, but also the production of new viral particles can lead to the infection of other neighboring cancer cells

• also get the production of an immune response, bc have lysed cancer cells floating around, and in some tissues it will give the immune response the ability to react to those cellular proteins and begin targeting that specific cancer and reduce/eliminate the cancer

  • the deliberate activation of the immune response to fight disease is also called immunotherapy

replication selectivity

• don’t target the surface proteins of the virus, but actually target the replication machinery

• these viruses can infect normal cells as well as cancer cells, but once they get inside a normal cell they don’t have the proper proteins or enzymes needed to initiate viral replication

using viruses as a delivery system

• can be used to deliver drugs and therapeutic genes

• essentially what they do without any modification, so this can be exploited to deliver what is needed to a particular cell type

• in the case of gene therapy, viruses are engineered to carry genes that will modify or replace the function of a defective gene inside of a host cell

• can also use these viruses to produce enzymes that will be toxic to the cell that they’ve infected

• by modulating not only the surface proteins to infect a specific cell type, we can also use the virus to deliver a toxic compound to the cell and kill it

  • currently, this treatment method is being explored as a targeted chemotherapy for certain cancers

  • however, this requires a very large virus, and so most of the viruses used for this type of delivery system are dsDNA viruses bc they have the genome capacity

altering the extracellular environment

• one of the primary ways that we do this is by altering angiogenesis, which is the production of blood vessels, or by eliciting immune responses

  • both of these have been some of the most successful ways that we’ve been able to use viruses to treat cancer

• in the case of angiogenesis, we can use viruses that infect a particular cancer cell and then cause that cell to secrete molecules into the extracellular environment that signal the body to stop production of new blood vessels

• angiogenesis is a common hallmark of cancer growth bc as a tumor grows, it needs nutrients, which are supplied by the blood, to fuel and support newly made tissue

  • if we stop angiogenesis, we can starve cancer

• these viruses have been used to reduce the size of some types of cancers, although they haven’t been able to fully eliminate them

• have been able to slow the cancer cell progression, making them good for use in combination therapy with other cancer killing treatments

immunotherapy

• treat cancers by modulating the immune response

• viruses get inside the cell and infect the cancer cell, but then they secrete molecules that specifically activate the immune response and recruit immune molecules to specifically target that cancer

• this treatment method has been so successful for some types of melanoma that it’s being looked at as a near cure for these cancers

gene therapy vs. oncolytic therapy

• for gene therapy, the goal is to keep the cells alive while inserting a functional gene that can be expressed

• for oncolytic therapy, the goal is to kill the tumor cells using the natural killing ability of the virus

oncolytic therapy then vs. now

• in the early years of oncolytic therapy, viruses were sometimes not modified at all, but just injected directly into the tumor

• there was success with this method, but also some large negative consequences, especially if the virus escaped from the tumor tissue and began causing disease elsewhere

• today, most oncolytic viruses used to treat cancer are genetically modified to specifically target and replicate in tumor cells

• now, even though oncolytic viral therapy is regarded as a relatively new and cutting edge treatment for cancers, the knowledge that viruses might be able to eliminate tumors has been around for quite a while

history of oncolytic therapy

• since the mid 1800s, there have been reports of patients with tumors that have regressed after viral infection

• at the time, doctors didn’t know what was causing the patient’s illness and didn’t even know what viruses were yet, but they noticed that tumors in their patients regressed or improved after acquiring an infectious disease like influenza or chickenpox

• for these cases, it was recognized that under the right circumstances, viruses would destroy tumors without causing harm to the patient

• this type of tumor regression was most often documented in cases where the patient was young and had a compromised immune system

  • so if the patient was suffering from leukemia or lymphoma, that would lead to the individual being immunocompromised

• these virus-induced remissions were very short lived, and tumor regression was incomplete, however, is was these observation that led to experimentation in the mid 1900s, using viruses as cancer treatments

• these initial studies were very high-risk and they did not end very well; viruses were not genetically modified, and doctors had little understanding of how viruses worked

  • chemotherapy and radiation surgery treatments were also being tested at the time, and were more successful

