Dendritic Cell Immunotherapy of Hepatitis C Virus Infection: Toxicology of Lipopeptide-Loaded...

Dendritic Cell Immunotherapy of Hepatitis C Virus Infection:

Toxicology of Lipopeptide-Loaded Dendritic Cells

David C. Jackson,1,4,

* Georgia Deliyannis,2

Emily Eriksson,1

Irene Dinatale,2

Michael

Rizkalla,2and Eric J. Gowans

3,4,*

(Accepted October 27, 2005)

Before carrying out a clinical trial in humans in which a cell-based therapeutic anti-hepatitis Cvirus lipopeptide vaccine candidate is to be evaluated, a limited toxicological study was carriedout. Murine bone marrow-derived dendritic cells (DCs) were loaded with lipopeptides

containing HLA A2.1-restricted epitopes recognised by cytotoxic T lymphocytes (CTL) andthen injected into C57BL6 mice by intradermal and intravenous routes. No significantbehavioural changes, clinical symptoms or changes in body weight were observed when

compared with a control group of animals receiving no treatment. One week after the thirddose of lipopeptide-pulsed DC, mice were killed and blood samples taken for biochemical andhematological analyses. The liver, spleen and skin at the injection site were also collected and

processed for histological analysis. Mild eosinophilia was observed at intradermal injectionsites of animals receiving untreated as well as lipopeptide-loaded DCs. Despite a slightdecrease in the size of livers of animals receiving lipopeptide-pulsed DCs, there was noevidence of inflammatory infiltrate or histological change. The only biochemical or

hematological abnormality associated with the injection of lipopeptide-pulsed DC was aslight reduction in potassium levels. The evidence indicates that the lipopeptide vaccines per seare not cytotoxic and do not induce adverse events. On this basis, the TGA has granted clinical

trial by exemption (CTX) approval for the proposed study using HCV lipopeptide-pulsedautologous DC to proceed in humans. This is the first approval of its kind in Australia settinga precedent for somatic cell immunotherapy of infectious disease.

KEY WORDS: Dendritic cells; lipopeptide vaccines; toxicology.

INTRODUCTION

Infection with hepatitis C virus (HCV) is unusualfor an RNA virus in that approximately 80% ofinfected individuals develop a lifelong persistentinfection that results in continued replication andexpression of the virus with concomitant liver diseasein a proportion of patients (Lavanchy and McMa-hon, 2001). As a result, it has been estimated thatthere are approximately 200 million HCV carriers inthe world. In the absence of effective control mea-sures, this figure continues to increase alarmingly(Hauri et al., 2004). The underlying liver diseaseranges from near normal liver to chronic hepatitis,

* Eric Gowans and David C. Jackson are equal senior authors.1 Department of Microbiology and Immunology, The University

of Melbourne, Parkville, VIC, Australia.2 VaxTX Pty Ltd., Level 1, 123 Camberwell Road, 3123, East

Hawthorn, VIC, Australia.3 Macfarlane Burnet Institute for Medical Research and Public

Health, GPO Box 22843001, Melbourne, VIC, Australia.4 Correspondence should be addressed to: David C. Jackson,Department of Microbiology and Immunology, The Universityof Melbourne, Parkville VIC, Australia. Tel: +61-3-8344-9940;Fax: +61-3-8344-9941; e-mail: [email protected] andEric J. Gowans, Macfarlane Burnet Institute for MedicalResearch and Public Health, GPO Box 2284, Melbourne, VIC3001, Australia. Tel: +61-3-9282-2204; Fax: +61-3-9282-2100;e-mail: [email protected]

International Journal of Peptide Research and Therapeutics, Vol. 11, No. 4, December 2005 (� 2005), pp. 223–235

DOI: 10.1007/s10989-005-9270-y

2231573-3149/05/1200–0223/0 � 2005 Springer Science+Business Media, Inc.

cirrhosis and hepatocellular carcinoma, which resultsin HCV being the current leading single indicator forliver transplantation in the Western world (Fishmanet al., 1996; Kerridge et al., 1996). It is still unclearwhy most patients fail to clear the acute infection,although a number of potential mechanisms havebeen proposed (reviewed in (Gowans et al., 2004).Among the proposed mechanisms is an impairmentof dendritic cell (DC) function (Kanto et al., 1999;Auffermann-Gretzinger et al., 2001; Bain et al.,2001), although it is still unclear if this is a cause oreffect of virus persistence because other studies(Rollier et al., 2003; Larsson et al., 2004; Longmanet al., 2004, 2005) in patients and chimpanzees havefailed to detect such impairment.

HCV is classified in the Flaviviridae and conse-quently, genome integration is not responsible forpersistence as HCV RNA replication has no DNAintermediate. Furthermore, HCV RNA replication,as measured by the HCV replicon system, is sensitiveto interferon (IFN)-a and IFN-c (Frese et al., 2002).This suggests that the direct antiviral and immuno-modulatory effects of IFN may have the potential toeliminate the virus. Therefore, and also due to a lackof any HCV-specific antiviral agents, the current goldstandard for treatment is a combination of IFN-aand ribavirin. This therapy is remarkably successfulfor patients infected with HCV genotypes other thangenotype 1 because approximately 70% of suchindividuals show a sustained response and eliminatethe virus (Manns et al., 2001). Patients who are in-fected with genotype 1 are, however, more resistant tothe combination therapy although approximately40% of these individuals do show a sustained viralresponse. In addition, IFN monotherapy of patientswith acute phase HCV infection has been reported toresult in a self-limited infection in a high proportionof patients (Santantonio et al., 2005). Many patientswith persistent infection, however, report a range ofside effects, including depression, associated with theuse of systemic IFN and consequently only a smallnumber of patients can be treated.

