The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Margolin, D. H.
Right arrow Articles by Letvin, N. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Margolin, D. H.
Right arrow Articles by Letvin, N. L.
The Journal of Immunology, 2006, 177: 1108-1119.
Copyright © 2006 by The American Association of Immunologists

Germinal Center Function in the Spleen during Simian HIV Infection in Rhesus Monkeys1

David H. Margolin2,*, Erika H. Saunders3,*, Benjamin Bronfin*, Nicole de Rosa4,*, Michael K. Axthelm{dagger}, Olga G. Goloubeva{ddagger}, Sara Eapen{ddagger}, Rebecca S. Gelman{ddagger} and Norman L. Letvin5,*

* Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; {dagger} Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR 97006; and {ddagger} Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Infection with HIV-1, SIV, or simian HIV is associated with abnormalities in the number, size, and structure of germinal centers (GCs). To determine whether these histopathologic abnormalities are associated with abnormalities in Ab development, we analyzed nucleotide sequences of Igs from splenic GCs of simian HIV-infected macaques. Virus-specific GCs were identified in frozen splenic tissue sections by inverse immunohistochemistry using rHIV-1 gp120 as a probe. B cells from envelope-specific GCs were isolated from these sections using laser capture microdissection. Their Igs were amplified from cDNA using nested PCR, then cloned and sequenced. Nucleotide sequences were recovered from nine multimember clonal lineages. Within each lineage, sequences had similar V-D-J or V-J junctions but differed by somatic mutations distributed throughout the variable domain. The clones were highly mutated, similar to that previously reported for HIV-1-specific human IgG Abs. The average clone had 37 mutations in the V region, for a frequency of 0.11 mutations/base. The mutational pattern was strikingly nonrandom, with somatic mutations occurring preferentially at RGYW/WRCY hotspots. Transition mutations were favored over transversions, with C->T and G->A replacements together accounting for almost one-third of all mutations. Analysis of replacement and silent mutations in the framework and CDRs suggests that the Igs were subjected to affinity selection. These data demonstrate that the process of Ab maturation is not seriously disrupted in GCs during the early stages of immunodeficiency virus infection, and that Env-specific Igs developing in GCs are subject to extensive somatic mutation and profound selection pressures.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Striking alterations in B lymphocyte populations occur in secondary lymphoid tissues of HIV-1-infected humans (1) and SIV-infected monkeys (2). B cells in lymph nodes and spleens become activated early in the course of infection, resulting in increases in the size and number of secondary lymphoid follicles and in hyperplasia of germinal centers (GCs)6. As immunodeficiency progresses, the GCs are reduced in size and number. Because lymphoid follicles are a crucial site for the clonal expansion and terminal differentiation of Ag-specific B cells, virus-induced abnormalities in secondary lymphoid tissues could have important consequences for the immune responses of these cells.

Critical processes occur in lymphoid follicles that lead to the generation of mature Ab responses. An iterative process of selection, expansion, mutation, and further selection culminates in the generation of high-affinity memory B lymphocytes and plasma cells. Specific features of the somatic mutations observed in expressed Igs have been well-characterized (3, 4) and are similar in humans and mice. Some disease states are associated with atypical patterns of somatic mutation in expressed Igs (5, 6, 7, 8, 9). In light of the follicular abnormalities seen in the setting of HIV/SIV infections, the processes that transpire in GCs may also show abnormalities in HIV/SIV-infected individuals.

The Ab response to the envelope glycoproteins (Env) of HIV-1 is of particular importance because of the ability of some anti-Env Abs to neutralize viral infectivity, protect against infection (10, 11, 12, 13, 14), exert selective pressure on HIV-1 and SIV following infection in vivo (15, 16, 17, 18, 19, 20, 21, 22, 23), and contribute to the control of viremia (24, 25, 26). Importantly, neutralizing Abs effective against a broad range of HIV-1 or SIV isolates are produced upon virus exposure only after a substantial delay, months or years following infection (16, 27, 28, 29, 30, 31, 32), suggesting that maturation of the immune response is necessary for the development of such broadly reactive Abs. Changes in the specificity of circulating neutralizing Abs continue to occur throughout at least the first 5 years of HIV-1 infection (33). The molecular mechanisms that underlie this evolution in virus-specific Abs have not been identified, but it has been suggested that GC processes may be critical for development and maintenance of virus-neutralizing Abs (34).

Macaques infected with recombinant viruses that express HIV-1 Env on an SIV backbone (simian HIVs (SHIVs)) have proven valuable for studying humoral immunity to HIV-1 (11, 12, 24, 35). A high degree of conservation of Ig sequences between humans and these monkeys increases their value as animal models for studying human Ab responses. Studies have shown that homologous human and macaque H (36, 37, 38, 39, 40) and L chain (41) V regions typically have >90% nucleotide identity, and even >95% identity for some V region genes.

In previous studies of SHIV-infected rhesus monkeys, we have shown that lymphoid GCs formed during primary and chronic immunodeficiency virus infection include a high frequency of Env-specific B cells (42). In the present report, we extend those studies with an analysis of Ig sequences from Env-binding GCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs and recombinant proteins

Recombinant HIV-1 89.6 gp120 (rgp120-89.6) was prepared by Dr. R. Doms (University of Pennsylvania, Philadelphia, PA) as previously described (43, 44) from supernatants of cells infected with recombinant vaccinia viruses expressing HIV envelope glycoproteins, and purified by lectin affinity chromatography. The rgp120-89.6 was biotinylated in 100-µg batches using the FluoReporter Minibiotin-XX protein labeling kit (Molecular Probes) according to the manufacturer’s instructions. Unreacted biotin was removed by filtration over a ChromaSpin LC-30 column (BD Clontech). Preservation of conformation-dependent epitopes in the biotinylated Env was documented as described (42).

SHIV clones, animal infections, and sample handling

The SHIVs used for this study are described elsewhere (45). In brief, the env, tat, and rev genes of HIV-1 HXBc2 were introduced into an infectious molecular clone of SIVmac239. The env gene of the resulting virus was replaced with the env gene of the HIV-1 clone 89.6 to generate SHIV-89.6 (46). Serial passage of SHIV-89.6 in monkeys generated the highly pathogenic SHIV-89.6P quasispecies, from which SHIV-KB9 was cloned. SHIV-KB9ct and SHIV-KB9(–305) are hybrid viruses that combine aspects of the parental –89.6 and more pathogenic –KB9 clones. These viruses were selected for the present study because they expressed a gp120 identical with or cross-reactive with that of the rgp120-89.6 used as an inverse immunohistochemical probe and show increased pathogenicity compared with the parental SHIV-89.6 (45, 47).

