1. GBV-C Viremia in Subjects Identified During Primary HIV Infection (PHI) UF Bredeek¹, T Kaiser², HL Tillman², S. Donfied³, G Hess 4, J Pitt 5, ES Daar 5 ¹West Los Angeles VA Medical Center, California, USA; ²Medizinische Hochschule, Hannover, Germany; ³Rho Inc., Chapel Hill, North Carolina, USA; 4 Roche Diagnostics, Mannheim, Germany; 5 Harbor-UCLA Research Education Institute, Los Angeles, California, USA. Background: GBV-C has been shown to inhibit HIV replication in vitro, and in some studies persistent viremia has been associated with delayed HIV clinical progression. Little is known regarding the prevalence and effect of GBV-C in subjects identified at time of PHI. Methods: Between 1997 and 2002 a cohort of 63 men and 2 women in Los Angeles, California with a mean age of 34 years were identified with PHI after sexual exposures. All subjects had serial HIV RNA and CD4 measures with quantitative GBV-C RNA (RT-PCR, lower limits of detection =3.7 log 10 c/ml) performed on stored specimens at baseline and in 63 at the most recent follow-up visit, at a median of 618 (range 13-1933) days. Results: GBV-C viremia was detected at baseline in 17 (27%) with a mean (+SD) viral load of 6.86 (+1.0) log 10 c/ml, with follow-up measure becoming undetectable in 1 and detectable in an additional 10, with mean GBV-C RNA in those detectable at follow-up being 7.16 (+1.0) log 10 c/ml. In fact, those GBV-C viremic both at baseline and follow up (n=16) had a mean increase of 1.17 (+1.04) log 10 c/ml (p=0.008). Being GBV-C detectable versus undetectable at baseline was not associated with differences in baseline mean CD4 cells (561+ 273 vs. 521+ 276 cells/uL) or HIV RNA (4.7 + 1.3 vs. 4.6 + 1.5 log 10 c/ml). 24 subjects (39%) had E2 antibodies at time of HIV seroconversion. Conclusions: GBV-C RNA is frequently detectable at the time of seroconversion in a Los Angeles cohort of PHI patients. A substantial number developed detectable GBV-C during follow-up. Only a minority of patients had no contact with GBV-C at the end of the observation period. In subjects persistently viremic there was a significant increase in GBV-C RNA at follow- up. Further study of the prevalence, incidence and change in GBV-C viremia in subjects identified with PHI will enhance our understanding of how co- infection influences the natural history of HIV disease Abstract Methods Conclusions Time- point analysis: Baseline [time of PHI] GBV-C E2 antibody positive: 24/61(39.4%) GBV-C RNA positive: 17/63 (27%) GBV-C plasma RNA: Mean (+STD) = 6.86 (+1.0) log 10 c/ml GBV-C RNA and E2 antibody negative: 21/61 (34.4%) Follow-up [median 618 (range 13-1933) days from PHI] GBV-C E2 antibody positive: 28/63 (44.4%) GBV-C RNA positive: 26/63 (41.3%) GBV-C plasma RNA: Mean (+STD) = 7.16 (+1.0) log 10 c/ml GBV-C RNA and E2 antibody negative: 10/63 (15.9%) Change of GBV-C status from PHI to follow-up Acquisition of GBV-C E2 antibodies: 5 subjects Loss of GBV-C E2 antibodies: 2 subjects Became GBV-C RNA positive: 10 subjects Loss of detectable GBV-C RNA: 1 subject Changes in HIV and GBV-C markers for entire cohort Mean CD4 count at baseline to follow-up: 529 + 256 to 622 + 242 cell/ul Mean HIV RNA at baseline to follow-up: 4.68 + 1.43 to 2.19 + 1.97 log 10 c/ml Mean GBV-C RNA at baseline to follow-up: 6.86 + 1.0 to 7.16 + 1.0 log 10 c/ml Mean GBV-C RNA if persistent viremic: 5.73 + 1.04 to 6.9 + 0.9 log 10 c/ml (p=0.008) Statistical analysis GBV-C RNA is not correlated with HIV RNA or CD4 count at the time of PHI or at follow-up visit In a linear regression model GBV-C RNA is not a predictor of HIV RNA or CD4 count at time of PHI or at follow-up Mean (STD)Neg-NegNeg-PosPos-NegPos-Pos N3610116 CD4 1542 (296)445 (197)240 (-)581(195) CD4 2603 (266)591 (257)558 (-)690 (173) HIV RNA 14.35 (1.69)5.36 (1.17)5.11 (-)4.87 (0.78) HIV RNA 21.97 (2.05)1.98 (2.10)2.63 (-)2.79 (1.77) GBV-C RNA 1n/a 7.85 (-)5.73 (1.04) GBV-C RNA 2n/a6.59 (0.36)n/a6.90 (0.90) GBV-C RNA is frequently detected in a cohort of subjects identified with PHI in Los Angeles, California. More than 25% of the subjects have detectable GBV-C viremia at the time of primary HIV infection and a