DC immunotherapy using human monocyte-de-rived DC (Mo-DC) has been used to treat cancerpatients with a range of different tumours. In thisprocess, DC from individual patients are generatedex vivo then exposed to tumour antigens, matured ifnecessary by the addition of appropriate cytokines,and then transfused back into the same patient. Tu-mour cell antigens can be delivered by a number ofmethods including tumour cell lysates or tumour cell-derived mRNA (reviewed in (Gilboa and Vieweg,

2004) or by purified antigen or peptides (Figdoret al., 2004). We have previously shown that lipo-peptide-based vaccines have the capacity to matureDC (Zeng et al., 2002) and have more recently dem-onstrated that a lipopeptide-based vaccine candidatebased on influenza virus epitopes when administeredsubcutaneously or intra-nasally is able to induce aprotective immune response against challenge withlive influenza virus (Jackson et al., 2004). We havepreviously argued a case for immunotherapy of HCVpatients with HCV antigen-or peptide-loaded DC(Gowans et al., 2004) and are currently in the processof conducting a clinical trial to determine if suchtherapy has the potential to influence the course ofHCV infection. During the pre-clinical work-up forthis trial, the Therapeutic Goods Administration ofAustralia (the Australian equivalent of the FDA)required that we prove that the lipopeptides which wepropose to use in the trial are not toxic to DC andthat the DC in turn are not toxic when administeredin vivo. Because it is not possible to perform thistoxicological analysis in patients, the aim of thecurrent study was to determine if murine bone mar-row-derived DC which are pulsed with HLA A2.1-restricted, HCV-specific, CTL epitopes, induce anysigns of toxicity following administration into syn-geneic mice.

MATERIALS AND METHODS

Peptide and Lipopeptide Synthesis

Peptides and lipopeptides were synthesised as described pre-

viously (Zeng et al., 2002) using Good Laboratory Practice. The

peptides were chosen because they represent HLA A2.1-restricted

CTL epitopes which are associated with a T cell response in pa-

tients with acute, self-limiting HCV infection (Ward et al., 2002).

The CTL epitopes were linked to a common helper T cell (Th)

epitope; KLIPNASLIENCTKAEL, derived from the fusion pro-

tein of the morbillivirus, canine distemper virus (Ghosh et al.,

2001). The MHC class 1-restricted CTL epitopes were;

• DLMGYIPLV-core protein

• FLLALLSCLTV-core protein

• YLLPRRGPRL-core protein

• KLVALGINAV-NS3 protein

• LLFNILGGWV-NS4 protein

• ILAGYGAGV-NS4 protein

Loading of DCs with Lipopeptides

Murine DC were derived either from splenocytes (Winzler

et al., 1997) or from bone marrow (Lutz et al., 1999) and lipo-

peptides added to the immature murine DC in equimolar amounts

224 Jackson, Deliyannis, Eriksson, Dinatale, Rizkalla, and Gowans

to a final concentration of 7.5 lM and incubated overnight with

the cells.

In vivo Cytotoxicity Assay

Analysis of CTL determinant-specific cytotoxicity in vivo was

performed according to the method of Coles et al. (2002). Target

cells were prepared by pulsing splenocytes of naı̈ve C57BL/6 mice

with 9 lM of the influenza virus CTL epitope (PA224–233, SSLEN-

FRAYV) derived from the viral RNA polymerase at 37�C for

90 min. The cells were washed 3 times and then labelled with a high

concentration (2.5 lM) of 5-(and 6) carboxyfluorescein diacetate

succinimidyl ester (CFSE; Molecular Probes, Eugene, OR). Non

peptide-pulsed syngeneic spleen cells were labelled with a low con-

centration (0.25 lM) of CFSE. Equal numbers (1�107) cells of highand low CFSE-labelled target cells were injected by the intravenous

(tail vein) route in a volume of 200 ll into mice that had been

inoculated with lipopeptide-pulsed DCs or non-pulsed DCs 7 days

previously. The lipopeptide vaccine contained the CTL epitope

(PA224–233, SSLENFRAYV) linked to the Th epitope; KLI-

PNASLIENCTKAEL (Ghosh et al., 2001). Naı̈ve mice, mice that

had been injected with saline and mice that had been infected with

influenza virus strain X-31 received a similar mixture of CFSE-la-

belled cells. After 16 h spleens were removed from these animals and

analysed for the presence of CFSE high and CFSE low cells by flow

cytometry. Up to a total of 1�104 CFSE positive cells were analyzed

from each sample of splenocytes. The following formulae were used

to calculate specific lysis: ratio = (percentageCFSE low/percentage

CFSE high) and percentage of specific lysis = [1)(ratio for unim-

munized mice/ratio immunized mice)]�100].

