Characterization of germline antibody libraries from human umbilical cord blood and selection of monoclonal antibodies to viral envelope glycoproteins: Implications for mechanisms of immune evasion and design of vaccine immunogens

2.1. Cord blood, viral proteins, human two-domain soluble CD4 (sCD4) and antibody

Cord blood was received from the National Disease Research Interchange (NDRI, Philadelphia, PA), and care was taken not to contaminate the cord blood samples with maternal blood. SARS CoV RBD [12] and human sCD4 were produced in our laboratory. HIV-1 Env gp120_Bal [13] was provided by T. Fouts (Institute of Human Virology, Baltimore; currently at Profectus, Baltimore, MD). Soluble Hendra virus envelope glycoprotein G (sG) [14] and gp140_JRFL were provided by C. Broder (Uniformed Services University of the Health Sciences, Bethesda, MD). The gp140_Con-s [15] was provided by B. Haynes and H. Liao (Duke University, Durham, NC). Horseradish peroxidase (HRP)-conjugated mouse anti-M13 antibody was purchased from Sigma–Aldrich (St. Louis, MO).

2.2. Library construction

As a source for amplification of antibody gene fragments, cDNA was prepared from the cord blood of two babies [16] and libraries were constructed using phagemid pComb3x as previously described [17].

2.3. Analysis for sequence diversity of the libraries

The heavy (VH) and light (VL) chain variable domains of antibodies were sequenced using the standard Sanger sequencing method. Resulting sequences were sorted and trimmed to produce independent pools of heavy and light chain sequences, and the latter were further sorted into kappa (V_κ) and lambda (V_λ) light chains by searching their translated sequences for a set of mutually-exclusive motifs. These sequences were analyzed using the program JoinSolver [18], and those which it found to be the result of productive rearrangement and likely to yield functional products formed the data sets for further analysis. Using the outputs from JoinSolver, germline diversity, the heavy chain complementarity determining region 3 (HCDR3) lengths, and the number of mutations compared to the closest germline match from the V(D)J families were obtained for each sequence. Translated heavy and light chain variable segment library sequences were aligned using ClustalX2 [19] and trimmed to equivalent lengths within each group: V_H codons 3–91, V_κ codons 9–88, and V_λ codons 3–88. Functional V_H, V_κ, and V_λ translated germline sequences in the NCBI IgBLAST database (http://www.ncbi.nlm.nih.gov/projects/igblast) were similarly treated. Separate identity matrices for each stream were generated using BioEdit [20] and the number of amino acid (AA) changes between each query sequence and its closest germline match, as found by JoinSolver, were determined.

2.4. Selection of antibodies against HIV-1 Envs, SARS CoV RBD and Hendra virus sG

The combined cord blood libraries were used for selection of antibodies against antigens conjugated to magnetic beads (Dynabeads M-270Epoxy; DYNAL Inc., New Hyde Park, NY) as described previously [8]. Antigen was present in the amounts of 5, 5, 2.5 and 1 μg for the first, second, third, and fourth rounds of panning, respectively. Specific enrichment was determined by using polyclonal phage-based ELISA (ppELISA). Briefly, ppELISA was performed by using Corning high-binding 96-well plates coated with 1 μg/ml of antigen and blocked with 3% non-fat dry milk in PBS (MPBS). The microplate wells were inoculated with 50 μl per well of MPBS containing 10¹⁰ PFU of pooled phage purified from each round of panning for 2 h at room temperature. Following 4 washes with PBS containing 0.05% Tween 20 (PBST), bound phage was detected by adding 50 μl of 1:5000 diluted HRP-conjugated mouse anti-M13 antibody (Sigma, St. Louis, MO) to each well. Following incubation for 1 h at room temperature, the plates were washed 4 times with PBST and the assay was developed at 37 °C with ABST substrate (Roche, Indianapolis, IN) and absorbance monitored at 405 nm. Clones that specifically bound to antigens were identified from the fourth round by using monoclonal phage ELISA (mpELISA) as described [8].

2.5. Expression, purification and binding of antibodies

Soluble antigen-binding fragments (Fabs) of antibodies were expressed, purified, and their binding activity was measured as previously described [21].

3.1. Construction of two human antibody libraries by using phagemid vector pComb3x

Two relatively large (6.7 × 10⁸ and 7.8 × 10⁸ members) phage-displayed human IgM Fab libraries (designated ml32 and ml33, respectively) were constructed from 50 ml of cord blood from each of two babies as described in Section 2. To estimate the sequence diversity of the libraries, 25 and 22 clones were randomly selected from ml32 and ml33, respectively, and sequenced. No identical sequences of heavy and light chains were found; more than 80% of the clones showed productive V(D)J rearrangements. Of the 25 m132 clones, 14 had V_κ light chains (families KV1–4) and 11 had V_λ light chains (families LV1–3, LV6–8 and LV10). Their heavy chains were also widely distributed (families HV1–5). Similar gene usage and family distribution were observed with m133. These results indicate that the cord blood libraries could have a high level of sequence diversity and contain antibody gene fragments from most of the families of both heavy and light chains.

3.2. High level of sequence diversity and low level of somatic diversification of antibodies randomly selected from the libraries

To more accurately estimate the diversity of the cord blood libraries, an additional 2112 clones from ml32 and ml33 were sequenced. Productive, full-length rearranged VH and VL sequences from each library were identified. Similarity between the sequences from the VH and VL repertoires was calculated, and 747 (99.2%) and 792 (99.5%) V_H sequences from m132and m133, respectively, were found to be unique (Table 1 ). The diversity of VL was significantly lower than that of VH; 70.1% and 88.5% for V_λ and V_κ of VL domains from m132, and 71.1% and 84.9% for V_λ and V_κ of VL domains from m133 were identified as unique. When the sequences from both libraries were combined, 1549 (99.3%), 780 (83.5%) and 745 (62.0%) sequences of V_H, V_κ, and V_λ, respectively, were unique. The combined cord blood antibody repertoire was then analyzed for gene usage, HCDR3 length, and somatic mutations compared to the closest germline counterparts. The heavy chain V (HV) regions were found to derive from all 7 HV germline families (Fig. 1 A), and contained representatives from 45 subfamilies found in the IMGT database [22]. All heavy chain D (HD) and J (HJ) gene families were found but in different proportions (Fig. 1B and C). The lengths of HCDR3 ranged from 6 to 26 amino acid residues and the mode was a HCDR3 length of 14 (Fig. 1D). For V_κ, the V regions, KVs, were identified from gene families KV1–6 but KV7 was absent (Fig. 1E); the J regions, KJs, were distributed among all five gene families, KJ1–5 (Fig. 1F). For V_λ, all the V regions, LVs, were present except for three LV gene families, LV4, LV10 and LV11 (Fig. 1G); the J regions, LJs, were mostly distributed in LJ1–3 gene families while LJ4 and LJ5 were absent (Fig. 1H).

To determine the level of somatic mutations of the antibodies, each sequence from the combined cord blood repertoire was compared to its closest germline and the number of amino acid (AA) mutations was calculated. Of the HVs, 21.5% were the same as germline sequences; 39.2% of the sequences contained only one AA mutation; the remaining 39.3% had two or more AA mutations, but very few sequences with more than 8 mutations were found (Fig. 2 A). In contrast, the light chains were significantly less mutated, with 57.6% and 73.0% of the KVs and the LVs, respectively, giving translations identical to germlines. The numbers of mutations occurring in the J regions of heavy and light chains were also calculated. Similarly to what was observed with the variable regions, relatively more mutations were observed in the HJs than in the KJs and LJs (Fig. 2B). The mutations in D regions were not estimated because of the difficulties in determining the D gene germlines for many antibodies due to the events of recombination involving insertion and deletion. These results suggest that the sequence diversity of the libraries is likely to be high although the light chains appear to be less diverse than the heavy chains, and the libraries exhibit low levels of somatic hypermutations.

3.3. Lack of human germline antibodies binding to the exposed epitopes on HIV-1 Envs

Previously, it was noted that the known bnAbs against HIV-1 were highly divergent from their germline sequences in contrast to bnAbs against SARS CoV and henipaviruses which were less diversified, possessing a limited number of mutations. In addition, it was found that germline-like predecessors of several HIV-1 bnAbs lack measurable binding to HIV-1 Envs [1]. Based on these findings, we hypothesized that there is a scarcity or absence of high affinity binders to HIV-1 Envs in the minimally somatically-hypermutated human cord blood antibody repertoire, whereas high affinity antibodies against other human infectious agents including SARS CoV and henipaviruses can be easily found. To test this hypothesis, the combined cord blood library of ml32 and ml33was panned against gp140_Con-s, a synthetic Env designed by aligning the consensus Env sequences of group M [15], and the Hendra virus protein sG in parallel. After four rounds of panning, enrichment and binding specificity of pooled phage were determined by ppELISA. Interestingly, significant and specific enrichment was observed with selection against sG only (Fig. 3 ). In a separate experiment, the mixed library was panned against another Env, gp120_Bal, and simultaneously the RBD. In line with previous observations, no marked enrichment was achieved with panning against gp120_Bal while considerable and specific enrichment was seen when the library was panned against the RBD (Fig. 3). These results support the hypothesis and suggest that HIV-1 Envs could have evolved strategies to avoid binding to the germline repertoire by presenting epitopes unsuitable for minimally somatically-diversified human antibodies to bind with, thereby denying them the chance to bind with high affinity. This strategy may represent a way exploited by the virus to escape from strong immune responses.

3.4. The selected antibodies against SARS CoV RBD are very close to germlines in sequence and have relatively high binding activity

To determine the extent to which antibodies against SARS CoV selected from the cord blood library are mutated, a number of individual clones with high affinity for the SARS CoV RBD were identified by mpELISA as described in Section 2. Sequencing of these clones revealed them to be identical. This unique clone, designated m390, contains gene products of HV3–30 and KV2–28 in the heavy and light chain V regions, respectively, (Fig. 4 A). M390 has only 2 AA mutations each in the V regions of both heavy and light chains and no mutations in the D and J regions. The purified soluble m390 Fab bound to SARS CoV RBD with an EC₅₀ of about 50 nM while no binding to an irrelevant antigen gp120_Bal was observed suggesting the specificity of the binding (Fig. 4B). Previously, vesicular stomatitis virus (VSV) specific antibodies with V_HQ52/V_κ19–28 gene combination elicited in a mouse model were found devoid of somatic mutations but showed high binding avidities [23]. These results are in line with our previous findings and provide supporting evidence for our hypothesis that extensive somatic hypermutations could be a unique feature for bnAbs against HIV-1 and perhaps other viruses causing chronic infections.

In summary, panning of a large phage-displayed minimally somatically-diversified antibody library derived from human cord blood against the HIV-1 Envs did not yield any significant binders. However, considerable specific enrichments were seen after panning against the SARS CoV RBD and Hendra virus sG, yielding an antibody against the RBD whose sequence is close to its parent germline. As we used the whole Envs in this study, one would expect the binding activity due to highly immunogenic epitopes such as the V3 loop which actually could bind and initiate immune response though it may be capable of diverting immune responses from the conserved epitopes. Therefore, the fact that there is no relatively high affinity binding of Envs might indicate that immune responses may not be so fast or in general are different than in the case of the other viruses. These results provide further evidence supporting and expanding the hypothesis that HIV-1 uses a strategy based on absent or weak binding of its conserved epitopes to germline antibodies for escaping neutralizing immune responses.