Effect of imiquimod as an adjuvant for immunotherapy of genital HSV in guinea-pigs

~ " 1 ~ T T I E IR W Q IR T H Ir~E I N E M A N N

Vaccine, Vol. 13, No. 1, pp. 72-76, 1995 Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0264-410)(/95 $10.00 + 0.00

Effect of imiquimod as an adjuvant for immunotherapy of genital HSV in gmnea-plgs Q

•

David I. Bernstein *~, Christopher J. Harrison*, Eric R. Tepe*, Ann Shahwan* and Richard L. Miller ~ Imiquimod, an immunomodulator which upregulates cell-mediated immune responses, was evaluated as an adjuvant for immunotherapy of recurrent genital herpes simplex virus (HSV) infection in guinea-pios. In two experiments at separate research centres, animals were immunized with H S V glycoprotein and either placebo, 1 or 5 days of imiquimod, or complete Freund's adjuvant, 14 and 35 days after genital HSV-2 infection. Recurrent lesion days were then evaluated from days 15-91. In both experiments, immunization with glycoprotein and imiquimod most effectively reduced recurrence compared with unimmunized controls (53-69%, p
Herpes simplex virus; immunotherapy; vaccine; adjuvant; recurrent HSV disease

Recurrent genital herpes simplex virus (HSV) infections are common, but, except for the daily administration of the antiviral acyclovir, there is no therapy available to decrease the recurrent attacks. Another strategy that has been used in animal models to reduce recurrence is to immunize animals with latent HSV infections to boost specific immune responses and thereby decrease recurrence 1-4. In previous trials, using the guinea-pig model of genital HSV-2, we have reported decreases in recurrent HSV disease of 5-47% after immunization with HSV glycoprotein and various adjuvants 1'2"5. This appears to be related to the boost in cell-mediated rather than humoral immune responses 5. • Imiquimod is an immunomodulator which upregulates selected cell-mediated immune responses presumably by inducing cytokines, especially alpha-interferon 6'7. When given as therapy for primary genital HSV-2 in the guinea-pig model, imiquimod 6-a appears to decrease the acute disease and vaginal viral shedding more effectively than acyclovir9-~2. Further, unlike acyclovir9, early treatment of the acute disease with imiquimod reduces subsequent recurrence 6'7. Imiquimod also increases the *The J.N. Gamble Institute of Medical Research, 2141 Auburn Avenue, Cincinnati, OH 45219, USA. *Creighton University and University of Nebraska Medical Center, Omaha, NE 68131, USA. t3M Pharmaceuticals, 3M Company, St Paul, MN 55144, USA. °°To whom correspondence should be addressed. (Received 15 February 1994; revised 18 May 1994; accepted 18 May 1994)

72 Vaccine 1995 Volume 13 Number 1

effectiveness of HSV glycoprotein immunization when it is given prior to challenge with HSV-213. Again, a significant decrease in subsequent recurrent disease was observed with the combination of glycoprotein immunization and imiquimod compared with glycoprotein immunization alone. Because the effectiveness of immunotfierapy appears to be at ]east partly related to the adjuvant used for immunization 2'4'14 we investigated the potential of using imiquimod as the adjuvant for glycoprotein immunotherapy of recurrent HSV-2 disease in the guinea-pig model. METHODS

HSV-2 glyeoprotein preparation The HSV-2 glycoprotein preparation was prepared using previously described methods 13. Briefly, HSV-2 strain MS-infected Vero cells were solubilized and the glycoprotein purified by lentil-lectin Sepharose chromatography. The glycoprotein preparation was diluted to contain 35 pg total protein per 0.1 ml and was used with either equal parts of saline or complete Freund's adjuvant (CFA). The glycoprotein preparation (0.2ml) was administered in the footpad.

