Flow cytometric antiviral drug susceptibility assays

CLINICAL

A d e n o v i r u s I n h i b i t i o n of I m m u n e . m e d i a t e d Apoptosis William S.M. Wold, Karoly Toth, Konstantin Doronin, Mohan Kuppuswamy, Drew L. Lichtenstein, and Ann E. Toilefson Saint Louis University School of Medicine, Department of Molecular Microbiology and Immunology, 1402 South Grand Blvd., St. Louis, MO 63104

s we all learned in biology, microorganisms are ubiquitous. Mammals can compete with microorganisms because they have developed a variety of anti-microbial defenses. As a general theme for microorganisms such as viruses that must replicate within cells, the mammalian host attempts to sacrifice the infected cell so that uninfected cells can survive. These anti-viral cell suicide defenses include intrinsic "programmed cell death" (apoptosis) and destruction of the infected cell by cytolytic cells of the immune system. What was not fully appreciated until about fifteen years ago is that microorganisms can compete with mammals because they, too, have defenses. Indeed, a surprisingly large portion of the microbial genome is devoted to combating the

A

host. In this article, we will discuss proteins synthesized by subgroup C human adenoviruses (Ad serotypes 1, 2, 5, and 6) that keep the infected cell alive so that the virus can replicate (also see refs. 1, 2). Of the 36 or so Ad-coded proteins, at least eight proteins are devoted to defense against the host. Subgroup C Ads cause relatively mild upper respiratory tract infections in young children, and by age two about 80% of the population has been infected.3 Strong antibody responses occur that appear to confer lifelong immunity. Adults have peripheral blood lymphocytes that proliferate in vitro in response to Ad virions, in particular to Ad structural proteins. In Phase I trials using replication-defective Ad vectors for gene therapy, strong

humoral and cellular responses were observed, as was the synthesis of the cytokine Interleukin-6 and the chemokine Interleukin-8.4 Thus, the acute Ad infection is nearly always kept well in check by the immune system. Ad, however, counters by forming persistent infections, probably in lymphoid cells, that may last the lifetime of the host) The Ad-coded anti-immune proteins are believed to give the virus an edge during the acute infection, prolonging replication, and allowing the virus to hide during persistent infections. The details of Ad infections in humans have not been extensively investigated, probably because Ad is generally not lifethreatening (except in immunocomproraised hosts). However, we can speculate Continued on page 2

Flow Cytometric Antiviral Drug Susceptibility Assays James J. McSharry, Ph.D. Department of Microbiology, Immunology & Molecular Genetics, The Albany Medical College, 47 New Scotland Ave., Albany, NY 12208

Introduction Human cytomegalovirus (HCMV) infections occur frequently in the general population usually with subclinical results. 2 However, HCMV infection is a major cause of morbidity and mortality in children with congenital infections and in both children and adults with acquired defects of the immune system such as CIMNDC 19(1/2)1-24

© 1999Elsevier

those with AIDS, cancer, and organ transplants. 2 Ganciclovir is the current drug of choice for treatment of HCMV disease.4 Long-term administration of ganciclovir can lead to the selection of ganciclovir resistant mutants with changes in the nucleotide sequence of the UL97 or in the viral DNA polymerase genes. 6'7 When therapy fails because of the selection of Con6nuedonpage 8

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Adenovtrus Inhibition of I m m u n e . m e d i a t e d Apoptosis ( C o n t i n u e d f r o m pg. l )

on what occurs from studies on Ad behavior in mouse models 5 as well as a general understanding of the immune response. Ad infects mouse cells reasonably well, but it does not replicate; thus, whereas the mouse immune response to Ad is very well understood, the model is limited by the lack of Ad replication. In general, a complete immune response to Ad is observed in mice, including an early inflammatory response mediated by macrophages, neutrophils, cytokines, chemokines, and probably natural killer (NK) cells. Following this innate response, an immune-specific response occurs involving CD4 + T-lymphocyte helper cells, CD8 ÷ cytotoxic T-lymphocytes (CTL), and antibodies. Cytokines and chemokines orchestrate the early inflammatory and late immune responses, inducing expression of immune-specific genes and causing lymphoid cells to migrate into the infected area, wall it off, and attack and lyse the infected cells. Macrophages and neutrophils phagocytose-infected cells, and NK and CTL destroy cells through apoptosis. CTL express a specific T-cell receptor on their surface, that interacts with major histocompatability (MHC) class I antigens complexed with viral peptide antigens expressed on the surface of the virusinfected target cell. This causes activation of the CTL, resulting in up-regulation of killer mechanisms. A major killing mechanism is the perforin-granzyme system, where perforin released from the CTL causes holes to form in the target and granzymes to be introduced. One of the granzymes, granzyme B (a protease), induces apoptosis by cleaving and activating the caspases, a unique set of aspartic acid-directed proteases that mediate apoptosis. There are multiple caspases that

Receptor:

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TNFR1

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Caspase 8 ~ ~

-

oo

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Caspases l

o

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n

co

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Figure 1. Death Receptors in the TNF receptor superfamily. The receptors are localized on the cell surface oriented with their N-terminal regions outside the cell and C-terminal regions in the cytoplasm. Both Fas and TNFR1 have a protein-protein interaction region known as the death domain (light stipple). In the case of Fas, Fas ligand binding causes Fas to oligomerize and form a complex with FADD, another death domain containing protein. The death effector domain of FADD (dark stipple) binds to the death effector domain of Procaspase 8, causing assembly of multiple Procaspase 8 molecules which undergo autocleavage and activation, resulting in cleavage of downstream effector caspases (e.g. Caspase 3), and the induction of apoptosis. A similar scenario applies to TNFR1, except that the TRADD protein is also involved. These receptors also signal through other pathways which are not shown in the figure. normally exist in an inactive procaspase fOFlll. 6 They are activated by cleavage by other caspases at a specific site involving aspartic acid. The caspase cascade is initiated by "initiator" caspases, e.g. Caspases 8 and 9, that cleave and activate downstream "effector" caspases, e.g.

Caspase 3. Activated CTL also express ligands that activate at least two so-called "death receptors" that are present on the surface of target cells, CD95/APO-I/Fas (Fas) and tumor necrosis factor type I receptor (TNFR1). 7 The ligands are Fas ligand and

CLINICAL IMMUNOLOGY NEWSLEITER (ISSN 0197-1859) is issued monthly in one indexed volume per year by Elsevier Science Inc., 655 Avenue of the Americas, New York, NY 10010. Subscription price per year: $260 (Dfl. 512) for institutional subscribers, $232 (Dfl. 457) for personal subscribers. Both of these include postage and handling. Periodical postage paid at New York, NY, and at additional mailing offices. Postmaster: Send address changes to Clinical InmmnologyNewsletter,Elsevier Science Inc., 655 Avenue of the Americas, New York, NY 10010. NOTE: No responsibility is assumed by tbe Publisher for any injury and/or damage to persons or property as a matter of prnducts liabilit)~ negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. No suggested test or procedure should be carried out unless, in the reader's judgment, its risk is justified. Because of rapid advances in the medical sciences, we recommend that the independent verification of diagnoses and drug doses should be made. Discussions, views and mcomn~ndations as to medical procedures, choice of drugs and drug dosages are the responsibility of the authors. I

0 1 9 7 - 1 8 5 9 / 9 9 (see frontmatter)

