Once the virus is in contact with host cellular receptors, the processes leading to viral infection are triggered. Glycoproteins in the lipid bilayer allow the viral envelope to fuse with the epithelial plasma membrane. Viral proteins are released into the cytoplasm and viral DNA enters the nucleus. This process triggers three phases of the innate response: (1) secretion of immune proteins, such as complement and natural antibodies; (2) an early-induced response, in which the main mediators are IFNs produced by the infected epithelial cells and resident DCs; and (3) the activation of inflammatory cells, such as neutrophils, macrophages, and NK cells [20]

At the site of mucosal contact the virus usually encounters several barriers, including mucus, normal bacterial flora, the glycocalyx, complement proteins, and natural immunoglobulin (IgM) antibodies [1, 20]. Although these substances act in concert to decrease the number of infected cells, HSV usually replicates successfully and triggers the early innate immune response. Humans may express different levels of natural antibodies to HSV, which is a reflection of their own past exposure experience [7, 20]

The early innate immune response has two goals: (1) limit viral replication and spread of the virus in uninfected cells, and (2) recruit other inflammatory cells [20]. The viral interaction with the epithelial cells may stimulate cell-surface TLR2 (e.g., in the genital mucosa) [1, 20]. With the activation of this receptor, the epithelial cells activate complement, chemokines, and IFN-α and -β soon (between 8 and 12 h) after the infection. Interferon-α and -β are produced by most cells types, but the DCs are responsible for the majority of their production. Interferon-α and -β are known to be two of the most important molecules to control HSV infection at this stage. Some studies suggest that resistance or susceptibility to HSV infection is directly correlated with the amount of IFN-α and -β produced [12].

Complement, chemokines, and IFN-α and -β activate the endothelial cells that express IL-8, tumor necrosis factor-α (TNF–α), IFN-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF), leading to neutrophil chemotaxis. Figure 17.1 illustrates the chemokine regulation of leukocyte movement [
21]. The inflammatory reaction alerts the DCs and resident macrophages to the presence of the virus and induces a “state of awareness” or “antiviral status” in the uninfected cells [7]. Infected macrophages that are able to survive acute HSV infection become a significant source of inflammatory chemokines and cytokines including TNF-α, IL-1, IL-6, IL-8, IL-12, IL-18, RANTES (regulated on activation, normal T-cell expressed and secreted; a chemokine that is a chemoattractant for eosinophils, monocytes, and lymphocytes), as well as IFN-α and -β. Subsequently, the immature DCs capture viral antigens and transport them to the regional lymph node to alert and stimulate the adaptive immune response [7].

Once the DC exits the mucosa, the late-induced innate response begins at the infection site. Initially, an influx of neutrophils, monocytes, and NK cells is established. These cells traverse the activated capillary endothelial cells guided by the chemokines present at the infection site. The neutrophils secrete α-defensins that insert into the virion lipid envelope and trigger the degradation of phagocytosed virions [22]. They also secrete TNF-α, which acts synergistically with IFN-α and -β, as well as IFN-γ to produce lysis of the infected cells inhibiting viral replication [22]. Simultaneously, NK cells are activated by the binding of immunoglobulins to the viral antigen (through their Fc receptor [CD16] located on the cell surface) and are recruited by chemokines and cytokines (IFN-α and -β, IL-12, IL-15, IL-18) produced by the activated infected cells within 2–3 days after infection. The NKs participate in cytolysis of virus-infected cells by perforin/granzyme-mediated processes that result in phagocytosis and destruction of infected cells and viral particles [20]. The accumulation of viral antigens is followed by complement activation, facilitating the uptake of viral peptides by phagocytes [23]. These phagocytic cells produce antiviral cytokines and defensins such as nitric oxide (NO), which enhance the immune response and promote phagocytosis and destruction of virus particles and infected cells [20].