Adjuvant Imiquimod hydrochloride salt (3M Pharmaceuticals, St Paul, MN, USA) was administered subcutaneously in

Adjuvant for immunotherapy of HSV: D.I. Bernstein et al. Table 1 Experimental procedure Group

Vaccine

Adjuvant

1 2 3 4 5

Placebo HSV-2 glycoproteins HSV-2 glycoproteins HSV-2glycoproteins HSV-2glycoproteins

None Saline CFA Imiquimod (1 day) Imiquimod (5 days)

Control Gly alone Gly+CFA Gly+imi x 1 day Gly + imi x 5 days

Gly, glycoprotein: imi, irniquimod

H F F were similar to those previously reported 5. Briefly, peripheral blood mononuclear cells (3.2x l0 s) were incubated for 14h with HSV-infected (HSV-1 strain Mclntyre) or uninfected H F F cells (4 x 104) labelled with 51Cr. Supernatants from triplicate wells were harvested and counted. Percentage lysis was calculated by standard formulae and the MHC-unrestricted HSV-specific cytolysis was defined as the lysis of HSV-infected H F F minus that of uninfected HFF.

Statistics the dorsum of the back at a dose of 3 mg kg-1 day- ~ Complete Freund's adjuvant (Sigma) was mixed with the glycoprotein at a ratio of 1:1.

Experimental procedure Hartley female guinea-pigs (Charles River Breeding Laboratory, Wilmington, MA, USA) weighing 250-300 g were purchased simultaneously from the same breeding colony by the two investigating laboratories and were inoculated intravaginaUy with the same pool of MS strain HSV-2 as previously reported 9. After the guinea-pigs had recovered from the initial infection (14 days after HSV-2 inoculation), animals that had developed acute vesicular disease were randomized (n=10 per group at each laboratory) by the severity of the acute disease to the groups shown in Table 1. Animals were immunized in the hind footpads on days 15 and 35 after HSV-2 inoculation with 35 #g of HSV-2 glycoproteins alone or mixed with complete Freund's adjuvant for the glycoprotein + CFA group. Imiquimod was administered simultaneously with immunization once or for five consecutive days beginning on the day of immunization. Animals were evaluated daily on days 15-91 for evidence of recurrent genital HSV-2 disease by an observer blinded to the study groups. Cumulative mean lesion days per animal were calculated by dividing the number of days with a lesion by the number of animals.

Proliferation Lymphoproliferative responses to HSV-2 were measured as previously described 5. Briefly, guinea-pig peripheral blood mononuclear cells (5x 105) were incubated in triplicate for 5 days with HSV-2 or control antigen. On day 5, aH-TdR (1/~Ci/well) was added and incorporated radioactivity was measured after an 18 h pulse. Results are expressed as the mean counts per minute for HSV-stimulated minus control-stimulated wells.

lnterleukin-2 (IL-2) assay As previously described 5, supernatants from wells stimulated as described above were collected after 2 days of incubation. The IL-2 activity in the supernatants was then assayed as 3H-TdR incorporation into 4 x 103 CTLL-2 cells after a 24 h incubation and a 4 h pulse and compared with known concentrations of r-IL-2 (Cetus).

Cytolytic assay Guinea-pig peripheral blood mononuclear cellmediated cytolytic assays of HSV-infected and uninfected

Analyses were performed by two-tailed analysis of variance with and without the Bonferroni correction to account for the multiple groups compared with controls. Data are expressed as means+s.e. (standard error). Comparisons of recurrent lesion days are expressed as percentage reductions, although statistical analysis and p values represent comparison of the means + s.e.

