Clearance of hepatitis C infection is associated with the early appearance of broad neutralizing antibody responses

Abstract

The contribution of humoral immune responses to spontaneous control of hepatitis C virus (HCV) infection remains unclear. We assessed neutralizing antibody (nAb) responses during acute HCV infection to determine whether infection outcome is associated with the nAb response, specifically, its timing or breadth (neutralization of multiple genotype-matched variants). A representative genotype 1 HCV pseudoparticle (HCVpp) library, consisting of 19 genetically distinct genotype 1 HCVpp that comprise the natural variability of genotype 1 E1E2 sequences, was used to assess anti-genotype 1 nAb responses during acute infection in at-risk persons followed prospectively. Neutralization of individual library HCVpp by the last viremic plasma sample obtained before clearance was compared to either 1-year post-initial viremia or clearance time-matched specimens obtained from subjects developing persistent infection. In persistently infected persons nAb responses were delayed then progressively broadened, whereas in persons who controlled viremia broader responses were detected early and contracted after clearance of viremia. Surprisingly, the breadth of anti-genotype 1 nAb responses was not dependent on subjects' infection genotype. Also, individual library HCVpp neutralization sensitivity was not associated with any known E2 sequence determinants. Interestingly, two single nucleotide polymorphisms in the HLA-DQ locus were associated with nAb breadth.

Conclusion: Control of HCV infection is associated with more rapid development of a broad nAb response, independent of the infection viral genotype, providing further evidence for the role of nAb in controlling HCV infection and the potential benefit of generating broad anti-HCV nAb responses by vaccination.

Figure 1

Representative genotype 1 HCVpp library. A) Neighbor-joining amino acid phylogenetic tree depicting amino acid diversity of distinct subtype 1a (closed circles) and subtype 1b (open circles) E1E2 sequences included in the genotype 1 HCVpp library. Reference sequences (text) were obtained from Genbank. Diversity plots depicting variability at each amino acid position of B) subtype 1a and C) subtype 1b HCVpp E1E2 sequences (grey line) compared to 374 subtype 1a or 260 subtype 1b reference sequences (black line) and D) divergence between subtype 1a and 1b reference sequences and subtype 1a and 1b library sequences. Amino acid position numbering is based on the H77 reference sequence.

Figure 2

The breadth of nAb responses was similar in Persistence subjects at one year of infection to Clearance subjects at last viremia or one year of infection. A) Heat map illustrating neutralization results against each HCVpp for Persistence and Clearance subjects. Each square represents negative (white) or positive neutralization (grey) of that particular HCVpp. HCVpp 1a154 denotes the H77 E1E2 clone included in the library. B) Breadth of anti-genotype 1 nAb responses in Persistence and Clearance subjects. Values represent the number of HCVpp neutralized by each subject. The median (line) breadth of nAb responses in Persistence subjects was similar to Clearance subjects (P = 0.757). C) Breadth of anti-genotype 1 nAb responses in subjects stratified by infection genotype. Values represent the number of HCVpp neutralized by each subject. The median breadth of nAb responses in each genotype was not different (P = 0.452).

Figure 3

Broader anti-genotype 1 nAb responses were observed in Clearance subjects compared to time-matched Persistence subjects. A) Heat map illustrating neutralization results against each HCVpp for Persistence and Clearance subjects. Each square represents negative (white) or positive neutralization (grey) of that HCVpp. HCVpp 1a154 denotes the H77 E1E2 clone included in the library. B) Breadth of anti-genotype 1 nAb responses in Persistence and Clearance subjects. Values represent the number of HCVpp neutralized by each subject. The median (line) breadth of nAb responses in Clearance subjects was greater than Persistence subjects (P = 0.007). C) Breadth of anti-genotype 1 nAb responses in subjects stratified by infection genotype. Values represent the number of HCVpp neutralized by each subject. The median breadth of nAb responses in each genotype was not different (P = 0.772).

Figure 4

nAb responses broaden in Persistence subjects during acute HCV infection and become more shallow in Clearance subjects following control of viremia. A) Increasing breadth of nAb responses in Persistence subjects over time. Values represent the number of HCVpp neutralized by each subject. The breadth of nAb response in each subject is connected by a line. The breadth of nAb responses was greater in the one-year samples than the time-matched subjects (P < 0.001). B) Decreasing breadth of detectable nAb responses in Clearance subjects following control of viremia. The breadth of nAb responses was greater in the last viremic samples than the one-year samples (P = 0.031).

Figure 5

E1E2 sequence variation versus neutralization sensitivity. A) Amino acid sequences of N-linked glycosylation sites in E2. Amino acid position numbering is based on the H77 reference sequence. CD81 binding residue amino acid sequences in B) subtype 1a and C) subtype 1b HCVpp E1E2 sequences. D) The presence of intact (white) or non-intact (grey) neutralizing epitopes in each HCVpp E1E2 sequence. E) Correlation between the number of non-intact epitopes and neutralization sensitivity. Values represent the number of non-intact epitopes in an HCVpp E1E2 sequence and the neutralization sensitivity of a HCVpp.

Figure 6

HLA-DQ A) rs2395522 and B) rs9275224 SNP genotypes are associated with an increased breadth of nAb responses. Values represent the number of HCVpp neutralized by each subject. * P < 0.05.