Front Immunol. 2014; 5: 171.

T Cell Responses to Viral Infections 每 Opportunities for Peptide Vaccination

Sietske Rosendahl Huber,1 Josine van Beek,1 Jørgen de Jonge,1 Willem Luytjes,1 and Debbie van Baarle1,*

Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, Netherlands

An effective immune response against viral infections depends on the activation of cytotoxic T cells that can clear infection by killing virus-infected cells. Proper activation of these T cells depends on professional antigen-presenting cells, such as dendritic cells (DCs). In this review, we will discuss the potential of peptide-based vaccines for prevention and treatment of viral diseases. We will describe features of an effective response against both acute and chronic infections, such as an appropriate magnitude, breadth, and quality and discuss requirements for inducing such an effective antiviral immune response. We will address modifications that affect presentation of vaccine components by DCs, including choice of antigen, adjuvants, and formulation. Furthermore, we will describe differences in design between preventive and therapeutic peptide-based vaccines. The ultimate goal in the design of preventive vaccines is to develop a universal vaccine that cross-protects against multiple strains of the virus. For therapeutic vaccines, cross-protection is of less importance, but enhancing existing T cell responses is essential. Although peptide vaccination is successful in inducing responses in human papillomavirus (HPV) infected patients, there are still several challenges such as choosing the right target epitopes, choosing safe adjuvants that improve immunogenicity of these epitopes, and steering the immune response in the desired direction. We will conclude with an overview of the current status of peptide vaccination, hurdles to overcome, and prospects for the future.

Keywords: DC, peptides, vaccination, virus, infection, chronic, acute

Viruses are small infectious agents that consist of nucleic acid that is coated in a simple protein shell or a cell-membrane-like protein casing, and need to infect host cells to replicate (1). Viruses can cause acute and chronic infections. In acute virus infections, such as a common cold, the virus is typically cleared from the body within a week. However, in some cases, an acute infection is followed by persistence of the virus in the host. Herpes simplex virus is an example of a virus causing a persistent infection, due to ability of the virus to hide in neurons. Often, these types of persistent infections do not cause any symptoms in healthy hosts (2). Chronic infections are a type of persistent infection often caused by an inefficient immune response of the host, leading to long-lasting symptoms. Especially, acute and chronic virus infections have a major general health impact. Annual influenza epidemics, for instance, result in about 3每5 million cases of severe illness and approximately 250,000每500,000 deaths worldwide (3). An example of a chronic infection causing major health impact is Human Immunodeficiency Virus (HIV). In 2012, more than 35 million people were living with an HIV infection and 1.6 million people died from an AIDS-related illness (4). Some persistent virus infections, such as Epstein每Barr virus (EBV) and Human Papillomavirus (HPV) can lead, under certain conditions, to the development of tumors (5, 6). Because viruses have such a major impact on health, strategies to limit or prevent virus infections are of major importance.

Mammals have developed a refined immune system to cope with all kinds of infections. Especially, the adaptive arm of the immune response is important in limiting and clearing viral infections. The humoral immune response consists of antibodies specific for the virus that can capture and neutralize virus particles before they enter the cell. However, if these antibodies are ineffective, viruses are able to infect host cells and can only be cleared by the cellular arm of the immune response. Once a virus infects a cell, the virus will use the protein-synthesis machinery of the host cell to synthesize its own proteins. During this process, some of the newly synthesized proteins will be degraded into peptide fragments and, if they have sufficient binding affinity, bind to MHC class I molecules. These MHC class I-peptide complexes will then be presented on the cell surface of an infected cell and activated CD8+ T cells, specific for the peptide, can recognize the MHC class I-peptide complex and induce apoptosis of the infected cell by releasing cytotoxic granules. Activation of these CD8+ T cells occurs in the draining lymph nodes, where antigen-presenting cells (APCs), such as dendritic cells (DCs), and naïve T cells encounter each other. In these lymph nodes, DCs and CD4+ T cells provide the co-stimulation necessary for proper activation of CD8+ T cells. This process is summarized in Figure ​Figure11 and will be further discussed in the next paragraphs.

