Correspondence
AIDS 2000,14:893±911
CD8 T cell anti-HIV activity as a complementary protective mechanism in vaccinated chimpanzees An Ad-HIV-1MN gp160 recombinant priming/HIV-
1SF2 gp120 boosting regimen protected three chimpan- zees against sequential low- and high-dose HIV-1SF2
challenges [1]. Subsequently, the chimpanzees, desig- nated 2PA, 1P and 3P, were boosted and challenged with the non-syncytium-inducing (NSI) heterologous primary isolate, HIV-15016. Chimpanzee 2PA was again protected, whereas 1P, 3P, and control chimpanzee C2 were infected [2]. Broad antibody reactivity with strong neutralizing activity, including the ability to neutralize the primary challenge virus, correlated with the protec- tion of 2PA. However, because neutralizing antibodies have not always been associated with vaccine protec- tion in non-human primates, other factors may also have contributed. Vaccine-elicited CD8 T cell antiviral activity (CD8AA) has been associated with the protec- tion of macaques from SIV infection [3±5] and was therefore investigated in the chimpanzees.
CD8AA was assayed as previously described [3]. Effec- tor cells were PHA-stimulated chimpanzee CD8 T cells cultured for 5 days with IL-2. PHA-stimulated
target cells were CD4 T cells from an HIV-1IIIB- infected chimpanzee (endogenous assay) or CD4 T cells from a seronegative human, infected with HIV- 1SF2 or HIV-15016 (acute assay). Effectors and acutely or endogenously infected targets were cultured for 7 days at ratios of 2 : 1, 1 : 1 and 0.5 : 1. Controls were CD4 cells alone. HIV-1 replication was assayed by p24 antigen capture.
Control chimpanzee C2 exhibited low, innate endogen- ous and acute CD8AA at boost and challenge (Fig. 1a, b). Among the immunized chimpanzees, only 2PA exhibited endogenous CD8AA at boost. Boosting in- creased endogenous CD8AA in 2PA and 1P. Peripheral blood mononuclear cells at boost for chimpanzee 3P were unavailable. Acute CD8AA was elevated post- boost only in chimpanzee 2PA using HIV-1SF2 targets and was not observed against HIV-15016-infected CD4 cell targets. Previously, however, at the time of chal- lenge the CD8 cells of chimpanzee 2PA suppressed HIV-15016 replication in macrophages by 61% at an effector to target ratio (E : T) of 0.5 : 1, whereas
Fig. 1. CD8 T cell antiviral activity (CD8AA) at boost and challenge with HIV-15016(a,b) and 10 weeks post-challenge (c) was assessed by the endogenous assay (j) or acute assays using CD4 cells infected with HIV-1SF2 (j) or HIV-15016 ( ).
Values represent means of triplicate determinations at CD8 : CD4 ratios of 0.5 : 1. Similar data were obtained at CD8 : CD4 ratios of 1 : 1 or 2 : 1.
60
40
20
0
60
40
20
0 2Pa 1P 3P C2
ND
2Pa 1P 3P C2 Percent suppression of HIV-1 replication
At boost At challenge
(a)
60
40
20
0
2Pa 1P 3P C2 ND
2Pa 1P 3P C2 Percent suppression of HIV-1 replication
60
40
20
0 SF2
5016
SF2 5016
At boost At challenge
(b)
60
40
20
0
2Pa 1P 3P C2 2Pa 1P 3P C2 2Pa 1P 3P C2 60
40
20
0
20
15
10
5
0 Percent suppression of HIV-1 replication
Endogenous SF2 5016
(c)
ISSN 0269-9370&2000 Lippincott Williams & Wilkins 893
chimpanzees 3P and 1P exhibited minimal suppression of 9 and 4%, respectively [2]. The assay of CD8AA against HIV-15016, which utilizes only the CCR5 co- receptor (not shown), is probably most sensitive on macrophages. Because the CD8AA of control chimpan- zee C2 suppressed HIV-15016replication 69% on macro- phages, we could previously not correlate CD8AA with the protection of chimpanzee 2PA [2]. Here, further results show that chimpanzee 2PA exhibited strong pre- challenge CD8AA in three out of four assay systems, whereas the infected animals had CD8AA of over 20%
in only one out of four assays. The broad, elevated pre- challenge CD8AA of chimpanzee 2PA may thus have contributed to its protection.
Ten weeks post-challenge, endogenous CD8AA in chimpanzee 2PA remained two- to nine-fold higher than levels in the other animals (Fig. 1c). Chimpanzee 2PA's acute CD8AA against HIV-1SF2 declined post-challenge, but low-level CD8AA against HIV- 15016-infected CD4 cells appeared, and was elevated compared with the other animals. Suf®cient effector cells at this timepoint were not available for assessment of CD8AA on HIV-15016-infected macrophages.
Two potentially protective mechanisms were thus present in 2PA at the time of and after challenge. Both may have contributed to protection. Our results imply that for greatest ef®cacy, vaccines should be designed to generate both viral-speci®c immunity and potent CD8AA. Reports of HIV-exposed but asymptomatic individuals who appear to control virus replication and
exhibit high levels of CD8AA [6] are consistent with this conclusion.
Michel Leno, Mark Kowalski and Marjorie Robert- Guroff, Basic Research Laboratory, National Cancer Institute, Bethesda, MD 20892-5055, USA.
Sponsorship: This study was partly supported by a Cooperative Research and Development Agreement be- tween NCI and Wyeth-Lederle Vaccines.
Received: 7 January 2000; accepted: 14 January 2000.
References
1. Lubeck MD, Natuk R, Myagkikh M,et al.Long term protection of chimpanzees against high-dose HIV-1 challenge induced by immunization.Nature Med1997,3:651±658.
2. Robert-Guroff M, Kaur H, Patterson L, et al.Vaccine protection against a heterologous, non-syncytium-inducing, primary human immunode®ciency virus.J Virol1998,72:10275±10280.
3. Leno M, Carter L, Venzon DJ,et al.CD8lymphocyte antiviral activity in monkeys immunized with SIV recombinant poxvirus vaccines: potential role in vaccine ef®cacy.AIDS Res Hum Retro- viruses1999,15:461±470.
4. Lehner T, Wang YF, Cranage M, et al. Protective mucosal immunity elicited by targeted iliac lymph node immunization with a subunit SIV envelope and core vaccine in macaques.
Nature Med1996,2:767±775.
5. Wang YF, Tao L, Mitchell E,et al.Generation of CD8 suppressor factor and beta chemokines, induced by xenogeneic immuniza- tion, in the prevention of simian immunode®ciency virus infection in macaques.Proc Natl Acad Sci USA1998,95:5223±5228.
6. Stranford SA, Skurnick J, Louria D,et al.Lack of infection in HIV- exposed individuals is associated with a strong CD8 cell noncytotoxic anti-HIV response.Proc Natl Acad Sci USA 1999, 96:1030±1035.
