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Departments of
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Pediatrics,
Microbiology and Immunology, and
Preventive Medicine, Vanderbilt University Medical Center, Nashville, TN 37232; and
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RV virus protein (VP) 6 is the major structural component of RV, and it elicits a large portion of the Abs observed following infection in humans. RV VP7 is an outer capsid protein that elicits neutralizing Abs. We used RV VLPs to isolate single RV VP6- or VP7-specific B cells from previously infected or recently infected healthy adults and determined the sequences of the Ab VH and VL regions. We identified five immunodominant H chain variable gene segments used in the adult Ab response to RV. We then compared adult RV-specific Ab sequences with those of single RV-specific B cells derived from RV-infected infants or of randomly selected adult B cells. Our results show that the RV-specific B cell repertoire of infants exhibits adult-like levels of combinatorial and junctional diversity. These data suggest that VH repertoire biases are not limiting for virus-specific Ab responses early in life.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We used a novel method of generating human RV-specific B cell clones in culture after single cell sorting using green fluorescent protein (GFP)-labeled VLP, as recently described (12). For the studies described here, we obtained whole blood from five types of donor groups: group A, infants with acute RV infection; group B, symptomatic adults with RV exposure; group C, healthy adults, RV-specific cells; group D, healthy adults, RV-specific IgD- cells; group E, healthy adults, randomly selected B cells. The origins of B cell clones and the number of Ab sequences analyzed for each donor group and RV Ag are shown (see Table I). B cell clones were obtained from four infants, aged 2, 3, 7, or 11 mo (donors 1–4). Blood was obtained from two adult patients (donors 5 and 6) who were naturally exposed to RV and reported symptoms of RV disease within 1 mo prior. Blood for RV-specific B cell analysis from eight healthy blood donors (donors 7–14) was collected from leukocyte reduction filters as described (13). For randomly selected B cell controls, we obtained blood cells from leukocyte reduction filters from three healthy blood donors (donors 15–17) by the same method. We isolated PBMC by density centrifugation on lymphocyte separation medium and separated CD19+ cells with paramagnetic beads according to the manufacturer’s instructions (Dynal Biotech, Lake Success, NY). GFP-VLPs for single cell sorting were produced by coinfection of Sf9 insect cells with recombinant baculoviruses as published previously (14). The specificity of binding of RV VLPs to RV-specific B cells was previously established by specific binding to RV-specific (but not nonspecific) murine hybridomas (our unpublished data), to murine B cells induced by RV infection of mice (14, 15), and to human B cells induced by natural RV infection (12, 15). We stained magnetically isolated B cells with anti-CD19-PE, VP2/6 or VP2/6/7 GFP-VLP, and anti-IgD-PerCP as described (12). We performed flow cytometric analysis and sorting, and single RV VLP+/CD19+ cells were collected one cell per well into 96-well cell culture plates.
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For the expansion of single B cells, we used a B cell culture system that we previously described to amplify and identify RV-specific or randomly selected B cell clones (12). Briefly, 50,000 irradiated EL-4-B5 mouse thymoma cells (kindly provided by Dr. R. H. Zubler, Geneva University Hospital, Geneva, Switzerland) per well of 96-well culture plates were used as feeder cells immediately following single cell isolation. A combination of 100 U of recombinant human IL-2, 5 ng/ml PMA, and 10% (v/v) of supernatant from pokeweed mitogen-activated human T cells (T cell replacing factor) was added. The culture plates were incubated for 7 days, then we removed 100 µl of supernatant, and added 10,000 irradiated fibroblastic L cells stably transfected with human CD154 to each well (16). This cell line was kindly provided by DNAX via the American Type Culture Collection (CRL 12095; Manassas, VA). We also added 5 ng/ml recombinant human IL-4 in addition to the B cell culture media described above. The cultures were kept for another 2 wk with a second addition of CD154 fibroblasts, cytokines, PMA, and T cell replacing factor on day 14. Secreted human Ig or RV-specific Abs were detected by ELISA on day 14 or 21, respectively, to confirm the specificity of the B cell clone for RV before RT-PCR, as described (12). To maximize the identification of human Ig-producing wells secreting RV-specific Abs, we used anti-Ig reagents that detected Abs of any isotype. Therefore, the isotype of the Abs secreted by cell lines derived from single B cells was not determined. Although the VLPs used to select RV-specific B cells by FACS contained GFP, we used viral particles derived from wild-type RV for the ELISA detection of Abs in B cell supernatants to eliminate the possibility of detection of GFP-specific Abs.
