The Cycle of Human Herpes Simplex Virus Infection: Virus Transport and Immune Control

HSV Infection Cycle • JID 2006:194 (Suppl 1) • S11

S U P P L E M E N T A R T I C L E

The Cycle of Human Herpes Simplex VirusInfection: Virus Transport and Immune Control

Anthony L. Cunningham,1 Russell J. Diefenbach,1 Monica Miranda-Saksena,1 Lidija Bosnjak,1 Min Kim,1

Cheryl Jones,2 and Mark W. Douglas1,a

1Centre for Virus Research, Westmead Millennium Institute, University of Sydney and Westmead Hospital, and 2Children’s Hospitalat Westmead, Sydney, Australia

After infection of skin or mucosa, herpes simplex virus enters the sensory nerve endings and is conveyed byretrograde axonal transport to the dorsal root ganglion, where the virus develops lifelong latency. Intermittentreactivation, which is spontaneous in humans, leads to anterograde transport of virus particles and proteinsto the skin or mucosa, where the virus is shed and/or causes disease. Immune control of viral infection andreplication occurs at the level of skin or mucosa during initial or recurrent infection and also within thedorsal root ganglion, where immune mechanisms control latency and reactivation. This article examines currentviews on the mechanisms of retrograde and anterograde transport of the virus in axons and the mechanismsof innate and adaptive immunity that control infection in the skin or mucosa and in the dorsal root ganglion—in particular, the role of interferons, myeloid and plasmacytoid dendritic cells, CD4+ and CD8+ T cells, andinterferon-g and other cytokines, including their significance in the development of vaccines for genital herpes.

Herpes simplex virus (HSV) type 1 infects 60%–80%

of people throughout the world, whereas the prevalence

of HSV-2 infection in adults varies markedly from

country to country, from as low as 7% up to 80%,

depending on sexual and, perhaps, contraceptive prac-

tices [1]. HSV-2 causes one of the most common sex-

ually transmitted diseases, genital herpes, occasionally

leading to neonatal herpes, which may result in severe

morbidity or death. In addition, severe morbidity may

result from recurrent genital herpes in immunocom-

promised patients. HSV-2 also appears to enhance the

risk of acquisition of HIV by 2- to 3-fold [2]. HSV-1

causes ocular herpes, a major cause of blindness in the

Western world, and is the most important cause of

sporadic encephalitis, usually resulting in severe mor-

bidity or mortality. HSV-1 is also causing an increasing

proportion of genital herpes, particularly in adolescents

Potential conflicts of interest: none reported.a Present affiliation: MRC Virology Unit, Institute of Virology, Glasgow, United

Kingdom.Reprints or correspondence: Dr. A. L. Cunningham, Westmead Millennium Institute,

Darcy Rd., Westmead NSW 2145, Sydney, Australia ([email protected]).

The Journal of Infectious Diseases 2006; 194:S11–18� 2006 by the Infectious Diseases Society of America. All rights reserved.0022-1899/2006/19406S1-0003$15.00

[3]. Furthermore, HSV-1 is being engineered for use

as a gene therapy vector to convey genes from the pe-

riphery to the central nervous system or for direct in-

fection of cerebral and other tumors, resulting in in-

fection and destruction of tumor cells but not of normal

brain tissue [4].

HSV-1 and -2 usually infect via the oral or genital

mucosa and replicate in stratified squamous epithelium;

this is followed by uptake into ramifying unmyelinated

sensory nerve fibers within the stratified squamous ep-

ithelium and then retrograde microtubule-associated

transport to the cell body of the neuron in the dorsal

root ganglion (DRG) adjacent to the spinal cord (or

the trigeminal ganglion for HSV-1). Here, acute infec-

tion is followed by lifelong latent infection of these cells.

Intermittent reactivation of the virus occurs sponta-

neously and results in anterograde microtubule-asso-

ciated transport of the virus, usually back to the original

infecting dermatome, where the virus crosses from the

nerve terminal into the stratified squamous epithelium

of skin or mucosa. Here, it replicates and is then shed

into oral or genital secretions. This process may or may

not result in clinical disease. Thus, the patient may

discover the characteristic vesicles or ulcers of genital

herpes, but, in most infected patients, small or atypical

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lesions are usually recognized only by expert clinicians or ed-

ucated patients. In another 20% of patients, shedding may be

entirely asymptomatic [5].