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TNF, respectively (Figure 1). Other death receptors are TRAIL receptor 1/DR4, TRAIL receptor 2/DR5, and Death Receptor 3. For the Fas system, Fas ligand on the CTL interacts with Fas on the target, resulting in trimerization of Fas. 7 As is the case with all the death receptors, Fas has a "death domain" in the cytoplasmic portion of the molecule (Figure 1); this is a region for interaction with other proteins that have death domains. The protein named FADD interacts through its death domain with the death domain of Fas. FADD has another protein-protein interaction domain, called the "death effector domain," which binds to the death effector domain of Procaspase 8, causing multiple Procaspase 8 molecules to concentrate about the Fas/FADD complex. Procaspase 8 has a small amount of intrinsic protease activity, and accordingly, the Pro-domain is cleaved from Procaspase 8, resulting in complete activation of the protease activity of Caspase 8. Caspase 8, an initiator caspase, cleaves and activates effector caspases such as Caspase 3. The end result is a cascade of caspase activity that cleaves cellular proteins and causes apoptosis. The interaction of TNF with TNFR1 causes a similar activation of the caspase cascade; however, the protein named TRADD is part of the TNFR1/FADD/Procaspase 8 complex (Figure 1). NK cells also express Fas ligand on their cell surface, and they likely lyse virus-infected cells in part through the Fas pathway. Before discussing how Ad affects these apoptotic pathways, we will review the fundamentals of relevant aspects of Ad molecular biology. Ad virions have an outer proteinaceous capsid that encloses a duplex DNA genome of 36,000 base pairs (bp) (Figure 2). The virion enters the cell by endocytosis, and the genome is extruded from endosomes into the cytoplasm and enters the nucleus where transcription occurs. The genes a r e arranged in transcription units which tend to encode proteins with similar functions. The first genes to be transcribed are in the "immediate early" E 1A region. There are two E1A proteins, of 289 and 243 amino acids, named 289R and 243R, that are coded by alternatively spliced mRNAs such that they differ only by a small protein domain. The function of

Virion structural proteins Major late transcription unit ~

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• Transcription • Pre-mRNA

processing

• Regulation of cellular

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• Apoptosis Figure 2. Schematic of the genome of Ad. The black bar represents the DNA genome of 36 kbp. Arrows indicate transcription units, i.e., groups of genes transcribed from a single promoter. The E1A proteins are expressed initially, and induce transcription of the genes in the E1B, E2, E3, and E4 transcription units. Viral DNA replication occurs, then late genes are expressed. Virions begin to assemble in the cell nucleus by about one day, and cells lyse and release virus particles after about two days. Functions associated with some of the genes in the transcription units are indicated.

289R is to induce transcription of the Ad "delayed early" genes in the EIB, E2, E3, and E4 transcription units (Figure 2). This is achieved by the 289R protein binding to a variety of cellular transcription factors, including the TATA box binding protein, and causing them to assemble onto promoters and stimulate transcription through RNA polymerase II. 8 Early gene expression is followed by viral DNA replication, expression of late virion structural genes, assembly of virus in the nucleus, and eventually lysis of the cell (in about two days) and release of virus from the cell. The function of the 243R protein is to force the quiescent epithelial cell into the S-phase of the cell cycle and synthesize the enzymes and metabolites required to synthesize viral DNA. 8 This is accomplished by the 243R protein binding to two cellular proteins, pRB and p300/CBP, pRB normally exists as a complex with the E2F family of transcription factors. E2F stimulates transcription of genes involved in S-phase by binding to E2F sites in promoters. In quiescent cells, the E2F/pRB complex is tethered to the E2F binding sites and represses transcrip© 1999 ElsevierScienceInc.

tion. Repression is achieved, in part, by the recruitment to these promoters of a histone deacetylase which removes negatively charged acetyl groups from histones; as a result, the histones bind more tightly to DNA, keeping the chromatin in a closed state and preventing transcription. 9 In S-phase, pRB is inactivated by phosphorylation (by cell cycle kinases), releasing E2F to induce transcription. Also, presumably, the histone deacetylase is released from the promoter. The 243R protein mimics phosphorylation of pRB, binding to pRB and causing release of E2E 8 The p300/CBP protein is a transcriptional coactivator that binds a large number of different transcription factors and through these it is tethered to promoters, some of which are active in S-phase. ~° p300/CBP has intrinsic histone acetyltransferase activity, catalyzing acetylation of histones and opening of the promoter to transcription .u p300/CBP is activated by phosphorylation by cyclin dependent kinases. As was the case with pRB, EIA binding to p300/CBP mimics the effect of phosphorylation, u Thus, E 1A stimulates tran019%1859/99(see frontmatter)

4 C L I N I C A L I M M U N O L O G Y Newsletter

scription of S-phase genes by inactivating pRB and activating p300/CBE Considering the multitude of transcription regulatory activities of the E1A proteins, it is perhaps not surprising that they induce cellular apoptosis. 2 In the context of this article, it is satisfying to consider this apoptosis to be a host defense against Ad infection. The mechanism by which E1A induces apoptosis is partially understood. The 243R protein functions in two ways to cause up-regulation of p53, the tumor suppressor protein that blocks the cell cycle and, under appropriate conditions, induces apoptosis. 12a3 p53 is a transcription factor that induces apoptosis, in part, by stimulating synthesis of proapoptotic proteins such as Bax. p53 stability is controlled by the protein named MDM2, a ubiquitin ligase that binds and destabilizes p53. p300/CBP interacts with both MDM2 and p53, resulting in degradation of p53.13 Binding of the 243R protein to p300/CBP is thought to prevent the degradation of p53.13 The second mechanism of E 1A-mediated p53 stabilization involves pRB. Binding of 243R to pRB causes release of E2F, resulting in induction of transcription of the gene encoding the protein named ARE12 ARF interacts with MDM2 and p53, stabilizing p53. E 1A-induced apoptosis also involves other mechanisms, the end result being the activation of caspases. Ad contends with E1A-induced apoptosis by synthesizing two proteins that inhibit intrinsic cellular apoptosis. 2 The Ad protein named E1B-55K (a 55 kDa protein encoded by the E1B transcription unit) binds to p53, preventing it from inducing apoptosis (Figure 3). As part of the mechanism, the E1B-55K/p53 complex is targeted to promoters containing p53-binding sites, where E1B-55K acts as a repressor of p53-mediated transcription. 14 The Ad protein named E1B-19K is a functional homolog of the Bcl-2 family of anti-apoptotic proteins. ~5'16These proteins inhibit apoptosis by preventing activation of caspases through the "stress pathway" which is mediated through mitochondria. The story is too complex to be discussed in detail here. In one model 16(Figure 3), the anti-apoptotic (e.g. Bcl-x L) and proapoptotic (e.g. Bax, Bik, Bak, BNIP1, BNIP3) members of the Bcl-2 family reside in the mitochondrial outer membrane. Bcl-XL sequesters the protein named

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Apaf- 1. Up-regulation of p53 (i.e. cellular E1A stress) induces synthesis of Bax, which in turn causes Apaf- 1 to dissociate from Bcl-XL and P53 I-- EIB-55K cytochrome c to be released from mitochondria. Apaf-1 and cytoMitochondria chrome c bind to Procaspase 9, resulting in Bcl-xJApafl ~ , Bax I--EIB-19K cleavage and activation of Caspase 9 (an initiator caspase). Caspase 9 cleaves and activates effector caspases, and Apafl + Cytochrome c apoptosis ensues. E IB19K binds to many of the pro-apoptotic members of the Bcl-2 family2'1s'17 and it may Procaspase 9 ~ Caspase 9 prevent them from stimulating release of Apaf1 and cytochrome c. Figure 3. Model depicting how E1B-55K and E1B-19K of Ad Whereas the E1Bmay inhibit intrinsic cellular apoptosis induced as a result of the 55K and E1B-19K profunctions of the E 1A proteins. teins block intrinsic cellular apoptosis, the apoptosis through stress effects on mitoproteins coded by the E3 transcription chondria, by binding to pro-apoptotic unit prevent apoptosis induced through members of the Bcl-2 family and preventcells of the immune system I (Figure 4). ing the activation of Caspase 9. E 1B-19K CTL are the major enemy, although NK also blocks the other major apoptotic pathcells are probably also important. The E3way that emanates from the death domain gpl9K protein (a glycoprotein of 19 kDa) receptors. 23'24It apparently prevents the is localized in the membrane of the endooligomerization of FADD about the plasmic reticulum (ER). Its role is to prereceptor/FADD/Procaspase 8 complex, vent recognition of Ad-infected cell by and accordingly prevents activation of CTL. gpl9K forms a complex with newly Caspase 8. 25 synthesized MHC class I antigens, retains RID is an integral membrane protein them in the ER, and prevents their transport to the cell surface) 8'19The gpl9K/ complex composed of two E3-coded proteins, RIDer and RIDI3.24'26RIDer and class I antigen complex is retained in the RIDB were previously named E3-10.4K ER by virtue of an ER retrieval signal and E3-14.5K, according to their moleculocated at the extreme C-terminus of lar masses, and the protein complex was gpl9K. Since the MHC class I/viral pepnamed E3-10.4K/14.5K. As seen by inditide complex does not appear on the cell rect immunofluorescence, RID localizes surface, the cells are not recognized and killed by CTL. 2° to the plasma membrane, ER, Golgi, and occasionally vesicles in the cell. 24 RIDB The Ad proteins named RID (Receptor is a type-I membrane polypeptide with a Internalization and Degradation), E3-14.7K, cleaved N-terminal signal, oriented with and E1B-19K are expected to inhibit its C-terminus in the cytoplasm.1 The Ncytolysis of infected cells by CTL should terminal luminal/extracellular domain is the gpl9K mechanism not be fully effective. 1 They might also be expected to O-glycosylated, and the cytoplasmic inhibit killing by NK cells. Both the Fasdomain is phosphorylated on one serine Fas ligand and TNF-TNFR1 pathways are residue. RIDct is composed of two forms, blocked by these Ad proteins 21-24(Figure 5). one with the N-terminal signal sequence As mentioned earlier, E1B-19K inhibits cleaved and the other with it uncleaved,

