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assay After 12-48 h of incubation (depending on the particular cytokme of mter-
est), supernatant fluids can be harvested and frozen at -20°C until ready to be
assayed for the presence of cytokines Although bloassays for some of the
cytokines exist, ELBA kits or palred antibodies are avallable commercially for
the detection of a wide variety of cytokmes
3.4. Mapping CTL Epitopes
1. Ideally lyophihzed peptides can be reconstituted and diluted appropriately m the
same media used for the CTL assay. However, this is sometimes not possible
since some peptldes are insoluble m these types of solutions In these cases a
stock solution of peptlde can be made m dlmethylsulfoxide (DMSO) and then
diluted with CTL media
2. Regardless of the solvent used, it 1sadvisable to prepare a peptlde stock solution
(e g ,2 mM works well) from which ddutlons can be made To prepare a 2-n-&J
stock solution of peptlde, one needs to calculate the molecular weight of the pep-
Cell-Mediated Immune Responses to HSV 339
tide. This is done by adding up the molecular weights of each of the mdtvidual
ammo acids from which the pepttde is comprised. Next, divide the actual amount
(m mg) of peptide synthesized by the molecular weight of the peptide to deter-
mine the number of moles of pepttde present For example, if the molecular
weight of a peptide with an ammo acid sequence of GGHQAAMQL is 1204, and
the actual amount ofpeptide synthesized is 2 mg, then 2 mg/l204 equals 1 66 p˜!4
of peptide present in a total weight of 2 mg. Thus, to prepare a 2-W stock solu-
tion of this pepttde, reconstitute the 2 mg of pepttde m 830 uL solvent.
3. To sensitize target cells with pepttde, 8-l 0 pL of pepttde (2 mM stock) is added to
l-3 x lo6 target cells m a total of l-2 mL of media also contaming 5™Cr (200 pet)
After 1 h mcubatton at 37°C the target cells are washed three to four tunes in CTL
medmm, and used to set up a standard CTL assay as descrtbed previously. It IS
important to wash away excess peptide from the target cells before addmg them to
the effector cells This prevents excess peptide present in the medium from bmdmg
to the effector cells themselves and resulting in effector cell.effector cell lysis (33)
4 The same steps detailed above can be used to identify T-helper epttopes. How-
ever, instead of CTL assays, proliferation and/or cytoktne assays are used to
detect antigen-specific proliferation and cytokme production

3.5. Murine Challenge Model
1 Before starting the actual experiments, pilot studies need to be performed to de-
termme the mmtmal dose of McKrae virus needed to kill the different strams of
mice This dose ˜111 vary between strains. For example, C3H/HeN mace are the
most susceptible to McKrae, followed by Balb/c, and then C57/BL6 mice, which
are the most resistant of these three strains
2 Once the dose of McKrae vnus has been established for each strain, the follow-
ing groups of mice (lo/group) should be injected as follows: group 1 represents
the experimental group to be injected with the test immunogen. For example, we
have tested the ability of a recombinant vaccmta vtrus expressing an herpes anti-
gen m this model by injecting both rear footpads with 1 x 10™ PFU of recombt-
nant vaccinia vnus Of course, other sites and other vector delivery systems can
be tested as well Group 2 1s a specificity control group. For example, if group 1
is injected with the recombinant vaccmia expressing an herpes antigen, then group
2 should be injected with a recombinant vaccmia virus expressing an irrelevant
(non-HSV) anttgen Group 3 animals represent the negative control group and
should be injected with PBS, whereas, m group 4 (postttve controls), the ammals
are inJected with HSV- 1 KOS strain.
3 Immunized mice are rested for approx 2 wk before challenge with the McKrae
vu-us Depending on the tmmunogen being tested, animals may be boosted once
or twice before challenge. Mice are challenged with an approprtate dose of
McKrae virus administered tp and then observed for 2 wk for signs of morbrdtty
and mortality Typically, all mice in group 4 survive, whereas all mice m groups
2 and 3 dte by approx d 10-12. The number of mace surviving m group 1 mdt-
cates the level of protectton afforded by the unmunogen.
