Exhaustive Study Guide on Viral Immunity and Immunological Mechanisms

Overview of Antiviral Immunity in Vertebrates

Antiviral immunity in higher vertebrate animals is characterized by a sophisticated and complex integration of innate and acquired (adaptive) immune response mechanisms. Although these are often categorized broadly as two separate systems, there is a considerable amount of interaction between them. Because viruses are obligate intracellular organisms, they have co-evolved alongside their respective hosts. Consequently, hosts have developed diverse and highly specialized capabilities to protect themselves from viral infections and the diseases associated with them. Simultaneously, viruses have evolved a remarkable variety of strategies to circumvent, evade, or overcome these host defenses.

To counter the constant risk of invasion by infectious agents, vertebrates have established a set of defensive measures known collectively as the immune system. During the initial encounter with a virus, the immune system identifies certain viral macromolecules, specifically proteins and carbohydrates, as foreign entities or antigens. Against these antigens, the body mounts various types of responses with the specific purpose of eliminating the virus or preventing subsequent reinfection.

Classification and Components of Innate and Acquired Immunity

Immunity is divided into innate and acquired systems, each possessing unique components and functions. Innate immunity includes physical barriers such as the skin, and mechanical actions like coughing, sneezing, vomiting, mucus production, diarrhea, normal flora, and urine flow. It also involves enzymes like lysozymes, intestinal enzymes, and bile. Molecular components of the innate system include types of interferons such as $IFN-I$ and $IFN-II$, which are produced by specific rapid-response cells. Other innate processes include inflammation, early apoptosis, phagocytosis (carried out by macrophages, neutrophils, and dendritic cells), and the complement system, which consists of antimicrobial serum proteins. Cellular components include Natural Killer ($NK$) cells that lyse infected cells and B1 lymphocytes which produce "natural" antibodies. Additionally, innate defense utilizes $RNAi$ (RNA interference) to obstruct viral replication and utilizes Pattern Recognition Receptors ($PRR$) to detect Pathogen-Associated Molecular Patterns ($PAMPs$). Specific receptors like Toll-like receptors ($TLRs$), such as $TLR3$, serve as ligands for double-stranded RNA ($dsRNA$). Cytokines are also crucial for the induction and regulation of both innate and adaptive immunity.

Adaptive or acquired immunity is characterized by antigen-specific reactions. It includes immune reactions mediated by antibodies and those mediated by cells. Unlike innate immunity, the adaptive system requires the recognition and binding of an antigen through specific receptors located on B and T lymphocytes. While innate immunity serves as the first line of defense, operates immediately upon viral entry, and lacks antigen specificity or memory, the adaptive system provides a tailored response that develops over time.

Interferons (IFN) and Viral Interference

Viral interference is a phenomenon where a cell infected by a specific virus resists a subsequent "superinfection" by the same or a different virus. This process prevents the replication of one virus through induction by another virus or through cellular products like interferons that result from the primary infection. There are two main mechanisms of interference: interference dependent on defective interfering mutants and interference mediated by interferon. Interferons were first reported in the year $1957$ by Isaacs and Lindenmann as new antiviral proteins capable of protecting uninfected cells against infection by the same or related viruses.

Currently, more than $30$ different interferons are known to exist in humans and animals. These are members of a large family of regulatory proteins called cytokines. Chemically, they are categorized into three distinct types. They are a family of cellular proteins produced and secreted in response to "exogenous" nucleic acids to protect other cells. In bovines, there are $14$ known Type I interferons and $7$ Type II interferons. Swine possess $27$ Type I interferons and $1$ Type III interferon.

Type I interferons include several subtypes: $IFN-\tau$ (tau), $IFN-\delta$ (delta), $IFN-\epsilon$ (epsilon), $IFN-\kappa$ (kappa), and $IFN-\omega$ (omega). Type II interferon is represented by $IFN-\gamma$ (gamma). Type III interferon includes $IFN-\lambda$ (lambda), often associated with $IL-10$.

Antiviral Activity and Signaling of Interferons

The induction of Type I interferon in virus-infected cells involves activation through cellular receptors called Pattern Recognition Receptors ($PRR$). These receptors detect pathogen-associated molecular patterns ($PAMPs$) which are highly specific to different virus types. Recognition can occur through receptors like Toll-like receptors ($TLRs$) or retinoic acid-inducible gene I ($RIG-I$), leading to the induction of genes encoding Type I $IFN$ in the infected cell. This is mediated by various distinct signaling pathways, including mitochondrial antiviral signaling and TNF-associated receptors.

