HLA-B, HLA-C and KIR improve the predictive value of IFNL3 for Hepatitis C spontaneous clearance

IFNL3 is the strongest predictor of spontaneous resolution (SR) of hepatitis C virus (HCV), however, consideration of IFNL3 genotype alone is of limited clinical value for the prediction of SR or chronic HCV infection. The objective of this study was to analyze the impact of HLA-B, HLA-C and KIRs on SR, as well as their additive effects on the predictive value of the IFNL3 genotype. We conducted a retrospective study of HIV patients that included both SR and chronic HCV patients. In our study, 61.6% of patients with IFNL3 CC achieved SR, and 81.5% with non-CC genotypes did not achieve SR. HLA-B*44, HLA-C*12, and KIR3DS1 were identified as predictive factors for SR, with percentages of 77.4%, 85.7% and 86.2%, respectively, for patients who did not experience SR. The presence of at least one of these three markers, defined as a genetically unfavorable profile (GUP), combined with the IFNL3 non-CC genotype showed a value of 100% for non-SR. The absence of the three markers, defined as a genetically favorable profile (GFP), in addition to the IFNL3 CC genotype showed a percentage of 74.1% for SR. The combination of these markers in addition to the IFNL3 genotype improves the predictive value of IFNL3 for SR of acute HCV infection in HIV patients, which would be clinically valuable.


Results
Baseline patient characteristics. Fifty-seven HIV-infected patients who experienced SR and 81 patients who developed chronic HCV infections were included in this retrospective study. All patients included were Caucasian. HCV genotype was not available for 89.7% of patients who achieved SR. The baseline characteristics of these patients are shown in Table 1. Due to the retrospective nature of the study, we were unable to ascertain the exact age when SR was reached or exclude the possibility that some individuals were exposed multiple times. The distribution of the HLA-B, HLA-C and KIR genotypes is shown in Supplementary Tables S1, S2, and S3, respectively.
Combination of HLA-B*44, HLA-C*12, KIR3DS1 and IFNL3 in the prediction of SR. The IFNL3 CC and non-CC genotypes, the presence/absence of the KIR3DS1 genotype, the presence/absence of the HLA-B*44 allele and the presence/absence of the HLA-C*12 allele were analyzed with regard to their impact on SR (Supplementary Table S4). The combination of the three favorable factors (absence of HLA-B*44, HLA-C*12 and KIR3DS1 genes) was defined as a genetically favorable profile for SR (GFP), whereas the presence of one of the three unfavorable factors (HLA-B*44, HLA-C*12 or KIR3DS1) was defined as a genetically unfavorable profile (GUP) for SR. Among those patients with a GFP, 29

Discussion
The association of the IFNL3 genotype with SR has been widely described and supports the importance of interferon lambda and innate immunity in the outcome of HCV infection 3-7 . The first evidence was reported by Thomas et al., who showed that patients with the IFNL3 CC genotype had a higher rate of SR (53%) than those with the T allele (28%) 3 [9][10][11] . Specific HLAs can increase or decrease the elimination of HCV-infected cells [12][13][14] . In the context of SR, two HLA-B alleles, HLA-B*27 and HLA-B*57, have been previously associated with self-limiting infections. First, two studies associated HLA-B*27 with SR 17,18 , although the association was limited only to patients with an HCV genotype 1, because no association was observed with other genotypes 19 . Second, HLA-B*57 was also associated with SR in a several studies 20,21 , though this association was not found   in another study 22 . HLA-C alleles have also been shown to affect the outcome of HCV infection. Two studies found HLA-C*05 to be associated with the viral persistence of HCV 20,23 . In our study, the presence of HLA-B*44 and HLA-C*12 were independent factors associated with HCV chronicity, showing higher frequencies among patients who developed chronic HCV infections (HLA-B*44: 77.4%; HLA-C*12: 85.7%). With respect to KIR genes and their associations with HCV chronicity, Dring et al. showed that the frequency of the KIR2DS3 receptor was significantly increased in patients who had chronic HCV infections 15 . Similarly, a study by De Re et al. also found that KIR2DS3 was the principal gene related to HCV viral persistence 16 . A study of risk for mother-to-child transmission of HCV found that KIR3DS1 was associated with development of chronic HCV infection 24 . Specifically, Ruiz-Extremera et al. found that the children of mothers with KIR3DS1 had a higher risk of HCV chronicity than the children of mothers without this receptor (75% and 19%, respectively) 24 . In our study, the presence of the KIR3DS1 receptor was also linked with chronic HCV infection, showing a higher frequency in patients who had chronic HCV infections (86.2%).
The combination of several immunological markers for the prediction of SR has been little studied 18,25-28 . Mangia et al. observed that the IFNL3 CC genotype in combination with DBQ1*0301 increased the accuracy of SR prediction from 63% to 69% 25 . Likewise, Duggal et al. found that the combination of DQB1*03:01 with the IFNL3 genotype could increase the positive predictive value for SR from 45% to 60% 26 . In contrast, Huang et al. found that two HLA alleles (A*02 and DRB1*1) together with IFNL3 did not significantly improve the prediction of the probability of SR (57.0% to 57.8%) 27 . In our study, we found that the combination of three markers, HLA-B*44, HLA-C*12 and KIR3DS1, improved the predictive value of IFNL3 for SR. In particular, no patient with the IFNL3 non-CC genotype (0%) achieved SR when they also had a GUP. Consequently, the consideration of these four genetic markers identified a large population of patients with low probabilities of achieving SR. Moreover, the percentage of SR in patients with an IFNL3 CC genotype significantly increased when a GFP is also considered (61% to 74.1%). In contrast, the likelihood of SR among patients with an IFNL3 CC genotype significantly dropped from 61% to 30.4% in combination with a GUP. These findings could significantly improve the predictive accuracy of IFNL3 for determining the probability of achieving SR. In patients with acute HCV infection, a dilemma exists regarding whether to delay or initiate HCV treatment while waiting to see if SR occurs. It is beneficial to delay treatment based on the likelihood of SR as it reduces unnecessary therapy in closely monitored populations 28 . Current HCV guidelines identify IFNL3 as an immunological marker for clinical decision making 8 . Nonetheless, by following this association, our study showed that almost 40% of patients were mismatched according to their IFNL3 CC genotype. Our results suggest that determining HLA-B*44, HLA-C*12 and KIR3DS1 would improve the predictive value of IFNL3 for clinical decision making in this setting. The real impact of the combination of these markers would be to enhance the prediction of SR in patients who have all of the unfavorable markers (IFNL3 non-CC genotype and a GUP), because the likelihood that such patients will experience SR is low and, consequently, they could benefit from immediate treatment implementation. In addition, in these patients, taking time to wait for SR would be unnecessary, thus significantly reducing the risk of HCV transmission. In contrast, in patients who have favorable markers (IFNL3 CC genotype and a GFP), HCV therapy could be deferred in the short-term because of the high probability that they will experience SR. Nevertheless, before this combination of markers can be implemented in clinical practice, the confirmation of our results in a validation cohort is mandatory.
Our study has several limitations that should be noted. First, this study included only HIV/HCV co-infected patients, and therefore, these results cannot be extrapolated to HCV mono-infected individuals. Second, HLA-A and all HLA-II genotyping was not available in this study; therefore, we do not know the role of all classic HLA class I and HLA class II molecules. Third, this study included a relatively small number of patients. Thus, we may not have enough statistical power to detect associations with other HLA alleles and KIR genotypes. Finally, due to the retrospective nature of the study, age at HCV infection, multiple exposure risk and viral genotype were unknown. Consequently, these factors, which have been associated with SR in other studies 29,30 , were not analyzed in this study.
In conclusion, our study identifies immunological factors, including the HLA-B*44 and HLA-C*12 alleles and the KIR3DS1 genotype, as independent factors associated with HCV chronicity in HIV patients. Furthermore, these factors improved the predictive capacity of IFNL3 for the outcome of acute HCV infection. A validation cohort is needed to confirm these results.