Stromal-derived factor-1 chemokine gene variant is associated with the delay of HIV-1 disease progression in two longitudinal cohorts
HIV-1 disease is multifactorial and is associated with a spectrum of clinical outcomes among infected persons [1]. Viral and host factors in¯uence HIV-1 disease progression. Chemokine receptors function as co-re- ceptors for viral entry, and studies have shown that viral isolates from long-term non-progressors (LTNP) maintain exclusive usage of the CCR5 co-receptor, whereas isolates from progressors generally broaden their co-receptor usage to include CCR2b, CCR3, and CXCR4 [2,3]. Host genetic factors, such as polymorphisms in the CCR2b receptor and a deletion in the CCR5 (CCR5Ä32) receptor, delay disease progression or prevent infection [4±6]. The later stud- ies emphasize the role of host genetic factors in the natural history of HIV-1.
No polymorphisms have been found in the CXCR4 gene that would explain why HIV isolates from LTNP maintain CCR5 as their predominant co-receptor [7].
Subsequent study of the gene encoding the CXCR4
ligand, stromal-derived factor (SDF-1) has shown a G to A polymorphism in the conserved 39 untranslated region (SDF1-39A), which was implicated in delaying HIV-1 disease progression [8,9]. Other studies have found an accelerated rate of HIV-1 disease progression [10] or death [11] in individuals with this polymorph- ism. To clarify this issue, we analysed for the presence of the SDF1-39A polymorphism in two longitudinal, HIV-1-infected cohorts.
The study participants included 90 HIV-seronegative individuals who comprised 52 normal blood donors and 38 high-risk, homosexual men, as well as HIV-1- infected individuals followed for more than 12 years [12,13]. The latter group was subdivided into 61 progressors and 31 LTNP. The progressors were characterized by AIDS-de®ning symptoms and a de- cline in CD4 T cell count to less than 200 in less than 8 years after infection, whereas the LTNP maintained CD4 T cell levels of over 400 for more than 8 years
after infection and had no clinical symptoms. None of the LTNP were on therapeutic interventions while enrolled in these studies. DNA was puri®ed from peripheral blood mononuclear cells and analysed for the presence of the SDF1-39A polymorphism [8] and the CCR5Ä32 base pair deletion [5] using polymerase chain reaction-based assays.
Among the HIV-seronegative individuals studied, the frequency of the SDF1-39A allele was 0.178 (Table 1).
Within this population, analysis of the blood donors and the homosexual men showed that the frequencies for SDF1-39A were 0.221 (n52) and 0.118 (n38), respectively. The progressors had a lower SDF1-3'A allele frequency of 0.148 when compared with the seronegative individuals. Of note is the fact that the allele frequency for the LTNP was 0.306, which was signi®cantly higher when compared with the frequen- cies for seronegative individuals (P0.046) and pro- gressors (P0.019) (Table 1). Interestingly, the CCR5Ä32 allele frequency for the LTNP was not signi®cantly higher than that of the seronegative in- dividuals, but was signi®cantly higher when compared with the progressors (P0.033) (Table 1).
Although these data are in agreement with the ®ndings from two previous studies [8,9], they differ from the results of two others [10,11]. Reasons for the discre- pancies between these studies may be the use of antiretroviral therapies [11], the inability to determine accurately the time of seroconversion [10], or the limited median follow-up time [11], all of which would impact the interpretation of these genetic polymorph- isms on the natural history of HIV-1.
The mechanism underlying the observed SDF1-39A protective effect is unknown. This polymorphism oc- curs in the 39 untranslated region of the gene [8], which may stabilize the RNA, thus affecting protein expression. SDF-1 binding of CXCR4 has been shown to result in rapid internalization of this receptor from the cell surface [14,15]. If increased protein expression occurs in vivo, it may explain the exclusive usage of
CCR5 by HIV-1 from the LTNP [3]. The lack of a strong association of the CCR5Ä32 bp deletion with disease progression in this study is intriguing. Previous work has shown that LTNP who are wild type for CCR5 are virologically and immunologically similar to persons who are heterozygous for the 32 bp deletion [16]. Although the virological and immunological responses of the HIV-1-infected individuals have not been studied, HIV-1-seronegative persons showed a similar spontaneous and induced production of â- chemokines, macrophage-in¯ammatory protein types 1áand 1â, or regulated upon activation, normal T cell expressed and secreted (data not shown), regardless of their SDF-1 genotypes. This implies that the SDF1- 39A polymorphism does not affect the production ofâ- chemokines, which could in¯uence the utilization of the CCR5 co-receptor by HIV-1.
In summary, it was demonstrated that LTNP in two HIV-1-infected cohorts have a higher frequency of the SDF1-39A polymorphism compared with the frequen- cies for HIV-seronegative individuals and progressors.
These data suggest that this polymorphism is associated with delayed HIV-1 disease progression.
Charlene S. Dezzuttia, Patricia C. Guenthnera, Timothy A. Greenb, Oren J. Cohend, Thomas J. Spirac and Renu B. Lala, aHIV and Retrovirology Branch, bOf®ce of the Director, andcHIV Immunology and Diagnostics Branch, Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA; and dLaboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA.
Received: 23 December 1999; 7 January 2000.
References
1. Fauci AS. Host factors and the pathogenesis of HIV-induced disease.Nature1996,384:529±534.
2. Connor RI, Sheridan KE, Ceradini D, Choe S, Landau NR.Change
Table 1. Frequency of the SDF1-39A polymorphism and the CCR5Ä32 deletion in two longitudinal cohorts of HIV-1-infected persons and HIV- seronegative individuals.
SDF-1 polymorphism (%) Frequency
WT /39A 39A/39A SDF1-39Aa CCR5Ä32b
Seronegative individuals (n90) 63 (70) 22 (24) 5 (6) 0.178 0.106
Progressors (n61) 45 (74) 14 (23) 2 (3) 0.148 0.082
LTNP (n31) 16 (52) 11 (35) 4 (13) 0.306 0.194
LTNP, Long-term non-progressors; SDF-1, stromal-derived factor type 1; WT, wild type.
Statistical signi®cance was determined using Fisher's exact test.
aLTNP versus seronegative individualsP0.046; LTNP versus progressorsP0.019.
bLTNP versus seronegative individualsP0.081; LTNP versus progressorsP0.033.
in coreceptor use correlates with disease progression in HIV-1 infected individuals.J Exp Med1997,388:296±300.
3. Xiao L, Rudolph DL, Owen SM, Spira TJ, Lal RB.Adaptation to promiscuous usage of CC and CXC-chemokine coreceptorsin vivo correlates with HIV-1 disease progression. AIDS 1998, 12:F137±F143.
4. Haung Y, Paxton WA, Wolinsky SM,et al.Homozygous defect I HIV-1 coreceptor accounts for resistance of some multiply- exposed individuals to HIV-1 infection.Nat Med1996,2:1240±
1243.
5. Eugen-Olsen J, Iversen AKN, Garred P,et al.Heterozygosity for a deletion in the CKR-5 gene leads to prolonged AIDS free survival and slower CD4 T cell fall in a cohort of HIV seropositive individuals.AIDS1997,11:305±310.
6. Smith MW, Dean M, Carrington M, et al. Contrasting genetic in¯uence of CCR2andCCR5 variants on HIV-1 infection and disease progression.Science1997,277:959±965.