One of the key cells for the innate response against HSV is the macrophage. The molecular mechanisms of the immune response within macrophages have been extensively studied. Once HSV infects macrophages, these cells release a series of cell mediators such as TNF-α and IL-12 (which subsequently stimulates the production of IFN-α and -β). Interferon-α and -β induce the NK and T cells to secrete IFN-γ, which eventually controls the HSV replication and initiates the adaptive response [24, 25]. At this stage, the site of origin of IL-12 production is controversial. The majority of investigators suggest that DCs are the main producers of IL-12 [7, 26]. However, other authors indicate that the main source of this molecule is the recruited inflammatory cells [27].

The adaptive immune response begins when the DCs migrate to the regional lymph nodes. These cells mature and display viral peptides coupled with MHC molecules, secrete cytokines, and regulate the expression of other inflammatory molecules. The cytokines produce differentiation of the Th0 cells to either Th1 or Th2. For HSV, the Th1 response is indispensable for clearance of the infection (particularly for HSV-2) [28]. Other cells such as NK cells and macrophages also stimulate the switch from a Th0 to a Th1 response [28] (Table 17.2).

One of the most studied T cells during HSV infection is the CD8+ T cell. These cells are capable of expressing the lymphocyte-associated antigen (cutaneous leukocyte antigen [CLA]) [29, 30]. During recurrent and symptomatic infection, antigen-specific CD4+ T cells and NK cells infiltrate the dermis by day 2 of the lesion formation, whereas CD8+ dermal infiltration (which implies cytotoxic activity resulting in viral clearance) occurs a few days later [29, 30]. Therefore, CD8 cells appear to play an important role in containing HSV infection. Posaved et al. showed that the levels of CD8+ cytotoxic T lymphocytes (CTLs) correlate inversely with the severity of HSV-2 infection and temporally with the local clearance of the virus in lesions [31]. The data from the study of Koelle et al. suggest that the expression of the homing receptor (CLA) by the CD8+ T cells is programmed at the site of the original antigen encounter and promotes migration and immune responses in reactivation [29]. Additional studies indicate that this specific cell has the capacity of T-cell memory and self-renewal that is crucial to control the infection and symptomatic recurrences [31].

Studies in vivo (mouse model) have demonstrated that CD8+ cells also play a crucial role in controlling the virus during the latent state [19, 32]. Latent herpes viral infection in humans in the trigeminal ganglia is accompanied by a chronic inflammatory process that involves elevated levels of cytokine transcripts such as IFN-γ, TNF-α (which affects viral replication), and chemokines, accompanied by persistent lymphocytic cell infiltration (CD8+ T cells, CD68+, and macrophages). It has been suggested that this phenomenon could be due to a low level of expression of viral genes during latency. The CD8+ T cells are capable of controlling the virus through cytokines, and this mechanism is likely to occur in response to the persistence of viruses that are prone to reactivate frequently, providing a survival advantage for both the host and the virus [19]. It is proposed that factors compromising CD8+ T cell function lead to virus escape from immune regulation and reactivation [33].

There appears to be a complex and well-balanced interaction between host cells and HSV. Success for the virus means a delicate balance between infection and latency. Therefore, evasive strategies must be carefully designed to permit viral replication at certain stages that could potentially lead to transmission to other susceptible hosts in order to maintain the viral progeny. Humans and the herpes viruses have evolved together in a symbiotic relationship that has allowed the adaption of the virus to the host and the environment, maintaining a “truce” with the immune system without any serious threats to human life. Some of the identified mechanisms to evade the immune system include the ability of HSV to alter the activity of neutrophils, macrophages, or NK cells [31]. It is known that HSV interferes with monocyte function, suppresses chemotaxis and phagocytosis, and reduces the secretion of IL-1 and TNF-α [7, 20, 34]. It has also been reported that HSV reduced the activity of NK cells upon contact with infected cells, due in part to the interference with IFN-α and -β production [20, 34]. Furthermore, HSV produces many antiapoptotic factors, such as cathepsins and latency-associated transcripts (LATs), which allow for maximal viral replications. LATs have been recovered from latently infected neurons and have been shown to inhibit granzyme-B mediated apoptosis and capsase-8-dependent apoptosis [17]. It has been proposed that LATs increase the efficiency of latency and subsequent reactivation [33] (Table 17.3).

