Antibody titers in all patients and two additional control samples were evaluated against six different recombinant HCV antigens, essentially derived from the whole proteome of HCV. A heat map, employing log10
-transformed antibody titers, was used to display the differing antibody responses toward the six antigens in individual samples from these subgroups (). As shown by the heatmap, LIPS profiling against these 6 HCV antigens clearly distinguished the 29 HCV-infected sera from the 2 uninfected control sera. The most useful antibody response was directed against the HCV core, in which all but one of the 29 HCV-HIV coinfected samples was positive. The second most useful antibody response was against NS3 (). The other 4 HCV proteins, E1, E2, NS4 and NS5 showed variable immunoreactivity with HIV-HCV-coinfected sera (). Of interest, one NR patient was completely negative for anti-core, anti-E1 and anti-E2 antibodies, but showed strong immunoreactivity to three other non-structural HCV proteins (). Antibody titers against the six HCV antigens correlated poorly with each other (rs
>0.60) suggesting marked heterogeneity in humoral responses (supplementary Table I
Figure 1 Baseline antibody titers to HCV antigens and p24. A. Heat map representation of patient antibody profiles to the 6 HCV antigens in pretreatment samples from HIV-HCV coinfected patients with differing response to HCV therapy. Antibodies titers were determined (more ...)
Antibody titers against these HCV antigens in pre-treatment sera showed no significant differences between the NR, RELAP and SVR groups (see supplementary figure 1
). For example, anti-core antibody mean antibody titer in the NR, RELAP and SVR subgroups was 497,200 LU (95% CI, 279,800-714,700); 483,400 LU (95% CI, 292,500-674,200); and 545,600 LU (95% CI, 299,400-791,900), respectively, and a Mann Whitney U
test showed no statistically significant differences (P
>0.65). These results suggest that anti-HCV antibody titers in the pretreatment HCV-HIV coinfected samples have no obvious predictive value for response to treatment.
Due to the known impact of HIV-HCV infection on HCV treatment, antibody responses to several HIV proteins were also evaluated in the 3 groups. As shown in , all 29 pre-treatment samples were robustly seropositive for anti-p24 HIV GAG antibodies as compared to the 2 uninfected controls and previously determined cut-offs [10
]. The mean anti-p24 antibody titers in the NR, RELAP and SVR were 2.77 × 106
, 2.17 × 106
and 1.71 × 106
LU, respectively. The anti-p24 antibody titer in the RELAP sub-group did not significantly differ from either the NR or SVR subgroup (Mann Whitney U
>0.47), the NR and SVR subgroups showed statistically significant differences in anti-p24 antibody titers (P
=0.023). The anti-p24 antibody titers did not correlate (P
>0.05) with HIV or HCV viral load, genotype or CD4 counts (data not shown). However, the anti-p24 antibody titers paralleled the cumulative group scores for the IFN-associated gene expression signature previously reported by Lempicki et al. [9
] on the same patients. Antibodies to TAT protein of HIV did not differ significantly (P
>0.26) between the NR, RELAP and SVR subgroups (data not shown). Since statistically higher anti-p24 antibody titers were detected in the NR versus the SVR group and correlated with failure to HCV therapy, a cut-off based on 2.2 million LU was determined to optimally separate these two groups. Using this approach, 9 of the 11 NR were above cut-off compared to only 2 of the 9 SVR (). Based on this analysis, the p24 antibody titers provide 82% positive predictive value in identifying the NR that would fail therapy. Although p24 antibody titers only had value in distinguishing NR from SVR, but not for the RELAP group, there is little practical predictive value for this test in HIV/HIV-coinfected individuals.
Antibody titers to the panel of HCV proteins were also evaluated in the pre and post-treatment for their value in monitoring HCV treatment. Wilcoxon sign ranked test revealed that four of the six HCV proteins (core, E1, E2 and NS4) showed statistically significant (P<0.05) decreases in antibody titer between the pre- and post-treatment samples (). In contrast, antibody responses to NS3 and NS5A antigens did not significantly change from the before and after treatment (P>0.44). Substratification by treatment outcome revealed that the SVR group showed the most consistent and largest decrease (P=0.02) in antibody titers to the 3 most informative antigens (core, E1, NS4) following treatment (). In contrast, the NR and RELAP groups had relatively stable antibody titers against these 3 antigens between the pre- and post-treatment (P=0.70 and P=0.43, respectively) (). Anti-p24 HIV and anti-BRLF2 EBV antibody titers also did not change with anti-HCV therapy (data not shown). There was heterogeneity in response to the different HCV antigens in the SVR, in which some patients showed the largest decrease in anti-HCV core antibodies, while other patients showed more pronounced decreases to ENV1 and NS4. Since decreasing antibodies to these 3 HCV antigens was a common feature of SVR compared to the NR and RELAP group, the relative decrease in antibody between the pre- and post samples was the most useful approach for distinguishing SVR from NR and REALP. Using an antibody titer decrease between the pre- and post-samples of greater than 1.5-fold as a marker of HCV therapy success, 6 of the 9 SVR were positive verses only 1 of the 9 RELAP, and none of the NR. Overall this LIPS test measuring differences in antibody titers to these 3 HCV from the pre and post-treatment samples showed 86% positive predictive value in identifying response to therapy.
Figure 2 Informative antibody titers pre- and post- HCV treatment in HIV-coinfected NR, RELAP and SVR subgroups. Anti-core, E1 and NS4 antibodies levels at baseline and post-treatment in individual patients from the NR, RELAP and SVR groups. The thick short solid (more ...)