1

LECTURE: 02

Title: THE IMMUNOLOGICAL PROTECTIVE MECHANISMS

AGAINST VIRAL INFECTIONS

LEARNING OBJECTIVES: The student should be able to: • Identify the types of immunity involve in the mechanisms of protection

against viral infections. - Cell-mediated immunity.

a. T-cytotoxic lymphocytes (CD8+ T-lymph). b. Natural killer cells (NK-cells).

- Humoral immunity (bacterial infection).

a. Antibody-mediated immunity:

1. Antibodies bind to virus site that binds to the host cell surface receptors.

2. Antibodies bind to virus to enhance the phagocytosis. 3. Antibodies bind to virus to activate complement system.

b. Cytokines-mediated immunity

1. IFN type 1(IFN- α, and β) inhibits viral replications, and also infected cell proliferations. 2. IFN- γ (called also immune interferon) produced by Th1.

• Enumerate some common serological techniques used in detection and diagnosis of viral infections, and provide some examples of infective viruses.

LECTURE REFRENCE: 1. TEXTBOOK: ROITT, BROSTOFF, MALE. IMMUNOLOGY. 6th edition. Chapter 14. pp 235-240 2. TEXTBOOK: INFECTION AND IMMUNITY . 2NDedition. Chapter 2. p 11-18. 3. HANDOUT.

2

IMMUNITY TO VIRUSES

Viruses are obligate intracellular parasites. They vary in their complexity and replication strategies. Some produce acute infection and are eliminated from the host, whereas others persist indefinitely producing late disease.

Innate immune mechanisms restrict the early stages of infection and delay spread of virus. These defenses include interferon and NK cells.

Antibody restricts the spread of virus to neighboring cells and tissues by neutralizing virus infectivity. This is a important defense mechanism in preventing reinfection.

Cytotoxic T cells recognize virus infected cells. They are able to destroy infected cells early in the virus replication cycle before new viral progeny appear.

Viruses have evolved strategies to avoid recognition by the host. These include latency, antigenic variation and the production of decoy proteins that interfere with the host’s antiviral defenses.

Viruses may directly disrupt the function of the immune system by initiating immunsuppression and immunodeficiency disorders, and by triggering autoimmune disease.

MODES OF VIRUS INFECTION

Viruses are obligate intracellular parasites, and require the host cell’s biochemical machinery to drive protein synthesis and metabolize sugars. They are extremely diverse in terms of their structure and genetic complexities – some have RNA genomes encoding only a few genes, and others have DNA genomes encoding up to 200 genes. Structurally, virus is little more than a bag of protein and nucleic acid. However, life-forms even simpler than this have been identified:

• Viroids are infectious agents of plants which consist of nucleic acid alone, encoding no protein.

• Prions are essentially ‘infectious proteins’ associated with degenerative neurological diseases of animals d man, including scrapie, bovine spongiform encephalopathies (BSE) and Creutzfeldt-Jakob disease (CJD). The infectious prion proteins (PrPsc) are thought to catalyze changes in normal prion protein (PrPc), resulting in the formation of protein complexes in neurons called fibrils, and leading to neuropathology.

A typical virus infection of a cell is shown in Figure-1. Viruses bind to host cells via specific receptors. This specificity identifies in part the tropism of a virus for a particular host or cell. Examples of cellular receptors used by viruses are shown in Figure-2. Following entry the virus uncoats, nucleic acid is released, and transcription occurs followed by the production of viral proteins. The viral genome is replicated and new ‘progeny’ virus particles (virions) are assembled and released to infect neighboring cells and tissues. The details of this process depend on the particular virus and on the metabolic state of the host cell. For example, picornaviruses (small RNA viruses) take around 8 hours to produce new virions, whereas human cytomegalovirus (a DNA virus) may take up to 48 hours.

