Immunology 1986 58 245-250

Cyclophosphamide and dimethyl dioctadecyl ammonium bromideimmunopotentiate the delayed-type hypersensitivity response to

inactivated enveloped viruses

R. H. SMITH & B. ZIOLA Immunovirology Research Group, Department of Microbiology, University of Saskatchewan,Saskatoon, Saskatchewan, Canada

Accepted for publication 9 January 1986

SUMMARY

Immunization of BALB/c mice with measles virus inactivated with fl-propiolactone and mixed with100 pg of the cationic surface-active lipid dimethyl dioctadecyl ammonium bromide (DDA) primesfor a strong virus-specific delayed-type hypersensitivity (DTH) response that peaks 1 week later.Optimal immunization and challenge doses were found to be 8 and 4 pg/mouse, respectively, andpretreatment with 200 mg of cyclophosphamide/kg 2 days prior to immunization significantlyenhanced the DTH response. When compared to Freund's complete and incomplete adjuvants,DDA was superior for induction of DTH to inactivated purified measles virus. As DDA could beadministered to animals at a site different from the measles virus antigens, or 1 day previously, andstill significantly enhance the DTH response, DDA is probably acting more as an immune modulatorthan as a simple adjuvant. The conditions for an optimal DTH response to measles virus were alsoshown to be applicable to other enveloped viruses, for example, a strong DTH response was similarlygenerated to inactivated purified influenza PR8 virus and to herpes simplex virus type 1 antigenspresent in plasma membranes isolated from infected Vero cells.

INTRODUCTION

Dimethyl dioctadecyl ammonium bromide (DDA) and cyclo-phosphamide (CY) have both been used to elicit predominantlyT-cell responses to antigens (Lagrange, Mackaness & Miller,1974; Snippe, Belder & Willers, 1977), even though it is unclearhow these compounds work. DDA is a surface-active lipid, andmodification of antigens by conjugation with fatty acids isknown to shift the immune response towards development of astrong delayed-type hypersensitivity (DTH) response (Coon &Hunter, 1973). It has been suggested that, when DDA and anantigen are mixed, the DDA associates with the antigen throughelectrostatic bonds, introducing lipid groups which lead toinduction of a strong cellular immune response (Dailey &Hunter, 1974). In contrast, CY is believed to remove suppressorcells involved in the regulation ofDTH (Lagrange et al., 1974;Leung & Ada, 1980; Liew, 1982), thus allowing development ofa strong DTH response.

One of our research interests concerns the extent to whichthere is T-cell cross-reactivity among paramyxoviruses. To-

Abbreviations: CY, cyclophosphamide; DDA, dimethyl dioctadecylammonium bromide; DTH, delayed-type hypersensitivity; FCA,Freund's complete adjuvant; FIA, Freund's incomplete adjuvant; HSV1, herpes simplex virus type 1; PBS, 0-02 M sodium phosphate, pH 7-4,containing 0-14 M NaCl.

Correspondence: Dr Barry Ziola, Immunovirology ResearchGroup, Dept. of Microbiology, A243 Health Sciences Building, Univer-sity of Saskatchewan, Saskatoon, Saskatchewan S7N OWO, Canada.

wards this end, we recently showed that a measles virus-specificDTH response is generated in mice after injection of live virus,and that this response is significantly augmented by pretreat-ment with CY (Smith & Ziola, 1984). However, a large amountof virus is required to generate a good DTH response, and, withdifficulties in keeping mice having different paramyxoviridaevirus infections in quarantine, we decided to investigate whethera strong DTH response could be induced in mice by immunizingwith purified viruses that had been inactivated with fl-propiolac-tone and mixed with DDA.

MATERIALS AND METHODS

MiceBALB/c female mice, 8-16 weeks old, were obtained from theanimal facilities in the Department of Microbiology, Universityof Saskatchewan, Saskatoon, Saskatchewan, Canada.