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with the two forms joined by a disulfide bond. The C-terminal regions of the two forms of RIDoc are oriented to the cytoplasm. 1 RID inhibits apoptosis induced through the TNFR122 and Fas 24'27'2spathways. This conclusion is derived from studies where cells were infected with Ad mutants that do or do not express RID, or with replication-defective Ad vectors that express only RID, followed by treatment of cells with either TNF or a monoclonal antibody to Fas that triggers apoptosis through the Fas pathway (cells stably expressing RID were also examined). The mechanism of action of RID is unique: it causes Fas to be internalized from the cell surface27 into endosomes which are transported to lysosomes where Fas is degraded. 24 It also causes TNFR1 to be internalized from the cell surface, but it is not clear whether this TNFR1 is degraded (unpublished results). Because Fas and TNFR1 are downregulated, they cannot interact with their respective ligands, and apoptosis does not occur. RID was shown to directly inhibit killing by CTL, in an experiment where CTL obtained from perforin knockout mice were examined for their ability to

lyse RID-expressing Ad-infected target cells. 24 Since these CTL lack perforin, they are expected to kill primarily through the Fas pathway. RID causes internalization and degradation not only of Fas, but also the receptor for epidermal growth factor.26 The RID effect is not global inasmuch as transferrin receptor, 6-1ymphotoxin receptor, and CD40 are not affected.24'27'2sIt is interesting to consider how RID, a small protein, can exert similar effects on members of the TNF receptor and protein tyrosine kinase superfamilies. It seems unlikely that RID binds directly with these receptors; rather, it probably interacts with a common component of the receptor sorting machinery. Interestingly, RIDtx and RIDI3 have two motifs in their cytoplasmic domains that are known to function in the endocytosis and lysosomal targeting of receptors, namely a dileucine motif (LL) and a tyrosine-based motif (YXX~, where ~ is a bulky, hydrophobic amino acid). Our working model is that RID, acting through these motifs, coerces receptors to enter endosomes and be targeted to lysosomes where the receptors are degraded. RID then recycles back to the cell surface

and repeats the process. The other anti-apoptotic E3 protein, E3-14.7K (14,700 Da), is a non-membrane protein localized in the cytoplasm and nucleus 29 (unpublished results). E314.7K is a potent inhibitor of TNF-induced apoptosis. 21'3°E3-14.7K is also reported to inhibit apoptosis through the Fas pathway. 31 In this latter study, E3-14.7K was shown to bind Procaspase 8 and inhibit apoptosis induced by transient transfection of Procaspase 8 FADD into cells, in another series of studies using the yeast or two hybrid system to identify proteins that interact with E3-14.7K, proteins named FIP- 1 (14.7K interacting protein1), FIP-2, and FIP-3 were identified. 29'32'33 FIP-2 is a member of a new family of proteins that may play a role in TNF signal transduction. FIP-2 co-localizes with E3-14.7K in the cytoplasm, especially the perinuclear region. 29 It overcomes the ability of E3-14.7K to inhibit apoptosis induced by overexpression of TNFR 1 or RIP, another death domain-containing protein that interacts with activated TNFR1 (Figure 5). FIP-2 does not induce apoptosis itself upon transient transfection into cells. FIP-3 is related to FIP-2. 33

RID Complex I

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• Inhibits:

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- F a s L apoptosis

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• Integral membrane proteins are indicated by black bars

• D e g r a d e s Fas

• hA: arachidonic acid

• Internalizes T N F R 1

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In vivo: • Inhibits inflammation • Inhibits inflammation and pathology

and pathology

Figure 4. Schematic illustration of the Ad E3 transcription unit. The proteins are indicated by the horizontal bars, and the functions known for the proteins are indicated. RID, receptor internalization and degradation; ADP, adenovirus death protection; FasL, Fas ligand; EGFR, epidermal growth factor receptor. © 1999 ElsevierScienceInc.

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Unlike FIP-2, FIP-3 binds directly to RIP. FIP-3 induces apoptosis in transfected cells, a response inhibited by E3-14.7K. 33 FIP-3 also inhibits TNF-induced activation of the transcription factor NFr,B. FIP-3 and FIP-2 may be part of TNF-activated apoptotic and NFr, B pathways that are induced through the TNFR1/TRADD/RIP complex. 33 E3-14.7K may inhibit TNFinduced apoptosis by binding the FIP proteins and blocking this pathway (Figure 5). FIP- 1 is a member of the Ras family of small GTPases; it is not known if FIP-1 is involved in TNF-induced apoptosis. 32 E3-14.7K and RID not only inhibit TNF-induced apoptosis, they also prevent TNF-induced synthesis of arachidonic acid (AA). 3°'~4 The AA is synthesized by cytosolic phospholipase A2 (cPLA2). Activation of cPLA 2 is believed to be important for TNF-induced apoptosis although the mechanism is not understood. 3a cPLA2 is localized in the cytosol in an inactive state; TNF causes cPLA2 to translocate to membranes where it cleaves AA from membrane phospholipids. RID prevents TNF-induced translocation of cPLA 2 to membranes, which could explain in part how RID inhibits TNF-induced apoptosis. 35 Inhibition of cPLA 2 translocation by RID occurs before TNFR1 is cleared from the cell surface by RID, implying that the RID effect on cPLA2 is independent of its ability to down-regulate TNFR1. It is not known how E3-14.7K inhibits TNF-induced synthesis of AA. AA is the precursor to the pro-inflammatory leukotrienes and prostaglandins. RID and E3-14.7K, by preventing TNFinduced synthesis of AA, may block a TNF-induced eicosanoid-mediated inflammatory response. Studies in animal models would be in accord with this possibility. In mice infected in the lung with Ad, there is moderate pathology and infiltration of inflammatory cells. 5 With Ad mutants that lack both RID and E3-14.7K, this pathology and inflammation is dramatically increased. Expression of either RID or E3-14.7K by Ad is sufficient to inhibit pathology and inflammation. 5 In a transgenic mouse that expresses E3-14.7K specifically in the lung, pathology and inflammation are much reduced as compared to wild-type mice following infection with an Ad vector that expresses luciferase but no Ad proteins. 36

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Further studies on these Ad proteins will be directed towards understanding their molecular mechanism of action. We expect that they will provide interesting clues to the mechanisms of death receptor signal transduction, apoptosis, and protein sorting from the cell surface to lysosomes. Further studies should also provide insights into how the host defends itself from attack by viruses, given that Ad would not have evolved a strategy to block host responses if these responses © 1999 Elsevier Science Inc.

were not important. These Ad proteins might also have practical uses. For example, Ad vectors for gene therapy suffer because they induce an inflammatory response that eliminates the transduced cells. Conceivably, expression of Ad anti-immune proteins from these vectors might hide the vector from the immune system and allow it to persist for longer periods. Indeed, such a study has been reported recently for an Ad vector that expresses gpl9K. 37