Viruses are extremely diverse in their ability to infect, persist and initiate disease in a host. Entry is commonly at mucosal surfaces; puncturing skin (e.g. by insect bites or needles) is another very efficient means of introducing virus directly into the blood stream. Replication usually occurs at epithelial surfaces, followed in some cases by viraemia (blood-borne spread) to infection other tissues. Recovery from the infection can involve the elimination of the virus from the host. Some viruses however, (e.g. herpes virus) persist in a latent (non-infectious) form after the acute infection is resolved, and can reactivate to produce new infectious virions. Other viruses can persist in an infectious form despite the presence of the immune

response (e.g. hepatitis B virus and lymphocytic chorio-meningitis virus). In scrapie and CID there is no acute stage; these agents persist as a slow infection, producing disease after many years. Unlike viruses, prions do not provoke an immune response nor is interferon produced following infection. However, dendritic cells can be infected by these agents and constitute an important step in the pathogenesis of infection, by transporting the agents from tissue sites (e.g. gut, skin) to the lymphoid system, where an amplification of prions takes place. Therefore through subsequent lymphocyte trafficking, the lymphoid system serves to aid in the transmission of the agent to the nervous system. A summary of the different forms of infection is shown in Figure-3.

Figure-1 Viruses must infect a host cell before they can replicate

3

Figure-2 Viruses attach to cells with via specific receptors and this partly determines which cell types become infected.

Figure-3 Virus infections can be acute or non-acute, and produce a variety of consequences.

INNATE IMMUNE RESPONSE TO VIRUSES

The early stage of an infection is often a race between the virus and the host’s defense system. The initial defense against virus invasion is the integrity of the body surface. Once breached, early ‘non-specific’ or innate immune defenses such as interferon, natural killer (NK) cells and macrophages become active.

Interferon (IFN) stimulates inhibition of viral replication

There are three types of interferon:

• IFN-α (leucocyte interferon) is encoded by a family or some 20 genes on chromosome 9.

• IFN-β (fibroblast interferon) is encoded by a single gene o chromosome 9.

• IFN-γ (immune interferon) is encoded by a single gene on chromosome 12.

Virus infection of a cell leads to the production of IFN-α/β, which activates antiviral mechanisms in neighboring cells enabling them to resist virus infection (Figure-4). Interferons activate number genes, including two with direct antiviral activity: a 67 kDa protein kinase which inhibits the phosphorylation of eIF.2 and blocks translation of proteins; anda 2’, 5’-oligoadenylate synthetase which activates a latent endonuclease (RNaseL) involved in degrading viral RNA.

4

Figure-4 The antiviral state develops within a few hours of interferon stimulation and lasts for 1-2 days.

Other antiviral mechanisms exist which have more specific action. The Mx proteins inhibit viral transcription of a range of RNA viruses, but have little effect on DNA viruses. In addition to the direct inhibition of virus replication, INF-γ and IFN-α/β enhance the efficiency of the adaptive immune response by stimulating increased expression of MHC class I and II, and both these interferons serve to activate macrophages and NK cells, promoting their antiviral activity.

The importance of interferons in vivo is underlined by the increased susceptibility of mice to virus infection following the depletion of interferons by specific antibody treatment.

Natural killer (NK) cells are cytotoxic for virally infected cells

Active NK cells are detected within 2 days of a virus infection. They have been identified as major effector cells against herpes viruses, and, in particular, cytomegalovirus (CMV). An absence or reduction of NK cell activity, as seen in Chediak-Higashi syndrome and being mutant mice, correlates with an increased susceptibility to CMV infection. It is still unclear which molecules the NK cells recognize on the surface of virus infected cells. However, there is inverse correlation between MHC class I expression and NK cell killing. This is an interesting feature since a number of viruses are now known to downregulate MHC class I expression; this is presumably a strategy to evade T-cell recognition. Interferon-γ activates NK cell function and provides an important mechanism for focusing and activating cells at sites of infection, NK cells are also one of the main mediators of antibody-dependent cellular cytotoxicity (ADCC).