Figure 1

Routes of presentation of viral peptides on DCs. Viruses can enter cells by two ways: some viruses can infect cells directly, leading to replication of virus inside the cells. During this process, some of the viral proteins will be degraded into peptide fragments, which will be presented on MHC class I molecules to CD8+ T cells (I). APCs, such as DCs can also take up viral particles or remnants of virally infected cells (II). During processing by professional APCs, viral peptides can be presented on MHC class I molecules via the cross-presentation pathway (III). In parallel, these extracellular-derived peptides will be presented on MHC class II molecules. The TCR of virus-specific CD4+ T can recognize MHC class II-peptide complexes on professional APCs. Next to the interaction of the MHC class II-peptide complex with the TCR, CD4+ T cells can activate DCs by interaction of CD40 with CD40 ligand on the DC (IV). This interaction activates DCs and results in upregulation of maturation markers CD80/CD86. CD80 and CD86 interact with CD28 on naïve CD8+ T cells (V). Together with the recognition of the MHC class I-peptide complex by the TCR, CD28 signaling will result in the activation of the CD8+ T cell (VI). These activated CD8+ T cells will differentiate into effector T cells that can recognize the MHC class I-peptide complex on virally infected cells. Binding of the TCR to the MHC class I-peptide complex leads to activation of the CD8+ T cell and the release of cytotoxic granules containing perforins and granzymes, and the production of cytokines such as TNF-汐 and IFN-污 (VII).

During the initial phase of a viral infection, there is a significant increase in the number of CD8+ T cells. Priming of these naïve T cells will not only occur through the classical pathway via infection of a cell, directly leading to presentation of peptides on MHC class I molecules, but also through cross-presentation. Cross-presentation enables the presentation of viral peptides, taken up from extracellular sources, on MHC class I molecules. Several different cell types have been demonstrated to cross-present antigens in vivo, including professional APCs such as macrophages and DCs (7). CD8+ T cells, activated either through the classical or cross-presentation pathway, induce apoptosis of virus-infected cells by the release of cytotoxic granules and the production of TNF-汐 and IFN-污 as depicted in Figure ​Figure1.1. The cytotoxic granules contain perforins, granzymes, and granulysin. Perforins aid in delivering contents of granules into the cytoplasm of the target cell. Granzymes, such as granzyme B, and granulysin activate apoptosis of the target cell. TNF-汐 can interact with the TNFR-I receptor, which induces apoptosis of infected cells. IFN-污 is an important cytokine in the immune response to various viral infections, since it can induce an antiviral state in uninfected cells and enhance the cytotoxic function of CD8+ T cells. By the classical antigen presentation pathway or by the cross-presentation pathway, any form of virus can be presented on MHC class I and MHC class II and thereby stimulate antiviral responses by both CD8+ T cells and CD4+ T cells, respectively, leading to a broad cellular response to infection (8). After infection, some of these activated T cells will develop into memory T cells. In the event that a secondary infection occurs, these cells can rapidly mature into effector cells and respond to infection.

Antigen-presenting cells that reside at the site of infection, can take up viral particles or remnants of virally infected cells from extracellular sources, and present them on MHC class II molecules. Subsequently, CD4+ T cells recognizing peptides in the context of MHC class II will be activated. These activated CD4+ T cells are capable of producing a wide range of cytokines and chemokines and can even exert cytotoxic functions themselves. Based on cytokine production, CD4+ T cells can be divided into several subsets, the most classical being Th1, Th2, and Tregs. Th1 cells are generally characterized by the production of IFN-污. Th2 cells, on the other hand, produce mainly IL-4, IL-5, and IL-13 and are important for providing an immune response against helminths by activating eosinophils, basophils, mast cells, and B cells. The third classical subset are the Treg cells, which are characterized by the production of IL-10 and TGF-b, and have mainly regulatory tasks such as dampening effector functions and limiting immunopathology (8, 9). In addition to their effector functions, activated CD4+ T cells can provide help to CD8+ T cells by CD40-CD40L interaction, which induces up regulation of ligands, such as CD80 and CD86, on DCs. These ligands interact with CD28 on naïve T cells, providing a co-stimulatory signal to activate CD8+ T cells (10). The mechanism by which CD4+ T cells can provide help to CD8+ T cells is shown in Figure ​Figure11.

In this review, we will discuss the value of T cell responses in both acute and chronic viral infections and how knowledge of these responses can help in designing effective vaccines.

