Conserved peptides within the E2 region of Hepatitis C virus induce humoral and cellular responses in goats

Abstract

The reason(s) why human antibodies raised against hepatitis C virus (HCV) E2 epitopes do not offer protection against multiple viral infections may be related to either genetic variations among viral strains particularly within the hypervariable region-1 (HVR-1), low titers of anti E2 antibodies or interference of non neutralizing antibodies with the function of neutralizing antibodies. This study was designed to assess the immunogenic properties of genetically conserved peptides derived from the C-terminal region of HVR-1 as potential therapeutic and/or prophylactic vaccines against HCV infection. Goats immunized with E2-conserved synthetic peptides termed p36 (a.a 430-446), p37(a.a 517-531) and p38 (a.a 412-419) generated high titers of anti-p36, anti-p37 and anti-P38 antibody responses of which only anti- p37 and anti- p38 were neutralizing to HCV particles in sera from patients infected predominantly with genotype 4a. On the other hand anti-p36 exhibited weak viral neutralization capacity on the same samples. Animals super-immunized with single epitopes generated 2 to 4.5 fold higher titers than similar antibodies produced in chronic HCV patients. Also the studied peptides elicited approximately 3 fold increase in cell proliferation of specific antibody-secreting peripheral blood mononuclear cells (PBMC) from immunized goats. These results indicate that, besides E1 derived peptide p35 (a.a 315-323) described previously by this laboratory, E2 conserved peptides p37 and p38 represent essential components of a candidate peptide vaccine against HCV infection.

Figure 1

Alignment of amino acid sequences of the E2 sub-genomic region among various HCV genotypes with special emphasis on subtype (4a). Predicted Peptides #38, #36 & #37 in this study are shown on the top of the aligned sequences. A hyphen indicates an amino acid residue identical to that of the HCV genotype 4a.EG.ED43.Y11604 sequence

Figure 2

Reactivity of human IgGs towards the conserved E2-peptides in chronic HCV genotype 4a patients. The corresponding titers of human IgGs against each tested peptide were determined in 100 HCV patients and 25 healthy control subjects. Levels of antibodies as detected with specific ELISA are depicted as scatter diagram. Cutoff value was calculated from the levels obtained from healthy controls (mean of negative values + 3×S.D).

Figure 3

Goat IgG levels against multiple doses of linear peptide p38 conjugated with KLH. (P35-KLH and saline treated goats served as positive and negative controls respectively.) Antibody titers were followed for a total of 145 days. The results shown represent the mean values of two goats at each time interval.

Figure 4

Comparison between titers of anti E2-peptide antibodies in chronic HCV patients and super-immunized goats. ELISA plates coated with p36, p37 or p38 were used for determining relevant antibody titers in both chronic HCV patients and goats super immunized with p36-KLH, p37-KLH or p38-KLH. Bars represent means of Ab titer from 100 HCV patients and from 2 goats who received subcutaneous injections of 1.5 mg at days 0, 14 and 28. Cut off values were calculated as means of anti p38-KLH in 25 healthy subjects and in 2 saline injected goats.

Figure 5

Viral neutralization by anti E2 peptide goat antibodies. Purified mono-specific polyclonal antibodies against p36–p38 epitopes were used at 300 to < 12.5 μg/tube to bind HCV from infected sera and the immune-capture activity of each Ab was assessed by RT-nested PCR amplification. The 174 bp amplicon denotes the presence of captured virus. The immune-capture experiment was repeated 3 times with different serum samples. Each Ab displayed the same immune-capture activity with different infected sera. Negative control and binding specificity were assessed by replacing the anti peptide Ab by PBS and anti HBV Ab respectively.

Figure 6

Effect of p38 epitope on Peripheral blood mononuclear cell proliferation in immunized goats. Goats were immunized with p38 epitope. PBMC derived from immunized (dark column) and from non immunized (light column) were cultured, stimulated with increasing concentrations (5–50 ug/ml culture) of p38 and analyzed by FACS for cell proliferation (A and C). Results were compared with p35 (B) as a positive control for peptide mediated cell proliferation.