7. Cohen OJ, Paolucci S, Bende SM, et al. CXCR4 and CCR5 genetic polymorphisms in long-term nonprogressive human immunode®ciency virus infection: lack of association with muta- tions other than CCR5-Ä32.J Virol1998,72:6215±6217.
8. Winkler C, Modi W, Smith MW, et al. Genetic restriction of AIDS pathogenesis by an SDF-1 chemokine gene variant.Science 1998,279:389±393.
9. Hendel H, HeÂnon N, Lebuanec H, et al.Distinctive effects of CCR5,CCR2, andSDF1genetic polymorphisms in AIDS progres- sion. J Acquired Immune De®c Syndr Hum Retrovirol 1998,
19:381±386.
10. van Rij RP, Broersen S, Goudsmit J, Coutinho RA, Schuitemaker H.The role of a stromal cell-derived factor-1 chemokine gene variant in the clinical course of HIV-1 infection. AIDS 1998, 12:F85±F90.
11. Mummidi S, Ahuja SS, Gonzalez E,et al.Genealogy of the CCR5 locus and chemokine system gene variants associated with altered rates of HIV-1 disease progression. Nat Med 1998, 4:786±793.
12. Fishbein DB, Kaplan JE, Spira TJ,et al.Unexplained lymphadeno- pathy in homosexual men.JAMA1985,254:930±935.
13. Pantaleo G, Menzo S, Vaccarezza M, et al. Studies in subjects with long-term nonprogressive human immunode®ciency virus infection.N Engl J Med1995,332:201±208.
14. FoÈrster R, Kremmer E, Schubel A,et al.Intracellular and surface expression of the HIV-1 coreceptor CXCR4/fusin on various leukocyte subtypes: rapid internalization and recycling upon activation.J Immunol1998,160:1522±1531.
15. Yang OO, Swanberg SL, Lu Z, et al. Enhanced inhibition of human immunode®ciency virus type 1 by met-stromal-derived factor 1âcorrelates with down-modulation of CXCR4. J Virol 1999,73:4582±4589.
16. Cohen OJ, Vaccarezza M, Lam GK, et al.Heterozygosity for a defective gene for CC chemokine receptor 5 is not the sole determinant for the immunologic and virologic phenotype of HIV-infected long-term nonprogressors. J Clin Invest 1997, 100:1581±1589.
HIV transmission between two siblings in Africa HIV infection in children is mainly acquired through vertical transmission from an infected mother. We report epidemiological and molecular evidence of horizontal transmission between two siblings. This is, to our knowledge, the ®rst documented case from Africa, where socioeconomic conditions may be more conducive to horizontal spread than in industrialized countries.
In 1996, a 312-year-old HIV-infected child (child A), with HIV-negative parents, was referred to our infec- tious diseases clinic. He initially presented with general- ized lymphadenopathy, hepatosplenomegaly and cryptosporidial diarrhoea. We con®rmed HIV infection by enzyme immunosorbent assay (ELISA) antibody detection and reverse transcriptase±polymerase chain reaction (RT±PCR) for HIV RNA detection. HIV negativity in the parents and sibling (child B) were also con®rmed. Child A had an uncomplicated normal birth and was breastfed by his mother for 4 months (surro- gate breastfeeding was denied). He had been hospita- lized at 3 years of age for diarrhoea and acute bacterial meningitis, and had never received a blood transfusion.
Seventeen months later, the sibling (child B), 28 months younger than his brother, presented with conjunctivitis and chronic otorrhoea. Cervical lympha- denopathy and hepatomegaly were noted. He was retested for HIV (ELISA and RT±PCR) and was found to be infected. Medical records disclosed an episode of gastroenteritis and herpes stomatitis 13 months earlier. Both parents remained negative by ELISA and RT±PCR. HLA genotyping con®rmed the
genetic lineage between the children and their parents with 99.9% certainty. The two brothers and their parents lived alone in the same household. There was no history or clinical evidence of sexual abuse. They were never immunized on the same day and the clinics do not re-use needles or syringes. In the period between child B's negative and positive serology, child A, who shared a bed with his sibling, had a few episodes of epistaxis, staining the pillow. Child B was also exposed to the purulent otorrhoea of child A, including during the period when child B had herpes stomatitis.
Phylogenetic analysis was performed on a 337 base pair region of the env gene, including the V3 domain of the viral strains from the two brothers. Distance calculation, tree construction methods and bootstrap analysis were performed using CLUSTAL W. The two strains differed by 3.2%, indicating horizontal trans- mission from child A to child B (Fig. 1). Contact with blood from epistaxis or purulent otorrhoea are implicated as a means of transmission. Child B's episode of herpes stomatitis may have contributed by compromising the mucosal integrity. Leitner et al. [1]
evaluated the use of phylogenetic methods in their study of HIV transmission patterns and found these methods to be very accurate. A few sporadic cases of horizontal transmission in the household involving children have been reported from the USA and Europe. In two of those cases molecular evidence supported horizontal transmission [2,3]. Epidemio- logical evidence indicated mucocutaneous exposure to infected blood.
Only one study from Africa (Zaire) has investigated the possibility of household transmission. Mann and collea- gues [4] surveyed 204 household contacts of 46 AIDS patients and 155 of 43 seronegative controls. Although the authors' conclusion negated horizontal transmission, their statistical methodology was questioned and on re- analysis the risk was thought to be real [5]. Razel [5]
estimated the risk of HIV infection through household contact to be 0.4 transmissions per 100 person-years of contact.
The origin of child A's infection still remains an open question because both parents were seronegative. Since the start of our investigation two other HIV-infected children, with unknown sources of infection (parents
also HIV negative), were detected at the same regional hospital. These children all come from poor socio- economic backgrounds where overcrowding is condu- cive to skin or mucous membrane contact with HIV-infected blood and other secretions. In conclu- sion, we have documented the possible horizontal spread of HIV between two siblings, supported by phylogenetic analysis, and have begun additional studies of this form of transmission.
Heidi Ortha, Susan Engelbrechta, Mark F. Cottonb, Brenda A. Robsona, Tracey-Lee Smitha, H. Simon Schaafb, Beryl Leibrandtc, Els Dobbelsc and Estrelita Janse van Rensburga,Departments of aMedical Virology andbPaediatrics and Child Health, University of Stellen- bosch and Tygerberg Hospital, Tygerberg 7505, South Africa; and thecDepartment of Pediatrics, Paarl Hospital, Paarl, South Africa.
Received: 16 December 1999; accepted: 22 December 1999.
References
1. Leitner T, Escanilla D, FranzeÂn C, UhleÂn M, Albert J.Accurate reconstruction of a known HIV-1 transmission history by phylo- genetic tree analysis.Proc Natl Acad Sci U S A1996,93:10864±
10869.
2. Fitzgibbon JE, Gaur S, Frenkel LD, Laraque F, Edlin BR, Dubin DT.Transmission from one child to another of human immuno- de®ciency virus type 1 with a zidovudine-resistance mutation.
N Engl J Med1993,329:1835±1841.
3. Salvatori F, De Martino M, Galli L, Vierucci A, Chieco-Bianchi L, De Rossi A. Horizontal transmission of human immunode®- ciency virus type 1 from father to child. AIDS Res Hum Retroviruses1998,14:1679±1685.