Currently, antiviral drugs are the main treatment option to combat viral diseases. However, antiviral treatment is associated with side effects and resistance through viral escape. Making use of the hosts own immune defense system by vaccination would be another powerful approach to combat viral diseases. However, many vaccination strategies are based on antibody-mediated protection and are only partially successful. Antibodies can be very efficient in preventing virus infection, but due to the variability of many virus surface proteins, the virus can escape and infect host cells. Once a virus has entered a cell, infection can only be cleared by a cellular response. We will highlight the history of synthetic T cell based vaccines as an important strategy to induce T cell responses and discuss current developments in this field. Then, we will discuss how the design of these vaccines, such as choice of antigen and adjuvant, influences their efficacy. Finally, we will conclude with potential pitfalls and recommendations for the design of effective peptide vaccines against virus infections.

T Cell Responses in Viral Infections
There are many viruses for which T cells, both CD8+ and CD4+, have been shown to play a role in protection, such as measles virus, cytomegalovirus (CMV), hepatitis C virus (HCV), and HIV (11每14). In general, an efficient antiviral adaptive response is thought to be of the Th1 type (15). However, many viruses can inhibit this Th1 response by downregulating the production of interferons (16, 17). This type of manipulation of the immune response can greatly influence the outcome of the infection. In infections caused by hepatitis viruses, manipulation of the immune response by the virus can lead to a persistent infection, in which the host is incapable of clearing the virus from the body. In mice, lymphocytic choriomeningitis virus (LCMV) is used as a model to study the role of CD8+ and CD4+ T cells in both acute and chronic infections. CD8+ T cell-deficient mice, which were infected with a LCMV-strain that normally causes acute virus infection, were not able to control infection and developed a persistent infection. In mice depleted of CD4+ T cells, infection with murine LCMV led to chronic infection, even in the presence of CD8+ T cells. This model shows that in acute infection, CD8+ T cells are sufficient to clear infection, but the help of CD4+ T cells is required (18).

The importance of T cell responses during acute viral infections in humans can be illustrated by research from Sridhar et al. describing that individuals with higher numbers of pre-existing CD8+ T cells specific for conserved CD8 epitopes, developed less severe illness after infection with pandemic H1N1 influenza virus (19). That not only CD8+ T cells mediate protection to influenza challenge, has been shown in a unique human challenge study by Wilkinson et al. In this study, healthy volunteers were challenged with influenza A virus, and antibody and T cell responses against influenza before and during infection were monitored. They showed that, in the absence of antibody responses, pre-existing CD4+ T cells responding to influenza internal proteins were associated with less severe illness and lower virus shedding. Further characterization of these CD4+ T cells showed that these cells had a cytotoxic function (20). These studies describe the importance of both CD8+ and CD4+ T cells in the immune response against influenza virus.

During chronic viral infections, when the host is not able to clear the virus, the main role of cytotoxic T cells is to limit disease severity and delay disease progression. This is exemplified by studies on HIV infection. Early during infection with HIV, there is a decline in viral replication as measured by the number of HIV RNA copies in plasma samples (21). In the first stages of HIV infection, it has been shown that patients with higher numbers of memory cytotoxic T cells show a much lower viral load in plasma than patients with a lower number of memory cytotoxic T cells, indicating that this decline is mediated by cytotoxic CD4+ T cells (22). In addition, cytotoxic CD4+ T cells are an immunological predictor of disease outcome. Patients that controlled HIV replication without antiretroviral therapy showed an increased number of CD4+ T cells specific for HIV proteins (23). The importance of CD8+ T cells in delaying HIV disease progression is shown in studies where a loss of CD8+ T cells coincides with disease progression (24, 25). Findings that HIV escape mutations often occur at HLA-binding sites specific for CD8 epitopes, the strong association of certain HLA-alleles with protection from HIV disease progression, the temporal relationship between viral load decline and increase in specific CD8+ T cells, and CD8+ T cell depletion studies in simian models, underline the importance of CD8+ T cell responses (11, 26每29). Knowledge on the mechanism of protection of T cell responses in immunity against viruses can be helpful in designing preventive and therapeutic therapies, such as vaccination.