RT-PCR amplification
For RT-PCR amplification of VH and VL regions we used a commercial oligo-dT based mRNA capture kit (mRNA Capture kit; Roche, Basel, Switzerland) and single tube RT-PCR method (Titan One Tube RT-PCR system) and a pooled primer mix. The VH and VL primer sequences used were designed to amplify all VH and VL gene segments in the VBASE complete database of genomic V gene sequences (17, 18). We further amplified the first-round PCR products using VH and VL nested primers hybridizing to framework regions (FR) 1 (forward) and FR4 (reverse) under the same conditions as above. The primers used and the amplification conditions are previously described (12).
DNA sequence analysis
We ligated gel-extracted PCR products into a TA cloning vector (Promega, Madison, WI), generated bacterial clones, then purified plasmid DNA from overnight bacterial cultures. Plasmid DNA was digested with restriction enzymes to identify clones with proper ligation of V regions. The nucleotide sequences of both strands of plasmid DNAs that contained a VH or a VL insert were determined using an automated DNA sequencer (Applied Biosystems, Foster City, CA).
Criteria for the analysis of Ab sequences
We analyzed VH or VL region sequences using DNAPLOT to search VBASE (http://www.mrc-cpe.cam.ac.uk) or the international ImMunoGeneTics database (http://imgt.cines.fr/) (18), reporting results with an updated nomenclature of the human Ig genes as recently summarized (19). The characteristics of individual sequences for VH and VL regions are shown in the supplemental material. 5 H chain complementarity determining region 3 (HCDR3) and L chain CDR3 (LCDR3) lengths were determined by counting amino acid residues starting at position 105 according to the ImMunoGeneTics numbering system. D gene segment assignments were performed by database alignment, and confirmed by manual inspection, using the genomic sequences of all D gene segments according to Corbett et al. (20). The assignment of D segments was defined using similarity with D segment germline sequences on the basis of 100% amino acid identity over a stretch of at least three consecutive amino acids. The reading frame was defined relative to the recombination signal sequence (21). For a definite assignment of a D or J segment, we required at least three consecutive amino acids to be identical to germline sequences. When two different D segment or D segment reading frame assignments were possible, based on these criteria, both segments and reading frames were used to calculate the frequency of use in this study. Two D assignments were possible in sequences from adult donors only in 11 of 100 instances. However, a D segment usage bias was not detected whether these assignments were included in the analysis or not. The number of altered amino acid residues in the N' or C' terminus of D segments within the HCDR3 was determined only in HCDR3 sequences in which a definite D segment assignment was possible. For the enumeration of mutated or added amino acid residues in the HCDR3 or LCDR3 regions, we included both nontemplated (N) and palindromic (P) nucleotides if these nucleotides encoded amino acids that differed from germline. Alterations that resulted solely in the loss of amino acids without mutated replacements were not included in the "Altered Amino Acids" category. However, such events would contribute to changes in CDR3 lengths, which were calculated separately. Insertions and deletions within the CDR3 were counted as changes if they resulted in altered amino acids in the CDR3.
Statistical analysis of genetic data
Fisher’s exact tests were used to examine overall differences in proportions of RV VP6- and VP7-specific frequencies of VH, D, JH, family use, VH gene segments, and V
, J, V
and J family use in random or RV-specific human B cell clones from adults and infants. Comparisons of LCDR3 and HCDR3 amino acid lengths were performed using ANOVA techniques. Logistic regression was used to examine the proportion of D assignments possible, number of VL gene segments analyzed, and the proportion of L chain used. Exact logistic regression techniques were required for analysis of the number of JH segments containing mutations because of the relatively small number of JH assignments possible. All analyses were performed using SAS version 8.2 (SAS Institute, Cary, NC).
