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. Author manuscript; available in PMC: 2022 Sep 15.
Published in final edited form as: Vaccine. 2021 Aug 13;39(39):5607–5614. doi: 10.1016/j.vaccine.2021.08.015

A large repertoire of B cell lineages targeting one cluster of epitopes in a vaccinated rhesus macaque

Liuzhe Li a, Ann J Hessell b, Xiang-Peng Kong c, Nancy L Haigwood b, Miroslaw K Gorny a
PMCID: PMC8449804  NIHMSID: NIHMS1731761  PMID: 34400018

Abstract

The repertoire of antibodies (Abs) produced upon vaccination against a particular antigenic site is rarely studied due to the complexity of the immunogens. We received such an opportunity when one rhesus macaque was immunized six times at 0, 4, 10, 16, 32, and 143 weeks with C4–447 peptide containing the 8-mer epitope for human monoclonal Ab (mAb) 447–52D specific to the V3 region of gp120 HIV-1. Strong anti-V3 antibody responses reached 50% binding titer in serum of 10−5 at week 10 that declined to 10−3 by week 70. After an additional boost of C4–447 peptide at week 143, titers rebounded to 10−5 at week 146, or 2.7 years after the first immunization. Using the blood sample at week 146, we produced 41 V3-specific recombinant mAbs by single B cell isolation and cloning. Sequence analysis revealed 21 B cell lineages, single and clonally related, based on immunoglobulin gene usage and CDR3s. The broad repertoire of Abs directed to a small antigenic site shows the targeting potency of a vaccine-elicited immune response in rhesus macaques.

Keywords: Repertoire of antibodies, vaccine-induced antibodies, V3 monoclonal antibodies, non-human primates’ immunization, rhesus macaque immunoglobulin genes

1. Introduction

The potency of antibody responses against an invading pathogen can be reflected, among the other parameters, by the number of B cell lineages targeting one particular antigenic site. Monoclonal antibodies (mAbs) are excellent tools to analyze the repertoire of specific Abs, and studies of anti-HIV-1 mAbs are providing an insight into this topic. Early studies using hybridoma technology produced a limited number of mAbs from one donor, and only a few mAbs were isolated against a particular antigenic determinant. From one HIV-1 infected individual, we isolated only two B cell lineages against conformational V2 epitopes (V2i) (1) and three mAbs against the V3 region and CD4bs (2). In another study, Lynch et al isolated three B cell lineages targeting a common epitope at the interface of the V1V2 (3).

In contrast to cellular methods, isolation and cloning from single B cells from the immune repertoire is very efficient, allowing the production of a large collection of mAbs from one donor (4, 7, 10). A notable early study using isolated memory B cells of six HIV-infected donors, Sheid et al reported B cell clones could be identified from each donor with specificity against particular regions in a range between 2 to 13 mAbs against the CD4bs, CD4i, and gp41 epitopes (4). The quantity of mAbs cloned likely depends on the number of antigenic sites in the region targeted by the vaccine, for example gp41 has at least six antigenic sites (5). In the setting of vertical transmission from mother to infant, Martinez et al recently reported from one to six neutralizing mAbs produced from each of seven HIV-1-infected mothers that ranged in specificity against CD4bs, V3, and V2 mAbs (6).

Several participants of the RV144 HIV vaccine trial were boosted throughout the 11-year vaccination period, and mAbs against HIV Env were selected using single-cell sorting and memory B cell cultures. Each of the ten vaccinees tested produced a different number of V2 mAbs ranging from 1 to 13 B cell lineages; however, their specificity against three antigenic sites, linear (V2p), conformational (V2i), and glycan N156-dependent (V2gly156), was not determined (7). The weak immunogenicity of gp41 MPER limits Ab development, as suggested in two recent reports where only two or three B cell lineages against MPER were developed using B cell sorting from single donors (8, 9).

In contrast, neutralizing Abs against respiratory syncytial virus (RSV) target the virus fusion (F) glycoprotein. A large number (364 mAbs) were produced from three healthy adult donors, and nearly half of the most potent mAbs were specific to six dominant antigenic sites on prefusion RSV F. Each antigenic site was targeted by 2 to 12 B cell lineages, single or clonally related, developed from each donor (10).

As shown in humans, rhesus macaques can produce several mAbs against one antigenic site. Two macaques infected with SHIVSF162P4 were used to produce four or five neutralizing mAbs each. All nine mAbs recognized quaternary epitopes (QNE) on the trimeric HIV-1 SF162 envelope spike. These mAbs exhibited different characteristics, and it was suggested they recognized overlapping QNE epitopes (11).

We analyzed a panel of 41 recombinant mAbs specific to the V3 region of gp120 HIV-1 produced from a rhesus macaque immunized six times over 2.7 years with a C4–447 peptide containing the 8-mer epitope for human anti-V3 mAb 447–52D. The mAbs were produced by antigen-specific B cell sorting using PBMCs collected at week 146 after the first immunization. Sequence analysis revealed 21 B cell lineages, including single and clonally related mAbs, based on immunoglobulin (Ig) gene usage and CDR3 sequences. The study demonstrates the development of a broad repertoire of Abs in a single vaccinated monkey against a well-defined small cluster of epitopes contrasting to other reports of smaller repertoires of Abs directed to more complex antigenic sites (4, 6, 7, 10, 11).

