AIDS RESEARCH AND HUMAN RETROVIRUSESVolume 8, Number 6, 1992Mary Ann Liebert, Inc., Publishers

Development of Antiviral Treatment Strategies in Murine Models

RUTH M. RUPRECHT,1'2 JOHN A. KOCH,13 PREM L. SHARMA,'3 and RONNIE S. ARMANY1

ABSTRACT

Murine models with type C murine leukemia viruses have been used to develop major new prophylactic andtherapeutic strategies in vaccination, drug therapy of acute virus exposure and chronic viremia, combinationtherapy, prevention of maternal transmission, and therapy targeted to the central nervous system. Transgenicmice expressing either the whole human immunodeficiency virus type 1 (HIV-1) provirus or subgenomicsequences allow the in vivo analysis of selected HIV-1 functions. The full replicative cycle of HIV-1 can bestudied in human/mouse chimerae which were created by transplanting human hematolymphoid cells into SCIDmice. The chimeric SCID mouse models have been used successfully to evaluate anti-HIV-1 drugs. The role ofthe various murine retrovirus systems in the development of anti-HIV-1 and anti-AIDS therapies issummarized.

INTRODUCTION

The pandemic OF the acquired immunodeficiency syn¬drome (AIDS), caused by the human immunodeficiency

virus type 1 (HIV-1),1·2 requires the rapid development ofeffective therapy and prophylaxis. Concerted efforts have beenundertaken by academia and by the pharmaceutical industry tofind inhibitors of HIV-1 replication. Presently, only 3'azido-3'-deoxythymidine (AZT, zidovudine)3-5 and dideoxyinosine(ddl)6 have been approved by the Food and Drug Administrationfor treating AIDS and related conditions. Several candidateantiviral agents have been entered into clinical trials, and manymore compounds have proven anti HIV-1 activity in cul¬tured cells. A practical small animal model system for HIV-1viremia and disease, in which candidate antiviral drugs couldbe evaluated rapidly for pharmacokinetics, toxicity, and antivi¬ral efficacy, would facilitate the selection of drugs for clinicaltrials.

In most animal/retrovirus systems known to date, a period ofclinically asymptomatic viremia precedes the development ofovert disease. In general, animal/retrovirus systems can be usedeither as models for viremia, models for virus-induced disease,or both.7 Thus far, no ideal animal model for human HIV-1infection exists, and depending on the system used, only one or

few aspects of HIV-1 viremia and disease in humans can beduplicated in animal models (Table 1).

Testing candidate antiviral agents in inbred laboratory micewould be desirable because a vast body of knowledge has beenaccumulated regarding murine genetics, immunology, cyto-kines, and metabolism, and because many immunological re¬

agents and cytokines are available. Breeding can be accom¬

plished easily because mice have a short generation time. Thesmall size of these animals greatly facilitates drug studies,especially if only limited amounts of investigational compoundsare available for analysis. This latter point is especially impor¬tant for drug development from natural product sources. Fur¬thermore, the small size of mice also assures relatively lowhousing costs.

The host range of HIV-1 is limited to humans, chimpan¬zees,8·9 and Gibbon apes. Neither cultured murine cells nor

mice can be infected by HIV-1. Even murine cells that had beentransfected with cDNA encoding the human CD4 receptor andwhich expressed adequate levels of receptor molecules were

refractory to HIV-1 infection.10 Apparently, one or severalpostreceptor binding steps, which are crucial for successful viralentry, are blocked in murine cells. When full-length HIV-1proviral DNA was transfected into cultured murine cells, prog¬eny virions were produced, thus demonstrating that directproviral DNA insertion techniques can overcome the earlybarrier of murine cells to HIV-1 replication. ' '

Using three different approaches, murine model systems havebeen developed and employed for testing antiretroviral therapy,

'Laboratory of Viral Pathogenesis, Dana-Farber Cancer Institute and Departments of 2Medicine and 'Biochemistry and Molecular Pharmacology,Harvard Medical School, Boston, MA 02115.

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998 RUPRECHT ET AL.

Table 1. Animal/Retrovirus Systems for Evaluation of Therapy

Model system for Therapeutic strategy to be tested

Viremia

-

Acute virus exposure

-

Maternal transmission

-

Chronic viremiaHIV-1-induced disease

-

Acquired immunodeficiency-

Opportunistic infection

-

Kaposi's sarcoma

-

Nervous system disease:centralperipheral

-

Thrombocytopenia

Prevention:-

vaccination

-

drug prophylaxis-

postexposure prophylaxis with passive immunotherapy and/ordrug therapy

Transplacental prophylaxis with passive immunization and/or drug therapy;suppression of maternal virus release

Suppressive therapy with single agents or drug combinations

-

antiviral therapy, immune restoration

-

antiviral therapy, antimicrobial therapy and prophylaxis-cytotoxic therapy, ? anti-tar therapy

-

antiviral therapy, ? anticytokine therapy-

antiviral therapy, ? other

-

antiviral therapy and/or therapeutic modalities for idiopathic thrombocy-topenic purpura (ITP)

even though HIV-1 is unable to replicate in mice. Each of thesethree experimental approaches has major advantages as well as

disadvantages, which will be discussed further in subsequentsections. In the first approach, Type C murine leukemia viruses(MuLVs) have been used in lieu of HIV-1 to study antiretroviraltreatment strategies in animals. The second approach involvesthe generation of transgenic mice which allow the in vivoanalysis of isolated HIV-1 functions in the absence of virusreplication. Lastly, chimeric animals now can be created thatpermit prolonged survival of human bone marrow-derived cellpopulations, which can sustain HIV-1 replication.