substantial number develop GBV-C viremia during follow-up. In persistently viremic subjects there was a significant increase in GBV-C RNA at follow-up. Only a minority of subjects (15.9%) have neither antibodies nor GBV-C RNA detectable during follow up, with 39.5% being GBV-C antibody positive at the time of HIV infection. The study group is very heterogeneous with regards to exposure to HIV treatment and this may influence the analyses. Further study of the prevalence, incidence and change in GBV-C viremia in subjects identified with PHI, as well as the relationship to HIV parameters will enhance our understanding of how GBV-C influences the natural history of HIV disease. Background RNA neg-negRNA neg-posRNA pos-negRNA pos-pos E2 Ab neg-neg98015 E2 Ab neg-pos4001 E2 Ab pos-neg0200 E2 Ab pos-pos22000 E2 Ab at PHI not done 1010 N3610116 Statistical evaluation: In a Linear regression model GBV-C subgroup (neg-neg, neg-pos, pos-neg, pos- pos) was not a predictor for HIV RNA or CD4 count In a t-test analysis no significant difference in CD4 count was seen in each of the 4 groups The increase of GBV-C RNA in the persistent viremic subgroup (pos-pos) was statistically significant with p=0.008 Treatment Grouped according to GBV-C RNA status at follow-up visit GBV-C pos GBV-C neg Number of subjects 26 37 not on treatment, first sample (%) 24 (92) 31 (84) not on treatment, second sample (%) 14 (54) 17 (46) No treatment or less than 3 months (%) 10 (39) 15 (41) Summary In this cohort of subjects with PHI, GBV-C viremia was detected at baseline in 17 (27%) with a mean (+SD) viral load= 6.86 (+1.0) log 10 c/ml. Follow-up measurements become undetectable in 1 of these subjects and 10 that were negative at baseline became viremic at follow-up, with mean GBV-C RNA in those detectable at follow-up being 7.16 (+1.0) log 10 c/ml. Those GBV-C viremic both at baseline and follow up (n=16) had a mean increase of 1.17 (+1.04) log 10 c/ml (p=0.008). Being GBV-C detectable versus undetectable at baseline was not associated with differences in baseline mean CD4 cells (561 + 273 vs. 521 + 276 cells/uL) or HIV RNA (4.7 + 1.3 vs. 4.6 + 1.5 log10 c/ml). Only a minority of subjects never had contact GBV-C, being both RNA and antibody negative (15.9%), with 39.4% of subjects having detectable GBV-C antibodies at the time of HIV seroconversion. References 1. Heringlake S, Ockenga J, Tillmann H, et al. 1998. GB virus/hepatitis G virus infection: a favorable prognostic factor in human immunodeficiency virus infected patients? J Infect Dis 177: 1723-6 2. Xiang J, Wunschmann S, Diekema D, Klinzman D, Patrick K, et al. 2001. Effect of coinfection with GB virus C on survival among patients with HIV infection. N Engl J Med 345: 707-14 3. Tillmann H, Heiken H, Knapik-Botor A, Heringlake S, Ockenga J, et al. 2001. Infection with GB virus C and reduced mortality among HIV-infected patients. N Engl J Med 345: 715-24 4. Aboulker J, Chams-Harvey V, Flandre P, Delarue S, Leonardo S, et al. 2003. Slower Disease Progression and Greater CD4 Response to Antiretroviral Treatment in HIV Patients Co-infected with GBV-C. Presented at 10th Conference on Retroviruses and Opportunistic Infections, Boston 5. Rodriguez B, Wooley I, Lederman M, Zdunek D, Hess G, Valdez H. 2003. Effect of GB virus C coinfection on response to antiretroviral treatment in human immunodeficiency virus-infected patients. J Infect Dis 187: 504-7 6. Xiang J, George SL, Wuenschmann S, Klinzman D, Stapelton J GBV-C infection inhibits CCR5 and CXCR4 HIV strains and alters chemokine and cytokine gene expression in PBMC cultures. Presented at 10th Conference on Retroviruses and Opportunistic Infections (CROI), Boston, MA 7. Björkman P, Flamholc L, Nauclér A, Wallmark E, Widell A. 2003. GB Virus C Viremia During the Natural Course of HIV-1 Infection: Viremia at Diagnosis Does Not Predict Mortality. Presented at 10th CROI, Boston, MA 8. Williams C, Klinzman D, Yamashita T, Xiang J, Polgreen P, et al. 2003. Persistent GBV-C virus Type C (GBV-C) Infection is Associated with Decreased Risk of Death in HIV-seroconvertors in the Multicenter AIDS Cohort Study (MACS). Presented at 10th