2. Materials and methods

2.1. Ethics Statement.

All animal experiments were performed following the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, the Office of Animal Welfare, and the U.S. Department of Agriculture. Experiments were reviewed and approved by the West Campus Institutional Animal Care and Use Committee at the Oregon Health & Science University (OHSU) and the New York University School of Medicine, New York, NY before the start of the study. The Oregon National Primate Research Center (ONPRC) is accredited by AAALACi.

2.2. Immunogen, peptide

The C4–447 immunogen is a 32-mer consisting of the C4 peptide and the cyclic peptide containing the epitope for a human anti-V3 HIV-1 monoclonal antibody (mAb) 447–52D (12) (Fig. 1A). The C4 peptide is a 16-amino acids part of the gp120MN HIV-1 (residues 428–443) that contains the helper T-cell epitope inducing T-cell immunity against gp120MN in mice (13). The C4–447 peptide, biotinylated C4MN peptide (Fig. 1A) and biotinylated cyclic V3MN peptide (CNKRKRIHIGPGRAFYTTKNC) were synthesized by Biopeptide Co, Inc, San Diego, CA.

Fig. 1.

Fig. 1.

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Immunogen, immunization protocol and binding activities of plasma and mucosal secretions antibodies of rhesus macaque 25328. (A) The C4–447 peptide, consisting of C4 peptide, 20 mer, and V3 mimotope, 16 mer (447 mimotope) for the 447–52D mAb with disulfide-constrained V3 crown. The 447–52D epitope is shown in red. (B) Animal 25328 was immunized 6 times at 0, 4, 10, 16, 32 and 143 weeks using 1 mg of C4–447 peptide with incomplete Freund’s adjuvant. (C) Plasma antibodies binding to C4–447 peptide (blue) and C4 peptide (green). (D) Binding of plasma samples to biotinylated cyclic V3MN peptide by ELISA. (E) Buccal and vaginal secretion antibodies binding to C4–447 peptide. Plasma samples and mucosal secretions were tested by standard ELISA.

2.3. Immunization of rhesus macaques

To understand the durability of antibody responses and collect additional blood samples for isolation of peripheral blood lymphocytes, we re-assigned to this project a single remaining rhesus macaque (ID 25328) from our original immunogenicity study (12) that was available at the Oregon National Primate Research Center. Animal 25328 was immunized six times at 0, 4, 10, 16, 32, and after re-assignment, a boost vaccination at week 143 with 1 mg of the C4–447 peptide (Fig. 1A–B). The C4–447 peptide was solubilized in 0.5 ml of phosphate-buffered saline (PBS) and then emulsified with an equal volume of incomplete Freund’s adjuvant (IFA). The peptide emulsion (1 ml) was inoculated intramuscularly in four sites on the upper back between the shoulder blades. Immediately before the immunization, a 10 ml blood draw was collected along with duplicate swabs of buccal and vaginal secretions. Blood and mucosal secretion collections were repeated at two-week intervals. Peripheral blood mononuclear cells (PBMCs) were prepared from each blood collection, and secretion samples were stored at −80°C for later processing.

2.4. Binding assay (ELISA)

Longitudinal plasma samples, buccal and vaginal mucosal secretions, and recombinant mAbs were tested against antigens by standard ELISA as described (12). We tested the binding activity to recombinant gp120MN, gp41MN (Immune Technology Corp.), biotinylated C4MN peptide (Biopeptide Co, Inc.). Briefly, the proteins and peptides were coated onto plastic plates at 1 μg/mL followed by incubation with blocking assay diluent (Phosphate buffer saline, PBS, containing 2.5% bovine serum albumin and 7.5% fetal bovine serum). Plasma samples were diluted by 10-fold dilutions ranging from 1:10 to 1:1,000,000 and incubated with coated antigens. Recombinant Abs were screened at IgG concentration of 2–10 μg/mL. The binding of Abs to antigens was detected by incubation with alkaline phosphatase (A.P.)-conjugated goat anti-human IgG (γ) (Southern Biotech) followed by incubation with the substrate to develop color. The plates were read at 405 nm. Biotinylated peptides were tested using streptavidin-coated plates (SreptaWell, Roche), which were incubated with biotinylated peptides (447 and C4 peptides) at 1 μg/mL. Mucosal buccal and vaginal secretions were screened by ELISA using the second antibody, the peroxidase-conjugated goat anti-human IgG (γ specific) (Jackson Immuno Research); the plates were read at 450/650 nm. The 50% titers of plasma Abs were determined by measuring the dilutions of plasma required for 50% maximal binding by linear regression (GraphPad Prism 8).

IgG quantitation of recombinant Abs in the culture supernatants was measured by ELISA as described with some modifications (2). Briefly, ELISA plates were coated with goat anti-human IgG (γ) Abs to capture recombinant IgG followed by the second Ab, AP-conjugated goat anti-human IgG (Fc) (both Southern Biotech). Affinity-purified human IgG (Sigma) was used to produce a standard curve, which then was used via interpolation to determine IgG concentrations of mAbs.