• in the last 30 years, we have learned a lot more about cancer biology, in part due to viral research

what we know now

• a collection of genetic changes in the cellular processes lead to cancer development

• some viruses are associated with development of some cancers by inducing mutations or cellular dysregulation through their normal infectious process

  • cancer development is is just a byproduct of viral infection

• viruses can hijack cells and use them for their own purpose, often resulting in cell death, which means that it’s possible they can also hijack cancer cells and destroy tumors as well

two primary ways that viruses are used to treat cancer

• they are modified to selectively target cancer cells

• used to activate the immune response so that the immune system will begin to target the cancer cell as well

• this two-pronged approach has been the most successful when using viruses to treat cancer

• the next evolution of oncolytic viral therapy is to develop a way that these viruses induce memory immunity against cancer cells that would prevent the cancer from reoccurring

viruses being used for oncolytic viral therapy

• there is a range of viruses with different families, replication strategies, sizes, and mechanisms for entering and killing cells

• however, almost all of them have been modified in some way for use as an oncolytic viral treatment

• there are a couple of wild-type, unmodified viruses that are used in clinical use as oncolytic viral treatments (like reovirus), but most that are used in treatment have been modified in some way

• virus families in clinical trials: herpesvirus, adenovirus, measles virus, vaccinia virus, reovirus, polio virus, coxsackievirus, VSV, parvovirus, retrovirus

reoviruses as oncolytic viruses

• a dsDNA virus that typically infect the gastrointestinal tract; rotavirus is a member of this family and it is estimated that 100% of adults have been infected with these viruses

• these viruses naturally show specificity for tumors with RAS activation, which is one of the genetic mutations that is associated with pancreatic cancer formation

• for reoviruses, infection of these cells ultimately leads to cell death by apoptosis

• use of the reoviruses as a cancer treatment works very well labs, and in combination with other modes of cancer treatment, like radiation therapy, however, it does not work well on its own

• one advantage of using this virus is that is has a limited pathogenic effect on the host, so side effects from the virus itself are very mild

• however, since 100% of adults have been exposed to this virus previously, it can also be quickly cleared by the immune system, and is usually cleared before it has time to remove or even significantly eliminate the tumor

RAS activated cells

• almost half of all tumors in humans have some sort of RAS gene that has been activated

• RAS activated cells expressed increased levels of a specific host protein called cathepsin, and these cells also block PKR

• cathepsin functions to help viral encoding so cancer cells with increased cathepsin expression can actually help viruses uncoat faster, which would lead to improved viral replication

• the normal function of PKR is to stop viral translation, but PKR is blocked by these RAS cells

• so in these cells, viral translation can continue, which then also leads to increased viral replication

T-Vec

• was the first oncolytic virus approved for use in cancer therapy

• this virus treats melanoma that can’t be removed by surgery

• it’s injected directly into the melanoma lesions to help selectively target the tumors

• this therapy is a modified HSV1, where two viral genes have been removed and one human gene has been added back

• the two viral genes removed are ICP34.5 and ICP46

  • ICP34.5 is a gene necessary for viral replication in normal cells but isn’t required for replication in cancer cells

  • ICP47 normally functions to suppress the immune response, but for oncolytic viral therapy, we want an immune response against the tumor

• the human gene added back is a gene that encodes for GM-CSF, which produces a cytokine the promotes recruitment and maturation of dendritic cells that ultimately leads to the creation of tumor specific T cells

  • this gene creates an immune microenvironment that allows for the host immune response to take part in the killing off of the tumor

tested versions on oncolytic viral therapies

• the ones being tested in the last couple of decades have been very safe

• there have been no oncolytic virus related deaths or severe reactions in any of the clinical trials where these treatments have been used

• so a major advantage to oncolytic viral therapy as a cancer treatment is that it has limited toxicity

• however, the reason that it has such a low toxicity is that the host immune response eventually recognizes and kills these viruses, so they likely have limited use as a treatment for cancer on their own

  • but they have been shown to be very effective in combination with other therapies

