Micro Week 5

Types of virus genome structure

+ssRNA: linear, of the same sense as mRNA. The genome can serve as mRNS, read AUG for methionine. -ssRNA: linear, antisense to mRNA. There are also examples of double strand linear RNA viruses and one circular single strand RNA virus, that happens to be of negative sense. DNA viruses can also be single strand, but the sense of the strand doesn't matter in this case because the single strand is rapidly converted to a double strand when the virus enters a cell. So for Parvovirus, either the + or - strand is packaged into genomes. Most common are linear double strand DNA genomes, but there are also circular dsDNA genomes as well. Most viruses have only a single piece of nucleic acid for their genomes, but some RNA viruses have multiple segments of nucleic acid for their genome. These segments function as mini-chromosomes

Strategy for making retroviral vectors

1) First, the genes encoding the structural proteins are deleted from the genome and cloned into a plasmid expression vector that is then transfected into a cell such that the cell can express the structural proteins, but no viral genomes. This is done in cell culture and the cell is then called a "packaging cell line". 2) To the produce a gene therapy vector, sequences from the virus that result in packaging of the genetic material into virus particles (called the packaging signals) are removed, cloned into a plasmid, and the therapeutic gene of interest is cloned between the packaging signals. 3) When this plasmid with the packaging signals and therapeutic gene is introduced into the packaging cell line, the structural proteins assemble the therapeutic gene into virus particles, which are then used as the gene therapy vehicle (or vector). This is now called the "producer cell line". 4) The virus particles (or vectors) that are released from the packaging cell can infect new cells (such as those removed from patients in an ex vivo strategy, or if given directly to a patient in an in vivo strategy), but because the genome lacks the genes encoding the structural proteins, they only deliver the therapeutic gene into cells, but no other infectious viruses are made since those cells (in the patient) do not encode structural proteins of the virus.

Therapeutic vs cytolytic strategies

1) Therapeutic strategies = Delivery of a gene that encodes a protein that is either defective or that is not present due to mutation(s) in the patients' endogenous gene(s). 2) Cytolytic is synonymous with "cell killing" = In cytolytic strategies a vector is designed to destroy or eliminate a diseased cell or tissue. The example here shows a virus carrying the gene for thymidine kinase (TK) from herpes simplex virus (HSV). Expression of HSV-TK converts the prodrug gancyclovir to the toxic product ganciclovir-phosphate which is a nucleoside analog that disrupts DNA replication. Many gene therapies applications for cancer involve cytolytic strategies.

Hep A vaccine

A killed vaccine and has played a major role in the reduction of hepatitis A that we've seen in recent years in the US.

Unplanned longevity

A related concept is unplanned longevity. The cells are not cleared by the immune system and forced to replicate, but rather the cells are immortalized and must replicate for long periods of time. The example here is for EBV and the associated Burkitt's lymphoma (BL), a B‐cell lymphoma. EBV immortalizes the B‐cells that it latently infects, and as these B‐cells replicate through the years, a chromosomal translocation event can occur between chromosomes 8 and 14. The translocation places a cellular oncogene, c‐myc (not a tumor suppressor gene), under the immunoglobulin heavy chain promoter. B‐cells, of course, "live" to produce antibodies, so the Ig heavy chain promoter is a very strong promoter, resulting in the overproduction of c‐myc protein. This results in a B‐cell lymphoma. BL is "endemic" in some parts of Africa and is always associated with EBV. However, BL also occurs outside of Africa, and in many of these cases, EBV is not involved. So, while EBV is clearly linked to BL, EBV infection is not the only cause for the chromosomal translocation.

Luxturna

A second gene therapy intervention (Luxturna) received FDA approval in December 2017. This is the first FDA approval of a gene therapy product for an inherited condition, namely Leber's congenital amarosis (an autosomal-recessive blinding retinal disease caused by biallelic mutations in the RPE-65 gene.) However, it will be expensive. Treatment will cost $425,000 per eye for the therapy, making it the most expensive drug ever marketed in the US. An initial safety trial was performed in which 3 young adults were treated by injection of an AAV vector carrying the RPE65 gene into subretinal region of one eye. This trial showed no adverse effects of the vector and additional trials were performed. The Phase 2/3 efficacy trials involved 12 patients ranging in age from 8-44. All the patients responded well to the treatment and the study subjects were able to walk through a low-light obstacle course such that vision was restored enough to where they were no longer classified as legally blind. This gene therapy product (called Luxturna) is now FDA-approved and available for patients with LCA.

Long latency retroviruses

A third and distinct group of cancer‐causing retroviruses is the nontransducing, long latency retroviruses. These are significant in that the lone human retrovirus that causes cancer belongs to this group - human T‐cell lymphotropic virus (HTLV‐1). There is a much lower rate of tumor formation and much slower rate (years) to tumor production.

3 ways that viruses lead to cancer

Activate signaling pathways to stimulate constitutive growth. Release cell cycle control to allow uncontrolled growth. Infected cell destruction/clearance leads to unplanned regeneration

Genome replication for RNA viruses general

All RNA viruses must encode their own RNA-dependent RNA polymerase. This is because animal cells generate RNA from a DNA template - animal cells do not make RNA using an RNA template. But the genome of RNA viruses is the only template they have, so they must have an RNA (template)-dependent RNA polymerase.

Viral entry into cells via endocytosis

An enveloped virus contains an attachment protein in the envelope, which binds to a receptor on the cell surface. Attachment triggers endocytosis, and as the membrane invaginates, it "captures" the virus. Eventually the invagination "pinches" off the membrane, forming an endosome. As the endosome matures to a late endosome, the pH drops to create an acid environment inside. The drop in pH triggers a conformational change to proteins on the surface of the virus, which allows them to fuse with the membrane of the vesicle, releasing the nucleocapsid to the cytoplasm. Thus, penetration via endocytosis is a pH-dependent process. If this were an RNA virus, the nucleocapsid would disassemble in the cytoplasm to allow genome replication. If this were a DNA virus, the nucleocapsid would migrate to the nucleus via microtubules and disassemble at the surface of the nucleus, releasing the genome into the nucleus. The example is of an enveloped virus, but this is also a common route of penetration for nonenveloped viruses as well.