Expression and purification of recombinant Abs
Detailed methods for the expression and purification protocols used were previously described (12). Briefly, RV-specific VH and VL segments were ligated in step-wise fashion into the linearized pComb3X
19 vector that contains both CH1 and C regions in-frame with the VH and VL inserts. The sequences of the constructs were verified by nucleotide sequence analysis and transformed into nonsuppressor strain HB2151 Escherichia coli cells to produce soluble Fab. To express the Fab Abs, a fresh bacterial colony containing a Fab phagemid was inoculated into 2XYT broth supplemented with 100 µg/ml ampicillin and 2% glucose and grown overnight at 30°C. The overnight culture was then diluted 1/10 into fresh medium and grown until it reached an OD600 of 0.6. Protein expression was induced by resuspension in fresh 2XYT broth supplemented with ampicillin and 50 µM isopropyl 
-D-thiogalactoside. The culture was grown at 30°C for 3 h and then cells were collected by centrifugation. To harvest the Fab, the periplasm was extracted by addition of TES buffer (10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS). Fabs were purified from the periplasmic extract using a Ni-NTA agarose system (Qiagen, Valencia, CA). The eluted purified Fab products were then dialyzed against PBS. The quantity of purified Fab in each preparation was determined by a sandwich ELISA in which Fab concentrations were determined from comparison of experimental OD values to that of varying dilutions of a purified Fab standard (The Jackson Laboratory, West Grove, PA).
RV-specific ELISA binding assay using recombinant Abs
To verify that the recombinant Fab Abs using the immunodominant VH segments bound RV Ag, we performed an ELISA using purified RV particles. Immulon II HB 96-well microplates (Dynex Technologies, Chantilly, VA) were precoated with either purified whole virus particles, or purified VLP 2/6 particles (particles lacking VP7 and VP4) 1/500 in carbonate buffer overnight at 4°C. The plates were blocked with 0.1% BSA and 0.1% Tween 20 in PBS for 2 h at room temperature. After the block was removed, the recombinant Abs were diluted serially 4-fold, as was a positive control human serum, and incubated for 1 h. After washing, a solution containing alkaline-phosphatase conjugated goat anti-human F(ab')2-specific polyclonal Abs (The Jackson Laboratory) at a 1/500 dilution in block was added and incubated for 1 h. Bound Abs were detected with p-nitrophenyl phosphate, disodium substrate (Sigma-Aldrich, St. Louis, MO) and the OD was determined using a Molecular Devices plate reader (Sunnyvale, CA).
Specific binding of recombinant Abs to RV-infected cells by immunofluorescence
We next demonstrated that recombinant human Fabs using each of three immunodominant VH gene segments bound to authentic RV proteins in infected cells but not to uninfected cells. Virus-infected cells used in the immunofluorescent staining procedure were obtained by infecting MA104 cell culture monolayers in 96-well tissue culture plates with 100 µl of serially 10-fold diluted suspensions of virus strain RRV after trypsin activation using 20 µg of trypsin/ml. Virus was adsorbed by centrifugation at 1000 x g at room temperature for 1 h. Wells were washed once with serum-free DMEM, and then 100 µl of serum-free DMEM containing 4 µg of trypsin/ml was added. The plates were incubated at 37°C for 15 h, after which the culture medium was removed by aspiration. The cells were fixed with 150 µl/well of ice cold 80% acetone for 20 min at -20°C. Acetone was removed and the wells were dried completely. Mock-infected (negative control) cell monolayers were processed in the same fashion. The staining reagent used as a positive control (RV-specific guinea pig serum, kindly provided by Dr. R. Ward, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH), was diluted 1/800 in 5% nonfat dry milk, added to the wells, and then incubated for 1 h. The wells were washed with PBS and an anti-guinea pig Ig Alexa 568 conjugate (Molecular Probes, Eugene, OR) was added at 1/500 in 5% nonfat dry milk for 1 h. Wells were washed again and fluorescence was visualized. The staining procedure for the recombinant Fabs was similar. The purified Fabs were diluted 1/4 in 5% nonfat dry milk and incubated for 1 h. After washing with PBS, a mouse anti-hemagglutinin tag Ab (Sigma-Aldrich) was diluted 1/500 in milk and incubated 1 h. After washing, an anti-mouse Ig Alexa 568 conjugate (Molecular Probes, Eugene, OR) Ab was added at a 1/500 dilution in milk for 1 h. A similarly prepared purified recombinant human Ab to respiratory syncytial virus with the same Ag tags was used in replicate wells as a control to rule out nonspecific binding of the Fab preparations (data not shown). Fluorescent foci were visualized on a Nikon Eclipse TE300 fluorescence microscope (Melville, NY). Photomicrographs were obtained at dilutions for each virus strain that resulted in large numbers of separated infectious foci.
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Results |
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The number of RV-specific B cells sorted and the yields of ELISA and RT-PCR methods are shown in Table I. We calculated the overall efficiency of amplification of variable gene sequences from Ig-positive and Ag-positive ELISA wells as a percentage of total sorted B cells for each Ag and donor population.