Banks, Hariharan, and Rouse
340
4. Notes
1. It 1s not neccesary to remove red blood cells (RBC) when setting up splenocyte
cultures. However&he absence of RBCs will make the cultures appear cleaner
and easier to visualize under the microscope during then 5-d incubation period.
Remove RBC as follows, pellet single-cell splenocyte suspension as described m
methods, but do not resuspend the pellet directly m CTL media Instead, resus-
pend cell pellet m a few miltliters of Trts-buffered ammonmm chloride (Tris-
NH&l) solution and mcubate for 2 min at 37°C Then add 10 mL of media
containing FCS to neutralize the activity of the ammonium chloride, centrifuge,
and resuspend cell pellet m CTL media for resttmulation with UV-irradiated
vnus. Note that lymph nodes do not contam RBC thus precluding the need for
Tris-NH&l treatment Tris-buffered ammonmm chlortde (Tris-NH&l) solutron
is prepared by first makmg stock soluttons of 0 16MNHJl (stock A) and 0 17M
Trts, pH 7 65 (stock B). These stocks are then mixed together to make the work-
ing solution (90 mL stock A and 10 mL stock B) The final pH of the solution is
adjusted to 7.2 with HCl (20)
2. Novice CTLers may find that then S™Cr-labeled targets demonstate high levels of
spontaneous lysis. This high background level is often the result of washmg the
labeled targets too vigorously Care must be taken to avoid this type of nonspe-
cific lysis of target cells. Furthermore, during washing, the target cells should be
gently but thoroughly resuspended in order to prevent cell clumping, a condttton
that will also adversely affect CTL-specific lysis
3 The success of LDA is pat-ocularly dependent on the quality of the supplements
added to the culture medium. For example, it IS critical that several lots of fetal
calf serum be tested m order to select the lot that correlates with the best assay
results. It IS also important to try different combmations of Con A-dertved T-cell
growth factor and recombmant IL-2. It also appears that the presence of 2-
mercaptoethanol m the medium is critical for the proper outgrowth of CTL.
4. Proliferation assays performed on splemc cultures often demonstrate high back-
ground counts (i e , substantial mcorporation of 3H-thymidme by negative con-
trol cultures). One solutton IS to set up proliferation assays using lymph-node
cultures, since, m many cases, these cultures do not exhibtt high background
counts However, if problems with high backgrounds persist, then the amount of
FCS m the culture medium should be reduced to levels below 5%.
5 Although the general gutdehnes given above should work for most peptides, a cer-
tam amount of trial and error is often necessary m order to find the optimal peptide
concentratton needed to sensitize targets for CTL lysts It is also recommended that
pepttde assays be performed m medium contammg reduced levels of FCS (2-5%
final). This reduces the potential for protease-mediated peptide breakdown.
6. When one is faced with a large number of pepttdes to screen, one can combme as
many as four pepttdes together before adding thts pool to target cells. Pepttdes
from positive pools can then be tested individually
7. In general, tt is advisable for screening purposes to synthesize peptides contain-
mg unblocked ammo and carboxyl ends. This holds trne for both CTL and Th
Cell-Mediated Immune Responses to HSV 341

epttope mapping. However, for Th epitope mapping, peptides wtth blocked ends
may be more effectrve especially when pepttdes less than 15 ammo actds are
used (34,3.5).

References
1. Nash, A A., Leung, K -N., and Wiley, P (1985) The T-cell mediated immune
response of mice to herpes simplex wus, in The Herpes Simplex Vzrus, vol. 4
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2. Martin, S and Rouse, B T (1990) The control of human herpesvuus infections
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MA, pp.73-98
3. Martin, S , Cantm, E., and Rouse, B. T. (1988) Cytotoxic T lymphocytes-their
relevance in herpesvirus infections Ann NY Acad Scz 532,257-272.
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induction and vaccmatlon. Rev Znfect Dzs 10, 16-33
5. Bonneau, R. H. and Jennings, S. R. (1989) Modulatton of acute and latent herpes
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6 Stevens, J G (1989) Human herpesviruses* a consideration of the latent state
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9 Banks, T A., Allen, E. A., Dasgupta, S., Sandra-Goldm, R , and Rouse, B T.