Once Type I $IFN$ binds to the interferon $\alpha$ receptor ($IFNAR$) on a neighboring uninfected cell, multiple downstream signaling pathways are induced. These pathways involve Janus kinase and Tyrosine kinase and lead to a wide range of biological effects mediated by interferon-stimulated genes ($ISGs$). Type I interferon is produced by many different cell types after infection. The released $IFN$ induces an antiviral state in adjacent cells. Innate immune cells can be activated to secrete interferon by viral infection, including non-productive infections, or by the "detection" of viral infection, which increases the local antiviral signaling state in the tissue. In many instances, this response can control or eliminate a viral infection before it becomes systemic or clinically manifest. If the virus manages to avoid this early innate response, systemic dissemination and clinical disease may follow.

Toll-Like Receptors (TLR) Signaling

Toll-like receptors ($TLRs$) are distributed on the cell membrane or within endosomes. Activation of $TLRs$ by ligand binding leads to downstream signaling that results in the production of Type I interferons and inflammatory cytokines. Transcription activators such as $NF-\kappa B$ (nuclear factor kappa-light-chain-enhancer of activated B cells) and $AP-1$ (activator protein 1) also contribute significantly to interferon production.

Specialized Cells of the Innate Immune System

Natural Killer ($NK$) cells are specialized lymphocytes that lack a specific antigen receptor. They are responsible for eliminating virus-infected cells, tumor cells, and cells under stress. Infected cells express multiple indicators of stress and inhibit the expression of standard cellular proteins. $NK$ cells activate and bind to these activators or inhibitors; when activating stimuli outweigh inhibitory ones, the process of cell death is initiated. This involves orienting cytotoxic granules toward the cell junction and releasing them. Perforin creates access to the target cell's cytosol, allowing the release of granzymes, which are serine proteases that induce cell death through multiple pathways.

Gamma Delta T cells ($\gamma \delta$ T cells) function in adaptive responses through antigen-specific interactions with the T-cell receptor ($TCR$), but they can also be activated non-specifically in response to cellular stress, such as that generated by a viral infection. Specifically, these cells can respond to infection by expressing $NKp46$ (Natural Cytotoxicity Receptors or $NCR$) and killing infected cells in a manner similar to $NK$ cells. Simultaneously, they produce strong cytokine responses, particularly the production of $IFN\gamma$, against viruses, bacteria, parasites, and tumor cells.

Dendritic cells ($DCs$) are fundamental to the initiation of the adaptive immune response and are also vital to innate immunity. Several subtypes exist with overlapping functions. The classical dendritic cell is of myeloid lineage from the bone marrow, expresses a high density of $MHC$ Class II proteins, and is highly phagocytic. These cells respond to $PAMPs$ by secreting large amounts of Type I interferon ($IFN\alpha$ and $IFN\beta$). Another myeloid subset, Langerhans cells, populates the skin's dermis and epidermis. Plasmacytoid dendritic cells ($pDCs$) arise from lymphoid lineage and have a distinct morphology. They were originally described as natural interferon-producing cells because they are remarkably effective at secreting Type I interferon in response to viral infections.

The Major Histocompatibility Complex (MHC) and Antigen Presentation

The Major Histocompatibility Complex ($MHC$) is a set of genes whose products are expressed on the surface of immune system cells. These genes are highly polymorphic, meaning there is a high degree of variation between individuals. The physiological importance of the $MHC$ was established approximately two decades after its discovery in $1940$, specifically regarding its role in immunization responses. Its major biological functions include antigen presentation, transplant immunobiology, the formation of the T-cell repertoire, and autoimmunity.

There are two primary pathways for $MHC$:

1. The Class I $MHC$ pathway involves cytosolic viral proteins that are unfolded and processed into peptides. These peptides are then presented on Class I $MHC$ molecules to $CD8+$ Cytotoxic T Lymphocytes ($CTL$).

2. The Class II $MHC$ pathway involves the endocytosis of extracellular viruses into endocytic vesicles. The viral proteins are then presented on Class II $MHC$ molecules to $CD4+$ T cells.

Cytokines: Functions, Nomenclature, and Sources

Cytokines are regulatory proteins secreted by immune cells that modulate and signal activities between cells. They are categorized by their source: lymphokines are derived from lymphocytes, and monokines are derived from monocytes.

Nomenclature within the cytokine family includes:

1. Interleukins ($IL$): Cytokines that regulate interactions between lymphocytes and other leukocytes.

2. Interferons ($IFN$): Synthesized in response to viral infections, immune stimuli, and chemical stimulants. They interfere with viral protein and RNA synthesis. Type I includes subtpes $IFN-\alpha$, $IFN-\beta$, $IFN-\delta$, $IFN-\omega$, and $IFN-\tau$. Type II consists of $IFN-\gamma$.