Mice Used for Toxicological Analyses

Three groups of 20 C57BL/6 mice were obtained from the

breeding facility at The University of Melbourne. The mice were

individually tagged by ear marking to permit unequivocal identi-

fication throughout the duration of the experiment. The mice were

treated as follows:

Group 1-no treatment

Group 2-injected with 2�106 syngeneic murine DC, by the

intradermal and intravenous routes (1�106 cells by each route).

Group 3-injected in a similar manner with lipopeptide-pulsed

murine DC.

Group 1 mice were 6 weeks old whereas Group 2 and Group 3

mice were 8 weeks old. Groups 2 and 3 were injected with the

DCs on days T=0, T=14 and T=28. An overview of the

experimental plan is shown in Table I.

Rationale for Cell Dose

The cell dose used in the toxicology studies corresponded to a

dose that is 38.5 fold that of the lowest and 4.4 fold that of the

highest dose proposed for the clinical trial in humans. These figures

were calculated using the formula, body surface area of a hu-

man = 1.71 m2 and that of a mouse =0.007 m2. The number of

doses and the routes of administration reflect those to be tested

clinically in the patients with the highest dose schedules in the

proposed trial.

Behavioural Observation of the Mice

The mice were examined on a daily basis for any behavioural

changes, clinical symptoms or any changes in body weight. Seven

days after the third injection, deemed to be the most appropriate

point to detect any acute phase reactions, the mice were killed and

examined as described below.

Necropsy Studies

The mice were killed by CO2 asphyxiation on day 35. The

thorax was opened and any gross abnormalities noted. Blood

samples were taken by cardiac puncture while the heart was still

beating. Blood samples were collected into plain sterile tubes for

biochemical analysis and into heparin tubes for hematological

analysis. Blood smears were also prepared. The organs were re-

moved from each mouse by experienced individuals who each re-

moved specific organs in an assembly line-type process that was

supervised by a qualified veterinary pathologist. The organs were

collected into pre-weighed formalin pots and the weight of each

organ calculated.

The liver, spleen and skin at the injection site were removed

and fixed in neutral buffered formalin for histological processing.

The histological assessment was performed by a veterinary

pathologist, who was blinded to the identity of the 3 groups of

mice. Hematological and clinical biochemical analyses were per-

formed on each of the 10 mice from each group by an accredited

veterinary pathology company using standard protocols. In

addition, the kidneys, lungs, gut mucosa and inguinal lymph

nodes from each group were fixed in formalin and archived in the

event that histopathological changes were noted in any of the

samples.

Statistical Analysis

Statistical analysis was performed on three sets of data, viz. the

biochemical, hematological, and the daily determined mouse

weights as well as the organ weights determined at necropsy. All

Table I. Adoptive Transfer Schedule for Murine DC

Time

Dose schedule

Group 1a

No treatment

Group 2Syngeneicmurine DC

Group 3Lipopeptide-

pulsed syngeneicmurine DC

Day 0 No treatment 3.25�105 IVb 2.8�105 IV3.25�105 ID 2.8�105 ID

Day 14 No treatment 4.2�105 IV 4.2�105 IV4.2�105 ID 4.2�105 ID

Day 28 No treatment 3.5�105 IV 3.5�105 IV3.5�105 ID 3.5�105 ID

Day 35 Mice killed forbiochemical,haematologicaland histologicalexamination

a 20 mice were used in each of groups 1, 2 and 3.b IV= intravenous administration via the tail vein; ID = intra-dermal administration.

Toxicological Study of Lipopeptide-pulsed Dendritic Cells as an Anti-viral Therapy 225

statistical analyses were performed using SAS version 8.2 (SAS

Institute Inc., Cary, NC. USA). Each outcome variable was ini-

tially assessed for normality. Groups of three were assessed for

equal means using analysis of variance (ANOVA) and further

validated using a non-parametric Kruskal–Wallis test. As the dif-

ference between test groups was of primary importance, differences

between two groups were assessed using student t-tests and vali-

dated using a non-parametric Wilcoxon rank sum test. Results are

reported as means (standard errors). Because a large number of

outcomes were considered, to reduce the probability of a type I

error being committed, a two-sided p-value of 0.01 was considered

to be statistically significant.

RESULTS

The current licensed treatment for hepatitis C isrestricted to combination therapy with pegylatedIFN-a and ribavirin. Because this therapy is noteffective in all cases additional and novel therapiesare urgently required. We are investigating the po-tential of loading, and at the same time maturing Mo-DC, with HCV-specific lipopeptides in an attempt toinduce new or amplify existing cell-mediated immuneresponses to viral proteins. Such treatment we hopewill influence the course of infection in HCV carriersfollowing autologous transfusion. Antigen loadedDC have been used to treat a variety of tumours, andare now being used to treat infections (Nair andBoczkowski, 2002) with reports indicating that suchDC immunotherapy generates specific humoral andcell-mediated immunity with no adverse events beingreported (Kundu et al., 1998; Fazle Akbar et al.,2004; Lu et al., 2004; Chen et al., 2005; Garcia et al.,2005). The product that we propose to use viz.autologous DC, loaded with HCV-specific lipopep-tide vaccine candidates, has not been previously usedin man and the Therapeutic Goods Administration,the relevant regulatory body in Australia, has re-quired that we perform restricted toxicological anal-ysis on the product prior to trial in humans. Becausethere is no small animal model of HCV infection inwhich to examine the safety and efficacy of new im-munotherapies we have examined any adverse eventsrelated to the administration of a large dose of HCV-lipopeptide loaded murine DC in C57BL/6 mice.