(1991) Herpes simplex vu-us type l-specific cytotoxic T lymphocytes recognize
mimedlate-early protein ICP27. J Vlrol. 65,3 185-3 19 1.
10. Banks, T. A., Natr, S , and Rouse, B. T (1993) Recognition by and m vitro mduc-
tion of cytotoxic T lymphocytes against predicted epitopes of the immediate-early
protein ICP27 of herpes simplex vu-us J Vwol 67,6 134 16.
11. Banks, T. A., Jenkins, F. J., Kanangat, S., Nair, S., Dasgupta, S., Foster, C. M.,
and Rouse, B T (1994) Vaccination with the immediate-early protein ICP47 of
herpes simplex virus-type 1 (HSV-1) induces virus-specific lymphoproliferation,
but fails to protect against lethal challenge. Vzrologv 200,236-245
12 Martin, S , Cantm, E., and Rouse, B. T. (1989) Evaluation of antiviral unmun˜ty
using vaccima virus recombinants expressing cloned genes for herpes simplex
virus type 1 glycoprotems. J Gen Vu-01 70, 1359-1370
13. Hanke, T., Graham, F L , Rosenthal, K. L., and Johnson, D. C. (199 1) Identifica-
tion of an immunodommant cytotoxic T-lymphocyte recognition site m glycopro-
tem B of herpes simplex virus by using recombinant adenovnus vectors and
synthetic peptides. J. Vwol. 65, 1177-l 186.
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Johnson, D C (1990) Cytotoxtc T lymphocytes specific for herpes stmplex virus
(HSV) studied using adenovirus vectors expressing HSV glycoprotems J Gen
Vu-01 71,387-396
15 Johnson, D. C., Ghosh-Choudhury, G., Smdey, J. R , Fallis, L , and Graham, F L
(1988) Abundant expresston of herpes simplex virus glycoprotem gB using an
adenovirus vector. Vzrology 164, l-14.
16 McDermott, M R., Graham, F. L , Hanke, T , and Johnson, D C (1989) Protectton
of mice agamst lethal challenge with herpes simplex virus by vaccmatton with an
adenovnus vector expressing HSV glycoprotem B Vzrology 169,244247
17 Irwin, M J., Laube, L S , Lee, V , Austin, M , Chada, S., Anderson, C G ,
Townsend, K , Jolly, D J , and Warner, J. F (1994) Direct inJectton of a recombi-
nant retrovtral vector induces human tmmunodefictency virus-specific nnmune
responses m mace and nonhuman prtmates J Vzrol 68,5036-5044.
18 Ulmer, J B , Donnelly, J. J., Parker, S. E., Rhodes, G H., Felgner, P. L., Dwarkr,
V. J., Gromkowskt, S. H., Deck, R. R., DeWttt, C. M., Friedman, A., Hawe, L. A.,
Leander, K R , Martmez, D , Perry, H C , Shiver, J W , Montgomery, D L , and
Liu, M. A. (1993) Heterologous protection against influenza by injection of DNA
encoding a vtral protein Sczence 259, 1745-l 749.
19. Tang, D. C., Devrt, M., and Johnson, S A. (1992) Genettc tmmunisatton is a
simple method for eliciting an immune response Nature 356, 152-l 54
Mtshell, B. B , Shngl, S M , Henry, C , Chan, E L , North, J , Galhly, R., Slomtch,
20
M., Miller, K., Marbrook, J , Parks, D , and Good, A H (1980) Preparatron of
mouse cell suspenstons, m SelectedMethods In Cellular Immunology (Mtshell, B
B. and Shiigt, S. M., eds ), Freeman, New York, p 23
Sherman, L A , Vittello, A, and Khnman, N R (1983) T cell and B cell re-
21
sponses to viral antigens at the clonal level. Annu Rev. Immunol 1,63-86.
Rouse, B T , Larsen, H S., and Wagner, H. (1983) Frequencey of cytotoxtc T-
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lymphocyte precursors to herpes simplex vnus type 1 as determined by hmitmg
dtlutton analysts. Infect Immun 39,785-792.