Viral and control antigensThe wild-type measles virus was obtained from Dr Aimo Salmi,Department of Medical Microbiology, University of Alberta,Edmonton, Alberta; the MacIntyre strain of herpes simplexvirus type 1 (HSV 1) was obtained from the American TypeCulture Collection, Rochville, MD, and influenza PR8 viruswas obtained from Connaught Laboratories, Toronto, Ontario.Propagation of HSV 1, and propagation and purification ofmeasles virus and influenza PR8 virus were done as described bySmith & Ziola (1984) and Ziola & Hader (1985), respectively.

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Preparation ofplasma membranes from uninfected and HSV 1-

infected Vero cells was done according to the procedure ofStone, Smith & Joklik (1974), except for omission of dimethyl-sulphoxide from the homogenizing buffer. All purified virus andplasma membrane antigens were treated with fl-propiolactoneto inactivate any remaining infectivity (Ziola & Hader, 1985).Protein levels were determined with the BioRad protein assay

using human gamma globulin as a standard.

ChemicalsCY was obtained from Bristol Laboratories of Canada, Belle-ville, Ontario, and dissolved in sterile 0-02 M sodium phosphate,pH 7 4, containing 0-14 M NaCl (PBS) just before use. CY was

used at 200 mg/kg and injections were given intraperitoneally 2days before immunization with virus (Smith & Ziola, 1984).Freund's complete (FCA) and incomplete (FIA) adjuvants wereobtained from Gibco Canada, Burlington, Ontario. DDA was

obtained from Eastman Kodak, Rochester, NY. Except wherenoted, 100 pg of DDA was used when DDA mixed with viruswas used for immunological priming.

Induction and measurement ofDTHThe footpad swelling test was carried out essentially as de-scribed by Cooper (1972). Mice pretreated with CY or PBS wereinjected intramuscularly 2 days later in each hind leg with 100 p1

of inactivated virus in PBS alone, PBS containing DDA or PBSemulsified with an equal volume ofFCA or FIA. On the desiredday post-immunization, groups of at least five mice were

challenged in the right hind footpad with inactivated virus inPBS. A Hamilton syringe was used for the 30-pl injections.Footpad swelling was measured 24 hr later with a Mitutoyoengineering micrometer. Depending on the age of the animals,the thickness of uninjected hind footpads varied from 1 60 to1-70 mm. Experimental results are expressed as percentagefootpad swelling according to the formula:% footpad swelling =

(right hind footpad thickness) -(left hind footpad thickness) x 100mean thickness of uninjected left hind footpads

Specificity of the DTH reactionMice pretreated 2 days previously with CY were immunizedwith 8 pg of inactivated measles virus, influenza PR8 virus or

plasma membranes from HSV 1-infected Vero cells mixed withDDA. Seven days later, groups of five mice were challenged inthe right hind footpad with 4 pg of inactivated measles virus,influenza PR8 virus, plasma membranes from HSV 1-infectedVero cells or plasma membranes from control Vero cells.Footpad swelling was measured 24 hr later.

Transfer ofDTHMice were treated with CY and immunized 2 days later with 8 pgof inactivated measles virus in DDA. Seven days later, draininglymph nodes were removed and pushed through a no. 60-meshwire screen to obtain a suspension of single cells. The cells werewashed twice with PBS, resuspended and counted. Cell viabilitywas determined by trypan blue dye exclusion. The cells were

sham-treated, treated with complement alone or treated withanti-Ly 1.2 serum plus complement as described by Wright &Ramshaw (1983). Viable lymph node cells remaining after eachtreatment were mixed with inactivated virus, and 106 cells plus 4

pg of virus were injected into the right hind footpads of fiverecipient mice. Footpad swelling was measured 24 hr later.

StatisticsThe Mann-Whitney U-test was used to assess statistical signifi-cance.

RESULTS

Immunization requirements and kinetics of the DTH response

Initial experiments determined the general effect of CY andDDA on immunological priming for DTH by inactivatedmeasles virus. Table 1 shows that CY (200 mg/kg) and DDA

Table 1. Influence ofCY and DDA on priming for DTH by inactivatedmeasles virus

% footpad swelling*

Priming Mice pretreated with PBS Mice pretreated with CYdose(pg) Virus in PBS Virus in DDA Virus in PBS Virus in DDA

0 6-3+4-4 5 0+5 0 8 3+4 5 8-6+3-01 8-8+56 407+71 tj 23-6+9-2 t 494+84tj8 15-0+7-1 475+ 10Ot 51 3+10-3t 774±+ 10t§

* 24 hr after a 4 pg challenge of inactivated measles virus given Day 7post-immunization.

tP < 005 compared to unprimed animals treated similarly or com-pared to animals that were PBS-pretreated and received the same primingdose in PBS.

tP< 005, compared to CY-pretreated mice primed with 1 pg ofinactivated measles virus in PBS.