2.5. Production of recombinant monoclonal antibodies

A blood sample from immunized macaque 25328 at week 146 was collected, PBMCs were frozen in liquid nitrogen and thawed for later analyses. PBMCs were washed with 1xPBS containing 1% BSA and stained with biotinylated cyclic V3MN peptide followed by streptavidin-APC and fluorescence-conjugated Abs against cell markers CD3-PE-CF594, CD20-PE-Cy7, CD19-APC-Cy7, IgG-FITC, and IgM-BV450 (Becton Dickinson). V3-specific single positive B cells were isolated in 96-well plates by FACS gating strategy as previously described (2) (Fig. 2). mRNA was isolated from single B cells and reverse transcribed in the 96-well plate by addition of random hexamer primers (Invitrogen), 0.35 mM dNTP mix (Invitrogen), 10 mM DTT, 2U RNAsin (Promega), 4U Superase-In (Ambion), 0.5% v/v Igepal CA-630 (Sigma-Aldrich) and 50 U superscript III reverse transcriptase (Invitrogen). The immunoglobulin (Ig) variable genes were amplified by nested PCR and cloned into plasmids containing a constant region as described (14, 15). Macrogen, Inc. sequenced the VH, Vκ, and Vλ chains. Recombinant Abs were produced by co-transfection of H/κ or H/λ plasmids into 293T Cells (2), and IgG production in culture supernatants was quantitated. Supernatants with recombinant Abs were screened for binding activity to V3MN, gp120MN, and gp41MN and mAbs specific to V3 and gp120, but not to gp41 were identified.

Fig. 2.

Fig. 2.

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Isolation of single B cells specific to the V3 mimotope peptide using FACS sorting. Single V3 specific IgG+ B cells were sorted from PBMCs collected after 6 immunizations at week 146. The PBMCs were stained with biotinylated V3MN peptide followed by streptavidin-APC and fluorescence-conjugated Abs against cell markers CD3-PE-CF594, CD20-PE-Cy7, CD19-APC- Cy7, IgG-FITC and IgM-BV450. The B cells were stepwise gated for single V3-specific IgG cells. The initial two gates with side scatter/yellow dye and side scatter/forward identified viable cells and lymphocytes, respectively. A forward scatter height (FSC-H) vs. forward scatter area (FSC-A) density plot was used to exclude doublets (data not shown). Cells stained with anti-CD3-PE-CF594 were excluded for T cells. B cells were further gated using anti-CD20-PE-Cy7 and CD19-APC- Cy7. IgG B cells were then selected by IgG and non-IgM staining. Finally, V3-specific B cells were sorted into a 96-well plate. Of 100,000 cells selected in the last gate, 460 V3+ cells (0.46%) were present.

2.6. Phylogenetic analysis for V3 mAbs

The Phylogenetic tree was built with MEGA software and used to estimate the relationships among the V3 mAbs. Firstly, all DNA sequences of VH region from the V3 mAbs were aligned using ClustalW. Then Maximum Likelihood (ML) model was chosen for analyzing the sequences. Bootstrap replicates number was set 500 to estimate the reliability of the nodes of the phylogenetic tree. The VH phylogenetic tree of V3 mAbs was generated with rectangular phylogram.

2.7. Statistical analysis

Comparison of percentage of mutations in the VH of V3 mAbs and pre-bleed VH sequences were determined by nonparametric Mann-Whitney test. Brown-Forsythe ANOVA compared the frequency of VH2, 3, and 4 gene family usage by two panels of mAbs and VH. sequences.

3. Results

3.1. Immunization of rhesus macaque with V3 mimotope peptide

This study is an extension experiment to the immunogenicity study described in Hessell et al. (12) that showed animals immunized with either of two mimotope V3 peptide immunogens, C4–447 and C4-VH5–51, developed strong antibodies in serum and buccal secretions to the respective homologous cyclic peptides. The mimotope peptide, C-447, was designed to mimic the epitopes of the human anti-V3 mAb 447–52D specific for the tip for the V3 loop on the HIV-1 Envelope (Env). To understand the durability of the antibody response and collect additional blood samples for isolation of peripheral blood lymphocytes to produce mAbs, we re-assigned for this experiment the single remaining rhesus macaque (ID 25328) from the original study that was available at the Oregon National Primate Research Center. Animal 25328 was immunized during the original study protocol (12) five times at weeks 0, 4, 10, 16, and 32, and then after two years as a boost vaccination at week 143. We used 1 mg of the C4–447 peptide 36-mer for each immunization (Fig. 1A). The timeline of the study is illustrated in Fig. 1B.

3.2. Plasma and mucosal secretions antibody response to antigens

We have evaluated the plasma antibody responses in animal 25328 during this boosting phase in ELISA against the C4–447 and C4 peptides (Fig. 1C). The Ab response against C4–447 increased over the first four weeks, reaching the 50% binding titer of 105 and then slowly declined to 103 at week 70. Residual 50% binding titers against the C4–447 peptide were at 103 before the boost at week 143. Ending titers at week 146 had increased steadily to 105, or nearly a 2 log10 increase from the pre-boost titers. In contrast, the C4 peptide was much less immunogenic and induced plasma Ab titers of only 102 four weeks after the first immunization that subsequently declined to below 101 at week 70. The anti-C4 peptide titers were not determined after boosting with the C4–447 peptide (Fig. 1C). We also titrated plasma samples for binding to the biotinylated cyclic V3MN peptide from different time points at week 12, 38, 58, 143, 144, 145, and 146. Unlike the C4 peptide titers, these titers were more similar to the pattern of Ab responses to the C4–447 peptide even at the latest timepoints tested (Fig. 1D).