RESULTS

Clinical The experiments were performed at two r e s e a r c h centres (experiments 1 and 2) under identical conditions using animals purchased simultaneously from the same breeding colony inoculated with the same viral pool. Ten animals were randomized to each group at each institution. The mean severity scores for the acute illness were similar for all groups (data not shown). As seen in Figure 1, glycoprotein immunization plus either 1 or 5 days of imiquimod most effectively reduced the number of recurrent lesion days in both experiments. Immunization with glycoprotein alone produced a small non-significant reduction in recurrent lesion days over the period of observation, while glycoprotein + imiquimod produced an overall reduction of 53-69% compared with controls (p <0.001-p<0.03) (Table 2). The single dose and 5-day regimen of imiquimod produced similar reductions of recurrent lesion days although 5 days was slightly more effective in both trials (not significant). When evaluated in 3-week intervals after the first immunization, protection from recurrent disease ,was highest following the second immunization. Peak reductions of 79 and 81%, in experiments 1 and 2 respectively, were seen in the imiquimod groups during this period. In experiment 1, the reduction in recurrence by immunization with 5 days of imiquimod appeared to persist longer than for immunization with 1 day of imiquimod, but this difference was not seen in experiment 2. The effectiveness also appeared to decrease over tiilae in all immunized groups in experiment 1 but not in experiment 2 and was absent in the final 3 weeks of observation in experiment I, perhaps due to the reduced recurrence rates in control animals in this experiment at this time. Immunization with complete Freund's adjuvant reduced recurrence by 26-41%, which is similar to that previously reportedl'2'L Compared to immunization with complete Freund's adjuvant, imiquimod further reduced recurrent lesion days by 37-52% over the entire observation period. This difference between the imiquimod groups and the complete Freund's adjuvant group in

Vaccine 1995 Volume 13 Number 1

73

Adjuvant for immunotherapy of HSV: D.I. Bernstein et al.

A 12

r~

0= --I

T

-+--Control --x--Gly ~ G l y + Imlqulmod Xld . | ~ Gly + ImlqulmodX~k:l " T "°~-GIyLCF-A .....

10 t

.H.va_t~: • ~:.+-H-" . . .~ . ~ " ,,,x.x-x-x +,+'~+'~" 0~ ' x ' x_' x"' xx,X~,~X.XX, _' x ' ~ ~ X-X ..... j . X ~ ~ . . . . . . .

f,+,e.

/

=. >=

=1 o

B

2~ ~ ~

III~:'----~--~"~

.

.

.

.

.

5O 45

¢'~ 40

--+-- Control Gly ÷ I ! Gly Imlqumod Xla

.~+,+,+~+~+ -~+'+' '"~'x'x'wx~ +,+.+, " .~,.x,x-~e'"

e-

p_ 3~ _~ 3o ¢.

m

25

~

2o

I0

o

....+."e+-'

$ 0 ~ + - H - + + - H - ~ - + + t - - H - + - H - F + - ~ - t - - ~ - + Day Day Day Day Day Day Day Day Day Day 22 29 36 43 50 57 64 71 78 15

+-+++J,-F.-

Day

Day

84

91

Day After HSV Inoculation Figure 1 The ettect of immunization on the cumulative mean number of days when recurrent herpetic lesions were noted. Experiments 1 (A) and 2 (B) were performed simultaneously in the two research laboratories. Animals were immunized with HSV-2 glycoprotein (Gly) alone or combined with complete Freund's adjuvant (CFA) in hind footpads on days 14 and 35 after vaginal HSV-2 infection. Groups that received glycoprotein immunization and imiquimod received either one dose of imiquimod on the day of immunization or one dose of imiquimod for live consecutive days beginning on the day of immunization. Animals were evaluated for recurrent lesions daily on days 15--91

experiment 2 achieved significance overall (p < 0.05) and at selected 3-week intervals (Table 2). I m m u n e response

HSV-2 antibody responses were increased by immunization (Fioure 2). The addition of imiquimod slightly increased antibody titres on day 49 (p < 0.05) but not day 63 compared to immunization without adjuvant. Antibody responses were highest in the group immunized with complete Freund's adjuvant (p<0.001 compared with any other group), Proliferation and IL-2 responses were variable (Fioure 3). No significant differences could be detected between groups, although the only immunized group to have an increased IL-2 response was the complete Freund's adjuvant group (not significant).

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Vaccine 1995 Volume 13 Number 1

The HSV-specific MHC-unrestricted cytolytic response was increased by immunization with adjuvants (Figure 4). Imiquimod and complete Freund's adjuvant produced equivalent increases in this response which were significantly greater than controls on days 42 and 60 (p < 0.05). Further, on the peak day of response (day 42), the HSV cytolytic activity was significantly greater in the groups immunized with imiquimod compared to the group immunized without adjuvant. This response remained elevated, but not significantly, through to day 90 in two of the three groups immunized with adjuvant. DISCUSSION Immunization of HSV-2 latently infected guinea-pigs with HSV-2 glycoproteins has been shown to reduce

Adjuvant for immunotherapy of HSV: D.I. Bernstein et al.

