Human herpesvirus 8 and Kaposi's sarcoma in the Amsterdam cohort studies. Disease association, transmission and natural history - Thesis

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Human herpesvirus 8 and Kaposi's sarcoma in the Amsterdam cohort studies.

Disease association, transmission and natural history

Renwick, N.M.

Publication date

2001

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Final published version

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Citation for published version (APA):

Renwick, N. M. (2001). Human herpesvirus 8 and Kaposi's sarcoma in the Amsterdam cohort

studies. Disease association, transmission and natural history.

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msterdamm Cohort Studies

Diseasee Association;

Transmissionn and

rall History

Humann Herpesvirus 8 and Kaposi's sarcoma

inn the Amsterdam Cohort Studies

©© Neil Renwick, 2001. All rights reserved. N o part off this publication may be reproduced, stored in a retrievall system, or transmitted, in any form or by anyy means, electronic, mechanical, photocopying, recordingg or otherwise, without prior permission off the author.

Cover:: Dr. Moritz Kaposi. Photograph: Dr. Szbekely. Thee photograph was kindly provided by the Wellcomee Trust, Medical Photographic library, London. .

Layout:: Scientific Text & Graphics, C.J. Witmans

Printer:: Febodruk BV, Enschede

Financiall Support:

HH

umann herpesvirus 8 and Kaposi's sarcoma

inn the Amsterdam Cohort Studies

Diseasee Association, Transmission and Natural History

ACADEMISCHH PROEFSCHRIFT

terr verkrijging van de graad van doctor aann de Universiteit van Amsterdam

opp gezag van de Rector Magnificus prof. dr J.J.M. Franse tenn overstaan van een door het college

voorr promoties ingestelde commissie, inn het openbaar te verdedigen

inn de Aula der Universiteit

op p

dinsdagg 18 december 2001, te 10.00 uur

door r

Neill Macdonald Renwick

Promotiee commissie:

promotor:: prof. dr. J. Goudsmit

overigee leden: prof. dr. J.M.A. Lange prof.. dr. P.A. Kager prof.. dr. F. Miedema dr.. D.C. Gajdusek dr.. W.A. Paxton dr.. P. Reiss

Faculteitt der Geneeskunde

Thee research described in this thesis was performed as part off The Amsterdam Cohort Studies on HIV-1 infection and AIDSS and was conducted at the Department of Human Retrovirology,, Academie Medical Center, University of Amsterdam,, Meibergdreef 15, Amsterdam 1105 AZ, The Netherlands. .

Thee studies described here were financially supported bv thee Dutch AIDS Fund (Stichting AIDS Fonds), the Nether-landss Foundation tor Preventive Medicine and the Dutch Ministryy of Public Health (VW'S) on advice of the Program Committeee for AIDS Research (PccAo) as part of the Na-tionall AIDS Research Stimulation Program.

Collarborativee projects were financially supported by the Medicall Research Council of Great Britain (69517856PB), thee NHS Biomedical research Fund (RDO/22/9), the Hu-ropeann Concerted Action on the Pathogenesis of Kaposi's sarcomaa (BMH 4-97-2302), the Swiss National Science-Foundationn (3100-064233.00), the Oxenhale Trust and the Northh West Cancer Research Fund.

Itt is easier to fashion a theory than to discover a phenomenon.

SantiagoSantiago Ramonj Cajal, Advice For A Young Investigator

I I

ablee of Contents

Tablee of Contents

Chapterr 1: Introduction

Parti i

Chapterr 2: Seroconversion for human herpesvirus 8 during HIV infection is highly

predictivee of Kaposi's sarcoma

NeilNeil Renwick, Teysir Halaby, GerritJ. Weverling, Nicole H.T.M. Dukers, GuyK Simpson,

RoelRoel A. Coutinho, Joep MA. Lange, Thomas F. Schulp and Jaap Goudsmit, Aids 1998; 2481-8

Addendum:: Timing of Human Immunodeficiency Virus type 1 and Human Herpesvirus 8 Infections

andd length of the Kaposi's sarcoma-free period in coinfected persons

NeilNeil Remviek, Gerritjan Weverling, Thomas Schulp and Jaap Goudsmit J Infect Vis 2001; 183: 1427

Chapterr 3:

Chapterr 4:

Clinicall symptoms related to primary human herpesvirus 8 infection in homosexual

menn with or at risk for HIV-1 infection

NeilNeil Renwick, Nicole H. Dukers, Peter Reiss, Gerritjan Weverling, Thomas F. Schulp

