scripts, see ref. 10). Shown in (C) are the punctate nuclear hybridization signals
(arrows) observed with a riboprobe to the 5â€™ leader sequence of the minor LAT. Hy-
bridization with an IE l-specific riboprobe is shown in (D) neuronal cultures during
the latent infection and (E) neuronal cultures during reactivation (12 h after NGF dep-
rivation). The scale bar in (A) is 50 mm and applies to all panels.
1. Wilcox, C. L. and Johnson, E. M. (1987) Nerve growth factor deprivation results
in the reactivation of latent herpes simplex virus in vitro. J. Viral. 61,23 1 l-23 15.
2. Wilcox, C. L. and Johnson, E. M., Jr. (1988) Characterization of nerve growth
factor-dependent herpes simplex virus latency in neurons in vitro. J. Virol. 62,
3. Wilcox, C. L., Smith, R. L., Freed, C., and Johnson, E. M. (1990) Nerve growth
factor-dependence of herpes simplex latency in peripheral sympathetic and sen-
sory neurons in vitro. J. Neurosci. 10, 1268-1275.
4. Smith, R. L., Escudero, J. L., and Wilcox, C. L. (1994) Regulation of the herpes
simplex virus latency-associated transcript during establishment of latency in sen-
sory neurons in vitro. Virology 202,4!%60.
5. Doerig, C., Pizer, L. I., and Wilcox, C. L. (1991) Detection of the latency-associ-
ated transcript on neuronal cultures during the latent infection with herpes sim-
plex virus type 1. Virology 63,423-426.
6. Smith, R. L., Pizer, L. I., Johnson, E. M., Jr., and Wilcox, C. L. (1992) Activation
Wilcox and Smith
of second-messenger pathways reacttvates latent herpes simplex virus m neuronal
cultures. Yzrology 188,3 1 l-3 18.
7 Baringer, J R. (1974) Recovery of herpes simplex vu-us from human sacral gan-
glions N Engl. J Med 291,828-830
8 Smith, R. L. and Wilcox, C. L. Gene transfer into neurons towards gene therapy
of neurologzcal diseases (Lowenstem, P and Enquist, L., eds.), Wiley, Sussex,
UK, m press
9 Sambrock, J , Frttch, E. F , and Maniatts, T (1989) Molecular Clomng* A Labo-
ratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)
10 Fraser, N I , Block, T M , and Sptvack, J G. (1992) The latency-associated tran-
scripts of herpes simplex virus: RNA in search of a function. Vzrology 191, l-8
11 Arthur, J., Efstathtou, S , and Simmons, A. (1993) Intranuclear foci contammg
low abundance herpes simplex virus latency-associated transcrtpts visualized by
non-tsotoptc zn satu hybrtdtzattons. J. Gen. Vtrol , 74, 1363-l 370.
Assessing Cell-Mediated Immune Responses to
HSV in Murine Systems
Theresa A. Banks, Mangala J. Hariharan, and Barry T. Rouse
Protective immumty against a majority of viral Infections is mediated by a
combination of both humoral and cell-mediated immune responses. However,
m the case of herpesvirus infections, where viral spread is largely cell-to-cell,
cell-mediated immune mechanisms (which facilitate the clearance of virally
infected cells) are parttcularly important (Z-4). Moreover, cell-mediated
immunity (CMI) has also been implicated m the establishment and/or reactiva-
tion of latent herpes simplex virus (HSV) infection (5,6). Thus, a major focus
of herpesvirus immunology continues to be the identification of those herpes-
virus antigens that serve as targets for CM1 and the means by which protective
responses can be optimally induced. Clearly this information is critical for the
rational development of effective vaccine strategies.
In the first sections of this chapter, we detail various in vitro assays com-
monly used to measure HSV-specific CMI. In later sections, we describe meth-
ods that allow for the identification of the viral proteins and the mmimal
epitopes within these proteins that serve as targets for CMI. Lastly, we discuss
a murine challenge model that allows herpesvirus antigens to be tested m viva
for then ability to confer protection from lethal doses of HSV. It should be
noted that m this chapter HSV refers specifically to HSV-1.