§P< 0-02 compared to all other groups.

(100 pg/immunization) each augmented the DTH response toinactivated measles virus. When CY and DDA together wereused in the same animal, the DTH response was higher than ifCY or DDA was used alone. Increasing the amount ofDDA upto 800 pg per CY-pretreated mouse did not increase the DTHresponse, while decreasing the amount to 50 or 25 pg per CY-pretreated mouse caused a progressive decrease in the DTHresponse (results not shown). Consequently, 100 pg ofDDA permouse was chosen for routine use.

Kinetic studies of the DTH response to inactivated measlesvirus administered in DDA to CY-pretreated mice showed thatthe DTH response increased rapidly, peaked on Day 7 andremained significantly elevated at least up to Day 15 post-immunization (Fig. 1). Based on these results the footpadchallenge dose was administered on Day 7 post-immunization insubsequent experiments.

The minimum amount of inactivated measles virus requiredto induce a significant level of DTH priming was 0-1 pug (Table2). An optimal DTH response required considerably more virusbe used. Although induction of DTH by inactivated measlesvirus had still not plateaued with an immunization dose of24 pg,we chose a priming dose of 8 pg for routine use. The reasons forthis were: (i) because the DTH induced by 8 pg is very

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Delayed hypersensitivity to enveloped viruses

100-

80-u

a1)3 60--0aQ.0 40-0

20-

100 -

80-.'Sa3 60-

a0 40-o0

20 -

I

I 3 5 7 9 I 13 15Days

Figure 1. Time-course ofthe DTH response to inactivated measles virus.Mice were treated with CY and immunized 2 days later with 5 Mg ofinactivated measles virus mixed with DDA. At various times thereafter,groups of five mice were footpad-challenged with 2 pg of inactivatedmeasles virus ( x x ), 2 jig of Vero cell plasma membranes (0) orPBS (0). Footpad swelling was measured 24 hr later.

Table 2. Dose of inactivatedmeasles virus required for opti-

mum DTH priming*

Priming dose % footpad(jg) swellingt

0 95+57002 137+5501 301+14210.5 41 6+±75 $2 485+11918 606+135T§

24 708±+119

* Inactivated measles virusmixed in PBS containing DDAwas administered to CY-pre-treated mice.

t 24 hr after a 4 pg challenge ofinactivated measles virus givenDay 7 post-immunization.

I P<0001 compared to un-primed animals.

§ P< 0-05 compared to ani-mals with 2 or 24 pg.

significant, and (ii) because using 8 pg rather than two or threetimes more immunizing antigen represents a considerable savingof purified measles virus.

This choice for the amount of inactivated measles virus usedto prime for a strong DTH response is indirectly supported bysimilar data for several other viruses, including inactivatedpurified mumps virus grown either in Vero cells or eggs,influenza PR8 virus grown in eggs, and respiratory syncytialvirus, parainfluenza virus 3 and canine distemper virus grown inVero cells. For these viruses, the dose-response relationships for

> 0 - 00 (fLUJ ) o- a-> r

MV

±f

> 0 - OD UE cr > cr CoLuV a- a->1

HSV

i

Ir >0

L U- a- a

>1PR8

Figure 2. Specificity of the DTH response. Mice were treated with CYand immunized 2 days later with 8 pg of inactivated virus antigen mixedwith DDA. Seven days later, seven mice were footpad-challenged with 4pg of inactivated virus antigen, 4 pg of Vero cell plasma membranes orPBS. Footpad swelling was measured 24 hr later. MV, measles virus;Vero, plasma membranes from uninfected Vero cells; HSV 1, plasmamembranes from herpes simplex virus type 1-infected Vero cells; PR8,influenza PR8 virus.

priming of DTH were similar to those shown in Table 2 formeasles virus (data not shown), but the difference in priminginduced by 8 pg and 16 or 24 pg of virus was not alwayssignificant as was the case for measles virus. Even though theslopes of the dose-response curves were comparable, the highestlevel ofDTH induced by a given virus could be lower than thatfor measles virus. For example, the maximum DTH levelinduced by influenza RP8 virus was approximately half that formeasles virus in any given experiment (see Fig. 2).

