Prevention & Treatment of Viral Diseases Abira Khan

History  Prevention of virus infections in practice since the 11th century without knowledge of agent  Survivors of smallpox protected against disease  Variolation: Inoculation of healthy individuals with material from a smallpox pustule (Lady Montagu)  1790s experiments by Edward Jenner in England establish vaccination

Prevention and Treatment of Viral infections  Viral vaccines  Antiviral chemotherapy  Interferons

Viral Vaccine General Properties  Immunity based on the antigens on the surface of the virus particles or virus infected cells  Surface glycoprotein for enveloped viruses  Antibodies against core, or non-structural proteins play little or no role in development of resistance to viral infection 

Vaccines Work by Maintenance of a critical level of immunity Herd immunity Virus spread stops when the probability of infection drops below a critical threshold The threshold is virus and population specific: Smallpox 80-85%, Measles:93-95% No vaccine is 100% effective When 80% of population is immunized with measles, 76% of population is immune

How to Make a Vaccine? Induction of an appropriate immune response- Th1/Th2 Vaccinated individual must be protected against disease caused by a virulent form of the specific pathogen

Types of Vaccine Active- Instilling into the recipient a modified form of the pathogen or material derived from it that induces immunity to disease Passive- Instilling the products of the immune response (antibodies or immune cells) into the recipient

Effective Vaccine Safety, no disease, minimal side effects Induce protective immunity in the population Protection must be long-­lasting Low cost (<$1, WHO); genetic stability; storage considerations; delivery (oral vs. needle)

Types of Viral Vaccine  There are two basic types of vaccines  Whole agent vaccines: contains whole non-virulent microorganisms  Subunit vaccines: contain some part or product of microorganisms that can induce an immune response in the recipient  Whole agent vaccine  Inactivated and killed  Attenuated live

Live Attenuated Virus Vaccines  Process of producing attenuated human virus by repeated transfers in cell culture. 1. First panel: isolation of the virus with cultured human cells (orange) 2. Second panel: passage of the new virus in monkey cells (purple). During the first few passages in nonhuman cells, virus yields may be low. Viruses that grow better can be selected by repeated passage as shown in the 3rd panel. 3. Thirdd panel: these viruses usually have several mutations, facilitating growth in nonhuman cells.  The last panel shows one outcome in which the monkey cell- adapted virus now no longer grows well in human cells. This virus may also be attenuated after human infection. Such a virus may be a candidate for a live vaccine if it will produce immunity but not disease.

‘Live’ Attenuated Vaccines  Viral replication occurs, stimulates immune response  Infection induces mild or inapparent disease  Intranasally administered influenza vaccine

Live Attenuated Virus Vaccines  Advantages: 1. The attenuated viruses replicate to some extent in the host 2. The attenuated viruses have a reduced capacity to spread from the site of replication 3. The attenuated viruses cause mild or inapparent disease  Disadvantages: 1. Virus shedding and infection of unvaccinated individuals 2. Arise of revertants due to compensatory mutations 3. Difficulty in prediction of behavior in individuals and the population: Elimination before induction of protective response, Infection of new niches in host, Initiation of atypical infections (e.g. triggering Guillain-Barré syndrome) 4. Ensuring purity and sterility

Inactivated Vaccines  Chemical procedures (e.g. formalin, β-propriolactone, nonionic detergents)  Infectivity is eliminated, antigenicity not compromised  Poliovirus treated with formalin to destroy infectivity  Stops spread from lymph node to blood  Inactivated influenza vaccine  Advantages: 1. non-infectious 2. relatively uncomplicated and inexpensive to produce 3. killed virus more easily stored than live-virus vaccines  Disadvantages: 1. Injection of large amounts necessary to elicit antibody response 2. Vaccine must be injected (no oral delivery) 3. Multiple rounds of immunisation required 4. Vaccination does not result in complete immunity