Major new prophylactic and therapeutic strategies have beenpioneered in MuLV model systems (Table 2). For instance, thefirst successful retroviral vaccine was discovered as early as

1959 when formalin-inactivated Friend virus was shown to

protect naive mice against disease after challenge with livevirus.12 Inhibitors of retroviral replication were first found inMuLV models, including AZT which exhibited activity againstFriend MuLV.13 Postexposure AZT prophylaxis was demon¬strated to prevent viremia and disease originally in MuLV-inoculated mice.14 Natural perinatal transmission of pathogenicretroviruses was first prevented pharmacologically in MuLVsystems.15 Pharmacokinetic studies revealed that AZT passeseffectively across the placental barrier and is excreted intomilk.1617 Therapy of in utero retroviral infection was tested

Table 2. Therapeutic Strategies Evaluatedin MuLV Models

Prophylaxis with vaccines or drugs-

Postexposure prophylaxis-

Therapy of chronic viremia

-

Transplacental therapy with drugs and antisera

-

Prevention of milk-bome maternal virus transmission

-

Quantitative analysis of antiretroviral drug interaction incombination regimens

Therapy of neurotropic virus infection

Source: From Refs. 12-20.

initially in two murine models, one involving direct MuLVinoculation of developing embryos16 and the other one, trans¬genic mice activating an infectious MuLV provirus duringgestation.1S In both model systems, transplacental AZT therapywas highly beneficial and caused neither embryo toxicity nor

increased losses of pregnancies.1618 Quantitative analysis ofantiretroviral drug interactions, using combination drug regi¬mens, was also evaluated first in vivo with MuLV models.19·20In these studies, AZT combined with interferon-a (IFNa)proved to be highly synergistic without significant toxicity to thehost animals at the effective dose ranges. While promising datahad been generated in MuLV systems, these models have theirlimitations due to the use of surrogate, nonlentiviral viruses. Thegeneration of transgenic and chimeric mice has extended the use

of mice to allow limited analysis of HIV-1 itself in vivo. Theindividual experimental approaches, the biology of the virusesused and any therapeutic trials will be discussed below.

TYPE C MURINE RETROVIRUSES

Murine retrovirologyThus far, no murine lentivirus is known to date; only Type A

(intracistemal A particles), Type (mouse mammary tumorviruses), and Type C (murine leukemia viruses, MuLVs) havebeen isolated from rodents. The latter have been used frequentlyin antiviral treatment evaluations. As members of the retroviralsubfamily Oncovirinae, Type C MuLVs encode gag, protease,pol, ribonuclease H, integrase, and env, but they lack theIentiviral regulatory genes such as tat, rev, vif, vpr, vpu, and nef.To enter susceptible target cells, MuLVs do not use the CD4receptor.

Murine retroviruses have been classified also according tohost range into ecotropic (i.e., viruses capable of replicating incells of the species of origin), xenotropic (endogenous viruses ofthe species of origin that cannot replicate well in cells of thatgiven species because of a block at the receptor level, but are

able to replicate in cells of distant species), and amphotropic

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ANTIVIRAL TREATMENT STRATEGIES 999

(viruses able to replicate in cells of the host species of origin as

well as those of distant species). A special subgroup of murineretroviruses comprises the dual tropic mink cell focus-forming(MCF) viruses that can replicate not only in murine cells but alsoin mink cells. Typically, ecotropic MuLVs are pathogenic inmice, with a long latency period. These viruses can be titrated bythe XC plaque assay, a test based on syncytium formation,21 incontrast to xenotropic viruses. Most murine ecotropic retrovi¬ruses replicate at low levels in cells derived from closely relatedrodent species, such as hamsters or rats, and cannot replicate incells of higher mammals because of a lack of appropriate cellsurface receptors. Many rodents carry complete or partiallydefective endogenous retrovirus genomes which upon activationexhibit a xenotropic host range. Thus far, no xenotropic murineretrovirus has been found to be pathogenic in any animal. This isin sharp contrast to ecotropic viruses or to the dual tropic MCFviruses (for review, see Ref. 22).

The key features of the pathobiology of the various MuLVsystems employed for therapeutic or prophylactic studies are

summarized in Table 3 and described below. Where available,animal model data are compared directly with the relevant datafrom clinical trials in humans.

MuLV pathobiologyRauscher Murine Leukemia Virus. Rauscher murine leukemia

virus (RLV)23 is a retrovirus complex that consists of a replica¬tion-competent murine leukemia virus, a defective spleen-focus-forming virus (SFFV), and possibly also MCF viruses.22RLV and the Friend virus (FV) complex (see below) are closelyrelated virus isolates with a nucleic acid identity of about 90%.24

Their pathobiology is also very similar; the typical RLV-induceddisease closely resembles that induced by the anemia strain ofthe FV complex. Both virus isolates are pathogenic in adultmice, in contrast to most other MuLVs. Eight days postinocula-tion, spleen colonies are formed, the relative number of whichreflects the initial virus titer. These erythroid precursor coloniescontinue to enlarge and increase in number. As a result, theinfected animal develops splenomegaly. Following this eryth¬roid proliferative phase, infected animals develop frank eryth-roleukemia and die within 4 to 6 weeks postinoculation.22

The RLV and FV systems have the unique advantage that theyrepresent quantitative systems of retroviral viremia. Both thenumber of spleen colonies formed on day 8 postinoculation as

well as the degree of splenomegaly measured two to three weekspostinoculation are proportional to the virus titer.25 This unique,easily measurable parameter has been exploited for assessing theactivity of antiviral agents. It was used also to calculate druginteractions26 in combination regimens involving AZT plusinterferon-a.19'20 A high degree of synergism for inhibition ofsplenomegaly was found, but interestingly, no toxicity was seen

at virus-inhibitory concentrations. Thus, these in vivo experi¬ments demonstrated a great advantage for the combinationregimen because antiviral synergism occurred at far lower dosesthan synergistic toxicity to bone marrow which was demon¬strated in vitro by Berman et al.27

As the results of clinical trials in patients with HIV infection invarious stages of disease progression become available, it is ofinterest to assess whether animal data could predict clinicalsuccess in humans. The available RLV data are summarized inTable 4 in the order of a decreasing therapeutic window andcompared with the outcome of human trials. Optimal results in

Table 3. MuLV Systems Used for Drug Development

Virus Virus characteristics Tropism Disease

Rauscher virus complex

Friend virus complex

Moloney (MuLV)

Mo-MSV complex

Cas-Br-E and Cas-Br-M

LP-BM5 complex

Complex of replication-competent helper virus anddefective spleenfocus-forming virus(R-SFFV)

Complex of replication-competent helper virus anddefective spleenfocus-forming virus(F-SFFV)

Replication competent

Complex of replication-competent helper anddefective virus carrying themos oncogene

Replication competent

Complex of replication-competent helper and MCFviruses as well as defectivepathogenic virus

Lymphocytes, erythroidprecursors, macrophages,fibroblasts

Lymphocytes, erythroidprecursors, macrophages,fibroblasts

cells, fibroblasts

cells, fibroblasts(transformation)

cells, endothelial cells,(including microglia),macrophages, neurons,fibroblasts

cells, macrophages,fibroblasts

glia

Erythroblastosis, erythroleuke-mia in adult mice

Erythroblastosis, erythroleuke-mia in adult mice;immunosuppression in somestrains

T-cell leukemia/lymphoma inneonatally inoculated mice

Fibrosarcomas in neonatallyinoculated mice

Hind-limb paralysis afterneonatal inoculation

Immunodeficiency in adult mice(MAIDS)

See Ref. 22 and text.