We collected buccal mucosal secretions during the original study and buccal and vaginal for this re-boost experiment from weeks 143 to 146 (Fig. 1E). The anti-C4–447 specific responses in buccal secretions increased rapidly in the three weeks following the first immunization then declined at week 70. No anti-C4–447 responses were detectable at the time of the boost immunization at week 143, but responses increased rapidly in buccal and vaginal secretions peaking at or above the activity measured in the original study (Fig. 1E). The pattern of binding activity of plasma and mucosal secretions Abs to the C4–447 peptide is similar despite comparing titers in the original study and measuring ODs here.

3.3. Production of monoclonal antibodies from immunized rhesus macaque 25328

Our goal in this study was to determine the repertoire of Ab responses against a short peptide (C4–447) in rhesus macaque 25328 after an extended longitudinal immunization protocol. Single B cells specific to the V3 region were isolated from PBMCs collected from 25328 at week 146 using FACS sorting. Of 100,000 B cells identified as IgG+, IgM−, 460 (0.46%) specific to the cyclic V3mn peptide, were sorted into 96-well plates (Fig. 2). We successfully cloned and expressed 46 recombinant mAbs identified by PCR with unique IgH-IgL pairings from one 96-well plate. Correct plasmid inserts for IgH and IgL were confirmed by sequencing before co-transfection of 293T cells and culture supernatants were screened by ELISA against the cyclic V3MN peptide, gp120MN and gp41MN resulting in the identification of 41 recombinant Abs, which were specific to V3mn and gp120MN, but not to gp41MN used as irrelevant control antigen (Table 2). The binding activity of recombinant Abs from culture supernatants was higher against gp120MN than V3mn peptide as measured by OD due to native expression of the V3 region in gp120. The VH gene usage of mAbs was determined from nucleotide sequences using the IMGT/V-QUEST/rhesus system (http://www.imgt.org). The percentage of mutations in the VH, JH, VL and JL regions was calculated from the percentage of identical nucleotides in the antibody’s V and J regions. The V region includes nucleotides up to the 2nd Cys 104 codon, which delimits the end of framework 3 (FR3) of all functional V genes of the VH and VL (16).

Table 2.

Percentage of mutations in the heavy and light chains of mAbs and their binding specificity.

# ID1 VH2 % mut JH2 % mut VL3 % mut JL3 % mut V3MN OD4 gp120MN OD4 gp41MN OD4
1 3.1 1.72 12.50 2.10 2.63 2.8 3.1 0.1
2 70.1 1.72 12.50 2.45 2.63 3.4 3.5 0.1
3 28.1 1.03 12.50 1.05 0.0 1.3 2.7 0.1
4 52.1 2.06 14.38 2.46 2.63 3.2 3.4 0.2
5 5.2 3.09 10.42 2.80 0.0 2.2 3.2 0.2
6 55.3 1.03 10.42 2.80 5.56 3.1 3.5 0.3
7 11.4 2.41 6.25 6.29 0.0 1.8 3.2 0.2
8 83.5 2.06 8.33 2.45 5.88 3.4 3.3 0.1
9 44.6 5.50 12.50 4.20 14.71 0.8 2.5 0.2
10 72.7 8.59 6.25 4.20 5.88 3.1 3.5 0.1
11 82.8 6.53 8.33 1.40 0.0 0.9 3.2 0.1
12 31.9 3.09 12.50 2.80 8.57 1.8 3.1 0.3
13 15.10 3.09 4.17 0.70 5.71 1.4 3 0.1
14 15/51.10 3.09 4.17 1.75 0.0 1.9 3.2 0.2
15 79.10 3.09 4.17 2.10 8.57 2.3 3.3 0.2
16 29.11 2.75 6.25 2.80 5.71 3.4 2.7 0.3
17 68.12 4.81 12.50 1.75 8.57 2.1 3.2 0.1
18 89.12 2.41 12.50 3.50 8.33 2.5 3.2 0.2
19 46.13 6.53 4.17 1.75 8.33 3.2 3.3 0.2
20 30.14 7.56 7.84 9.32 5.71 3.4 3.3 0.2
21 12.14 9.97 13.73 1.40 2.78 3.5 3.3 0.1
22 57.14 7.56 5.88 6.57 13.89 3.5 3.2 0.1
23 54.14 7.22 5.88 1.40 0.0 3.3 3.2 0.1
24 17.14 6.19 7.84 0.35 0.0 3.3 3.2 0.1
25 93.15 18.06 12.50 2.69 5.26 1.8 3.4 0.1
26 69.16 9.72 3.92 4.21 13.16 1.4 3.2 0.2
27 69/25.16 9.72 3.92 3.15 13.16 1.1 3 0.2
28 69/26.16 9.72 3.92 2.46 10.53 1.1 0.8 0.2
29 62.17 11.11 8.33 5.38 2.63 2.1 3.1 0.6
30 88.17 12.20 8.33 4.30 2.63 2.9 3.2 0.1
31 21/71.18 4.76 0.0 5.0 5.41 2.5 3.3 0.2
32 71.18 5.78 2.08 5.0 5.41 2.1 3.2 0.1
33 9.19 10.07 9.80 9.86 2.78 3.5 3.2 0.1
34 67.20 4.17 8.33 2.02 2.86 3 3.3 0.1
35 19.21 7.37 5.77 4.30 0.0 1.3 1.4 0.1
36 18.21 8.77 5.77 4.66 0.0 1.5 0.7 0.2
37 41.21 8.42 5.77 5.73 8.82 1.5 2.2 0.1
38 50.21 13.33 7.84 8.24 0.0 2.9 3.5 0.1
39 87.21 9.10 5.77 4.68 0.0 1.9 1 0.1
40 87/92.21 9.10 5.77 5.38 0.0 1.2 2.4 0.1
41 20.21 13.33 5.77 6.81 5.88 2.1 1.9 0.1
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1