RoelRoel A. Coutinho, Jaap Goudsmit, Manuscript submitted

Kaposi'ss sarcoma (KS) and human herpesvirus 8 (HHV8) infection do not protect

HIV-11 infected homosexual men from AIDS dementia complex (ADC)

NeilNeil Remviek, Gerritjan Weverling, Teysir Halaby, PeterPortegies, Margreet Bakker,

ThomasThomas F. Schulp Jaap Goudsmit, Manuscript in press

Partt II

Chapterr 5: Risk factors for HHV8 seropositive and HHV8 seroconversion in a cohort of

homosexuall men

NicoleNicole H.TM. Dukers, NeilRenwick, Maria Prins, Ronald' B. Geskus, Thomas F. Schulp

GerritjanGerritjan Weverling, Roel A. Coutinho, Jaap GoudsmitAm JEpidemiol2000; 151:213-24

7 7

11 1

31 1

33 3

45 5

47 7

55 5

63 3

65 5

Chapterr 6: Lack of evidence for blood-borne transmission of human herpesvirus 8 during

100 year follow-up (1985-1996) of drug users in the Netherlands 83

MariaMaria Prins, Roel A. Coutinho, Jaap Goudsmit, Manuscript submitted

Partt III 95

Chapterr 7: Two distinct gamma-2 herpesviruses in African green monkeys: a second gamma-2

herpesviruss lineage among old world primates? 97

ThomasThomas K Schulp J Virol 2000; 74:1572-7

Chapterr 8: Human herpesvirus 8 infections in the Amsterdam Cohort Studies (1984 -1997):

analysiss of seroconversions to ORF65 and ORF73 111

MafgreetMafgreet Bakker, Thomas F. Schulp , Marion Co melissen, and Genit J. W'everling, ProcProc Natl Acad Sci USA 2000;97: 4838A 3

Chapterr 9: VEGF levels in serum do not increase following HIV-1 and HHV8 seroconversion

andd lack correlation with AIDS-KS 125

JaapJaap Goudsmit, Manuscript submitted

Chapterr 10: General Discussion 131

Summaryy 141

Samenvattingg 142

Curriculumm Vitae 144

Dankwoordd 145

Moritzz Kohn was born in Kaposvar, Hungary on 23 October, 1837.. He matriculated at the University of Vienna in 1856 and graduatedd with the degrees of Doctor of Medicine in 1861 and Doctorr of Surgery in 1862. His interest in dermatology began whilee working in the Department of Syphilology at the Allgemei-ness Krankenhaus and he transferred to Ferdinand von Hebra's Departmentt of Dermatology in 1866. Kohn changed from the Jewishh to the Roman Catholic faith in 1869, changed his surname too Kaposi in 1871 and married von Hebra's daughter. Among his manyy distinguished contributions to dermatology, Kaposi was the firstfirst to describe xeroderma pigmentosum and idiopathic multiple pigmentedd sarcoma. Moritz Kaposi died in 1902.

(Oberr W. Kaposi: The Man and the Sarcoma. In: Kaposi's sar-coma:: A Text and Atlas. 1988. Edited by GJ Gottlieb, AB Ackerman.. Lea and Febiger Publishers, Philadelphia, USA).

Introduction n

Epidemiologyy of Kaposi's sarcoma

Kaposi'ss sarcoma (PCS) is a vascular tumor that was first de-scribedd as "idiopathic multiple pigmented sarcoma of the skin"" by the Hungarian dermatologist, Dr Moritz Kaposi, in 18722 (1). Four epidemiological variants of KS are now re-cognised;; the original report detailed the rare Classic variant off KS which is found in elderly Mediterranean men and Jewishh people born in Eastern Europe (2). Endemic KS has beenn observed in children and young black adults in equato-riall Africa and Transplant KS affects organ transplant recip-ientss and patients who have previously received immunosuppressivee therapy (3-7).*

AIDS-KSS is the most recent epidemiological variant to be recognisedd and was first noted in immunocompromised homosexuall men from New York and California (8). As AIDS-KSS was found to be more common in persons who acquiredd HIV-1 through homosexual contact than by het-erosexuall contact, injection-drug use or contaminated bloodd products, it was suggested that this vascular tumor is causedd by an unidentified sexually transmitted agent (9).

Clinicall presentation of Kaposi's sarcoma

trointestinall tract with fatal consequence (1). The clinical coursee of KS is, however, not always commensurate with thatt of a malignant process; KS has been reported to regress followingg the administration of antiretroviral therapy and to remitt on reduction or cessation of immunosuppressive therapyy (10,11).