This cycle of infection is subject to immune control at several

levels. Elucidation of these various mechanisms should help

guide the development of therapeutic and better prophylactic

vaccines. At entry, innate and adaptive immune mechanisms

control the level of replication in the mucosa, thus determining

the amount of virus that enters neurons, becomes latent, and

reactivates from the DRG neurons. At the level of the DRG

neuron, immune control determines control over latency and

reactivation. After anterograde transport of virus to the axon

terminals, adaptive immune mechanisms at the interface with

mucosal epithelial cells may determine the level of replication

and whether clinical disease or asymptomatic shedding occurs.

ENTRY OF HSV INTO THE SKIN/MUCOSA

HSV-1 and -2 have difficulty penetrating intact stratum cor-

neum except when cracks or abrasions exist or this layer is very

thin or nonexistent (labium minora and vagina in females,

prepuce in males), allowing access to surface receptors on the

epidermal keratinocytes and perhaps on Langerhans cells, re-

sulting in infection. Both epidermal keratinocytes and dendritic

cells (DCs) have been shown to express HSV receptors, nectin-

1, and herpesvirus entry mediator (HVEM), on their surface

[6, 7]. Recently, it has been shown that epidermal keratinocytes

can also be infected via endocytosis (as well as via neutral fusion

at the cell surface) [8]. HSV infection of keratinocytes results

in the production of numerous cytokines, including interferon

(IFN)-a, IFN-b, interleukin (IL)-1, IL-6, and b-chemokines,

all present within vesicle fluid and shown to be liberated from

infected keratinocytes in vitro [9]. Infection of Langerhans cells

resident within the epidermis is also likely to liberate cytokines,

as has been found in the supernatants of HSV-infected DCs in

vitro [10].

RETROGRADE TRANSPORT OF HSV-1IN NEURONS

HSV is rapidly taken up by neurons in the mucosa or skin in

guinea pig models, although amplification in keratinocytes

probably provides the bulk of virus entering sensory nerve

terminals. After entry into cells, including neurons, the surface

glycoproteins of the virus are left behind in the cell membrane

as a consequence of virus–cell membrane fusion. Many of the

outer tegument proteins are phosphorylated and dissociate

from the virion [11]. Indeed, most of the tegument proteins

appear to be lost from the internal core or nucleocapsid. The

exact tegument proteins remaining on the capsid have yet to

be defined, but this process allows the capsids with a comple-

ment of only a few or even 1 tegument protein to bind to

molecular motors associated with microtubules and to be trans-

ported rapidly in a retrograde direction to the cell body [12].

There is evidence that the principal retrograde molecular motor

dynein, in association with dynactin, is involved in this trans-

port [13]. The viral protein(s) binding to this molecular motor

have yet to be fully defined. In the related alphaherpesvirus

pseudorabies virus, at least 3 inner tegument proteins appear

to remain on the capsid during retrograde transport: US3,

UL36, and UL37 [14, 15]. However, this may not necessarily

be the case with HSVs (authors’ unpublished data). It is likely

that the viral proteins binding to dynein are such inner tegu-

ment proteins or outer capsid proteins. In fact, we have shown

that VP26, a small 12-kDa protein perched externally on the

capsid that binds to the major capsid protein VP5 in its hexon

configuration, also binds to dynein as a recombinant protein

[16]. Furthermore, when HSV capsids are assembled in vitro

from their constituent proteins with or without VP26, only the

capsid with VP26 on its surface is transported to the nuclear

membrane after deposition of both types of capsids into the

cytoplasm by microinjection [16]. It is possible that the external

position of VP26 may allow it to bind to dynein during ret-

rograde transport in vivo if not covered by any of the other

inner tegument proteins. Nevertheless, there must be another

(possibly inner tegument) protein binding to dynein, because

VP26 can be deleted from the virus without completely im-

pairing retrograde transport in mice [17]. Herpesviruses often

show redundancy of important functions, and it seems likely

that this will also be the case with retrograde and anterograde

transport.

