Microbicidal peptides have broad-spectrum antiviral effects

The innate immune response to viruses involves complex interactions between soluble factors and cells. For example during influenza virus infection mucins, gp-340 and pentraxins compete with the virus for its receptor, sialic acid, and cause aggregation of virus particles. Respiratory secretions are also rich in the collectin surfactant proteins (SP)-A and SP-D. These molecules bind to carbohydrates on a range of pathogens, including influenza virus where they adhere to the hemagglutinin protein (HA) resulting in virus neutralization. Some strains of influenza virus fail to be recognized by collectins due to reduced levels of glycosylation of HA. An example of this was the H1N1 virus that caused the 1918 pandemic.

Other families of antimicrobial peptides with antiviral activity include the defensins and the related cathelicidins. Alpha-defensin and the cathelicidin LL37 are produced by epithelial cells and neutrophils in reponse to infection. They have broad-spectrum direct antiviral activity, and also modulate the inflammatory response at sites of infection.

Type I interferons have critical antiviral and immunostimulatory roles

The activation of the IFN system is arguably the most important defence for containing the initial stages of virus infection. There are three major families of IFNs:

Other types of IFN exist, including IFN-ω, -τ, -δ, and -κ, some of which play a role during pregnancy. Here we will focus on the IFNs with antiviral activity. Of these, it is the type I and type III IFNs that are induced directly following virus infection, whereas IFNγ is produced by activated T cells and NK cells. Type III IFNs are much less well characterized than type I IFNs, but their functions are thought to be similar.

Type I IFN production typically starts to be induced within the first few hours after virus infection. Type I IFNs can be produced by almost any cell type in the body if it becomes infected with a virus. There are also specialized interferon-producing cells, plasmacytoid DCs, which can be triggered to produce high levels of type I IFN following exposure to virus without themselves becoming infected. This is important because, as discussed below, many viruses have evolved strategies for impairing type I IFN production in the cells they infect. Plasmacytoid DCs typically make at least half of the type I IFN produced during a virus infection.

Type I IFN production is triggered following recognition of molecular patterns characteristic of viral but not host components (Fig. 13.1). Host pattern-recognition receptors involved in detecting the presence of virus infections include:

Fig. 13.1 Pathways by which type I IFN production can be triggered following virus infection

Most cells in the body express cytoplasmic pattern-recognition receptors such as RIG-I. If the cell becomes infected, these detect the presence of viral nucleic acids in the cytoplasm and stimulate IRF3 and NF-κB activation, leading to transcription (txn) of type I IFN and inflammatory cytokine genes and production of these factors. Plasmacytoid DCs can detect the presence of virus and up-regulate type I IFN production without becoming infected. Following virion uptake, viral nucleic acids are detected by TLRs 7 and 9 in endosomal compartments. This stimulates IRF7 and NF-κB activation, leading to production of type I IFNs and inflammatory cytokines.

Triggering of pattern-recognition receptors initiates signaling along pathways that culminate in the activation of transcription factors including IFN regulatory factor (IRF)3 and NFκB, which translocate into the nucleus and activate the transcription of type I IFNs and inflammatory cytokines, respectively (see Fig. 13.1). Plasmacytoid DCs also have a unique signaling pathway for induction of type I IFN production in response to TLR7 or TLR9 ligation that involves the transcription factor IRF7.

The IFN released acts on both the cell producing it and also neighboring cells where it establishes an antiviral state, enabling them to resist virus infection (Fig. 13.2).

Fig. 13.2 Type I IFNs mediate direct antiviral effects and play an important role in activating other antiviral defenses

Type I IFNs produced by infected cells and plasmacytoid DCs (pDCs) detecting virion components up-regulate the expression of antiviral genes in both infected and uninfected cells, thereby helping to eradicate infection and block its spread. Type I IFNs also activate cells participating in the innate response, including pDCs, conventional DCs (cDCs), NK cells and macrophages (mØs) and promote the activation of adaptive responses not only via DC activation but also by acting directly on T and B cells.

IFNs mediate their activity by up-regulating the expression of a large number of genes known as IFN-stimulated genes (ISGs), some of which encode proteins that mediate an antiviral response. These include the key dsRNA-dependent enzymes protein kinase R (PKR) and 2′,5′-oligoadenylate synthetase.

Another inhibitor of transcriptional activation is the Mx protein, which is active against variety of RNA viruses, most notably influenza virus. Although some ISGs have broad activity against multiple viruses there are also other ISGs that mediate antiviral activity against selected classes of viruses, e.g. apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC)s, which combat infection with retroviruses including HIV-1.