3. Tumor Necrosis Factors ($TNF$): Polymeric cytokines derived from macrophages ($TNF-\alpha$) and T lymphocytes ($LT-\alpha$ and $LT-\beta$, formerly known as $TNF-\beta$/Lymphotoxins). They can cause apoptosis in some tumor cells.

4. Growth Factors: Also known as colony-stimulating factors, they induce cell multiplication to ensure the organism has enough cells for defense.

5. Chemokines: Small proteins that induce inflammation by acting as chemotactic factors or activators of leukocytes.

Cytokine production is stimulated by various signals, most importantly antigens or $MHC$-antigen complexes through $TCR$ or $BCR$, antigen-antibody complexes through $Fc$ receptors, superantigens, and microbial components like Lipopolysaccharides ($LPS$). When cytokines bind to receptors on target cells, they can induce cell division or differentiation, stimulate the production of new receptors, or inhibit these effects. The concept of redundancy refers to the ability of different cytokines (such as $IL-2$, $IL-4$, $IL-5$, and $IL-6$) to act on a single target cell like B lymphocytes.

Exhaustive List of Cytokines and Their Properties

Below is a detailed list of various cytokines, their molecular weights ($kDa$), primary sources, and main effects:

• $IL-1\alpha, \beta$ ($17\,kDa$): Sourced from monocytes, macrophages, B cells, and dendritic cells. Effects include T lymphocyte proliferation, expression of $IL-2R$, and antibody production.

• $IL-2$ ($15\,kDa$): Sourced from Th1 cells. Effects include T lymphocyte proliferation and differentiation.

• $IL-3$ ($25\,kDa$): Sourced from T cells, $NK$ cells, and mast cells. Effects include hematopoiesis, mast cell regulation, and histamine release.

• $IL-4$ ($20\,kDa$): Sourced from Th2 cells, mast cells, and $NK$ cells. Effects include proliferation and differentiation of B cells, T cells, and macrophages, and isotype switching from $IgM$ to $IgG$.

• $IL-5$ ($18\,kDa$): Sourced from Th2 cells and mast cells. Effects include B cell proliferation and $IgA$ production.

• $IL-6$ ($26\,kDa$): Sourced from Th2 cells and macrophages. Effects include B cell proliferation, differentiation into plasma cells, and fever induction.

• $IL-7$ ($25\,kDa$): Sourced from bone marrow and thymocytes. Effects include proliferation of pre-T and pre-B cells and expression of $IL-12$ and its receptor.

• $IL-8$ ($8\,kDa$): Sourced from macrophages and endothelial cells. Effects include chemotaxis, adhesion, and diapedesis of neutrophils.

• $IL-9$ ($39\,kDa$): Sourced from Th cells. Acts as a mitogen in the absence of antigen.

• $IL-10$ ($35\,kDa$): Sourced from Th2 cells. Inhibits cytokine production by macrophages.

• $IL-11$ ($23\,kDa$): Sourced from the bone marrow stroma. Effects include growth and differentiation of pre-B cells and megakaryocytes.

• $IL-12$ ($75\,kDa$): Sourced from macrophages and B cells. Acts in synergism with other cytokines to promote $LTc$ differentiation.

• $IL-13$ ($10\,kDa$): Sourced from Th cells. Inhibits macrophage activation and the release of inflammatory cytokines.

• $IL-15$ ($15\,kDa$): Sourced from T cells, intestinal epithelium, $NK$ cells, and B cells. Effects include growth and proliferation of intestinal epithelium and T cells.

• $IL-16$ ($13\,kDa$): Sourced from $LTc$, macrophages, and eosinophils. Effects include Th cell chemotaxis and $MHC-II$ expression.

• $IL-17$ ($17\,kDa$): Sourced from Th cells and targets fibroblasts.

• $IL-18$ ($18\,kDa$): Sourced from macrophages and targets Th1 and $NK$ cells.

• $TNF-\alpha, \beta$ ($25\,kDa$ on average): Sourced from macrophages, Th1, $LTc$, and mast cells. Effects include antiviral activity, B and T cell proliferation, activation of $NK$ and fibroblasts, fever, and cytotoxicity.

• $IFN-\alpha, \beta$ ($16-25\,kDa$): Sourced from leucocytes and other cells. Primary effects are antiviral and fever induction.

• $IFN-\gamma$ ($20-25\,kDa$): Sourced from Th1, $LTc$, and $NK$ cells. Effects include antiviral activity and activation of Th2, macrophages, and $NK$ cells.