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Acknowledgments Our research was supported by grants CA21470, CA58538, and CA71704 from the National Institutes of Health. We apologize to authors whose work could not be fully cited because of space limitations. References 1. Wold WSM, Tollefson AE: Adenovirus E3 proteins: 14.7K, RID, and gpl9K inhibit immuneinduced cell death; adenovirus death protein promotes cell death. Semin Virol 8:515-523, 1998. 2. Chinnadurai G: Control of apoptosis by human adenovirus genes. Semin Virol 8: 399-408, 1998. 3. Horwitz, MS, Adenoviruses. In Fields Virology (BN Fields, DM Knipe, and PM Howley, Eds.), p. 2149-2171, Lippincott-Raven Publishers, Philadelphia, 1996. 4. Sterman DH, Treat J, Litzky LA, Amin KM, Coonrod L, Molnar-Kimber K, Recio A, Knox L,Wilson JM, Albelda SM, Kaiser LR: Adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir gene therapy in patients with localized malignancy: results of a phase I clinical trial in malignant mesothelioma. Human Gene Ther 9:1083-1092, 1998. 5. Sparer T, Tripp RA, Dillehay DL, Hermiston TW, Wold WSM, Gooding LR: The role of adenovirus early region 3 proteins (gpl9K, 10.4K, 14.5K, and 14.7K) in a marine pneumonia model. J Virol 70:2431-2439, 1996. 6. Thornherry NA, Lazebnik Y: Caspases: enemies within. Science 281:1312-1316, 1998. 7. Ashkenazi A, Dixit VM: Death receptors: signaling and modulation. Science 281:1305-1308, 1998. 8. Nevins JR: Adenovirus EIA: transcription regulation and alteration of cell growth control. Curr Topics Microbiol Immunol 199:25-32, 1995.

CLINICAL IMMUNOLOGY Newsletter 7

Molecular Cell 2:405-415, 1998. 14. Martin IVIED,Berk AJ: Adenovirus E1B 55K represses p53 activation in vitro. J Virol 72:3146-3154, 1998. 15. Han J, Sabbatini P, Perez D, Rao L, Modha D, White E: The E1B 19K protein blocks apoptosis by interacting with and inhibiting the p53inducible and death-promoting Bax protein. Genes Dev 10:461-477, 1996. 16. Adams JM, Cory S: The Bcl-2 protein family: arbiters of cell survival. Science 281:13221326, 1998. 17. Boyd JM, Malstrom S, Subramanian T, Venkatesh LK, Schaeper U, Elangovan B, D'Sa-Eipper C, Chinnadurai G: Adenovirus EIB 19 kDa and Bcl-2 proteins interact with a common set of cellular proteins. Cell 79:341351, 1994. 18. Andersson M, Paabo S, Nilsson T, Peterson PA: Impaired intracellular transport of class I MHC antigens as a possible means for adenoviruses to evade immune surveillance. Cell 43:215-222, 1985. 19. Burger HG, Kvist S: An adenovirus type 2 glycoprotein blocks cell surface expression of human histocompatibility class I antigens. Cell 41:987-997, 1985. 20. Rawle FC, Tollefson AE, Wold WS, Gooding LR: Mouse anti-adenovirus cytotoxic T lymphocytes. Inhibition of lysis by E3 gpl9K but not E3 14.7K. J Immunol 143:2031-2037, 1989. 21. Gooding LR, Elmore LW, Tollefson AE, Brady HA, Wold WS: A 14,700 MW protein from the E3 region of adenovirus inhibits cytolysis by tumor necrosis factor. Cell 53:341-346, 1988. 22. Gooding LR, Ranheim TS, Tollefson hE, Aquino L, Duerksen-Hughes P, Horton TM, Wold WS: The 10,400- and 14,500-dalton proteins encoded by region E3 of adenovirus function together to protect many but not all mouse cell lines against lysis by tumor necrosis factor. J Virol 65:4114-4123, 1991.

9. DePinho RA: Transcriptional repression. The cancer-chromatin connection. Nature 391:533536, 1998. 10. Giles RH, Peters DJM, Breuning MH: Conjunction dysfunction: CBP/p300 in human disease. Trends Genet 14:178-183, 1998. 11. Ait-Si-Ali S, Ramirez S, Bane E Dkhissi F, Magnaghi-Jaulin L, Girault JA, Robin P, Knibiehler M, Pritchard LL, Ducommun B, Trouche D, Harel-Bellan A: Histone acetyltransferase activity of CBP is controlled by cycledependent kinases and oncoprotein EIA. Nature 396:184-186, 1998.

23. Gooding LR, Aquino L, Duerksen-Hughes PJ, Day D, Horton TM, Yei SP, Wold WS: The E1B 19,000-molecular-weight protein of group C adenoviruses prevents tumor necrosis factor cytolysis of human cells but not of mouse cells. J Virol 65:3083-3094, 1991. 24. Tollefson hE, Hermiston TW, Lichtenstein DL, Colle CF, Tripp RA, Dimitrov T, Toth K, Wells CE, Doherty PC, Wold WSM: Forced degradation of Fas inhibits apoptosis in adenovirusinfected cells. Nature 392:726-730, 1998. 25. Perez D, White E: EIB 19K inhibits Fas-mediated apoptosis through FADD-dependent sequestration of FLICE. J Cell Biol 141:1255-1266, 1998.

12. Prives C: Signaling to p53: breaking the MDM2-p53 circuit. Cell 95:5-8, 1998. 13. Grossman SR, Perez M, Kung AL, Joseph M, Mansur C, Xiao Z, Kumar S, Howley PM, Livingston DM: p300/MDM2 complexes participate in MDM2-mediated p53 degradation.

26. Tollefson hE, Stewart AR, Yei SP, Saha SK, Wold WS: The 10,400- and 14,500-dalton proteins encoded by region E3 of adenovirus form a complex and function together to down-regulate the epidermal growth factor receptor. J Virol 65:3095-3105, 1991.

© 1999 Elsevier Science Inc.

27. Shisler J, Yang C, Walter B, Ware CF, Gooding LR: The adenovirus E3-10.4K/14.5K complex mediates loss of cell surface Fas (CD95) and resistance to Fas-induced apoptosis. J Virol 71:8299-8306, 1997. 28. Elsing A, Burgert H-G: The adenovirus E3/10.4K-14.5K proteins down-modulate the apoptosis receptor Fas/Apo- 1 by inducing its internalization. Proc Natl Acad Sci USA 95:10072-10077, 1998. 29. Li Y, Kang J, Horwitz MS: Interaction of an adenovirus E3 14.7-kilodalton protein with a novel tumor necrosis factor alpha-inducible cellular protein containing leucine zipper domains. Mol Cell Biol 18:1601-1610, 1998. 30. Krajcsi P, Dimitrov T, Hermiston TW, Tollefson AE, Ranheim TS, Vande Pol SB, Stephenson AH, Wold WSM: The adenovirus E3-14.7K protein and the E3-10.4K/14.5K complex of proteins, which independently inhibit tumor necrosis factor (TNF)-induced apoptosis, also independently inhibit TNF-induced release of arachidonic acid. J Virol 70:4904-4913, 1996. 31. Chert P, Tian J, Kovesdi I, Bruder JB: Interaction of the adenovirus 14.7K protein with FLICE inhibits Fas ligand-induced apoptosis. J Biol Chem 273:5815-5820, 1998. 32. Li Y, Kang J, Horwitz M: Interaction of an adenovirus 14.7-kilodalton protein inhibitor of tumor necrosis factor alpha cytolysis with a new memher of the GTPase superfamily of signal transducers. J Virol 7 i :1576-1582, 1997. 33. Li Y, Kang J, Friedman J, Tarassishin L, Ye J, Kovalenko A,Wallach D, Horwitz M: Identification of a Cell Protein (FIP-3) as a Modulator of NF-kB Activity and as a Target of an Adenovirus Inhibitor of Tumor Necrosis Factor c~induced Apoptosis. Immunology 96:1042-1047, 1999. 34. Thorue TE, Voelkel-Johnson C, Casey WM, Parks LW, Laster SM: The activity of cytosolic phospholipase A2 is required for the lysis of adenovirus-infected cells by tumor necrosis factor. J Virol 70:8502-8507, 1996. 35. Dimitrov T, Krajcsi P, Hermiston TW, Tollefson AE, Hannink M, Wold WSM: Adenovirus E310.4K/14.5K protein complex inhibits tumor necrosis factor-induced translocation of cytosolic phospholipase A2 to membranes. J Virol 71:2830-2837, 1997. 36. Harrod KS, Hermiston TW, Trapnell BC, Wold WSM, Whitsett JA: Lung-specific expression of E3-14.7K in transgenic mice attenuates adenoviral vector-mediated lung inflammation and enhances transgene expression. Human Gene Ther 9:1885-1898, 1998. 37. Bruder JT, Jie T, McVey DL, Kovesdi I: Expression of gp 19K increases the persistence of transgene expression from an adenovirus vector in the mouse lung and liver. J Virol 71:7623-7628, 1997.