1.1. Cytotoxic T-Cell (CTL) Assay
A major player m the cell-mediated immune response against herpesvirus
infections is the cytotoxtc T-cell (CTL). In general, virus-specific CTL are CDF
and recognize small pepttdes derived from viral proteins that are presented by
class I major histocompattbhty complex (MHC) molecules on the surface of
From Methods m Molecular Medtnne, Vol 10 Herpes S/mp/ex L&us Protocols
Edlted by S M Brown and A A MacLean Humana Press Inc , Totowa, NJ
Banks, Hariharan, and Rouse
virally infected cells (7). Thus, the primary function of CTL is to first recognize
and then lyse cells m an antigen-specific manner. Moreover, cell lysis is not the
only way in which CTLs mediate their antiviral effects. CTLs also appear to
stimulate mtracellular enzymes that can degrade viral genomes, and CTLs can
induce the secretion of cytokines with apparent interferon activity (8). To study
CTLs in vitro, CTLs are mixed with 51Cr-labeled target cells which express the
appropriate viral antigens. Lysis of the target cells releases 5â€™Cr and serves to
indicate the activity of CTL against these target antigens. To induce herpesvirus-
specific CTL in VIVO,murme systemsexist m which mice can be mnnumzed
with human strains of HSV. Alternattvely a vartety of viral and nonvnal systems
m which individual HSV antigens are expressed (e.g., recombinant vaccima
viruses, recombinant adenovnuses, recombinant retrovnuses, and plasmid DNA)
can also be used to m-tmumzemice m order to generate CTL (948). However,
for the purposes of this chapter, the primary focus will be the use of HSV and
recombinant vaccima vn-uses expressing HSV antigens as tools to study viral-
specific cell-mediated immune responses.
1.2. Limiting Dilution Analysis
to Determine CTL Precursor Frequency
Although the standardCTL assayas describedm the prevtous sectiondoes detect
the presence of antigen-specific CTLs, tt does not allow for then quantttatton.
For this reason, it is not possible to directly compare on a per-cell basis the
CTL responses generated from different effector populations. In contrast,
limiting dilution analysis (LDA) allows one to quantitate the CTL present m
a given effector cell population. LDA is a retrospective process based on the
measurement of the number of activated CTL clones generated from a given
population of ceils. Since each of these clones is originally derived from an
mdividual precursor clone, one can retrospectively determine the frequency
of precursors that led to the generatton of the active clones, Thus, LDA lets
you determine how many CTLs in a given population have participated in
response to a particular antigen, and allows the number of such precursors to
be measured at the clonal level.
In particular, LDA has proven to be invaluable for assessing immune
responses to various immunization protocols, For example, LDA can be
used to make decisions regarding the most effective vector delivery sys-
tems (e.g., recombmant vaccmla virus vs recombinant adenovnuses), as
well as doses of tmmunogen, frequency of admmtstratton, and routes of
immunization. It should also be noted that LDA can be used to determine
precursor frequencies for B cells and for helper T cells as well (21). How-
ever, m this section we have confined our dtscussion to LDA of CTL di-
rected against HSV (22,231.
Cell-Mediated Immune Responses to HSV 329
1.3. T-Helper Cell-Proliferation Assay
Helper T cells (Th) are an important group of cells that not only provide
the help needed for B cells to mount antigen-specific humoral immune
responses, but they also provide help for the differentiation and maturation
of CTL precursors mto effector CTL. Th cells are generally of the CD4+
phenotype and are MHC class II restricted. Two distinct subsets of Th cells,
Th, and Th2, have been well documented and characterized in both the human
and murine systems (2.5-29). This separation mto either Th, or Th, subsets is
based on their distinct cytokine secretion patterns. In most cases, Thl cells
secrete interferon-y (IFN-y), tumor necrosis factor (TNF-P), interleukin-2
(IL-2), IL-3, and granulocyte-macrophage colony-sttmulating factor (GM-
CSF), whereas Thz cells express IL-3, IL-4, IL-5, IL-6, IL-g, IL- 10, and GM-
CSF. Then ability to express different cytokine profiles enables Th, and Thz
cells to mediate different effector functions. For example, Th, cells appear
most effective in mediating antiviral and antitumor immune responses,
whereas Thz cells predominate in the immune clearance of extra cellular
pathogens (e.g., parasites and bacteria).