Correction of ADA-SCID by stem cell gene therapy

Another trial for ADA-SCID was reported in 2002. ADA-SCID causes the "bubble-boy syndrome" resulting from a purine metabolic defect which causes impaired lymphocyte development and function. This trial involved only 2 patients and also used a retrovirus-derived vector containing the ADA cDNA in an ex vivo strategy for transduction of CD34+ stem cells. Like the first trial, the therapy was successful and both children were reported to be living normal lives.

Antigenic variation in an immunodominant structure

Antigenic variation in an immunodominant structure also interferes with vaccine strategy. In the case of hepatitis C virus (HCV), there is a stretch of 30 amino acids in one of the viral glycoproteins that mutates at a high frequency. Because this is an immunodominant structure, we make antibody to the region, but as it mutates in an individual, the initial antibody response is no longer effective at neutralization. As the region constantly mutates, we must continually mount a new immune response to the new antigen. This facilitates chronic infection by HCV, which occurs in 85% or more of patients. Thus, the glycoprotein containing the hypervariable region, even though it is immunodominant, is not a good candidate for a vaccine

Where are nucleocapsids assembled?

Assembly of RNA nucleocapsids occurs in the cytoplasm of cells, and the assembly of DNA nucleocapsids occurs in the nucleus of cells.

Steps in virus replication

Attachment, penetration, uncoating, early transcription and synthesis of nonstructural proteins (viral polymerase, auxillary proteins involved in replication), genome replication, late transcription and synthesis of structural proteins, assembly, release.

Chimeric Antigen Receptor (CAR) design

CARs consist of portions of three different molecules. The recognition domain consists of the variable region of an antibody in which the variable region gene segments are cloned to construct a single-chain variable fragment (scFv). The scFv is then genetically fused to a spacer region that often consists of the extracellular domain of CD8. This is then joined to sequences encoding the transmembrane and cytoplasmic domains of CD3-zeta, which as you know from Dr. Miller's lectures is the signaling domain of CD3 on T cells. To get the CAR into a patient's T cells, a viral vector (generally a retrovirus vector, which I'll discuss shortly) is used to transduce the T cells ex vivo. Retroviruses are used because the CAR gene will integrate into the genome of the T cell resulting in long-term, stable expression.

Rabies vaccine

Can be used prophylactically, but is even more important for post-exposure purposes

Persistent viral infections

Chronic productive infections are those in which the virus and our immune system have reached somewhat of an equilibrium. Virus particles are continually produced, but at levels low enough not to cause overt disease symptoms, at least not at all times. Examples of chronic productive infections are hepatitis B and hepatitis C infections. In contrast to chronic productive infections, viruses such as the herpesviruses establish latent infections. In latent infections, there are periods of time in which no virus particles are produced - the virus genome is still present, but it is quiescent and does not contribute to a lytic infection. When lapses in immunity occur, the virus reactivates, produces virus particles, and a recurrent infection occurs. The virus genome will still be detected in the patient, just no particles. Transforming infections are cancer-causing persistent infections. An example is papilloma virus

Complementation

Complementation is a sharing of proteins, and can occur for both RNA and DNA viruses. In the example shown, the virus in the upper left encodes for two proteins, A and B. The virus in the upper right makes a wild type copy of protein A, but contains a mutation in gene B so that no protein B is made - a lethal result for the virus. If both of these viruses infect the same cell, the B protein synthesized by the wild type virus can be used by both viruses to successfully produce infectious virus, even though the virus on the right still contains a defective B gene. In contrast, if the defective virus infects a cell by itself, it will have no source of protein B, and will not produce infectious virus, reaching a dead-end pathway. Thus, complementation allows the defective virus to be propagated and maintained in the lysate/infection. However, the defective virus has not been permanently corrected because only proteins, not genetic material, has been shared or exchanged

Flu antigenic DRIFT

During the replicative cycle of flu in another person, or in a non-human host such as a pig or fowl, the hemagglutinin and/or neuraminidase genes accrue mutations that alter the determinants that were recognized by protective antibody responses in human hosts. Typically, this type of antigenic variation in influenza results in a new pandemic that is not terribly serious, probably because many of the determinants recognized by flu-specific T cells in previously infected/immune patients have not been altered, so generating a new B cell response is very efficient and happens relatively quickly

Human papilloma virus

Dysregulation of the cell cycle, then, is a cause of tumorigenesis. Proteins normally involved in DNA tumor virus replication can directly disrupt cell cycle controls during non‐lytic replication. This is a by‐product of virus replication, and is not required for virus replication. A good example of this is human papillomavirus (HPV). Some HPV genotypes cause benign warts and are considered low risk for causing cancer. Other genotypes cause anogenital and head‐and‐neck cancers and are considered high risk for causing cancer. HPV infections are common, and fortunately, our immune system clears most of the infections, but cancer can arise later in a person's life if the infections are not cleared. The first anticancer vaccine is directed against HPV, although it can be argued that the earlier developed hepatitis B vaccine is also an anticancer vaccine.

Eclipse / latent period

Eclipse period: The period of time in which intracellular virus cannot be observed is termed the eclipse period, and marks that time between uncoating of the infecting virus and the assembly of progeny virus particles. Latent period: that period of time in which no extracellular virus can be detected. Thus the eclipse period ends before the latent period. The eclipse period best applies to non-enveloped viruses, while the latent period applies to both nonenveloped and enveloped viruses.

How do distinguish whether a virus is a passenger or a perpetrator of a tumor

Epidemiologic Criteria: Coincident geographic distribution of infection, cancer. Higher incidence of viral markers in cases vs control references. Viral markers should precede cancer. Reduction in infection rates should reduce cancer. Virologic criteria: Virus should transform cells in vitro. Virus genome present in tumor but not normal cells. Tumor induction in experimental animals

Adenovirus as a vector

Episomal, high transduction efficiency, infects replicating and non-replicating cells, elicits an immune response, insert capacity 8-36 kb. Non-enveloped viruses with a large ds DNA genome that can accommodate a large gene inserts (up to ~36,000 bp). There are over 51 different serotypes of adenoviruses in humans and adenoviruses are responsible for 5-10% of upper respiratory infections in children and adults. Hence all of us have seen adenoviruses and have an immunological memory to them. Adenovirus enters cells by binding to specific receptors and following virus uncoating the genome is delivered into the nucleus, but in contrast to retroviruses, adenovirus genomes do not integrate, but instead replicate episomally. Like retrovirus vectors, adeno-vectors have the genes for the structural proteins and proteins that induce host cell division (such as the E1A gene product) deleted. Sometime these are called "gut-less" vectors because they lack almost all viral genes and carry only the therapeutic gene of interest.