ROLE OF IFN AND THE INNATE IMMUNERESPONSE IN INITIAL INFECTION BY HSV

IFNs, macrophages, NK cells, and gd T cells have all been shown

to play a role in innate immunity [18–20] (figure 1A). In mu-

rine models, the role of IFN-a and -b in protection against

HSV-1 infection has long been known. However, recently, Toll-

like receptors (TLRs) have also been shown to be important

mediators of innate immune responses in HSV infection in the

genital tract and, perhaps, systemically. HSVs interact with both

TLR-2 and -9 [21]. The interaction of HSV-1 and -2 with TLR-

2 appears to be at the surface [21], whereas the interaction of

HSV-2 with TLR-9 appears to be via viral DNA within the

endosomes, particularly of plasmacytoid DCs [22]. This latter

interaction is a potent stimulus to the production of IFN-a.

Indeed, plasmacytoid DCs are the main effectors of the pre-

viously well-described stimulation of IFN-a production from

human blood mononuclear cells by HSV antigen [23]. Whether

plasmacytoid DCs are present within human lesions is currently

under investigation.

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Figure 1. A, Innate immune mechanisms induced by initial herpes simplex virus (HSV) infection. B, Role of Langerhans cells (LC), keratinocytes (K),and T cells in connecting innate and adaptive immune control of recurrent herpes simplex. IFN, interferon; IL, interleukin; M, macrophage; MHC, majorhistocompatibility complex; NO, nitric oxide; pDCs, plasmacytoid dendritic cells; TLR, Toll-like receptor.

ROLE OF THE IMMUNE SYSTEMIN MAINTENANCE OF LATENCYAND PREVENTION OF VIRUS REACTIVATION

Immune control of latency or reactivation from neurons cannot

be studied in humans, so accurate animal models of latency

and reactivation are needed. In humans, both spontaneous and

induced (e.g., by UV light) reactivation from latency followed

by recurrent disease occurs, but spontaneous reactivation does

not occur in the mouse [24]. In guinea pigs and rabbits, in

which such spontaneous reactivation resulting in disease does

occur, a full range of reagents to characterize immune cells is

not available. Nevertheless, careful examination of murine DRG

neurons during the phase of HSV latency shows that latency

is somewhat “leaky,” as shown by the presence of HSV RNA

and proteins in occasional neurons [25]. Latency needs to be

maintained by the immune system, particularly by noncytolytic

CD8+ T cells (specific for HSV structural proteins), which lie

in apposition to neurons and secrete IFN-g [24]. Experiments

with IFN-g (or receptor) knockout mice and/or subsequent

addition of IFN-g within 24 h of ex vivo culture support its

importance in preventing reactivation, possibly through inhi-

bition of the function of the key immediate early viral protein

ICP0. CD4+ T cells are also present and may help maintain

latency [24].

ANTEROGRADE TRANSPORT OF HSVFROM DRG NEURONS TO PERIPHERY

The process of alphaherpesvirus assembly and egress in the cell

body of DRG neurons appears to be similar to that in cultured

cell lines [26, 27]. The study of anterograde transport of HSV

from the cell bodies of neurons in the DRG to the periphery

is difficult. Early studies relied on empirical sampling of HSV-

infected mice or rabbits or of neurons cultured in vitro at

different time points and the identification of a small number

of enveloped or unenveloped capsids in the DRG and peripheral

nerves. The region of the axon in which these enveloped capsids

were observed was either proximal (close to the cell body) or

uncertain [28].

To study the process of anterograde transport more exactly,

we adapted the 2-chamber systems of Lycke and colleagues [28,

29], as shown in figure 2. Intact DRGs were placed in the central

chamber, while autologous epidermal explants were placed in

the external chamber. The addition of nerve growth factor re-

sulted in the outgrowth of axons from the DRG, which then

penetrated an agarose plug in a barrier between the 2 chambers,

extended into the exterior chamber, and interacted with epi-

dermal cells. Inoculation of virus into the inner chamber re-

sulted in infection of the DRG and anterograde transport of

the virus within axons. Indeed, the only route of virus particles

from the inner to the outer chamber was via these axons. Virus

crossed from the axons into the epidermal explants, as observed

by serial fixation and confocal microscopy for viral antigen.