Since Th, responses appear critical for the mduction of effective antiviral
immune responses, one important goal of herpesvirus mnnunology is to iden-
tify herpesvirus antigens capable of inducing Thi cells. By the same token, it
may be equally important to identify viral antigens that induce Thz cells, since
the inclusion of such antigens could impact negatively on the ability of an
immunogen to induce protective immune responses.
When antigen-specific Th cells are stimulated with antigen in the presence of
the appropriate antigen-presenting cells, cellular proliferation and cytokine secre-
tion is induced. Thus T-helper responsescan be determined by measuring antigen-
specific cellular proliferation and/or cytokine production. Specifically, cellular
proliferation can be quantitated by measuring the amount of tritiated thymidine
incorporated into dividing cells, whereascytokme levels can be measuredby using
bioassaysor enzyme-basedimmunoassays(ELISA, ELISPOT). Typically, detec-
tion of IL-2, IFN-y and TNF-P is regarded as indicative of a Thi-type response,
whereas detection of IL-4 and IL-6 indicates a Th,-type response. What follows
are the details needed to assayfor HSV-specific lymphoproliferatton. These meth-
ods have been adapted from a protocol originally described by Horohov et al. (30).
1.4. Mapping CTL Epitopes
A variety of methods can be used to identify the mmimal antigemc regions
(epitopes) within a viral protein that serve as targets for CMI. In this section we
will focus on mapping CTL epitopes, but the same approaches are applicable
to mapping Th epitopes as well.
Banks, Hariharan, and Rouse
Once a viral protein has been Identified as a CTL target, several options can
be pursued. One option is to scan the amino acid sequence of the protein for the
presence of allele-specific pepttde-bmdmg motifs (31,32). These motifs, which
allow one to identify putative CTL epitopes, reflect the actual sequence of natu-
rally processed peptides eluted from MHC class-1molecules. For example, pep-
tides eluted from class-I molecules of the H-2Kd haplotype are invariably 9-l 0
ammo acids m length, wtth a tyrosme always at ammo acid posmon 2 and one of
five favored, predommantly hydrophobic ammo acids at positton 9 Thus, an
appropriate protem sequencecan be most easily scanned by identifying the loca-
tions of all tyrosme restdues and fixing this as position 2 of a putative epttope 9
ammo acids m length. Peptides representing these regions can then be synthe-
sized and tested for their ability to sensitize appropriate targets for CTL lysis.
An alternative strategy is to synthesizea series of overlappmg peptides that
span the entire length of the protem of mterest. One recommended strategy for
CTL epitope screening is to synthesizeoverlapmg peptides that are 15 ammo acids
in length (15 mer) with 10-mer overlaps This ensuresthat all putative CTL epitopes
of < 10 ammo acids will be represented.Although this approach ISextremely rigor-
ous, it can be costly depending on the sizeof the protein For example, to screen a
protein of 512 amino acids m length usmg 15 mers with a lo-mer overlap, 86
peptides need to be synthesized! However, once positive peptides are identified,
the mmrmal CTL epttope can be quickly defined by sequentially deletmg ammo
acids from both the amino and carboxyl ends of the pepttde.
The identtfication of CTL epitopes in herpesvirus protems continues to be
an important goal for several reasons. From a research perspective, peptides
representing CTL epitopes remain a powerful tool. In particular, pepttdes can
be used to restimulate CTL cloned lines in vitro, thus ehmmatmg the need to
contmually set up bulk effecters from the spleens or lymph nodes of immu-
nized mice From a therapeutic standpoint, CTL peptides can be used alone or
in conjunctton wtth Th and neutralizing antibody epttopes to test the efficacy
of subunit vaccine strategies for the mductton of protective immune responses.
1.5. Murine Challenge Model
Several animal models exist for assessmgthe protective m-ununeresponses
induced by herpesvirus antigens (36-38). In particular, the mouse has proven
useful smce many different immunization protocols can be tested, allowmg
one to evaluate a wide variety of munune responses (38,39). In this section we
will detail a murine challenge model that is easy to perform and can be adapted
for use with other viruses besides HSV. However, we encourage the reader to
explore the use of other models as well. For example, the zosteriform model
(39) may be used to mimic recurrent infection and thus allows one to test the
ability of immun˜zatton to protect against peripheral neurologtc spread of HSV
Cell-Mediated Immune Responses to HSV
2.1. Cyfotoxic T-cell (CTL) Assay
1. Inbred mouse strams (ideally 5-9 wk old) to immumze with HSV Three com-
monly used strains of mice with different MHC class I haplotypes are C3H/HeN
(H-2k), C57BL/6 (H-2b), and BALB/c (H-2d).