Subunit Vaccines- rDNA  Break virus into components, immunize with purified components  Clone viral gene, express in bacteria, yeast, insect cells, cell culture, purify protein  Antigen usually a capsid or membrane protein  HBsAg protein produced in yeast  Advantages 1. Recombinant DNA technology 2. No viral genomes or infectious virus  Disadvantages 1. Expensive 2. Poor antigenicity 3. Injected

Subunit Vaccines- Synthetic Peptides  ~20 amino acids in length  Identification of immunogenic viral protein  Cloning and sequencing of gene encoding this protein  Synthesis of short peptides corresponding to sections in sequence  Test for immune response  Advantages: Extremely safe, well-defined  Disadvantages: Expensive; weak and short-lived antibody response; requires adjuvant; single-epitope vaccine will readily select mutants

Disadvantages of Inactivated & Subunit Vaccines  Viral proteins don’t replicate or infect  Don’t send out ‘Danger signal’ to the immune response  Pure proteins often require adjuvant to mimic inflammatory effects of infection

Adjuvants  Stimulate early processes in immune recognition  Produce a more robust acquired immune response with less antigen- Slow release of antigen at site of inoculation, Inflammation  Adjuvants work in at least 3 ways: 1. By presentation of antigen as particles 2. By localisation of antigen to the site of inoculation 3. By direct stimulation of the immune response  Aluminum hydroxide or phosphate

New Vaccine Technology- Viral Vectors  Use nonpathogenic virus to immunize host against a pathogenic virus.  Merges subunit vaccine and live attenuated virus technologies.  Provides “benefit” of viral infection with respect to the immune response without the pathogenesis associated with the virulent virus.  Vaccinia virus: smallpox eradication; continued use as viral vector  Problems: 1. Host is immunized against viral vector as well as the vaccine antigen subsequent uses of vector may result in weak/no response, or an immunopathological response 2. Immunocompromised individuals may be infected within the vaccinated population with adverse consequences

New Vaccine Technology- DNA vaccines  DNA vaccines consist simply of a DNA plasmid encoding a viral gene that can be expressed inside a cell.  The system is based on a double-stranded plasmid, typically from E. coli, carrying its own origin of replicn and a selectable marker (amp).  A key component is a strong eukaryotic promoter element (CMV).  The antigen-encoding gene is placed downstream of this strong promoter and expression may be enhance by including an intron sequence (to facilitate mRNA transport from the nucleus) and a 3’ UTR providing a polyadenylation signal as well as stability to the antigen-encoding mRNA.  The plasmid DNA produced in bacteria can be prepared free of contaminating protein and has no capacity to replicate in the vaccinated host.  Remarkably, unlike the requirements for standard protein subunit vaccines, no adjuvants are necessary to stimulate an immune response- all that’s needed is for the viral protein encoded by the DNA to be recognised by the immune system.  Delivery is by injection into muscle or skin tissue, or by using a gene gun that shoots DNA-coated microspheres through the skin into dermal tissue

Antiviral Chemotherapy  Criteria  Antiviral agent must be capable of selectively inhibiting viral functions without damaging the host  Reduce disease symptoms without modifying the viral infection  For the viruses for which vaccines are not available  To reduce morbidity and economic loss due to viral infections  Treat immunosuppressed patients who are at increasing of infections

Antiviral Chemotherapy (Cont.)  Theoretically, any stage in the virus replicative cycle could be a target for antiviral therapy  Reality, it is very difficult  The mechanisms of action vary among antivirals  Some antivirals need to be activated by cellular enzymes

Why Less Antiviral Drugs?  Compounds interfering with virus growth can adversely affect the host cell. Side effects are common (unacceptable). Every step in viral life cycle engages host functions  Many medically important viruses can’t be propagated, have no animal model, or are dangerous- HBV, HPV, norovirus, Smallpox, Ebola, Lassa  A compound must block virus replication completely! It must be potent  Many standard pharmaceuticals can be effective if enzyme activity is partially blocked Partial inhibition is not acceptable for an antiviral drug- resistant mutants will arise  Many acute infections are of short duration