Signaling: EBV

Epstein‐Barr virus is a herpesvirus, and 90% of people are infected by late adolescence. A primary infection can cause infectious mononucleosis. EBV will also cause a latent infection in B‐cells, causing life‐long infection - almost all of you harbor EBV genomes in your B‐cells. EBV has been clearly linked to several cancers. There are many factors that can lead to tumorigenesis by EBV, but one key player is latency membrane protein‐1 (LMP1). LMP‐1 is a transmembrane protein that is always "on" even in the absence of bound ligand. Thus, in the same pathway shown on the previous slide, the v‐ onc protein is acting at the level of membrane signaling, but ultimately leads to the constitutive activation of NF‐kB, which then leads to immortalization of the B‐cell.

Requirements and challenges of gene therapy

Gene identification and cloning of a functional cDNA clone that can be expressed in cells, delivery (viral vectors, liposomes, or gene gun technology), controlled gene expression (making the correct amount of therapeutic protein at the right time. Maintaining long-term expression of the gene), host immune responses.

Nucleocapsid

Genome + capsid = a nucleocapsid. For nonenveloped, "naked" viruses, the nucleocapsid is the mature virus. Shown in the top panel is an electron micrograph of an adenovirus, where the icosahedron is visible, with spike proteins emerging. The spike proteins mediate adenovirus attachment to cells

Cell cycle disruption: HPV and SV40

HPV E7 protein (as well as SV40 large T‐antigen) can bind to cellular Rb protein, which is a "brake" in the cell cycle. Rb is a cellular tumor suppressor protein, and if inhibited, will allow E2F activation and uncontrolled cell proliferation. Similarly, HPV E6 (as well as SV40 large T‐antigen again) can bind p53 and lead to p53's degradation. In the absence of p53, another cellular tumor suppressor protein, uncontrolled cell division will occur. Again, because viral genome integration disrupted the HPV E2 repressor protein, E6 and E7 are expressed at high levels, and this high level expression is believed to contribute to cancer formation, and not simply benign wart formation.

HPV cell cycle disruption

HPV is related to polyoma viruses and contains a circular double‐strand genome. A linear representation of the genome is shown the circular genome. The genome contains a number of early genes whose products are needed for genome replication in so‐called permissive cells (because these cells permit lytic infection). There are two late genes encoding the capsid proteins, and it is the L1 protein that is expressed in yeast to make the HPV vaccine. In so‐called non‐permissive cells, the viral late genes are not expressed and no virus particles are produced. However, the virus genome is maintained in these cells - extrachromasomally as circular DNA in benign tumors but as a linear integrated genome in host chromosomes in cancers (bottom figure). Significantly, if the integration event disrupts the E2 gene (the figure shows E2 split in half at each end of the integrated genome), then E2 loses function. E2 normally represses the expression of E6 and E7, two key mediators of cancer.

Signaling: HTVL-1

HTLV‐1 can cause ATL (adult T cell leukemia) by transforming CD4 positive T cells. Each box in the figure represents a gene, some with overlapping sequences. A key player in causing ATL is the tax gene. Tax protein activates the IkK complex (represented here as Nemo‐alpha‐beta complex) in the absence of a "trigger" such as a ligand bound on the cell surface. Activated IkK complex phosphorylates IkB, the inhibitor of NF‐kB (RelA‐p50). When phosphorylated, IkB no longer binds to NF‐kB, allowing NF‐kB to enter the nucleus and activate genes necessary for CD4 cell proliferation. This unregulated proliferation leads to cancer.

Human cancer viruses

Human T-lymphotropic virus type 1 (HTVL-1, a retrovirus). Human herpesvirus 8 (KSHV, causes Kaposi's sarcoma). Epstein-Barr virus (EBV, another herpesvirus). Human papillomavirus. Hep B virus. Hep C virus. SV40 is not a human virus, but it will be discussed because it has been a useful model system for studying cancer production.

Immortalized vs transformed cells

Immortalized cells retain original properties but grow indefinitely. Transformed cells are immortalized but lose many growth properties: Reduced need for serum growth factors, Loss of contact inhibition, Anchorage independent (can grow in soft agar), Appear round as opposed to typical morphology, May cause tumors when introduced into suitable animal

Recombination

In recombination, we have an exchange of nucleic acid, so that the recipient can be permanently repaired. In this example, we have two viruses, each with a lethal defect in either gene A or gene B. If these viruses infect the same cell, we could have a reciprocal recombination event that swaps the B genes of the two viruses. As a result, we have a virus defective for both A and B genes, but we also have a fully wild type virus genome as well. Both viruses will be released from the cell because of complementation, but whereas both viruses were originally defective, the infection is now releasing a fully infectious wild type virus who had its B gene repaired. This is an exchange of genetic material, not simply a sharing of proteins. Strictly speaking, when we are talking about true recombination with the formation of Holliday structures, etc. that you learned about in your molecular biology course, recombination is limited to DNA viruses. However, RNA viruses can achieve the same outcome using a mechanism known as strand switching in which the RNA polymerase "hops" from one template to another to effectively exchange nucleic acid.

Self-assembly of isocahedral capsomeres

Individual protomers self-assemble to form triangular capsomeres. The triangles then assemble to form a pentamer - so called because it exhibits 5-fold axis of symmetry. The pentamers then self-assemble to form procapsids - loose structures that resemble the final capsid, but which have gaps so that the genome can be inserted. Thus, these hollow shells are first assembled, and then the genome is inserted once it has fully replicated. This occurs in a sequence rather than at the same time as in concerted assembly, hence the phrase sequential assembly. Once the genome is inserted, the procapsid completely seals to generate the mature procapsid. The capsid can only contain a fixed size of genome (again, in contrast to helical assembly) because of the internal pressure exerted by the genome. This is known as headful packaging - too large a piece of nucleic acid, and the capsid would "explode." One genome equals one "head full" for the icosahedral capsid.