The rhesus monkeys used in this study were maintained at the Oregon National Primate Research Center in accordance with the guidelines of the committee on Animals for the Harvard Medical School and the Guide for the Care and Use of Laboratory Animals (48). Healthy rhesus monkeys were inoculated i.v. with cell-free SHIV. Subject Mm18284 was infected with SHIV-KB9ct, and subject Mm19941 was infected with SHIV-KB9(–305). Some immunologic and virologic data from these monkeys have been previously reported (42, 45, 47). Infection of both animals was confirmed by serologic and virologic assays (45, 47). Before infection, subject Mm18284 had a CD4+ cell count of 1200/µl. Following infection the CD4+ cell count fell to a nadir of 350 on postinfection day (p.i.d.) 17 before stabilizing at ~500 by p.i.d. 28. Plasma viral load as measured by ELISA detection of the SHIV Gag protein p27 peaked at 1.4 ng/ml on p.i.d. 14, and declined to below the limit of detection by p.i.d. 21 (47). Mm19941 had a baseline CD4+ cell count of 2000/µl. Following infection the CD4+ count fell to 831 by p.i.d. 21 and rose gradually to stabilize at ~950 on p.i.d. 78. Cumulative p27 antigenemia in this animal through p.i.d. 21 was 0.95 ng/ml; viral p27 was below the limit of detection beyond that date (45). The sustained >50% decrease in absolute CD4 counts in the blood of both animals following infection is evidence of the pathogenicity of the SHIV types used in these studies

Monkeys were anesthetized with ketamine-HCl 5 mg/kg, i.m. for all blood sampling, and were euthanized by an overdose of sodium pentobarbital administered i.v. at 3 mo (Mm19941) and 9 mo (Mm18284) after SHIV infection. These time point were chosen to assess GC functions during the early stages of chronic infection, at a time when Env-specific GCs are plentiful (42). Pieces of spleen were frozen in Tissue-Tek cryoembedding medium (Fisher Scientific) immediately upon removal, and stored at –80°C until use. Frozen sections were cut at 10-µm thickness, thaw-mounted onto Superfrost Plus slides (Fisher Scientific), immediately refrozen, and then stored at –80°C.

Inverse immunohistochemistry

Slide-mounted tissue sections of spleen from SHIV-infected monkeys were fixed in cold acetone for 10 min, air dried, and rehydrated in TBS (pH 8). Slides were then processed using the Sequenza immunostaining workstation (Shandon Lipshaw). Inverse immunostaining was enhanced using catalyzed signal amplification with the Renaissance TSA-Indirect kit (PerkinElmer). Each slide was first treated with Tris-NaCl buffer with a proprietary blocking reagent (Renaissance kit) to reduce nonspecific staining, and then incubated for 1 h at room temperature with biotinylated rHIV-1 envelope gp120-89.6 at a concentration of 0.2 mcg/ml (1.67 nM). Slides were rinsed three times with TBS containing 0.05% Tween 20 (TBST) after each subsequent staining step. To catalyze amplification of the biotin signal, the slides were incubated with streptavidin-HRP (1/100 dilution, Renaissance kit) for 30 min at room temperature and subsequently incubated with biotinyl tyramide (1/50 dilution) for 10 min. The amplified biotin signal, indicating bound biotinylated rgp120, was detected histochemically using alkaline phosphatase-conjugated streptavidin-biotin complex (DakoCytomation) and Vector Blue chromogenic substrate (Vector Laboratories) together with levamisole (DakoCytomation) to suppress endogenous alkaline phosphatase activity. After counterstaining with either nuclear fast red (Vector Laboratories) or Gill’s hematoxylin (Fisher Scientific), slides were covered with Crystal/Mount permanent mounting medium (Fisher Scientific) and air dried.

Microdissection of Env-binding GCs

Slide-mounted splenic frozen sections stored at –80°C were fixed in cold acetone for 10 min and air dried. Sections were rehydrated in nuclease-free water, stained in nuclear fast red (Vector Laboratories), dehydrated through graded alcohols and xylenes, and air dried. The targeted GCs were microdissected using an Arcturus PixCell II laser microdissecting workstation (Arcturus Engineering) and the following method. After Env-binding GCs were identified by inverse immunohistochemical staining, the same GCs were located on adjacent tissue sections using landmarks such as position within the section, orientation relative to other GCs, and distinctive features such as GC contour or the presence of vessels within the structure. Cells from each targeted GC were isolated from the surrounding tissue using laser capture microdissection (49) with CapSure HS membranes (Arcturus Engineering). Multiple overlapping laser pulses of 30-µm diameter spot size were used to capture substantial portions of each GC from the slide. Care was taken to avoid capturing cells from the surrounding mantle zones and other regions outside the targeted zone. Digitized images of immunostained tissue sections and the microdissection process were prepared using the Arcturus Engineering workstation and formatted using Adobe Photoshop version 4 software.

Cloning and sequence analysis of Ig genes from Env-binding GCs

mRNA present in the captured cells was purified using the RNeasy kit (Qiagen) according to the manufacturer’s instructions. cDNA was prepared using the AMV Reverse Transcription System (Promega). The resulting cDNA (1 µl/reaction) was amplified using nested or seminested PCR catalyzed by a proofreading enzyme, PfuTurbo DNA Polymerase (Stratagene) to provide high copying fidelity and optimal yield from the minute samples obtained using microdissection. Each round of the PCR was performed in 50 µl using oligonucleotide primers designed to selectively amplify rearranged Ig H or L chains (Table I) for 30 cycles using the following parameters: 20 s at 94°C, 30 s at 55°C, 120 s at 72°C. Each PCR was concluded with an extension step at 72°C for 10 min. Products of the second-round PCRs were size selected by electrophoresis in 1.5% agarose, visualized by ethidium bromide staining and cut from the gel. The PCR products were purified using the Qiaex II Gel Extraction System (Qiagen), and cloned into Escherichia coli using the TopoBlunt cloning kit (Invitrogen Life Technologies). Plasmids containing inserts were isolated from expanded cultures using QIAprep Spin Miniprep kit (Qiagen), and sequenced on both strands using an Applied Biosystems Prism 377XL Automated DNA Sequencer and primers that bound outside the insert.


View this table:
[in this window]
[in a new window]
  		Table I. Oligonucleotide primers for amplification of macaque Ig H and L chain V regionsa

 
Nucleotide sequence data from both strands were reconciled using the Seqman module of the Lasergene2000 software package (DNAstar). Sequences of all clones were submitted to GenBank (accession numbers AY452535-AY452600). The set of individual sequences was analyzed to identify clusters of sequences with similar overall length, CDR3 length, and junctional nucleotide sequence; each cluster might represent descendants of a common ancestor clone.

Somatic mutations in expressed Ig V regions are usually identified with reference to the sequence of their probable precursor in the germline. Because the macaque Ig loci have not been comprehensively catalogued, macaque Ig cDNA sequences were compared with their nearest homologs among the human germline variable (V), diversity (D), and joining (J) region minigenes, which are closely related to those of macaques (36, 37, 38, 39, 41). The nearest homolog minigenes were identified from the complete dataset of human germline Ig sequences based on sequence similarity (50). To facilitate analysis of the CDR3 regions, the germline segments were trimmed and joined to create hypothetical junctional sequences; these junctions were optimally assembled to minimize the number of nucleotide differences with the macaque clones. When V, D, and J segments were insufficient to generate a CDR3 of the observed length, N and P nucleotides were not added; these positions are shown as blanks in the alignment.

Somatic mutations were tallied twice, under different assumptions. First, any nucleotide difference when compared with the nearest human germline V region and hypothetical junctional sequence was assumed to result from a somatic mutation (assumption 1). Second, it was assumed that any nucleotides present in every member of a lineage were encoded in the macaque germline, so that only intraclonal sequence differences were counted as somatic mutations (assumption 2). Statistical software packages SAS version 8.2 and Splus version 3.4 were used to read in the nucleotide sequence data and determine the number of occurrences for each possible type of substitution (e.g., from nucleotide X to nucleotide Y) for each clone under both assumptions 1 and 2.

Because the genealogical relationships among the clones were inferred but not known with certainty, statistical analyses were based on a population model: the incidence of mutation at each nucleotide position was tallied cumulatively for each occurrence, even when the same substitution occurred in multiple members of the population.