Infants (donors 1–4).
This method generated a total of 11 VP7-specific H chain, 11 VP7-specific L chain, 3 VP6-specific H chain, and 2 VP6-specific L chain V region Ab genes from RV-infected infants for analysis. The overall efficiency for RV VP7-specific VH segments was 11 confirmed clones amplified of 2359 B cells sorted (0.5%) and for RV VP6-specific VH segments 3 confirmed clones amplified from 2359 sorted B cells (0.1%). The efficiency data for RV VP7- or VP6-specific VL segments were 11 of 2359 sorted B cells (0.5%) and 2 of 2359 sorted B cells (0.1%), respectively. Infant B cells did not appear to proliferate or differentiate in culture as well as adult B cells in this study. The decreased proliferation likely reflects the fact that some infant samples were rested in vitro for several hours or overnight, because those patients presented to the emergency department at a time of day when sterile flow cytometry sorting services were not immediately available. In contrast, all adult cell selections were performed with cells immediately after phlebotomy. It is possible that the difference in efficiency between adults and infants may be due to intrinsic limitations in the ability of infant B cells to proliferate in culture. However, subsequent experiments for the expansion of neonatal or young infant B cells of another specificity that were performed only with freshly isolated cells showed no difference in efficiency of proliferation and differentiation of infant and adult cells (data not shown).
Adults (donors 5–17).
The frequency of isolated RV VP6- or VP7-specific sequences is shown in Table I. The frequency of RV-specific B cells was low in healthy adult volunteers who were not recently infected (5 in 10,000 B cells). In contrast, RV-specific cells were found at an 
10-fold higher frequency in infants or adults who were recently infected, or in IgD- cells of healthy volunteers. The recovery of RV VP7-specific VH and VL segments following expansion was four to five times more efficient in recently RV-exposed adults than in healthy adult blood donors or RV-infected infants. We analyzed a total of 30 RV-specific VH and 39 RV-specific VL segments from recently RV-exposed adults (donors 5 and 6) and 62 RV-specific VH and 46 RV-specific VL segments from healthy adult blood donors (donors 7–14). For the control group (donors 15–17), a total of 83 VH and 46 VL segments from randomly selected (not virus-specific) B cell clones was analyzed. For the analysis of somatic mutations in the CDR1 and CDR2 and the three FR regions in randomly selected B cell clones, 77 VH segments were examined.
The repertoire in randomly selected adult B cells was VH3-dominated
We first expanded randomly selected CD19+ B cells from three healthy donors and determined the nucleotide sequences of VH segments from 83 B cells. In randomly selected B cells, 50% of all VH segments belonged to the VH3 family (Fig. 1, A and B), a frequency that was very similar to the VH frequencies of directly amplified randomly selected B cells reported by others (20, 22). The distribution of D and JH gene segment use in randomly selected B cells (Fig. 1, C and D) was similar to that described previously (20, 22). As shown in Fig. 2A, the most frequently observed VH segment in randomly selected B cells in our study was VH3–23 (average of 10% (range 3–19%)). These data show that the culture amplification method used does not significantly bias or alter the frequency of VH segments recovered.
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We found that RV-specific B cells exhibited a strong VH bias that differed from that of randomly selected B cells. Forty-one percent (11 of 27) of RV VP7-specific B cells from recently RV-exposed and 50% (8 of 16) of RV VP7-specific B cells from healthy blood donors expressed VH gene segments belonging to the VH4 family. The frequency of all VP7-specific adult clones belonging to VH4 was 44% (19 of 43; Fig. 1A). We also observed a marked VH4 bias for RV VP6-specific B cells with a frequency of 71% (22 of 31; Fig. 1B). This difference was statistically significant for both RV VP7- and VP6-specific clones in comparison with the 20% (17 of 83) VH4 frequency in randomly selected, nonspecific B cells (p = 0.030 and p < 0.001, respectively). Therefore, an overall VH4 family bias was observed in RV-specific clones (Fig. 1, A and B). In addition to the VH4 family predominance, the most frequently used individual VH segment in VP7-specific adult B cells overall was segment VH1–46 (14%, 6/43) (Fig. 2A). The most frequently used VH segment in RV VP6-specific adult B cells was VH4–31 (23%. 7/31) (Fig. 2B). These VH segments were used infrequently in randomly selected B cells. Thus, adult RV-specific B cells showed a strong VH1/VH4 bias that greatly differed from the random VH usage.