Interferon-alpha (IFN) pathway

Infected cells release IFN, and the IFN is then able to bind to surrounding cells, both infected and uninfected. There are 3 major pathways that are induced upon IFN binding to a cell. IFN-induced pathways are expressed in all cells bound by IFN, but only in the presence of viral dsRNA (ie, virus infection) are the pathways activated. In this way, infections can be contained by cell "suicide" and uninfected cells are not harmed. The major outcome of the activated PKR pathway is inhibition of all protein synthesis in the cell, and the major consequence of the activated 2-5A pathway is cleavage of all RNA in the cell. The Mx pathway is less well understood, and we will not discuss it

Flu antigenic SHIFT

Influenza virus has a segmented genome that has either 7 or 8 segments of ssRNA. During co-infection of a non-human host (pig or fowl, typically), two different viruses can undergo their replicative cycle simultaneously in the same cell. As two flu viruses replicate simultaneously inside the same cell, it is possible for the resulting virions to be packaged with a shuffled version of the genome segments. Therefore, the resulting antigenically shifted virus can cause a new pandemic that causes much more severe disease than a viral strain that arose via antigenic drift.

Adeno-associated virus (AAV) as a vector

Integrates genome into specific region on human chromosome 19, low immunogenicity, no associated disease, infects both dividing and non-diving cells, limited insert capacity ~5 kb. One of the most widely used vectors for in vivo therapeutic strategies is based on adenoassociated virus (AAV), which is a small virus that infects humans and some other primate species, but does not cause disease and results in only a very mild immune response. This is a major advantage for a gene therapy vector. AAV belongs to the parvovirus family, which are non-enveloped viruses that have singlestranded DNA genomes. There are 12 human serotypes of AAV and more than 100 serotypes from nonhuman primates. AAV can infect both dividing and non-dividing cells and persists in an extra-chromosomal state but it can integrate into the genome of the host cell, although this is relatively inefficient.

Cell cycle disruption: KSHV

KSHV can also directly disrupt the cell cycle. KSHV produces a cyclin homolog (v‐cyclin) that activates CdK6. The v‐ cyclin/CdK6 complex is not inhibited by Cip or Ink4 as the normal cellular complex is, and this leads to uncontrolled activation of the cell cycle.

Signaling: KSHV

Kaposi sarcoma‐associated herpesvirus (KSHV) is latent predominantly in B cells, but in other lymphocytes as well. KSHV has been clearly linked to KS (Kaposi's sarcoma, lymphatic endothelial cancer), pleural effusion lymphoma (non-Hodgkin's body cavity lymphoma), and Castleman's disease (benign lymph node tumors, not strictly a cancer), which is not strictly a cancer because the lymph nodes contain benign tumors. KSHV leads to cancer by a sophisticated process, encoding cytokine and chemokine homologs to stimulate cell proliferation and apoptotic inhibitors. A key protein is vGPCR, a constitutively active G protein‐coupled receptor that is not dependent on ligand binding to a receptor for activation. Thus, even in the absence of an extracellular first messenger, vGPCR will lead to the production of a signaling pathway and second messengers in the cytoplasm that will lead to uncontrolled cell division.

Herpesvirus as a vector

Large insert capacity, broad host range, infects dividing and non-dividing cells

Latency

Latency is a non-replicative state that some viruses can achieve in host cells. The viral genome integrates into host cell nucleic acids (either by integrating into the host cell chromosomal DNA as a provirus or by existing in the host cell nucleus in a circularized episomal form). While in one of these states, there is no way for the immune system to recognize infected host cells.

MMR vaccine

Live, measles mumps and rubella. The MMR vaccine has been in the news for not being used frequently enough to obtain herd immunity, and we have had outbreaks of measles and mumps that should not be occurring

Varicella-zoster vaccine

Live. Chickenpox, shingles. There is a childhood vaccine to protect against varicella (chickenpox), which presumably helps in preventing zoster (shingles) later in life. However, there is also an adult vaccine, for those over 60 years, that is used as a booster to help prevent shingles in the elderly who had a natural infection as a child.

Small pox vaccine

Live. No longer in routine use, but first responders can still receive it.

Killed vs live vaccines

Many live viruses can be administered by their normal route of infection, say orally or by inhalation. This is a big advantage over the required injection of killed vaccines. Live vaccines generally require a lower dose, and sometimes only a single administration, because they will produce a subclinical infection, thereby self-amplifying the dose in the host. Killed vaccines almost always require multiple injections to boost the immune response because they cannot self-amplify in the host. Immunity typically lasts longer with a live vaccine because it better simulates a natural infection. If the live vaccine is given via the oral or respiratory route, a mucosal secretory IgA response can be obtained in addition to an IgG response. The secretory IgA response provides key additional protection. Killed vaccines will only produce an IgG response. There are several disadvantages to live vaccines. They tend to be heat labile, and many of the infections we are trying to fight with vaccines are found in the tropics, making transport and storage difficult. Killed vaccines can be lyophilized ("freeze dried") and reconstituted on site for use. The biggest drawback to live vaccines is that they can revert, rarely, to a virulent form that causes disease in recipients - killed vaccines obviously cannot revert.

+ssRNA replication

Most viruses do not use the 5' CAP structure of eukaryotic mRNAs, but a ribosome can bind to the 5' end of the genome at an internal ribosome entry site (IRES). Because the +ssRNA genome can function as an mRNA, a protein is synthesized from the genome. Often in +ssRNA genomes, the protein is one big "polyprotein" rather than individual proteins. The polyprotein is cleaved into its component parts, usually by a combination of host and self-cleaving viral proteases. The viral proteases are sometimes targets of antiviral drugs. Cleavage of the polyprotein frees the viral RNA polymerase, which can now bind to the virus genome and synthesize a complementary, antisense copy of the genome. The antisense strand is then used as an RNA template to make lots and lots of copies of the +ssRNA genome. These copies will serve as progeny virus genomes and as additional sources of mRNA to produce more viral proteins, particularly the capsid proteins and other structural proteins. The RNA progeny genomes will be encapsidated by the structural proteins to produce. Thus, for +ssRNA viruses, the first molecular event is translation of the genome to produce viral proteins.