0197-1859/99 (see frontmatter)

8 C L I N I C A L I M M U N O L O G Y Newsletter

Flow Cytometric Antiviral Drug Susceptibility Assays (Continuedfrompg. 1) ganciclovir resistant mutants, foscarnet or cidofovir can be used for treatment of life threatening disease if the clinical isolate is susceptible to these antiviral drugs. 22'25 However, adequate procedures for determining drug susceptibility of HCMV isolates in a clinically relevant time frame are not currently available.

Drug Susceptibility Assays for HCMV Clinical Isolates When immunocompromised patients with HCMV infection are treated with ganciclovir, foscarnet or cidofovir there is a significant potential for selecting drug resistant viruses. 6"9Both genotypic and phenotypic methods are available for determining drug susceptibilities of HCMV clinical isolates, t Genotypic assays that detect specific mutations known to be associated with resistance to a particular drug are rapid and often automated. However, they depend on prior knowledge of the specific nucleotide changes that lead to drug resistance or of the gene targeted by the antiviral agent. The variety of possible genetic changes both within the targeted gene and within other viral genes not directly targeted by the antiviral compound that could lead to resistance makes the availability of genetic probes for all possible mutations a difficult task. In contrast, phenotypic assays determine drug susceptibility irrespective of genetic changes and may be more useful for detecting drug resistant viruses. Currently used phenotypic assays for HCMV drug susceptibility testing involve isolation of infectious virus from clinical specimens by inoculation of cell cultures and the production of cytopathic effect (CPE) characteristic of HCMV infection. Confirmation of the virus causing the CPE is usually performed by treating infected cells with a fluorochrome labeled monoclonal antibody directed against an antigen specific for HCMV followed by observation of specific immunofluorescence using a fluorescence microscope. Clinical isolates of HCMV remain cell associated when grown in cell culture. Once a sufficient number of virus infected cells are available, a drug susceptibility 0197-1859/99 (see frontmatter)

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assay can be performed. However, most drug susceptibility assays require cell free virus that can be obtained by ultrasonic disruption or freeze thaw cycles to release the virus from the infected cells. The down side of these disruption procedures is the loss of infectious virus. Alternatively, the virus infected cells can be successively passed for several weeks until they begin to release infectious virus. Once cell free virus is available and the amount of infectious virus has been determined by a plaque assay, then the drug susceptibility of the clinical isolate can be determined by the plaque reduction assay (PRA), 5'26 or by a dye release assay? Each of these procedures are very time consuming taking several weeks for plaques to form in the PRA or for enough cells to be killed in the dye release assays. Furthermore, any assay involving the production of plaques must be read using a microscope to count the plaques, making the assay very labor intensive and often subjective. A more rapid PRA was developed by Lurain et al. 12that used virus infected cells from cultures of HCMV clinical isolates instead of cell free virus. Medium containing various concentrations of antiviral drugs was placed in different wells of 24well plates and then approximately 100 virus infected cells were added to each well. After seven days of incubation at 37°C, the plaques were counted under a microscope. This assay can be completed within a few weeks after obtaining the clinical sample. In addition to saving time, the assay is performed on fresh clinical isolates that have not been repeatedly passed in culture, a procedure that could lead to the introduction of mutations in the virus population that could influence the phenotype of the isolate. In another attempt to shorten the time required to determine drug susceptibility of HCMV clinical isolates, a number of phenotypic assays have been developed that rely on the detection of viral antigens or viral DNA that are produced during infection of cell cultures with HCMV clinical isolates. The assays involved inoculation of cell cultures with the patient's peripheral blood leukocytes in the absence and presence of inhibitory concentrations of antiviral drugs. After four to six days of incubation, infected cells were treated with a fluorochrome labeled monoclonal antibody to the © 1999 ElsevierScienceInc.

HCMV immediate early (IE) or late antigens followed by detection by fluorescence microscopy. TM These assay systems require less time than the PRA. However, they remain labor intensive, they do not lead to the determination of an IC50 value, and they are only useful when a relatively large amount of virus infected cells is present in the clinical sample. The in situ DNA hybridization assay that measures virus replication by detection of de novo viral DNA synthesis has also been developed for drug susceptibility testing; however, it does not save time in sample preparation and is not very sensitive. ~ The use of fluorochrome labeled monoclonal antibodies and flow cytometry to detect and count virus infected cells has been reviewed. 15 Recent publications have reported the use of this technique for the rapid phenotypic determination of susceptibility of HCMV laboratory strains and clinical isolates to ganciclovir.11'16-21 The flow cytometry based drug susceptibility assay has several advantages over the currently used phenotypic assays. First, a large number of cells can be analyzed in less than one minute allowing for the determination of a robust statistical difference between susceptible and resistant clinical isolates. Second, when cell associated HCMV clinical isolates are used as the inoculum, the time required to obtain enough infected cells for analysis is reduced to approximately one week after virus isolation. Third, this drug susceptibility assay requires only 72 to 96 hr of incubation in the presence of drug before analysis of the percentage of cells synthesizing IE antigens and only 144 hr of incubation in the presence of drug before analysis of the percentage of cells synthesizing late antigens. Fourth, since a large percentage of the cells in the clinical sample are analyzed, there is a better chance of detecting a small number of drug resistant mutants that may be present among the population of virus infected cells. Fifth, the IC50 values of HCMV clinical isolates for ganciclovir determined by the flow cytometry assay are similar to those obtained with the PRA and the CPE assays. 11'16'18"19Sixth, the assay of virus infected cells can be performed on fresh clinical isolates that have not been repeatedly passed in culture, thus avoiding the possibility of introducing mutations into

C L I N I C A L I M M U N O L O G Y Newsletter 9

Vol. 19, No. 1/2, 1999

the virus population that may influence the phenotype of the original clinical isolate. In summary, flow cytometric analysis of drug susceptibility of HCMV clinical isolates is automated, rapid, objective, and not labor intensive. This procedure has also been used to determine the susceptibility of herpes simplex virus type 1 to acyclovir, foscarnet, and ganciclovir. ]623 This report will review the published papers that have used flow cytometry to determine the drug susceptibility of HCMV clinical isolates for ganciclovir. This will be followed by a detailed description of the flow cytometry based drug susceptibility assay as it is currently used in my laboratory for determining drug susceptibility of HCMV clinical isolates to a number of clinically used and experimental antiviral drugs that inhibit their replication.

Flow Cytometric Determination of Ganciclovir Susceptibility of HCMV Clinical Isolates The first report of the use of fluorochromelabeled monoclonal antibodies and flow cytometry to determine drug susceptibility was an analysis of the effect of ganciclovir on the synthesis of IE and late antigens in cells infected with the AD169 laboratory strain of HCMV) 6 MRC-5 cell monolayers were infected with AD 169 at an MOI of 10 in the presence of various concentrations of ganciclovir. Virus infected cells were harvested at various times post infection, permeabilized with 90% methanol, treated with either an FITClabeled monoclonal antibody to an IE antigen or an FITC-labeled monoclonal antibody to a late antigen and the percentage of antigen positive cells was determined by flow cytometry. At this MOI, where only a single round of virus replication occurs, inhibitory concentrations of ganciclovir had no effect on the percentage of IE antigen positive cells whereas the percentage of late antigen positive cells was reduced by greater than 90%. This study showed that fluorochrome labeled monoclonal antibodies in conjunction with flow cytometry could be used to determine the effect of ganciclovir on the synthesis an HCMV late antigen. The fact that ganciclovir had no effect on the synthesis of the IE antigen in cells infected at high MOI is consistent with its mode of action.