Pathologicall features of Kaposi's sarcoma

Thee pathology of KS is similar for all variants; lesions tend too progress through patch, plaque and nodular stages on the skin.. Patch lesions are characterised by irregularly dilated lymphatic-likee spaces lined by endothelial cells and perivascularr infiltrates of lymphocytes and plasma cells. The plaquee stage is notable for more extensive infiltration of bloodd vessels and the presence of short fascicles of spin-dle-shapedd cells. In the nodular stage, sheets of spindle cells, withh ovoid but seldom mitotic nuclei, are seen and vascular spacess are more prominent (12). Although spindle cells ap-pearr to be the proliferative component of the KS lesion, theree is considerable debate as to their endothelial origin andd whether their proliferation represents a hyperplastic polyclonall process rather than an oligo- or monoclonal ma-lignancyy (13).

Thee original description of KS showed that this tumor not onlyy affects the skin but also may involve the lungs and

Humann herpesvirus 8 (HHV8) / Kaposi's

sarcoma-associatedd herpesvirus (KSHV)

Discoveryy and classification

off HHV8 as a rhadinovirus

Twoo herpesviral D N A sequences were discovered using Representationall Difference Analysis that preferentially identifiedd the genomic differences between KS lesions and normall skin (14). These sequences were present in 25 (93%) off 27 AIDS-KS tissues compared with 6 (15%) of 39 lvmph nodess and lymphomas from AIDS patients without KS. Thiss agent was named Kaposi's sarcoma-associated herpes-viruss (KSHV) and is also known as Human Herpesvirus 8 (HHV8)) for taxonomie reasons. Preliminary phylogenetic analysiss of a genomic subfragment placed HHV8 in the Rhadinoviruss (gamma2) lineage of the gammaherpesvirinae alongg with Herpesvirus Saimiri (HVS) (15). The closest hu-mann relative is Epstein-Barr Virus (HBV), which belongs to thee Lymphocryptovirus (gammal) herpesvirus subfamily (Figuree 1).

Thee nucleotide sequence of HHV8 provides

insightss into KS pathogenesis

Thee genome of H H V 8 (based on two samples with a differ-encee of 0.4°/o) has a 140.5-kb-iong unique region (LUR) whichh is flanked by multiple 801bp terminal repeat se-quencess (Figure 2) (16,17). Within the LUR, 81 potential Openn Reading Frames (ORFs) with greater than 100 amino acidss have been identified and several additional spliced geness have since been added to this list. The overall G + C contentt in the LUR is 53.5% and 84.5%> in the terminal re-peatt sequence. The numbering of HHV8 ORFs is based on positionall homologies with HVS due to substantial col-linearityy between these genomes wThereas those ORFs with-outt positional homologues are numbered consecutively

withh a K prefix. The LUR consists of conserved herpesviral geness that are involved in herpesvirus replication and struc-turee and non-conserved genes that may provide insights intoo the pathogenetic mechanisms of this virus (16,17). The presencee in the viral genome of ORFs with significant homologyy to mammalian genes involved in cellular growth controll indicates that "molecular mimicry,..of cell cycle reg-ulatoryy and signaling proteins is a prominent feature of this virus"" (16). The type, function and expression of these viral geness are discussed below.

Serologicall detection and seroepidemiology of

HHV8 8

Thee presence of HHV8 DNA sequences in all epidemiolog-icall variants of KS implies that antibodies to HHV8 should bee detectable at the time of KS diagnosis and sera from KS patientss should thereby serve as positive reference material (18).. Currently negative reference sera are chosen from a populationn that has a low risk for KS. Sensitivity and speci-ficityficity data for serological assays can be calculated from these referencee materials.

Latentt antigens

Antibodiess to a high molecular weight (224-234kDa) latent nuclearr antigen (LNA or LAN A) can be detected by West-ernn Blot (WB) or immunofluoresence assays (I F A) on B cell liness that were established from patients with body cavity basedd lymphoma (BCBL)/ primary effusion lymphoma (PEL)) with latent HHV8 infection (19-22). LAN A has been shownn to be encoded by ORF73 following expression of thiss gene in bacterial and mammalian expression systems andd subsequent demonstration of the 222-234kDa doublet

B B

Neww World y2-herpesvirus s

Oldd World 2-herpesvirus s

Figuree 1a. DNA maximum likelihood tree for a 1802-bp fragment of herpesviral DNA polymerase. Sequences were aligned by using CLUSTALXX and analysed by using the DNAML program (PHYLIP version 3.5c; J. Felsenstein and the University of Washing-ton). .

b.. Neighbor-joining protein distance tree for a 454-bp fragment of herpesviral DNA polymerase. Sequences were aligned as abovee and analysed by using the PROTDIST and NEIGHBOR programs in PHYLIP. These findings have been published