There are currently two different groups of vaccines targeting HSV: prophylactic and therapeutic. Prophylactic vaccines aim to protect seronegative patients against HSV infection. The goal of therapeutic vaccines is to reduce symptoms and infectivity (outbreaks and shedding) in patients who are already seropositive for HSV [35]. In the recent decades, the focus has been primarily on developing a prophylactic vaccine with varying results in trials. Two studies were carried out by GlaxoSmithKline, using a recombinant vaccine preparation containing HSV glycoproteins. The first enrolled patients who were seronegative for both HSV-1 and HSV-2. The second study allowed inclusion of HSV-1–seropositive patients [36]. The studies’ primary end point was protection against genital HSV-2 clinical disease and the secondary end point was HSV-2 seroconversion. The first study did not show any difference in the primary end point between the group that received vaccine and the group that received a placebo. However, when a gender-stratified analysis was performed, the vaccine proved protective in females only (73 %; 95 % confidence interval [CI], 19–91 %). The sex differences found in these studies might be explained by innate anatomic differences of the genital epithelial layers between men and women. The epithelial layer of male genitals consists mostly of stratum corneum, which is lacking in the genitals of women at the vaginal-cervical mucous membrane. Additionally, the secretions produced by the vagina and the genital mucous membranes contain antibodies and migratory leukocytes. Therefore, women appear to have enhanced immune responses mediated by helper T cells exhibiting a Th1 response, compared to men [37]. The Herpevac Trial for Woman was conducted to evaluate this sex difference in vaccine protection. Although the primary endpoint of HSV-1 and HSV-2 genital disease prevention was not achieved, the vaccine was found to be effective in decreasing infection caused by HSV-1 but not HSV-2 [38]. Further studies are needed to evaluate this unexpected outcome.

Recent HSV vaccine research has transitioned away from prophylactic vaccines to therapeutic vaccine candidates. There are currently three novel HSV vaccine candidates in phase I or II trials. The HerpV vaccine is composed of recombinant human heat shock protein-70, 32 different HSV-2 antigens and an adjuvant. This vaccine increased HSV-2 CD4+ and CD8+ T cell activity in HSV-2 seropositive patients in a phase I trial [39]. It is currently being evaluated for efficacy in prevention of shedding and lesions in a phase II trial [40]. The GEN-003 vaccine consists of the gD2 glycoprotein, ICP4 (a potent CD8+ T-cell immunogen) and Matrix M adjuvant. In animal models, this vaccine candidate decreased recurrence of lesions and viral shedding [41]. The GEN-003 vaccine has completed a phase I/II trial evaluating its efficacy as a therapeutic vaccine but the results of this trial have not been published [42]. Finally, the HSV529 vaccine is composed of a DNA replication defective HSV virus. This candidate vaccine has demonstrated efficacy in animal models and is in a phase I/II trial as a therapeutic and prophylactic vaccine [43]. Research on a prophylactic HSV vaccine needs to focus on inducing a stronger antibody response. In contrast, research on a therapeutic HSV vaccine needs to focus on prompting a more potent T-cell response [43].

Initial clinical observations indicate that the primary site of inoculation is the respiratory tract. A rash usually develops after an incubation period of 10–12 days [11, 45, 46]. The entry of the VZV into a host cell requires fusion of the viral envelope with the host cell plasma membrane. This phenomenon is mediated by interactions of viral oligosaccharides with the heparan sulfate proteoglycans (via N-linkage) on the host cell surface. The fusion permits the entry of the viral proteins into the host cell cytosol and then to the nucleus where the nucleocapsid fuses with the outer nuclear membrane, releasing the viral DNA genome into the nucleus [47]. The spread of the virus is controlled by the innate and adaptive responses. The host cell membrane has specific glycoproteins such as gH, gL, gB, and gE that permit the fusion process between cells. These proteins are used by VZV to quickly spread to adjacent epidermal cells by inducing the fusion of the infected cells with noninfected ones [32]. Varicella-zoster virus has a special tropism for three major cell types: the peripheral blood mononuclear cells (PBMCs), skin cells, and sensory neurons [32]. This virus not only infects these cells, but also is capable of replicating inside them. This phenomenon of viral replication can be observed in intraepidermal vesicular lesions of the skin, which are loaded with free virus [11, 45, 47].