RV-specific B cell clones from infants also demonstrated a VH1/VH4 bias and a relative paucity of VH3 family usage
The relative underrepresentation of VH3 family members in RV-specific adult B cells compared with randomly selected B cells was also observed in infant B cells. The VH4 bias in infants was similar to that in adults, with 44 vs 36% VH4 (for RV VP7) or 71 vs 67% VH4 (for RV VP6) in adults vs infants, respectively. As shown in Fig. 1A, 55% (6 of 11) of RV VP7-specific VH segments in infants belonged to the VH1 family, a strong bias that was consistent with the highly increased use of segment VH1–46 in RV VP7-specific VH segments in adults (Fig. 2A).
Infants and adults both showed a frequent usage of the VH1–46 segment in RV VP7-specific B cells
The VH1–46 gene segment, rarely used in randomly selected B cells (0 of 83 randomly selected adult B cells in our study), was highly overrepresented in adult RV VP7-specific B cells. Infants also used the VH segment 1–46 more often than expected by chance alone, with 2 of 11 (18%) infant RV VP7-specific B cell clones using that segment (Fig. 2A). The VH1–46 segment was not overrepresented in randomly selected B cells in previously reported second trimester liver or cord blood cDNA libraries (9). These data suggest that the VH1–46 segment is a dominant component of both adult and infant RV-specific responses. Other dominant VH gene segments were observed in RV VP6- or VP7-specific B cells in adults, such as VH4–31, VH4–61, VH4–39, and VH4-30-4 (Fig. 2).
The distribution of RV-specific D and JH gene segments was similar in adults and infants
We analyzed in detail the D and JH gene segment usage in all populations, but significant differences between groups were not detected. In nonspecific adult B cells or RV-specific B cells from adults or infants, we observed a uniform bias toward the D3 and JH4 families (Fig. 1, C and D). These data are consistent with previously identified usage frequencies of randomly selected B cells in other studies.
Infants and adults shared a similar distribution of Ab L chain gene segments in RV-specific B cells
We analyzed 11 RV VP7- and 2 RV VP6-specific L chain sequences from RV-infected infants. These sequences were compared with 53 RV VP6- or RV VP7-specific L chain sequences from recently RV-exposed or healthy adults, 24 RV VP6-specific L chain sequences from adult memory (IgD-) B cells, and 46 L chain sequences from randomly selected B cells. The distribution of V, J, and J families in randomly selected B cells did not differ from that of RV-specific B cells from infants or adults (all p values 
0.05). RV-specific B cells from infants showed a trend toward overrepresentation of the J family 4 in contrast to RV-specific B cells from adults and randomly selected B cells that demonstrated a V family 1 bias. The V distribution was different between randomly selected and RV-specific B cells from adults (Fig. 3C, p = 0.018), suggesting a possible minor selection bias associated with particular V segments but a specific segment association could not be discerned.
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We analyzed the adult and infant RV-specific V-D and D-J junctional sequences for evidence of the functional maturity of insertion or excision mechanisms in infants (Table II). The mean frequency of mutations at the N- and C-terminal ends of D segments was similar in all donor groups. Templated P insertions were observed in both infant and adult junctional sequences. The mean amino acid length of D segments did not differ between adult and infant sequences. Similarly, we found that the mean total HCDR3 length and the mean frequency of the hydrophobic reading-frame usage for D segments did not differ between adults and infants, when analyzed as a group (all p values 0.05; Table II). Interestingly, however, all three assignable D segments of B cells derived from the youngest infant (donor 1, 2-mo-old) were encoded by the hydrophobic D reading frame, which is less commonly used in functional Abs in the expressed repertoire (23).
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vs L chain usage was similar in infant and adult RV-specific B cells.
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The highly significant association of particular gene sequences with RV-specific B cells, which differed from those of random B cells, suggested that these segments encoded RV-specific Abs. To verify this association, we expressed and purified recombinant Fab Abs from representative adult clones (donor groups C or D) for each of three immunodominant VH gene segments, VH1–46, VH4–31, and VH4–61 and tested whether they bound to RV Ags in infected cells or to virus particles. We found that each of the Abs bound specifically to purified RV particles, in a dose-dependent manner, using ELISA (Fig. 4). These Abs also bound to RV-infected cell monolayers in an immunofluorescent assay (Fig. 4) but not to uninfected cell monolayers (Ref.12 and data not shown); similarly prepared Abs to another virus did not bind RV-infected monolayers (Ref.12 and data not shown). These studies showed that the physical sorting, ELISA binding confirmation, and molecular cloning methods used to determine immunodominant gene segments that were amplified from putative RV-specific B cells actually did specify RV-specific Abs.
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Discussion |
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RV-specific repertoire in infants
We showed that human infants use sufficiently diversified Ab repertoires to make adult-like Ag-specific B cell responses. The combinatorial diversity of infant RV-specific clones was similar to that of adults, in that they exhibited a VH1 and VH4 dominance. Infants shared a high frequency of use of the dominant VH segment used by adults, VH1–46. Thus, the analysis of the VH family and VH segment use suggests that infants share the strong Ag selection bias seen in adults. It was previously speculated that infant combinatorial diversity was restricted, reflecting repertoire skewing similar to that noted in fetal repertoires. Such repertoire restriction has been hypothesized to limit the structural and functional quality of human infant Abs. Our data show that combinatorial diversity did not appear to be limiting for RV-specific responses in these four infants who ranged from 2 to 11 mo of age. This finding suggests that improved immunogens or multiple antigenic exposures might induce Ab responses with enhanced protective capacity in this age group. Previous work on random B cell repertoires suggests that variable gene repertoire biases noted during fetal life may resolve during the first few months of life. Therefore, future studies of virus-specific responses following fetal or neonatal infection are warranted. Nevertheless, given that disease caused by RV infection occurs predominantly in the age group that we studied, the lack of the repertoire limitations in the RV-specific response that we describe in the age group studied is significant.
RV-specific repertoire in adults
To compare infant and adult Ag-specific repertoires, we first determined the dominant VH gene segments used by adults in the response to RV VP6 and VP7 proteins, which were found to be VH1 and VH4 family members. For RV VP7-specific B cells, the predominant gene segment used was VH1–46 in both adults and infants. Segment VH1–46 was observed in only 1 of 31 RV VP6-specific IgD-unselected B cell clones from adults and was not observed in any of the nonspecific B cell clones, and thus may represent an important variable gene for RV VP7-specific Ab responses. However, 6 of 18 IgD- RV-specific clones obtained by sorting with VP2/6 VLPs also used the VH1–46 segment. This finding suggests that the use of this segment may also be associated with VP6 binding if somatic hypermutations are present. This possibility is supported by the apparent binding of an Fab expressed from cDNA obtained from a VP2/6-sorted VH1–46 Ab to VP2/6 double-layered particles in ELISA (see the VH1–46 Fab in Fig. 4). There is limited previous data to support the association of particular VH segments with preferential binding to Ags, such as the Haemophilus influenzae, Streptococcus pneumoniae, or Cryptococcus neoformans polysaccharides (24, 25, 26, 27, 28). All of these associations of polysaccharide Ags with particular VH gene segments were made with VH3-origin Abs, mostly with the VH3–23 segment that is also the dominant segment in randomly selected B cells.
We found a strong association of RV specificity with VH1 and VH4 segments. The only other published sequences of human RV-specific Abs were derived from phage display combinatorial libraries (29). In that work, investigators described two RV VP6-specific libraries with distinct epitope recognition, one of which derived from the 4–31 germline gene. Although our methods were different, it is interesting that we also identified VH4–31 as a dominant VH segment in RV VP6-specific B cell clones. In contrast to our finding of VH1 and 4 associations with RV specificity, we did not find a particular association of D, JH, V, J, V, or J segment usage in RV-specific clones that was shared by adults and infants.
Junctional diversity is similar in adult and infant RV-specific Abs
Junctional diversity, representing P or N insertions, the latter mediated by TdT, or excision events, were observed at similar frequencies in infants and adults. Both populations used CDR3s of similar lengths to make RV-specific Abs, consistent with recent analyses of random B cell HCDR3 length in which premature infant B cell repertoires showed progression of HCDR3 length during the third trimester (30). Infants appear to achieve maturation of HCDR3 length in random B cells by 2 mo of age (11), and we show the same applies to Ag-selected B cells. Marked developmental differences have been noted in the past between fetuses and adults in these characteristics. Adults use larger Ag binding site structures (specified by longer HCDR3s) than those used in fetal tissues or early in infancy (11, 31, 32). Fetal B cells use short D segments and a paucity of N additions (11, 33, 34). In human fetal or cord blood B cells, N-region addition can be observed but only at low frequency, and these additions increase with age (11, 32, 35, 36). CDR3 length is of interest because longer D and JH segments lead initially to more complex HCDR3 regions (9), and previous studies suggest high affinity is linked to optimal CDR3 length (37). Previous investigators showed that human fetuses and preterm infants preferentially use short D segments (11, 31, 32, 35). Infants did not use unusually short D segments or high frequencies of the hydrophobic or germline stop reading frames to encode virus-specific Abs in our study, except for the clones derived from the youngest infant (donor 1, see supplemental Table S II). We also analyzed junctional diversity in RV-specific L chain genes. We found that infant sequences differed from those of RV-specific adult IgD- B cells only in that the infant sequences were one amino acid longer, a minimal difference. Thus, maturity of the mechanisms mediating junctional diversity, D segment selection, and CDR3 diversification were not limiting in infants. Our studies cannot rule out limited diversity mediated by these mechanisms in neonates (the first month of life), because the infant B cell clones we obtained were from subjects of at least 2 mo of age. Nevertheless, given that disease caused by RV infection occurs predominantly in the age group that we studied, the lack of these repertoire limitations in this age group is significant.
In conclusion, we determined the molecular basis of human B cell responses to RV, and found an unusual VH1 and VH4 family dominance, with particular segments within these families especially preferred. We demonstrated that human infant antiviral responses are not limited by repertoire skewing or lack of junctional diversity. In this regard, our data show that infant B cell repertoires, in contrast to previous speculation, are much closer in character to adult repertoires than fetal repertoires.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
3 Address correspondence and reprint requests to Dr. James E. Crowe, Jr., Department of Pediatrics, Division of Pediatric Infectious Diseases, Vanderbilt University Medical Center, D-7235 Medical Center North, 1161 21st Avenue South, Nashville, TN 37232-2581. E-mail address: james.crowe{at}vanderbilt.edu
4 Abbreviations used in this paper: RV, rotavirus; VLP, virus-like particle; VP, virus protein; GFP, green fluorescent protein; FR, framework region; CDR3, complementarity determining region 3; HCDR3, H chain CDR3; LCDR3, L chain CDR3.
5 The on-line version of this article contains supplemental material.
Received for publication February 20, 2003. Accepted for publication August 18, 2003.
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47 integrin expression but does not require IgA antibody production. J. Immunol. 166:1894.47. Virology 305:93.[Medline]
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 K. Kolibab, S. L. Smithson, B. Rabquer, S. Khuder, and M. A. J. Westerink Immune Response to Pneumococcal Polysaccharides 4 and 14 in Elderly and Young Adults: Analysis of the Variable Heavy Chain Repertoire Infect. Immun., November 1, 2005; 73(11): 7465 - 7476. [Abstract] [Full Text] [PDF]
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J.-H. Weitkamp, N. L. Kallewaard, A. L. Bowen, B. J. LaFleur, H. B. Greenberg, and J. E. Crowe Jr VH1-46 Is the Dominant Immunoglobulin Heavy Chain Gene Segment in Rotavirus-Specific Memory B Cells Expressing the Intestinal Homing Receptor {alpha}4{beta}7 J. Immunol., March 15, 2005; 174(6): 3454 - 3460. [Abstract] [Full Text] [PDF]
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N. Parez, A. Garbarg-Chenon, C. Fourgeux, F. Le Deist, A. Servant-Delmas, A. Charpilienne, J. Cohen, and I. Schwartz-Cornil The VP6 Protein of Rotavirus Interacts with a Large Fraction of Human Naive B Cells via Surface Immunoglobulins J. Virol., November 15, 2004; 78(22): 12489 - 12496. [Abstract] [Full Text] [PDF]
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, donor group E) or RV-specific clones from adults (
, donor groups B, C, and D combined) or infants (
, donor group A). The number of B cell clones and donors for each of the groups analyzed is shown in Table I
) or infants (
. Above the dotted line is the positive control, which was human immune serum diluted 1/100 and then serially diluted 4-fold. The dilution is shown in log scale on the x-axis. Below the dotted line are the recombinant Fab Abs expressed using three of the immunodominant VH segments, followed by a negative control Fab Ab that is directed against a heterologous virus. The concentration of recombinant Fab Ab present is shown in log scale along the x-axis.