00 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 kb ll 1 1 1 1 1 1 1 1 1 i I I I l l ' l i

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Classs I transcripts Class II transcripts Class III transcripts Terminall repeat regions

Figuree 2. The HHV8 genome is 140.5kb long and is flanked by multiple terminal repeat sequences which are depicted as hatched squares.. Three classes of transcript are recognised (136); class I transcripts are constitutively expressed in BCBL/PEL cells andd these latent transcripts are coloured black, class II transcripts are expressed at low levels during latency but can be in-ducedd chemically and are shaded grey and class III transcripts are only present following chemical induction and are repre-sentedd in white. It should be noted that the nut-1 transcript is not translated. Blocks of structural genes that are conserved betweenn most gammaherpesviruses are labelled l-V. This diagram shows "non-conserved" genes that are discussed in the textt and the map is not to scale.

byy WB of the nuclear extract (23-25). LAN A WB and IFA formatss have high sensitivity and specificity; HHV8 anti-bodiess are detected in the majority (71-100%) of AIDS-KS andd Classic KS sera but only in the minority (0-4%) of bloodd donors (19-22).

Lyticc antigens

Lyticc antigens have been identified to increase the sensitiv-ityy of HHV8 immunoassays. Antibodies to lytic antigens cann be detected by IFA on BCBL/PEL cell lines that have beenn pre-treated with phorbol esters or sodium butyrate (26-29).. Lytic antigens can be detected by WB or radioim-munoprecipitationn of chemically induced BCBL/ PEL cell liness and also by whole-virus enzyme immunoassay (ElA) ass H H V 8 virions can be produced by treating the same cells withh tetradecanoylphorbol acetate (TPA) (27-31). An EIA thatt uses lysed HHV8 virion as the target antigen has high sensitivityy (94%) and specificity (93%) (32). Recombinant structurall antigens such as a capsid-related protein (ORF65) andd membrane glycoprotein gp35-37 (ORFK8.1) are also usefull for seroepidemiological purposes (22,30, 33-35).

Generally,, lytic IFAs have a sensitivity close to 100% how-everr the specificity varies between 69%) and 100% in differ-entt laboratories (36). Recombinant ORF65 proteins and ORFK8.11 give sensitivities in the range of 75-90%, with somee variability if different expression vectors or purifica-tionn methods are used (22,30,33-35). The specificity of re-combinantt ORF65 and ORFK8.1 based assays is generally higherr than 80% and may approach 100% when assay con-ditionss are varied (22,30, 33-35,37).

Geographicc distribution of HHV8

Itt is possible to demonstrate that certain geographic regions havee comparatively high or low seroprevalences of HHV8

despitee the variety of HHV8 serological tests and blood do-norss are a useful group in which to demonstrate this varia-tion.. In Northern Europe, HHV8 appears to be rare in bloodd donors, with seroprevalence rates using LAN A or ORF655 less than 5% in the United Kingdom, Sweden and Switzerlandd (22,38,39). HHV8 seroprevalence rates rise dramatically,, using the same serological tests, in countries in Southernn Europe; rates range from 2% to 32% in regions of Italyy (19,40-42). Use of a lytic IFA also increases the seroprevalencee in blood donors to 20% in Sweden and to 28%% in Italy (39,41).

Inn North America, less than 5% of blood donors have anti-bodiess to ORF65 and/or LAN A (19-22,26). This seropre-valencee rate ranges from 0-14% when seven IFAs and EI As aree used to study interlaboratory and interassay variation on aa panel of aliquoted sera (36). Anti-LANA antibodies were virtuallyy undetectable in other studies on US blood donors (23,31,33).. Use of a lytic IFA to detect antibodies against an undefinedd antigen increases the seroprevalence (range: 8-29%)) among US blood donors (26,33,36). ORFK8.1 WB andd whole-virus EIA detect similar proportions (8-11%>) of bloodd donors (31,33). Only 4% blood donors were seropos-itivee with the whole-virus EIA or lytic/ LAN A IFA in Ja-maicaa (43). HHV8 antibodies were detected by whole-virus EIAA in 5%) or less blood donors from the United States, Ja-maicaa and Trinidad (44).

Seroprevalencee data are limited for blood donors from otherr regions. The seroprevalence of HHV8 among former bloodd donors in Israel was 22% using LAN A IFA and only 22 (0.2%) of 1000 blood donors were LAN A IFA positive in Japann (45,46).The seroprevalence of HHV8, using LAN A

IFA,, was 20% in black blood donors and 5% in white blood donorss in South Africa (47). It is salient to note that HHV8 iss considered an endemic infection in regions of Italy, Africa andd South America (48-50).