Proof of Principle for Lipopeptides-pulsed DC

Anti-viral Therapy

Because there is no economical and easilyaccessible animal model for hepatitis C virus withwhich to evaluate anti-viral treatment, we assessedthe lipopeptide-pulsed dendritic cell therapeutic

approach using an in vivo cytotoxicity model basedon influenza virus (Jackson et al., 2004) and using theinfluenza-specific epitope SSLENFRAYV which isderived from the RNA polymerase of the virus.

Dendritic cells isolated from the spleens of micewere loaded with an SSLENFRAYV epitope-basedlipopeptide and transfused back into syngeneic ani-mals. These mice were then challenged with syngeneicspleen cells which had been fluorescently labelled andthen either loaded or not loaded with the CTL epitope.In this way, animals were challenged with target cellseither expressing or not expressing the viral epitope.

The results of the experiment (Fig. 1) demon-strate that animals that had been vaccinated withlipopeptide pulsed DCs were able to efficiently killtarget cells loaded with the viral epitope. In fact, theefficiency of target cell removal in these animalsparalleled that observed in mice which had beenpreviously infected with live influenza virus and thenallowed to recover. There was little or no removal oftarget cells from those animals which received un-treated DCs.

Fig. 1. Summary of specific lysis of epitope-pulsed target cells.Groups of three mice were inoculated intravenously with either 106

lipopeptide-pulsed DCs; 106 non-pulsed DCs; PBS only or with104 pfu X-31 influenza virus which was administered intranasally.Equal numbers of (i) syngeneic spleen cells pulsed with epitopeSSLENFRAYV and labelled with a high concentration of CFSEand (ii) syngeneic spleen cells labelled with a low concentration ofCFSE but not exposed to epitope, were injected into inoculatedmice and into naive mice 7 days later. After 16 h mice were killedand CFSE-labelled splenocytes were enumerated. Specific lysis ofthe two cell populations was calculated from the ratio non-pulsed/epitope-pulsed target cells in vaccinated or virus-infected micecompared with animals which received no treatment. Data arepresented as the mean and standard deviation for the three mice ineach group.


Toxicological Consequences of Vaccination

with Lipopeptide-pulsed DCs: Behavioural Changes

and Clinical Symptoms

The experimental result described above providesstrong evidence for the concept that viral infectionsare treatable using dendritic cell therapy.We thereforecarried out toxicological studies on recipient mice todetermine if any detrimental effects resulted from suchtherapy. In our clinical trial, we will use escalatingdoses of lipopeptide-loaded DC. The single total doseof syngeneic DC administered to mice therefore cov-ered the range of approximately 40–times the mini-mum dose to 4-times the maximum dose that isplanned for patients. Furthermore the murine DCwere inoculated by the same routes that are plannedfor the trial i.e. intravenous and intra-dermal andfurthermore followed a similar dosing schedule. Aftereach dose, we compared the behaviour and bodyweights of the three groups of mice on a daily basis.Those mice that received the lipopeptide-loaded DCshowed no adverse events or behavioural changeswhen compared with mice that received untreated DCor mice that received no treatment.

Daily Mouse Weights

The weights of mice in Groups 1, 2 and 3 weremeasured daily. The average weights of the mice in

each group did not differ significantly (Fig. 2). Thedata showed a normal distribution (Fig. 2 inset)permitting statistical analysis and show that thegroup which received no treatment (Group 1) and thegroup that received DCs only (Group 2) were statis-tically different (P=0.697). Furthermore the groupreceiving lipopeptide-pulsed DCs (Group 3) andGroup 1 were also statistically different (P=0.0036).These data are in all likelihood explained by thedifference in the age of the mice in Group 1. Therewas, however, no statistically significant differencebetween animals of Groups 2 and 3.

Procedure for Necropsies and Macroscopic

Examination

After each mouse was killed by CO2 asphyxiationit was pinned to a dissection board that contained themouse identification number prior to removal of anyorgan. This board and number accompanied themouse through each step of the procedure to ensurethat there were no errors in the identification ofsamples. All containers for biochemical, hematologi-cal and histological analyses were pre-labelled withthe mouse identification number and a double checkprocedure ensured that the identification numbers onthe dissection board and the sample containers wereidentical. In the case of organ weight determinations,containers were weighed prior to and after receiving

Fig. 2. Weights of individual mice measured on a daily basis following the three treatment paradigms. On the indicated days individualanimals were weighed and the results plotted as an average of members of the individual groups and showing standard error of the mean. Theinset shows the weights as a normal distribution indicating the validity of the statistical analysis.


the organ and weights determined by difference. Everymouse was examined macroscopically after openingand prior to dissection by a Veterinary Pathologist.No gross pathological changes were apparent.

