HOST DEFENSE AGAINST VIRAL INFECTION- ANIMALS • Primary defenses- physical and chemical barriers -skin -mucous secretions -tears -acid pH -surface cleansing mechanisms (swallowing, blinking)

IMMUNE DEFENSES • Three critical processes in the immune defense: -recognition -amplification -control • The immune response to viral infection consists of: Innate (nonspecific) defense: first line of immune defense, responds to any infection, recognizes characteristics common to microbial invaders,consists of interferons, complement, natural killer cells, dictates the adaptive response Adaptive (specific) defense: antibody response and the lymphocyte-mediated response also called the humoral and cell-mediated responses

Innate and adaptive immune responses:

The innate immune response: • Can be activated rapidly and functions within hours of a viral infection. • Continued activity is damaging to the host. • Considerable interplay occurs between the adaptive and innate immune defenses. Important components are: -cytokines -complement -collectins -natural killer (NK) cells

The adaptive immune response: • Differentiates self from nonself, tailored to the particular invader Has ‘memory’; subsequent infection by the same agent are met with a robust and highly specific response that stops the infection • Consists of the: antibody response - humoral response lymphocyte mediated response- cell-mediated response

O’Neill, Scientific American, Jan 2005 pp. 38-45

The inflammatory response: Essential in initiating immune defenses Cell and tissue damage caused by infection induces the inflammatory response Provides communication with the components of the immune system Characterized by redness, heat, swelling and pain

The inflammatory assault is initiated by Toll-like receptors

Inflammation can be initiated in several ways: By interferon released by immature dendritic cells Locally produced cytokines, such as interleukin-1, tumor necrosis factor- a and interferon-g control the reactions that occur during inflammation Inflammatory cytokines also activate B and T cells that are needed for the adaptive response

Cytokines: • Regulatory proteins that mediate intercellular communication during an antiviral defense. • Their presence is one of the first indicators that the host has been infected. • They act locally, near the cells that make them. • They control inflammation, induce and antiviral state in cells and regulate the adaptive immune response. • They exert their activities by binding to specific receptors and activating gene expression. • Three types of interferons are the most important cytokines in the innate response to viral infection.

Interferons Ifn-g is induced only when certain lymphocytes are stimulated to replicate and divide after binding a foreign antigen Ifn-a and Ifn-b are induced by viral infection of any cell type

Interferons • Ifn is induced by accumulation of double stranded RNA (dsRNA). • Ifn induces gene expression at the transcriptional level after binding to specific cell surface receptors. • A cell that is bound to interferon and responds to it is in an antiviral state. • Ifn induces expression of more that 100 genes, products of many of these genes possess broad spectrum antiviral activity. • They lead to cell death by apoptosis or programmed cells death, limiting cell to cell spread of virus. • Production of large amounts if Ifn causes common symptoms such as fever, chills, nausea, etc.

Interferon induced antiviral responses: • Both viral and cellular protein synthesis stops in Ifn treated cells. • This is dues to two cellular proteins, ds-RNA activated protein kinase (Pkr) and ribonuclease L (RNase L). • Pkr is a serine/threonine kinase that has antiviral properties, as well as antiproliferative and antitumor functions. • Activated Pkr phosphorylates the alpha subunit of the translation initiation factor eIF2, inhibiting translation. • RNase L is a nuclease that can degrade cellular and viral RNA; its concentration increases after Ifn treatment.

Interferon induced antiviral responses: • RNase L concentration increases 10-1,000 fold after Ifn treatment, but is inactive unless 2’-5’-oligo(A) synthetase is produced. • 2’-5’-oligo(A) synthetase produces 2’, 5’ oligomers of adenylic acid, only when activated by dsRNA. • These poly(A) oligomers then activate RNase L, which degrades all host and viral mRNA in the cell. • RNase L participates not only in Ifn-mediated antiviral defense, but also in apoptosis. • Ifn is a broad spectrum, highly effective antiviral agent. However, viruses have developed numerous mechanisms for inhibiting interferon action.

The adaptive immune response: • Humoral response Consists of lymphocytes of the B-cell lineage Interaction of a specific receptor on precursor B lymphocytes with antigens promotes differentiation into antibody secreting cells (plasma cells). • Cell-mediated response Consists of lymphocytes of the T-cell lineage Cytotoxic T cells (Tc cells) and T-helper cells (Th cells) are the key effectors of this response.

Cell-mediated response cont. • T lymphocytes recognize antigens on the surface of self cells. • The antigens on self cells can be recognized only by a receptor on the surface of T cells when they are bound to the MHC family of membrane proteins. • The Th cells recognize antigens bound to MHC class II molecules and produce powerful cytokines that affect other lymphocytes (B and T cells) by promoting or inhibiting cell division and gene expression. • Once activated by Th cells, Tc cells differentiate into CTLs that can kill virus infected cells.

The antigen receptors on the surface of B and T cells B cells have about 100,000 molecules of a single antibody receptor per cell, which has specificity for one antigen epitope. T cells bearing the surface membrane protein CD4 always recognize peptides bound to MHC class II proteins and function as Th cells. T cells bearing the surface membrane protein CD8 always recognize peptide antigens bound to MHC class I proteins and function as cytotoxic T cells.