Diseasee Association

HHV88 in populations with or at risk of AIDS-KS

Inn the United States of America and Europe, antibodies to LANN A and/or ORF65 are more common among HIV-1 infectedd homosexual men (23-39%) than among HIV-1 in-fectedd drug users, heterosexuals, transfusion recipients or personss with hemophilia (0-7%) (19-22, 26, 41,51). Higher seropp re valence rates are seen using lytic IFA however the distributionn pattern remains the same; over 90% HIV-1 in-fectedd homosexual men have HHV8 antibodies compared too 2 3 % HIV-1 infected injection-drug users and 2 1 % HIV-11 infected women (26). These studies have all demon-stratedd high prevalences of HHV8 antibodies in patients withh AIDS- KS (range: 52-85%) compared with HIV-in-fectedd controls without KS (range:18-35%). As a result, sig-nificantt odds ratios (range: 2-19) have been found, where calculable,, for the presence of HHV8 antibodies between KSS cases and controls (19-22,26,38).

HHV88 in populations with or at risk for Classic KS

HHV88 seroprevalences using LANA or ORF65 range from 94-100%% among persons with Classic KS and from 4-19%

Sexuall transmission

Inn a cohort of Danish homosexual males, the presence of antibodiess to HHV8 LANA and ORF65 at study entry was independentlyy associated with the number of receptive anal intercoursess and sex with men from the United States of Americaa (57). HHV8 seroconversion in this study was inde-pendentlyy associated with visits to homosexual

communi-forr matched or blood donor controls with odds ratios, whenn calculable, ranging from 130-257 (19,22,40,51).

HHV88 in populations with or

att risk for Transplant KS

Seroconversionn against HHV8 ORF65 occurred within the firstfirst year after transplantation in Switzerland (52). Case-controll studies from different geographic regions havee again demonstrated a strong association between HHV88 antibodies and Transplant KS. In Italy, 10 (91%) of 111 transplantation recipients with KS were seropositive to antibodiess to LANA and ORF65 compared to 2 (12%) of 177 organ recipients who served as controls (OR=75) (53). Highh odds ratios were also seen in case-control studies from Francee using LANA and ORF65 (OR=28) and Saudi Ara-biaa using the lytic antigens, p40 and sVCA (OR=34) (54,55).

HHV88 in populations with or at risk fo Endemic KS

Dataa on Endemic KS are scarce; a combined total of 44 pa-tientss with Endemic KS were all positive by lytic/LANA IFAA (19,26,56).

tiess in the United States of America and HIV-1 seropositivity. .

Inn San Francisco, anti-LANA antibodies were detected in 40%% exclusively homosexual men, 13% men who reported mostlyy homosexual activity and were not detected in exclu-sivelyy heterosexual men (58). HHV8 seropositivity was stronglyy associated with HIV-1 infection and self-reported

historyy o f sexually t r a n s m i t t e d disease in this study and H H V 88 s e r o p r e v a l e n c e increased linearly with the n u m b e r o ff male i n t e r c o u r s e p a r t n e r s in t h e preceding two years. H H V 88 a n t i b o d i e s , as d e t e c t e d using L A N A / O R F 6 5 or lytic I F A ,, h a v e b e e n linked to p a r a m e t e r s of sexual b e h a v i o u r suchh as n u m b e r o f p a r t n e r s a n d oroanal c o n t a c t in Sydney andd San F r a n c i s c o (59,60).

Seraa f r o m patients in a L o n d o n sexually transmitted disease clinicc w e r e tested with latent I F A and 198 (7%) o f 2718 pa-tientss w e r e s e r o p o s i t i v e ; i n d e p e n d e n t risk factors w e r e h o m o -- a n d bisexuality, b i r t h in Africa, a history of syphilis, H S V - 22 and H I V - i n f e c t i o n (61). H H V 8 seroposttivity, as de-t e r m i n e dd by lyde-tic I F A , was linked de-to de-the n u m b e r o f sexual p a r t n e r ss and a history o f o t h e r sexually transmitted diseases inn Swedish w o m e n (62). Similar associations with sexually t r a n s m i t t e dd diseases w e r e seen in N o r t h American w o m e n withh o r at risk o f H I V - 1 infection using either O R F 6 5 and O R F K 8 . 11 F.I A o r a c o m b i n a t i o n o f lytic/latent IFA (63,64). Inn W e s t e r n Sicily, H H V 8 seroprevalences using a lytic IFA w e r ee h i g h e r in H I V - 1 positive and negative h o m o s e x u a l m e n ,, m e n w h o h a d sex w i t h prostitutes, female prostitutes a n dd clients at a sexually t r a n s m i t t e d disease clinic than in the generall p o p u l a t i o n (65). T h e seroprevalence o f H H V 8 was h i g h e rr in female c o m m e r c i a l sex w o r k e r s than control p o p -ulationss in C a m e r o o n a n d H o n d u r a s (66-68).