Targets of Antivirals

Types of antiviral drugs  Nucleoside analogs  Nucleotide analogs  Protease inhibitors  Others

Nucleoside Analogs How do analogs work? Analogs inhibit nucleic acid replication by inhibition of enzymes of metabolic pathways Analogs inhibit nucleic acid replication by inhibition of enzymes of metabolic pathways Inhibition of polymerases for NA replication Inhibition of polymerases for NA replication Some analogs can incorporate into nucleic acid and block further synthesis or alter its functions Some analogs can incorporate into nucleic acid and block further synthesis or alter its functions Analogs can inhibit cellular enzymes also Analogs can inhibit cellular enzymes also Use depends on a high therapeutic ratio Use depends on a high therapeutic ratio Resistant to drugs arise over time Resistant to drugs arise over time Use of combination drugs can delay the emergence of drug resistance Use of combination drugs can delay the emergence of drug resistance

Nucleoside Analogs (Cont.) Acyclovir (Acycloguanosine)  Analog of guanocine or deoxyguanocine  Phosphorylated by virus encoded thymidine kinase  Inhibit virus encoded DNA polymerase  Incorporate into growing viral DNA chain and terminates DNA synthesis  Used for the herpes viruses that encode their own thymidine kinase  Mutant herpes virus lacking thymidine kinase fail to phosphorylate the drug and are resistance to it  Ester is taken up after oral administration, acyclovir is released when the ester is cleaved by cellular enzymes

Nucleoside Analogs (Cont.) Gancyclovir - Methyl guanine derivative related to acyclovir - Inhibit DNA polymerase - Block viral DNA chain elongation - Active against cytomegalovirus, good for transplant patients with severe CMV infections - but not active against latent infection - Good for retinitis - Can cross the blood brain barrier and the placenta

Nucleoside Analogs (Cont.) Lamivudine (3TC) NS analog with antiretroviral activity NS analog with antiretroviral activity Used against HIV since 1995 Used against HIV since 1995 Also used against HBV Also used against HBV Needs to be phosphorylated Needs to be phosphorylated Inhibit RTase and viral DNA synthesis Inhibit RTase and viral DNA synthesis Resistance develops by mutation at codon 184 of the reverse transcriptase gene Resistance develops by mutation at codon 184 of the reverse transcriptase gene This mutation inhibits resistance to zidovudine This mutation inhibits resistance to zidovudine

Nucleoside Analogs (Cont.) Ribavirin Synthetic analog related to guanosine Synthetic analog related to guanosine Effective against both RNA and DNA viruses Effective against both RNA and DNA viruses Inhibit GTP synthesis which may affect viral mRNA synthesis Inhibit GTP synthesis which may affect viral mRNA synthesis Approved for aerosol treatment of RSV infection in infants Approved for aerosol treatment of RSV infection in infants Intravenous treatment is effective against Intravenous treatment is effective against Lassa fever

Nucleoside Analogs (Cont.) Stavudine (d4T) Synthetic thymidine NS analog Synthetic thymidine NS analog Activity depends on its phosphorylation by cellular kinases. Activity depends on its phosphorylation by cellular kinases. Inhibits reverse transcriptase enzyme of HIV Inhibits reverse transcriptase enzyme of HIV Also inhibit viral DNA synthesis Also inhibit viral DNA synthesis Approve for use against HIV in 1994 Approve for use against HIV in 1994

Nucleoside Analogs (Cont.) Vidarabine Purine analog used as ophthalmic antiviral drug Purine analog used as ophthalmic antiviral drug Block viral DNA synthesis by inhibiting viral DNA polymerase Block viral DNA synthesis by inhibiting viral DNA polymerase Active against HSV, VZV, CMV, and HBV Active against HSV, VZV, CMV, and HBV Can be used topically to treat corneal lesions by HSV Can be used topically to treat corneal lesions by HSV