Furthermore, somewhat surprisingly and controversially, ultra-

thin sections taken from behind the advancing front of viral

antigen were examined by transmission electron microscopy

and showed the presence of unenveloped nucleocapsids in these

axons [30].

Follow-up observations of infected axons in this system by

transmission immunoelectron microscopy showed unenvel-

oped capsids adjacent to microtubules immunolabeled for cap-

sid (VP5) and surrounded by tegument proteins (VP16), where-

as glycoproteins and other tegument proteins were observed

within axonal vesicles in distal axons [31, 32]. Unenveloped

capsids were demonstrated in infected axons during antero-

grade transport but not in uninfected axons by rigorously con-

trolled and blinded electron microscopic observations and by

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Figure 2. Two-chamber system for examining anterograde axonaltransport of herpes simplex virus in human or rat dorsal root ganglion(DRG) neurons.

both transmission immunoelectron microscopy and scanning

immunoelectron microscopy. In our laboratory, previous stud-

ies have observed, usually in cross-sections, 1100 unenveloped

capsids but no enveloped capsids in the mid- or distal axon

[26, 30–32] (authors’ unpublished data). These studies were

further supported by the examination of capsid, tegument, and

envelope protein localization by serial fixation and immuno-

fluorescence, demonstrating differences in the kinetics of the

appearance of glycoprotein and capsid protein in axons. Fur-

thermore, the tegument protein US11 was found to directly

interact with the heavy chain of the anterograde molecular

motor kinesin [33]. The above observations suggested separate

capsid and glycoprotein transport and assembly at the axon

terminus (although the latter has not been visualized). Other

laboratories have also provided further evidence for such an-

terograde axonal transport of unenveloped HSV-1 capsids [34].

Our most recent studies of anterograde transport of HSV-1

in human fetal DRG axons showed, by confocal microscopy,

antigenic colocalization of all 3 classes of HSV-1 components

(capsid, tegument, and envelope proteins), mainly in axonal

varicosities (axonal swellings usually at branch points) and

growth cones at the axon terminus. These findings correlated

with the presence of enveloped capsids in vesicles, which were

observed in clusters at these sites by transmission electron mi-

croscopy. In these growth cones, tegument (VP22) and envelope

(gD) proteins, alone or together, were detected on vesicles by

transmission immunoelectron microscopy, consistent with the

suggestion that some tegument and envelope proteins may be

transported separately or together in association with axonal

vesicles [35]. This is consistent with real-time fluorescence stud-

ies of pseudorabies virus in chick DRG neurons [14, 26]. Early

availability of these proteins on vesicles in the distal axon could

allow later assembly and envelopment of virions in the vari-

cosities and growth cones, by a process similar to that in the

trans-Golgi network.

In contrast, a recent report has challenged this “separate

transport” or “subassembly” hypothesis. In a 3-chamber sys-

tem, enveloped particles within vesicles were observed in the

proximal and mid-axon [36]. How can these different obser-

vations be reconciled? At present, there are no clear answers.

Different alphaherpesviruses, such as HSV, pseudorabies virus,

and varicella-zoster virus, may vary markedly in the proteins

used for various functions, but it seems unlikely that they would

use completely different processes for axonal transport [37].

Quantitative differences in the relative importance of the unen-

veloped versus enveloped capsid transport pathways are more

likely.