• $GM-CSF$ ($18/22\,kDa$): Sourced from T cells, macrophages, and endothelium. Regulates hematopoiesis of granulocytes and monocytes.

Viral Counter-Defense Against Cytokines

Viruses have developed strategies to combat cytokines, which are fundamental to both innate and adaptive antiviral responses. Some viruses have acquired and modified cellular genes to create viral proteins that are homologs of cytokines or their receptors. Viral cytokine homologs can be functional (called virocines), mimicking the biological effect of the authentic molecule, or non-functional, acting to simply bind and block specific cytokine receptors to neutralize their activity. Similarly, viral receptor homologs bind and neutralize relevant cytokines. Other viral proteins interfere with PRR signaling pathways activated by $dsRNA$ (like $TLR3$ or $RIG-I$) which normally trigger the production of Type I interferon and other antiviral cytokines. These proteins modulate cytokines like Interleukins ($IL-1$, $IL-6$, $IL-8$), interferons (Type I and II), and Tissue Necrosis Factor to benefit viral replication.

Antibodies and the Complement System

Antibodies are glycoproteins resulting from the activation and maturation of B lymphocytes. They react with epitopes present on the antigens that triggered their production. While the antigen-binding site is highly variable, the constant fraction is highly conserved and possesses effector functions such as complement fixation, adhesion to phagocytic cells, and placental transfer or colostrum transfer. They operate in various environments including blood, milk, and body surfaces.

The complement system is a cascade involving proteins $C1$ through $C9$ (specifically pathing through $AbC1 \rightarrow AbC142 \rightarrow C3 \rightarrow AbC1423b \rightarrow AbC1423b5 \rightarrow AbC1423b5b67 \rightarrow AbC1423b5b6789$). Activation occurs through the classical pathway (involving antibodies and the $Fc$ region) or the alternative pathway (without antibodies). Biological results include the release of histamine, chemotaxis, accumulation of polymorphs, inflammation, convertase activity, adhesion to phagocytes (leading to viral phagocytosis), and damage to viral membranes.

Immunity Against Reinfection and Immunological Memory

The first line of defense against reinfection is the antibody. In natural systemic infections, antibodies persist for years, and the degree of acquired immunity generally correlates with the serum titer. Transfer of antibodies, whether natural or artificial, provides robust protection. After antigenic stimulation and subsequent lymphocyte clonal expansion, a subpopulation of memory cells emerges and persists indefinitely. When re-exposed to the same antigen, these populations respond faster and more vigorously than during the primary encounter.

Passive Immunity and Maternal Transfer

Passive immunity is categorized as artificial (injection of antibodies) or natural (physiological transfer from mother to fetus/neonate). Natural passive immunity is essential for protecting newborns during their first days of life. However, maternal antibodies can also interfere with active immunity processes and immunization schedules. The type of placentation determines the level of prenatal and postnatal transfer:

• Cattle, Swine, Horses: Epitheliochorial placentation ($3$ maternal and $3$ fetal layers). Prenatal transfer is $0$, and postnatal transfer via the gut is highly significant ($+++$). The cut-off time for translocation is within $2$ days for cattle/swine and $1$ day for horses.

• Dogs, Cats: Endotheliochorial placentation ($1$ maternal and $3$ fetal layers). Prenatal transfer is low ($+$, about $5-10\%$), and postnatal transfer is significant ($++$) for $1-2$ days.

• Humans: Hemochorial placentation. Prenatal transfer is significant ($+++$), and postnatal transfer is zero.

Immunoglobulin concentrations in colostrum and milk vary by species:

• Cattle: Colostrum contains $36-77\,g/L$ of $IgG$, $4-5\,g/L$ of $IgA$, and $3.2-4.9\,g/L$ of $IgM$. Milk contains $1.0-1.8\,g/L$ of $IgG$.

• Swine: Colostrum contains $62\,g/L$ of $IgG$, $10\,g/L$ of $IgA$, and $3.2\,g/L$ of $IgM$. Milk contains $1.4\,g/L$ of $IgG$ and $3.0\,g/L$ of $IgA$.

• Humans: Colostrum contains $0.3\,g/L$ of $IgG$ and $120\,g/L$ of $IgA$. Milk contains $1.5\,g/L$ of $IgA$.

Failure of passive transfer (FPT), whether total or partial, is the most common immunodeficiency disease in domestic animals, predisposing newborns to viral infections, particularly enteric and respiratory diseases. Immunizing the mother to ensure adequate passive immunity for the neonate is a primary strategy in veterinary medicine.