In an independent study, monoclonal antibody to the HCMV IE antigen and flow cytometry were used to determine drug susceptibility of a number of ganciclovir or foscarnet susceptible or resistant laboratory strains and clinical isolates of HCMV.n Tube cultures of MRC-5 cell monolayers were inoculated with 100 tissue culture infectious dose 50s (TCID50s) of cell free virus in the presence of various concentrations of either drug. After 7 days of incubation, the cells were removed, permeabilized and treated with a murine monoclonal antibody to the HCMV IE antigen followed by FITC-labeled goat antimouse IgG. Then the cells were analyzed by flow cytometry for forward and right angle light scatter to separate cells from debris and the intact cells were gated and analyzed for events versus FITC fluorescence intensity to determine the percentage of antigen positive cells. PRA were done in parallel. Drug susceptible clinical isolates had a mean IC50 value of 18 laM ganciclovir and 80 }aM foscarnet with the flow cytometry assay and 9 }aM ganciclovir and 56 }aM foscarnet with the PRA. Resistant viruses had a mean IC5o value of 47 }aM ganciclovir and >200 laM foscarnet by the flow cytometry assay and 49 }aM ganciclovir and >200 }aM foscarnet by the PRA. These results showed that the flow cytometry drug susceptibility assay can determine IC50 values for both HCMV laboratory strains and clinical isolates and that the results were qualitatively similar to those obtained by the PRA. For the drug sensitive strains, the IC50 values for ganciclovir were approximately 1.5 to 2 fold higher by the flow cytometry assay than by the PRA, but both assays clearly identified drug susceptible and drug resistant strains of HCMV. In a third study, the utility of using flow cytometry to determine drug susceptibilities of the ganciclovir susceptible laboratory strain of HCMV, AD 169, a ganciclovir resistant derivative of AD 169, D6/3/1,13 and a number of drug susceptible and resistant clinical isolates was tested in a ~8 multicenter study. These authors used a three color flow cytometric analysis to simultaneously measure the effect of ganciclovir on the synthesis of IE and late antigens. Monolayer cultures of HFF, MRC-5, or lung cells were infected with AD169 or its ganciclovir resistant deriva© 1999 ElsevierScienceInc.

tive, D6/3/1, at an MOI of 1 to 10. After 72 to 96 hr of incubation, the cells were harvested, permeabilized, and treated with a cocktail consisting of FITC-labeled monoclonal antibody to the HCMV IE antigen and PE-labeled monoclonal antibody to an HCMV late antigen. After incubation and washing, 7-amino-actinomycin D (7-AAD) was added to the cells to label the DNA with a fluorochrome and the cells were analyzed for three color fluorescence by flow cytometry. Initially the cells were analyzed for 7-AAD versus forward angle light scatter to distinguish intact cells from debris. The intact cells (7-AAD positive) were gated and analyzed for the late antigen PE versus IE antigen FITC fluorescence intensities. At these MOIs with AD169 or D6/3/1, ganciclovir had no effect on the synthesis of the IE antigen but reduced the percentage of cells synthesizing the late antigen from 47% in the absence of drug to 0.7% in the presence of 12 }aM ganciclovir. For cells infected with the ganciclovir resistant derivative, D6/3/1, ganciclovir (12 }aM) had no effect on the percentage of cells synthesizing either the IE or the late antigen, confirming that this isolate is resistant to ganciclovir) 3 The IC50 values for AD169 determined by five different laboratories were between 1.5 and 3 }aM ganciclovir, well within the accepted value " M V . lo In for this laboratory strain of H,.. the same multicenter study, the ganciclovir susceptibilities of 19 HCMV clinical isolates were determined using an FITClabeled monoclonal antibody to a late antigen to label the virus infected cells and 7-AAD to label the DNA in intact cells in a two-color flow cytometry assay. For analysis of clinical isolates, 105 virus infected cells were added directly to medium containing various concentrations of ganciclovir and the flasks were incubated at 37°C for 144 hr. Under these circumstances the MOI was 0.1 (105 infected cells/106 uninfected cells) and every cell in the culture was not infected at the beginning of the experiment. This assay measured the ability of input virus infected cells to spread infection to adjacent cells in the monolayer in the absence or presence of various concentrations of ganciclovir. PRA were performed in parallel. The results showed that two clinical isolates were resistant with IC50 values of 0197-1859/99(see frontmatter)

10 C L I N I C A L I M M U N O L O G Y Newsletter

>96 laM ganciclovir by both assays and in 14 clinical isolates were sensitive by both assays with average IC5o values of 2.79 laM ganciclovir by flow cytometry assay and 2.80 laM ganciclovir by the PRA, a remarkable correlation. Three of the clinical isolates had IC5o values by flow cytometry that were discordant with the PRA data; the IC50 values with the flow cytometry assay were at least two fold greater than the values obtained with the PRA. Despite the less than perfect correlation between the IC50 values derived from each assay, the flow cytometry assay accurately identified the clinical isolates that were susceptible or resistant to ganciclovir. This multicenter study showed that the flow cytometry drug susceptibility assay is transportable to other laboratories and that different laboratories could obtain similar IC50 values for AD169 strain of HCMV. A second publication from the multicenter group reported on a simplified and more rapid procedure to determine drug susceptibilities on HCMV clinical samples. 19Medium containing various concentrations of ganciclovir was added to HFF cell monolayers that were infected with HCMV infected cells at an MOI of 0.1, harvested at 96 hr post infection and treated with an FITC-labeled monoclonal antibody to the HCMV IE antigen followed by 7-AAD and analysis by flow cytometry. PRA were done in parallel. Of 25 HCMV clinical isolates, four were shown to be resistant by both assays, two were partially resistant by both assays, and the remainder were susceptible by both assays. The flow cytometry assay gave slightly higher ICs0 values than the PRA for the sensitive and partially resistant clinical isolates, but the ICso values for both assays had an acceptable correlation (r2 = 0.473; P = 0.001). These authors suggest that this rapid flow cytometry drug susceptibility assay should replace the labor intensive, subjective PRA. In summary, these studies showed that flow cytometry is useful for a rapid quantitative determination of ganciclovir susceptibility of HCMV laboratory strains and clinical isolates. The assay readily distinguished between drug-susceptible and drug-resistant laboratory strains and clinical isolates. For cell-free, drug-susceptible and drug-resistant, laboratory 0197-1859/99 (see frontmatter)

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strains, the ICso values strains for ganciclovir were obtained over a range of MOIs (1 to 10 for AD169) when the effect of the drug on the percent of cells synthesizing the late antigen was examined. For cell associated drug susceptible and resistant HCMV clinical isolates, IC50 values were obtained when the effect of drug on the percent of IE antigen synthesis was analyzed after infection of cells at MOIs of 0.01 to 0.1. In each case, the average IC50 values obtained with the flow cytometry assay were approximately two fold higher than those obtained with the PRA. However, both assays clearly distinguished drug susceptible from drug resistant laboratory strains and clinical isolates. Similar findings were reported for HSV type 1 for acyclovir.23 For herpes simplex viruses, IC50 values can be determined within 24 hr after obtaining the clinical isolate. Since the assay can be performed over a wide range of MOI, a virus titration is not required before performing the drug susceptibility assay. These results suggest that the flow cytometry drug susceptibility assay should replace the time consuming and labor intensive PRA for determining drug susceptibility of HCMV to ganciclovir. With the availability monoclonal antibodies to human T-cell leukemia viruses (HTLV I and II), human immunodeficiency viruses (HIV 1 and 2), varicelia zoster virus (VZV), Epstein Barr virus (EBV), human herpesviruses 6, 7, and 8 (HHV6, 7, and 8), similar phenotypic flow cytometry based drug susceptibility assays could be developed for antiviral compounds that inhibit the replication of these viruses.