Liposomes/naked DNA as a vector

No limit to the size of genes that can be delivered, low immunogenicity, poor levels of gene transfer. Formulation of synthetic lipids that bind to and encapsulate plasmid DNA which then fuse with cell membranes to deliver the genetic payload into cells. Liposomes do not bind to specific cell receptors and therefore can deliver therapeutic genes into many different cell types, but generally the efficiency is much lower than for viral vectors.

Retrovirus as a vector

Non-pathogenic in humans, stably transduces dividing but not non-dividing cells, inserts genome into host cell's DNA, long term expression, insert capacity of 8 kb, inactivated by human complement. Enveloped viruses that have an RNA genome that, upon infection of a cell, is converted to dsDNA by the enzyme reverse transcriptase. For infection, virus binds to a specific receptor on the host cell membrane and after uncoating, the RNA genome is reverse transcribed and then the ds DNA genome is delivered into the cell nucleus where it integrates and therefore becomes a part of the host cell genome. This ability to integrate (in theory) allows for long-term expression and allows the therapeutic gene to be maintained during cell division. To generate a gene therapy vector with retroviruses the viral genes encoding the structural proteins (receptor binding protein and capsid proteins) are deleted and replaced with the therapeutic gene of interest. One of the disadvantages of murine-based retrovirus vectors is that they require replicating cells for genome integration, which limits their use. Lentiviruses (or HIV-based gene therapy vectors, which are a sub-type of retrovirus) can integrate into both replicating and non-replicating cells, so most gene therapy vectors used today are based on lentivirus vectors

Nontransducing retroviruses

Nontransducing retroviruses do not contain a v‐onc gene, and will not cause tumors in 100% of infections. Nonetheless, they lead to a high rate of tumor formation, and the time to tumor production is intermediate - weeks to months. MMTV is murine mammary tumor virus, and it is depicted as flanked by its LTRs - long terminal repeat sequences at each end of the virus. The virus has integrated into a mouse chromosome upstream of a proto‐oncogene - a silent progenitor to a c‐onc. The proto‐oncogene does not have a sufficiently strong promoter to express the proto‐oncogene and cause cancer. However, the LTRs of MMTV contain a strong promoter that leads to transcription "to the right" in this figure. Thus, the left LTR transcribes viral genes, but the right LTR transcribes the downstream proto‐oncogene at a rate suitable for tumor development

How are viruses released from cells?

Once assembled, viruses are released from infected cells. A naked virus is most likely released by lysing the cell. Enveloped viruses are released by "budding," in which the nucleocapsid pushes into a cellular membrane (containing viral glycoproteins) and pinches off the membrane to seal around the nucleocapsid and form the mature virus. Typically, the membrane is the plasma membrane, but intracellular membranes are used by some viruses. Note that the lipid portion of the envelope is from the cell, but the glycoproteins in the envelope are from virally-encoded genes.

Phenotypic mixing

One form of complementation is known as phenotypic mixing. When two different enveloped viruses infect the same cell, the envelope glycoproteins of one virus can be incorporated into the envelope of the other virus and vice versa. Because some envelope glycoproteins serve as virus attachment proteins, this can result in a new cell tropism for the "phenotypically mixed" virus. Note, however, that this is still an exchange of protein only, not genomic material, and is therefore a change in phenotype that will be lost once a mixed virus infects a subsequent cell in the absence of a different virus

Oncoviruses activation of signaling pathways

One of the principal ways that RNA and DNA viruses can cause tumors is through the activation of signaling pathways. Often the consequence is an increase in kinase (phosphorylation) cascades that increase gene expression and cell division. V‐oncs can substitute at many of the stages depicted in the figure to upregulate expression. This is achieved because the v‐onc protein are always on (constitutively expressed) in the pathway. Alternatively, the v‐onc may be an alternative transcription factor that leads to increased expression.

Passive immunization

Passive immunization with immunoglobulin can be used when there is not time to wait for immunity to build from active immunization (vaccination). Passive immunization can last up to 3 months. Immunoglobulin can also be used post-exposure. Anyone suspected of rabies exposure will receive immunoglobulin. Babies born to mothers with active cases of hepatitis B will receive passive immunization. Hep A/B, measles, rabies, chickenpox

Capsids

Protein shells. 3 basic types: helical (stick), isocahedral (spherical), complex

Reassortment

Reassortment is limited to those few viruses that have segmented genomes - the presence of multiple pieces of nucleic acid ("mini-chromosomes") in a single capsid. In this hypothetical situation, we have a segmented virus where the capital and lower case letters represent different alleles of the same gene found on the segments. If these two viruses infect the same cell, mixing of the segments can occur so that progeny viruses can be released that now have A,b and a,B allelic combinations in addition to the original viruses (not shown). This is exchange of genetic material that does not involve recombination, but nonetheless alters the genetic make up of the genome

Syncytium formation

Recall that some enveloped viruses bud from the plasma membrane, and at the sites of budding, viral glycoproteins are inserted in the plasma membrane. For viruses that enter cells via direct fusion with the plasma membrane, one of the glycoproteins inserted is a pHindependent fusion protein. This fusion protein functions in the virus envelope to mediate virus entry of a cell. However, when expressed on the infected cell surface in anticipation of budding, the fusion protein can fuse with an adjacent cell, uniting their contents and effectively becoming one large cell with two nuclei. The fused cells are known as a syncytium. As more and more cells fuse, we can observe large, multinucleated cells, and these can be used as a diagnostic marker for some infections, notably herpes, respiratory syncytia virus, and HIV. Syncytia promote localized spread without the requirement for extracellular virus release. In this way, the virus can avoid neutralizing antibodies that may be circulating in the region.