MODULE 13

bacteriophage

• a virus that infects bacteria

• the most abundant organisms on earth

• the most numerous type of virus, bc they infect the other most abundant organism on the planet

• the most well-known have dsDNA genomes

  • when depicted, the most commonly used is myoviridae or T4 phage

• extremely diverse in structure and other ways, like genome type, replication strategies, and cell types they target

structure of bacteriophages

• all of these viruses have an icosahedron capsid head that encases their genetic material

  • exception - filamentous phage and the blobby plasmaviridae; know very little about

• can be categorized as tailed or non-tailed

• for the tailed phages, the tails can be fairly long like with siphoviridae, or they can be small like for T7 or podoviridae phages

• all of the tails have some sort of additional protruding structure, like tail fibers for T7 and lamba phages, or star-like structures for AG3 phages

• both the tail and protruding fibers are used in attachment to the bacterial cell and injection of the DNA into the host cell

• all of these tailed viruses have dsDNA genomes, phages can have different types of genomes as well

  • filamentous phage like M13 have ssDNA genomes, while cystoviridae have dsRNA genomes

  • leviviridae or MS2 phage have ssRNA genomes

  • while the dsDNA phages are the most widely studied phage, the ssDNA phages, particularly the microviridae are the most abundant phages in out environments, especially in the oceans

bacteriophage role in the environment

• play an important role in ecology and evolution of bacteria

• bc of the critical role that the bacteria play in carbon and nitrogen cycling, the ability of phage to infect and potentially change these bacteria, means these viruses also greatly impact geochemical cycles

diversity of bacteriophages

• the majority of viruses belong to the siphoviridae family, even though the best known and most widely studied bacteriophage are the myoviridae

• like eukaryotic viruses, bacteriophage genomes vary widely in size, and most have relatively small genomes, but there are some that have very large genomes

lytic vs lysogenic

• a phage can have multiple phase of replication, lytic and lysogenic phases, but this isn’t true for all

• when viruses go through the lytic cycle, they are categorized as virulent phages since this cycle will result in the death of a bacterial cell

• when phage undergo the lysogenic cycle, they are referred to as avirulent

• there are some phages that can use both of these cycles as part of the replication and infection strategy, like going latent with eukaryotic viruses; however, there are many phage that only undergo lytic replication, and there are some that readily enter the lysogenic cycle upon infection

• the lytic cycle always ends in cell death bc the cell breaks open and releases all the newly made phage particles, and in the lysogenic phase the virus is maintained as a prophage incorporated into the genome

• lysogenic phase cna also exist as an extra chromosomal element, similar to a plasmid

• these prophage genomes then get passed on to new bacteria when the bacterial cell grows and divides somewhat analogous to retroviruses; so while viruses are in this pahse, they are avirulent, but they can be induced to virulence and then enter either the lytic or the chronic cycle

chronic cycle

• there is actually a third life cycle called the chronic cycle, but only filamentous phage use this type of life cycle

• the chronic cycle of phage infection is between lytic and lysogenic phase

• where the phage infects and is maintained as an extra chromosomal element, and instead of resulting in lysis of the cell, it buds out of the cell continuously

• there aren’t any tailed phages that use this type of cycle, mostly bc of the tail structure, which needs to be actively assembled inside of the cell prior to viral egress

differences in infectious cycles

• infectious cycles vary widely, depending on which phase of infection used, and also the details of viral attachment, entry, replication, and egress from the cell

  • some of these differences are due to the phage itself, like whether the virus has a tail or an envelope, and some are due to the bacterium it infects

  • gram(-) bacteria have a different membrane structure than gram(+); (-) has an inner and outer membrane, (+) has a thick cell wall

• modes of entry include membrane fusion, permeabilization, pilus retraction, or a combination of these

tail contraction entry method

• the most famous mech of entry is the one used by T4 phages and is the tail contraction method, in which the phage binds and uses its tail fibers to bind to the lipopolysaccharide, its cellular receptor

• this binding triggers a conformational change where the tail undergoes a rearrangement, and it punctures the outer membrane of the cell, forming a pore