4. Mann JM, Quinn TC, Francis H,et al.Prevalence of HTLV III/
LAV in household contacts of patients with con®rmed AIDS and controls in Kinshasa, Zaire.JAMA1986,256:721±724.
5. Razel M.Casual household transmission of human immunode®- ciency virus.Med Hypotheses1998,51:115±124.
Widespread circulation of a B/F intersubtype recombinant form among HIV-1-infected individuals in Buenos Aires, Argentina
Argentina has, after Brazil, the second largest HIV-1- infected population in South America, with 120 000 estimated cases of infection by the end of 1997 [1] and almost 15 000 reported AIDS cases by the end of 1998, with a high proportion (76%) from the Buenos Aires metropolitan area [2]. Previous phylogenetic studies in Argentina, based on V3 [3,4], on V3 and gag[5], and on envand protease [6] sequences indicated the presence of viruses grouping with B or F subtypes, as well as a minority of B/F recombinant viruses. In a recent study, based onenvand protease sequences [6], it was reported that 55% of 58 individuals from Buenos Aires harboured B subtype viruses, 40% F subtype viruses, and 5% had either B/F mosaic or dual infections. Here we report the results of the phylogenetic analysis of HIV-1 reverse
transcriptase (RT) sequences from 52 adult individuals from the Buenos Aires metropolitan area. DNA from peripheral blood mononuclear cells was used to amplify and directly sequence a segment of approximately 0.6 kb in RT, comprising most resistance-associated codons, using the polymerase chain reaction (PCR).
The phylogenetic neighbour-joining tree, constructed using Kimura's two-parameter distances, showed clus- tering in two groups: the ®rst, consisting of 31 samples (60%), clustered with the B subtype reference sequence, and the second, consisting of 21 samples (40%), clustered with a high bootstrap value (73%) as outliers of the F subtype branch. Analysis of the second group with the recombinant identi®cation program (RIP) [7] suggested that all were intersubtype recombinants (59F/B39) with
0.1
O-ANT70
D-ELI B-RF
B-HXB2 H-CA13
H-VI557 G-92RU131
G-92UG975 A-SF1703 C-92BR025
B A F-BZ126A F-BZ163A E-93TH253 E-90CR402
C-ETH2220 C-UG268 C-ZAM18
Fig. 1. Phylogenetic tree analysis of children A and B HIV-1 sequences and Los Alamos HIV database reference se- quences (A-SF1703, B-HXB2, B-RF, C-ZAM18, C- 92BR025.8, C-ETH2220, C-UG268, D-ELI, E-90CR402.1, E-93TH253.3, F-BZ126A, F-BZ163A, G-92RU131.9, G- 92UG975.10, H-CA13, H-V1557, O-ANT70). An indication of the degree of sequence dissimilarity is shown on the horizontal axis.
breakpoints at the same approximate location. To investigate the presence of additional breakpoints down- stream in RT, the remaining portion of RT was PCR- ampli®ed in 10 putative recombinant and in four B subtype samples, obtaining a near-full-length RT se- quence (except 90 nucleotides at the 59 end). RIP analysis indicated that all 10 recombinant samples had a second intersubtype breakpoint approximately 0.7 kb downstream from the ®rst, delimiting a segment with high similarity scores to the B subtype consensus,
¯anked by sequences with high similarity scores to the F subtype consensus. Bootscan analysis [8] with SimPlot [9] con®rmed the presence of the two breakpoints, with the 59F/B/F39subtype pattern supported by high boot- strap values (up to 100%). Protease and the N-terminal portion of RT were ampli®ed and sequenced in three recombinant samples, and the assembled protease±RT sequences were analysed by bootscanning (Fig. 1a), which supported the branching of protease with the F subtype consensus sequence, except for a short stretch across protease±RT, for which bootstrap values sup- ported branching with the B subtype consensus.
Informative site analysis using the maximum chi- squared method [10,11] was consistent with RIP and bootscanning, identifying two intersubtype breakpoints, the ®rst between RT nucleotides 424 and 453, and the second between RT nucleotides 1152 and 1181, with signi®cance at the level of P,0.001, in eight of 10 samples analysed, with slight variations within the mentioned ranges. In two samples breakpoints were slightly displaced relative to the rest: in one, the ®rst breakpoint was between nucleotides 498 and 507, and in another, the second breakpoint was between nucleo- tides 1128 and 1129.
Separate phylogenetic trees of the three RT segments delimited by breakpoints (Fig. 1b) con®rmed the grouping of recombinant samples with the F subtype reference in the 59 and 39 segments and with the B subtype reference in the middle segment, in all cases with high bootstrap values. Nucleotide distances in the middle segment were signi®cantly lower between B/F recombinants (3.00.7%) than between B subtype viruses (6.9 1.1%), which is consistent with a more recent common ancestor of the former.
The phylogenetic tree of the V3 loop and ¯anking sequences of ®ve B subtypes and ®ve B/F recombinant viruses showed that samples of subtype B in RT were also of subtype B in V3, whereas samples B/F recombi- nant in RT were of subtype F in V3, with bootstrap values of 100% in all cases. Nucleotide distances were 11.92.1% for the B subtype viruses and 9.13.2%
for the B/F recombinants.
The prevalence of HIV-1 genetic forms varied with risk groups and sex. All six injecting drug users (IDU)
(three men and three women) harboured B/F recom- binants, whereas four of ®ve (80%) homosexual men harboured B subtype viruses. Among 28 individuals with heterosexually acquired infection (13 men and 15 women), there was a clear predominance of B subtype viruses among men (85%) and of B/F recombinants among women (67%).
These results therefore indicate that B/F intersubtype Fig. 1. Analysis for intersubtype recombination of HIV-1 protease and reverse transcriptase (RT) sequences in indivi- duals from Buenos Aires. (a) Bootscan analysis of protease and RT sequences from one individual. Consensus se- quences of the presumed parental subtypes, B and F, were used, plus an A subtype sequence as the outgroup. Plots represent bootstrap values, based on 100 resamplings, sup- porting branching with the reference sequences within a 400 nucleotide window moving in steps of 20 bases. Values in the x axis represent the position of the centre of the window from nucleotide 1 of protease. (b) Neighbour-joining phylo- gentic trees of RT segments delimited by intersubtype break- points in 14 HIV-1-infected individuals. Bootstrap values (out of 100 replicates) supporting branching of the sample se- quences with subtype reference sequences are shown.
Branches corresponding to B/F recombinant viruses (10 sam- ples) are labelled with ®lled circles, and branches corre- sponding to B subtype viruses (four samples) are labelled with un®lled circles.
110 100 90 80 70 60 50 40 30 20 10 0 210
0 200 400 600 800 1000 1200 1400 1600 1800 Position of centre of window
% of permuted trees
(a)
Protease RT polymerase RNase H
F B A
0.1 F
D B J C H A G
99 81
94 100
78
J G
C A H F D
B F
C A
G J H D
B RT nt 91-442 RT nt 443-1164 RT nt 1165-1680 (b)
recombinant viruses are widely circulating among HIV-1-infected individuals in Buenos Aires (40% of our cases), and are predominant among IDU and among women with sexually acquired infection. Vir- tually identical breakpoints in RT (with slight varia- tions expected from mutations accumulated along the years) are strongly suggestive of a common origin.