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1000 RUPRECHT ET AL.

Table 4. Relative Effectiveness of Candidate Antiviral Agents in the RLV Model

TherapyOptimal dose

schedule

Optimalinhibition

ofsplenomegaly Observations in mice

Clinical experiencein humans

AZT combined 15 mg/kg/day p.o.with interferon-a 5 x 10 U/kg(rHulFN-aA/D)20 i.p., qd

AZT1 165/mg/kg/day p.o.

Interferon-a20 5 x 105 U/kgi.p., bid

Castanospermine33 300 mg/kg/day p.o.

6-O-butanoyl- 346 mg/kg/day p.o.castanospermine34

Suramin36 40 mg/kg/day i.V.,q3d

97%

98%

94%

79%

55%

56%

Combination is highlysynergistic in vivo andnontoxic. 100% effective inpreventing viremia whengiven as postexposureprophylaxis.

Anemia and leukopeniadeveloped in some mice.Effective as postexposurechemoprophylaxis and fortreatment of chronic viremia.

Linear dose-response.

Therapeutic window appearsrelatively narrow.

Therapeutic window appearsrelatively narrow.

Therapeutic window appearsrelatively narrow.

ACTG trial 068 ongoingcombination therapyagainst KS safe andeffective29"32

Effective in humans withHIV-1 infection;FDA-approved. Doserange in mice iscomparable to humandoses.

Used in KS patients(reviewed in32).

Trials with anotherinhibitor of glycosylationare ongoing; some

toxicity was seen35Too toxic and ineffective

in AIDS patients.37Abbreviations. KS, Kaposi's sarcoma; p.o., oral; i.p., intraperitoneal; qd, daily; bid, twice daily; i.V., intravenous; q3d, every

third day; ACTG, AIDS Clinical Trial Group.

the animal model were obtained with the combination of AZTand interferon-a,20 followed by single-agent AZT,14 and single-agent interferon-a.20 Data from direct comparisons of single-agent AZT versus the combination of AZT plus interferon-a inhuman HIV-1 disease are not available, but both regimens areeffective.3"5·29-32 A moderately narrow therapeutic windowwas found for the inhibitors of glycosylation castanospermine33and its 6-O-butanoyl analogue in RLV-infected mice.34 Interest¬ingly, clinical trials with another inhibitor of glycosylation,/V-butyl-deoxynojirimycin,35 revealed toxicity as well. In theRLV system, suramin, a reverse transcriptase (RT) inhibitor thatblocks the enzyme competitively with regard to template/primer, showed only modest activity,36 whereas in AIDS pa¬tients, neither clinical improvement nor a fall in the level of p24antigen were seen.37 Overall, the RLV system clearly identifiedtreatment regimens found to be active against human HIV-1infection in clinical trials (i.e., AZT,3~5 IFNa, and AZTcombined with IFNa28"32).

Friend Murine Leukemia Virus. The Friend virus (FV) com¬

plex consists of a mixture of viruses, the replication-competenthelper virus F-MuLV, and different strains of replication-defective spleen focus-forming viruses (SFFV), causing eitheranemia (SFFVJ or polycythemia (SFFVp).22 FV is predomi¬nantly B-cell tropic,38 but infects also macrophage precursors.39The disease produced by F-MuLV + SFFVa is biphasic; earlyon, FV infection gives rise to foci of erythroid proliferation;liver and spleen are infiltrated by immature erythroblasts re¬

sulting in hepatosplenomegaly and severe anemia; thymus andlymph nodes typically remain unaffected. Later on, the ani¬mals develop frank leukemia and succumb within 8 to 12weeks. The oncogenic potential of the FV complex is closely

linked to the truncated envelope gene (gp55) of the defectiveSFFV component.40 Neonatal as well as adult mice are suscep¬tible to the disease. Depending on the genotype of the hostanimal, the FV complex can also induce acquired immunodefi¬ciency.

Friend virus infection of (BIO.A x A/WySn) Fl mice41shares several of the characteristics of HIV-1 infection inhumans, including immunosuppression of the host concomitantwith the development of antiretroviral antibodies and the contin¬ued presence of low levels of infectious virus. Infected mice alsoexhibit inhibition of splenic helper and suppressor/cytotoxic cells, low CD4:CD8 ratios and a depressed response to phyto-hemagglutinin (PHA).

The rapid progression of FV disease as well as the variousvirologie and immunologie parameters available for assay facil¬itate the evaluation of candidate antiviral and immunomodula-tory therapies. For example, treatment of mice with AZT earlyafter FV infection led to a marked inhibition of splenomegaly,elimination of infectious centers and circulating FV, and pre¬vented depression of total cells in infected mice.42 Althoughtreatment with AZT inhibited FV-induced splenomegaly anddeath, low levels of virus could still be detected in some micewhich may be latent or below a critical threshold to cause

disease. Immunomodulatory agents have been shown to affectthe course of FV infection by altering T- and B-cell function. Forexample, imexon treatment improved T-cell function as mea¬

sured by PHA-induced blastogenesis while reducing splenome¬galy and viral titers.43

Moloney Murine Leukemia Virus. Moloney murine leukemiavirus (MoMuLV), a T-cell tropic virus, is pathogenic only inneonatally inoculated animals which develop T-cell leukemia/

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ANTIVIRAL TREATMENT STRATEGIES 1001

Iymphoma after 3 to 6 months.22 More than 40 transgenic mouse

strains carrying the MoMuLV provirus, designated Mov strains,have been derived by exposing mouse embryos to MoMuLVduring ontogeny.44"48 Four different patterns of virus expres¬sion among these strains have been observed: virus activationduring embryogenesis, soon after birth or in adult life andfinally, no virus expression at all. In Mov mice, MoMuLV geneexpression begins in a few cells and eventually infectious virionsare released which spread from the site of activation to othercells. In animals activating the provirus early in life, viremia isfollowed by the development of T-cell leukemia/lymphomatypical of exogenous MoMuLV infection. Mov strains withprenatal virus activation have been used as model systems for inutero infection.18 In contrast, postnatal virus activation mimicsthe natural route of infection via the milk or experimentalinoculation of neonatal mice, since virus expression and spreadoccur in newborns. These transgenic mice have been used as a

rapid test system for evaluating perinatal and transplacentalantiretroviral therapy.18'49