Weights of Mouse Organs at Necropsy

The weights of the different organs are shown inFig. 3 and the normal distribution shown in the insetto the figure panels. The weights of the organs in miceof Group 1 were generally lower because these micewere younger. The data generally show a normaldistribution for all organs except in the case of theinguinal lymph nodes. Difficulties were encounteredin weighing this organ because it was so small andthese data are therefore not included in this assess-ment. When the three groups were compared, thedata show that there were significant differences inthe weights of the lungs, colon, liver and spleen, butnot in the kidneys. However, when only Groups 2and 3 were included in the comparison, only theweights of the liver and colon were found to be sig-nificantly different. In both cases, the organs of micereceiving lipopeptide-pulsed DCs (Group 3) werefound to be smaller than those in the group receivingDC only (Group 2). Although the liver was smaller,this difference was not reflected in the associatedhistology and liver function (see below).

Clinical Biochemistry Results

The raw data for Groups 1, 2 and 3 are shown inTables II–IV. Most markers showed a normal dis-tribution (data not shown), indicating that the datawere of sufficient quality to provide reliable statisticaldata. The three groups were analysed by ANOVAand Kruskal analysis; in these analyses, a figure of<0.01 was considered to be significant. The datashow that the only differences which were significantwere albumin, potassium and ALT (Table V). How-ever, when Group 2 (DC only) and Group 3 (lipo-peptide-loaded DCs) were compared directly, onlyone indicator, potassium, was found to be signifi-cantly different.

Mice from Group 2 and Group 3 showed elevatedlevels (9.667 mmol/l and 8.678 mmol/l respectively)of potassium over published normal values, but micein Group 1 also showed an elevated (9.74 mmol/l)reading. In this case, it appears that the addition ofthe lipopeptide resulted in a reduction of potassiumlevels (Table V).

Fig. 3. Weights of various mouse organs obtained from the dif-ferent groups at necropsy. Individual organs were weighed and themean plotted with the standard error of the mean. Group 1 (un-treated) open histogram; Group 2 (animals which received DCsonly) light grey histogram; Group 3 (lipopeptide-pulsed DCs) darkgrey histograms.


Table

II.Biochem

icalParametersofAnim

alsReceivingnoTreatm

ent,Group1

Na

KCl

Creat

Ca

Ict

Lip

Haem

Prot

Alb

Glob

TBil

Alk

Phos

ALT

GGT

AST

Mouse

#mmol/l

mmol/l

mmol/l

Na/K

mmol/l

mmol/l

Index

Index

Index

g/l

g/l

g/l

lmol/l

U/l

U/l

U/l

U/l

1151

8.7

104

17.4

0.04

2.96

Clear

+Clear

61

45

16

3199

33

272

20.04

58

43

15

139

30

<1

72

3149

9.7

106

15.4

0.03

3.1

Clear

Clear

Clear

73

42

31

2126

42

<1

103

40.04

55

41

14

109

29

<1

125

50.04

63

46

17

157

35

<1

103

60.04

62

45

17

215

39

<1

252

7151

9.3

107

16.2

0.04

3.07

Clear

Clear

Clear

58

43

15

4159

29

<1

96

8152

11.1

111

13.7

0.03

2.97

Clear

Clear

Clear

54

42

12

3147

25

179

9149

9.9

107

15.1

0.03

3.11

Clear

Clear

Clear

60

44

16

2144

27

180

10

0.04

59

43

16

134

29

<1

101

Ave

150.4

9.7

107

15.6

0.04

3.0

60.3

43.4

16.9

2.8

152.9

31.8

1.3

108.3

S.D

.1.3

0.9

2.5

1.4

0.00

0.1

5.3

1.6

5.2

0.8

32.2

5.4

0.6

53.2

Norm

alrange

156–165

5.0–7.6

92–106

0.04–0.05

46–54

28–33

47–85

26–54

74–232

Na:sodium;K,postassium;Cl,chloride;

Creat,Creatinine;

Ca,Calcium;Ict,Icterus;Lip,Lipaem

ia;Haem

,Haem

olysis;Prot,protein;Alb:albumin;Glob,globulin;

TBil:Totalbilirubin;Alk

Phos:alkalinephosphatase;ALT:alanineaminotransferase;GGT:gammaglutamyltransferase;AST:aspartate

aminotransferase.

Thedata

are

incomplete

dueto

thesm

allvolumeofbloodthatcould

becollectedfrom

each

mouse.

Table

III.