2. Needles (18,26, and 30 gage) and syringes (1 and 3 mL) for mouse nnmunizattons
3. Sterile dissection mstruments to remove spleens and lymph nodes from tmmu-
mzed animals* scissors, forceps, and scalpels Sterile wtre-mesh screens to pre-
pare single-cell suspenstons of spleens and lymph node cells.
4 MHC class I-compatible permanent cell lines to serve as targets in the CTL assay
LTA-cells (H-2k), C57SV40 (H-2b), and EMT6 (H-2d) (9)
5. Complete culture medium for growing permanenT-cell lines. DMEM supple-
mented with (final concentrations shown): 10% heat-inactivated fetal calf serum
(FCS), 2 mM L-glutamine, 100 IU/mL pemcillm, 100 pg/mL streptomycin
6 CTL medium for lymphocyte cultures. RPMI- 1640 medium contammg 10% heat-
mactivated FCS, 10 mA4 L-glutamine, 100 IU/pemcillm, 100 pg/mL streptomy-
cin, and 5 x lOPsA 2-mercaptoethanol
7. Tissue-culture grade trypsin/EDTA solution (0.5 g trypsm and 0 2 g EDTA/L)
used to detach adherent cell lines for routme passage
8. EDTA (0.02%) solutton to detach cell lines used as targets m CTL assay.
9 Hemocytometer and trypan blue solution to determine live cell counts.
10 Multichannel (12) pipettor (50-200 pL) and an adjustable ptpettor (50-200 pL).
11 Sterile plasttcware. 15- and 50-mL polypropylene tubes, various sizes of vented
tissue culture flasks, 96-well round-bottom tissue-culture plates, 6-well plates,
tips for ptpettors (200 and 1000 IJL capacity), 5- and IO-mL pipets, and Petri
dishes (6 and/or 10 cm)
12. Ultraviolet (UV) light sauce (e.g., germicidal lamp)
15. Table-top centrifuge with carrters that hold tubes as well as mtcrottter plates.
16. 37Â°C Incubator with humtdified atmosphere of 5% C02/95% an.
17 37Â°C Water bath
2.2. Limiting Dilution Analysis
to Determine CTL Precursor Frequency
1. Mice immunized with HSV or with other vector systems as decrtbed prevtously.
2. Spleens from naive, ummmumzed, syngenetc mice to serve as feeder cells during
in vitro culture
3. Same materials as required for CTL assays.
4. CTL medium as described previously is also used for LDA, but it is addtttonally
supplemented with the following (final concentrations) 5% concanavalm A-
derived T-cell growth factor (Collaborative Research, Bedford, MA), 50 mM cx-
methyl mannostde, human recombinant interleukm-2 (5 U/well of 96-well plate,
Banks, Hariharan, and Rouse
Collaborative Research), 1 mA4 oxaloacetic acid, 0 2 U/mL bovine insulin, and 5
x 1tY5A4of 2-mercaptoethanol
5 Computer program(s) for data analysis based on formulas provided by Taswell(24).
2.3. T-Helper Cell-Proliferation Assay
1 Mice unmunized with HSV or recombmant vaccmia viruses expressing HSV pro-
teins as described previously
2 Spleens from naive, unimunized mice to serve as stimulator cells. These mice
must be the same stram as the immunized mice (1 e , same haplotype)
4 Concanavalm A.
5 RPMI-1640 medmm supplemented with 10% heat-inactivated FBS, 25 mM
HEPES buffer, 5 x 10-5M2-mercaptoethanol, 100 U/mL pemcillm, and 100 pgi
6 96-well U-bottom plates
7. Cell harvester and glass fiber filter strips.
8 Liquid scintillation counter, scintillation fluid, and scmttllation vials
2.4. Mapping CTL Epitopes
1 Mice immumzed with HSV or other vectors systems from which HSV antigens
2 Approprrate target cells (MHC class I compatible) to which peptides are
added (target sensitization) Positive controls set up m parallel should m-
elude target cells infected with HSV or with recombmant vaccima viruses
expressing HSV antigens This ensures that the effector cells are capable of