Patients. HIV patients from the Infectious Diseases Unit at the Hospital Reina Sofía de Córdoba (Spain)
were included in this retrospective study between 2013 and 2016. The patients were included in two groups. The first group corresponded to SR patients and all those who fulfilled the following criteria were included: i) positive HCV antibodies confirmed by recombinant immunoblot assay and negative HCV RNA without the patient ever having received anti-HCV therapy, or ii) HCV RNA detected only in the first 6 months after HCV seroconversion. Patients who were active intravenous drug users (IDU) were excluded from the study. The second group comprised patients with chronic HCV infection, and the criteria for inclusion were: i) persistent detection of HCV RNA for more than 6 months, and ii) HCV treatment-inexperienced patients.
Variable collection and definition. The variables collected included the genotypes of IFNL3, KIR, HLA-B, and HLA-C; age; gender; baseline CD4 + cell count (cells/mL); HIV viral load; antiretroviral therapy (ART) usage; acquired immunodeficiency syndrome (AIDS) condition; and HCV genotype.
IFNL3 SNP rs129679860 was genotyped using a custom TAQMAN assay (Applied Biosystems, Foster City, California, USA), according to the manufacturer's instructions. The IFNL3 genotype was defined as CC or non-CC (TT/CT). KIR genotyping was performed using sequence-specific primers to detect the presence of 16 different KIR genes as described previously by Gomez-Lozano et al. 31 . This method provided a high degree of resolution since each primer pair identifies two linked, cis-located polymorphic sites (Invitrogen, Thermo Fisher Scientific, Massachusetts, USA). Only KIR genes with frequencies greater than 5% were included in the analysis. HLA-B genotyping was performed with the INNO-LIPA HLA-B Multiplex kit (Innogenetics N.V., Ghent, Belgium), using HLA-B multiplex primers for amplification of the second and fourth exon of the HLA-B locus. This technique was based on the PCR-SSO reverse method. HLA-C genotyping was performed according to the method previously described by Turner et al. for amplification of the second and third exon of the HLA-C locus 32 . Only HLA-B and HLA-C alleles with frequencies greater than 5% were included in the analysis. Statistical Analysis. Categorical variables are expressed as numbers of cases and percentages. Frequencies were compared using the χ 2 or Fisher's exact tests to analyze associations for SR or chronic HCV. Significance was set at a p value of less than 0.05. A logistic regression model for predicting SR was performed and tested by the bootstrap method (1,000 replicates). The area under the receiver operating characteristic (AUROC) curve was used to evaluate sensitivity and specificity for IFNL3 and additional genetic markers, in accordance with the explanation and elaboration of studies of diagnostic accuracy described by Bossuyt et al. 33 . These analyses were performed using the SPSS statistical software package, version 18.0 (IBM Corporation, Somers, NY, USA) and GraphPad Prism version 6 (Mac OS X version; GraphPad Software, San Diego, CA, USA).
Ethical statement. This study was designed and performed according to the Helsinki Declaration. All patients signed an informed consent form that gave permission for the storage and processing of material from routine diagnostic processes at the Biobanco del Hospital Universitario Reina Sofía de Córdoba (ISCIII reference: B.0000419), which is integrated into the Biobanco del Sistema Sanitario Público de Andalucía. The CEIC (Clinical Trial and Ethical Committee) of the Hospital Universitario Reina Sofía de Córdoba approved the study protocol. Data availability. All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).