CAR-T cell immunotherapy for Acute Lymphoblastic Leukemia (ALL)

Recently (August, 2017), the first gene therapy intervention for the treatment of acute lymphoblastic leukemia (ALL) for pediatric and young adult patients was approved by the FDA. This product goes by the trade name Kymriah and consists of T cells collected from a patient, which is then modified, or re-programmed to express a Chimeric Antigen Receptor (CAR) that specifically recognizes the B-cell marker CD19. For this strategy, T cells are collected by apheresis and then infected (transduced) by a viral vector expressing the CAR. Those cells are then reintroduced into the patient via T cell adoptive transfer. There are ~ 3,100 patients aged 20 and younger diagnosed with ALL each year. ALL can be of either T- or B-cell origin, but Kymriah is approved for use in patients with B-cell ALL and is intended for patients whose cancer has not responded to or has returned after initial treatment, which occurs in an estimated 15-20% of patients. Upon binding to the leukemic B cells via CD19, the CAR activates the T cells which then destroy the B cells. CD19 is found on the surface of differentiated B cells but is not expressed on hematopoietic stem cells or other essential cell types, which makes it specific; however, normal B-cells also express CD19, so please think about potential adverse effects that could occur

Genome replication for retroviruses

Retroviruses must encode their own reverse transcriptase, because as you probably know, retroviruses have a step in their replication cycle where they convert RNA back to DNA, going against the standard dogma of DNA>>RNA>>protein. Animal cells do not have reverse transcriptase activity. Like -ssRNA viruses, retroviruses must physically provide their own polymerase when entering a cell, even though technically they are +ssRNA viruses. The first event is for the reverse transcriptase to synthesize a complementary strand to the RNA genome, but this complementary strand is DNA, not RNA, hence reverse transcription. The original RNA strand (genome) is degraded, and a second strand of DNA is synthesized to form a double strand DNA molecule. This DNA is then integrated into the host chromosome where the genome basically functions like our own genes, being expressed by host RNA polymerase II. This will ultimately lead to the production of +ssRNA progeny genomes.

Signaling: SV40

SV40 was present in the early polio vaccines, but there have been no reports of those vaccine preps causing cancer in humans. SV40 is a papova virus, specifically a polyoma virus. It is a circular, double‐strand DNA virus of about 5000 bp - small by DNA virus standards. SV40 encodes for 6 proteins, and these are indicated in the figure by the thick black arrows. I want to call your attention to the two proteins that begin in the lower left quadrant, small t‐antigen and large T‐ antigen. The "t" stands for transforming because we know these proteins are key to the transformation of cells infected by SV40. One mechanism leading to transformation is that small t‐antigen inhibits cellular protein phosphatase 2A, a serine/threonine phosphatase. If we think back to the aforementioned signaling cascades, one way to sustain cell division is by having the cascades always on. However, another way to sustain cell division is to never let the cascades reset to baseline levels. This is what small t‐antigen is doing by inhibiting the phosphatase - by never removing the phosphate groups placed on signaling proteins by kinases, the cascade is sustained even in the absence of additional ligand binding/stimulation.

Genome replication for DNA viruses

Small DNA viruses produce proteins that alter the host DNA-dependent DNA polymerase. These proteins redirect the host DNA polymerase to viral origins of replication, away from the host origins of replication. In this way, they ensure replication of the virus genomes. Large DNA viruses encode their own DNA-dependent DNA polymerases, although in theory they do not need to. Making their own DNA polymerase ensures replication from viral origins of replication, but they also provide us with some of our best targets for antiviral therapy. So, in some ways, the large viruses are too sophisticated for their own good

Viral envelopes

Some viruses are surrounded by a lipid membrane, the envelope. The lipid membrane is derived from cellular membranes, but the proteins inserted in the membrane are virus-encoded glycoproteins (proteins modified with sugars). The viral glycoproteins mediate virus attachment, penetration, and release from cells. Therefore, the virus is only infectious if the envelope is intact - the nucleocapsid portion alone is not infectious

Example of antigenic variation

Streptococcus pneumonia uses an antigenic variation mechanism that is fairly standard in the microbial world. There are 84 distinct serotypes of S. pneumo which all differ in the structure of their capsular polysaccharides.

Subunit vaccines

Subunit vaccines are currently available for hepatitis B and human papilloma (HPV) viruses. Virus proteins, but not the intact virus, are produced in yeast and subsequently purified. In the case of HPV, the expressed protein is a capsid protein, which self-assembles to form antigenic procapsids. At one time, an HBV vaccine was derived from blood and contained the same viral glycoprotein that we produce today in yeast. But blood-derived products carry the risk of infection from other agents, so having the subunit vaccines produced in yeast is safer. Subunit vaccines, like killed virus, do require multiple injections. Both of the subunit vaccines can be considered anti-cancer vaccines as both HBV and HPV has significant links to cancer

Measuring virus growth

The basic method for measuring virus growth is a plaque assay. A plaque is the hole in a monolayer of cells that results from the virus replicating and killing the host cells. If conditions are such that only one virus infects a cell, then one plaque equals one infectious particle. After the virus replicates and lyses the first cell, the progeny will infect the surrounding cells, and so on. This leads to a fairly circular shape for plaques as the progeny radiate from the original infection. A lysate is the suspension of virus particles in the culture medium. As the infection spreads from cell to cell, the monolayer will eventually be obliterated, leaving a lysate behind.

Unplanned regeneration

The final mechanism of cancer causation by viruses is unplanned regeneration of cells. Hepatitis B and C viruses (HBV and HCV, respectively) are the leading cause of liver cancer. The cancer develops typically in the sixth decade following infection, and is the result of chronic infection. Over the years, as our immune system clears the infection, it is removing infected hepatocytes. Hepatocytes do not "want" to replicate so much, and over time mutations accumulate, leading to "conventional" cancer mechanisms such as chromosomal translocations and the deletion of tumor suppressor genes. These mechanisms are independent of v‐oncs, which HBV and HCV don't appear to contain. Having said that, HBV encodes a transcription factor, the X antigen ("X" as in mystery or unknown, such as the old TV show "The X‐Files). The X antigen promotes transcription in hepatocytes, and some studies have suggested that it is sufficient to cause cancer in experimental animals. However, the evidence for this is not nearly as clear‐cut as for some of the other examples we've discussed today.