In addition to the direct inhibition of virus replication, IFNs also activate macrophages and NK cells and enhance their antiviral activity (see Fig. 13.2). In addition, they help to promote the activation of adaptive responses. They act on antigen presenting cells including conventional DCs to stimulate increased expression of MHC class I and II, along with components of the antigen processing machinery; and they also act directly on T and B cells to promote an antiviral response (see Fig. 13.2).

The importance of type I IFNs in vivo is underlined by the increased susceptibility of mice lacking the IFNα/β receptor to virus infection. Similarly, depletion of IFNs by specific antibody treatment also augments virus infection.

NK cells are cytotoxic for virally-infected cells

Activated NK cells can typically be detected within 2 days of virus infection. Since viruses require the replicative machinery of live cells to reproduce, NK cells act to combat virus replication directly by recognizing and killing infected cells; they also produce cytokines such as IFNγ and TNFα and mediate important immunomodulatory effects, stimulating the activation of macrophages via IFNγ and regulating DC responses.

NK cells are non-specifically activated by innate cytokines including type I IFNs, IL-12, IL-15, and IL-18, but their activation state and effector activity are also regulated by signaling through multiple activating and inhibitory receptors.

NK cells are important in combating herpes virus infections

The NK response to murine cytomegalovirus (MCMV) is especially well-characterized (Fig. 13.w1) and has given important insights into virus-NK interactions.

Fig. 13.w1 The NK cell response to MCMV in C57BL/6 mice

In uninfected mice, host cells express ligands (e.g. MHC class I) that engage inhibitory receptors on NK cells and prevent NK activation. When mice are infected with murine cytomegalovirus (MCMV), production of innate cytokines such as IL-12, IL-15, and IL-18 is upregulated, which drives nonspecific NK cell activation and proliferation. MHC class I expression is actively reduced on MCMV-infected cells and viral proteins including m157 are expressed on the cell surface. NK cells expressing the activating receptor Ly49H⁽⁺⁾ are activated (emboldened cell surface) on contact with infected cells, as few of their inhibitory receptors are engaged but they receive activating signals, most importantly as a consequence of Ly49H binding to m157. This results in triggering of NK effector functions (leading to lysis of the infected cell) and also drives enhanced activation and proliferation of the Ly49H-expressing NK population. After the infection has been cleared, Ly49H-expressing NK cells retain an enhanced ability to combat virus replication if reinfection occurs.

In the early stages of MCMV infection NK cells respond to locally-produced innate cytokines by undergoing non-specific activation and proliferation. In C57BL/6 mice, specific recognition of MCMV-infected cells is mediated by NK cells expressing the activating NK receptor Ly49H, which interacts with the MCMV protein m157 on infected cells. This results in a clonal expansion of m157-specific NK cells, which play a critical role in controlling virus replication. Mouse strains which lack Ly49H or other activating receptors that can specifically recognize MCMV are highly susceptible to infection with this virus. Notably, passage of MCMV in C57BL/6 mice leads to selection of viruses bearing mutations in the m157 gene, the replication of which is not well-controlled in these mice.

If the m157 protein of MCMV targets infected cells for recognition by NK cells, why does the virus express this protein? Although m157 enables mouse strains expressing the activating NK receptor Ly49H to recognize and destroy MCMV-infected cells, certain other inbred mouse strains express an inhibitory receptor, Ly49I, which also binds to m157 and protects the virus by inhibiting NK cell responses in these animals.

The m157-driven activation and expansion of Ly49H-expressing NK cells in C57BL/6 mice bears similarities to clonal expansion of antigen-specific T cells. Following virus clearance, the frequency of Ly49H-expressing NK cells declines – but the remaining population of cells retains the capacity to mediate a more efficient response on secondary exposure to MCMV, reminiscent of the heightened responsiveness of memory T cells. These recent findings of antigen specificity and memory (defining features of adaptive responses) in NK populations suggest that there can be overlap between features of the innate and adaptive response during virus infections – nonetheless the rapid response to naive NK cells to infection and multiple ‘generic’ mechanisms for NK activation demonstrate that their principal role is as an innate effector subset.

Macrophages act at three levels to destroy virus and virus-infected cells

Macrophages are ever present in the tissues of the body and act as a first line of defense against many pathogens. In virus infection they act at three levels to destroy virus and virus-infected cells:

Phagocytosis of infected cells and virus complexes is part of the normal housekeeping role of macrophages at a site of infection.

As with many pathogens the phagolysosome represents a hostile environment for viruses in which oxygen-dependent and oxygen-independent destructive mechanisms prevail. The induction of nitric oxide synthetase and the generation of nitric oxide is a potent inhibitor of herpesvirus and poxvirus infection.