Moloney Murine Sarcoma Virus. Moloney murine sarcoma

(MSV)22 is a virus complex consisting of the replication-competent Moloney murine leukemia virus and a defective agentthat arose when the mos oncogene was inserted into the pol andenv regions of the helper virus. The MSV complex inducesrhabdomyosarcomas in newborn and adult mice. Induction ofneoplasia is due to the defective virus containing the mos

oncogene. When newborn mice are inoculated with MSV, a

tumor forms at the site of inoculation with a latent period of only4 to 5 days. The disease progresses rapidly, ultimately killing themice within 10 to 12 days. In adult mice, the majority of tumorseventually regress, whereas those induced in mice renderedimmunologically hyporesponsive do not.50 Tumor regression isdue to cellular immune responses, especially those mediatedby CD4+ T-helper cells. Functional depletion of CD4+ cells facilitates virus spread and growth of MSV-inducedtumors.51·52 This mouse model system has been utilized re¬

cently to evaluate the activity of antiviral agents by measur¬

ing increased survival of neonatally inoculated mice giventherapy.53·54

The Neurotropic Retroviruses Cas-Br-E and Cas-Br-M. Theneurotropic virus Cas-Br-E was isolated from the brain of a

paralyzed wild mouse caught in a remote area in California,where a sequestered population of wild mice was found to havea high prevalence of Iymphoma and hind-limb paralysis.55'56The wild mouse virus isolates contain a mixture of amphotropicand ecotropic MuLVs.57 Amphotropic viruses have been asso¬

ciated with induction of lymphomas, whereas the ecotropiccomponent can cause either Iymphoma or paralysis, dependingon the strain of mice used. Cas-Br-E (for review, see Ref. 58)and Cas-Br-M59 represent two different isolates of the ecotropiccomponent which is responsible for the development of hind-limb paralysis. Neoplasms induced by Cas-Br-M include T- andB-cell lymphomas, myelogenous and megakaryocytic leuke-mias.59 When injected into neonatal mice, Cas-Br-E induceshind-limb paralysis in all animals of susceptible laboratorystrains. Cas-Br-E has been molecularly cloned by Jolicoeur etal.60 By mutational analysis, the potential to cause hind-limbparalysis was mapped to sequences within the gene encoding theenvelope glycoprotein gp70.61 ·62 The target cells of Cas-Br-E or

Cas-Br-M are thought to be lymphocytes, macrophages, fibro¬

blasts, glial cells, including microglia, as well as endothelialcells and neurons. Cas-Br-E can be passed through semen andcontaminated milk. In the initial stages, the virus replicates inthe spleen and subsequently spreads to the central nervous

system (CNS).In paralyzed animals, Cas-Br-E causes spongiform polioen-

cephalomyelopathy in the anterior homs of the spinal cord,the dentate nucleus of the cerebellum, and the brain stem.63"66The white matter shows less damage, and characteristically,there is no inflammatory reaction. Cas-Br-E-induced spinal cordlesions resemble certain HIV-1-induced myelopathies; the lat¬ter, however, show a greater involvement of white matter.67Cas-Br-E and Cas-Br-M have been used to study antiviraltherapy targeted to the CNS during gestation and in the neonatalperiod.16·68

The immunosuppressive Murine Leukemia Virus LP-BM5.Susceptible young adult mice inoculated with the LP-BM5 viruscomplex develop a severe acquired immune deficiency syn¬drome termed murine AIDS or MAIDS. This syndrome sharesseveral clinical features with human AIDS, such as loss ofT-cellfunction, opportunistic infections, and the development oflymphomas late in the course of disease.69-70

The LP-BM5 virus complex consists of replication-competentecotropic and MCF viruses, as well as defective virus genomessuch as BM5d or Du5H.71 This mixture of retroviruses wasderived from the Duplan-Laterjet strain72 of radiation-inducedMuLVs. In contrast to the replication-competent viruses whichare apparently nonpathogenic, the replication-defective ge¬nomes are associated with the development of immunodefi¬ciency. LP-BM5 can replicate in cells, macrophages, andfibroblasts. On the cell surface, infected cells express mutantPr60*"* encoded by the defective virus. Expression of thisabnormal virus product leads to massive stimulation of normalCD4+ cells, indicating that this gag-encoded protein may actas a superantigen.73 The stimulated CD4+ T-cells releasecytokines, which further expand the B-cell targets for virusinfection. This in turn augments T-cell stimulation and repre¬sents a positive feedback loop (Fig. 1). Infected animals developT- and B-cell dysfunction, polyclonal B-cell proliferation,lymphadenopathy, splenomegaly, hypergammaglobulinemia,and enhanced susceptibility to opportunistic infections. Late inthe course of disease, oligoclonal B-cell proliferation and B-celllymphomas are seen.

LP-BM5 infection of susceptible mice has been used tostudy the efficacy of antiviral drugs as well as immunomodula-tors. Several laboratories have confirmed the efficacy of AZTtherapy.74"78 None of these studies suggested the completeprevention of LP-BM5 infection after acute virus exposure, incontrast to AZT postexposure prophylaxis in RLV-inoculatedmice.14 More recently, continuous oral AZT therapy ofC57BL/10 mice exposed to a low dose of virus preventedLP-BM5 immunodeficiency.77'78 An interesting therapeuticeffect was seen when cytotoxic therapy with cyclophosphamidewas used.79 Paradoxically, therapy of LP-BM5-infected micewith this immunosuppressive drug prevented virus-induceddisease, presumably by killing the expanding B-cell populationexpressing the abnormal PróO8"* viral protein. Therapy with theimmunomodulators diethyldithiocarbonate (DTC)80 and cyclo-sporin A81 both attenuated the course of LP-BM5-induceddisease (Fig. 1).

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1002 RUPRECHT ET AL.

*LP-BM5Virus Beeil

Expansion oftarget cellpopulation

CD4+Tcell © proposed mechanism of Cyclosporin A

inhibition of cytokine release.(D inhibition of target cell expansion by

cyclophosphamide.FIG. 1. Proposed mechanism for LP-BM5-induced immunodeficiency. The individual steps are explained in the text.