Features of an Effective Response
To induce an effective response against viral infections, there are several requirements that should be met. One important requirement is that there is a sufficient number of T cells available to kill virus-infected cells. The need for an appropriate magnitude of T cells in order to clear virus was elegantly shown by Thimme et al. in a CD8+ T cell depletion study in chimpanzees. Chimpanzees were depleted of CD8+ T cells, and subsequently infected with HBV, complete depletion of CD8+ T cells in the chimpanzees resulted in the inability to clear virus. When CD8+ T cells reappeared in the animal, 98% of viral DNA was eliminated from the liver. However, while the number of CD8+ T cells remained suppressed, the animal was not able to clear virus completely. Only when the number of CD8+ T cells was able to expand further, the virus was completely eliminated (57). Furthermore, an increased breadth of T cell responses can be beneficial. Analysis of CD8+ T cell responses in untreated HIV-infected individuals showed that an increasing breadth of Gag-specific responses is associated with decreased viremia (58). In parallel with these findings, vaccination of mice with a vaccine containing multiple epitopes, were more effective in generating a response to influenza infection than vaccination with single epitopes (49). These findings indicate that a broad response is more effective than a response dedicated to only one peptide. Another advantage of induction of a broad response is that small mutations of the virus will not lead to escape of the virus from the immune response. Next to a broad response, T cell responses of high avidity also contribute to an antiviral response. Ex vivo screening of T cell responses in HIV-infected patients showed that controllers reacted to lower antigen concentrations compared to non-controllers, indicating that controllers have T cell responses of higher functional avidity and that this higher avidity is advantageous (59).

A fourth requirement is that an effective antiviral response should be of proper functionality to enable control or clearance of the virus. CD8+ T cells are the main cell type that is involved in clearance of viral infections. These cells are characterized by the production of Th1-cytokines such as IFN-污 and by the expression of degranulation marker CD107a (60). CD107a is an indicator of cytotoxic functions such as the production of granzymes and perforins. IFN-污 increases expression of both MHC class I and II molecules and enhances the antigen-presenting function of MHC class I by stimulating loading of peptides onto this molecule. Thereby, IFN-污 can induce the cytotoxic function of CD8+ T cells and promote the production of other cytokines such as TNF-汐, IL-2, and type I interferons. TNF-汐 induces apoptosis of virus-infected cells and IL-2 is an important growth factor for T cells. Type I interferons, such as IFN-汐 and IFN-汕, can induce resistance to viral infections in uninfected cells, increase MHC class I expression and antigen presentation and activate both DCs and macrophages (8, 61). Activated macrophages in their turn produce chemokines such as MIP-1汕 to attract more T cells. Together, these cytokines, chemokines, granzymes, and perforins enable control or clearance of the virus from the host. In HIV infection, a polyfunctional CD8+ T cell response is observed in non-progressors, while progressors show a more limited response (62). As reviewed by Seder et al., a polyfunctional response, characterized by production of IFN-污, TNF-汐, and IL-2, was indeed shown to induce more robust T cell proliferation and protection against several viral infections (63).

However, elevated amounts of inflammatory cytokines can also lead to immunopathology as has been shown in H5N1 influenza A virus infection (64). The immune system normally has its own regulatory mechanisms, such as the production of anti-inflammatory cytokines including IL-10 and TGF-汕. IL-10 is produced by a wide range of cells, including T cells, macrophages and neutrophils. The main function of IL-10 is to act as a negative feedback loop to suppress the production of IFN-污 and other pro-inflammatory cytokines (65). TGF-汕 acts by inducing apoptosis of CD8+ T cells, which regulates T cell homeostasis and prevents immune inflammation (66). These feedback loops are a way of the immune system to regulate itself, however viral factors can negatively impact this balance as is illustrated in HCV infection. Patients with progressive liver injury showed upregulation of Th1-cytokines IFN-污 and IL-2 and down regulation of the regulatory cytokine IL-10 (67). Another regulatory mechanism is the upregulation of inhibitory receptors such as PD-1, LAG-3, and CTLA-4, which leads to decreased activation potential of T cells and the activation of inhibitory genes in T cells (68). However, upregulation of these receptors has also been shown to be responsible for the exhaustion of T cells and thereby a diminished response in chronic viral infections (69). Summarizing, an effective antiviral response consists of a broad variety of antigen-specific T cells of sufficient magnitude, affinity, and appropriate polyfunctionality. Furthermore, these T cells should be capable of performing cytotoxic functions, but should not induce immunopathology. Such a response greatly depends on the way antigen is presented to the T cells, emphasizing the important role APCs play in antiviral responses.