Biochem


alsReceivingDendriticCellsOnly,Group2

Na

KCl

Creat

Ca

Ict

Lip

Haem

Prot

Alb

Glob

TBil

Alk

Phos

ALT

GGT

AST

Mouse

#mmol/l

mmol/l

mmol/l

Na/K

mmol/l

mmol/l

Index

Index

Index

g/l

g/l

g/l

lmol/l

U/l

U/l

U/l

U/l

1150

9.3

108

16.1

0.03

3.0

Clear

Clear

Clear

59

43

16

4117

44

<1

109

20.04

63

111

39

<1

183

3 4149

10.0

109

14.9

0.03

2.9

Clear

Clear

Clear

62

43

19

4121

28

<1

90

5150

9.3

106

16.1

0.04

3.0

Clear

Clear

Clear

60

44

16

3106

38

1167

6150

9.0

107

16.7

0.03

3.0

Clear

+Clear

61

43

18

3106

23

578

70.04

62

44

18

142

42

<1

143

8165

10.8

113

15.3

0.04

3.1

Clear

Clear

Clear

71

49

22

4152

46

2161

90.04

61

43

18

137

33

<1

183

10

150

9.6

106

15.6

0.04

3.1

Clear

Clear

Clear

62

44

18

3109

35

1159

Ave

152.3

9.7

108.2

15.8

0.04

3.0

62.3

44.1

18.1

3.5

122.3

36.4

2.3

141.4

S.D

.6.2

0.7

2.6

0.6

0.01

0.1

3.5

2.0

1.9

0.5

17.1

7.5

1.9

39.6

Norm

alrange

156–165

5.0–7.6

92–106

0.04–0.05

46–54

28–33

47–85

26–54

74–232

Mouse

3diedpriorto

theorganharvestdate,hence

theabsence

ofdata.


Creat,Creatinine;


ia;Haem

,Haem

olysis;Prot,protein;Alb:albumin;Glob,globulin;TBil:Total

bilirubin;Alk


aminotransferase.

Thedata

are

incomplete

dueto

thesm


becollectedfrom

each

mouse.


Table

IV.Biochem


alsReceivingDendriticCellsPulsed

withLipopeptide,

Group3

Na

KCl

Creat

Ca

Ict

Lip

Haem

Prot

Alb

Glob

TBil

Alk

Phos

ALT

GGT

AST

Mouse

#mmol/l

mmol/l

mmol/l

Na/K

mmol/l

mmol/l

Index

Index

Index

G/l

g/l

g/l

lmol/l

U/l

U/l

U/l

U/l

1152

8.4

112

18.1

0.03

3Clear

Clear

Clear

65

43

22

4141

31

1252

2150

9.1

106

16.5

0.04

3.03

Clear

+’-

Clear

64

43

21

4101

29

<1

123

3151

8.6

109

17.6

0.04

3.02

Clear

Clear

Clear

59

42

17

3128

28

1168

40.04

61

42

19

143

33

<1

153

5150

8.9

109

16.9

0.03

2.79

Clear

Clear

Clear

55

38

17

4132

20

2107

6150

8.6

109

17.4

0.03

3Clear

Clear

Clear

62

43

19

3141

31

2192

7150

8.9

109

16.9

0.03

2.94

Clear

+Clear

60

42

18

3140

31

<1

136

8153

7.7

109

19.9

0.03

3.11

Clear

Clear

Clear

61

43

18

3165

34

2153

9148

9.2

107

16.1

0.03

2.87

Clear

Clear

Clear

58

39

19

3105

21

161

10

148

8.7

105

17

0.04

3Clear

Clear

Clear

58

42

16

2168

27

1135

Ave

150.2

8.7

108.3

17.4

03.0

60.3

41.7

18.6

3.2

136.4

28.5

1.4

148

S.D

.1.6

0.4

2.1

1.1

00.1

3.0

1.8

1.8

0.7

21.7

4.7

0.5

50.98

Norm

alrange

156–165

5.0–7.6

92–106

0.04–0.05

46–54

28–33

47–85

26–54

74–232


Creat,Creatinine;


ia;Haem

,Haem

olysis;Prot,protein;Alb:albumin;Glob,globulin;

TBil:Totalbilirubin;Alk


aminotransferase.

Thedata

are

incomplete

dueto

thesm


becollectedfrom

each

mouse.

Table

V.MeanParametersin

MicewhichShowed

StatisticallySignificantDifferencesbetweenGroups

Parameter

Norm

alrange

Group1

Group2

Group3

Whitebloodcells(�109/l)

6.5–13.8

8.1

(2.8)*

14.5

(3.0)

16.3(4.6)

Neutrophils(�109/l)

0.4–1.6

0.7

(0.3)

1.7

(0.4)

1.8

(0.7)

Neutrophils(%

)7–13

11.2

(3.9)

9.4

(3.8)

10.1

(3.4)

Lymphocytes(�109/l)

4.5–12.3

7.3

(2.7)

12.3

(2.7)

13.6

(4.2)

Lymphocytes(%

)72–94

87.2

(4.0)

85.8

(3.2)

84.7

(5.7)

Albumin

(g/l)

28–33

43.4

(1.6)

44.1

(2.0)

41.7

(1.8)

Potassium

(mmol/l)

5.0–7.6

9.7

(0.9)

9.7

(0.7)

8.7

(0.4)

*Standard

deviationsare

shownin

parentheses.


Table

VI.

Hem

atologicalParametersofAnim

alsReceivingnoTreatm

ent,Group1

Mouse

#RBC

�1012/l

Hb

g/l

PCV

MCV

fLMCH

pg

MCHC

g/l

WCC

�109/l

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

Platelets

�109/l

%�1

09/l

%�1

09/l

%�1

09/l

%�1

09/l

%�1

09/l

110–13

11

84

23

27–10

20

74

24

310–13

10

90

47–10

889

35

4–7

12

87

16

7–10

16

83

17

4–7

17

81

11

84–7

10

89

19

4–7

14

86

10

4–7

990

111

9.75

155

0.56

57

16

279

3.9

11

0.4

87

3.4

20.1

952

12

10.7

166

0.62

58

15

268

5.7

90.5

91

5.2

00

13

10.2

154

0.59

58

15

262

6.8

80.5

90

6.1

10.1

10.1

1311

14

10.3

152

0.58

57

15

261

6.4

10

0.6

89

5.7

10.1

1017

15

10.2

156

0.58

57

15

270

6.2

18

1.1

78

4.8

00

40.2

990

16

10.3

161

0.6

58

16

269

11.9

81

92

10.9

00

17

10.7

165

0.61

58

16

269

9.0

10

0.9

90

8.1

00

1022

18

10.3

152

0.58

57

15

261

9.6

80.8

91

8.7

00

10.1

19

9.61

151

0.58

61

16

259

12.6

81

91

11.5

10.1

673

20

10.2

157

0.6

59

15

262

9.2

60.6

91

8.4

30.3

895

Ave

10.2

156.9

0.6

58

15.4

266

8.1

11.2

0.7

87.2

7.3

1.1

0.1

30.2

10.1

980

S.D

.0.3

5.4

0.02

1.2

0.5

6.1

2.8

3.9

0.3

4.9

2.7

10.1

1.4

00

189.3

Norm

al

range

9.40–11.80

130–190

0.40–0.63

47–51

11–20

240–360

6.5–13.8

7–13

0.4–1.6

72–94

4.5–12.3

1.5–3.7

<0.4

1–2.5

<0.3

908–1792

RBC,Red

bloodcells;Hb,haem

oglobin;PCV,packed

cellvolume;

MCV,meancellvolume;

MCH,meancellhaem

oglobin;

MCHC,meancellhaem

oglobin

concentration;WCC,whitecellcount.

Thedata

are

incomplete

dueto

thesm


becollectedfrom

each

mouse.


Table

VII.Hem


alsReceivingDendriticCellsOnly,Group2

Mouse

#RBC

�1012/l

Hb

g/l

PCV

MCV

flMCH

pg

MCHC

g/l

WCC

�109/l

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

Platelets

�109/l

%�1

09/l

%�1

09/l

%�1

09/l

%�1

09/l

%�1

09/l

17–10

11

83

62

10–13

492

13

3 47–10

786

16

510–13

687

16

613–18

690

47

13–18

586

27

813–18

784

45

910–13

586

27

10

10–13

588

16

11

9.9

159

0.61

62

16

260

17

12

285

14.4

30.5

738

12

10.2

162

0.59

58

16

273

18.1

12

2.2

87

15.7

10.2

13

10.4

165

0.61

59

16

269

15.7

91.4

83

13

20.3

60.9

14

10.4

157

0.59

57

15

267

19.2

13

2.5

86

16.5

10.2

15

9.82

156

0.56

57

16

277

13

11

1.4

88

11.4

00

10.1

16

10.1

158

0.59

59

16

268

9.1

19

1.7

81

7.4

00

17

10.3

152

0.59

57

15

257

12.5

13

1.6

78

9.8

10.1

81

18

10.1

157

0.6

59

16

261

12.8

11

1.4

84

10.8

00

50.6

19

10.1

151

0.58

57

15

262

13.3

11

1.5

88

11.7

10.1

20

10

149

0.58

58

15

255

14.4

11

1.6

88

12.7

10.1

Ave

10.1

156.6

0.6

58.3

15.6

264.9

14.5

9.4

1.7

85.8

12.3

1.3

0.2

5.4

0.7

738

S.D

.0.2

4.9

0.01

1.6

0.5

7.1

3.0

3.8

0.4

3.2

2.7

1.0

0.16

1.9

0.4

Norm

al

range

9.40–11.80

130–190

0.40–0.63

47–51

11–20

240–360

6.5–13.8

7–13

0.4–1.6

72–94

4.5–12.3

1.5–3.7

<0.4

1–2.5

<0.3

908–1792

Mouse

3diedpriorto

theorganharvestdate,hence

theabsence

ofdata.

RBC,Red

blood

cells;

Hb,haem

oglobin;PCV,packed

cellvolume;

MCV,mean

cellvolume;

MCH,mean

cellhaem

oglobin;MCHC,mean

cellhaem

oglobin

concentration;

WCC,whitecellcount.

Thedata

are

incomplete

dueto

thesm


becollectedfrom

each

mouse.


Table

VIII.

Hem


alsReceivingDendriticCellsPulsed

withLipopeptide,

Group3

Mouse

#RBC

�1012/l

Hb

g/l

PCV

MCV

fLMCH

pg

MCHC

g/l

WCC

�109/l

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

Platelets

�109/l

%�1

09/l

%�1

09/l

%�1

09/l

%�1

09/l

%�1

09/l

112

84

13

24

91

14

36

94

49

85

15

512

85

12

67

88

14

78

86

15

817

76

25

97

84

27

10

986

511

9.69

149

0.59

61

15

251

24.8

92.2

91

22.6

00

12

9.91

157

0.59

60

16

263

21.2

17

3.6

74

15.7

10.2

81.7

13

10.5

163

0.61

58

16

266

20.4

91.8

90

18.4

10.2

14

10.6

155

0.61

58

15

252

13.9

10

1.4

89

12.4

10.1

15

9.95

158

0.6

60

16

266

12.3

10

1.2

89

10.9

10.1

16

9.81

158

0.58

59

16

272

16

11

1.8

79

12.6

20.3

81.3

17

10.1

154

0.59

58

15

262

16.5

91.5

75

12.4

11

0.2

15

2.5

18

9.79

153

0.58

60

16

264

14

12

1.7

78

10.9

10.1

91.3

19

9.44

149

0.57

60

16

262

13.7

15

2.1

84

11.5

10.1

20

9.9

153

0.58

59

16

264

10

90.9

85

8.5

10.1

50.5

Ave

9.97

154.9

0.6

59.3

15.7

262.2

16.3

10.1

1.8

84.7

13.6

1.8

0.1

6.2

1.5

S.D

.0.4

4.3

0.01

1.1

0.5

6.3

4.6

3.4

0.7

5.7

4.2

2.4

0.1

3.4

0.7

Norm

al

range

9.40–11.80

130–190

0.40–0.63

47–51

11–20

240–360

6.5–13.8

7–13

0.4–1.6

72–94

4.5–12.3

1.5–3.7

<0.4

1–2.5

<0.3

908–1792

RBC,Red

bloodcells;

Hb,haem

oglobin;PCV,packed

cellvolume;

MCV,meancellvolume;

MCH,meancellhaem

oglobin;MCHC,mean

cellhaem

oglobin

concentration;

WCC,whitecellcount.

Thedata

are

incomplete

dueto

thesm


becollectedfrom

each

mouse.


Hematological Results

The raw data for Groups 1, 2 and 3 are shown inTables VI–VIII respectively. Again the valuesshowed normal distributions (data not shown), indi-cating that the data are of sufficient quality to providereliable statistical data. Analysis of the data byANOVA and Kruskal analysis showed that therewere no significant differences between the 3 groups.In these analyses, a figure of <0.01 was considered tobe significant and on this basis, the values for whiteblood cells, neutrophils and lymphocytes were con-sidered to be significantly different. However, manyof these values result from differences between theGroup 1 and the Group 2/3 mice, probably becausethe Group 1 mice were younger, and when the Group2 (DC only) and Group 3 (DC plus lipopeptide) micewere compared directly, only the albumin levels werefound to be significantly different.

The levels of albumin in each of the three groupswere higher than the published normal range of val-ues (28–33 g/l). The reason for this is unclear. Thedata show that Group 2 mice (which received the DConly) had a slightly more elevated reading than theGroup 3 mice (which received lipopeptide-loadedDCs), indicating that the lipopeptides per se had notoxic effect. The parameters which were statisticallysignificant are highlighted in Table V.

Histological Results

The histological diagnoses for Groups 1, 2 and 3were made by a veterinary pathologist. As might beexpected, the histology in Group 1 (naı̈ve mice) wasnormal whereas, although the liver and spleen sam-ples from the Group 2 (DC only) and Group 3 (DCplus lipopeptide) mice were normal, the injection sitesin these mice showed a focal mild eosinophilia thatwas similar in both groups (data not shown). Thus,the mild eosinophilia resulted from the injection ofthe DC per se but not from the addition of the HCVlipopeptides to the cells prior to injection.

We conclude that neither lipopeptides per se norlipopeptide-loaded DC are toxic. This conclusion issupported by results of our earlier studies (Zenget al., 2002; Jackson et al., 2004) where no adverseeffects were apparent in animals which received avariety of lipopeptide vaccine candidates.

CONCLUDING REMARKS

DC immunotherapy is an exciting and relativelynovel treatment regimen and is currently at a cross-road facing considerable challenges in order to ad-vance further and realize its potential (Steinman andMellman, 2004). We believe that the results of thepresent study provide optimism for a next develop-mental step in immunotherapeutic approaches usingDCs. The capacity of the lipopeptide vaccines thathave been used here are capable of not only targetingDCs through Toll-like receptors that are present onthe surface of DCs, but also of activating them(Jackson et al., 2004). These two properties providethis vaccine strategy with an edge over existing DC-based therapies which do not incorporate deliverystrategies specific for dendritic cells and which mayrequire additional and extraneous components inorder to induce maturation of DCs. The results of thecurrent toxicological study has paved the way for ourproposed study in HCV positive patients and there issome optimism for the elimination of virus from or-gans, especially the liver, which is accessible to infil-trating lymphocytes elicited in an epitope-specificmanner by lipopeptide-loaded DCs.

Conflict of Interest

Georgia Deliyannis, Irene Dinatale and MichaelRizkalla are employees of VacTX Pty. Ltd. a bio-technology company that is seeking to commercialiselipopeptide technology for use in vaccines. David C.Jackson is retained as a consultant to VacTX Pty.Ltd.

ACKNOWLEDGMENTS

We thank many members of the staff in the Department of

Microbiology and Immunology, The University of Melbourne for

invaluable help in processing the mouse samples. Our special

thanks go to Maria Kaparakis, Brendon Chua, Katherine Ked-

zierska and Joleen Rose. David Woollard of the Burnet Institute

also provided valuable help. We also thankMichael Bailey, Dept of

Epidemiology and Preventative Medicine, Monash University for

the statistical analysis. This study was funded by grant number 1

RO1A1054459-01 from the National Institutes of Health, Beth-

esda, USA.


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