Failure to detect non-recombinant F subtype viruses and the grouping of V3 of the recombinant viruses with subtype F viruses suggest that most, if not all, HIV-1 isolates of Buenos Aires previously identi®ed as being of subtype F are in fact B/F recombinants.
Analysis of the homology of V3 sequences with Genebank sequences using BLAST [12] shows the highest similarity scores with F subtype viruses from Brazil. RT sequences from Brazil, analysed by us (unpublished data) and by others [13], however, show a low prevalence of B/F recombination, with those recombinants detected having breakpoints different from the viruses from Argentina. Taken together, these data are consistent with an individual dually infected with B and F subtype viruses, with the F subtype virus originating in Brazil, who transmitted the recombinant form in Argentina, as the most likely source of the B/F recombinant form of Buenos Aires.
Although numerous HIV-1 intersubtype recombinant forms have been reported, only three, the A/E(CM240) of southeast Asia, the A/G(IbNG) of Africa, and the A/
B(Kal153) of Kaliningrad, Russia, have spread to epi- demic proportions comparable to the prevalent B/F recombinant form of Buenos Aires reported here [14,15]. Sequencing of the full-length genome of this virus is underway further to validate its recognition as a major HIV-1 circulating recombinant form.
Michael M Thomsona, MarõÂa Luisa Villahermosaa, Elena VaÂzquez-de-Pargaa, MarõÂa Teresa Cuevasa, Elena Delgadoa, Nuria ManjoÂna, Leandro Medranoa, LucõÂa PeÂrez-AÂlvareza, Gerardo Contrerasa, Manuel GoÂmez Carrillob, Horacio SalomoÂnb and Rafael NaÂjeraa, aAÂrea de Patogenia Viral, Centro Nacional de BiologõÂa Funda- mental, Instituto de Salud Carlos III, Madrid, Spain; and
bCentro Nacional de Referencia para el SIDA, Departa- mento de MicrobiologõÂa, Facultad de Medicina, Buenos Aires, Argentina.
We thank Isabel Herrero and Gustavo Kijak for technical assistance, and Saladin Osmanov for his contribution to
implementing the UNAIDS program that made this work possible. This work was supported by grant VI1236 from Plan Nacional del SIDA, Ministerio de Sanidad y Con- sumo, Madrid, Spain.
Received: 7 January 2000; accepted: 14 January 2000.
References
1. UNAIDS (online). Epidemiological fact sheet on HIV/AIDS and sexually transmitted diseases. http://158.252.21.5/wsite/ 00core- frame.html.
2. Ministry of Health and Social Action, Buenos Aires, Argentina.
BoletõÂn sobre el SIDA en la RepuÂblica Argentina. July 1999.
3. Campodonico M, Janssens W, Heyndrickx L,et al. HIV type 1 subtypes in Argentina and genetic heterogeneity of the V3 region.AIDS Res Hum Retroviruses1996,12:79±81.
4. Marquina S, Leitner T, Rabinovich RD, Benetucci J, Libonatti O, Albert J.Coexistence of subtypes B, F, and B/Fenvrecombinant of HIV type 1 in Buenos Aires, Argentina. AIDS Res Hum Retroviruses1996,12:1651±1654.
5. FernaÂndez-Medina RD, Jansson M, Rabinovich RD, Libonatti O, Wigzel H.Identi®cation of human immunode®ciency virus type 1 subtypes B and F, B/F recombinants and dual infections in Argentina.Scand J Infect Dis1999,31:235±242.
6. Masciotra S, Livellara B, Belloso W, Lal R, Pieniazek D.Molecu- lar epidemiology of HIV-1 in Argentina: predominance of subtype F infections in a heterosexual population. 6th Confer- ence on Retrovirus and Opportunistic Infections. Chicago, Janu- ary 1999 [Abstract 714].
7. Siepel AC, Halpern AL, Macken C, Korber BT. A computer designed to screen rapidly for HIV type 1 intersubtype recombi- nant sequences. AIDS Res Hum Retroviruses 1995, 11:1413±
1416.
8. Salminen M, Carr JK, Burke DS, McCutchan F.Identi®cation of breakpoints in intergenotypic recombinants of HIV type 1 by bootscanning.AIDS Res Hum Retroviruses1995,11:1423±1425.
9. Ray SC. Simplot for Windows (version 1.6). Baltimore, MD.
Distributed by author. http://www.welvh.jhu.edu, 1998.
10. Maynard Smith J.Analyzing the mosaic structure of genes.J Mol Evol1992,34:126±129.
11. Maynard Smith J. Maximum chi-squared 1.0. 1995. Available at http://biomix.biols.ac.uk/biols/Biochem/Molbiol/maximum-chi- squared.html.
12. HIV Sequence Database (online). Los Alamos National Labora- tory, Los Alamos, NM. http://hiv-web.lanl.gov.
13. Brindeiro R, Vanderborght B, Caride E,et al.Sequence diversity of the reverse transcriptase of human immunode®ciency virus type 1 from untreated Brazilian individuals.Antimicrob Agents Chemother1999,43:1674±1680.
14. Carr JK, Foley BT, Leitner T, Salminen M, Korber B, McClutchan F.
Reference sequences representing the principal genetic diversity of HIV-1 in the pandemic. In: Human retroviruses and AIDS 1998: a compilation and analysis of nucleic acid and aminoacid sequences. Korber B, Kuiken C, Foley B, et al. (editors). Los Alamos: Theoretical Biology and Biophysics Group, Los Alamos National Laboratory; 1998. pp. III-10±III-19.
15. Carr JK, Laukkanen T, Salminen MO, et al.Characterization of subtype A HIV-1 from Africa by full genome sequencing.AIDS 1999,13:1819±1826.
Valproic acid induces human herpesvirus 8 lytic gene expression in BCBL-1 cells Kaposi's sarcoma is a multifocal, highly vascularized
tumor that is the most common neoplasm associated with HIV infection [1,2]. Kaposi's sarcoma herpes virus or human herpesvirus 8 (HHV8) is present in Kaposi's sarcoma lesions from all risk groups, and appears to be
essential for the development of Kaposi's sarcoma [3±
6]. The virus resides in monocytes and B cells in asymptomatic hosts [7±10], whereas individuals with clinical Kaposi's sarcoma have HHV8 present in the spindle cells and in microvascular, lymphatic or im-
mature endothelial cells in Kaposi's sarcoma lesions [11±15]. The development of Kaposi's sarcoma may be partly dependent on the lytic production of infectious virions and the subsequent infection of endothelial cells and related spindle cells with HHV8 [16]. Some macrophages in Kaposi's sarcoma lesions are lytically infected with HHV8 [17], potentially providing infec- tious HHV8 virions.