Nucleotide differences between the clones were tallied for the entire V region excluding positions that overlap the PCR primers, which correspond with the initial 22 nt of the IgH framework region 1 (FR1) region or 18 nt of the Ig{lambda} and Ig{kappa} FR1. FR and CDR in the cloned Igs were identified based on standard Kabat criteria (E. A. Kabat, Kabat database of sequences of proteins of immunological interest, National Institutes of Health, <www.kabatdatabase.com/>), summarized online at <www.rubic.rdg.ac.uk/abeng>.

Nucleotide mutations that result in amino acid substitutions (replacement mutations, R) or do not change the amino acid sequence (silent mutations, S) were counted separately for FR1, 2, and 3 and CDR1, 2, and 3 of each macaque clone. Enumeration of R and S mutations was performed using an automated algorithm that incorporates the methods of Nei (51, 52), implemented online at <http://hiv-web.lanl.gov/content/hiv-db/SNAP/WEBSNAP/SNAP.html> (B. T. Korber, Synonymous Non-Synonymous Analysis Program, Los Alamos National Laboratory, 53). The excess or paucity of replacement mutations in the FRs or CDRs of each sequence was compared with that expected by chance using a multinomial distribution model, implemented online at <www-stat.stanford.edu/Ig> (54).

For analysis of purine or pyrimidine transition or transversion mutations, the test for whether the rates of mutation from T to C, T to A, and T to G were all equal given that T was mutated (and similarly for mutations from C, from A, and from G) was an exact Poisson test (i.e., the multinomial test of ratios after conditioning on the sum of the three mutations). Comparisons between pairs of mutation rates used the exact Poisson test (i.e., the binomial test of ratios after conditioning on the sum). The tests of differences between lineages within chain (H or L) and animal used the exact Zelen test for homogeneity of odds ratios. An adjustment was made for multiple comparisons using Holm’s method. That is, when there were n relevant comparisons, the smallest of the n p values was compared with 0.05/n, the next smallest p value was compared with 0.05/(n-1), and so on until one of the p values was not smaller than its bound (i.e., 0.05/n), in which case that comparison and all comparisons with larger p values were declared nonsignificant. The multiple comparison adjustment was done separately for each different initial nucleotide.

Within the motif RGYW and its reverse complement WRCY, only the underlined G or C was considered a potential hot spot preferentially targeted for mutation (55, 56). Comparison of mutation frequency for hot spot and non-hot spot sites was made using the Fisher exact text.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Identification and microdissection of GCs containing Env-binding cells, and cloning of their Ig sequences

Splenic tissue frozen sections derived from two rhesus monkeys chronically infected with moderately pathogenic variants of SHIV-89.6 were stained by inverse immunohistochemistry using biotinylated recombinant HIV-1 envelope gp120 (Env) as an Ag probe. Splenic tissues from both monkeys demonstrated numerous GCs containing cells that bound Env. For Mm19941, 35% of >1500 examined GCs contained Env-binding cells, while for Mm18284, 3% of >400 evaluated GCs contained such cells. Both intensely stained and entirely unstained GCs were frequently apparent on a single tissue section in close proximity (data not shown), indicating that the Ag probe was bound to B cells or Abs produced locally rather than to serum Abs nonspecifically bound to GC follicular dendritic cells. One Env-binding GC from each animal, shown in Fig. 1, A and B, was selected for further evaluation.


Figure 1
View larger version (131K):
[in this window]
[in a new window]
  		FIGURE 1. Localization and microdissection of HIV-1 Env-binding GCs in splenic tissues of SHIV-infected rhesus monkeys. Frozen sections were stained by inverse immunohistochemistry using biotinylated rgp120-89.6 as an Ag probe and counterstained. The Env-binding GCs depicted in A and B correspond to GCs 2S and 4S, respectively, in the text. C, The microdissection of the GC shown in B from four serial sections, counterstained only. Upper row, Tissues after counterstaining; middle row, tissues after microdissection; bottom row, tissues recovered by microdissection. Care was taken to avoid capturing cells from a blood vessel passing through GC 4S.

 
Cells from each targeted GC were isolated from multiple, serial sections using laser capture microdissection. The capture of tissue from the Env-binding GC shown in Fig. 1B is depicted in the panels of Fig. 1C. The upper row presents the same GC as it appeared in several adjacent sections. The next row shows the same tissue sections after microdissection. The bottom row shows the GC cells isolated by microdissection.

Samples from each tissue section were processed separately. mRNA present in the captured cells was purified, reverse transcribed into cDNA, and amplified using nested PCR with oligonucleotide primers designed to selectively amplify rearranged Ig H or L chains (Table I). Products of the second-round PCRs were size selected, cloned, and both strands were sequenced.

Sequence analysis of Igs cloned from microdissected GCs

The recovered nucleotide sequences from each GC could be grouped on the basis of sequence similarity into multimember clonal lineages (Table II). Within each lineage, all sequences have similar V-D-J or V-J junctions but differ by somatic mutations distributed throughout the variable domain. The V-D-J-C and V-J-C joins were all "in frame" (i.e., productive rearrangements), and no premature termination codons were found in any of the clones. Data from nine clonal lineages were combined to create an aggregate data set that was analyzed statistically.


View this table:
[in this window]
[in a new window]
  		Table II. Germline similarity and replacement and silent mutations in the macaque Ig H and L chain cDNA clones

 
Ig clones from GC2

H chain. cDNA from GC2 was amplified using oligonucleotide primers designed to amplify Ig H chain V regions encoding IgG or IgM Abs. The IgG-primed reactions revealed an amplicon of the expected size only for the VH3-primed reaction. Nucleic acid sequencing of VH3-IgG clones yielded a clonal lineage, designated 2S1H3, with nine distinct members (Fig. 2A).


Figure 2
View larger version (55K):
[in this window]
[in a new window]
  		FIGURE 2. Nucleotide sequences of Ig V regions from Env-binding macaque GC B cells. All sequences were cloned from cDNA from the microdissected GCs depicted in Fig. 1. The sequences are here aligned to the most similar human germline V and J sequences. For simplicity, the germline V and J have been trimmed and concatenated to form CDR3 sequences that resemble those of the GC clones. Nucleotides identical with the germline are represented by a period ("."). Nucleotide substitutions are shown in lower case if they encode a silent mutation and upper case if they encode a replacement mutation. The inferred amino acid sequence of the germline is presented in single letter format. The residues that form the CDRs are indicated. Each alignment shows one lineage, corresponding to the text as follows: A, 2S1H3. B, 2S2L5. C, 4S1H3. D, 4S2H3. E, 4S3H3. F, 4S4L5. G, 4S5L2. H, 4S6L8. I, 4S7K2.

 
The IgM-primed reactions also yielded an amplicon of the expected size only for VH3-primed products. The VH3-IgM PCR product was ligated and cloned, and 30 clones were sequenced. They were all identical, with no intraclonal variation. This IgM may represent a B cell resident in the mantle zone outside GC2, inadvertently collected during laser capture microdissection.

L chain. cDNA from GC 2 was amplified using oligonucleotide primers designed to amplify Ig L chain V regions of either {kappa} or {lambda} isotype. Electrophoresis of PCR products revealed an amplicon of the expected size only among the VL5-primed reaction. Nucleic acid sequencing of clones from GC2 yielded one multimember clonal lineage, designated 2S2L5, with six distinct members (Fig. 2B).