For drug development, the LP-BM5 system can be used as a

model for retroviral viremia, provided that the caveats outlinedbelow are taken into account. When the system is used to studytherapy of acquired immunodeficiency, the immunopathogene-sis of LP-BM5 disease needs to be considered, especially ifimmunomodulators are tested as candidate agents for the treat¬ment of human AIDS. Unlike HIV-1-induced AIDS, which ischaracterized by a massive loss of CD4+ cells, LP-BM5-induced MAIDS is associated with abnormal stimulation andincreased numbers of CD4+ cells, and the virus itself does notreplicate in cells. Consequently, it is unclear whether LP-BM5-induced MAIDS can be used as a general model forHIV-1-induced disease in humans.

The use of MuLV systems for drug developmentPrior to selecting an animal/retrovirus system for assessing in

vivo efficacy, the possible mechanism of viral inhibition by a

candidate antiviral agent needs to be considered. Does the noveldrug directly block certain steps during the replicative cycle ofHIV-1? Is it targeted against one of the six common retroviralfunctions, or does it inhibit an important Ientiviral regulatory

gene? Alternatively, does the candidate drug act indirectly byeliciting certain cytokine responses? Does it act as an immuno-modulator?

Evaluation ofDrugs Directly Inhibiting Virus Replication. Toanalyze a candidate antiviral drug in vivo in an MuLV system,the following criteria need to be fulfilled: (1) The drug mustblock similar functions of both HIV-1 and the surrogate MuLV;(2) in cultured cells, the investigational drug must inhibit HIV-1replication to approximately the same degree as that of theMuLV; (3) drug metabolism in human and mouse cells must becomparable; (4) the pharmacokinetics in mice and humansshould be similar; and (5) if oncogenic murine retroviruses are

used for testing, control experiments must be conducted to ruleout antineoplastic activity as to the cause of a favorable treat¬ment response, instead of solely antiviral activity.

The potential role that MuLV/animal model systems can playin the development of antiviral agents is depicted in Figure 2.The schema is intended for candidate drugs exhibiting directantiviral activity and does not apply for prodrugs. The latter mayneed to be evaluated directly in one of the available animalmodel systems for retroviral viremia and/or disease, since theiractivity may be evident only after enzymatic activation in certain

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ANTIVIRAL TREATMENT STRATEGIES 1003

Inhibitor of HIV-1 in vitro

targeted againstIentiviral

regulatory genes

targeted againstcommon retroviralgenes/functions

£"in vitro activityagainst MuLV

,®drug specific

for HIV-1

in vivo in SCIDmouse system,

efficacy and toxicity

7\l

in vivo againstMuLV

efficacy and toxicity

0too toxic0^ ^0

VIC too toxic

1clinical

trialFIG. 2. Proposed schema for in vivo testing of novel anti-HIV-1 drug for efficacy and toxicity in murine models

0VIC

iclinical

trial

organs. Because MuLVs.only encode the common retroviralgene products gag, protease, polymerase, ribonuclease H,integrase, and env, their use for in vivo drug development islimited. Antiviral agents targeted against Ientiviral regulatorygenes cannot be tested in MuLV model systems. The same holdstrue for compounds inhibiting a common retroviral functionsuch as a reverse transcriptase if they are highly selective for theHIV-1 enzyme, such as the TIBO compounds.82 Furthermore,because MuLVs use cellular receptors other than CD4 and differsignificantly in their pathobiology from HIV-1, data obtainedwith MuLVs must be interpreted with caution. On the otherhand, the low cost, lack of biohazard, and ease of measuringvirological parameters as well as the short assay times required,make the MuLV systems valuable tools, as long as the caveatsdiscussed above are considered. Thus far, MuLV systems are

the only mouse models that allow testing the efficacy of viralinhibitors targeted to the CNS or during gestation.

Evaluation of Immunomodulators. Immunomodulatoryagents may need to be evaluated in an animal model of acquiredimmune deficiency. Infection with several MuLVs leads to

immunological dysfunction, and in the case of LP-BM5, a

clinical syndrome develops reminiscent of human AIDS. Be¬cause destruction of CD4 cells by virus infection is not theunderlying mechanism in any MuLV-induced immunodefi¬ciency, data obtained in such model systems must be interpretedwith caution. Confirmatory experiments may need to be carriedout in monkeys infected with the simian immunodeficiencyvirus (SIV). The latter animal system was considered by a panelof experts convened by the World Health Organization as thegold standard among the various animal/retroviral model sys¬tems for HIV-1 infection and disease.83

THE SEARCH FOR A PUTATIVERODENT LENTIVIRUS

An immunosuppressive lentivirus in inbred laboratory micewould provide a valuable research tool. Lentiviruses have beenfound in humans, nonhuman primates, sheep, goats, cows,

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horses, and cats, and it is likely that they also occur in mice. Forthese reasons, several groups are probing systematically forputative rodent lentiviruses.84

Our search is focused on geographical neighbors of the housemouse population (Mus musculus), from which laboratory micewere derived. We reasoned that the putative lentivirus, whilepathogenic for laboratory mice, might silently infect otherrodent species that inhabit sites favored by house mice. Presum¬ably, a prevalent parasite cannot maintain a stable associationwith the host population if severe, AIDS-like pathology were toensue from the virus infection. SIV, for example, produces asilent infection in African green monkeys, the naturally infectedhost for this agent,85 but causes severe disease in other species.Extending this reasoning further, we suggest that the searchshould focus on well-adapted rodents that are abundant, in partbecause they can defend their territories against competitors. Werejected an alternative hypothesis which depended on searchingfor the putative lentivirus in naturally sick mice. Indeed, in thecourse of examining some 30,000 wild rodents caught in NewEngland, Dr. Andrew Spielman has never trapped such an

animal, presumably because predators would rapidly remove

any impaired animals (personal communication).We are attempting to isolate rodent lentiviruses directly by

tissue culture methodology from rodents caught in the wild.Additionally, putative Ientiviral sequences are being screenedusing the polymerase chain reaction (PCR) with degenerativeprimer pairs from highly conserved sequences obtained byscanning known Ientiviral sequences with multisequence align¬ing techniques.86'87 In parallel, the rodent sera are analyzed byWestern blot analysis for cross-reactivity with known Ientiviralantigens. Thus far, we have found one serum sample withcross-reactivity to a known lentivirus. The corresponding ge-nomic DNA isolated from the spleen of this wild-caught rodentshowed discreet bands with the degenerate primer pairs, andsequence analysis of some of these products demonstrated some

homology with known Ientiviral sequences. Presently, the rele¬vance of these results are under investigation.