Table2

Effect of immunization and adjuvant on reduction of recurrent HSV lesion days over time compared with the control group (no immunization, no adjuvant)

G ro u p

150' I-'-1 control 125. . RZi ely + Imkluimod x5

1:SDGly+ CFA

Percentage reduction in recurrent lesion days

Total:

i 100,

Days 15-35

Days 35-56

Days 57-77

Days 78-91

Days 15-91

75,

30 60* 62* 35

43 79t 78t 39

0 13 70 0

0 0 0 20

17 53* 59* 26

14 58~,§ 65t, § 21"

6 71 t 73** 54t

5 73** 81t, § 51 t

18 67t 56~ 44*

12 64:, ~ 69t, ~ 41 ~

50.

Experiment 1 Gly alone Gly+ imi x 1 day G l y + i m i ×5 days Gly + CFA

25

Day 1¢

Experiment 2 Gly alone Gly+ imi x 1 day Gly+imi x 5 days Gly+CFA

Statistical analysis compares the mean plus s.e. of lesion days over time although the percentage reduction is presented. Analysis was initially performed by ANOVA followed by the Bonferroni correction for multiple groups compared with controls. Those comparisons marked, or ~: remained significantly different after this correction: *p<0.03 versus control; tp<0.01 versus control; ~tp<0.001 versus control; §p<0.05 versus CFA Gly, giycoprotein; imi, imiquimod

Day 4-9

Day 63

60o o o

40,

X

:E CL O

20.

6- 1---3 Control 5-

Gly 2~] Gly + Imiquimod x l Gly + Imiquirnod x5 Gly + CFA

###:~

"-~ 4(.-9

o _J 3-

v I-0

2-

1111i Day 14

Day 4 2 - 4 9

× x X x × x × X × X × X X

Day 49 Days After HSV Inoculation Figure 3 Effect of immunization on (A) HSV lymphoproliferative response and (B) IL-2 response. Periperal blood mononuclear cells from animals in experiment 1 were stimulated with ultraviolet-inactivated HSV for 5 days to measure lymphoproliferation; supernatants were removed after 48 h for measurement of IL-2 using CTLL-2 cells. The results shown are the mean counts per minute (CPM) __+s.e. for HSV-stimulated minus control-stimulated wells. No significant differences were found

Do 6 0 - 6 3

Days A f t e r HSV Inoculation

Figure 2 Effect of immunization on HSV-2 antibody titres. The geometric mean antibody titre (GMT)_+s.e. determined by enzyme-linked immunosorbent assay against HSV-2 glycoprotein is shown. All statistical comparisons shown involved comparison of titres during that period after the Bonferroni correction for multiple groups: *p < 0.051 comparing the control and immunized groups and p < 0.05 comparing immunization with glycoprotein plus imiquimod to immunization with glycoprotein alone; **p<0.001 comparing the complete Freund's adjuvant group to any other group; #p < 0.05 comparing the control and immunized groups

50¢0-

Control Gly I;~;~ Gly + Irniquirnod x l k ~ Gly + Imiquimod x5 l ~ Gly + CFA

O9 U3 O O > O9 -r-

#

30-

###

2010-

clinical recurrence s-* and asymptomatic viral shedding in animals Is. Effective immunization requires potent adjuvants 2-~ and appears to be related to the boost of specific cell-mediated immune responses 5. This strategy has now been applied to patients with frequently recurring genital HSV infection in preliminary investigations with encouraging results. Initially, Straus e t al. 16 reported that administration of a recombinant glycoprotein D vaccine with alum to HSV-immune individuals boosted HSV-2 antibody titres. More recently, a recombinant glycoprotein D vaccine given with alum was shown to significantly reduce recurrent disease in patients