Ig clones from GC4

H chains. cDNA from GC4 was amplified using oligonucleotide primers designed to amplify Ig H chain V regions encoding IgA, IgD, IgE, IgG, or IgM Abs. Only IgG-specific PCRs produced an amplicon of the expected size. Sequencing of clones from the IgG-specific PCR revealed three multimember clonal IgG lineages, designated 4S1H3, 4S2H3, and 4S3H3, whose sequences are presented in Fig. 2, C–E, respectively.

L chains. cDNA from GC4 was amplified using oligonucleotide primers designed to amplify Ig L chain V regions of either {kappa} or {lambda} isotype. {lambda} L chain PCRs yielded Ig sequences that belonged to three distinct multimember clonal lineages, designated 4S4L5, 4S5L2, and 4S6L8, whose sequences are presented in Fig. 2, F–H. {kappa} L chain PCRs yielded Ig sequences that could be assigned to a single multimember clonal lineage.4S7K2, presented in Fig. 2I.

Frequency and rate of somatic mutations in V regions of the Ig clones

The somatic mutation rate was calculated based upon two different assumptions, as described in Materials and Methods. Under assumption 1, the 66 Ig sequences cloned from microdissected GCs of SHIV-infected monkeys have an average of 37.1 apparent somatic mutations per sequence, with a range of 24.3–56.8. This corresponds to an average mutation frequency of 0.11 mutations/nucleotide. No significant difference was found between H and L chain clones in this regard. Under assumption 2, the Ig sequences have an average of 16.5 apparent somatic mutations per sequence, with a range of 2.0–37.9. The corresponding mutation frequency is 0.05 mutations/nucleotide. Under assumption 2, the mutation frequency of H chains (0.015) was significantly less than that of L chains (0.07; p < 0.02).

Bias in somatic mutation of Ig V regions

Some disease states are associated with atypical patterns of somatic mutation in expressed Igs (5, 6, 7, 8). We therefore analyzed the pattern of mutations observed in GC B cells from these SHIV-infected monkeys to determine whether they are indicative of abnormalities.

The frequency with which mutations were targeted to nucleotides within the previously described RGYW/WRCY "hot spot" motif was statistically analyzed for the 66 Ig clones obtained from the SHIV-infected monkey spleens. Within and across animals, for both H and L chains, there was a higher probability of mutation at hot spots than elsewhere (p < 0.0001). Preferential targeting of hot spots was also evident within each lineage (Table III), despite the smaller sample size. Overall, the percentage of hot spot sites that were mutated was more than twice as large as that seen in non-hot spots.


View this table:
[in this window]
[in a new window]
  		Table III. Targeting of mutations to RGYW/WYCY hot spots

 
As noted earlier, transversion mutations should be twice as common as transition mutations in a purely stochastic process. To assess the substitution pattern in the macaque Igs, the frequency of mutation from each of the four nucleotide species to each of the other bases was tallied by lineage. The frequency of transition and tranversion mutations was calculated both under assumptions 1 and 2.

Under assumption 1, there were striking deviations from randomness in the mutational pattern, which was similar to that of normal human B cells in showing a disproportionate (increased) frequency of transition mutations. For example, tests were done evaluating the relative likelihood of pairs of specific mutations (e.g., from T to C vs T to A). Such comparisons were only performed for lineages where, given that a nucleotide of a particular type (T, C, A, or G) mutates, it was not equally likely to mutate to each of the other three nucleotides. After pairwise comparisons, it was found that there were 28 (of a possible 93) significant differences, and 20 of these favored transition mutations. The other eight significant differences occurred within a nucleotide group (e.g., from T to A vs T to G). In all comparisons between transition and transversion mutations showing a statistically significant difference, transition mutations were favored. Overall, 58% of mutations were transitions (Table IV), similar to the normal human pattern but contrasting with the stochastic prediction of 33% transitions.


View this table:
[in this window]
[in a new window]
  		Table IV. Dysproportionate occurrence of transition mutations among expressed macaque Ig V regions

 
Similarly, under the more conservative assumption 2, it was found that there were 23 (of a possible 42) significant differences, and transition mutations were favored for 18 of them, while the other 5 occurred within a nucleotide group. Altogether, transition mutations comprised half of observed mutations, rather than one-third as predicted for a random process.

Mutational bias attributable to cytosine deaminase (57) was specifically examined. Under assumption 1, for eight of the nine lineages, the nucleotide C when mutated was most often replaced by T. Mutational bias in mutations of C was statistically significant for seven of nine lineages (range of p values, 0.0008-<0.0001). Similarly, eight of nine lineages showed preferential replacement of G by A, and the deviation from randomness was statistically significant for all of these (range of p values, 0.021-<0.0001). Combining the results for all lineages, the C->T and G->A replacements together constituted almost one-third of all mutations (Table IV). Under assumption 2 also, the nucleotide C when mutated was most often replaced by T, and G was most often replaced by A. Thus, calculations based on either assumption support the conclusion that somatic mutational processes are operating normally in these GCs.

Selection pressures on somatic mutations in Ig V regions

To assess the influence of positive and negative selection on the Ig sequences cloned from the Env-binding GCs of SHIV-infected monkeys, the ratio of replacement to silent mutations was determined for the FRs and CDRs of each V(D)J region by comparison with the nearest human germline homologs (Table II). The excess or paucity of replacement mutations in the FRs or CDRs of each sequence, tallied both under assumptions 1 and 2, was compared with that expected by chance using a multinomial distribution model (54). The fraction of clones from each lineage that showed statistically significant evidence for a selection bias under assumption 1 is shown in Table II. Remarkably, 63 of 66 clones had a statistically significant (p < 0.05) paucity of R mutations in the FRs. This indicates that nearly all the clones had survived substantial negative selection pressures in vivo. The absence of premature termination codons, out-of-frame V(D)J junctions and other inactivating mutations among the cloned sequences provides additional evidence of negative selection. A majority of the clones (37 of 66) also had a statistically significant excess of R mutations in the CDRs, reflecting positive selection pressures in vivo.

We repeated the analysis under assumption 2 (data not shown). Although this assumption drastically reduced the total number of apparent somatic mutations, 35 of 66 clones still showed a statistically significant paucity of R mutations in their FRs, and 27 clones had a statistically significant excess of R mutations in the CDRs. Therefore, calculations based on either assumption support the conclusion that positive and negative selection processes are operating normally in these GCs.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Abnormalities in the number, size, and structure of lymphoid GCs are common in individuals infected with HIV-1, SIV, or SHIV. Because GCs are an important anatomic site for the maturation of Ab responses, these abnormalities could have important consequences for the development of virus-specific Abs. To determine whether immunodeficiency virus infection is associated with abnormalities in Ab development in GCs, we analyzed the nucleotide sequences of Igs in B cells obtained from the HIV-1 Env-binding splenic GCs of SHIV-infected monkeys. The types of observed nucleotide mutations and the targeting of base substitutions in both H and L chain clones support the conclusion that the same Ag-driven GC processes observed in normal humans and mice are also operating in the GCs of SHIV-infected monkeys. The Ig sequences from SHIV-infected monkeys had an average of 16.5–37.1 apparent somatic mutations per sequence. This is close to the mutation frequency previously reported for human IgG Abs recognizing HIV-1 gp120 (58, 59, 60, 61, 62, 63, 64), and substantially higher than the average of 9.5 somatic mutations in IgG produced by unselected human tonsillar GC B cells (65).