Considerable progress has been made in understanding the switch between latency and the lytic life cycle of HHV8 [18±20]. Although both 12-O-tetradecanoyl phorbol (TPA) and butyrate can induce lytic replica- tion, we and other authors have shown that butyrate leads to higher levels of viral DNA replication and lytic gene expression than TPA [21,22]. In a primary effusion lymphoma cell line, BCBL-1, we have also shown that butyrate, but not TPA, induces apoptosis in a dose-dependent manner [22]. Furthermore, apoptosis is delayed when lower concentrations of butyrate are used, thereby permitting the effective production of infectious virions.
Valproic acid (2-propylpentanoic acid) is an eight carbon branched chain fatty acid that has considerable structural similarities to butyric acid, a four carbon fatty acid. Its sodium salt, valproate, is a commonly pre- scribed mood stabilizing and broad spectrum anti- seizure substance that has proven ef®cacy in controlling partial and generalized epileptic seizures, bipolar dis- orders, schizophrenia, and neuropathic pain [23,24].
Because valproate is sometimes used in individuals who might be infected with HHV8, we investigated whether this agent might induce HHV8 to enter into the lytic cycle in a manner analogous to butyrate.
BCBL-1 cells were incubated with either valproate or with butyrate, and changes in HHV8 gene expression were compared by Northern blot analysis. Valproate was as effective as butyrate at inducing the expression of multiple HHV8 encoded transcripts that are asso- ciated with entry into the lytic phase of replication, including vIL-6, vGPCR, vMIP I, vBcl-2 and ORF 26 (Fig. 1). Both valproate and butyrate showed comparable dose dependence for gene induction, with the highest concentration of inducer (3 mM) leading to the greatest increase in gene expression. By densito- metric analysis and with correction for glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) levels, vIL-6 messenger RNA levels at 24 h in response to either valproate or butyrate were increased more than 14-fold in response to 3 mM inducer and more than sixfold in response to 0.3 mM inducer. Peak levels of ORF 26 occurred later than the other genes examined, with ORF 26 levels at 46 h being at least sixfold greater than control in response to 3 mM inducer. A steeper dose±
response curve was found for v-Bcl2 and v-MIP I than for v-IL-6, v-GPCR, or ORF 26. For example, no detectable v-Bcl2 expression was found in control cells,
and responses to 3 mM inducer were more than sixfold higher than responses to 0.3 mM inducer when exam- ined at 24 h. The latency associated gene, v-cyclin, showed only a minor transient change in expression in response to butyrate or valproate, as has previously been reported for butyrate [22]. Decreases in the cellular GAPDH mRNA levels occurred in response to high concentrations of either valproate or butyrate, as Fig. 1. Induction of human herpesvirus 8 (HHV8) gene ex- pression by valproate and butyrate in BCBL-1 cells. BCBL-1 cells were grown in unsupplemented medium (control) or medium supplemented with sodium valproate or sodium butyrate at the indicated concentrations for either 24 or 46 h.
Total cellular RNA was extracted using RNAzol B and 20ìg/
lane was size fractionated on a 1% agarose formaldehyde gel. Northern analysis was performed as previously described [22] using double stranded DNA probes representing the full- length open reading frames for the indicated genes. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.
has previously been shown with butyrate [21,22]. The similar chemical structure and responsiveness seen with valproate and butyrate suggest a shared mechanism of action. Butyrate is well recognized for its ability to inhibit histone deacetylase [21,25], thereby affecting chromatin structure and gene expression.
Valproate is used to treat many neurological diseases that occur in individuals infected with HIV and HHV8 [26,27]. Although valproate stimulates HIV replication in some HIV-infected cell lines in culture [28,29], it also enhances zidovudine levels in patients treated with both agents [30], potentially overcoming any deleter- ious effect that valproate might have with respect to HIV infection. The results presented here suggest that the use of valproate in patients infected with HHV8 might be detrimental. Plasma concentrations during chronic oral treatment with valproate typically range from 0.28 to 0.69 mM [24], concentrations that effec- tively induced lytic gene expression in our studies. The induction of lytic replication by valproate might en- hance viral load, potentially triggering the development or exacerbation of Kaposi's sarcoma. Furthermore, several genes that are induced by valproate, using concentrations encountered clinically, could have pro- found biological effects. For example, vIL-6 is biologi- cally active and has been implicated in the pathogenesis of some forms of Castleman's disease [31±36]. v-MIP I and vGPCR are angiogenic [31,37,38] and vGPCR is oncogenic [37], suggesting that increased expression of these genes could exacerbate Kaposi's sarcoma. These results should thus be considered when selecting therapy for neurological disease in patients who have HHV8, especially those who have diseases such as Kaposi's sarcoma and Castleman's disease.
Renee N. Shawa, Jack L. Arbiserb and Margaret K.
Offermannac, aWinship Cancer Center, Atlanta, GA 30322, USA;bDepartment of Dermatology andcDivision of Hematology/Oncology, Department of Medicine, Em- ory University, Atlanta, GA 30322, USA.
Sponsorship: This work was supported by NIH grants RO1 CA67382, KO8 AR 02096 and RO3 AR44947.
Received: 13 January 2000; accepted: 19 January 2000.
References
1. Dupon M, Marimoutou C, Cazorla C, et al. Incidence of acquired immunode®ciency syndrome (AIDS)-related Kaposi's sarcoma in the Aquitaine Cohort, France, 1988±1996. Groupe d'Epidemiologie Clinique du SIDA en Aquitaine.Int J Epidemiol 1998,27:1078±1082.
2. Brodt HR, Kamps BS, Helm EB, Schofer H, Mitrou P. Kaposi's sarcoma in HIV infection: impact on opportunistic infections and survival.AIDS1998,12:1475±1481.
3. Schulz TF. Epidemiology of Kaposi's sarcoma-associated her- pesvirus/human herpesvirus 8. Adv Cancer Res 1999, 76:
121±160.
4. Chang Y, Cesarman E, Pessin MS, et al. Identi®cation of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma.Science1994,266:1865±1869.
5. Chang Y, Moore PS. Kaposi's Sarcoma (KS)-associated herpes- virus and its role in KS.Infect Agents Dis1996,5:215±222.
6. Moore PS, Chang Y. Detection of herpesvirus-like DNA se- quences in Kaposi's sarcoma in patients with and without HIV infection.N Engl J Med1995,332:1181±1185.
7. Blackbourn DJ, Ambroziak J, Lennette E, Adams M, Ramachan- dran B, Levy JA.Infectious human herpesvirus 8 in a healthy North American blood donor.Lancet1997,349:609±611.
8. Humphrey RW, O'Brien TR, Newcomb FM, et al. Kaposi's sarcoma (KS)-associated herpesvirus-like DNA sequences in peripheral blood mononuclear cells: association with KS and persistence in patients receiving anti-herpesvirus drugs. Blood 1996,88:297±301.
9. Kikuta H, Itakura O, Taneichi K, Kohno M. Tropism of human herpesvirus 8 for peripheral blood lymphocytes in patients with Castleman's disease.Br J Haematol1997,99:790±793.
10. Kikuta H, Itakura O, Ariga T, Kobayashi K.Detection of human herpesvirus 8 DNA sequences in peripheral blood mononuclear cells of children.J Med Virol1997,53:81±84.
11. Dupin N, Fisher C, Kellam P, et al. Distribution of human herpesvirus-8 latently infected cells in Kaposi's sarcoma, multi- centric Castleman's disease, and primary effusion lymphoma.