Concluding, in addition to antibody responses, T cell responses are of major importance in limiting and clearing virus infections. Effective therapeutic and preventive vaccines should therefore be able to induce both antibody and T cell responses. Peptide-based vaccines can meet these demands and induce both antibody and T cell responses. Furthermore, because peptides are synthetic, they are safe and relatively easy to produce. Currently, several peptide-based vaccines, for viruses such as EBV, HBV, and influenza virus, are evaluated in clinical trials (101). Hurdles to overcome are choosing the right target epitopes and choosing adjuvants that improve immunogenicity of these epitopes and steer the immune response in the desired direction. Adjuvants for peptide-based vaccines should target antigen to DCs, or other APCs capable of cross-presentation, and provide stimuli to ensure efficient presentation of the antigen. In addition, an overstimulation resulting in immunopathology should be avoided. Providing, these criteria are met, the future of peptide-based vaccines is very promising.

T Cell Responses to Viral Infections 每 Opportunities for Peptide Vaccination

Immune Defense Against Viruses

The Empire Strikes Back: Host Response to Virus Infection
Your body doesn't simply sit idly by as viruses come in and kill off all your cells. For millions of years, we have been exposed to viruses, and have developed various ways of defending ourselves against virus infections, since punching viruses never works. This may not seem obvious, but viruses don't infect us with the goal of killing us or causing us disease. Many of the symptoms we see in a virus infection 每 runny nose, fever, skin redness 每 are a function of the immune response to virus infection. So, the immune system is a double-edged sword. We need it to protect ourselves from virus infections, but it makes us miserable when it works. However, viruses have evolved so that some symptoms of diseases 每 sneezing, diarrhea 每 actually help virus spread from host to host.

Immune Defense Against Viruses
The immune system is far too complicated to stick in one section on a chapter on viruses, but we will talk about the arm of the immune system that handles virus infection. There are two types of immunity: innate and adaptive immunity. Innate immunity is basically the first line of defense of a host against an infectious agent. Because it is so basic, it includes such minimal defenses as the skin and mucous membranes, proteins that non-specifically degrade foreign cells, and a giant "Beware of Dog" sign. Obviously, this defense is not well tailored to stopping a virus infection.

On the other hand, the adaptive immune system is a branch of immunity evolved in mammals that is like "smart bomb" technology. We are exposed to a pathogen that makes us sick, then our adaptive immunity "remembers" this, so the next time we see that pathogen, it disposes of it like week old fishsticks.

The adaptive immunity has two important cells: B and T cells (B means cells that mature in the bone marrow, while T cells develop in the thymus). B cells make antibodies, and are not as useful in defending against virus infection as T cells. T cells are highly important in the immune response to virus infection because they do a few things:

Kills virus-infected cells
Activate interferon that inhibits virus replication
Activates cells that kills virus-infected cells
The major defense against virus-infected cells is the CD8+ T-cell, also called cytotoxic T cell (CTL), killing of virus-infected cells. In immunology, all proteins are called "CD" followed a number, because immunologists have no imagination, and they never got on board with the whole "mp3" phenomenon. So, CD8 is just a protein that is found on the surface of certain T cells that kill infected cells (either virus, bacteria, or parasite).

Activation of T Cells (Source)

How do CTL cells recognize that a cell is infected? All T cells have a T-cell receptor (TCR in Figure 1), which recognizes a protein complex in infected cells, also called antigen-presenting cells, called MHC. MHC complexes continuously display short peptides of proteins in the cell. And if a cell is infected with a virus, then many of the peptides will be virus peptides. The T cell sees these virus peptides and says "WHAT IS THIS????" and then kills that cell. This is why we can't take CTLs to Europe. Croissants just freak them out.

CTLs kill infected cells by activating apoptosis in the infected cell. Apoptosis is programmed cell death, sort of like cell euthanasia. Other cells, such as natural killer (NK) cells, can be activated by T cells to kill infected cells. NK cells work similar to CTLs, but they don't respond to virus antigens, like CTLs do. So, they are more a part of innate immunity than adaptive immunity, even though they are activated by T cells.

The cool thing about adaptive immunity is that once a virus infection has been controlled, the cells that were responsible for clearing the virus are rewarded, and more cells like those virus-killing cells are made. It's like CTLs are treated as war heroes, and everyone wants to be like them. The best part about this is that next time that virus tries to get into a cell there are several more types of cells that are ready to kill that specific virus. This is an important fact that we'll come back to in the vaccines section.

T cells can also inhibit virus replication by activating cells to make a compound called interferon, which does what the name implies: interfere with virus replication. Interferon is mostly responsible for activating two genes, protein kinase R (PKR) and oligo-A-synthetase (OAS). Interferon activates several other genes that have antiviral function, but PKR and OAS are the most significant as they completely shutoff protein synthesis in an infected cell.

Immune Defense Against Viruses