The abundance of mutations in the Igs indicates that the B cells were subjected to a profound degree of antigenic stimulation in the GC microenvironment. Although it was not formally demonstrated that the studied Igs actually bind Env or another SHIV protein, the abundance of Env-binding cells in the studied GCs provides direct evidence that the viral envelope is a potent immunogen for B cells in fully functional GCs.

Somatic mutation among GC B cells typically occurs at a rate of 10–3 mutations per base pair per cell per generation (66, 67). The cell cycle of GC B cells has been calculated at between 6 (68) and 12 h (69). Assuming the same rate of mutation applies to the tissues from which the clones were derived, calculations show that the average clone in our dataset underwent between 50 and 113 cycles of replication. Based on the 6–12 hour cycling time, this number of generations could arise in 2–8 wk of continuous antigenic stimulation. Because the tissues were obtained at least 13 wk after SHIV infection, it cannot be determined whether diversification of the clones we studied occurred rapidly during acute infection or accumulated more gradually over subsequent weeks.

Using the lower estimate of 50 generations, this number of cell divisions is sufficient to produce nearly 800 trillion cells, if all survived. However, the largest GCs contain a few million cells at most. The difference between these values highlights the stringency of negative selection enforced during clonal expansion in these GCs.

In the present study, we found that GC function was preserved in two monkeys in the steady state stage of SHIV infection who had ~50% reductions in CD4+ T cell counts. This corresponds with the protracted lymphadenopathy stage of HIV-1 infection in the early years following infection, although the subset of CD4+ T lymphocytes lost in these monkeys differs from those targeted for destruction in HIV-1-infected humans (70). However, GC function likely declines with more advanced immunodeficiency. HIV-1-infected individuals with very low numbers of CD4+ cells often develop follicular involution, with GCs depleted of lymphocytes and follicular dendritic cells.

The analysis of data in this study was subject to several limitations. First, any characterization of GC B cell lineages will necessarily be incomplete because the iterative process of mutation and selection will delete intermediate ancestors, branches, and entire lineages as an indispensable component of the process leading to affinity maturation. Other B cells present in the GC will be missed through sampling error or because mutations in their Ig sequence disrupt homology with the PCR primers. In any case, because the H and L chain lineages characterized in this study all showed a consistent pattern of nucleotide substitution and selection, it is unlikely that any missing sequences would have altered our conclusions.

A more serious limitation in any analysis of macaque Ig sequence data stems from the substantial gaps in our knowledge of macaque Ig germline sequences. These gaps preclude the use of macaque Ig germline sequences as a basis for comparison with the Ig clones sequenced in this study. Instead, the sequenced macaque Ig cDNAs were compared with the human genomic Ig repertoire. This germline repertoire is known to be very similar but not identical with that of the macaques (36, 37, 38, 39, 41). As a result, where a difference from the human germline sequence was shared by all clones in a macaque lineage, we could not determine whether the differences were introduced by somatic mutation in an ancestral clone or instead were encoded in the macaque germline.

To rule out possible bias introduced by such errors of assignment, we performed most analyses under two different assumptions. First, it was assumed that any nucleotide difference between a macaque sequence and its nearest human germline homolog resulted from a single somatic mutation. This assumption will tend to overestimate the frequency of somatic mutations. Second, it was assumed that any substitutions present in every member of a lineage were encoded in the macaque germline, and were therefore excluded from analysis. This more conservative assumption will undercount somatic mutations, including those retained because they contribute to the specificity of all Igs of a lineage. Importantly, the conclusion that somatic mutation and selection were operating as they should be in the GCs of the SHIV-infected monkeys was supported under either assumption. There also is little published information regarding the normal pattern of somatic mutation in macaques. The lack of a formal baseline or comparator would be of greater consequence had our results suggested that macaque GCs introduce somatic mutations according to some novel pattern. However, because the pattern was the same in normal humans and rodents as in the SHIV-infected macaques we studied, it is likely that the pattern in normal macaques is also the same.

There are few published studies that address the role of GCs during immune responses to viral infection in humans or nonhuman primates. Analyses of somatic mutations in mAbs derived from individuals exposed to viruses suggest that the processes of somatic mutation and Ag-affinity selection can play a major role in shaping the Ab response to viruses, including HIV-1 (58, 59, 71, 72, 73). The present study directly demonstrates that GCs are a major site for somatic mutation and selection of antiviral Abs in primates. Additional studies are needed to determine the contribution of somatic mutation outside of GCs (74, 75, 76) to shaping the Ab response to an infecting virus.


   Acknowledgments
 
We thank Robert Doms (Philadelphia, PA) for providing rgp120-89.6 produced in his laboratory; and Randy Todd and David Wong (Harvard School of Dental Medicine) for access to the laser capture microdissection workstation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


Figure 2
View larger version (57K):
[in this window]
[in a new window]
  		FIGURE 2A. Continued

 

Figure 2
View larger version (55K):
[in this window]
[in a new window]
  		FIGURE 2B. Continued

 

   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 D.H.M. was supported by Public Health Service (PHS) Grants AI-01587 and AI-45370 from the National Institute of Allergy and Infectious Diseases (NIAID). E.H.S. participated in these studies as a Medical Student Fellow of the Howard Hughes Medical Institute. M.K.A. was supported by Grant AI-054292 from the NIAID, Grant CA-75922 from the National Cancer Institute, Grants RR-016025 and RR018107 from the National Center for Research Resources, and a PHS institutional grant from the National Center for Research Resources supporting the Oregon National Primate Research Center, Grant RR-000163. N.L.L. was supported by PHS Grant AI-067854 from the NIAID Center for HIV/AIDS Vaccine Immunology and Grant AI-20729 from the NIAID. O.G.G. and R.S.G. were supported by PHS Grant AI-28691 to the Dana-Farber Cancer Institute Center for AIDS Research from the NIAID. Back

2 Current address: Massachusetts General Hospital, Wang Ambulatory Care Center-835, Boston, MA 02114. Back

3 Current address: University of Michigan Hospital Psychiatry 9d9812 University Hospital 0120, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0118. Back

4 Current address: Department of Surgery, Mount Sinai School of Medicine, 5 East 98th Street, New York, NY 10029. Back

5 Address correspondence and reprint requests to Dr. Norman L. Letvin, Viral Pathogenesis Division, RE-113/Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. E-mail address: nletvin{at}bidmc.harvard.edu Back

6 Abbreviations used in this paper: GC, germinal center; SHIV, simian HIV; p.i.d., postinfection day; FR, framework. Back