Rouse, B. T. and Wagner, H. (1984) Frequency of herpes simplex virus-specific
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cytotoxic T lymphocytes precursors in lymph node cells of infected mice. Zmmu-
nology 51, 57-64
24. Taswell, C. (1981) Ltmrting dilutton assays for the determmation of
immunocompetenT-cell frequenctes 1. Data analysis. J Zmmunol 126,161&1620.
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25
(1986) Two types of murme helper T-cell clones I Definition according to profiles
of lymphokme acttvttres, and secreted proteins J Immunol 136,2348-2357
Mosmann, T R and Coffman, R L (1989) Th 1 and Th2 cells Dtfferent patterns
26
of lymphokine secretion lead to different functtonal properttes Annu Rev
Immunol. 7, 145.
Romagnam, S (1991) Human Thl and Th2 subsets: doubts no more. Immunol
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Today 12,256.
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Cell-Mediated immune Responses to HSV
29. Mosmann, T. R. and Coffman, R. L. (1989) Heterogeneity and cytokme secretion
patterns and functions of helper T-cells. Adv Immunol 46, 11 l-147
30 Horohov, D W., Moore, R N , and Rouse, B. T (1985) Herpes simplex vnus-
specific lymphoprohferatron: an analysrs of the mvolement of lymphocyte sub-
sets. Immunobiol 170,460-473
3 1. Falk, K , Rotzschke, O., Stevanovic, S., Jung, G , and Rammensee, H G (1991)
Allele specific motifs revealed by sequencing of self peptides eluted from MHC
molecules Nature 351,290-296
32 Elliot, T., Smith, M., Dnscoll, P., and McMichael, A. (1993) Peptide selection by
class I molecules of the maJor histocompatibility complex. Current Bzol. 3,854-866
33. Burrows, S. R., Suhrbier, A., Khanna, R., and Moss, D. J. (1992) Raprd visual
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cell klllmg. Immunology 76, 174,175
34. Chen, W , McCluskey, J., Rodda, S., and Carbone, F. R. (1993) Changes at pep-
tide residues burred m the maJor htstocompatibility complex (MHC) class I bmd-
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35. Bednarek, M. A., Sauma, S Y., Gammon, M. C , Porter, G., Tandrankar, S ,
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the mflueza virus matrrx protein. extra and intra cellular loadmg of HLA-A2 J
Immunol. 147,4047-4053
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studying latent and recurrent herpes simplex infection. Infect Immun 12, 162-l 65
37 Meyers-Elliot, R. H and Chitjan, P (1981) Immunopathogenesis of cornea1
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23
Assays for HSV Gene Expression
During Establishment and Maintenance
of Latent Infection
Peter Speck and Stacey Efstathiou


1. Introduction
Assays in use for the analysis of herpes simplex virus (HSV) gene expres-
sion during the establishment and maintenance phases of infection m the ner-
vous system include:
1 The use of reporter genes, for example, the 1acZ gene from Eschenchza ˜011,
which is inserted by homologous recombmatton mto the vu-al genome, and which
may be driven either by viral promoters or by an exogenous promoter, such as the
major immediate early (IE) promoter of cytomegalovnus. In our hands, the
detection of ZacZ activity m neuronal tissue infected with recombinant HSV con-
structs has proven to be a simple and effective means of monitormg viral activity
in the peripheral nervous system.
2. Analysis of virally encoded RNA transcripts, either by zn sztu hybrtdtzatton (ISH)
using radioactive or nonradioactive indicator molecules, or by Northern analysis
(this technique is described m Chapters 13 and 24)
3. Immunohistochemistry to demonstrate the presence of viral proteins, which tech-
nique can also be used m combination with ISH (dual labeling)

2. Methods
2.1. Use of the Reporter Gene IacZ
This bacterial gene encodes the enzyme P-galactostdase, a substrate for
which is 5-bromo-4-chloro-3-indolyl-/3-n-galactopyranoside (X-gal, NovaBio-
them, Nottmgham, UK), which is cleaved by the enzyme to yield an msoluble
blue product. This assay for j3-galactosidase m infected animal tissue IS an
adaptation of a standard method (2) for the detection m cell monolayers of
From Methods m Molecular MedIcme, Vol 10 Herpes 8mplex Wrus Protocols

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