MURINE MODELS INVOLVING HIV-1

Transgenic mice

Transgenic mouse-model systems involving HIV-1 se¬

quences are summarized in Table 5. Transgenic mice have beencreated carrying either the intact provirus,88 subgenomic frag¬ments,89"91 or reporter genes linked to the HIV-1 long terminalrepeat (LTR).91"96

Transgenic Mice Carrying Full-Length HIV-1 Transgenes.Leonard et al.88 inserted the full-length HIV-1 provirus intotransgenic mice. Detectable levels of HIV-1 expression or signsof disease were not found in any founder animals, even thoughintegrated proviral copies were demonstrated. One founderfemale mated to a normal syngeneic male produced F, litters thatsurvived only about 25 days postnatally. These F, animals hadstunted growth, perivascular pulmonary lymphocytic infiltrates,epidermal hyperplasia involving tail, ears, nose, and feet andwere found to have splenomegaly, lymphadenopathy, and invo¬lution of the thymus. No selective destruction of CD4+ lympho¬cytes, the characteristic manifestation in human AIDS, was

found. Only affected F, animals, but no normal litter mates,carried the HIV-1 provirus. Attempts to isolate HIV-1 fromaffected F, mice were successful and yielded HIV-1 virionsinfectious for human CD4+ cells but not for murine fibroblasts.HIV-1 was unable to replicate in affected F, mice, even thoughvirus release was demonstrated.

The relevance of these HIV-1 transgene-carrying mice tohuman HIV-1 infection and AIDS is unclear. First, only latesteps of the viral replication cycle occurred in these transgenicmice; virions released into the bloodstream were unable to infectnew murine target cells which are nonpermissive for viral entry.Second, the question still remains whether the severely affectedF¡ mice exhibited lesions characteristic of human AIDS. Theskin disease seen in these animals does not resemble the skinlesions occurring in AIDS patients such as seborrheic-likedermatitis, psoriasis, or ichthyosis.·97"99 In contrast, the pulmo¬nary interstitial lymphoid infiltrates resemble those seen innonspecific interstitial pneumonitis of adult AIDS patients.100The lethal congenital disease of these F, transgenic mice may bedue to the expression of HIV-1 gene products, especially sincethe HIV-1 LTR has demonstrable transcriptional activity insome of the tissues affected (see below). Alternatively, this F,phenotype may have resulted from insertional mutagenesis andthe disruption of important murine genes, similar to the trans¬

genic animals described by Jaenisch.101 In any event, shortlyafter the publication of this model system,88 all transgenic miceperished in a laboratory accident.102

Transgenic Mice Carrying Defective HIV-1 Proviruses.Three lines of transgenic mice were generated with an HIV-1provirus containing an in frame deletion of gag and pol sequenc¬es.89 This defective provirus encoded the complete sequencesfor env, tat, rev, nef, vpu, and vif as well as a pl7/p34 fusiongene product. Offspring of the founder mice exhibited renaldisease characterized by proteinuria and interstitial nephritis andhad a high mortality rate. Electron microscopy of renal tissuerevealed ultrastructural features consistent with glomeruloscle-rosis. Immunocytostaining detected the presence of HIV-1proteins in the glomeruli of affected mice. HIV-1-specificmRNAs and proteins were expressed to various degrees inseveral other tissues.89

Transgenic Mice Expressing HIV-1 tat. Transgenic micecontaining the HIV-1 -tat gene fused to the HIV-1 LTR were

created by Vogel et al.90 Male animals developed skin lesionsconsisting of spindle-cell proliferation and tumor formationthought to resemble Kaposi's sarcoma. Even though femalemice synthesized similar levels of tat mRNA, they failed to

develop lesions.Khillan et al.91 have generated a line of transgenic mice

carrying the chloramphenicol acetyl transferase (CAT) geneunder the transcriptional control of the HIV-1 LTR. These micewere mated with transgenic animals of the opposite sex carryingthe HIV-1 tat gene fused to a mouse -crystalline A transcrip¬tional control element. In the F, offspring, transactivation of theCAT gene was seen only in the eye, the tissue in which the -crystalline A promoter/enhancer element exhibits preferentialactivity. These data demonstrate that the HIV-1 tat gene isfunctional in mice.

Transgenic Mice Carrying the HIV-1 LTR Linked to ReporterGenes. Several groups have demonstrated the transcriptionalactivity of the HIV-1 LTR in transgenic mice. Leonard et al.92

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ANTIVIRAL TREATMENT STRATEGIES 1005

Table 5. HIV-1 Sequences in Transgenic Mice

HIV-1 sequence Virus replicationTissues with

gene expression Disease

Complete provirus8

Defective HIV-1 provirus8

HIV-1 LTR-tar-39

HIV-1 release only, no

replication in mouse cells

None

None

HIV-1 LTR linked to reporter gene:-HIV-1 LTR linked to CAT

gene *

HIV-1 LTR linked to CAT

HIV-1 LTR linked to CAT

None

None

Nonegene

Strain a: HIV-1 LTR linkedto CAT91Strain b: murine -crystallineA transcriptional controlelements fused to HIV-liar91

-HIV-1 LTR controllingfirefly luciferase or

ß-galactosidase genes95-HIV-1 LTR controlling SV40 and t antigen genes96

None

None

None

Tail, ears, skin, and a fewscattered cells in the liverand gastrointestinal tract.

Skin, skeletal muscle, kidney,brain, eye, GI tract, andspleen

Skin

Thymus, tail, eye, heart,spleen. Among monocyte-macrophage derived cells,maximal activity inLangerhans cells.

Glial cells of cerrebral cortex,basal ganglia, andcerebellum.

Thymus, spleen, and spinalcord; after morphineadministration, high CATexpression in brain(hypothalamus, midbrain),spinal cord and eyes.

Strain a x strain b: CATtransactivation in the eyes ofF, offspring carrying bothtransgenes

Skin; activation of HIV-1 LTRin epidermis upon exposureto UV, psoralen, andsunlight

Thymus, lymph nodes, spleen,and skin

Epidermal hyperplasia,pulmonary lymphoidinfiltrates, lymphadenopathy,runting, death at 25 days.

