Breakdown of strain diversity in the study cohort based on sequencing of three hypervariable CMV genes.

<p>* The two women with two strains each had one unique strain (based on UL55/UL144) and one shared strain.</p

primers used in sequencing study.

<p>primers used in sequencing study.</p

Alignment of gB subtypes.

<p>Amino acid alignment encompassing the proteolytic cleavage site of gB subtypes detected in the 53 CMV-infected women. Variants with amino acid changes within a subtype are depicted: gB1 (1 and 1**), and gB3 (3 and 3*). The gB1 prototype is Towne, gB2 prototype is AD169, and gB3 prototype is Toledo. The gB1* and the two variants of gB2 are associated with nucleotide changes only. Considering the five major gB subtypes, the two women with multiple strains had gB5/gB1, and gB4/gB1. The subtype distribution among the 51 women who shed one strain was as follows: 15 women shed gB1 (including variants), 9 women - gB2, 10 women - gB3, 4 women - gB4 and 13 women -gB5.</p

Number and source of CMV isolates from 53 CMV-infected women.

<p>Number and source of CMV isolates from 53 CMV-infected women.</p

Phylogenetic tree of UL55 and UL144 subtypes.

<p>Left- UL55 DNA, right- UL144 DNA subtypes. Colored in red are -vaccine recipients, and in black- placebo recipients.</p

Interval (in months) between first and last sample tested by PCR.

<p>Samples available for PCR sequencing were collected from CMV-infected women at different times following seroconversion.</p

alignment of UL146 subtypes.

<p>Amino acid alignment of UL146 subtypes. Subtype definition by Dolan et al (10) appears in parentheses. Several variants in Dolan's subtypes G8, G9, and G11 have been detected in this cohort and are designated E1, E2, G1, G2, H1, and H2 based on common patterns of a small number of nucleotide and amino acid polymorphisms. Among 37 women the distribution of UL146 subtypes was: 14 women - G9, 1- G9*, 5 - G7, 5 - G13, 3 - G11, 2 - G11*, 2 - G8, 1- G8*, 2 - G5, 2 - G1, 1 - G12, and 1- G14. The women with multiple strains had G8*/G11, and G13/G14.</p

Image_3_Convergent antibody responses are associated with broad neutralization of hepatitis C virus.pdf

IntroductionEarly development of broadly neutralizing antibodies (bNAbs) targeting the hepatitis C virus (HCV) envelope glycoprotein E2 is associated with spontaneous clearance of infection, so induction of bNAbs is a major goal of HCV vaccine development. However, the molecular antibody features important for broad neutralization are not known.MethodsTo identify B cell repertoire features associated with broad neutralization, we performed RNA sequencing of the B cell receptors (BCRs) of HCV E2-reactive B cells of HCV-infected individuals with either high or low plasma neutralizing breadth. We then produced a monoclonal antibody (mAb) expressed by pairing the most abundant heavy and light chains from public clonotypes identified among clearance, high neutralization subjects.ResultsWe found distinctive BCR features associated with broad neutralization of HCV, including long heavy chain complementarity determining region 3 (CDRH3) regions, specific VH gene usage, increased frequencies of somatic hypermutation, and particular VH gene mutations. Most intriguing, we identified many E2-reactive public BCR clonotypes (heavy and light chain clones with the same V and J-genes and identical CDR3 sequences) present only in subjects who produced highly neutralizing plasma. The majority of these public clonotypes were shared by two subjects who cleared infection. A mAb expressing the most abundant public heavy and light chains from these clearance, high neutralization subjects had features enriched in high neutralization clonotypes, such as increased somatic hypermutation frequency and usage of IGHV1-69, and was cross-neutralizing.DiscussionTogether, these results demonstrate distinct BCR repertoires associated with high plasma neutralizing capacity. Further characterization of the molecular features and function of these antibodies can inform HCV vaccine development.</p

Table_1_Convergent antibody responses are associated with broad neutralization of hepatitis C virus.docx

IntroductionEarly development of broadly neutralizing antibodies (bNAbs) targeting the hepatitis C virus (HCV) envelope glycoprotein E2 is associated with spontaneous clearance of infection, so induction of bNAbs is a major goal of HCV vaccine development. However, the molecular antibody features important for broad neutralization are not known.MethodsTo identify B cell repertoire features associated with broad neutralization, we performed RNA sequencing of the B cell receptors (BCRs) of HCV E2-reactive B cells of HCV-infected individuals with either high or low plasma neutralizing breadth. We then produced a monoclonal antibody (mAb) expressed by pairing the most abundant heavy and light chains from public clonotypes identified among clearance, high neutralization subjects.ResultsWe found distinctive BCR features associated with broad neutralization of HCV, including long heavy chain complementarity determining region 3 (CDRH3) regions, specific VH gene usage, increased frequencies of somatic hypermutation, and particular VH gene mutations. Most intriguing, we identified many E2-reactive public BCR clonotypes (heavy and light chain clones with the same V and J-genes and identical CDR3 sequences) present only in subjects who produced highly neutralizing plasma. The majority of these public clonotypes were shared by two subjects who cleared infection. A mAb expressing the most abundant public heavy and light chains from these clearance, high neutralization subjects had features enriched in high neutralization clonotypes, such as increased somatic hypermutation frequency and usage of IGHV1-69, and was cross-neutralizing.DiscussionTogether, these results demonstrate distinct BCR repertoires associated with high plasma neutralizing capacity. Further characterization of the molecular features and function of these antibodies can inform HCV vaccine development.</p

Image_1_Convergent antibody responses are associated with broad neutralization of hepatitis C virus.pdf

IntroductionEarly development of broadly neutralizing antibodies (bNAbs) targeting the hepatitis C virus (HCV) envelope glycoprotein E2 is associated with spontaneous clearance of infection, so induction of bNAbs is a major goal of HCV vaccine development. However, the molecular antibody features important for broad neutralization are not known.MethodsTo identify B cell repertoire features associated with broad neutralization, we performed RNA sequencing of the B cell receptors (BCRs) of HCV E2-reactive B cells of HCV-infected individuals with either high or low plasma neutralizing breadth. We then produced a monoclonal antibody (mAb) expressed by pairing the most abundant heavy and light chains from public clonotypes identified among clearance, high neutralization subjects.ResultsWe found distinctive BCR features associated with broad neutralization of HCV, including long heavy chain complementarity determining region 3 (CDRH3) regions, specific VH gene usage, increased frequencies of somatic hypermutation, and particular VH gene mutations. Most intriguing, we identified many E2-reactive public BCR clonotypes (heavy and light chain clones with the same V and J-genes and identical CDR3 sequences) present only in subjects who produced highly neutralizing plasma. The majority of these public clonotypes were shared by two subjects who cleared infection. A mAb expressing the most abundant public heavy and light chains from these clearance, high neutralization subjects had features enriched in high neutralization clonotypes, such as increased somatic hypermutation frequency and usage of IGHV1-69, and was cross-neutralizing.DiscussionTogether, these results demonstrate distinct BCR repertoires associated with high plasma neutralizing capacity. Further characterization of the molecular features and function of these antibodies can inform HCV vaccine development.</p