0 Day 14

Day 42

Day 60

Days After HSV Inoculation

iin Day 90

Figure 4

Effect of immunization on HSV-specific MHC-unrestricted cytolysis. Peripheral blood mononuclear cells from the animals in experiment 2 were incubated with HSV-infected or uninfected HFF cells in a 14h SlCr-release assay. HSV-specific lysis is the percentage cytolysis of HSV-infected minus uninfected HFF targets. All statistical comparisons shown performed after applying the Bonferroni correction for multiple groups: *p <0.05 comparing the immunized groups to either controls or Gly alone; #p<0.05 comparing the immunized groups to controls

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75

A d j u v a n t for i m m u n o t h e r a p y of HSV: D.I. Bernstein et al.

with frequent HSV recurrences compared with a placebo group given alum alonea 7. In previous experiments using the guinea-pig model, vaccination with alum as an adjuvant has not significantly reduced recurrence 14'18. It therefore appears that vaccination using more potent adjuvants could further reduce recurrent disease in patients as it has in guinea-pigs. In this report, we have evaluated the immunomodulator imiquimod as the adjuvant for immunization. Immunization with HSV-2 glycoprotein and imiquimod significantly reduced recurrent lesion days by 53-69%. A peak reduction of 70-80% was seen in the weeks following the second immunization. This reduction is greater than the 5-47% previously detected using glycoprotein immunization and other adjuvants including complete Freund's adjuvant, Ribi triple mix or muramyl dipeptide L2'5. In fact, in the experiment reported here, the reduction in recurrence was 37-52% greater in the imiquimod groups compared with the group receiving complete Freund's adjuvant. The mechanism(s) whereby immunization decreases recurrent HSV disease is poorly understood. Antibody titres do not appear to correlate with the reduction in recurrent disease 2'4'19, although the induction of antibody to critical epitopes of HSV not recognized during infection cannot be ruled out. In contrast, increased cell-mediated immune responses have consistently been shown to correlate with reduced recurrence in animal models 3-5'19. These responses have included lymphoproliferative responses to HSV, and IL-2 and ~,-interferon production, as well as MHC-unrestricted cytolytic activity against HSV-infected targets. In this report, we could not detect a correlation between lymphoproliferation or IL-2 responses and decreased recurrence, although the HSV-specific MHC-unrestricted cytolytic responses were increased in immunized animals that received either imiquimod or complete Freund's adjuvant. Imiquimod appears to be an effective adjuvant for a vaccine designed to reduce recurrent HSV disease. The availability of an orally administered preparation of imiquimod 2° should further increase the interest in this immunomodulator.

2

3 4

5

6

7 8

9

10 11

12 13 14

15

16

17

ACKNOWLEDGEMENTS This work was supported by 3M Pharmaceuticals and NIH Grant AI23482. The authors are grateful to Teresa Schneider for preparation of the manuscript.

18

19

REFERENCES Stanberry, L.R., Burke, R.L. and Myers, M.G. Herpes simplex virus glycoprotein treatment of recurrent genital herpes. J. Infect. Dis. 1988, 167, 156-163