  • for gram(-), the pore will go through both the inner and outer membranes, for (+), the process is similar and the pore extends through the entire cell wall

• once the pore is formed, an additional conformational change occurs and releases the plug protein, which completes the formation of this channel

• once the channel is stably opened, the virus will fuse with the inner membrane and then release its viral NDA into the bacterial cytoplasm

• so after the bacterial NDA is released into the cytoplasm, then viral replication begins

final stages of replication for tailed phage

• after the DNA has been inserted into the bacterial cell, expression of these viral genes leads to formation of viral proteins

• similar to eukaryotic viruses, the genome replication and production of capsid proteins occurs in close proximity to each other inside of the cell, to allow for those components to come together and the viral capsid to form

• the other external structures are assembled in other areas of the cell, but all of the components will come together to form the complete virus, and then it will exit the cell

• this cell lysis can occur in a couple different ways; some bacteriophage encode proteins that can poke holes in the membrane, called endolysins, holins, and spanins

• there are also other cell wall biosynthesis inhibitors, and these work by stopping the production of the cell wall components or peptidoglycan

  • so when the bacterial cell eventually tries to divide, it doesn’t have enough of these components to build new cellular membranes, so it just ends up releasing its internal contents, which now includes viruses, into the environment

immunity of bacteria against viruses

• bacteria have systems analogous to our innate and adaptive immune responses

• in terms of innate immunity, bacteria have restriction enzymes, which are enzymes that cleave DNA but aren’t specific in what DNA they cut (viral vs bacterial); this innate immune response isn’t very strong, bc if it was they would lead to a lot of degradation of the bacteria as well

• bacteria also have an adaptive immune response called the CRISPR/Cas system

  • stands for clustered regularly interspaced short palondromic repeats; refers to short pieces of DNA that are in these areas that the bacteria directs the viral DNA towards

  • has been adapted by scientists and developed into an efficient and targeted system for introducing changes in more complex genetic systems like animal and humans

• they way is works is that after the virus infects the cells and injects the genome, the genome gets degraded, and then pieces of it will get inserted into this CRISPR locus of the bacterial genome

• so once the viral DNA gets incorporated, it becomes part of the CRISPR RNA when the gene is expressed

• after these RNAs are transcribed, they interact with the Cas proteins (short from CRISPR associated proteins), and this RNA is now referred to as a guide RNA

• when guide RNA encounters phage DNA in the cell, it will bind to its complementary areas of that DNA , and the Cas enzyme will cut the area where the RNA is bound and degrade the viral DNA

  • the cleavage will either prevent the DNA from being replicated properly or if the DNA is on its way to getting incorporated into new viral particles, and the new virus won’t function properly in the next cell it infects

uses of bacteriophage

• use these to combat pathogenic bacteria

• felix d’herelle is credited with spearheading work into using phage as a treatment for bacterial infections, known as phage therapy

  • phage therapy is most often used in the context of treating infections, but there are some other uses

• phage therapy was widely studied in the early 1900s prior to the invention of antibiotics, but with the discovery of antibiotics in combination with the ease and cost-effective nature of antibiotic production, the use and interest in phage as a means of treating disease has declined

• however, now we are in an era of antibiotic resistant bacteria, so phage therapy has garnered new attention

• its being tested in clinical trials to treat burn wound infections, ear infections, and infections against antibiotic resistant bacteria

• its also being used by the FDA and USDA to combat the presence of bacterial pathogens and commonly contaminated food products

reasons for studying bacteriophage

• first, bacteria have a limited ability to generate resistance to lytic phages since the phage kills the bacterial cell, but resistance can be generated by bacteria, and this results in another advantage to phage therapy

• a characteristic of bacteriophage is that they have a very narrow host range, but if a bacterium develops resistance to a specific phage bc the host range of the phage is so narrow, resistance will only occur in a very small population of bacteria rather than to a wide range of bacteria, which is what we see with antibiotics

• there’s also a lack of cross resistance; since the mechanism of infection differ bw phage where they use specific cell receptors, we don’t commonly see, or have not seen yet, any cross resistance bw different types of phage