Childhoodd transmission

T r a n s m i s s i o nn b e f o r e p u b e r t y a p p e a r s to be rare in the U n i t e dd States b u t d o e s o c c u r in countries w h e r e H H V 8 is m o r ee w i d e s p r e a d ; H H V 8 a n t i b o d i e s are detectable in chil-d r e nn from C a m e r o o n , Flgypt, S o u t h Africa, U g a n chil-d a anchil-d It-alyy (40, 6 7 , 69-77). Similarly H H V 8 antibodies are found in c h i l d r e nn o f individuals o f African origin in F r e n c h G u i a n a a n dd also in Brazilian A m e r i n d i a n s (49,76). Age distribution o ff H H V 8 a n t i b o d i e s s h o w e d that the H H V 8

seroprevalencee for adults was reached well before puberty inn Ugandan children but that H H V 8 infection was also rare beforee t h e age o f 2 (72). Similar results w e r e found in Cam-eroonn w h e r e there was a steady increase in seroprevalence fromm 2 8 % at 4 years o f age to 4 8 % a b o v e 15 years (74). Cor-relationn with hepatitis B infection suggests that H H V 8 is transmittedd horizontally in c o n d i t i o n s of close contact and crowdingg (72). I I H V 8 seroprevalence was almost equal in spouses,, children and siblings of K S patients in Sardinia and indicatess intrafamilial transmission (70). H H V 8 appears to bee transmitted within families in Israel and significant mother-childd and sib-sib correlations t o r H H V 8 seropositivityy were seen in I Tench G u i a n a (45,76).

Inn South Africa, the majority (88%) of H H V 8 seropositive childrenn had a seropositive m o t h e r indicating that mother-childd transmission exists (71). A similar conclusion wass reached in Italy and 5 children with KS all had H H V 8 seropositivee m o t h e r s in Z a m b i a (78,79). Vertical transmis-sionn of H H V 8 was n o t seen in m o t h e r - i n f a n t pairs from Haiti,, N o r t h e r n Italy o r Africa (80,81).

Parenterall transmission

Inn Switzerland, a country w h e r e H H V 8 is not e n d e m i c , the appearancee o f IgM and IgG antibodies to H I 1 V 8 lytic capsidd ( O R F 6 5 ) antigen within m o n t h s or transplantation indicatess that H11V8 is transmitted t h r o u g h renal allografts orr blood transfusion (52). H H V 8 is m o r e likely t o be reacti-vatedd in transplant recipients w h o orginate in countries wheree H H V 8 is endemic (53,54,82-85). F v i d e n c e for both transplantation-associatedd transmission and reactivation hass been seen in a c o h o r t or renal transplant recipients in Belgiumm (86).

H H V 88 has been transmitted experimentally from the C D 19 cellss of an H H V 8 seropositive b l o o d d o n o r to H H V 8

nega-tivee CD 19 cells (87). Transmission through contaminated bloodd products appears to be rare; of 14 individuals who re-ceivedd blood products from HIV-1 and HHV8 co-infected donors,, 10 developed antibodies to HIV-1 but none seroconvertedd to HHV8 (88). Similarly in Jamaica, recipi-entss of blood products from HHV8 seropositive donors re-mainedd seronegative (89). Anti-LANA antibodies were foundd in the minority (1%) of Romanian children who were HIV-11 infected as a result of parenteral transmission (90). Seroprevalencee rates in haemophiliacs vary from 0-28% de-pendingg on whether a lytic or latent IFA is used (22,26,91).

Nucleicc acid based detection of HHV8

Nucleicc acid based studies have not been sensitive enough too determine the prevalence of HHV8 infection within pop-ulations.. Detection of HHV8 by polymerase chain reaction (PCR)) may also depend on the severity of clinical disease (92-95).. Nonetheless, PCR studies have demonstrated the strongg association between HHV8 and KS; HHV8 is de-tectablee by PCR in peripheral blood mononuclear cells (PBMCs)) of AIDS-KS patients (range: 35-91%) and HIV-infectedd controls (range 0-19%)) with odds ratios rang-ingg from 2-440 (96-100). Sequence variation in the ORF26, ORFK1,, ORF73 and ORF75 genes can be used to study the molecularr epidemiology of HHV8, however this burgeon-ingg field is not covered in this thesis.