Nucleoside Analogs (Cont.) Zidovudine (AZT)  Synthetic thymidine analog  Inhibits replication of HIV by blocking synthesis of proviral DNA  Phosphorylated by cellular enzymes  Viral RTase is 100 times more sensitive to AZT then the cellular polymerase  The drug incorporate into growing DA chain in place of thymidine First antiviral drug against approve for use against HIV since 1987 First antiviral drug against approve for use against HIV since 1987 Also used against HBV, and EBV Also used against HBV, and EBV Resistance develops by mutation in the reverse transcriptase gene Resistance develops by mutation in the reverse transcriptase gene This mutation inhibits resistance to zidovudine This mutation inhibits resistance to zidovudine

Nucleotide Analog Cidofovir Contain one extra PO4 group Contain one extra PO4 group Persist in cells for long periods Persist in cells for long periods Have increased potency Have increased potency Active against CMV, HSV Active against CMV, HSV Inhibits viral DNA polymerase and terminates the growing DNA chain Inhibits viral DNA polymerase and terminates the growing DNA chain Approved for treatment of CMV retinitis in 1996 Approved for treatment of CMV retinitis in 1996

Protease Inhibitor Indinavir Inhibit the proteases of both HIV-1 and HIV-2 Inhibit the proteases of both HIV-1 and HIV-2 Approve for treatment of HIV in 1996 Approve for treatment of HIV in 1996Ritonavir Has improved bioavailability Has improved bioavailability Competitive inhibitor of both HIV-1 and HIV-2 Competitive inhibitor of both HIV-1 and HIV-2 Approve in 1996 for treatment of HIV Approve in 1996 for treatment of HIV

Protease Inhibitor (Cont.) Saquinavir First protease inhibitor to be approved for the treatment of HIV First protease inhibitor to be approved for the treatment of HIV Designed by computer modeling Designed by computer modeling Inhibit viral protease needed at the last stage of viral replication cycle Inhibit viral protease needed at the last stage of viral replication cycle Noninfectious virus particles are produced Noninfectious virus particles are produced Often used in combination with other antiretroviral drugs Often used in combination with other antiretroviral drugs Reduce viral loads and extending survival of infected patients Reduce viral loads and extending survival of infected patients Approve for treatment of HIV in 1995 Approve for treatment of HIV in 1995

Other Types of Antiviral Agents  Amantadine – Synthetic amine, inhibit Influenza A viruses by blocking viral uncoating. Protective against influenza A but not against influenza B or other viruses  Rimantadine – Derivative of amantadine with same spectrum of infectivity. Less toxic and have fewer side effect  Foscarnet- Organic analog of inorganic pyrophosphate, inhibit replication of most herpesviruses, and polymerase of HBV, Retroviruses. Selectively inhibit viral DNA polymerase and reverse transcriptase at pyrophosphate binding site  Influenza virus NA inhibitors- Zanamivir, Oseltamivir  Methesazone- First antiviral drugs to be described, used against pox virus

Drug Resistance  RNA viruses: Error prone RNA polymerase, no correction mechanism  DNA viruses evolve more slowly than RNA viruses because they have less diversity  Genetic analysis of resistance provides insight into antiviral mechanism  May reveal new strategies to reduce or circumvent problem

Mathematics of Drug Resistance

INTERFERONS  Interferons (IFN) are host encoded proteins of that inhibit viral replication.  Belongs to cytokine family  Produced by live animals or cultured cells in response to viral infection or other inducers.  Body’s first line of defense against viral infection.

Properties of IFNs  Categorize into 3 general groups: IFNα, IFNβ, and IFNγ  IFNα and IFNβ are type I interferons; IFNγ is a type II interferon  Normal cells do not synthesize interferons unless they are induced  Different classes of interferons are produced by different cell types  IFNα and IFNβ are produced by different cell types but IFNγ is produced mainly by lymphocytes

Induction of interferons Virus Titer Interferon titer Ab titer