Glybera

The first commercial gene therapy vector that received approval in Europe was an AAV vector carrying the lipoprotein lipase gene. Patient's with LPL deficiency cannot breakdown fats. This is a rare disease affectting only 1 out of 1 million. Symptoms of familial LPL deficiency usually begin in childhood and include abdominal pain, acute and recurrent pancreatitis, eruptive cutaneous xanthoma and hepatosplenomegaly. This gene therapy product (called Glybera) was approved in November, 2012, but the company withdrew renewal of its marketing authorization in October, 2017 after not getting FDA approval in the US. The main reason the company decided not to pursue Glybera was because LPLD is extremely rare (1 in 106 people) and therefore they said it was not "economically feasible" to continue pursuing the product.

First gene therapy trial

The first gene therapy trial in humans occurred in 1990 in which an ex vivo strategy was used to treat children with adenosine deaminase deficiency > causes severe combined immunodeficiency (SCID). The term ex vivo simply means that cells were removed of patients, exposed to a gene therapy vector in culture and the infused back into the patient.

First in vivo gene therapy treatment

The first report of an in vivo gene therapy treatment was in 1993 for cystic fibrosis. In vivo means the vector was infused directly into the patient, with the goal (hope) that it would be able to target the correct cell type responsible for the disease.

Early studies of RNA tumor viruses

The first studies were on RNA tumor viruses - avian leukosis virus and Rous sarcoma virus. These are now known to be transducing retroviruses. The are considered to be transducing viruses because they contain cancer‐causing genes (oncogenes) in their genomes. These genes are known as v‐onc genes for viral oncogenes. V‐oncs are related to cellular oncogenes (c‐oncs) that can cause cancer in humans as well. The top figure shows a c‐onc, c‐src, that can lead to cancer under certain circumstances. The middle figure depicts a standard retrovirus genome, which we have not yet covered in detail in the curriculum, lacking a v‐onc. The bottom figure is the genome of the Rous sarcoma virus, a standard retrovirus genome with an inserted oncogene closely related to c‐src. The viral oncogene is called v‐src. Because the v‐src is part of the virus genome, RSV can cause tumors rapidly and in 100% of infections

Gene therapy of human SCID-X1 disease

The first was reported in the journal Science in 2000 and was for treatment of infants who had mutations in the common gamma-subunit of the IL-2,-4, -7, -9 and -15 receptors, which as you know or will learn from Dr. Miller's lectures causes an early block in T and NK cell differentiation. This therapy used an ex vivo strategy in which CD34+ hematopoietic stem cells were transduced with a retrovirus-derived vector carrying the gene encoding the IL receptor gamma subunit. Remarkably, 10 of the 11 infants had full immune system reconstitution and were no longer immunodeficient. Another serious setback involved 5 of the 11 patients from the SCID-X1 trial reported in the Science article from 2000. Here, the patients developed a T cell leukemia-like illness 3 years after the gene therapy procedure and one of them died. There is now strong evidence that the leukemia resulted from "insertional oncogenesis" in which the vector-derived gene sequences were integrated next to a gene (LMO-2) known to induce tumor in animals. All the patients received chemotherapy and 4 are still in remission

Self-assembly of helical capsomeres

The helical protein shell spirals around the RNA helix. The proteins are not forming a helix because they are bound to the RNA helix, rather, the shape of the proteins (called capsomeres) automatically lead to a helical shape when they gather. This is the principle of self-assembly. Significantly, the capsomeres will continue to assemble around the nucleic acid for the length of the genome. Therefore, the longer the genome, the longer the helix. The capsomeres assemble around the nucleic acid as it is being synthesized. This is known as concerted assembly, because packaging is occurring in concert with genome replication

Reassortment and flu

The individual genes on flu segments can mutate and accumulate point mutations just as any stretch of nucleic acid can, and this alters the antigenicity of the virus somewhat. This is known as antigenic "drift" and is the reason why we must get a seasonal flu virus vaccine each year. However, if reassortment occurs, a fundamentally different allele of the antigen has been introduced to the virus, and this results in a more significant change in antigenicity. This is known as antigenic "shift" where a shift is considered greater than drift. Antigenic shift is responsible for pandemic flu - worldwide epidemics with increased morbidity

Viral pathogenesis general

The routes of viral infection are the same as those for bacterial infections, with the respiratory route playing an even larger role. Initial infection is followed by localized spread, dictated by virus tropism (somewhat akin to plaque formation). Viruses can disseminate infection via the blood (viremia), the lymph, or reach the central nervous system via the CSF, or in some cases, via direct uptake of the virus by neurons and transport to the CNS. Disseminated infection is more significant than localized infection, nonetheless many still resolve on their own.

Types of diseases in gene therapy protocols

There are 4 main types of disease states that are being targeted for gene therapy intervention. The vast majority are for the treatment of cancers followed by heritable, monogenic diseases such as lipoprotein lipase deficiency causing severe pancreatitis due to hyperlipidemia; adenosine deaminase deficiency causing SCID; and Leber's congenital amaurosis, which causes progressive, incurable blindness. Next are infectious diseases and cardiovascular diseases

Superantigens

There are several bacterial and viral pathogens that produce superantigens.These proteins are able to crosslink the MHC class II molecule to the T cell receptor in a non-specific way, by binding simultaneously to the outer surfaces of the MHC class II alpha chain and the beta chain of the T cell receptor. This non-specific interaction results in a pseudo-activation of T cells (between 1-20% of the total repertoire) resulting in a cytokine storm that disregulates normal immune responses. This cytokine storm can cause systemic toxicity that closely resembles septic shock.