Renal disease, muscle wasting,runting, thymic atrophy,papillomatous skin lesions.

Dermal lesions in transgenicmale mice resemblingKaposi's sarcoma.

None

None

None

None

None

Respiratory insufficiencysecondary to thymichyperplasia.

*CAT gene-chloramphenicol acetyl transferase gene.

generated four lines of transgenic mice containing the CAT genefused to the HIV-1 LTR. In all transgenic animals, tissue-specific CAT expression was noted in the eye, heart, spleen,thymus, and tail. Cocultivation of circulating lymphocytes andmonocytes in the presence of mitogens or various cytokinesresulted in CAT expression. Among cells of the monocyte-macrophage lineage, Langerhans cells of the skin exhibited thehighest levels of CAT activity.

Two more reports were published describing transgenic micecarrying the HIV-1 LTR linked to the CAT gene. Harlan et al.93reported the generation of transgenic mice to assess the relativeactivity of the HIV-1 LTR in brain tissue by immunocytochem-ical staining techniques. Labeling was observed in glial cellswith morphological features of astrocytes or microglia in thecerebral cortex, basal ganglia, and the cerebellum. Prakash etal.94 demonstrated in their line of transgenic mice carrying theHIV-1 LTR linked to CAT gene that the transgene was ex¬

pressed in thymus, spleen, and spinal cord. After morphine

administration, high CAT expression was noticed in the brain,especially in the hypothalamus and midbrain, the spinal cord,and the eyes. Morrey et al.95 constructed transgenic micecontaining the HIV-1 LTR linked to either the firefly luciferaseor the ß-galactosidase reporter genes. In this system, the HIV-1LTR was activated in vivo by exposure to sun light or ultraviolet(UV) radiation. Topical application of the photosensitizingagent psoralen to the skin of these mice shortened the timerequired for UV activation of the LTR.

Skowronski et al.96 generated three lines of transgenic micewhich carried the HIV-1 LTR linked to the S V40 and t-antigengenes (pHIV-1 Tag). The only consistently occurring pathologywas enlargement of the thymus. The transgene was transcribedactively in skin, thymus, spleen, and lymph nodes in alltransgenic lines. The highest level of T- and t-antigen transcriptsamong various lymphocyte cell populations were found in cells, while total cells and T-cell subpopulations contained5-10-fold fewer transcripts.

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Table 6. SCID Mouse Model Systems Involving HIV-1

Model feature SCID-hu' hu-PBL-SCID'

Reconstitution

Surviving human cellpopulations

Maximal length of graftsurvival

Humoral immunity

Cellular immunity

HIV-1 isolates capable ofreplication

SCID-hu Thy/-: transplantation with humanfetal thymus under the renalcapsule108

SCID-hu Thy/Liv: transplantation with humanfetal thymus and liver109,110

SCID-hu LN: transplantation of human lymphnodes into the mammary fatpads111

SCID-hu Bone: subcutaneous insertion ofhuman bone112

Proliferation of multilineage hematopoieticcells, differentiation of human and cells

Phenotypically and functionally normalmature human lymphocytes

SCID-hu Thy/-: 3 monthsSCID-hu Thy/Liv: > 12 monthsSCID-hu LN: 3 monthsSCID-hu Bone: 4-5 monthsProduction of human IgG, primary and

secondary responses

Normal appearing lymphoid follicles, T-cellcompartment functionally intact includingprimary responses110"3·"4

Only primary patient isolates but notlaboratory-adapted strains1 ' '

Adult human PBL, from EBV+orEVB", andCMV" aswell as HBV" donors

CD4+ and CD8+ human cells, cells, and to a lesserdegree macrophages

Phenotypically and functionallynormal mature humanlymphocytes

22-48 weeks as measured byhuman IgG production

Production of human IgG,secondary responses totetanus toxoid

Secondary responses; T-cellproliferation to mitogen andalloantigens detectable up to6 weeks

Many laboratory strains (IIIB,MN, RF, SF2, SF13, SF33,DFCI-HT1), primary patientisolates and AZT-resistant

Route of HIV-1 inoculation SCID-hu Thy/-: intrathymic injectionSCID-hu LN: i.v.

i.p.

The pattern of constitutive expression of genes controlled bythe HIV-1 LTR in transgenic mice is thought to arise from theinteraction of the LTR with cellular transcription factors. Suchtransgenic mice may prove useful for the analysis of tissue-specific transcription factors capable of interacting with theHIV-1 LTR. They may be useful also to evaluate environmentalfactors or pharmacological agents acting through the HIV-1LTR either as potentiators or inhibitors of gene expression.

Chimeric mouse models for HIV-1 infectionBecause the full replication cycle of HIV-1 cannot be studied

in mice, human/mouse chimerae were created by transplantingcongenitally immunodeficient C.B-17 mice with human hema-tolymphoid cells. The recipient animals, homozygous scid/scid,so-called SCID mice,103104 lack functional T- and cellsbecause of a faulty VDJ recombinase mechanism.105J06 Severalexperimental systems have been established using either fetalorgan grafts or peripheral blood leukocytes from normal adult

human donors. Prolonged graft survival is observed in bothsystems. The major features of the two models are summarizedin Table 6.

SCID-hu Mice.'07~>15 To create human/mouse chimerae,human fetal tissues originating from liver, thymus, lymphnodes, and bone were transplanted into SCID mice.107""4Thymic and hepatic tissue were transplanted under the renalcapsule, whereas lymph nodes were transplanted into the mam¬

mary fat pads, and human bone fragments"2 were transplantedsubcutaneously. Four different heterospecies systems have beencreated, depending on the type and combination of tissue(s)transplanted (Table 6). The various models offer unique oppor¬tunities to study normal differentiation of human hematopoieticprogenitors and the maturation of functional and cells. Afterengraftment in the SCID-hu mice, differentiation of maturehuman cells and cells was observed. In the chimerae, humanhematopoietic cells of different lineages were able to proliferate,and differentiation of human and cells resulted in phenotyp¬ically and functionally normal mature human lymphocytes.

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ANTIVIRAL TREATMENT STRATEGIES 1007

SCID-hu mice transplanted with all three different tissue types(i.e., fetal thymus, liver, and lymph nodes), produced humanIgG, and primary as well as secondary antibody responses couldbe elicited. Transplanted lymph nodes exhibited normal appear¬ing lymphoid follicles, and the T-cell compartment was func¬tionally intact. Even primary T-cell responses can be obtained(Dr J.M. McCune, personal communication).