Received for publication July 18, 2005. Accepted for publication April 19, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Racz, P., K. Tenner-Racz, F. van Vloten, H. Schmidt, M. Dietrich, J. C. Gluckman, N. L. Letvin, G. Janossy. 1990. Lymphatic tissue changes in AIDS and other retrovirus infections: tools and insights. Lymphology 23: 85-91. [Medline]
  2. Chalifoux, L. V., N. W. King, N. L. Letvin. 1984. Morphologic changes in lymph nodes of macaques with an immunodeficiency syndrome. Lab. Invest. 51: 22-26. [Medline]
  3. Dorner, T., S. J. Foster, H. P. Brezinschek, P. E. Lipsky. 1998. Analysis of the targeting of the hypermutational machinery and the impact of subsequent selection on the distribution of nucleotide changes in human VHDJH rearrangements. Immunol. Rev. 162: 161-171. [Medline]
  4. Neuberger, M. S., M. R. Ehrenstein, N. Klix, C. J. Jolly, J. Yelamos, C. Rada, C. Milstein. 1998. Monitoring and interpreting the intrinsic features of somatic hypermutation. Immunol. Rev. 162: 107-116. [Medline]
  5. Levy, Y., N. Gupta, F. Le Deist, C. Garcia, A. Fischer, J. C. Weill, C. A. Reynaud, B. Bertocci, L. Quint, F. Delbos, et al 1998. Defect in IgV gene somatic hypermutation in common variable immuno-deficiency syndrome. Proc. Natl. Acad. Sci. USA 95: 13135-13140. [Abstract/Free Full Text]
  6. Manheimer-Lory, A. J., G. Zandman-Goddard, A. Davidson, C. Aranow, B. Diamond. 1997. Lupus-specific antibodies reveal an altered pattern of somatic mutation. J. Clin. Invest. 100: 2538-2546. [Medline]
  7. Dorner, T., S. Kaschner, A. Hansen, A. Pruss, P. E. Lipsky. 2001. Perturbations in the impact of mutational activity on V{lambda} genes in systemic lupus erythematosus. Arthritis Res. 3: 368-374. [Medline]
  8. Revy, P., T. Muto, Y. Levy, F. Geissmann, A. Plebani, O. Sanal, N. Catalan, M. Forveille, R. Dufourcq-Labelouse, A. Gennery, et al 2000. Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the hyper-IgM syndrome (HIGM2). Cell 102: 565-575. [Medline]
  9. Zeng, X., D. B. Winter, C. Kasmer, K. H. Kraemer, A. R. Lehmann, P. J. Gearhart. 2001. DNA polymerase eta is an A-T mutator in somatic hypermutation of immunoglobulin variable genes. Nat. Immunol. 2: 537-541. [Medline]
  10. Mascola, J. R., G. Stiegler, T. C. VanCott, H. Katinger, C. B. Carpenter, C. E. Hanson, H. Beary, D. Hayes, S. S. Frankel, D. L. Birx, M. G. Lewis. 2000. Protection of macaques against vaginal transmission of a pathogenic HIV- 1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nat. Med. 6: 207-210. [Medline]
  11. Shibata, R., T. Igarashi, N. Haigwood, A. Buckler-White, R. Ogert, W. Ross, R. Willey, M. W. Cho, M. A. Martin. 1999. Neutralizing antibody directed against the HIV-1 envelope glycoprotein can completely block HIV-1/SIV chimeric virus infections of macaque monkeys. Nat. Med. 5: 204-210. [Medline]
  12. Mascola, J. R., M. G. Lewis, G. Stiegler, D. Harris, T. C. VanCott, D. Hayes, M. K. Louder, C. R. Brown, C. V. Sapan, S. S. Frankel, et al 1999. Protection of Macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies. J. Virol. 73: 4009-4018. [Abstract/Free Full Text]
  13. Emini, E. A., W. A. Schleif, J. H. Nunberg, A. J. Conley, Y. Eda, S. Tokiyoshi, S. D. Putney, S. Matsushita, K. E. Cobb, C. M. Jett, et al 1992. Prevention of HIV-1 infection in chimpanzees by gp120 V3 domain-specific monoclonal antibody. Nature 355: 728-730. [Medline]
  14. Conley, A. J., J. A. Kessler, II, L. J. Boots, P. M. McKenna, W. A. Schleif, E. A. Emini, G. E. Mark, III, H. Katinger, E. K. Cobb, S. M. Lunceford, et al 1996. The consequence of passive administration of an anti-human immunodeficiency virus type 1 neutralizing monoclonal antibody before challenge of chimpanzees with a primary virus isolate. J. Virol. 70: 6751-6758. [Abstract/Free Full Text]
  15. Wei, X., J. M. Decker, S. Wang, H. Hui, J. C. Kappes, X. Wu, J. F. Salazar-Gonzalez, M. G. Salazar, J. M. Kilby, M. S. Saag, et al 2003. Antibody neutralization and escape by HIV-1. Nature 422: 307-312. [Medline]
  16. Richman, D. D., T. Wrin, S. J. Little, C. J. Petropoulos. 2003. Rapid evolution of the neutralizing antibody response to HIV type 1 infection. Proc. Natl. Acad. Sci. USA 100: 4144-4149. [Abstract/Free Full Text]
  17. McKeating, J. A., J. Gow, J. Goudsmit, L. H. Pearl, C. Mulder, R. A. Weiss. 1989. Characterization of HIV-1 neutralization escape mutants. AIDS 3: 777-784. [Medline]
  18. Burns, D. P., C. Collignon, R. C. Desrosiers. 1993. Simian immunodeficiency virus mutants resistant to serum neutralization arise during persistent infection of rhesus monkeys. J. Virol. 67: 4104-4113. [Abstract/Free Full Text]
  19. Nara, P., L. Smit, N. Dunlop, W. Hatch, M. Merges, D. Waters, J. Kelliher, W. Krone, J. Goudsmit. 1990. Evidence for rapid selection and deletion of HIV-1 subpopulations in vivo by V3-specific neutralizing antibody: a model of humoral-associated selection. Dev. Biol. Stand. 72: 315-341. [Medline]
  20. Cheng-Mayer, C., A. Brown, J. Harouse, P. A. Luciw, A. J. Mayer. 1999. Selection for neutralization resistance of the simian/human immunodeficiency virus SHIVSF33A variant in vivo by virtue of sequence changes in the extracellular envelope glycoprotein that modify N-linked glycosylation. J. Virol. 73: 5294-5300. [Abstract/Free Full Text]
  21. Evans, D. T., R. C. Desrosiers. 2001. Immune evasion strategies of the primate lentiviruses. Immunol. Rev. 183: 141-158. [Medline]
  22. Arendrup, M., A. Sonnerborg, B. Svennerholm, L. Akerblom, C. Nielsen, H. Clausen, S. Olofsson, J. O. Nielsen, J. E. Hansen. 1993. Neutralizing antibody response during human immunodeficiency virus type 1 infection: type and group specificity and viral escape. J. Gen. Virol. 74: 855-863. [Abstract/Free Full Text]
  23. Chackerian, B., L. M. Rudensey, J. Overbaugh. 1997. Specific N-linked and O-linked glycosylation modifications in the envelope V1 domain of simian immunodeficiency virus variants that evolve in the host alter recognition by neutralizing antibodies. J. Virol. 71: 7719-7727. [Abstract]
  24. Rasmussen, R. A., R. Hofmann-Lehmann, P. L. Li, J. Vlasak, J. E. Schmitz, K. A. Reimann, M. J. Kuroda, N. L. Letvin, D. C. Montefiori, H. M. McClure, R. M. Ruprecht. 2002. Neutralizing antibodies as a potential secondary protective mechanism during chronic SHIV infection in CD8+ T-cell-depleted macaques. AIDS 16: 829-838. [Medline]