Flow Cytometry Drug Susceptibility Assay: 1999 Version The remainder of this review will present some unpublished data using our current procedures for performing the flow cytometry drug susceptibility assay. The procedures for performing the assay on cell free virus and cell associated virus are presented in Tables 1 and 2, respectively. Figure 1 illustrates a typical analysis of the effect of foscarnet on the percentage of cells synthesizing the IE (A-C) or late (D-F) antigen. The monoclonal antibody to the HCMV IE antigen (E-13, M-810) used in these experiments recognizes an epitope encoded by exon 2 that is shared by © 1999 Elsevier Science Inc.

both IE-1 and IE-2 antigens of HCMV. 14 It has been widely used for identification of HCMV infected cells and seems to detect clinical isolates obtained throughout the world. Monoclonal antibodies to both IE and late antigens are commercially available from Chemicon International, Inc., Temecula, CA, as well as other monoclonal antibody companies. For the experiment shown in Figure 1, HFF cell monolayers were infected with a foscarnet sensitive HCMV clinical isolate. When 90% of the cells in the monolayer exhibited CPE, the cells were harvested and counted to determine the number of cells/ml. Medium containing various concentrations of foscarnet (0, 50, 100, 200, 400, and 800 laM) were added to 25 cm 2 flasks containing confluent HFF cell monolayers. Then 103 tO 104 HCMV infected cells were added to each flask. After incubation at 37°C for 144 hr, the cells were removed from the flasks and prepared for flow cytometric analysis as described in Table 1. The data in Figure 1 show the effect of increasing concentrations of foscarnet on the percentage of cells synthesizing the IE or late antigens. In the absence of foscarnet (panels B and E), 34.7% of the cells are positive for the IE antigen and 24.7% of the cells are positive for the late antigen. In the presence of 100 laM foscarnet (panels C and F), 16.7% of the cells are positive for the IE antigen and 11.6% are positive for the late antigen. Using the percent reduction data obtained from all six foscarnet concentrations tested, the IC5o values for this clinical isolate were 99.58 laM foscarnet based on the reduction in the percentage of IE antigen positive cells and 102.06 laM foscarnet based on the reduction in the percentage of late antigen positive cells. These two IC5o values are in excellent agreement and demonstrate that, under conditions of low MOI, IC5o values can be determined using either the IE antigen or late antigen. Furthermore, the ability to use the IE antigen to measure IC5o values lends itself to analysis after 72 to 96 hr of incubation with the antiviral drug.~9 The broad applicability of this assay for susceptibility testing of HCMV was demonstrated when it was used to determine the IC50 values of six different anti HCMV compounds for a set of 35 HCMV clinical isolates. 2°'21 The data comparing II

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CLINICAL IMMUNOLOGY

Newsletter

11

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F i g u r e 2. E f f e c t o f foscarnet on the percentage of HCMV IE a n d l a t e a n t i g e n positive cells. Panels D-F are cells treated with an FITClabeled monoclonal antibody to an H C M V late antigen. D) Uninfected H F F cells; E) H C M V i n f e c t e d cells with no foscarnet; F) H C M V infected cells treated with 100 IJM foscamet. At i00 NM foscarnet, the percentage o f IE antigen positive cells is reduced f r o m 34.7% to 16.7%, and the percentage o f late antigen positive cells is reduced from 24.7% to 11.6%.

Vol. 19, No. 1/2, 1999

the average ICs0 values obtained by the flow cytometry assay and the PRA for these six drugs are p r e s e n t e d in Table 3. The data s h o w that, on a m i c r o m o l a r basis, the antiviral activity o f these c o m p o u n d s is R P R C M V 4 2 3 > R P R 1 2 7 0 2 5 >GW126W94 >Cidofovir >Ganciclovir >Foscarnet. T h e r e is e x c e l l e n t c o r r e l a tion b e t w e e n IC50 v a l u e s obtained using the IE and late antigens and b e t w e e n the f l o w c y t o m e t r y assay and the P R A for individual drugs. A s was s h o w n by the p r e v i o u s l y published papers for drug susceptibility o f H C M V for ganciclovir, the a v e r a g e IC50 values for g a n c i c l o v i r and foscarnet obtained w i t h the f l o w c y t o m e try a s s a y are h i g h e r t h a n those obtained with the P R A . H o w e v e r , that difference

TABLE 1. PROCEDURE FOR DRUG SUSCEPTIBILITY ASSAY FOR CELL FREE VIRUS 1. Infect MRC-5 or HF'F cell monolayers with virus at an MOI of 0.01 to 0.1 PFU/cell 2. After adsorbing virus to cells at 37°C for 2 hr, remove the inoculum and add medium containing various concentrations of antiviral drug. 3. Incubate at 37°C until the virus has under gone at least 1 round of replication (18 hr for HSV and 96 to 144 hr for HCMV). 4. Remove cells from flasks with trypsin/versene, resuspend the ceils in medium with 10% FBS, wash the cells 2X with PBS without Ca++and Mg++and incubate on ice for 1 hr. 5. Permeabilize the cells by the addition of absolute methanol (-70°C), dispense the cells into microfuge tubes at 1 X 106 cells/ml and store at -70°C (the cells in methanol can be stored at -70°C for many months without losing the ability to interact with antibody). 6. Remove the methanol, wash IX with PBS without Ca++and Mg++, resuspend the pellet, and add 1-10 lag of fluorochrome labeled monoclonal antibody diluted in Evans blue to the antigen of interest. 7. Incubate at 37°C for 1 hr, wash the cells 3X with PBS containing 0.01% Tween 20. 8. Resuspend the pellet in 1% paraformaldehyde and analyze the sample for the percent of antigen positive cells by flow cytometry. 9. Controls should include uninfected and virus infected cells treated in the same manner as the experimental cells. Isotype control antibodies should also be included with each experimental sample.

CLINICAL I M M U N O L O G Y Newsletter 13

does not follow for cidofovir and 1263W94 w h i c h s h o w e d a closer c o r r e l a t i o n between the two assays. D e t e r m i n a t i o n of the drug susceptibility o f H C M V clinical isolates was independent o f M O I for m o s t o f the drugs tested. H o w e v e r , for s o m e drugs, increasing the M O I a b o v e 0.01 had a drastic effect on the ability o f the drug to inhibit the synthesis o f either IE or late antigens. Therefore, we strongly suggest that the drug susceptibility assay be perf o r m e d at an M O I of 0.001 to 0.01 virus infected cells per uninfected cell. In our hands, M O I less than 0.001 does not lead to a sufficient n u m b e r o f virus infected cells in the absence o f drug to perform the susceptibility assay. In summary, determination o f drug susceptibility by f l o w c y t o m e t r y is accurate, rapid, quantitative, and automatable. It can be used to measure the susceptibility o f H S V or H C M V to s e v e r a l antiviral c o m p o u n d s that m a y have different m o d e s o f action. T h e f l o w c y t o m e t r y assay should replace the time c o n s u m i n g , subj e c t i v e , and labor intensive P R A for the determination o f drug susceptibilities o f H C M V and HSV. Acknowledgments

The author wishes to thank A n n O g d e n M c D o n o u g h and Betty Olson for technical assistance and N e l l L u r a i n for p r o v i d i n g s o m e o f the clinical samples. This w o r k was supported in part by grants A I 4 1 6 9 0 f r o m the National Institutes o f Health and contracts R P 59500 and R P R 127025 f r o m R h o n e Poulenc-Rorer.

TABLE 2. PROCEDURE FOR DRUG SUSCEPTIBILITY ASSAY FOR VIRUS INFECTED CELLS 1. Add medium containing various concentrations of antiviral drugs to flasks containing monolayers of HFF or MRC-5 cells. 2. Obtain virus infected cells from cell cultures with approximately 90% CPE. 3. Add between 1000 to 10,000 virus infected cells directly to the medium in each flask. 4. Incubate at 37°C for at least one round of replication (96 to 144 hr for HCMV). 5. Follow the steps 4 through 9 outlined in Table 1.

References

1. Boeckh M, Boivin, G: Quantitation of cytomegalovirus: methodologic aspects and clinical applications. Clin Microbiol Rev 1 i :533-554, 1998. 2. Britt WJ, Alford CA: Cytomegalovirus. In Fields Virology, Third Edition, BN Fields, DM Knipe, PM Howley et al. Lippincott-Raven Publishers, Philadelphia, 1996, p. 2493-2523. 3. Denizot F, Lang, R: Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Meth 89:271-277, 1986. 4. Crumpacker C: Ganciclovir. N Engl J Med 335:721-729, 1996. 5. Drew WL, Miner R, Saleh E: Antivirai susceptibility testing of cytomegalovirus: criteria for detecting resistance to antivirals. Ciin Diag Virol 1:179-185, 1993. 6. Erice A, Gil-Roda C, Perez J-L et al.: Antiviral susceptibilities and analysis of UL97 and DNA polymerase sequences of clinical cytomegalovirus isolates from immunocompromised

TABLE 3. COMPARISON OF AVERAGE ICso VALUES FOR HCMV CLINICAL ISOLATES

Drug Foscarnet

Flow Cytometry IE Ag laMa

SIP ±

Flow Cytometry Late Ag laMc

SD ±

PRA pM d

SD ±

241.00

90.92

191.08

82.50

140.67

47.24

Ganciclovir

4.93

1.43

3.76

1.65

2.77

1.47

Cidofovir

0.33

0.09

0.71

0.28

0.74

0.29

1263W94

0.44

0.18

0.45

0.15

0.35

0.13

RPR 127025

0.069

0.053

0.035

0.03

ND5

ND

RPR CMV423

0.038

0.021

0.018

0.010

ND

ND

qCsa valuesdeterminedusing the effectof drugs on the percentIE antigenpositivecells. bSD = Standard Deviation qc~o valuesdeterminedusing the effectof drugs on the percent late antigen positivecells. ~PRA= plaque reductionassay "ND = not determined

© 1999 Elsevier Science Inc.