CROI, Boston, MA 9. Grant R, Kropp J, Liegler T, Osmond D, Jacobson M GB Virus C Viral Load decreases during HIV-1 infection. Presented at 10th CROI, Boston. Contact information: Fritz Bredeek, M.D., PO Box 691575, West Hollywood, CA 90069 Results GBV-C is a Flavivirus, identified in 1995. The closest related human pathogen is Hepatitis C Virus, another RNA virus. So far GBV-C has not been associated with any disease. The natural history of infection is either to persist with detectable plasma RNA, or to be cleared with seroconversion to envelope protein E2 antibodies (anti-E2). Subsequently, immunity is acquired against GBV-C. Persistent viremia rates vary in studied populations. Approximately 2% of healthy blood donors in the United States are GBV-C viremic, and in some studies up to 35% of HIV infected individuals present with detectable GBV-C RNA. While no disease has been associated with the presence of the virus several clinical studies have demonstrated that GBV-C viremia is associated with a delayed clinical HIV progression in co-infected individuals. Both a lower plasma HIV-RNA and a higher CD4 count (1-4), as well as a more favorable response to HAART (5) have been associated with this observation. In contrast, the loss of GBV-C viremia has been associated with HIV disease progression. In tissue culture GBV-C reduces HIV replication by different mechanisms including the down regulation of CCR5 on the cell surface and by increasing the expression of Chemokines such as RANTES, MIP 1-alpha, MIP 1-beta and SDF-1 (6). Recent studies suggest that the persistence of viremia and not infection alone has the strongest influence on HIV progression (7,8). It is not known how often subjects with PHI are GBV-C viremic, nor is it clear how many of these subjects have pre-existing immunity to GBV-C infection. A small study of 18 recent HIV seroconverters in San Francisco found 7 out of 18 GBV-C viremic at baseline with no incident cases during a total of 102 person years of follow-up. No association between HIV and GBV-C viral load was seen in that study (9). Table 1: Antibody and GBV-C RNA at PHI and follow-up Patients: Between 1997 and 2002 63 men and 2 women were identified within 90 days of seroconversion in Los Angeles, California. All acquired HIV through sexual exposure, and had a median age at time of HIV infection of 35 (18-52) years. The population included 50 (77%) Caucasians, 8 (12%) Hispanic, 4 (6%) African American, and 3 (5%) other. 55 (87.3%) of subjects were not taking antiretroviral medication at the time of first blood draw, with 31 (49.2%) not taking treatment at the time of follow up testing. 25 (39.7%) study subjects received either no therapy, or less than three months of antiretroviral medications during the period of observation. Methods: Frozen plasma specimens from within 90 days of HIV infection were tested for the presence of GBV-C RNA and antibodies against GBV-C envelope (E2). A follow-up specimen from 63 subjects was tested at a second time point a median of 618 (13-1933) days later. Screening for the presence of GBV-C RNA was done by nested polymerase chain reaction (PCR) with the use of the Titan One Tube RT PCR System (Roche Diagnostics, Mannheim, Germany). Lower limit of detection was 3.7 log 10 (5000) copies per milliliter. Screening for the presence of envelope E2 antibody was done by enzyme immunoassay (GBVenv uplate EIA, Roche Diagnostics, Mannheim). All subjects had serial HIV RNA and CD4 measurements, on average every 3 months, after enrollment in the cohort study. Subgroup analysis according to the GBV-C RNA status Subjects were placed into four different groups according to their GBV-C RNA status at PHI and follow-up 1. “RNA neg-neg”: subjects with no detectable GBV-C RNA at both time points n=36 2. “RNA neg-pos”: subjects with detectable viremia only at follow-up n=10 3. “RNA pos-neg”: subjects with detectable viremia only at the baseline visit n=1 4. “RNA pos-pos”: subjects with detectable GBV-C RNA at both time points n=16 Table 2: Grouping according to GBV-C RNA status at PHI and follow-up 1, measurement at baseline; 2, measurement at follow-up visit