Proc Natl Acad Sci U S A1999,96:4546±4551.
12. Boshoff C, Schulz TF, Kennedy MM, et al. Kaposi's sarcoma- associated herpesvirus infects endothelial and spindle cells.
Nature Med1995,1:1274±1278.
13. Foreman KE, Bacon PE, Hsi ED, Nickoloff BJ.In situ polymerase chain reaction-based localization studies support role of human herpesvirus-8 as the cause of two AIDS-related neoplasms:
Kaposi's sarcoma and body cavity lymphoma.J Clin Invest1997, 99:2971±2978.
14. Li JJ, Huang YQ, Cockerell CJ, Friedman-Kien AE.Localization of human herpes-like virus type 8 in vascular endothelial cells and perivascular spindle-shaped cells of Kaposi's sarcoma lesions by in situ hybridization.Am J Pathol1996,148:1741±1748.
15. Staskus KA, Zhong W, Gebhard K, et al. Kaposi's sarcoma- associated herpesvirus gene expression in endothelial (spindle) tumor cells.J Virol1997,71:715±719.
16. Offermann MK. Consideration of host-viral interactions in the pathogenesis of Kaposi's sarcoma. J Acquired Immune De®c Syndr1999,21:S58±S65.
17. Blasig C, Zietz C, Haar B,et al.Monocytes in Kaposi's sarcoma lesions are productively infected by human herpesvirus 8.J Virol 1997,71:7963±7968.
18. Sun R, Lin SF, Staskus K, et al. Kinetics of Kaposi's sarcoma- associated herpesvirus gene expression.J Virol1999,73:2232±
2242.
19. Sun R, Lin SF, Gradoville L, Yuan Y, Zhu F, Miller G.A viral gene that activates lytic cycle expression of Kaposi's sarcoma- associated herpesvirus. Proc Natl Acad Sci U S A 1998, 95:
10866±10871.
20. Lukac DM, Renne R, Kirshner JR, Ganem D. Reactivation of Kaposi's sarcoma-associated herpesvirus infection from latency by expression of the ORF 50 transactivator, a homolog of the EBV R protein.Virology1998,252:304±312.
21. Miller G, Heston L, Grogan E, et al.Selective switch between latency and lytic replication of Kaposi's sarcoma herpesvirus and Epstein±Barr virus in dually infected body cavity lymphoma cells.J Virol1997,71:314±324.
22. Yu Y, Black JB, Goldsmith CS, Browning PJ, Bhalla K, Offermann MK. Induction of human herpesvirus-8 DNA replication and transcription by butyrate and TPA in BCBL-1 cells.J Gen Virol 1999,80:83±90.
23. Bowden CL.New concepts in mood stabilization: evidence for the effectiveness of valproate and lamotrigine. Neuropsycho- pharmacology1998,19:194±199.
24. Loscher W. Valproate: a reappraisal of its pharmacodynamic properties and mechanisms of action. Prog Neurobiol 1999, 58:31±59.
25. Vettese-Dadey M, Grant PA, Hebbes TR, Crane-Robinson C, Allis CD, Workman JL.Acetylation of histone H4 plays a primary role in enhancing transcription factor binding to nucleosomal DNA in vitro.EMBO J1996,15:2508±2518.
26. Cohen BA. Neurologic complications of HIV infection. Prim Care1997,24:575±595.
27. Khouzam HR, Donnelly NJ, Ibrahim NF.Psychiatric morbidity in HIV patients.Can J Psychiatry1998,43:51±56.
28. Witvrouw M, Schmit JC, Van Remoortel B, et al. Cell type- dependent effect of sodium valproate on human immunode®- ciency virus type 1 replicationin vitro. AIDS Res Hum Retro- viruses1997,13:187±192.
29. Moog C, Kuntz-Simon G, Caussin-Schwemling C, Obert G.
Sodium valproate, an anticonvulsant drug, stimulates human immunode®ciency virus type 1 replication independently of glutathione levels.J Gen Virol1996,77:1993±1999.
30. Akula SK, Rege AB, Dreisbach AW, Dejace PM, Lertora JJ.
Valproic acid increases cerebrospinal ¯uid zidovudine levels in a patient with AIDS.Am J Med Sci1997,313:244±246.
31. Aoki Y, Jaffe ES, Chang Y,et al.Angiogenesis and hematopoiesis induced by Kaposi's sarcoma-associated herpesvirus-encoded interleukin-6.Blood1999,93:4034±4043.
32. Staskus KA, Sun R, Miller G, et al.Cellular tropism and viral interleukin-6 expression distinguish human herpesvirus 8 invol- vement in Kaposi's sarcoma, primary effusion lymphoma, and
multicentric Castleman's disease.J Virol1999,73:4181±4187.
33. Parravinci C, Corbellino M, Paulli M,et al.Expression of a virus- derived cytokine, KSHV vIL-6, in HIV-seronegative Castleman's disease.Am J Pathol1997,151:1517±1522.
34. Burger R, Neipel F, Fleckenstein B,et al.Human herpesvirus type 8 interleukin-6 homologue is functionally active on human myeloma cells.Blood1998,91:1858±1863.
35. Moore PS, Boshoff C, Weiss RA, Chang Y.Molecular mimicry of human cytokine and cytokine response pathway genes by KSHV.
Science1996,274:1739±1744.
36. Molden J, Chang Y, You Y, Moore PS, Goldsmith MA.A Kaposi's sarcoma-associated herpesvirus-encoded cytokine homolog (vIL- 6) activates signaling through the shared gp130 receptor sub- unit.J Biol Chem1997,272:19625±19631.
37. Bais C, Santomasso B, Coso O,et al.G-protein-coupled receptor of Kaposi's sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator.Nature1998,391:86±89.
38. Boshoff C.Kaposi's sarcoma. Coupling herpesvirus to angiogen- esis.Nature1998,391:24±25.
Antiretroviral drug removal by haemodialysis Triple therapy is the current standard of therapy for HIV-infected patients. With the availability of new antiretroviral agents, it was soon recognized that in some patients quadruple therapy was necessary to reduce and maintain plasma HIV-RNA levels below the level of detection [1]. Because the majority of these agents underwent relatively rapid development pro- cesses, little is known about the pharmacokinetic considerations in special populations. Consequently, there is a lack of recommendations on the dosing for most antiretroviral drugs in patients with renal impair- ment treated by haemodialysis [2]. The results obtained in one patient treated with a quadruple antiretroviral therapy, including stavudine, nevirapine, ritonavir and saquinavir, are reported.