IMMUNE RESPONSE TO HUMAN RECURRENTHERPES IN THE SKIN

Several laboratories, including our own, have concentrated on

studying immune control of HSV infection in the human skin,

especially in recurrent oral or genital herpes, demonstrating the

importance of the cell-mediated immune response to HSV in

control and clearance of recurrent infection. The increased se-

verity and persistence of recurrent herpes as a presenting syn-

drome of AIDS reflects the key role of T cells, especially CD4+

T cells [38]. In the Merigan laboratory in 1983, we used biopsy

samples of recurrent herpetic lesions and immunohistochem-

ical methods to demonstrate the importance of CD4+ T cells,

both as a source of IFN-g and as cytotoxic effectors early in

the course of infection, followed by a later influx of CD8+ T

cells [39, 40]. This was later confirmed by Koelle et al. [40] in

studies of T cell clones derived from such biopsy samples. We

also demonstrated that the IFN-g secreted by CD4+ T cells

restored major histocompatibility complex (MHC) class I ex-

pression on infected epithelial cells, thus overcoming the HSV

ICP47–induced blockade of MHC class I expression [41] and

allowing recognition by CD8+ cytotoxic T cells [42]. IFN-g also

stimulated MHC class II expression on keratinocytes through-

out the lesion, allowing recognition by CD4+ T cells. Therefore,

it is not surprising that the frequency of recurrent herpes was

found to correlate with levels of IFN-g produced by blood CD4+

T cells [43]. b-Chemokines, IL-12, and IFN-a, -b, and -g pro-

duced by both epithelial cells and the infiltrating immune cells

within herpetic lesions are critical for the control of this highly

cytopathic virus [44, 45]. b-Chemokines attract monocytes and

T cells into the lesions, and IL-12 entrains CD4+ T cell secretion

to a Th1 pattern (especially IFN-g), which activates cytotoxic

CD8+ T cells, through IL-12 and IFN-a (figure 1B). The later

infiltration with CD8+ cytotoxic T cells corresponds with clear-

ance of virus from the lesions [40]. Study of the immunology

of human recurrent herpetic lesions has helped guide the de-

velopment of prophylactic as well as therapeutic vaccine can-

didates, suggesting the need to induce Th1 rather than Th2

patterns of response [46].

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Homing and infiltration of monocytes and T cells.

Although it now seems clear that earlier infiltration of CD4+

T cells and monocytes than of CD8+ T cells into herpetic lesions

is an important sequence, the mechanisms responsible for the

differential homing of the T cell subsets are unclear. Recent

investigations have demonstrated up-regulation of E-selectin

on cutaneous venule endothelial cells in recurrent herpetic le-

sions, as expected in any inflammatory lesions [47]. This could

be a result of secretion of IL-1b by keratinocytes and, as the

lesion progresses, by IFN-g from CD4+ T cells. In blood, HSV-

2–specific CD4+ and CD8+ T cells express the E-selectin ligand

and/or the similar cutaneous T cell–associated antigen at dif-

fering levels: 50%–70% for CD8+ lymphocytes and ∼20% for

CD4+ T cells. However, CD4+ T cells markedly up-regulate

cutaneous lymphocyte-associated antigen and E-selectin ligand

after in vitro stimulation with HSV-2. Cutaneous lymphocyte-

associated antigen and/or E-selectin ligand may be up-regulated

by cytokines such as IFN-a, IL-12, and transforming growth

factor–b secreted in blood or lesions by immune cells and

keratinocytes [48]. Further work is needed to fully understand

these mechanisms of differential T cell homing and entry into

lesions and their relative importance.

Viral antigens recognized by T cells. The next important

question is that of which are the key immunogenic proteins

within the HSV virion. Using blood lymphocytes from HSV-

2–infected patients that were restimulated with whole HSV in

vitro and incubated with IFN-g–stimulated epidermal cells in-

fected with recombinant vaccinia virus containing a repertoire

of HSV proteins, we showed that the surface glycoproteins gD

and gB (produced late in the replication cycle) are key targets

for CD4+ T cells in most patients, and ICP27 (a protein present

only at early stages in infected cells) is the target for CD8+ T

cells in most patients [49] (both gB and ICP27 are also rec-

ognized by CD8+ T cells in mice [50, 51]). However, the im-

munogenicity of individual proteins in mice and humans does

not always correlate. Koelle et al. [52] cloned T cells out of

recurrent herpes simplex lesions and reacted them against B

cells infected with HSV-1, HSV-2, and recombinants of the two.

In this system, several proteins from the tegument of the virus

have been found to be important targets for type-specific im-

munity (HSV-2 but not HSV-1). The differing results may be

complementary (i.e., HSV-1 and HSV-2 may have been cross-

reactive and type-specific epitopes), but testing of these 2 sys-

tems in vivo is required. Although these studies were conducted

in humans with recurrent herpes simplex, glycoprotein D seems

to be a good candidate for prophylactic vaccines, and here CD4+

T cell responses are still likely to be important [53].