  25. Schmitz, J. E., M. J. Kuroda, S. Santra, M. A. Simon, M. A. Lifton, W. Lin, R. Khunkhun, M. Piatak, J. D. Lifson, G. Grosschupff, et al 2003. Effect of humoral immune responses on controlling viremia during primary infection of rhesus monkeys with simian immunodeficiency virus. J. Virol. 77: 2165-2173. [Abstract/Free Full Text]
  26. Binley, J. M., B. Clas, A. Gettie, M. Vesanen, D. C. Montefiori, L. Sawyer, J. Booth, M. Lewis, P. A. Marx, S. Bonhoeffer, J. P. Moore. 2000. Passive infusion of immune serum into simian immunodeficiency virus-infected rhesus macaques undergoing a rapid disease course has minimal effect on plasma viremia. Virology 270: 237-249. [Medline]
  27. Arendrup, M., C. Nielsen, J. E. Hansen, C. Pedersen, L. Mathiesen, J. O. Nielsen. 1992. Autologous HIV-1 neutralizing antibodies: emergence of neutralization-resistant escape virus and subsequent development of escape virus neutralizing antibodies. J. Acquir. Immune Defic. Syndr. 5: 303-307. [Medline]
  28. Moog, C., H. J. Fleury, I. Pellegrin, A. Kirn, A. M. Aubertin. 1997. Autologous and heterologous neutralizing antibody responses following initial seroconversion in human immunodeficiency virus type 1-infected individuals. J. Virol. 71: 3734-3741. [Abstract]
  29. Clements, J. E., R. C. Montelaro, M. C. Zink, A. M. Amedee, S. Miller, A. M. Trichel, B. Jagerski, D. Hauer, L. N. Martin, R. P. Bohm, et al 1995. Cross-protective immune responses induced in rhesus macaques by immunization with attenuated macrophage-tropic simian immunodeficiency virus. J. Virol. 69: 2737-2744. [Abstract]
  30. Cole, K. S., J. L. Rowles, B. A. Jagerski, M. Murphey-Corb, T. Unangst, J. E. Clements, J. Robinson, M. S. Wyand, R. C. Desrosiers, R. C. Montelaro. 1997. Evolution of envelope-specific antibody responses in monkeys experimentally infected or immunized with simian immunodeficiency virus and its association with the development of protective immunity. J. Virol. 71: 5069-5079. [Abstract]
  31. Pilgrim, A. K., G. Pantaleo, O. J. Cohen, L. M. Fink, J. Y. Zhou, J. T. Zhou, D. P. Bolognesi, A. S. Fauci, D. C. Montefiori. 1997. Neutralizing antibody responses to human immunodeficiency virus type 1 in primary infection and long-term-nonprogressive infection. J. Infect. Dis. 176: 924-932. [Medline]
  32. Wrin, T., L. Crawford, L. Sawyer, P. Weber, H. W. Sheppard, C. V. Hanson. 1994. Neutralizing antibody responses to autologous and heterologous isolates of human immunodeficiency virus. J. Acquir. Immune Defic. Syndr. 7: 211-219. [Medline]
  33. Quinnan, G. V., Jr, P. F. Zhang, D. W. Fu, M. Dong, J. B. Margolick. 1998. Evolution of neutralizing antibody response against HIV type 1 virions and pseudovirions in multicenter AIDS cohort study participants. AIDS Res. Hum. Retroviruses 14: 939-949. [Medline]
  34. Zhang, Z. Q., W. A. Schleif, D. R. Casimiro, L. Handt, M. Chen, M. E. Davies, X. Liang, T. M. Fu, A. Tang, K. A. Wilson, et al 2004. The impact of early immune destruction on the kinetics of postacute viral replication in rhesus monkey infected with the simian-human immunodeficiency virus 89.6P. Virology 320: 75-84. [Medline]
  35. Joag, S. V.. 2000. Primate models of AIDS. Microbes Infect. 2: 223-229. [Medline]
  36. Helmuth, E. F., N. L. Letvin, D. H. Margolin. 2000. Germline repertoire of the immunoglobulin VH3 family in rhesus monkeys. Immunogenetics 51: 519-527. [Medline]
  37. Andris, J. S., A. B. Miller, S. R. Abraham, S. Cunningham, F. Roubinet, A. Blancher, J. D. Capra. 1997. Variable region gene segment utilization in rhesus monkey hybridomas producing human red blood cell-specific antibodies: predominance of the VH4 family but not VH4-21 (V4-34). Mol. Immunol. 34: 237-253. [Medline]
  38. Link, J. M., M. A. Hellinger, H. W. Schroeder, Jr. 2002. The Rhesus monkey immunoglobulin IGHD and IGHJ germline repertoire. Immunogenetics 54: 240-250. [Medline]
  39. Meek, K., T. Eversole, J. D. Capra. 1991. Conservation of the most JH proximal Ig VH gene segment (VHVI) throughout primate evolution. J. Immunol. 146: 2434-2438. [Abstract]
  40. Bible, J. M., W. Howard, H. Robbins, D. K. Dunn-Walters. 2003. IGHV1, IGHV5 and IGHV7 subgroup genes in the rhesus macaque. Immunogenetics 54: 867-873. [Medline]
  41. Ermert, K., H. Mitlohner, W. Schempp, H. G. Zachau. 1995. The immunoglobulin {kappa} locus of primates. Genomics 25: 623-629. [Medline]
  42. Margolin, D. H., E. F. Helmuth Saunders, B. Bronfin, N. de Rosa, M. K. Axthelm, X. Alvarez, N. L. Letvin. 2002. High frequency of virus-specific B lymphocytes in germinal centers of SHIV-infected rhesus monkeys. J. Virol. 76: 3965-3973. [Abstract/Free Full Text]
  43. Baik, S. S., R. W. Doms, B. J. Doranz. 1999. HIV and SIV gp120 binding does not predict coreceptor function. Virology 259: 267-273. [Medline]
  44. Broder, C. C., P. L. Earl, D. Long, S. T. Abedon, B. Moss, R. W. Doms. 1994. Antigenic implications of human immunodeficiency virus type 1 envelope quaternary structure: oligomer-specific and -sensitive monoclonal antibodies. Proc. Natl. Acad. Sci. USA 91: 11699-11703. [Abstract/Free Full Text]
  45. Etemad-Moghadam, B., D. Rhone, T. Steenbeke, Y. Sun, J. Manola, R. Gelman, J. W. Fanton, P. Racz, K. Tenner-Racz, M. K. Axthelm, et al 2001. Membrane-fusing capacity of the human immunodeficiency virus envelope proteins determines the efficiency of CD+ T-cell depletion in macaques infected by a simian-human immunodeficiency virus. J. Virol. 75: 5646-5655. [Abstract/Free Full Text]
  46. Reimann, K. A., J. T. Li, R. Veazey, M. Halloran, I.-W. Park, G. B. Karlsson, J. Sodroski, N. L. Letvin. 1996. A chimeric simian/human immunodeficiency virus expressing a primary patient HIV-1 isolate env causes an AIDS-like disease after in vivo passage in rhesus monkeys. J. Virol. 70: 6922-6928. [Abstract/Free Full Text]
  47. Karlsson, G. B., M. Halloran, D. Schenten, J. Lee, P. Racz, K. Tenner-Racz, J. Manola, R. Gelman, B. Etemad-Moghadam, E. Desjardins, et al 1998. The envelope glycoprotein ectodomains determine the efficiency of CD4+ T lymphocyte depletion in simian-human immunodeficiency virus-infected macaques. J. Exp. Med. 188: 1159-1171. [Abstract/Free Full Text]
  48. Guide for the Care and Use of Laboratory Animals 1996 National Research Council, National Academy Press, Washington, DC.
  49. Bonner, R. F., M. Emmert-Buck, K. Cole, T. Pohida, R. Chuaqui, S. Goldstein, L. A. Liotta. 1997. Laser capture microdissection: molecular analysis of tissue. Science 278: 1481-1483. [Free Full Text]
  50. Lefranc, M. P.. 2001. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. 29: 207-209. [Abstract/Free Full Text]
  51. Nei, M., T. Gojobori. 1986. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol. Biol. Evol. 3: 418-426. [Abstract]