An HIV-positive patient on antiretroviral therapy was treated by haemodialysis for end-stage renal failure related to Alport syndrome. The daily doses were as follows: stavudine 15 mg a day (evening dose); nevir- apine 200 mg twice a day; saquinavir 600 mg twice a day; and ritonavir 200 mg twice a day. Antiretroviral drug concentrations were monitored before and after dialysis to check for drug accumulation. On the day of the study, the 27th day of therapy, ritonavir, saquinavir and nevirapine were administered 11 h before starting haemodialysis, and stavudine was administered the day before (once daily administration) 19 h before starting haemodialysis. Haemodialysis was performed for 4 h, using a double-needle access to an arteriovenous ®stula, polysulfone high permeability membrane dialyser (BLS 624, Belco) and a single-pass dialysate delivery system, with a constant dialysate (bicarbonate ¯uid) ¯ow rate of 500 ml/min: the blood ¯ow entering the dialyser was approximately 250 ml/min and the ultra®ltration rate was 1350 ml/h. Venous blood samples were drawn before drug intake, 3 h after drug administration and hourly during dialysis. Arterial and venous blood entering and leaving the dialyser was sampled simulta-
neously in mid-dialysis. All antiretroviral drug concen- trations were assayed by high-performance liquid chromatography according to a method previously described [3]. The dialyser extraction ratio (E) was calculated fromCaand Cv, which are, respectively, the plasma concentrations entering (`arterial') and leaving (`venous') the dialyser: E(CaÿCv)/Ca.
The 4 h haemodialysis clearance (ClHD) was calculated using the following formula [4]:
ClHDQpCaÿ(QpÿQUF)Cv Ca
where Qp is the plasma ¯ow entering the dialyser derived from the blood ¯ow, Qb, and the haematocrit Ht: QpQb(1ÿ Ht); QUF is the ultra®ltration rate displayed on the dialyser.
Drug concentrations at the beginning and the end of dialysis, and parameters measured during dialysis are listed in Table 1. Unfortunately, the ritonavir dose was too low to provide plasma concentrations above the limit of quanti®cation of the assay. The measured dialysis parameters indicate that stavudine and nevira- pine but not saquinavir are removed during a 4 h haemodialysis session.
Clinical success in non-nucleoside reverse transcriptase inhibitor and protease inhibitor treatment requires the maintenance of the plasma trough level in a pharmaco- logically effective range, well above the in-vitro viral IC90 [5,6]. Interestingly, the plasma concentrations reported above at the end of haemodialysis were below the reported trough concentration range.
The accumulation of drugs in patients with renal dysfunction and haemodialysis removal are dependent on their respective pharmacokinetic properties. Stavu-
dine is eliminated mainly unchanged through the kidney and accumulates in patients with renal impair- ment, which leads to dosing-adjustment as a function of creatinine clearance [7]. Saquinavir and nevirapine are biotransformed by the liver, so no drug accumula- tion is anticipated in patients with end-stage kidney disease [8]. The observed dialysis clearance and extrac- tion ratios of stavudine, nevirapine and saquinavir are in agreement with their respective pharmacokinetic properties: volume of distribution (approximately 1 l/
kg for stavudine and probably for nevirapine, although not reported, and 10 l/kg for saquinavir), and plasma protein binding was low for stavudine (,5%) and nevirapine (660%) but high for saquinavir, mainly on alpha1-acid-glycoprotein ranging around 98±99% [8].
Consequently, the haemodialysis index [9] de®ned as the ratio of unbound fraction to the volume of distribution were 95 and 40 for stavudine and nevir- apine, respectively, which are .20, and in the range of drugs removed by haemodialysis, and 0.1 for saquinavir, which is in the range of poorly removed drugs. It should be pointed out that in this patient the half-life of the three drugs studied during dialysis are shorter than that reported in patients with renal disease or patients with no organ failure [7,8].
In conclusion, the poor removal of saquinavir during a haemodialysis session indicates that a supplemental dose of saquinavir need not be routinely given to patients after haemodialysis. In contrast, the dosing of stavudine and nevirapine is warranted at the end of a haemodia- lysis session. This study clearly demonstrates that the disposition of each drug during a haemodialysis session should be considered to maintain effective concentra- tions throughout dialysis and interdialysis sessions.
However, patient to patient variability could occur, therefore further data should be collected before de®ni- tive dosage recommendations can be proposed.
Anne-Marie Tabureta, Laurence Gerardb, Mayeule Legrandc, Guy Aymardc and Jean-Michel Berthelotd, Departments of aClinical Pharmacy, Hospital BiceÃtre,
bImmunology and Hematology, Hospital Saint-Louis,
cPharmacology, Hospital PitieÂ-SalpeÂtrieÁre, Assistance Publique±HoÃpitaux de Paris and AURA, Hemodialysis, Paris France.
Received: 16 December 1999; accepted: 22 December 1999.
References
1. Kirk O, Katzenstein TL, Gerstoft J, Mathiesen L, Nielsen H, Pedersen C, Lundgren JD. Combination therapy containing ritonavir plus saquinavir has superior short term antiretroviral ef®cacy: a randomized trial.AIDS1999,13:F9±F16.
2. Hilts AE, Fish D.Drug watch: antiretroviral dosing in patients with organ dysfunction. The AIDS Reader 1998, Nov/Dec:
179±184.
3. Aymard G, Legrand M, Trichereau N, Diquet B.Simultaneous determination of twelve antiretroviral agents in human plasma sample using reverse-phase high performance liquid chromato- graphy.J Chromatogr B2000, (in press).
4. Gotch FA.Haemodialysis: technical and kinetic considerations.
In: The kidney. Brenner BM, Rector FC (editors). Philadelphia:
WB Saunders; 1976. pp. 1672±1704.
5. Delfraissy JF. Prise en charge theÂrapeutique des personnes infecteÂes par le VIH: recommandation du groupe d'experts.Paris:
MeÂdecine-Sciences, Flammarion; 1999.
6. Havlir D, Cheeseman SH, McLaughlin M, Murphy R, Erice A, Spector SA,et al.High dose nevirapine: safety, pharmacokinetics and antiviral effect in patients with human immunode®ciency virus infection.J Infect Dis1995,171:537±545.
7. Lea AP, Faulds D.Stavudine: a review of its pharmacodynamic and pharmacokinetic properties and clinical potential in HIV infection.Drugs1996,51:846±864.
8. Barry M, Mulcahy F, Merry C, Gibbons S, Back D.Pharmacoki- netics and potential interactions amongst antiretroviral agents used to treat patients with HIV infection. Clin Pharmacokinet 1999,36:289±304.
9. Gwilt PR, Perrier D. Plasma protein binding and distribution characteristics of drugs as indices of their hemodialyzability.
Clin Pharmacol Ther1978,24:154±161.
Protease inhibitor therapy-associated lipodystrophy, hypertriglyceridaemia and diabetes mellitus Intensive combination therapy including a protease
inhibitor is considered a standard of care in HIV-1 infection. Recently, an association has been noted
between protease inhibitors and a syndrome consisting of peripheral lipodystrophy, hypertriglyceridaemia and diabetes mellitus [1±3]. Protease inhibitor-induced
Table 1. Concentrations and dialysis pharmacokinetic parameters of three antiretroviral drugs in an HIV-infected patient treated with haemodialysis.
Stavudine Nevirapine Saquinavir
Concentrations:
· Before dialysis 37ìg/l 2.8 mg/l 0.076 mg/l
· End dialysis 15ìg/l 1.4 mg/l 0.035 mg/l
Parameters at mid-dialysis:
· Coef®cient of extraction (%) 100 43 24
· Clearance (ml/min) 137 59 33
Half-life during dialysis (h) 3.1 4.0 3.6