Role of DCs. Human herpes simplex, whether initial or

recurrent, is an epidermal disease. Therefore, the primary DC

likely to be involved in HSV antigen uptake is the Langerhans

cell, and, indeed, recently we have demonstrated HSV structural

antigens within these cells during recurrent herpes simplex (au-

thors’ unpublished data). Involvement of dermal DCs in the

upper region of the dermis has not been excluded. Older and

more recent studies of Langerhans cells and other DCs pre-

senting antigen in lymph nodes initially appear to be contra-

dictory. Earlier studies suggested that depletion of Langerhans

cells from skin, after HSV-1 infection of mice via the footpad,

led to increased HSV virulence [54]. However, 2 recent studies

have demonstrated that Langerhans cells are not the cells pre-

senting HSV antigen to CD8+ T cells in lymph nodes. After

epidermal scarification with the virus, it was observed that CD8+

DCs present HSV antigens to CD8+ T cells in draining lymph

nodes 2 h after infection [55], and after vaginal inoculation of

HSV, it was found that dermal DCs present HSV antigen to

CD4+ T cells [56]. The apparent paradox of initial HSV antigen

uptake by Langerhans cells but presentation by another DC sub-

type may be explained by transfer of the antigens from one DC

subtype to another.

In view of the difficulty in obtaining sufficient numbers of

immature human Langerhans cells, monocyte-derived DCs have

been used as a model. Both immature and mature monocyte-

derived DCs express the HSV receptors nectin-1, nectin-2, and

HVEM and can be infected with HSV-1 and HSV-2, but only

immature monocyte-derived DCs (which more closely resem-

ble the immature sessile Langerhans cells in epidermis) pro-

duce virus at low levels. HSV infection results in asynchro-

nous down-regulation of the key costimulatory molecules CD40,

CD80, CD83, and CD86, preventing proper maturation of the

DCs [10, 57]. Interestingly, MHC class I is not down-regulated

on infected monocyte-derived DCs, unlike most other infected

cells. Similarly, in most other cell types, HSV-1 produces anti-

apoptotic effects, whereas in DCs, both HSV-1 and HSV-2 in-

duce apoptosis progressively throughout the cell sheet over 24

h [57, 58]. However, these apoptotic cells are preferentially

taken up by uninfected bystander cells, and the HSV antigens

contained within these apoptotic cells are then cross-presented

(on MHC class I) to CD8+ T cell clones [58]. This suggests

a mechanism for the antigen transfer between DC subtypes

observed in murine models and suggests that the immuno-

evasive mechanisms of costimulatory molecule down-regu-

lation and apoptosis of DCs by HSV can be counteracted by

uptake by bystander DCs. Preliminary experiments with im-

mature Langerhans cells in vitro show a similar down-regu-

lation of costimulatory molecules, but the other effects need

to be verified in this cell type (authors’ unpublished data).

Whether bystander Langerhans cells or subjacent dermal DCs

respond in human infection in a fashion similar to that in the

murine models remains to be elucidated. In addition, unin-

fected immature monocyte-derived DCs undergo partial mat-

uration after uptake of inactivated HSV. Inactivated HSV is also

a potent stimulator of IFN release by blood plasmacytoid DCs

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and is responsible for the majority of IFN-a released by pe-

ripheral blood mononuclear cells stimulated with HSV antigen,

a long-standing observation (figure 1A) [23]. Whether such

plasmacytoid DCs infiltrate herpetic lesions as they do cuta-

neous psoriasis lesions remains an open question.

Future studies. Longitudinal studies of recurrent genital

herpes and asymptomatic genital shedding of HSV in humans

indicate a decrease in frequency initially over 3 months and

then again over years, suggesting a maturation of the immune

response. The reasons for this are unclear and may reflect an

effect on memory cells or even on maturation of homing re-

sponses. In addition, studies of innate and adaptive immune

responses in initial human HSV infection are difficult and

therefore infrequently undertaken. More work needs to be done

on this topic to compare with the abundant studies in murine

models.