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Stanberry, L.R., Harrison, C.J., Bernstein, D.I., Burke, R.L., Shukla, R., Ott, G. and Myers, M.G. Herpes simplex virus glycoprotein immunotherapy of recurrent genital herpes: factors influencing efficacy. Antiviral Res. 1989, 11,203-214 Ho, R.J., Burke, R.L. and Merigan, T.C. Antigen presenting liposomes are effective in treatment of recurrent herpes simplex virus genitalis in guinea pigs. J. Virol. 1989, 63, 2951-2958 Ho, R.J.Y., Burke, R.L. and Merigan, T.C. Liposome4ormulated interleukin-2 as an adjuvant of recombinant HSV glycoprotein gD for the treatment of recurrent genital HSV-2 in guinea-pigs. Vaccine 1992, 10, 209-213 Bernstein, D.l., Harrison, C.J., Jenski, L.J., Myers, MG. and Stanberry, L.R. Cell-mediated immunologic responses and recurrent genital herpes in the guinea pig. J. Immunol. 1991, 146, 3571-3577 Harrison, C.J., Jenski, L., Voychehovski, T. and Bernstein, D.I. Modification of immunological responses and clinical disease during topical R-837 treatment of genital HSV-2 infection. Antiviral Res. 1988, 10, 209-223 Bernstein, D.I. and Harrison, C.J. Effects of the immunomodulating agent R-637 on acute and latent herpes simplex virus type 2 infections. Antimicrob. Agents Chemother. 1989, 33, 1511-1515 Bernstein, D.I., Miller, R.L. and Harrison, C.J. Effects of therapy with an immunomodulator (imiquimod, R-837) alone and with acyclovir on genital HSV-2 infection in guinea-pigs when begun after lesion development. Antiviral Res. 1993, 20, 45-55 Bernstein, D.I., Stanberry, L.R., Harrison, C.J., Kappas, J.C. and Myers, MG. Antibody response, recurrence patterns and subsequent HSV-2 reinfection following initial HSV-2 infection of guinea pigs: effect of ecyclovir. J. Gen. Virol. 1986, 67, 1601-1612 Kern, E.R. Acyclovir treatment of experimental genital herpes simplex virus infections. Am. J. Med. 1982, 73(Suppl.), 100-198 Landry, M.L., Lucia, H.L., Hsiung, G.D., Pronovost, A.D., Dann, P.R., August, M.J. and Mayo, D.R. Effects of acyclovir on genital infection with herpes simplex virus types 1 and 2 in the guinea pig. Am. J. Med. 1982, 73(Suppl.), 143-150 Myerson, D. and Hsiung, G.D. Prophylactic and therapeutic treatment with acyclovir of genital herpes in the guinea pig. Proc. Soc. Exp. Biol. Med. 1983, 174, 147-152 Bernstein, D.I., Miller, R.L. and Harrison, C.J. Adjuvant effects of imiquimod on a herpes simplex virus type 2 glycoprotein vaccine in guinea pigs. J. Infect. Dis. 1993, 167, 731-735 Berman, P.W., Vogt, P.E., Gregory, T., Lasky, L.A. and Kern, E.R. Efficacy of recombinant glycoportein D subunit vaccines on the development of primary, recurrent and latent genital infections with herpes simplex type 2 in guinea pigs. J. Infect. Dis. 1988, 157, 197 Myers, MG., Bernstein, D.I., Harrison, C.J. and Stanberry, L.R. Herpes simplex virus glycoprotein treatment of recurrent genital herpes reduces cervicovaginal virus shedding. Antivira/Res. 1988, 10, 83-88 Straus, S.E,, Savarese, B., Tigges, M., Freifeld, A.G., Krause, P.R., Margolis, D.M. et al. Induction and enhancement of immune responses to herpes simplex virus type 2 in humans by use of a recombinant glycoprotein D vaccine. J. Infect. Dis. 1993, 167, 1045-1052 Meier, J., Corey, L., Burke, R.L., Savarese, B., Barnum, G., Kost, R. et al. Immunotherapy of genital herpes with a recombinant herpes simplex virus type 2 glycoprotein D (gD2) vaccine: A placebocontrolled trial. Clin. Res. 1993, 41, 199A Burke, R.L., Sekulovich, R., Tigges, M., Langenberg, A., Adair, S., Dekker, C. et al. The increased immunogenicity of a HSV glycoprotein subunit vaccine combined with the novel adjuvant MF59 as compared with alum as adjuvant. In: Abstracts of the 19th International Herpes Virus Workshop, 1993, Abstract C-37 Weinberg, A., Konrad, M. and Merigan, T.C. Regulation by recombinant interleukin-2 of protective immunity against recurrent herpes simplex virus type 2 genital infection in guinea pigs. J. Virol. 1987, 61, 2120-2127 Borden, E., Witt, P., Kvam, D., Wick, K., Ritch, P., Anderson, T. et a/. An effective oral inducer of interferon in humans. J. Interferon Res. 1991, 11(Suppl. 1), S92