The virus infects the immature DCs of the respiratory mucosa, and these cells transport the virus to the T-cell–draining lymph nodes. From these nodes VZV is subsequently transported to the reticuloendothelial system and then is capable of gaining access to the bloodstream, causing a primary viremia. During this first viremic phase, VZV is able to reach the reticuloendothelial organs where it undergoes a phase of viral amplification. It was previously thought that VZV reached the skin during a second viremic episode that occurred in the late incubation period [47]. However, studies of viral infection in mouse models now suggest that infected T cells from the tonsil area are capable of transferring VZV to the skin immediately after entering the circulation during the primary viremia and that the prolonged interval between exposure and the skin rash reflects the time required for the virus to become recognized by potent innate immune barriers such as IFN-α [44, 47].

Varicella-zoster virus preferentially infects the active memory CD4 T cells that express skin homing markers such as CLA and the chemokine (C-C motif) receptor 4 (CCR4). These T cells are usually programmed for immune surveillance and then may facilitate the transfer of VZV into the skin [44]. It appears that VZV does not trigger any early inflammatory response that might block the appearance of virus-filled vesicles at the skin surface [44, 47]. The initial viremia is necessary to ensure that enough cutaneous lesions are formed to transmit the virus efficiently to other individuals as a viral survival mechanism. The T cells trafficking through the skin activate VZV replication at this site, and this process permits infection of more T cells that will return to the circulation [44].

The innate immune response is induced during the acute VZV infection. This response involves the release of IFN-α and IFN-γ and the secretion of cytokines such as IL-6 (by monocytes) via TLR2 [48]. This cell-mediated immune response contains VZV replication and prevents the host from a systemic disease (including severe compromise of organs such as lungs, kidneys, and spleen). The adaptive immune response begins with the presence of CD4+ T cells and the release of IL-2, IL-10, IL-12, and IFN-γ by Th1 and Th2 cells [49]. These cytokines cause the proliferation of VZV specific CD4+ and CD8+ T cells, which express MHC class I and II molecules and recognize the viral glycoproteins gE, gH, and gI, with the subsequent killing of the VZV-infected cells [50]. During the presence of rash, many mononuclear infiltrating cells are present. Most of these cells express CD4 and CD8, including CD45RO+ memory cells, skin homing CLA, and CCR4+ T cells. During this period, the skin shows expression of E-selectin (a cell adhesion molecule and CD antigen that mediates neutrophil, monocyte, and memory T-cell adhesion to cytokine-activated endothelial cells), intercellular adhesion molecule 1 (ICAM-1, a cell-surface ligand involved in leukocyte adhesion and inflammation), and vascular cell adhesion molecule-1 (VCAM-1, a cytokine- induced cell adhesion molecule present on activated endothelial cells, tissue macrophages, and DCs), allowing the migration of the inflammatory cells. The humoral immune response has a reduced role in controlling the virus. Immunoglobulin G (IgG), IgM and IgA appear to respond to some viral proteins. It seems that these antibodies neutralize cell-free virions and contribute in the lysis of infected cells [44] (Table 17.4).

During the resolution of varicella, VZV establishes latency in the trigeminal and dorsal root ganglia where it remains latent through the lifetime of its host. Approximately 20 % of infected people develop herpes zoster. Figure 17.2 illustrates the latent virus in a dorsal root ganglion and reactivated virus causing acute vesicular zoster rash [
51]. In this period, the VZV multiplies and spreads centrifugally down the sensory nerve, causing neuronal necrosis and intense neuritis. Recent studies have shown that VZV possesses multiple open reading frames (ORFs) that encode proteins present in the cytoplasm of neurons during latency and are localized in the cell nucleus during reactivation [52]. Some of these genes are involved in VZV assembly, replication (such as ORF2) and latency. ORF47 encodes a protein that is part of the VZV virion tegument and is essential for VZV growth in differentiated skin and T cells [53]. Studies of this peptide indicate that the blockage of the kinase function of this protein decreases the VZV virulence in the skin, suggesting interference with the production of complete VZV virions. This peptide has also been implicated as a latency- associated protein along with the products of the ORF4 and ORF63 [54–57]. Additionally, the mechanisms of latency appear to be controlled LATs. However, the exact mechanisms are not well understood [58].