VACCINES AND IMMUNITY

Until recently, a search for a vaccine candidate to prevent genital

herpes had been unsuccessful, partly because of low antigen

concentrations and a focus solely on neutralizing antibodies.

The use of a vaccine candidate that incorporated high concen-

trations of recombinant soluble glycoprotein D, which is widely

recognized in human populations [49], and the adjuvant de-

acylated monophosphoryl lipid A derived from the cell walls

of bacteria showed partial efficacy in the prevention of genital

herpes and a trend toward prevention of infection with HSV-

2. Although these results remain to be confirmed by the on-

going HERPEVAC trial, it is of particular interest that the vac-

cine showed an efficacy of 73%–74% only in women (not men)

and only in those who were seronegative for both HSV-1 and

HSV-2 [53]. The reasons for this sex bias and apparent evidence

of cross-protection of HSV-1 against disease are of considerable

interest.

The success of this vaccine candidate has been attributed to

the role of the adjuvant in inducing Th1 patterns of immune

and cytokine response, especially induction of IFN-g, in both

guinea pig models and human phase 1 trials [59] as well as

during the trial itself (L. R. Stanberry, A. L. Cunningham, S. L.

Spruance, M. Denis, G. Dubin, and D. I. Bernstein, unpublished

data). No induction of T cell cytotoxicity was demonstrated.

Neutralizing antibody was induced, but not to the very high

levels induced by another vaccine candidate (from Chiron), con-

taining HSV-2 glycoprotein D and glycoprotein B with MF59

adjuvant, in clinical trials [60] (L. R. Stanberry, A. L. Cunning-

ham, S. L. Spruance, M. Denis, G. Dubin, and D. I. Bernstein,

unpublished data). The sex bias in the immune response may

be due to local effects of this Th1 response in enhancing genital

mucosal T cell responses, which therefore enhances resistance of

the female genital mucosa so that it is more similar to that of

male genital mucosa. The latter has greater intrinsic resistance

because of the presence of a thick intact stratum corneum in

penile skin (with the exception of the prepuce). However, women

also display greater systemic Th1 responses than do men, which

could partly explain the sex difference. Recently, murine studies

showing gender differences in immune responses to HSV have

been published [61].

The trial results with the GlaxoSmithKline candidate vaccine

Simplirix seem to demonstrate several key principles for a vac-

cine against HSV. First, it is possible to obtain substantial pro-

tection against disease with a single recombinant viral protein

combined with an adjuvant that induces the correct form of

immune response. If these correlates of protective immunity

can be confirmed as CD4+ T cell Th1 responses (especially pro-

ducing IFN-g) in the current HERPEVAC trial, they will be

helpful in the design of surrogate markers for evaluating other

vaccine candidates, such as DNA vaccines and replication-de-

fective mutants [62]. Nevertheless, the current vaccine can-

didate eventually needs to be improved to achieve 195% effi-

cacy in both males and females.

For example, the design of future vaccine candidates must

also be aimed at inducing innate immune responses and at

controlling viral infection at the level of both the genital mucosa

and the DRG. The former is probably more important to the

prevention or reduction of the viral inoculum entering cuta-

neous sensory nerve endings and, thence, the DRG, thus re-

ducing the number of latently infected DRG neurons, which

determines subsequent reactivation rates and the frequency of

recurrent herpes. Failure to stimulate innate immunity by the

current vaccine candidate might be partly responsible for the

discrepancy between prevention of genital herpes and genital

HSV-2 infection; that is, stimulation of the adaptive immune

response may reduce levels of virus in the DRG and subsequent

disease but may not prevent symptomatic infection and shed-

ding. Alternatively, induction of CD8+ T cell immunity and/or

neutralizing antibody with different HSV-2 antigens and ad-

juvants may synergize with the induced CD4+ T cell Th1 re-

sponse, as in natural infection [39, 40, 42]. Mucosal immu-

nization is also a worthwhile strategy and could be aimed at

stimulating adaptive and/or innate immune mechanisms after

oral or nasal delivery [63]. Finally, the effects of all such vaccine

candidates on asymptomatic genital shedding and subsequent

transmission are of great importance epidemiologically.

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