0197-1859/99 (see frontmatter)

14 CLINICAL I M M U N O L O G Y Newsletter

patients. J Infect Dis 175:1087-1092, 1997. 7. Field AK, Biron KK: "The end of innocence" revisited: resistance of herpesviruses to antiviral drugs. Clin Microbiol Rev: 7:1-13, 1994. 8. Gerna G, Sarasini A, Percivalle E et al.: Rapid screening for resistance to ganciclovir and foscarnet of primary isolates of human cytomegalovirus from culture positive blood samples. J Clin Microbiol 33:738-741, 1995. 9. Jabs DA, Enger C, Forman Met al.: Incidence of foscarnet resistance and cidofovir resistance in patients treated for cytomegalovirus retinitis. Antimicrob Agents Chemother 42:2240-2244, 1998. 10. Jokela J, Erice A, Stanat Set al.: A standardized plaque reduction assay for CMV antiviral susceptibility. Abs 2nd National Conference of Human Retroviruses and Related Infections, Washington DC, Jan. 29 - Feb. 2, 1995, Abstract #283. 11. Lipson SM, Soni M, Biondo FX et al.: Antiviral susceptibility testing-flow cytometric analysis (AST-FCA) for the detection of cytomegalovirus drug resistance. Diagn Microbiol Infect Dis 28:123-129, 1997. 12. Lurain NS, Ammons, HC, Kapel KS et al.: Molecular analysis of human cytomegalovirus strains from two lung transplant recipients with the same donor. Transplantation 62:497-502, 1996. 13. Lurain NS, Spafford LE, Thompson KD:

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Mutation in the UL97 open reading frame of human cytomegalovirus strains resistant to ganciclovir. J Viro168:4427-4431, 1994. 14. Mazeron MC, Jahn G, Plachter B: Monoclonal antibody E-13 (M-810) to human cytomegalovirus recognizes an epitope encoded by exon 2 of the major immediate early gene. J Gen Virol 73:2699-2703, 1992. 15. McSharry JJ: Uses of flow cytometry in virology. Clin Microbiol Rev 7:576-604, 1994. 16. McSharry JJ: Flow cytometry based antiviral resistance assays. Clin Immunol Newslett 9:113119, 1995. 17. McSharry JJ: Flow cytometric analysis of virally infected cells: in vitro and in vivo studies. In Rapid Detection of Infectious Agents, Steven Specter, Mauro Bendinelli, and Herman Friedman, eds., Plenum Press, New York, 1998, p. 39-56. 18. McSharry JJ, Lurain NS, Drusano GL et al.: Flow cytometric determination of ganciclovir susceptibilities of human cytomegalovirus clinical isolates. J Clin Microbiol 36:958-964, 1998. 19. McSharry JJ, Lurain NS, Drusano GL et al.: Rapid ganciclovir susceptibility assay using flow cytometry for human cytomegalovirus clinical isolates. Antimicrob Agents Chemother 42:2326-2331, 1998. 20. McSharry JJ, Luarain NS, McDonough AC et al.: Drug susceptibilities of human cytomegalovirus (HCMV) clinical isolates as determined by flow

cytometry. Abs of 38th ICAAC, San Diego, CA, Sept. 24-27, 1998, Abstract # H-106. 21. McSharry JJ, Talarico C, Davis M et al.: Comparison of phenotypic assays for determining susceptibility of human cytomegalovirus (HCMV) to 1263W94 and ganciclovir. Abs 38th ICAAC, San Diego, CA Sept. 24-27, 1998, Abstract #H- 107. 22. Palestine AG, Polis MA, DeSmet MD et al.: A randomized controlled trial of foscarnet in the treatment of cytomegalovirus retinitis in patients with AIDS, Ann Intern Med 115:665-673, 1991. 23. Pavic I, Hartmen A, Zimmermann Aet al.: Flow cytometric analysis of herpes simplex virus type 1 susceptibility to acyclovir, ganciclovir, and foscarnet. Antimicrob Agents Chemother 41:2686-2692, 1997. 24. Pepin J-M, Simon E Dussault Aet al.: Rapid determination of human cytomegalovirus susceptibility to ganciclovir directly from clinical specimen primocultures. J Clin Microbiol 30:2917-2920, 1992. 25. Polis MA, Spooner KM, Baird BF et al.: Anticytomegalovirai activity and safety of cidofovir in patients with human immunodeficiency virus infection and cytomegalovirus viruria. Antimicrob Agents Chemother 39:882-886, 1995. 26. Stanat SC, Reardon JE, Erice EJ et al.: Ganciclovir-resistant cytomegalovirus clinical isolates: Mode of resistance to ganciclovir. Antimicrob Agents Chemother 35:129 l-1297, 1981.

Flow Cytometric Testing of Susceptibilities of Mycobacterium tuberculosis Ronald F. Schell, 1'2'3 Andrea V. M o o r e , 1'2 Renee M. Vena, 1'2 Scott M. Kirk, 4 and Steven M. Callister s'6 Wisconsin State Laboratory of HygieneI and Departments of Medical Microbiology and Immunology2 and Bacteriology, S University of Wisconsin, Madison, Wisconsin 53706; Microbiology Research Laboratory5 and Department of lnfectious Diseases,6 Gundersen Lutheran Medical Center, LaCrosse, Wisconsin 54601; and Bio-Rad Laboratories, Hercules, California 945474 Introduction A f t e r steadily d e c r e a s i n g f r o m 1952 to 1985, the n u m b e r o f cases o f tuberculosis increased by 2 0 % b e t w e e n 1985 and 1992. In r e s p o n s e to the r e s u r g e n c e o f tuberculosis and increases in resistance to antim y c o b a c t e r i a l agents t5 the Centers for D i s e a s e C o n t r o l and P r e v e n t i o n ( C D C ) stated that rapid and accurate susceptibility testing o f Mycobacterium tuberculosis is essential and should be p e r f o r m e d for control o f the disease. 2 R e d u c i n g the t i m e r e q u i r e d for susceptibility testing w o u l d greatly i m p r o v e the care o f patients and

0197-1859/99 (see frontmatter)

the control o f the disease. Classically, susceptibility testing o f M. tuberculosis has been p e r f o r m e d by g r o w i n g the tubercle bacillus on m e d i u m in the presence or absence of antituberculosis agents for two or three w e e k s o f incubation before obtaining results. 6 This is called the proportion m e t h o d and it is the " g o l d standard" for susceptibility testing o f M. tuberculosis. A n u m b e r o f methods, however, are practiced or h a v e been p r o p o s e d that greatly decrease the time r e q u i r e d to o b t a i n s u s c e p t i b i l i t y test results. The m o s t frequently used method, B A C T E C - 4 6 0 , requires four to 12 days o f

© 1999 Elsevier Science Inc.

incubation before results are available. 71° A p p r o x i m a t e l y nine days after initiation o f testing p r o c e d u r e s B A C T E C results are reported f r o m our laboratory. To decrease further the time required for susceptibility testing, n e w e r m e t h o d s h a v e u t i l i z e d m e t a b o l i c activity detected by fluorescent dyes, tl'12 q u a n t i f i c a t i o n o f total m y c o bacterial - R N A , 1 3 h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y m y c o l i c acid analysis TMand a b i o l u m i n e s c e n c e assay for d e t e c t i o n o f m y c o b a c t e r i a l A T E 15 M o r e recently, Jacobs et al. 16 described a rapid m e t h o d for drug susceptibility test-