Varicella-zoster virus utilizes several mechanisms to overcome the immune response. Major histocompatibility complex class II expression is restricted to APCs and is required to present the viral peptides to CD4 T cells. Usually, the host immune response to the virus produces the release of IFN-γ, which stimulates the expression of MHC II molecules. To evade the immune response and ensure replication, VZV produces a protein that interferes with Stat1 (a signal transducer and activator of transcription that mediates cellular responses to interferons). Stat1 interacts with P53 tumor suppressor protein and regulates expression of genes involved in growth control and apoptosis activation and thereby inhibits antiviral IFN-γ production in foci of infected skin cells [44]. Inhibition of IFN-γ results in decreased MHC II expression, which subsequently affects the efficiency of VZV specific immune responses. Varicella-zoster virus takes advantage of these circumstances, and as a consequence, the initial formation of VZV lesions in skin (varicella or during the VZV reactivation as zoster) is facilitated [44, 58, 59]. Additionally, VZV has mechanisms to delay clearance of virus-infected cells by interfering with the expression of MHC class I proteins that are necessary for CD4 and CD8 T-cell recognition [58]. This mechanism allows VZV to evade CD8 T-cell lysis during the viremic phase. The precise molecular event is related to the downregulation of MHC I by VZV via interference of its transport from the Golgi compartment to the plasma membrane [44] (Table 17.5)

Before the introduction in 1995 of Varivax, a live attenuated Oka strain of VZV, millions of children developed primary varicella (chickenpox). Varivax is indicated for vaccination against varicella in individuals 12 months of age and older and it is recommended as part of the childhood immunization schedule. Its effectiveness is greater than 95 % for varicella prevention. Herpes zoster is the result of the reactivation of latent VZV infection residing in sensory ganglia. In the United States, herpes zoster affects approximately 1,000,000 persons annually, with an incidence of 2.2 cases per 1000 persons of age or older [60, 61]. Results from several clinical trials have determined that a live, attenuated VZV vaccine using the Oka strain (Zostavax) is safe, elevates VZV- specific cell-mediated immunity, and significantly reduces the incidence of herpes zoster and postherpetic neuralgia in immunocompetent people over the age of 50. This vaccine is 14-fold more concentrated than VARIVAX and has been approved for use in the United States. It is expected to reduce the risk for herpes zoster by >50 % [61]. There have been concerns about the decline in Zostavax efficacy however. Although vaccine efficacy was maintained through year 5 after vaccination in the Short-Term Persistence Substudy, it steadily declined during that time period [62]. Figure 17.3 illustrates how the administration of zoster vaccine to older persons may prevent VZV-specific T cells from dropping below the threshold for zoster occurrence [
63].

Human papillomavirus has a special tropism for the epithelial cells (they are epitheliotropic), infecting keratinocytes at a wide range of body sites where they cause aberrant cellular proliferation leading to benign warts or cervical cancer [3, 64, 75]. Initially, the virus infects primitive basal keratinocytes and starts DNA replication and transcription of the early genes at very low levels [76]. The peak of productive synthesis of virions is reached once the keratinocyte reaches higher strata of the epithelium. Thus, high levels of viral proteins and viral assembly occur only at the upper layers of the squamous epithelia (stratum spinosum and granulosum) [11]. At this point the cycle of replication and patterns of gene transcription are dependent on the stage of differentiation of the keratinocyte [76].

A significant amount of information regarding the immune response has been gathered from observations in animal models. Once the virus has reached the skin, it establishes itself at the basal cells of the epithelia where it starts replication. At this point the replication is minimal but becomes greater once the keratinocyte matures toward desquamation [77]. At the basal layer, there is practically no immune response against the virus (two to several months), but once it is detected by the immune system, replication increases and results in clinically detectable lesions [78, 79]. After a few months (which varies depending on the host), the infected keratinocytes express the late viral genes (encoding proteins L1 and L2) that trigger a full immune response [77–79].

The keratinocytes are able to secrete cytokines, growth factors, and chemokines upon viral stimuli. The host immune response dictates the emergence and characteristics of clinical lesions [76]. Some of the cytokines involved are transforming growth factor-β (TGF-β), TNF-α, and IFNs. TGF-β has been shown to inhibit the growth of nontumorigenic HPV-16 and HPV-18 immortalized cells, and it seems to control the expression of E6 and E7 (although some controversy exists on these facts) [76]. In normal cervical cells, TGF-β inhibits viral growth, but HPV can evade this control mechanism [76]. TNF-α, which is another product of the keratinocyte, may have an antiproliferative effect on the HPV-16–infected cells through cell cycle arrest (between the G0 and G1 phases of the cell cycle) [76, 80]. It has also been suggested that TNF-α acts as a repressor on the expression of E6 and E7 in the immortalized human keratinocytes [81]. The effects attributed to IFNs may be virus-type specific. These molecules also appear to inhibit the production of viral proteins [76].

The time between HPV infection and the development of a clinical lesion could vary from weeks to more than 9 months. This is an indication that HPV could modulate the immune system as evidenced by the following: (1) HPV infects mainly keratinocytes, which are cells programmed to die and desquamate in a specific and timely manner (apoptosis); and (2) the intracellular location of HPV in these cells allows the virus to hide and avoid immune surveillance. As a consequence, the apoptotic keratinocyte infected with HPV does not trigger a significant inflammatory reaction, eventually resulting in a persistent chronic infection [78, 82].

The adaptive immune response involves two phases: the recognition of antigen and the response to it. This adaptive immune response involves the LCs that capture the virus and its antigen for transport to local lymph nodes and presentation to naive T cells. The T cells return to the infected epithelial tissues via mechanisms that include secretion of chemokines and expression of adhesion molecules to clear the infection. In the recognition phase, the LCs are the major APCs. However, in some HPV infections, a depletion of these cells has been documented, which is associated with enhanced survival of HPV, prolonged courses of infection, and possibly malignancy [83–85]. At this stage, the infected keratinocytes produce IL-1α and TNF, which promote LC migration.

If the immune response is appropriate, the regression of warts usually follows. At this stage, a large infiltrate of T cells (CD4+ and CD8+) and macrophages is present, and the cytokines involved exhibit the Th1 pattern (IL-12, TNF-α, and IFN-γ plus the expression of the adhesion molecules for lymphocyte trafficking) (Table 17.6). A systemic T-cell response to E1 and E6 viral proteins is effective only at the peak of wart regression (although activity is present during the whole infective cycle) [78, 79].

In summary, HPV is capable of escaping the immune response for several months by modulating the inflammatory reaction from the keratinocytes after invasion. Nonetheless, in the majority of cases, the immune system finally catches up and is capable of detecting the virus, (probably when the replication turnover is highest) and thus is able to resolve the infection.

The reasons for the failure of the immune system to recognize HPV are not well understood. It appears that HPV is able to escape detection by the APCs. To survive and have enough time to replicate without detection, HPV has developed many strategies to evade the immune system. The special tropism for keratinocytes (as discussed earlier) is one of the first mechanisms of immune evasion [78].

Keratinocytes are powerful immune cells. They constitute almost 95 % of the cervical and skin epithelium and can express MCH II and co-stimulatory signals to T cells (such as ICAM-I) during inflammation [92]. If the immune response is activated, the HPV-infected keratinocytes release TNF-α, which negatively affects the replication of HPV [70, 92]. TNF-α upregulates the expression of ICAM-1 levels, which decrease the levels of IFN-γ. In CIN, the keratinocytes express less TNF-α, decreasing the stimulus to the LCs. This alteration also affects the expression of MHC II, and as a consequence the presentation of antigens is altered and the expression of the suppressive cytokine IL-10 is increased [68, 92]. These changes in the immunologic microenvironment (with inappropriate T-cell presentation) may contribute to the persistence and progression of the viral infection and development of a CIN lesion [84, 92, 94].