Challenge dose requirements

The challenge amount of inactivated measles virus givingmaximum footpad swelling after immunization with 8 Mg ofvirus was determined (Table 3). Since the DTH responseplateaued with challenge doses of 2 25-9 jug, a challenge dose of4 ug was selected as representing an optimal amount. In the caseof the other six viruses for which priming dose curves have beencompleted, challenge requirements for optimum DTH werefound to be identical to that for measles virus (data not shown).

Transfer of DTH

The cell-type involved in the DTH response induced afterinjection of inactivated measles virus mixed in DDA into CY-pretreated mice was determined by cell transfer experiments. ADTH response was transferred if the recipient mice receivedimmune lymph node cells that were sham-treated or treated withcomplement alone before being mixed with inactivated measlesvirus and injected (percentage footpad swelling of2 22 + 3-4 and20-0 + 5-2, respectively). Treating the immune cells with anti-Ly1.2 serum and complement abolished transfer ofDTH (percent-age footpad swelling of 6-3 + 4-4; P < 0-02 compared to the othertwo treatments), clearly indicating that the response is mediatedby T cells.

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R. H. Smith & B. Ziola

Table 3. Challenge doseof inactivated measlesvirus required to evokean optimum DTH reac-

tion*

Challenge % footpaddose (Mg) swellingt

0 63+450 56 41 5+8-21-13 50 9+7-6225 569+6-745 60-1+4121t9 655±+10-2t

* 8 pg of inactivatedmeasles virus was admin-istered to CY-pretreatedmice.

t 24 hr after chal-lenge on Day 7 post-immunization.

I Do not significantlydiffer from each other(P> 0-05).

Specificity of the DTH response

Specificity of the DTH response was tested by immunizing CY-pretreated mice with inactivated measles virus, influenza PR8virus or plasma membranes from HSV 1-infected Vero cells andthen challenging seven mice from each group with inactivatedmeasles virus, influenza PR8 virus and plasma membranes fromHSV 1-infected or uninfected Vero cells (Fig. 2). For miceimmunized with each of the three antigens, only challenge withthe homologous antigen gave a significant DTH response

compared to the DTH response seen following challenge withVero plasma membranes (P< 0 0 1). For mice immunized withinactivated measles virus, the DTH response obtained followingchallenge with Vero cell plasma membranes was significantlyabove swelling seen after injection of PBS alone (P < 0 05). Formice immunized with plasma membranes from HSV 1-infectedVero cells, challenge with either inactivated measles virus or

Vero cell plasma membranes gave a significantly elevated DTHresponse compared to footpad swelling after injection of PBS(P < 0 025).

Immunopotentiation by FCA, FIA and DDA

Immunopotentiation by DDA of the DTH response to inacti-vated measles virus was compared to the immunopotentiationobtainable with FCA and FIA (Table 4). Immunization withvirus in FIA or in DDA significantly increased the DTHresponse to measles virus in PBS-pretreated mice; however,augmentation of the DTH response provided by DDA was

significantly higher than that provided by FIA. Irrespective ofwhich immunopotentiating compound was used, the DTHresponse increased if CY-pretreated mice were used, but onlythe increase seen after immunization with virus in DDA was

Table 4. Influence ofDDA, FCA and FIA on priming for DTHby inactivated measles virus*

Compound mixed % footpad swellingiwith virus for

priming PBS-pretreated mice CY-pretreated mice

Control 20-0+ 5-3 36 0+ 14-4FIA 34-4+6 2t 43 5 + 5 3FCA 18-0+7-6§ 25 8 + 3 4 §DDA 51 3 + 6-8t§ 78 8±131t¶¶T

* Two days after treatment with PBS or CY, mice wereimmunized with 8 pg of inactivated measles virus in PBS(control) or PBS mixed with FIA, FCA or DDA.

t 24 hr after a 4 pg challenge of inactivated measles virusgiven Day 7 post-immunization.

t P <0-02 compared to control animals pretreated thesame way.