HHV88 nucleic acid sequences

inn cells and body fluids

PCRR studies have also been invaluable in determining the distributionn of HHV8 in cells and body fluids. Viral DNA sequencess have been detected in the following cells; endo-theliall and spindle cells of KS lesions, B cells, CD8+ T cells, macrophages,, monocytes, prostatic giandular epithelium, circulatingg endothelial cells, brain tissue, lung tissue, skin,

bonee marrow, non-neoplastic lymph nodes and gastrointes-tinall mucosa from HIV-1 seropositive individuals (101-113).. HHV8 sequences were not detected in the faeces off HIV-1 infected individuals (96).

HHV88 DNA sequences have been detected in body fluids andd provide some insight into the mode(s) of HHV8 trans-mission;; HHV8 has been detected in saliva from HIV-1 in-fectedd persons with or without KS (96, 114-118). HHV8 hass been detected by PCR in saliva and nasal secretions of HIV-11 infected individuals and HHV8 seropositive persons butt not in control subjects (119, 120). HHV8 is also detect-ablee in bronchoalveolar lavage samples from AIDS-KS pa-tientss (121-124). Reports on the proportions of HHV8 in semenn and prostate glands of HIV-1 infected and unin-fectedd individuals range dramatically from 0-100% (125). Thesee differences are probably due to variations in the assay orr reflect geographic and population differences; a recent multicenterr study has shown that it is easy to contaminate thee HHV8 PCR and in those that were not contaminated, HHV88 DNA was detected infrequently (8%)) in semen from HIV-11 infected and uninfected subjects (126). HHV8 is rarelyy detectable in cervical and vaginal secretions (127). In-terestinglyy the number of HHV-8 copies per microgram of tissuee or body fluid from KS patients was highest in PBMCs followedd by saliva and semen and undetectable in faeces (128).. HHV8 is also detectable, albeit rarely, in cerebrospinall fluid and urine (118,129,130).

Presencee and location of HHV8 and expression of

HHV8-encodedd genes Kaposi's sarcoma lesions

HHV88 DNA sequences have been demonstrated by in situ hybridisationn and/or amplification in spindle cells and en-dotheliall cells that line the vascular spaces of patch, plaque andd nodular KS lesions, however these sequences are not presentt in normal endothelial cells (131-134). In early KS

le-sions,, HHV8 is present in less than 10% of cells forming the ectaticc vessels whereas it is present in greater than 90% of spindlee cells in nodular KS (135). HHV8 transcripts can be groupedd in the following three classes; Class I and Class II transcriptss are constitutively produced in BCBL/PEL cells howeverr Class II transcripts are induced by TPA treatment whereass Class 111 transcripts are only produced following TPAA treatment (136). Class I and III transcripts correspond too the latent and lytic phases of the herpesviral lifecycle whereass Class II transcripts do not conveniently fit this par-adigm,, ("lass 1 transcripts are expressed in KS spindle cells, ass judged by in situ hybridisation or immunohistochemistry andd indicate latent persistence of HHV8 in the majority of KSS spindle cells (137-149).

Pathogenesiss of Kaposi's sarcoma

KSS used to be considered solely as a "cytokine-driven" pro-liferationn because IL-6 enhances the proliferation of spindle cellss in culture and IFN-gamma induces endothelial cells to acquiree the phenotypic features of spindle cells (150-152).Thee pathogenesis of KS can now also be consid-eredd in terms of the functions of HHV8-encoded proteins, whichh are grouped below on the basis of their correspond-ingg transcript (136, 153).

Proteinss that are translated

fromm Class I transcripts

Cellss that express viral Fas-ligand interleukin converting en-zyme-likee caspase (v-FLIP, encoded by ORFK13/ORF71) aree protected against apoptosis induced by the CD95 death receptorr and other related death receptors (154).

Virall cyclin (v-cyclin, encoded by ORF72) binds to cyclin-dependentt kinase (cdk) 6 and results in phosphorylation of thee retinoblastoma-tumor suppressor protein and histone HII (155-157). It should be noted that phosphorylated

reti-noblastomaa protein is unable to repress transcription fac-torss such as E2F which regulate cellular DNA synthesis (153).. Viral cyclin/cdk6 is also resistant to inhibition by cel-lularr CDK inhibitors such as pi6, p21 and p27 (158).

Latencyy associated nuclear antigen (LANA-1, encoded by ORF73)) is abundantly expressed in KS spindle cells (25, 144,159).. LANA-1 colocalizes with the viral episome in interphasee nuclei and along mitotic chromosomes indicat-ingg that this protein is involved in episome maintenance du-ringg cell division (160). This protein also inhibits the ability off the p53 tumor-suppressor protein to induce cell death (161). .