• also don’t see cross resistance bw phage and antibiotics; since chemical antibiotics use very different mechs to kill bacteria, there’s no cross resistance bw antibiotics and phage, which also opens the door for using phage and antibiotics in combination therapies that could improve their effectiveness in treating disease

• another advantage is that phage also have a low toxicity; since they are extremely specific to bacteria, they aren’t going to harm the human or animal hosts that you’re treating

  • in addition, since phage have a narrow host range, they also only result in minimal disruption of the bacteria within the microbiome

• a major challenge with the use of antibiotics is that they are fairly non-specific, and they wipe out the good bacteria along with killing the bad ones, but since phage have extreme host specificity and are sometimes so specific that they only infect certain strains of a given bacterium, this ultimately keeps the natural flora of the host intact during treatment

advantages and disadvantages of phage treatment

• since phage are living organisms that replicate within their host and make more of themselves upon infection, there’s also the potential of only needs a single dose of phage treatment to treat active infections compared to the multiple doses that are always required for using antibiotics

• also some disadvantages, first is the narrow host range, bc bacteria mutate rapidly and phage only infects sometimes a few strains of a given bacterium

• treatment with a phage must target a specific bacterium that’s infecting a person, and this is a challenge if you don’t know exactly what bacterium is causing the infection; whereas for antibiotics, if a general type of bacterium is known, then an antibiotic can be prescribed bc those will target a wide range of bacteria

  • a way around this is to give a phage cocktail, which is a combination of phages that treat many strains of a given bacterium; these can be targeted with just one dose of phage

• another disadvantage of using phage is that they have a small potential to interact with the host immune response; they are live biological agents, and unlike a drug, they don’t always behave predictably

  • they are going to evolve like all viruses, and there’s a potential for them to evolve during the manufacturing process, so even the best controls and testing might not be able to account for mutations that could occur and potentially impact the person being treated

• regardless of the disadvantages, phages are likely the next waves of drug that will be used for treating bacterial infections

the microbiome

• refers to the microorganisms that colonize a particular environment, including external environments like soils and estuaries, as well as the human body

  • refers to both bacteria, archaea, fungi, and viruses, and is not just within the host

  • however, bacteria are the best and most widely studied and characterized component of the microbiome

• in our bodies, human cells and bacterial cells are about 1:1, with even slightly more bacteria

• there are about 30 trillion cells in our body, but are estimated to be colonized by about 38 trillion bacteria

  • most of the bacteria belong to the same six phyla: firmicutes, bacteroidetes, actinobacteria, fusobacteria, cyanobacteria, and proteobacteria

• bacteria that make up the microbiome vary depending on what area of the body it is; some areas close in proximity to each other still have very unique bacterial communities associated with them

• the gut is mostly bacteroides, and some firmicutes and proteobacteria, whereas hair is majority actinobacteria; the gastrointestinal tract has some actinobacteria

changes in the microbiome

• slight changes in the microbiome can have drastic changes in host health; even though the different areas of the body share bacterial communities in common, the proportion to which these bacteria are present have a lot to do with your particular heath in that area

• each bacterial community is distinct and changes in communities can be associated with either healthy states or disease states

• the microbiome can change over time due to many factors that influence microbiome composition, such as your genetics, diet, medications, where you live, etc.

• at birth, the majority of the bacteria within the infant gastrointestinal tract belong to the firmicute phyla, then the first major shift occurs when solid foods are introduced; at about 161 days, there is a much larger increase in the proportion of bacteroides

• another shift occurs when the child is completely transitioned to adult food; with the full adult diet, there is a bit of variability within the first few months, but then you get a well-established microbiome, and there will be changes over time

• time is an important factor to the types of bacterial communities that colonize your gut, but if you extend this over the life of the child into adolescence and adulthood, you’ll see a leveling off of the bacteria in those bacterial communities for that particular individual

geography and the microbiome

• genetics plays a role, but geography is a really big influencer of the microbiome compositiion

• people who live in the same geographical area tend to share greater microbiome similarities with other people that live in the same area irrespective of other factors