§ P < 005 compared to FIA animals pretreated the sameway.

1¶ P < 0-02 compared to all other groups.

significant. Immunization of CY-pretreated mice with virus inDDA, but not in FIA or FCA, gave a DTH responsesignificantly above that obtained in CY-pretreated mice immu-nized with virus in PBS alone. Immunization with virus in FCAresulted in DTH responses significantly lower than thoseobtained with virus in FIA or DDA.

Effect of DDA pretreatment

Whether DDA could augment the DTH response to inactivatedmeasles virus if pre-administered or administered the same day,but at a site different from the virus, was also investigated. CYwas not used in these experiments. Treatment with DDA 2 daysbefore immunization with virus in PBS did not increase theDTH response significantly (Table 5), but injection of DDA

Table 5. Influence of pre-administeredDDA on DTH priming by inactivated

measles virus

Time DDA given relative % footpadto priming dose* swellingt

Control (no DDA given) 139±+4-12 days before 19-9+6-81 day before 23 6+7721Same day, different site 23-6 + 6 2TSame day, mixed with virus 35-1 + 84§

* 8 pg of inactivated measles viruswas used for priming.

t 24 hr after a 4 pg challenge ofinactivated measles virus given Day 7post-immunization.

t P< 001 compared to controlanimals.

§ P <0-01 compared to all othergroups.

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Delayed hypersensitivity to enveloped viruses

either 1 day before immunization with virus or on the same daybut at a site different from immunization caused a significantincrease in the DTH response. However, immunization withvirus mixed in DDA resulted in a DTH response significantlyhigher than that obtained with any other DDA utilizationprotocol.

DISCUSSION

Earlier, we showed that BALB/c mice injected with live measlesvirus were primed immunologically for a virus-specific DTHresponse (Smith & Ziola, 1984). However, a large amount oflivevirus was required to get a good DTH response, probably due tomeasles virus replicating poorly in mouse tissues (Griffin et al.,1974; Neighbour, Rager-Zisman & Bloom, 1978). We have nowshown that a very strong measles virus-specific, T-cell depen-dent DTH response can also be obtained in mice that have beenpretreated with CY and immunized with inactivated purifiedmeasles virus mixed with the cationic surface-active lipid DDA.The fact that CY pretreatment significantly enhanced the DTHresponse to inactivated measles virus was not surprising becauseCY pretreatment had similarly increased the DTH responseafter injection of live virus (Smith & Ziola, 1984). CY augmen-tation of DTH to measles virus may be due to the removal ofsuppressor cells that control the DTH response (Lagrange et al.,1974; Leung & Ada, 1980; Liew, 1982). This suggestion isdirectly supported by the recent demonstration by Cowens et al.(1984) that the 4-hydroperoxy derivative of CY inhibits thedevelopment of suppressor cells in vitro.

In an earlier study, Kraaijeveld et al. (1980) showed thatDDA enhances the DTH response to inactivated Semliki Forestvirus. A unique aspect ofour findings is that CY pretreatment ofmice and immunization with inactivated virus mixed with DDAtogether provides a DTH response to inactivated virus that ishigher than that provided by either procedure alone. WhileKraaijeveld et al. (1983) have reported that a CY pretreatmentof 8 hr can augment the murine DTH response to infectiousSemliki Forest virus, these authors have not reported using CYpretreatment to enhance the DTH response to inactivated virusgiven as a mixture with DDA.