SCID-hu mice are susceptible to HIV-1 infection. For infec¬tion with HIV-1, SCID-hu Thy/- and SCID-hu LN mice havebeen used. While mice transplanted only with human thymusrequired intrathymic inoculation of HIV-1, SCID-hu LN micecan be infected intravenously with primary patient isolates.Interestingly, laboratory-adapted strains of HIV-1 do not repli¬cate in these chimerae. ' ' '

Antiviral drug therapy can be evaluated in HIV-1 inoculatedSCID-hu mice. For instance, postexposure prophylaxis withAZT was shown to prevent viremia in treated SCID-hu LN mice,provided that treatment was initiated promptly."5 A time-dependent decrease in the percentage of animals protectedagainst viremia was noted, but when AZT was started 1 hpostinoculation, all animals were protected by a 2-week course

of therapy. Even two weeks after cessation of AZT therapy, no

virus could be detected. These data confirm earlier resultsobtained in RLV-inoculated BALB/c mice14 and in FeLV-inoculated cats given AZT as postexposure prophylaxis."6Because the SCID-hu mouse model provides a quantitativeparameter for HIV-1 infection, namely, the percent of HIV-positive, treated animals 2 weeks postinoculation, the relativeefficacy of drug therapy can be assessed in vivo. The system can

be used also to calculate the drug interactions in combinationregimens.

hu-PBL-SCID Mice."7-"9 A different experimental ap¬proach to create human/mouse chimerae was used by Mosier etal."7 Intraperitoneal injection of peripheral blood leukocytes(PBL) obtained from adult human donors resulted in stableengraftment of a functional human immune system that survived22-48 weeks posttransplantation as measured by secretion ofhuman IgG. Transplanted human lymphocytes were phenotypi¬cally and functionally normal, and a specific secondary humanantibody response could.be elicited following challenge withtetanus toxoid. All major human cell populations present nor¬

mally in PBL could be isolated from blood and lymphoid tissuesof the chimerae, so called hu-PBL-SCID mice. hu-PBL-SCIDmice can support HIV-1 replication,"8,119 and in contrast toSCID-hu mice, they can support not only the replication ofprimary patient isolates, but also laboratory-adapted strains suchas III-B, MN, RF, SF2, SF13, SF33, DFCI-HTI, and AZT-resistant virus (Dr. D. Mosier, personal communication). Vire¬mia can be obtained reproducibly by intraperitoneal injection.

Chimerae of the bglnulxid Genotype. Immunodeficient micewith the triple mutation bglnulxid were transplanted with normalhuman bone marrow cells following irradiation.120 In thesemice, the nu mutation results in a loss of thymic function, the bgmutation greatly reduces the number of natural killer (NK) cells,and the xid mutation results in low numbers of lymphokine-activated killer cells (LAK). Following transplantation, pro¬longed survival and differentiation of human macrophage pro¬genitor cells was noted. These chimerae may become useful to

analyze the suceptibility of human macrophages to HIV-1 in

vivo, but to date, no data regarding HIV-1 infection of thesechimerae have become available.

The Use of Chimeric Mouse Models for Drug Development.Both the SCID-hu and the hu-PBL-SCID mouse systems lendthemselves for evaluating inhibitors of HIV-1 replication invivo. The obvious advantages of these systems are the small sizeof the host animals, the reproducibility with which HIV-1infection can be obtained experimentally, and the relativelyshort assay times. Whether HIV-1 pathogenesis can be exam¬ined in these models is unclear presently. In hu-PBL-SCIDmice, uninhibited HIV-1 replication results in the loss of CD4+human target cells and a concomitant decrease in human IgGproduction."8 Nevertheless, many aspects of HIV-1 infectionin humans cannot be studied in SCID mouse models. CNSinfection in chimeric animals has not been reported, and mater¬nal transmission cannot be evaluated. Other potential drawbacksare the relatively limited lifespan of human xenografts and thepotential to create viruses with altered host range.121 Thegenome of laboratory mice is known to harbor integratedsequences of xenotropic and ecotropic viruses.22 If xenotropicviruses become activated, human target cells could potentiallybe coinfected by HIV-1 as well as the mouse xenotropic virus,leading to pseudotype formation and the creation of novelretroviruses with altered host range and pathobiology. In exper¬iments conducted to date, no evidence has emerged that geneticrecombination or pseudotype formation occurs in the SCIDmouse systems.122 On the other hand, HIV-1 with alteredtropism has been isolated from a human T-cell leukemia cell linethat had become infected by a xenotropic murine leukemia viruswhen transplanted into irradiated nude mice.123 When conduct¬ing experiments in SCID mouse systems with HIV-1, thepotential of forming pseudotypes must be kept in mind, andadequate biosafety containment facilities and procedures are

essential.

SUMMARY

The role of different murine model systems in the preclinicalevaluation of antiviral or anti-AIDS therapies, summarizedpreviously,7·82·124,125 was updated. These models involve ei¬ther Type C MuLVs, transgenic mice expressing complete or

incomplete HIV-1 proviruses or chimeric SCID mice trans¬planted with human HIV-1 target cell populations. Each exper¬imental model offers some unique advantages or drawbacks.The selection of the appropriate model for the in vivo analysis ofa given antiviral drug requires careful consideration of theantiviral target, mechanism of inhibition, and drug metabolismin various target cells. When studying the efficacy and toxicityof therapeutic agents aimed at treating acquired immune defi¬ciency as opposed to viremia in murine models, the underlyingimmunopathogenesis of the model system must be consideredcarefully.

ACKNOWLEDGMENT

The authors thank Angela Chrisis and George Pearson for thepreparation of this manuscript. Work by RMR described in thisarticle was supported in part by NIH Contract #N01-AI-72664,

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NIH Grants #2U01AI24845, #R01-AI-25715, #R01-AI-29797 to RMR, #R43-AI-31740 to JAK, and by the Center forAIDS Research (CFAR) Core Grant #IP30 28691 awarded tothe Dana-Farber Cancer Institute to support the Institute's AIDSresearch efforts. RMR is the recipient of a Faculty ResearchAward from the American Cancer Society.

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Address reprint requests to:Ruth M. Ruprecht

Laboratoty of Viral PathogenesisDana-Farber Cancer Institute

44 Binney StreetBoston, MA 02115

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