Endogenous antigen processing: MHC class I peptide presentation Intracellular proteins of host and virus are marked for degradation by ubiquitination and are degraded by the Proteasome. The resulting viral peptides are transported into the ER lumen by the Tap1-Tap2 heterodimeric transporter. In the ER lumen, viral peptides associate with newly synthesized MHC class I molecules. MHC class I-peptide complex is transported to the cell surface via the golgi compartments. On the cell surface, the MHC class I-peptide complex interacts with the T- cell receptor of a Tc cell carrying the CD8 coreceptor.

Endogenous antigen processing: MHC class I peptide presentation

Exogenous antigen processing: MHC class II peptide presentation MHC class II complex is prevented form binding to viral peptides in the ER by association with the invariant chain. The complex is transported through golgi where the invariant chain is removed, activating the MHC class II complex. The peptides are derived from extracellular proteins that enter the cell by endocytosis. Viral proteins are degraded in the lysosomes by proteases that are activated by low pH. Endosomes fuse with vesicles containing MHC class II. On the surface of the cell the MHC class II complex interacts with the T cell receptor of a Th cell carrying the CD4 coreceptor.

Exogenous antigen processing: MHC class II peptide presentation

ANTIVIRAL DRUGS Because viruses are obligate intracellular parasites, identification of safe and effective antiviral therapies is difficult. The best antiviral drugs inhibit a specific step in viral replication or pathogenesis. Drug discovery can be accomplished by screening or rational design. The emergence of virus mutants resistant to antiviral drugs is a serious problem. Combination of targeted delivery strategies to control toxicities and resistance.

The pathway for drug discovery

Antiviral compounds Many well-known antiviral compounds are nucleoside and nucleotide analogs: • Acyclovir is a nucleoside analog similar to guanosine, but contains an acyclic sugar group • AZT is a nucleoside analog similar to thymidine, but contains an azide group

Acyclovir: Close to a perfect antiviral drug (specific, nontoxic). Highly effective against herpes simplex virus (HSV), less so against varicella-zoster virus (VZV). Higly selective and extremely safe. Acyclic guanine derivative that inhibits viral DNA synthesis. It is a prodrug, a precursor of the antiviral compound. Activation of the drug requires three kinase activities to be present in the cell to convert acyclovir to a triphosphate derivative, the actual antiviral drug.

Activation of acyclovir: • Acyclovir has no effect on host DNA replication because the first kinase activity is not found in an noninfected cell. • Cellular enzymes complete the synthesis. • Incorporated into viral DNA, lacks the 3’ OH of the sugar ring. • Growing DNA chain terminates, replication is blocked.

Chemical structures of effective antiviral compounds:

Commercially available antiviral drugs:

Steps in replication of HIV targeted by antiviral drugs:

Antivirals against HIV: • Azidothymidine (AZT) Dideoxy analog of thymidine Inhibits viral DNA synthesis • Efficiently phosphorylated to triphosphate by cellular kinases • AZT monophosphate competes with thymidine monophosphate • Much less selective than acylovir and has side effects Does not eliminate previously incorporated provirus

Protease inhibitors Uncleavable mimics of gag-pol polyprotein Inhibits HIV protease Does not eliminate previously incorporated provirus but does prevent further spread Resistance due to protease alterations

Prevention & Treatment of Influenza: Beyond Chicken Soup Zanamivir (Relinza): Blocks active site of all NA proteins. Must be inhaled as a spray, but prevents flu symptoms if given before infection & reduces symptoms if given shortly after infection. Has no side effects. Other Anti NA Drugs in development can be taken as pills and appear to be very effective in reducing illness if taken early in infection. These drugs look very promising in clinical trials and have no adverse effects.

Neuraminidase Inhibitors: Zanamivir & Oseltamivir Mechanism: blocking of the active site of neuraminidase; prevents removaal of sialic acid residues and results in clumping of viral progeny Effective against influenza A & B. Effective when flu symptoms are < 2 days old. Inhibitors reduce disease syndrome by 1 day. May decrease influenza secondary complications Antiviral resistance can occur, but much less frequently than with the ion channel blockers amantadine or rimantadine

Influenza Treatment with Ion Channel Blockers Amantadine & Rimantadine Effective against influenza A but not B. Mechanism: inhibit ion channels & viral uncoating. Can reduce severity & duration of illness if started within 48 hrs of onset of symptoms. Treated persons may shed resistant virus after 5-7 days of treatment (sometimes as early as 2-3 days). Treatment should be discontinued after 3-5 days of treatment or within 24-48 hrs after disappearance of signs/symptoms).

Antiviral Agents for Influenza Neuraminidase inhibitors appear to have similar efficacy to the amantidine & rimantidine ion channel blockers for prevention & treatment of influenza Neuraminidase inhibitors have Less Central Nerveous System side effects, but more Gastro-Intestinal effects Neuraminidase Inhibitors are more expensive, but there is less risk of inducing virus resistance. Very rapidly growing field of research.