Herpes simplex virus spreads rapidly in human foreskin, partly driven by chemokine-induced redistribution of Nectin-1 on keratinocytes.

HSV infects keratinocytes in the epidermis of skin via nectin-1. We established a human foreskin explant infection model to investigate HSV entry and spread. HSV1 entry could only be achieved by the topical application of virus via high density microarray projections (HD-MAPs) to the epidermis, which penetrated beyond one third of its thickness, simulating in vivo microtrauma. Rapid lateral spread of HSV1 to a mean of 13 keratinocytes wide occurred after 24 hours and free virus particles were observed between keratinocytes, consistent with an intercellular route of spread. Nectin-1 staining was markedly decreased in foci of infection in the epidermis and in the human keratinocyte HaCaT cell line. Nectin-1 was redistributed, at the protein level, in adjacent uninfected cells surrounding infection, inducible by CCL3, IL-8 (or CXCL8), and possibly CXCL10 and IL-6, thus facilitating spread. These findings provide the first insights into HSV1 entry and spread in human inner foreskin in situ.

Herpes Simplex Virus type 1 infects Langerhans cells and the novel epidermal dendritic cell, Epi-cDC2s, via different entry pathways.

Skin mononuclear phagocytes (MNPs) provide the first interactions of invading viruses with the immune system. In addition to Langerhans cells (LCs), we recently described a second epidermal MNP population, Epi-cDC2s, in human anogenital epidermis that is closely related to dermal conventional dendritic cells type 2 (cDC2) and can be preferentially infected by HIV. Here we show that in epidermal explants topically infected with herpes simplex virus (HSV-1), both LCs and Epi-cDC2s interact with HSV-1 particles and infected keratinocytes. Isolated Epi-cDC2s support higher levels of infection than LCs in vitro, inhibited by acyclovir, but both MNP subtypes express similar levels of the HSV entry receptors nectin-1 and HVEM, and show similar levels of initial uptake. Using inhibitors of endosomal acidification, actin and cholesterol, we found that HSV-1 utilises different entry pathways in each cell type. HSV-1 predominantly infects LCs, and monocyte-derived MNPs, via a pH-dependent pathway. In contrast, Epi-cDC2s are mainly infected via a pH-independent pathway which may contribute to the enhanced infection of Epi-cDC2s. Both cells underwent apoptosis suggesting that Epi-cDC2s may follow the same dermal migration and uptake by dermal MNPs that we have previously shown for LCs. Thus, we hypothesize that the uptake of HSV and infection of Epi-cDC2s will stimulate immune responses via a different pathway to LCs, which in future may help guide HSV vaccine development and adjuvant targeting.

HBV vaccination and HBV infection induces HBV-specific natural killer cell memory.

OBJECTIVE: Vaccination against hepatitis B virus (HBV) confers protection from subsequent infection through immunological memory that is traditionally considered the domain of the adaptive immune system. This view has been challenged following the identification of antigen-specific memory natural killer cells (mNKs) in mice and non-human primates. While the presence of mNKs has been suggested in humans based on the expansion of NK cells following pathogen exposure, evidence regarding antigen-specificity is lacking. Here, we demonstrate the existence of HBV-specific mNKs in humans after vaccination and in chronic HBV infection. DESIGN: NK cell responses were evaluated by flow cytometry and ELISA following challenge with HBV antigens in HBV vaccinated, non-vaccinated and chronic HBV-infected individuals. RESULTS: NK cells from vaccinated subjects demonstrated higher cytotoxic and proliferative responses against autologous hepatitis B surface antigen (HBsAg)-pulsed monocyte-derived dendritic cells (moDCs) compared with unvaccinated subjects. Moreover, NK cell lysis of HBsAg-pulsed moDCs was significantly higher than that of hepatitis B core antigen (HBcAg)-pulsed moDCs (non-vaccine antigen) or tumour necrosis factor α-activated moDCs in a NKG2D-dependent manner. The mNKs response was mediated by CD56dim NK cells coexpressing CD57, CD69 and KLRG1. Further, mNKs from chronic hepatitis B patients exhibited greater degranulation against HBcAg-pulsed moDCs compared with unvaccinated or vaccinated patients. Notably, mNK activity was negatively correlated with HBV DNA levels. CONCLUSIONS: Our data support the presence of a mature mNKs following HBV antigen exposure either through vaccination or infection. Harnessing these antigen specific, functionally active mNKs provides an opportunity to develop novel treatments targeting HBV in chronic infection.

Relay of herpes simplex virus between Langerhans cells and dermal dendritic cells in human skin.

The mechanism by which immunity to Herpes Simplex Virus (HSV) is initiated is not completely defined. HSV initially infects mucosal epidermis prior to entering nerve endings. In mice, epidermal Langerhans cells (LCs) are the first dendritic cells (DCs) to encounter HSV, but it is CD103(+) dermal DCs that carry viral antigen to lymph nodes for antigen presentation, suggesting DC cross-talk in skin. In this study, we compared topically HSV-1 infected human foreskin explants with biopsies of initial human genital herpes lesions to show LCs are initially infected then emigrate into the dermis. Here, LCs bearing markers of maturation and apoptosis formed large cell clusters with BDCA3(+) dermal DCs (thought to be equivalent to murine CD103(+) dermal DCs) and DC-SIGN(+) DCs/macrophages. HSV-expressing LC fragments were observed inside the dermal DCs/macrophages and the BDCA3(+) dermal DCs had up-regulated a damaged cell uptake receptor CLEC9A. No other infected epidermal cells interacted with dermal DCs. Correspondingly, LCs isolated from human skin and infected with HSV-1 in vitro also underwent apoptosis and were taken up by similarly isolated BDCA3(+) dermal DCs and DC-SIGN(+) cells. Thus, we conclude a viral antigen relay takes place where HSV infected LCs undergo apoptosis and are taken up by dermal DCs for subsequent antigen presentation. This provides a rationale for targeting these cells with mucosal or perhaps intradermal HSV immunization.

Advances in understanding the mechanism of action of adult vaccines.

The occurrence of herpes zoster (HZ) correlates with declining memory T cells that had responded to earlier infection with varicella-zoster virus (VZV). There are especially lower T cell responses to the single immunodominant VZV protein glycoprotein E (gE) in people over 50 years of age, although antibody responses to VZV persist. Therefore, a live attenuated zoster vaccine (ZVL) aimed at restoring T cell responses was developed. Surprisingly, a recombinant zoster vaccine (RZV) consisting of gE combined with the AS01B adjuvant system proved superior in efficacy and durability. In this issue of the JCI, Laing, Ford, and colleagues showed that both vaccines stimulated preimmunization naive CD4+ T cells, not just memory CD4+ T cells, to gE, and recruited these naive responses into the overall memory response. However, compared with ZVL, RZV stimulated this response to a much greater degree. These results will help guide development of more effective and durable vaccines for older individuals.

Cytokines and chemokines: The vital role they play in herpes simplex virus mucosal immunology.

Herpes simplex viruses (HSV) types 1 and 2 are ubiquitous infections in humans. They cause orofacial and genital herpes with occasional severe complications. HSV2 also predisposes individuals to infection with HIV. There is currently no vaccine or immunotherapy for these diseases. Understanding the immunopathogenesis of HSV infections is essential to progress towards these goals. Both HSV viruses result in initial infections in two major sites - in the skin or mucosa, either after initial infection or recurrence, and in the dorsal root or trigeminal ganglia where the viruses establish latency. HSV1 can also cause recurrent infection in the eye. At all of these sites immune cells respond to control infection. T cells and resident dendritic cells (DCs) in the skin/mucosa and around reactivating neurones in the ganglia, as well as keratinocytes in the skin and mucosa, are major sources of cytokines and chemokines. Cytokines such as the Type I and II interferons synergise in their local antiviral effects. Chemokines such as CCL2, 3 and 4 are found in lesion vesicle fluid, but their exact role in determining the interactions between epidermal and dermal DCs and with resident memory and infiltrating CD4 and CD8 T cells in the skin/mucosa is unclear. Even less is known about these mechanisms in the ganglia. Here we review the data on known sources and actions of these cytokines and chemokines at cellular and tissue level and indicate their potential for preventative and therapeutic interventions.

The Role of Tissue Resident Memory CD4 T Cells in Herpes Simplex Viral and HIV Infection.

Tissue-resident memory T cells (TRM) were first described in 2009. While initially the major focus was on CD8+ TRM, there has recently been increased interest in defining the phenotype and the role of CD4+ TRM in diseases. Circulating CD4+ T cells seed CD4+ TRM, but there also appears to be an equilibrium between CD4+ TRM and blood CD4+ T cells. CD4+ TRM are more mobile than CD8+ TRM, usually localized deeper within the dermis/lamina propria and yet may exhibit synergy with CD8+ TRM in disease control. This has been demonstrated in herpes simplex infections in mice. In human recurrent herpes infections, both CD4+ and CD8+ TRM persisting between lesions may control asymptomatic shedding through interferon-gamma secretion, although this has been more clearly shown for CD8+ T cells. The exact role of the CD4+/CD8+ TRM axis in the trigeminal ganglia and/or cornea in controlling recurrent herpetic keratitis is unknown. In HIV, CD4+ TRM have now been shown to be a major target for productive and latent infection in the cervix. In HSV and HIV co-infections, CD4+ TRM persisting in the dermis support HIV replication. Further understanding of the role of CD4+ TRM and their induction by vaccines may help control sexual transmission by both viruses.

Vaccines for Herpes Simplex: Recent Progress Driven by Viral and Adjuvant Immunology.

Herpes simplex viruses (HSV) types 1 and 2 are ubiquitous. They both cause genital herpes, occasionally severe disease in the immunocompromised, and facilitate much HIV acquisition globally. Despite more than 60 years of research, there is no licensed prophylactic HSV vaccine and some doubt as to whether this can be achieved. Nevertheless, a previous HSV vaccine candidate did have partial success in preventing genital herpes and HSV acquisition and another immunotherapeutic candidate reduced viral shedding and recurrent lesions, inspiring further research. However, the entry pathway of HSV into the anogenital mucosa and the subsequent cascade of immune responses need further elucidation so that these responses could be mimicked or improved by a vaccine, to prevent viral entry and colonization of the neuronal ganglia. For an effective novel vaccine against genital herpes the choice of antigen and adjuvant may be critical. The incorporation of adjuvants of the vaccine candidates in the past, may account for their partial efficacy. It is likely that they can be improved by understanding the mechanisms of immune responses elicited by different adjuvants and comparing these to natural immune responses. Here we review the history of vaccines for HSV, those in development and compare them to successful vaccines for chicken pox or herpes zoster. We also review what is known of the natural immune control of herpes lesions, via interacting innate immunity and CD4 and CD8 T cells and the lessons they provide for development of new, more effective vaccines.

Mechanisms of Immune Control of Mucosal HSV Infection: A Guide to Rational Vaccine Design.

Herpes Simplex Virus (HSV) is a highly prevalent sexually transmitted infection that aside from causing cold sores and genital lesions, causes complications in the immunocompromised and has facilitated a large proportion of HIV acquisition globally. Despite decades of research, there is no prophylactic HSV vaccine ready for use in humans, leaving many questioning whether a prophylactic vaccine is an achievable goal. A previous HSV vaccine trial did have partial success in decreasing acquisition of HSV2-promising evidence that vaccines can prevent acquisition. However, there is still an incomplete understanding of the immune response pathways elicited by HSV after initial mucosal infection and how best to replicate these responses with a vaccine, such that acquisition and colonization of the dorsal root ganglia could be prevented. Another factor to consider in the rational design of an HSV vaccine is adjuvant choice. Understanding the immune responses elicited by different adjuvants and whether lasting humoral and cell-mediated responses are induced is important, especially when studies of past trial vaccines found that a sufficiently protective cell-mediated response was lacking. In this review, we discuss what is known of the immune control involved in initial herpes lesions and reactivation, including the importance of CD4 and CD8 T cells, and the interplay between innate and adaptive immunity in response to primary infection, specifically focusing on the viral relay involved. Additionally, a summary of previous and current vaccine trials, including the components used, immune responses elicited and the feasibility of prophylactic vaccines looking forward, will also be discussed.

Identification of HIV transmitting CD11c+ human epidermal dendritic cells.

Langerhans cells (LC) are thought to be the only mononuclear phagocyte population in the epidermis where they detect pathogens. Here, we show that CD11c+ dendritic cells (DCs) are also present. These cells are transcriptionally similar to dermal cDC2 but are more efficient antigen-presenting cells. Compared to LCs, epidermal CD11c+ DCs are enriched in anogenital tissues where they preferentially interact with HIV, express the higher levels of HIV entry receptor CCR5, support the higher levels of HIV uptake and replication and are more efficient at transmitting the virus to CD4 T cells. Importantly, these findings are observed using both a lab-adapted and transmitted/founder strain of HIV. We also describe a CD33low cell population, which is transcriptionally similar to LCs but does not appear to function as antigen-presenting cells or acts as HIV target cells. Our findings reveal that epidermal DCs in anogenital tissues potentially play a key role in sexual transmission of HIV.

Dendritic cells in the cornea during Herpes simplex viral infection and inflammation.

Herpes simplex keratitis is commonly caused by Herpes simplex virus type 1, which primarily infects eyelids, corneas, or conjunctiva. Herpes simplex virus type 1-through sophisticated interactions with dendritic cells (DCs), a type of antigen-presenting cell)-initiates proinflammatory responses in the cornea. Corneas were once thought to be an immune-privileged region; however, with the recent discovery of DCs that reside in the cornea, this long-held conjecture has been overturned. Therefore, evaluating the clinical, preclinical, and cell-based studies that investigate the roles of DCs in corneas infected with Herpes simplex virus is critical. With in vivo confocal microscopy, animal models, and cell culture experiments, we may further the understanding of the sophisticated interactions of Herpes simplex virus with DCs in the cornea and the molecular mechanism associated with it. It has been shown that specific differentiation of DCs using immunohistochemistry, flow cytometry, and polymerase chain reaction analysis in both human and mice tissues and viral tissue infections are integral to increasing understanding. As for in vivo confocal microscopy, it holds promise as it is the least invasive and a real-time investigation. These tools will facilitate the discovery of various targets to develop new treatments.