1ID of rhesus macaques anti-V3 mAbs which are divided alternately by color for 22 unique and clonally related mAbs

2

Percentage of mutations in nucleotide sequences of the V and J regions of the heavy chains, and

3

light chains is deduced from the V- and J-Region identity, respectively, which is analyzed using IMGT/V-QUEST.

4

Elisa reactivity of mAbs to V3MN peptide, recombinant gp120MN and gp41MN reported as optical density (OD), antigens were coated onto plate at 1 μg/mL. OD – 0.6–2.0 orange, 2.1->3.0 red.

3.4. Multiple recombinant Abs derived from 21 B cell lineages.

Among the 41 V3-specific mAbs, eight were clonally related Abs, each containing two to seven member Abs, and 13 single mAbs, and based on sequence analysis, the Abs derived from 21 B cell lineages (Table 1). Clonality of Abs was determined by the usage of the same VH, JH and DH genes for heavy and VL and JL genes for light chains. Similar sequences of CDR H3 and L3 with the number of amino acids changes not exceeding 50%, were the same length as three CDRs and the same number of nucleotides for VH and VL or the same insertion of 3 nucleotides in the FR2 for one clonal Ab family (#21). These parameters differ to some degree among the 13 single mAbs (Table 1). The phylogenetic tree analyzing VH nucleotide sequences only, confirmed the clusters of clonally related mAbs and three groups of mAbs using VH2, VH3 and VH4 family genes (Fig. 4).

Table 1.

Vaccine-induced macaque recombinant antibodies specific to the epitope defined by human anti-V3 mAb

U1 ID2 VH JH DH CDRs3 HCDR3 nt4 VL JL CDRs3 LCDR3 nt4 Ins5
1 3.1 2–1*01 4*01 2–4*01 10.7.14 CARTNKYFSGSYYYYW 367 L1–7*01 3*01 8.3.12 CQSYDTRLSAHVLF 334
70.1 2–1*01 4*01 2–4*01 10.7.14 CARTNKYFSGSYYYYW 367 L1–7*01 3*01 8.3.12 CQSYDTRLSAHVLF 334
28.1 2–1*01 4*01 2–3*01 10.7.14 CARTSEYYSGSYYYYW 367 L1–7*01 3*01 8.3.12 CQSYDSSLSAHVLF 334
52.1 2–1*01 4*01 2–3*01 10.7.14 CARTRKYYSGSYYYYW 367 L1–7*01 3*01 8.3.12 CQSHDSNLNLEVLF 334
2 5.2 2–1*01 4*01 4–2*01 10.7.14 CARTGLYSGYSFYDYW 367 L1–7*01 3*01 8.3.10 CQSYDSGLSVLF 328
3 55.3 2–1*01 4*01 4–4*01 10.7.14 CARPTVMNTVSPYDYW 367 L1–7*01 2/3*01 8.3.10 CQSYDISFSVLF 328
4 11.4 2–1*01 4*01 6–6*01 10.7.13 CARMQILSSSSFDYW 364 L1–7*01 1*01 8.3.10 CCSFGSGNPYIF 328
5 83.5 2–1*01 4*01 2–1*01 10.7.15 CTRTFYSNPNYYLNDYW 370 K1–9*01 1*01 6.3.8 CQQYDDFPTF 319
6 44.6 2–1*01 4*01 2–1*01 10.7.15 CTRTYYNFLTTNYEDSW 370 L2S9*01 2/3*01 9.3.10 CCSYGSGSNWIF 331
7 72.7 2–1*01 4*01 3–1*01# 10.7.15 CARGSFGISNYFTFDYW 370 L1–7*01 2/3*01 8.3.10 CQSYDSNLSALF 328
8 82.8 2–1*01 4*01 6–1*01 10.7.15 CARGDIAAAGTSAFDYW 370 L1–7*01 1*01 8.3.10 CQSYDSSLSAIF 328
9 31.9 2–1*01 4*01 2–2*01 10.7.11 CARTYYSGGYDYW 358 L1–7*01 3*01 8.3.10 CQSYDSSLSVLF 328
10 15.10 2–1*01 4*01 4–3*01 10.7.11 CARSSLTSSFDYW 358 L1–7*01 2/3*01 8.3.10 CQSYDSNLNPVF 328
15/51.10 2–1*01 4*01 4–3*01 10.7.11 CARTSLTSSFDYW 358 L1–7*01 2/3*01 8.3.10 CQSYDNNLNPVF 328
79.10 2–1*01 4*01 4–3*01 10.7.11 CVRSSLTSSFDYW 358 L1–7*01 2/3*01 8.3.10 CQSYDSNLNPVF 328
11 29.11 2–1*01 4*01 2–3*01 10.7.18 CARTREYCSSTYCTSPFDYW 379 L1–7*01 3*01 8.3.12 CQSYDISLSAHVLF 334
12 68.12 2–1*01 4*01 3–3*01 10.7.12 CVRTGVIVSGFDFW 361 L1–7*01 3*01 8.3.10 CQSYDSRLSAVF 328
89.12 2–1*01 4*01 3–3*01 10.7.12 CARTGVIVSGFDFW 361 L1–7*01 3*01 8.3.10 CQSYDSSLSAVF 328
13 46.13 2–1*01 4*01 3–1*01# 10.7.19 CVRGAYYEDDYNYSFFYFDYW 382 L1–7*01 6*01 8.3.10 CQSYDSTLIPVF 328
14 30.14 2–1*01 5–2*02 2–2*01 10.7.15 CARYCSGSACYWSMDVW 370 L1–7*01 3*01 8.3.10 CQSYDSSLSAVF 328
12.14 2–1*01 5–2*02 2–5*01 10.7.15 CARYCAASGCYWSMDVW 370 L1–7*01 3*01 8.3.10 CQSYDSNLSAVF 328
57.14 2–2*01# 5–2*02 2–5*01 10.7.15 CARYCTGSGCYWSLDVW 370 L1–7*01 3*01 8.3.10 CQSYDSSLSAVF 328
54.14 2–2*01# 5–2*02 2–5*01 10.7.15 CARYCTGSGCYWSLDVW 370 L1–7*01 3*01 8.3.10 CQSYDTNLSVVF 328
17.14 2–2*01# 5–2*02 2–1*01 10.7.15 CARYCTGSTCYWSLDVW 370 L1–7*01 3*01 8.3.10 CQSYDSSLSVVF 328
15 93.15 3–14*01 4*01 2–2*01 8.8.15 CARGCSRGVCYADLDFW 367 K2S17*01 2*01 12.3.9 CMQALEFPYSF 340
16 69.16 3–14*01 5–1*01 2–2*01 8.8.18 CARDRSNVGVVVATNRFDVW 376 L1–15*01 2/3*01 8.3.12 CAAWDDRLGGYWVF 334
69/25.16 3–14*01 5–1*01 2–2*01 8.8.18 CARDRSNVGVVVATNRFDVW 376 L1–15*01 2/3*01 8.3.12 CAAWDDSLGGNWVF 334
69/26.16 3–14*01 5–1*01 2–2*01 8.8.18 CARDRSNVGVVVATNRFDVW 376 L1–15*01 2/3*01 8.3.12 CAAWDDSLSGNWVF 334
17 62.17 3–14*01 4*01 4–4*01 8.8.12 CSKPMSLVTDSDSW 358 K1–22*01 4*01 6.3.9 CLQCSISPLTF 322
88.17 3–14*01 4*01 4–4*01 8.8.12 CAKPMDIITDSDDW 358 K1–22*01 4*01 6.3.9 CLQYNSSPLTF 322
18 21/71.18 3–18*01 4*01 4–2*01 8.10.17 CTSLHPYSDDSPWDYFDYW 379 L3S1*01 1*01 6.3.11 CNSWDSSTKHYIF 325
71.18 3–18*01 4*01 4–2*01 8.10.17 CTSLRAYSEYSPWDYFDHW 379 L3S1*01 1*01 6.3.11 CNSWDSSTKHYIF 325
19 9.19 3–7*01 5–2*02 3–1*01# 8.8.18 CTRGEQYEEDYFLGGALDVW 376 K2S20*01 4*01 11.3.9 CMQGKQFPLTF 337
20 67.20 3–9*01 4*01 2–3*01 8.8.12 CARANWEVNLYDYW 358 K2S14*01 4*01 12.3.8 CMQYIHTPTF 337
21 19.21 4–2*01 1*01 2–4*01 8.8.18 CAKNGYCSGIYCYAGYFEFW 376 K3S3*01 1*01 6.3.7 CLQSSNWTF 316 FR2
18.21 4–2*01 1*01 2–4*01 8.8.18 CVKNGYCSGIYCYAGYFEFW 376 K3S3*01 1*01 6.3.7 CLQSSYWTF 316 FR2
41.21 4–2*01 1*01 2–4*01 8.8.18 CAKNGYCSGIYCYAGYFEFW 376 K3S3*01 1*01 6.3.7 CLQSSNWAF 316 FR2
50.21 4–2*01 1*01 2–4*01 8.8.18 CAKNGFCSGIYCYAGYLEFW 376 K3S3*01 1*01 6.3.7 CLQSDNWTF 316 FR2
87.21 4–2*01 1*01 2–4*01 8.8.18 CAKNGYCSGIYCYAGYFEFW 376 K3S2*01 1*01 6.3.7 CLQSSNWTF 316 FR2
87/92.21 4–2*01 1*01 2–4*01 8.8.18 CAKNGYCSGIYCYAGYFEFW 376 K3S3*01 1*01 6.3.7 CLQSSYWTF 316 FR2
20.21 4–2*01 1*01 2–4*01 8.8.18 CARNGYCYGLYCYAGYFEFW 376 K3S2*01 1*01 6.3.7 CLQSSNWRF 316 FR2
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1

U – A panel of 22 unique antibodies, single and clonally related, determined by the usage of the same VH, JH, DH, VL and JL immunoglobulin genes, the same length of CDRs, CDR1, 2 and 3, similar nucleotide sequence length and similar sequence of heavy and light chains CDRs; unique antibodies are divided alternately by color

2

ID – name of 41 recombinant antibodies

3

CDRs – amino acids length of CDR1, 2 and 3

4

nt – nucleotide sequence length

5

Ins – insertion; # - ORF.

Fig. 4.

Fig. 4.

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Phylogenetic tree for VH of the V3 mAbs. Evolutionary analyses were conducted in MEGA X. All VH DNA sequences were aligned with ClustalW. The phylogenetic tree was generated by Maximum Likelihood model. The branches and nodes, indicating the sequence relationships, were obtained from 500 bootstrap replicates. This analysis involved 37 nucleotide sequences. The basic shapes mark the clonally related mAbs and usage of the VH family genes.

In terms of Ig gene usage, the recombinant Abs used three VH gene families: VH2 (2–1 and 2–2), VH3 (3–7, 3–9, 3–14, and 3–18), and VH4 (4–2). The length of CDR H3 ranged from 11 to 19 amino acids, while CDR L3 length was 7 to 12 amino acids. The majority of Abs (total of 28) used lambda and 13 Abs used kappa light chains (Table 1).

We also analyzed the percentage of mutations in the V and J regions of the heavy and light chains; these were in a broad range from 1.03% to 18.06% in the VH, and 0.0% to 14.38% in the joining region (IGHJ) (Table 2). The recombinant Abs with a low percentage of mutations in the VH, for example, 1%−3%, probably were induced by the vaccine boost at week 143 (blood sample collected at week 146). The higher percentage of mutations, 4%−18%, most likely was induced by all six immunizations. The percentage of the VL mutations in the majority of Abs was lower but corresponded to % mutations in the VH region.

3.5. Analysis of VH sequences produced from pre-vaccination of animal 25328

To evaluate the clonal diversity of unknown specificity present in macaque 25328 before the C4–447 peptide immunizations, we characterized the VH sequences from PBMCs isolated two weeks prior to the first immunization (12). We stained the PBMCs with fluorochrome-labeled Abs against CD20-PE, IgG-FITC, and CD27-APC to sort single IgG+ memory B cells. The VH sequences from single B cells were generated using molecular techniques described here and previously (2).

From the pre-immunization sample, we produced 122 VH sequences, as shown in Supplementary Table 1. Six clonal VH sequences were defined based on identical Ig gene usage and similar CDR3 sequences, although 4 of them have identical CDR3 sequences suggesting that they might combine with different VL regions. Identifying only six clonal VH sequences from these 122 VH sequences is much lower than nine clonal families identified out of 41 V3-specific mAbs produced from the same animal at week 146 (Table 2). Also, the pre-immunization sequences contained 23 insertions and three deletions compared to only one insertion in 21 B cell lineages obtained from the week 126 sample (Supplementary Table 1), suggesting a more affinity maturation in Abs over a longer period of time resulting from the presence of various unknown antigens.

The percentage of VH mutations is significantly higher in pre-bleed VH sequences than in the VH of cloned V3-specific mAbs (mean 10.7% versus 6.5%, respectively, p<0.0001) (Fig. 3A). The unequal distribution of mutations in the two panels of VH sequences is expected because the V3-specific mAbs contain Abs occurring early after the late vaccine boost that can be expected to have a low percentage of mutations ranging from 0%−3% were detected in 21.9% in the V3 mAbs versus 4.9% in pre-bleed VH sequences, whereas mutations above 10% were detected in 14.6% in the V3 mAbs compared to 52.4% in pre-bleed VH sequences. It means that pre-bled Abs are more mature and have a higher percentage of Abs with mutations above 10% in the VH sequence (Table 1, Supplementary Table 1). The percentage of mutations in the joining fragment, IGHJ, of both V3-specific Abs and the pre-bleed Abs is comparable because the protein encoded by this gene has little involvement in the Ab-epitope interaction (Fig. 3B).

Fig. 3.

Fig. 3.

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Percentage of mutations in immunoglobulin genes and gene usage for V3 mAbs derived from rhesus macaque 25328. (A) Percentage of mutations in the variable IGHV genes, and (B) in J fragment IGHJ genes of V3 mAbs and Ig sequences from pre-bleed B cells. Nonparametric Mann-Whitney test. (C ) VH gene family usage of the V3 mAbs produced from B cells collected at week 146 and from pre-bleed B cells at week 0. Three sets of data, % of VH2, VH3 and VH4, were compared between two panels by Brown-Forsythe ANOVA, p<0.0001. (D) VH individual gene usage (detailed); clonal Abs are represented by one VH gene.

3.6. Immunoglobulin gene usage by recombinant antibodies

We also found a difference in the pattern of VH gene family usage between V3 and pre-bleed mAbs (Table 1, Supplementary Table 1). The V3 mAbs used only three VH gene families with dominant VH2, then VH3, and some VH4 gene family (63.6%, 27.1%, and 4.5%, respectively). The pre-bleed sequences used all seven VH gene families, and the frequency of usage of the VH2, VH3, and VH4 genes was significantly different compared to V3 mAbs: 0.8%, 53.9%, and 28.7% (p<0.0001, Brown-Forsythe ANOVA test) (Fig. 3C).

In terms of individual VH gene usage, the V3 mAbs used seven genes, whereas pre-bleed sequences were encoded by 18 genes (Fig. 3D). V3 mAbs usage of the VH2.1 gene was almost exclusive (63.6% versus 0.8% for pre-bleed), while pre-bleed sequences were encoded dominantly by the VH4.2 gene (28.7% versus 4.5% for V3 mAbs). The remaining VH genes were used with various frequencies of 0.8% to 13.7% (Fig. 3D). This exceptional pattern of VH genes used by V3 mAbs suggests some antigenic restrictions.

4. Discussion

The study showed that rhesus macaque 25328 immunized with the C4–447 mimotope peptide responded with a broad repertoire of V3-specific mAbs that included 21 B cell lineages. The immunogen C4–447 contains a 16-mer truncated V3 peptide with a short 8-mer epitope RIHIGPGR for anti-V3 mAb 447–52D located at the tip of the native V3MN loop (17, 18). The macaque Ab response was most likely directed to the 447–52D epitope because the surrounding N-terminal amino acids (aa) RC and C-terminal AFYACG are not targeted by V3 mAbs (19).

The V3 immunogenic target in the C4–447 peptide could be different from the native V3 loop, which is partially shielded by glycans, resulting in human V3 mAbs that bind mainly to the V3 crown of the loop but not to its base. Therefore, the macaque mAbs may react to the 447-epitope and a few aa in the C-terminal part (AFYACG) making the cluster of epitopes a little longer than the 8 mer, at least theoretically. Either way, if mAbs bind only to epitope RIHIGPGR or to its extension RIHIGPGRAFYACG, the cluster of epitopes are overlapping, taking into account that the epitope is usually 5–8 aa long. This does not mean that the V3 Abs will compete with each other because each Ab can bind to a different virion.

The V3 region forms a loop due to the disulfate bridge connecting two Cysteines and has two clusters of epitopes recognized by human V3 mAbs. One cluster is located at the tip of the V3 loop consisting of approximately 13 aa, which includes the 447-epitope. The second cluster of epitopes is discontinuous and present in the N- and C-terminal sides of the V3MN loop consisting of seven key residues: RK-IHI-FY. This epitope is recognized only by mAbs using the VH5–51 gene (20). The 447 mimotope peptide is recognized by human V3 mAbs encoded by VH1, VH2, VH3, and VH4 family genes, but not by V3 mAbs using the VH5–51 genes (12). The macaque mAbs were encoded by the VH2, VH3, and VH4 genes corresponding to human mAbs gene usage, suggesting that they bind to 447 epitopes, and none of them used the VH5–51 gene.

The 21 macaque V3-specific B cell lineages directed to a small single antigenic site are larger than reported in other studies, which used, however, different biological materials. Various papers have described the generation of several recombinant mAbs using similar methods from one donor against one region or antigenic determinant larger than peptides (4, 6, 7, 10, 11). For example, 12 mAbs were produced against the RSV Fusion protein, and 13 mAbs against V2 region from a single donor (7, 10).

The reason for an extended repertoire of V3 mAbs in our experiment could be related to longitudinal immunization of six times over three years, a dose and nature of immunogen that is de-glycosylated, and the relatively strong immunogenicity of the C4–447 peptide used for vaccination. Ab maturation is based on point mutations in nucleotide sequences and is similar in humans and nonhuman primates (NHP). However, we observed an individual difference in a high number of insertions/deletions in mAbs derived from pre-bleed PBMCs, indicating a persistent increase in the process of natural affinity maturation in the immune repertoire of rhesus macaques.

This study additionally analyzed the VH germline gene usage by two groups of mAbs. Comparing the VH gene usage by V3 mAbs and pre-bleed mAbs with unknown specificity from the same animal, we observed that the frequency of usage of the VH2, VH3, and VH4 family genes was significantly different. This observation confirmed our previous observation of biased usage of VH genes in human mAbs against HIV envelope antigens produced from one donor compared to other mAbs with undefined specificities, a difference that was significant (2). These two observations indicate that selecting the VH genes coded for Abs is not random for certain antigens but could be preferential due to antigen restriction.

5. Conclusion

We described an extended repertoire of 21 B cell lineages produced from a single immunized rhesus macaque that targeted one antigenic site in the small cyclic C4–447 peptide. The definition of B cell lineages, which include single and clonally related recombinant Abs, was based on Ig and CDR3 heavy chains. This number of B cell lineages is higher than reported in other human or NHP studies and reflects the exceptional repertoire and potency of NHP Ab response to a particular peptide.

Supplementary Material

1

Acknowledgments

We thank Lily Liu for her technical assistance.

Funding

The study was supported by NIH grant R01 AI112546 (MKG), and ONPRC resource support from P51-OD011092, and U42-OD010246.

Footnotes

Declaration of Competing Interest

None of the authors declares conflicts of interest.

Declaration of interests

The authors declare that they have no known competing financial relationships that may influence the research work as a potential conflict of interest.

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