• studies have shown that siblings who grew up together, ate similar foods, but are living in different environments have very different microbiomes compared to people living in their same neighborhood

aging and the microbiome

• as people age, there are other factors, such as hormones during puberty, even hormones later in life as people get older

• women hit menopause and their hormones begin to change

• age also plays a role in microbiome composition, but these factors can also be confounded, especially when talking about elderly people by things like medications, changes in hormones, and probably changes in geographic area bc people can move when they retire

• it has been difficult to separate the exact role of age versus all other factors in microbiomes changes

why we need bacteria

• have done a lot of research, particularly in germ free animal models, meaning that these animals, usually mice, are raised in a completely sterile environment, so they are never colonized by bacteria

• one of the biggest takeaways is the fact that the immune response doesn’t develop normally in the absence of commensal bacteria

• people are actually really deficient in a lot of their specific T cell responses, as well as some innate immune responses when they aren’t colonized with the microbiome at birth

• the vast majority of bacteria are not pathogenic, and we have evolved to need these bacteria

genetic disorders

• there are certain genetic disorders that can have a large impact on microbiome composition

• genes mutations that are known to lead to reduced microbiome health: MEFV, APOA1, MYD88, NOD2

• some of the genes that can have the biggest impact on microbiome are those that are associated with the innate immune response, one of the reasons being bc we know that the microbiome plays a large part in immune development

• the microbiome impacts the development of our immune response, and genetic deficiencies in our immune response impact the microbiome composition; don’t have a great understanding of this linkage yet, but our immune responses and intestinal microbiome are closely tied together

• dysbiosis refers to an imbalance of the microbiome composition

• intestinal diseases like inflammatory bowel disease or diseases that are influenced by diet like type 2 diabetes, can result in drastic changes in the microbiome composition

• the onset of some disorders like inflammatory bowel disease and type 1 diabetes is linked to viral infection; don’t fully understand the cause or effect, but it may be the key to resolving or preventing some of these chronic disorders

probiotics

• the observed associated bw microbiome composition and health is the idea behind probiotics; if we understand what a healthy gut looks like, we can maybe introduce healthy bacteria to resolve diseases like inflammatory bowel disease

• not as straightforward since the microbiome is impacted by so many different factors, and a healthy microbiome for one person might not be for someone else

studying the microbiome

• one problem with measuring the bacteria in the microbiome is the fact that only about 1% of bacteria are curable; if we can’t grow them in the lab, we can’t study them

• so the way we do this for bacteria and all microbes is through genetic sequencing and metagenomics

• there are a few types of metagenomics methods that are used, but the most common are targeted metagenomics and shotgun metagenomics

targeted metagenomics

• take you community of bacteria and then isolate the DNA from the sample

  • ex. stool sample for the intestinal microbiome

• next, amplify a sequence using PCR, and since it’s the bacterial community, it would be the 16S rRNA gene, which is a highly conserved gene among bacteria

  • use this gene bc there’s enough difference bw bacterial species that they can be distinguished

• after amplification, all of the amplified 16S rRNA genes are then sequences, and you can compare these sequences to databases of bacterial genomes, telling you the identity of the bacteria in the sample

• this method is precise, and also fairly inexpensive bc you’re only sequencing one gene

• use this method if the goal is to confidently determine which bacteria are present in the sample and in what quantity

shotgun metagenomics

• another sequence-based method, but differs in that instead of targeting a specific gene, you sequence all the DNA in your sample

• this method is more expensive as you are sequencing everything, but it also provides much more data

• from this you can get more functional information, not just what bacteria are present, but also what these bacteria are capable of doing, bc this method will show metabolic genes and other functional aspects of the bacteria that are present

• however, with all this information, data analysis is also much more complex

• since so much data is being sequenced, you don’t get the same coverage, meaning that you might miss some bacterial species

• if you have bacteria that are present in lower abundance, you might not be able to identify them just bc you’re not getting enough sequence

• use if the goal is to get a good idea of what bacteria are the major ones in a given community and what function they are providing