Proteinss that are translated

fromm Class II transcripts

Virall IL-6 (vIL-6, encoded by ORFK2) activates the Janus tyrosinee kinase (JAK)-signal transducers and activators of transcriptionn (STAT) pathway that is known to have multi-plee downstream effects on cellular survival, proliferation, differentiationn and apoptosis (162,163). Viral IL-6 induces B-celll proliferation and prevents apopotosis in susceptible celll lines (164-166). This cytokine also promotes haemato-poiesis,, plasmacvtosis and angiogenesis in athvmic mice (167). .

Virall Bcl-2 (vBcl-2, encoded by ORF16) has anti-apoptotic propertiess in yeast and baby hamster kidney cells although thee ability of vBcl-2 to form a dimeric protein with human Bcl-22 is disputed (168,169).

Threee viral macrophage inflammatory proteins (vMIPs) havee been identified namely; vMlP-I (ORFK4), vMIP-II (ORF4.1)) andvMIP-III (ORF6) (16,17,164,165). All three proteinss belong to the CC chemokine family and are agonistss of the chemokine receptors CCR3, CCR4 and CCR88 which selectively chemoattract Th2 lymphocytes

(170-174).. These chemokines are highly angiogenic in the chorioallantoicc assay and also block the entry of HIV-1 into cellss that express CCR3 amd CCR5 (164, 167, 170, 173, 175). .

Virall interferon regulatory factor (vIRF-1, encoded by ORFK9)) inhibits interferon-responsive gene expression andd prevents interferon-induced growth arrest (176-179). Virall IRF-1 also transforms NIH3T3 cells and induces tu-morss when injected into nude mice (176,177). Two other vIRFss are recognised but further functional analysis is re-quiredd (180,181).

O R F K ll 2 encodes a small protein termed Kaposin whose transcriptt (TO.7) is expressed in abundance in unstimulated BCBL/PELL cells and is detectable in the majority of KS tu-morss of differing epidemiological variants (139, 182). Ex-pressionn of this protein in Rat-3 cells results in focal trans-formationformation and subcutaneous injection into nude mice pro-ducess highly vascular and undifferentiated sarcomas (183).

Theree are two variants of the latency associated membrane proteinn (LAMP, encoded by ORFK15) which have several srcc homology 2-like motifs and potential tumor necrosis factorr receptor-associated factor binding sites in its cyto-plasmicc tail. It is postulated that this protein is similar to the EBVV latent membrane proteins which are involved in cellu-larr transformation and maintenance of viral latency in B cellss (184,185).

Thee epidemiology of AIDS-KS suggested that this vascular tumorr was caused by an unidentified sexually transmitted infection.. Following the identification of HHV8 DNA se-quencess in AIDS-KS tissue, an argument rapidly ensued as

Proteinss that are translated

fromm Class III transcripts

O R F K ll posseses an immunoreceptor tyrosine activation motiff involved in B-cell and T-cell antigen receptor signal-ingg and is the positional analogue of saimiri transforming proteinn (STP) and one of the EBV latent membrane pro-teinss (186). A role in cellular transformation is surmised fol-lowingg the demonstration that rodent fibroblasts that ex-presss ORK1 show evidence of transformation and that O R F K ll can replace STP in the HVS genome and induce lymphomaa in the common marmoset (187).

Virall G protein-coupled receptor (vGPCR, encoded by ORF74)) signals through the phosphoinositide-inositol-triphosphate-proteinn kinase C pathway and stimulates rat kidneyy fibroblasts to proliferate (188). Viral GPCR appears too be expressed only in a subpopulation of KS spindle cells, whichh presumably undergo lytic replication (189). Expres-sionn of vGPCR in a focus-formation assay led to focal transformationn of NIH3T3 cells and subsequent injection off transformed cells into nude mice caused tumors that consistedd of spindle-shaped cells (190). Transgenic mice thatt express v-GPCR within haematopoietic cells develop angioproliferativee lesions (191).

ORFK33 and ORFK5 are proteins encoded by Class III and Classs II transcripts respectively that inhibit the expression off MHC class I antigens by enhancing endocytosis of sur-facee MHC molecules (192).

too whether HHV8 was the cause of KS or a "passenger" vi-rus.. We developed an Enzyme Immunoassay for the rapid detectionn of antibodies to recombinant HHV8 encoded lytic-phasee capsid (ORF65) and latent phase nuclear

(ORF:73)) antigens. In this thesis, we examined the disease association,, transmission and natural history of HHV8

in-fectionn in the homosexual and drug user cohorts of the Am-sterdamm Cohort Studies on HIV-1 infection and AIDS.

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