Influenza vaccine

There is an inhaled live vaccine and a killed injected vaccine

Staph aureus and GAS subversion of host responses

This figure shows that Staphylococcus aureus uses a wide array of mechanisms to interfere with normal host immune responses. As you can see, these bugs produce a number of molecules that interfere with neutrophil function, the complement cascade, antibody opsonizaton, and that provide resistance to phagocyte killing (via interference with anti-microbial peptides and the oxidative burst). This is a well-studied pathogen, enabling the identification of each of these different virulence factors. Careful study of other bugs would likely identify a similar array of virulence factors that interfere with immune effector mechanisms. GAS (strep pyogenes) is another well-studied bacterial pathogen, and as you can see, it has developed an array of strategies for defeating the effector responses of its hosts. It is important for you to understand that none of these mechanisms provide complete protection against the specific effector mechanism they are meant to interfere with, but they each reduce the efficiency of immune clearance, giving the bug more time to replicate within the host and extending its opportunity for transmission to another host

Viral entry into cells via plasma membrane fusion

This is almost exclusively reserved for enveloped viruses only. Attachment of the virus to its receptor triggers a conformational change to a "fusion" protein in the virus envelope that is pH-independent (normal physiologic pH). This allows the virus envelope to fuse with the plasma membrane, resulting in release of the nucleocapsid into the cytoplasm, and disassembly and genome replication will ensue as described for endocytic penetration.

Varicella zoster virus latency

This is another example of a latent virus infection. This is latent chickenpox (varicella-Zoster virus) that has been reactivated, a condition known as the shingles. VZ remains latent after chickenpox infection in the dorsal root ganglia that serve one side of the trunk or one side of the face. When reactivation occurs, the lesions are typically confined to that side of the trunk or face, often from front midline to back midline. It has been thought for some time that only one reactivation of this latent virus was possible, but there is mounting evidence that one person can have the shingles more than once.

Example of ex vivo gene therapy

This slide shows an example of an ex vivo gene therapy strategy in which hepatocytes from a section of liver from a patient with familial hypercholesterolemia are collected, the cells of interest are isolated and then treated with a retrovirus-based vector carrying the wild-type gene for the low-density lipoprotein (LDL) receptor, which is defective in these patients. The cells that successfully integrated the LDL receptor gene into the genome are then reimplanted back into the patient with the hope that the wild-type gene will be expressed and reduce the high levels of circulating cholesterol in these patients.

CRISPR

This system is called CRISPR/Cas9. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats which are segments of bacterial DNA containing short, repetitive base sequences that form a base-paired hairpin structure. The hairpin is preceded by a sequence that confers sequence specificity as is referred to as the target sequence and together these for what's called the Guide RNA. When transcribed into RNA, the target sequence helps the Cas proteins, which are endonucleases, recognize and cut exogenous DNA. The CRISPR/Cas9 system therefore has the ability to edit genomic DNA of any species by delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell. The cell's genome can be cut at a desired location, allowing existing genes to be removed or new ones added. CRISPR/Cas genome editing techniques have many potential applications

Titers of virus lysates

Titers of virus lysates are generally expressed as the number of plaque-forming units (pfu) per ml of lysate. This is a biological assay - only infectious particles will be recognized in this assay. However, not all virus particles released from an infected cell are infectious. Many will be defective because they failed to package the entire genome, may contain a lethal mutation, or may be missing a key protein. The ratio of the number of virus particles produced to the number of pfu produced is the particle-to-pfu ratio. For animal cells, this can range from as low as 10 particles: 1 infectious virus to as high as 10,000:1. With as much synthesis and assembly that is going on in infected cells, there is much room for error. Lysate titers are too high to be able to infect a Petri dish monolayer and have only one virus infection per cell, a prerequisite for the "one plaque equals one virus" assumption. So, lysates are serially diluted 10-fold, and based on prior experience of typical titers, a subset of the dilutions is used to infect monolayers

Trypanaosome

Trypanasomes use still another version of antigenic variation. These pathogens have genes that encode over 1000 distinct "variant-specific glycoproteins" using a sort of cassette system. During any trypanosome infection, most of the newly generated trypanosomes will express the predominant VSG (in this example, VSGa). Once the host has begun to make immune responses directed at VSGa, some of the daughter trypanosomes begin to express a different VSG protein (in this case, VSGb). Now, the new VSGb-bearing trypanosomes are able to escape the pre-formed immune response of the host, and their numbers increase in the host until the host makes a new VSGb-specific immune response. Now the trypanosome switches VSG expression again, allowing the bug to escape the preformed immune response, and the whole cycle repeats again. This is why African sleeping sickness is a chronic, episodic condition. Ultimately, the inflammation caused by the recurrent immune responses and immune complex formation/clearance causes damage to host tissues, including neural tissue, and ultimately results in coma. This is another form of antigenic shift.

Viral mutations

Viral mutations occur at a relatively high frequency because of the large number of genome copies that must be made inside each infected cell, and in the case of RNA viruses, because RNA polymerases tend to lack proofreading function. Therefore, base substitutions are not corrected as often in RNA viruses, giving them a higher mutation frequency than DNA viruses, whose polymerases have proofreading function. The 3 types of exchanges we will discuss for viruses within the same infected cell are complementation, recombination, and reassortment.

Polio vaccine

We have both a live (oral) and killed (injected) vaccine

-ssRNA replication

When the genome is released into cells, it does not function as a mRNA for the immediate production of viral proteins. There is no IRES for ribosome binding, and no codons are present - everything is in the wrong sense. Therefore, it is not sufficient for -ssRNA viruses to simply encode an RNA-dependent RNA polymerase, they must physically package one in the virus and have it enter the cell along with the genome (it is generally already bound to the genome). The polymerase will immediately transcribe a complementary strand that will be of the positive sense and which will function as a mRNA. This will produce new RNA polymerase, and the +ssRNA will also serve as a template for the generation of progeny -ssRNA genomes. In contrast to +ssRNA genomes, the proteins in this instance are not typically synthesized as a polyprotein. As the cycle continues, the structural proteins accumulate and package the -ssRNA progeny genomes. However, in addition, a copy of the viral RNA polymerase itself, not just its gene, is also packaged. This way, when the progeny virus infects a cell, it will have available an RNA polymerase before new translation can occur in the newly infected cell. Thus, the first molecular event is transcription, not translation.

Somatic vs germline gene therapy

When we discuss gene therapy for humans you should recognize that we are referring to somatic cell gene therapy in which the therapy would be corrective for the patient, but the modification is not inherited by the next generation. For comparison germline intervention is strictly restricted to animal models and involves genetic modification that can be passed on to the next generation. As you're probably aware, we have the technology to create genetically modified individuals using germline gene therapy, but for many reasons this is strictly forbidden by all ethical practitioners of molecular medicine.