The fact that DDA was found to be better than FCA or FIAfor immunopotentiation of the DTH response to inactivatedmeasles virus agrees with the findings of others working withdifferent types of antigens (Snippe et al., 1977; van der Meer,Hofius & Willers, 1979; Snippe et al., 1982). Even so, the modeof action of DDA is not clear. Daily & Hunter (1974) haveshown with autoradiographic studies that immunization ofguinea-pigs with '251I-labelled bovine serum albumin mixed withDDA results in the antigen being localized in the paracortexarea of draining lymph nodes. As Coon & Hunter (1973) hadearlier shown that conjugation of bovine serum albumin withthe lipid dodecanoic acid leads to the same localization ofantigen in the thymus-dependent area of lymph nodes, it wassuggested that DDA binds strongly to antigens, causing them tobe preferentially localized in areas of the immune systemprimarily involved in priming for DTH responses. However,Bloksma, de Reuver & Willers (1983) have more recently shownthat DDA impairs macrophage function by inhibiting lyso-some/phagosome fusion, suggesting that the influence of DDAon immune responses is through altered antigen processing.Irrespective of its exact mode of action, what is clear is that

DDA is not acting as a simple adjuvant, since both Snippe et al.(1982) and we have shown that the administration of DDA tomice prior to immunization can still enhance immunologicalpriming for an antigen-specific DTH response.

From the results presented here, it is clear that the treatmentof mice with 200 mg of CY/kg 2 days before adminstration ofantigen mixed with 100 Mg of DDA causes an enhanced DTHresponse compared to either treatment used alone. The fact thatthe combination of CY pretreatment and use of DDA issynergistic suggests that DDA and CY act independently on theimmune system. Similar synergistic influences on the ability ofthe mouse immune system to mount a DTH response have beenreported for the combination of CY pretreatment and thepolyanionic molecule dextran (Battisto, Beckman & Yen-Lieberman, 1985). It appears that dextran interferes with theproduction of the macrophage suppressor factor involved indown-regulation of DTH responses. The similarity in resultsarising from using DDA and dextran suggests another mecha-nism for DDA modulation of the immune system.

Our results showing a synergistic effect between CY pre-treatment and DDA on priming for a DTH response differconsiderably from the results of Willers and co-workers (Snippeet al., 1977; van Houte et al., 1981). Using a CY dose of 300 mg/kg administered 8 hr before antigen was given in DDA, theseinvestigators showed that the CY treatment enhanced DTHresponses to sheep red blood cells and dinitrophenyl-conjugatedbovine serum albumin. However, the peak DTH response of theCY-pretreated mice was 6 days out ofphase with the response ofuntreated mice. This CY pretreatment regimen was also foundto impair the development of a DTH response to haptenatedliposomes and to eliminate the difference seen between theability of DDA and FCA to enhance priming for a DTHresponse. Since we did not see similar effects ofCY pretreatmenton the development of DTH to inactivated measles virusfollowing adminstration as a mixture with DDA, we concludethat these differences may be explained by our using 200 mg,rather than 300 mg, ofCY/kg and our giving the CY 48 hr ratherthan just 8 hr before the mice received antigen mixed in DDA.Support for this contention comes from Lagrange et al. (1974),who found that the timing of a CY treatment alters the effectseen on DTH responses.

Although inactivated measles virus was used in most of theexperiments reported here, conditions described for generatinga strong measles virus-specific DTH response have also beenused to generate similar responses to several other inactivatedpurified paramyxoviridae viruses, including respiratory syncy-tial virus, mumps virus, canine distemper virus and parain-fluenza virus 3. As shown here, we have also generated specificDTH responses to the orthomyxovirus, influenza PR8 virus,and to viral antigens in purified plasma membranes from HSV1-infected Vero cells, further documenting the wide applicabilityof the described protocol. As the DTH responses obtained havebeen both high and specific, we have been able to use this assaysystem to analyse the way in which the mentioned paramyxovir-idae viruses are immunologically related at the T-cell level (R.H. Smith and B. Ziola, manuscript in preparation).

ACKNOWLEDGMENTSWe thank Mrs Brenda Karvonen and Mrs Mildred Shukin for growingand purifying the virus material used. This study was supported by

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250 R. H. Smith & B. Ziola

Development Grant 204 and Operating Grant MA 7813 from theMedical Research Council of Canada. R. H. Smith was alternativelysupported by a Research Studentship from the Multiple SclerosisSociety of Canada and a Graduate Scholarship from the University ofSaskatchewan.

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