MOST DEFENCE INVOLVING B AND T CELLS

An absence of T cells renders the host highly susceptible to virus attack. For example, cutaneous infection of congenital athymic ‘nude’ mice (which lack mature T cells) with herpes simplex virus (HSV) results in a spreading lesion; the virus eventually travels to the central nervous system, resulting in the death of the animal. The transfer of HSV-specific T cells shortly after infection is sufficient to protect the mice. The significance of T and B cells countering viral infections will now be discussed.

5

Antibodies and complement can limit viral spread or reinfection

Antibodies can neutralize the infectivity of viruses As infection proceeds, the adaptive (specific) immune response unfolds, with the appearance of cytotoxic T cells, helper T cells and antiviral antibodies. Antibodies provide a major barrier to virus spread between cell and tissues and are particularly important in restricting virus spread in the blood stream. IgA production becomes focused at mucosal surfaces where it serves to prevent reinfection. Antibodies may be generated against any viral protein in the infected cell, although only those directed against glycoproteins that are expressed on the virion envelope or glycoproteins that are expressed on the virion envelope or on the infected cell membrane are of importance in controlling infection. Antibody-mediated immunity can be achieved in a member of ways, involving quite diverse mechanisms. Defence against free virus particles involves neutralization of infectivity, which can occur in various ways (Figure-5). Such mechanisms are likely to operate in vivo, since injection of neutralizing monoclonal antibodies is highly effective at inhibiting virus replication. Clearly the presence of circulating virus-neutralizing antibodies is an important factor in the prevention of reinfection.

Figure-5 Antibody acts to neutralize virus or kill virally infected cells.

Complement is involved in the neutralization of some free viruses Complement can also damage the virion envelope, a process known as virolysis. Some viruses can directly activate the classical and alternative complement pathways. However, complement is not considered to be a major factor in the defense against viruses since individuals with complement deficiencies are not predisposed to severe viral infection. Antibodies mobilize complement and/or effector cells to destroy virus-infected cells Antibodies are also effective in mediating the destruction of virus infected cells. This can occur by antibody-mediated activation of the complement system, leading to the assembly of the membrane attack complex and lysis of the infected cell. This process requires a high density of viral antigens on the membrane (about 5 x 106 /cell) to be effective. In contrast, ADCC mediated by NK cells can recognize as few as 103 IgG

6

7

molecules in order to bind and kill the infected cell. The IgG coated cells are bound using the Fc-γRIII (CD16), and are rapidly destroyed by a perform-dependent killing mechanism. Just how important these mechanisms are in vivo is difficult to resolve. The best evidence in favour of ADCC comes from studying the protective in favor of ADCC comes from studying the protective effort of non-neutralizing monoclonal antibodies in mice. Although these antibodies fail to neutralize virus in vitro, they can protect C5-deficient mice from a high-dose virus challenge. (C5-deficient mice are used in this study to eliminate the role of the late complement components). T-cells mediate viral immunity in several ways T cells exhibit a variety of functions in antiviral immunity. Most of the antibody response is thymus dependent, requiring the presence of CD4+ T cells for class switching and affinity maturation CD4+ T cells also help in the induction of CD8+ cytotoxic T cells and in the recruitment and activation of macrophages at sites of virus infection. CD8 T cells are also effective in prevention of re-infection. CD8 T cells are also effective in prevention of re-infection (following vaccination) by viruses such as influenza virus and respiratory syncytial virus. However, even, memory T cells need time to evolve a response to a re-infecting virus, and therefore antibodies assume a more dominant role by neutralizing incoming virus and containing the infection by preventing spread to other tissues. CD8+ cytotoxic T cells The principal T-cell surveillance system operating against virus is high efficient and selective. MHC class I restricted, cytotoxic CD8+ T cells focus at the site of virus replication and destroy virus-infected cells. Virtually all cells in the body express MHC class I molecules, making this an important mechanism for identifying and climinating virus-infected cells. Processing and presentation of virus proteins Virtually any viral protein can be processed in the cytoplasm to generate peptides, which are then transported to the endoplasmic reticulum and are associated with MHC class I molecules. This has particular advantages for the host, since viral proteins expressed early in the replication cycle can be targeted, enabling T-cell recognition to occur long before new viral progeny are produced. For example, T-cell-mediated immunity against murine CMV is mediated by immediate early protein pp89. The epitope has been identified as a monomer peptide presented by the MHC class- I molecule Ld. Immunization of mice with a recombinant vaccinia containing pp89 is sufficient to confer complete protection from murine CMV-induced disease; deletion of the DNA sequence encoding the nonapetide abolishes the protective effect of the protein. The importance of T cell mechanisms in vivo have been identified using various techniques:

• The adoptive transfer of specific T-cell subpopulations or T cell clones to infected animals and monitoring of viral clearance.

• Depletion of T cell populations in vivo using monoclonal antibodies to CD4 or CD8.

• Creation of ‘gene knockout’ mice, in which genes such as CD4, CD8, and β2 microglobulin are removed from the germline.

The continued ability of knockout mice that lack particular lymphocyte populations to mount a response against virus infections is a good illustration of the redundancy that can occur in the immune system. For example, in the absence of CD8+ T cells, CD4+ T cells or other mechanisms are able to compensate and bring the infection under control.

CD4+ T cells can have important effector functions against virus infections

CD4+ T cells are a major effector cell population in the immune response to HSV-1 infection of epithelial surfaces. In this instance recruitment of macrophages occurs as in delayed-type hypersensitivity and an accelerated clearance of virus results. Macrophages are an important component in this process, inhibiting virus infection probably through the generation and action of nitric oxide. Key cytokines in this response include IFN-γ, important in the activation of monocytes, and tumor necrosis factor (TNF-α). TNF-α has several antiviral activities, including the induction of intracellular interferon defence mechanisms and apoptotic cell death following interaction with the apoptotic TNF receptor.

CD4+ cytotoxic T cells

In measles virus infection, cytotoxic CD4+ T cells are generated which recognize and kill MHC class II positive cells infected with the virus. This suggests that measles virus peptides are generated by normal pathways of antigen presentation (i.e. following phagocytosis and degradation). However, other pathways have been implicated in which some measles proteins/peptides enter class II vesicles from the cytosol by an unknown mechanism.

A summary of antiviral defense mechanisms is illustrated in Figure-7, and the kinetics of their induction is shown in Figure-8.

Figure-7 Entry of virus at mucosal surfaces is inhibited by IgA. Following the initial infections, the virus may spread to other tissues via the blood stream. Interferons produced by the innate (IFN-α, and IFN-β) and adaptive (IFN-γ) immune responses make neighboring cells resistant to infection by spreading virus. Antibodies are important in controlling free virus, whereas T cells and NK cells are effective at killing infection cells.

8

Figure-8 Kinetics of host defenses in responses to a typical acute virus infection. Following an acute virus infection, for example by influenza or herpes virus, NK cells and interferon are detected in the blood stream and locally in infected tissues. Cytotoxic T cells (Tc) then become activated in local lymph nodes or spleen, followed by the appearance of serum in neutralizing antibodies. Although activated T cells are absent by the second to third week, T-cell memory is established and lasts for many years.

Figure-9 The major surface antigens of influenza virus are hemagglutinin and neuraminidase. Hemagglutinin is involved I attachment to cells, and antibodies to hemagglutinin are protective. Antibodies to neuraminidase are much less effective. The influenza virus can change its surface slightly (antigenic drift) or radically (antigenic shift). Alternation in the structure of the hemagglutinin antigen render earlier antibodies ineffective and thus new virus epidemics break out. The diagram shows strains that have emerged by antigenic shift since 1933. The official influenza antigen nomenclature is based on the type of

9

10

hemagglutinin (H0, H1 etc.) and neuraminidase (N1, N2 etc.) expressed on the surface of the virion. Note that although new strains replace old strains, the internal antigens remain unchanged.

Dr. MUSTAFA HASAN LINJAWI