  52. Ota, T., M. Nei. 1994. Variance and covariances of the numbers of synonymous and nonsynonymous substitutions per site. Mol. Biol. Evol. 11: 613-619. [Abstract]
  53. Korber, B. T.. 2000. HIV signature and sequence variation analysis. A. G. Rodrigo, Jr, and G. H. Learn, Jr, eds. Computational and Evolutionary Analysis of HIV Molecular Sequences 55-72. Kluwer Academic Publishers, Dordrecht.
  54. Lossos, I. S., R. Tibshirani, B. Narasimhan, R. Levy. 2000. The inference of antigen selection on Ig genes. J. Immunol. 165: 5122-5126. [Abstract/Free Full Text]
  55. Rogozin, I. B., N. A. Kolchanov. 1992. Somatic hypermutagenesis in immunoglobulin genes. II. Influence of neighbouring base sequences on mutagenesis. Biochim. Biophys. Acta 1171: 11-18. [Medline]
  56. Betz, A. G., M. S. Neuberger, C. Milstein. 1993. Discriminating intrinsic and antigen-selected mutational hotspots in immunoglobulin V genes. Immunol. Today 14: 405-411. [Medline]
  57. Rada, C., G. T. Williams, H. Nilsen, D. E. Barnes, T. Lindahl, M. S. Neuberger. 2002. Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice. Curr. Biol. 12: 1748-1755. [Medline]
  58. Barbas, C. F., T. A. Collet, W. Amberg, P. Roben, J. M. Binley, D. Hoekstra, D. Cababa, T. M. Jones, R. A. Williamson, G. R. Pilkington, et al 1993. Molecular profile of an antibody response to HIV-1 as probed by combinatorial libraries. J. Mol. Biol. 230: 812-823. [Medline]
  59. Toran, J. L., L. Kremer, L. Sanchez-Pulido, I. M. de Alboran, G. del Real, M. Llorente, A. Valencia, M. A. de Mon, A. C. Martinez. 1999. Molecular analysis of HIV-1 gp120 antibody response using isotype IgM and IgG phage display libraries from a long-term non-progressor HIV-1-infected individual. Eur. J. Immunol. 29: 2666-2675. [Medline]
  60. Cavacini, L. A., C. L. Emes, A. V. Wisnewski, J. Power, G. Lewis, D. Montefiori, M. R. Posner. 1998. Functional and molecular characterization of human monoclonal antibody reactive with the immunodominant region of HIV type 1 glycoprotein 41. AIDS Res. Hum. Retroviruses 14: 1271-1280. [Medline]
  61. Bagley, J., P. J. Dillon, C. Rosen, J. Robinson, J. Sodroski, W. A. Marasco. 1994. Structural characterization of broadly neutralizing human monoclonal antibodies against the CD4 binding site of HIV-1 gp120. Mol. Immunol. 31: 1149-1160. [Medline]
  62. David, D., M. Zouali. 1995. Variable region light chain genes encoding human antibodies to HIV-1. Mol. Immunol. 32: 77-88. [Medline]
  63. Moran, M. J., J. S. Andris, Y.-I. Matsumato, J. D. Capra, E. M. Hersh. 1993. Variable region genes of anti-HIV human monoclonal antibodies: non-restricted use of the V gene repertoire and extensive somatic mutation. Mol. Immunol. 30: 1543-1551. [Medline]
  64. Kunert, R., F. Ruker, H. Katinger. 1998. Molecular characterization of five neutralizing anti-HIV type 1 antibodies: identification of nonconventional D segments in the human monoclonal antibodies 2G12 and 2F5. AIDS Res. Hum. Retroviruses 14: 1115-1128. [Medline]
  65. Pascual, V., Y.-J. Liu, A. Magalski, O. de Bouteiller, J. Banchereau, J. D. Capra. 1994. Analysis of somatic mutation in five B cell subsets of human tonsil. J. Exp. Med. 180: 329-339. [Abstract/Free Full Text]
  66. Clarke, S. H., K. Huppi, D. Ruezinsky, L. Staudt, W. Gerhard, M. Weigert. 1985. Inter- and intraclonal diversity in the antibody response to influenza haemagglutinin. J. Exp. Med. 161: 687-704. [Abstract/Free Full Text]
  67. Jacob, J., J. Przylepa, C. Miller, G. Kelsoe. 1993. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. III. The kinetics of V region mutation and selection in germinal center B cells. J. Exp. Med. 178: 1293-1307. [Abstract/Free Full Text]
  68. Liu, Y. J., J. Zhang, P. J. Lane, E. Y. Chan, I. C. MacLennan. 1991. Sites of specific B cell activation in primary and secondary responses to T cell-dependent and T cell-independent antigens. Eur. J. Immunol. 21: 2951-2962. [Medline]
  69. Jacob, J., R. Kassir, G. Kelsoe. 1991. In situ studies of the primary immune response to (4-hydroxy-3- nitrophenyl)acetyl. I. The architecture and dynamics of responding cell populations. J. Exp. Med. 173: 1165-1175. [Abstract/Free Full Text]
  70. Feinberg, M. B., J. P. Moore. 2002. AIDS vaccine models: challenging challenge viruses. Nat. Med. 8: 207-210. [Medline]
  71. Andris, J. S., P. H. Ehrlich, L. Ostberg, J. D. Capra. 1992. Probing the human antibody repertoire to exogenous antigens: characterization of the H and L chain V region gene segments from anti-hepatitis B virus antibodies. J. Immunol. 149: 4053-4059. [Abstract]
  72. Sakurai, H., R. A. Williamson, J. E. Crowe, J. A. Beeler, P. Poignard, R. B. Bastidas, R. M. Chanock, D. R. Burton. 1999. Human antibody responses to mature and immature forms of viral envelope in respiratory syncytial virus infection: significance for subunit vaccines. J. Virol. 73: 2956-2962. [Abstract/Free Full Text]
  73. Ivanovski, M., F. Silvestri, G. Pozzato, S. Anand, C. Mazzaro, O. R. Burrone, D. G. Efremov. 1998. Somatic hypermutation, clonal diversity, and preferential expression of the VH 51p1/VL kv325 immunoglobulin gene combination in hepatitis C virus-associated immunocytomas. Blood 91: 2433-2442. [Abstract/Free Full Text]
  74. William, J., C. Euler, S. Christensen, M. J. Shlomchik. 2002. Evolution of autoantibody responses via somatic hypermutation outside of germinal centers. Science 297: 2066-2070. [Abstract/Free Full Text]
  75. Matsumoto, M., S. F. Lo, C. J. Carruthers, J. Min, S. Mariathasan, G. Huang, D. R. Plas, S. M. Martin, R. S. Geha, M. H. Nahm, D. D. Chaplin. 1996. Affinity maturation without germinal centres in lymphotoxin-{alpha}-deficient mice. Nature 382: 462-466. [Medline]
  76. Takahashi, Y., P. R. Dutta, D. M. Cerasoli, G. Kelsoe. 1998. In situ studies of the primary immune response to (4-hydroxy-3- nitrophenyl)acetyl. V. Affinity maturation develops in two stages of clonal selection. J. Exp. Med. 187: 885-895. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Int ImmunolHome page
U. Hershberg, M. Uduman, M. J. Shlomchik, and S. H. Kleinstein
Improved methods for detecting selection by mutation analysis of Ig V region sequences
Int. Immunol., May 1, 2008; 20(5): 683 - 694.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Margolin, D. H.
Right arrow Articles by Letvin, N. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Margolin, D. H.
Right arrow Articles by Letvin, N. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS