University of Groningen

Anti-cytomegalovirus applications of the intrinsically active drug carrier lactoferrinvan der Strate, Barry Willem Albertus

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173

9

Treatment of Cytomegalovirus-infected rats with

recombinant human Lactoferrin.

B.W.A. van der Strate1, L. Beljaars1, H.I. Bakker1, C. Smit1,A.M. van Loenen-Weemaes1, D.K.F. Meijer1, G. Molema1,2 and M.C. Harmsen2

1. GUIDE, Dept. Pharmacokinetics & Drug Delivery.2. GUIDE, Dept. Pathology and Laboratory Medicine, Medical Biology Section

University Hospital Groningen.

Submitted

Chapter 9174

ABSTRACT

Lactoferrin (LF) is a glycoprotein that plays an important role in the non-specific immune system because of its antimicrobial properties. In this study, weevaluated the antiviral activity of LF employing two in vivo models for ratcytomegalovirus (RCMV) infection.

In our cell free virus (CFV) infection model, rats were treated weekly withsingle doses of ganciclovir or cidofovir intraperitoneally, or three subsequentdoses of recombinant human LF (rHLF) intravenously, for 4 weeks. In ourrecently developed leukocyte mediated dissemination (LMD) model for RCMV,rats were treated with cidofovir i.p., rHLF i.v., or a combination of cidofovir i.p.with rHLF i.v. for 4 weeks.

At least a thousandfold reduction in virus titres in salivary glandsconfirmed antiviral activity of cidofovir in both models. In the CFV-model,treatment with 40 and 160 mg/kg rHLF i.v. resulted in a tenfold reduction in virustitres, whereas ganciclovir displayed no antiviral activity. However, in the LMD-model, no antiviral activity of rHLF was observed. In addition, combinationtherapy of cidofovir with rHLF in this model did not show synergistic activity. AnELISA was used to detect rHLF-specific antibodies in the sera of all animalstreated with rHLF. Immunohistochemical studies provided further evidence forantigenicity of rHLF in rats. This induction of rHLF-antibodies may contribute tothe low antiviral effect of rHLF.

In conclusion, rHLF displayed moderate antiviral activity in the CFV-model. However, in both models therapy with rHLF was complicated by thedevelopment of antibodies in the treated animals.

Chapter 9 175

INTRODUCTION

Human cytomegalovirus (HCMV), a ß-herpes virus, is a complicatingfactor in immunocompromised subjects such as AIDS patients and transplantrecipients. In these patients, reactivation or primary infection with HCMV cancause severe morbidity or even mortality 1,22,38,39. Currently, HCMV-infections aretreated with conventional antiviral drugs, such as ganciclovir, cidofovir orfoscarnet. However, when continuous therapy is required, therapeutic success islimited due to severe side effects and the development of drug resistance2,6,9,21,28,40. Therefore, combination therapy, in which antiviral drugs with differentmechanisms of action are used together, may be beneficial for these patients. Thesimultaneous use of different drugs may lead to a more efficient inhibition of viralreplication and may also prevent or at least delay the development of drugresistance. As shown previously, proteins can be modified in order to obtainvarious several drug carriers for the selective delivery of antiviral drugs into targetcells. Such sugar- or charge-modified proteins may even exhibit antiviral effectsthemselves. This dual approach may lead to a more specific delivery of drugs,resulting in fewer side effects and possibly synergism 13,16,20.

Lactoferrin (LF), an 80 kD glycoprotein, is thought to play an importantrole in the innate immune system since it comprises antimicrobial properties.Antibacterial and antifungal effects of LF in vitro and in vivo have been described17,18,36. In addition, LF has been found to be antivirally active against a wide rangeof viruses, including HIV 12,23,33.

LF has antiviral activity against CMV of different species in vitro and invivo. The antiviral activity against HCMV in vitro has been described extensively4,12,27,33. In a mouse model for CMV, therapy with LF proved to be protectiveagainst a lethal challenge with this virus. In this model, LF not only exerted adirect antiviral effect; but also stimulated NK-cell activity, which proved essentialto clear the virus infection 27. Similar augmenting effects of LF on the immunesystem have been described previously 5,7,25.

Earlier in vitro experiments have demonstrated a synergistic effect of thecombination of LF with cidofovir against HCMV 35. Synergy implies that thecombination of different drugs at a certain concentrations leads to a more efficient

Chapter 9176

virus inhibition as expected from the individual dose-response curves of the singledrug.

In studies on the pharmacokinetic characteristics of rHLF in rats, weshowed that rHLF distributes to regions that are prone to CMV infection 3. rHLFdistributed to endothelial cells in liver as well as in other organs. In addition,accumulation in the lymphatic system and binding of rHLF to membranes ofinfiltrating leukocytes was observed.

The aim of our study was to evaluate the antiviral activity of LF in twodifferent models for rat CMV (RCMV) infection. The first model, adapted fromearlier studies 29, employs intraperitoneal injection of cell free virus (CFV). In thesecond model, which was recently developed in our group 34, RCMV-infectedleukocytes serve as the inoculum. This leukocyte mediated dissemination model(LMD-model) is possibly a more physiological model for virus dissemination,since during active HCMV infections infected granulocytes and monocytes arebelieved to be vehicles for virus particles, mediating dissemination of the virus10,11,14,24,37. In both models, animals were subjected to immune suppression by totalbody irradiation (TBI). At 4 weeks post infection, animals were sacrificed and theantiviral effect of rHLF and the conventional antiviral was evaluated using aplaque assay and histological staining.

MATERIALS & METHODS

Production of virus.Virus stocks were produced as described earlier 19. In brief, male specified

pathogen free AO rats (Harlan, Zeist, The Netherlands), 8 weeks of age, receivedtotal body irradiation (5 Gy) and were infected i.p. with 106 plaque forming units(pfu) of the Maastricht strain RCMV, 6 h after irradiation. Animals were held atthe Central Animal Facility of the Groningen University and received standardchow and water ad libitum. Four weeks after the administration of virus, rats weresacrificed and the submandibular salivary glands were removed. The salivaryglands were homogenised, sonified and centrifuged as described 19. Virus stockswere stored at –80°C. A plaque assay was used to quantify the titre of virus in the

Chapter 9 177

homogenate. All animal experiments described in this study were approved by thelocal Ethical Board for Animal Studies.

Antiviral compounds.Cidofovir (Vistide, Pharmacia & Upjohn S.A., Luxembourg),

ganciclovir (Cymevene, Roche Nederland B.V., Mijdrecht, The Netherlands)and recombinant Human Lactoferrin (rHLF, Numico Research B.V., Wageningen,The Netherlands) were reconstituted according to manufacturer’s advice anddiluted in pyrogen free saline for injection.

Infection of rats with cell free RCMV.Male F344-rats (Harlan), at the age of 4 weeks, were injected i.p. with 1x

106 pfu of the AO virus stock with, or without 5 Gy TBI. The virus was injected16 h after irradiation of the rats. The animals were treated with ganciclovir (50mg/kg, three times weekly i.p., n = 4), cidofovir (25mg/kg, once weekly i.p., n =4), or rHLF (4, 40, 160 mg/kg, three times weekly i.v., n = 3) A control group ofanimals were not treated after infection with RCMV (n = 3).

At 4 weeks post infection, the animals were sacrificed. Sera were obtainedby heart puncture and the submandibular salivary glands and spleen were removedfor determination of the virus titres, using a plaque assay, and for histology.Furthermore, the pancreas, heart, lung, liver, colon and kidney were removed forhistological evaluation.

LMD-model: Generation of RCMV-infected mononuclear cells (MNC).The rat lung fibroblast cell line RFL-6 (ECACC, UK) was cultured in 162

cm2 culture flasks (Corning Costar, Cambridge, UK), using DMEM/HAM F12(Biowhittaker Europe, Verviers, Belgium), supplemented with 20% Fetal CalfSerum (FCS) (Brunschwig Chemie, Maarssen, The Netherlands), 200mM L-glutamin (Gibco BRL, Paisley, Scotland), 60 µg/ml gentamycine sulphate (Gibco)and 1% Non Essential Amino Acids (Gibco) at 37°C and 5% CO2. Prior toinfection, cells were seeded in 6-wells plates (Coring Costar) and gradually serumstarved and maintained on culture medium with 5% FCS. Subsequently, RCMVwas added at a multiplicity of infection of 1.0.

Chapter 9178

Male PVG rats (Harlan), 8 weeks of age, were anaesthetised and amaximal amount of blood was obtained with a heart puncture. Subsequently, theanimals were terminated. Blood was collected in heparinised tubes, mixed in equalvolumes with Phosphate Buffered Saline (PBS) pH 7.4 and loaded on lymfolyteRat (Cedarlane Laboratories, Ontario, Canada). Cells were centrifuged at 2,500rpm for 30 min. The MNC fraction was retrieved and kept on ice. Contaminatingerythrocytes were removed by hypotonic lysis with ice-cold buffer, consisting of155mM NH4Cl, 10mM KHCO3, and 0.1mM Na2EDTA.

The MNCs were washed and cocultivated with infected RFL-6 cells at37°C, 5% CO2 and 100% relative humidity for 2 h. The MNCs were removedfrom the infected fibroblast layer by gentle aspiration and transferred to emptytrans-well plates (Corning Costar). After migration for 16 h on a chemotacticgradient of 1.0 µM histamin (Sigma, St. Louis, MO), the transmigrated MNCs inthe lower chamber were washed twice with saline to remove cell-free virus,counted in trypan blue, diluted in physiological saline and stored on ice untilfurther use.

LMD-model: infection protocol.Male PVG rats (Harlan) received 5 Gy TBI and were injected in the tail

vene with 40,000 RCMV-infected MNCs 16 h after irradiation. Animals weretreated with cidofovir (25 mg/kg, once weekly, i.p.), rHLF (40 mg/kg, three timesweekly, i.v.) or a combination of cidofovir (25 mg/kg, once weekly, i.p.) plusrHLF (40 mg/kg, three times weekly, i.v.). As a positive control, one group ofanimals was injected i.v. with vehicle three times weekly, after infection withRMCV-infected MNCs. All experimental groups consisted of 4 animals. Theanimals were sacrificed at 4 weeks post infection. Sera were obtained by heartpuncture. Subsequently, submandibular salivary glands and spleen were removedand homogenised for determination of the virus titres using the plaque assay, andfor histology. Furthermore, the pancreas, heart, lung, liver, colon and kidney wereremoved for histological evaluation. The organs were frozen in isopentane andsubsequently stored at –80°C until further use.

Chapter 9 179

Histochemical evaluation.Organs were fixed in 3.7% formalin solution in PBS, pH 7.4 for 24 h, and

subsequently embedded in paraffin, according to standard methods. Sections of4µm were incubated with monoclonal antibody (Mab) 8 or 35 8 to assess thepresence of viral antigens. Mab 8 was used to detect Early antigens, whereas Mab35 detects Late antigens. Subsequently, Rabbit-anti-Mouse peroxidase (DAKO,Glostrup, Denmark) was added and detection was achieved using amino-ethyl-carbazole (Sigma). Counterstaining was achieved with hematoxillin. In addition,sections of 4 µm were stained with hematoxillin and eosin, according to standardmethods.

Standard indirect immunohistochemical staining with monoclonalantibodies directed against CD4 (w3/25, ), CD8 (EG-8, ), CD3 (OX-19,), B-cells(His-24) or LF (DAKO) were performed on 4 µm cryosections of the collectedorgans. The sections were counterstained with hematoxillin and embeddedaccording to standard procedures.

Quantification of virus titres using plaque assay.The RCMV titres in salivary gland and spleen homogenates were

quantified using a plaque assay as described before 26. In brief, tenfold serialdilutions of tissue homogenates were added to a confluent layer of Rat EmbryonalFibroblasts (REF)32, which were cultured in 24-well plates, and incubated for 1 h.Subsequently, the inocula were removed and replaced by culture mediumsupplemented with 1% hydroxypropyl methyl cellulosum (Brocacef, Maarssen,The Netherlands). Plates were cultured at 37°C and 5% CO2 for 7 days, followedby fixation of the cells with 3.7% formalin in PBS. The cells were stained withmethylene blue and the number of virus plaques was evaluated microscopically.The virus titre was expressed as plaque forming units (pfu) per gram tissue.

Detection of LF antibodies in serum of RCMV-infected rats.Microtitre plates (Maxisorb, Costar) were coated with 10µg/ well of rHLF

in 0.1M Carbonate coating buffer (pH 9.6) and incubated at room temperature for16 h. Subsequently, the plates were washed five times with wash buffer (0.015MNaCl, 0.05 M Tris/HCl and 0.05% Tween-20, final pH 8.0). Twofold serialdilutions of sera, starting at a tenfold dilution, in incubation buffer (0.05M

Chapter 9180

Tris/HCl, 0.05% Tween-20 and 2% bovine serum albumin) were added to a finalvolume of 100 µl per well and incubated at room temperature for 1 h.Subsequently, plates were washed with wash buffer, and 100 µl Rabbit-anti-Rattotal Ig-HRP (DAKO), 4000-fold diluted in incubation buffer, was added to eachwell. The plates were incubated at room temperature for 30 min., after whichstaining was performed with 100µl of ortho-phenylene-diamine (OPD) solution(Sigma) per well. The staining reaction was stopped by adding 100 µl of 1 MH2SO4 to each well. Finally, the optical density at 490nm was measured with aplate reader, equipped with SOFTMAX software.

Statistics.Mann-Whitney U tests were performed to detect significant differences in

virus titres between the different groups of animals. Differences were consideredstatistically significant when p < 0.05.

RESULTS

Virus titres in salivary glands: Cell Free Virus (CFV) model.Virus titres in the salivary glands of rats infected with cell free virus were

determined in a plaque assay and expressed as the number of pfu/g tissue (Fig. 1).In the group of irradiated animals, treatment with the conventional anti–CMV drugcidofovir resulted in a significant decrease of virus titres (p < 0.001), whereasganciclovir treatment did not affect the virus titres, as compared to the controlgroup. In the group of irradiated rats that was treated with rHLF a dose dependenteffect of rHLF was observed. Administration of 4 mg/kg rHLF was not successful,whereas therapy with 40 or 160 mg/kg resulted in a significant reduction of virustitres in comparison with the non-treated group (p < 0.01).

Chapter 9 181

gcv

Vis

non lf4 lf40

lf160 gcv

Vis

non lf4 lf40

lf160

102

103

104

105

106

107

108

109

+ TBI - TBI* **

***

# pf

u/ m

l

Fig. 1: Virus titres in salivary glands of RCMV-infected rats in the cell-free virus model. Rats, withor without total body irradiation (TBI) were infected with 1x 106 pfu RCMV intraperitoneally. The rats weretreated with ganciclovir (50 mg/kg), cidofovir (25 mg/kg), or rHLF (4, 40, 160 mg/kg). As a control, one group ofrats was not treated after infection with RCMV.

In the non-irradiated animals the virus titres in all groups of animals wereconsiderably lower as compared to the irradiated animals. In these animals, noneof the conventional antivirals that were used in this study were capable ofdecreasing virus titres in the salivary glands. Although variation in virus titres inthese groups was rather high, treatment with rHLF in all doses did not exert anyantiviral effect. Probably, the virus titres in the untreated animals were not highenough for a proper evaluation of an antiviral effect of the tested compounds.

Virus titres in salivary glands: Leukocyte Mediated Dissemination (LMD)model.

The virus titres of the animals that were infected with RCMV-infectedMNCs are shown in Fig. 2. Again, therapy with the conventional anti-CMV drugcidofovir resulted in a significant decrease in virus titres (p < 0.01). Virus titres inthe cidofovir treated group were approximately 10,000-fold lower as compared tothe non-treated group. Although in the CFV-model treatment with 40 mg/kg rHLFresulted in a tenfold reduction in salivary gland virus titres, treatment with thisdose in the LDM-model did not affect viral replication. In addition, combinationof rHLF with cidofovir did not result in a more potent inhibition of virus, ascompared to treatment with cidofovir alone.

Chapter 9182

NON Vistide LF Vistide +LF102

104

106

108

***

# pf

u/ g

tiss

ue

Fig. 2: Virus titres in salivary glands in RCMV-infected rats in the leukocyte-mediateddissemination model. Rats were injected with RCMV-infected MNCs, 16 h after TBI. The rats were treatedwith cidofovir (Vistide, 25 mg/kg), rHLF (40 mg/kg), or a combination of cidofovir and rHLF. As a control, onegroup of rats was not treated after infection with RCMV.

(Immuno)histochemical evaluation.Histochemical evaluation of hematoxillin/eosin stained paraffin embedded

sections of salivary glands revealed the presence of dense leukocyte infiltrates and“owl’s eyes”, which are a characteristic feature for a CMV-infection (Fig. 3a).Staining with Mab 8 or 35 did not reveal infected sites other than the secretorytubules in the salivary glands. The leukocyte infiltrates were further characterisedusing specific antibodies on cryosections of salivary glands. The majority of theleukocyte infiltrates consisted of CD8-positive cells (Fig. 3b). Also B-cells wereprominent in the infiltrate (Fig. 3c). Monocytes and granulocytes were virtuallyabsent in these sections. No differences in composition of infiltrating cells in thedifferent groups of rats were found, although the total amount of “owl’s eyes” andinfiltrates was lower in the cidofovir treated groups and in all non-irradiatedanimals.

Chapter 9 183

Fig. 3: (Immuno)histochemical evaluation of RCMV infection. A: Hematoxillin/eosin staining of aparaffin embedded section, showing RCMV-infected cells (“Owl’s eyes”, indicated by arrows) in the secretorytubules of a salivary gland. Magnification 200x. B: Immunohistochemical staining (EG-8) on a cryosection forthe presence of CD8-positive cells in the inflammatory infiltrate in the salivary glands of RCMV-infected rats.Magnification 200x. C: Immunohistochemical staining (His-24) on a cryosection for the presence of B-cells cellsin the inflammatory infiltrate in the salivary glands of RCMV-infected rats. Magnification 200x. D:Immunohistochemical staining for hLF on a cryosection of a kidney from a RCMV-infected rat, treated withrHLF. A typical mesangial pattern is observed. All animals were sacrificed 48 h after the last gift of rHLF.Magnification 400x. E & F: Immunohistochemical staining on a serial cryosection of a spleen from an RCMV-infected rat, treated with rHLF for the presence of LF (E) and B-cells (F). Note the typical colocalization in thefollicle of both LF and B-cells. Magnification 400x.

Chapter 9184

Using immunohistochemical staining, LF was detected on the membranesof infiltrated leukocytes, a result that is consistent with earlier data 3. Furthermore,in the glomeruli of rHLF treated rats, LF bound to the mesangial cells (Fig. 3d).This staining pattern was not observed in an earlier set of experiments wheresingle doses of LF were injected for pharmacokinetic studies 3. In addition,staining of serial sections of spleen for B-cells and LF revealed presence of bothantigens in follicles in the same region of the spleen (Fig. 3e & f). This stainingpattern was observed in all animals that were treated with rHLF, in both models.However, the staining for LF in the glomeruli and spleen follicles was morepronounced in the non-irradiated group.

Detection of anti-rHLF antibodies in serum of RCMV-infected rats.Since immunohistochemical staining indicated antigenicity of rHLF after

repeated dosage in both models, an ELISA was developed to detect the presenceof antibodies in the sera of RCMV-infected rats. Sera of uninfected and untreatedrats, or rats treated with cidofovir or ganciclovir, did not react with rHLF that wascoated in the microtitre plates, indicating that cross reacting antibodies wereabsent in these sera. However, in the sera of all rats treated with rHLF, antibodiesagainst rHLF were detectable (Fig. 4). Interestingly, the titres of sera of irradiatedrats were comparable to titres of non-irradiated animals. This implies that within 4weeks after irradiation, the immune system was properly reconstituted to mount ahumoral response to rHLF. Furthermore, 4 mg/kg rHLF already elicited themaximal humoral response, because the levels of anti-LF antibodies were similarin animals treated with 4, 40 or 160 mg/kg rHLF respectively.

Chapter 9 185

0 1 2 3 40

1

2BlancoNON +NON -GCV +GCV -

Log dilution

OD

490

nm

0 1 2 3 40

1

2Lf 40 +Lf 40 -

Log dilution

OD

490

nm

Fig. 4: Anti-rHLF antibodies in serum of RCMV-infected rats treated with rHLF. An ELISA wasused to detect serum antibodies against rHLF in rats. Antibodies in sera of all the rats treated with rHLF weredetectable, whereas serum antibodies of uninfected rats and rats that were not treated with rHLF, did not showany reactivity.

DISCUSSION

Side effects and the development of drug resistance complicate continuoustreatment with anti-CMV drugs 2,6,9,21,28,40. Combination therapy or the use ofintrinsically active carrier molecules, coupled with conventional antivirals, may bea solution to this problem. LF has antiviral activity against CMV in vitro 4,12,27,33

and may be a useful candidate for combination therapy. Furthermore, its preferredlocalisation at the endothelial cells after i.v. administration 3 may be exploited fordrug targeting strategies 13,16,20. The antiviral effect of rHLF, either alone or incombination with conventional antiviral drugs against RCMV was evaluated invivo, using two different models for RCMV infection.

In both models described in this study, the antiviral effect of cidofoviragainst RCMV 15,29 could be confirmed. However, the antiviral effect ofganciclovir, a conventional antiviral that is used for active HCMV infections,could not be established. These data are in contrast with another study, whereganciclovir, administered in combination with hyper immune serum, was claimedto be antivirally active against RCMV 30,31. The major differences with thisexperiment and ours were the fact that BN-rats, instead of F344- or PVG-rats,

Chapter 9186

were challenged with a lethal RCMV infection and received a higher amount ofTBI.

In our cell free virus (CFV) model, treatment with rHLF resulted in atenfold decrease in virus titres. This moderate, yet statistically significant, antiviraleffect of rHLF could be explained by the fact that the dosage regimen may nothave been optimal. Pharmacokinetic studies with 125I-labeled rHLF in rats haveshown that with a dose of 160 mg/kg rHLF saturating kinetics enable theestablishment of prolonged high plasma levels, at least for a few hours 3. Itremains to be ascertained however, that rHLF levels in plasma upon multipledosing will be maintained at high enough concentrations throughout theexperiment to inhibit RCMV optimally.

In addition, our antiviral therapy with rHLF appeared to be complicated bythe development of antibodies against rHLF. More evidence for the antigenicity ofrHLF was found after immunohistochemical staining for rHLF. Rats and dogs arethe only known mammals that do not excrete LF in breast milk, and have onlysmall amounts of LF in their granulocytes 17,18,36. This virtual absence of LF inrats, besides the human origin of the LF, may make rHLF additionally antigenic.It is possible that the developed serum antibodies against rHLF bind to rHLF afteri.v. administration. This binding of antibodies to rHLF could result in theformation of complexes that precipitate in the glomerulus, causing the mesangialstaining pattern that we observed in the present study. Moreover, the binding ofantibodies to rHLF will certainly change the pharmacokinetic behaviour of rHLF,as a result of binding and uptake of the antibody complexes by macrophages. Thismay result in plasma levels that are not sufficient to inhibit RCMV in vivo.

As discussed above, pharmacokinetic studies after repeated dosage ofrHLF may reveal this altered pharmacokinetic behaviour of rHLF. Plasma LFlevels were measured in the sera obtained during obduction of the rats (72 h afterthe last injection of rHLF) and rHLF was still present in the ng/ml range (data notshown).In the LMD-model, rHLF did not protect against the development of systemicRCMV-infection. It is possible that the antiviral effect of rHLF in this model iscomplicated by the fact that infectious virus is cell-bound. Although in vitrostudies have shown that LF, in a concentration of 1 mg/ml, is capable of inhibitingcell-bound viral infection by 50% 14, this effect is probably not sufficient to result

Chapter 9 187

in a decrease of virus titers of the infected animals. In addition, the local rHLFconcentrations could have been too low to ensure adequate inhibition of virustransmission. It is likely, that infected MNCs, after i.v. administration, circulatethrough the body via the bloodstream. However, it cannot be excluded that theMNCs also migrate through the lymphatic system. Although HLF can be detectedin the lymphatic system after i.v. administration 3, the concentrations may notreach a level that is sufficiently high to inhibit virus transmission by infectedMNCs in the lymphatic system for a longer time period. In addition, it remains tobe established whether the development of anti-LF antibodies interferes with theaccumulation of rHLF in the lymphatic system.

Although earlier in vitro experiments have demonstrated a synergisticeffect of the combination of LF with cidofovir against HCMV 35, in our in vivostudy in the LMD-model no additional decrease of virus titres was observed aftercombination therapy of rHLF with cidofovir: the inhibition of RCMV withcidofovir alone was similar to inhibition with the combination of cidofovir andrHLF. Since therapy with rHLF alone failed to result in a decrease in virus titres, itis reasonable to assume that no synergistic activities of rHLF with cidofovir areobserved.

In conclusion, rHLF only exhibited a moderate inhibitory effect in vivoagainst RCMV infection in one of our models tested. Further pharmacokineticstudies will have to be performed to investigate whether the chosen dosageregimen was optimal in relation to these in vivo effects. However, it is conceivablethat the development of anti-rHLF antibodies in all animals treated with rHLFplayed a major role in the failure of rHLF therapy in these RCMV-infected rats.The antigenic effect of rHLF in these studies could in principle be evaded by usingrats that are transgenic for rHLF.

Chapter 9188

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