De novo identification of VRC01 class HIV-1-neutralizing antibodies by next-generation sequencing of B-cell transcripts.

Next-generation sequencing of antibody transcripts provides a wealth of data, but the ability to identify function-specific antibodies solely on the basis of sequence has remained elusive. We previously characterized the VRC01 class of antibodies, which target the CD4-binding site on gp120, appear in multiple donors, and broadly neutralize HIV-1. Antibodies of this class have developmental commonalities, but typically share only ∼50% amino acid sequence identity among different donors. Here we apply next-generation sequencing to identify VRC01 class antibodies in a new donor, C38, directly from B cell transcript sequences. We first tested a lineage rank approach, but this was unsuccessful, likely because VRC01 class antibody sequences were not highly prevalent in this donor. We next identified VRC01 class heavy chains through a phylogenetic analysis that included thousands of sequences from C38 and a few known VRC01 class sequences from other donors. This "cross-donor analysis" yielded heavy chains with little sequence homology to previously identified VRC01 class heavy chains. Nonetheless, when reconstituted with the light chain from VRC01, half of the heavy chain chimeric antibodies showed substantial neutralization potency and breadth. We then identified VRC01 class light chains through a five-amino-acid sequence motif necessary for VRC01 light chain recognition. From over a million light chain sequences, we identified 13 candidate VRC01 class members. Pairing of these light chains with the phylogenetically identified C38 heavy chains yielded functional antibodies that effectively neutralized HIV-1. Bioinformatics analysis can thus directly identify functional HIV-1-neutralizing antibodies of the VRC01 class from a sequenced antibody repertoire.

Monoclonal and polyclonal gammopathy measured by serum free light chain and immunofixation subdivide the clinical outcomes of diffuse large B-cell lymphoma according to molecular classification.

Elevated serum free light chain (FLC) is known to be an adverse prognostic factor for diffuse large B-cell lymphoma (DLBCL). We hypothesized that monoclonal gammopathy (MG; elevated kappa [κ] or lambda [λ] FLC with an abnormal κ/λ ratio or a positive IF [immunofixation]) and polyclonal gammopathy (PG; elevated κ and/or λ FLC with a normal κ/λ ratio and a negative IF) would have different clinical outcome according to the molecular classification of DLBCL. In addition, MG would be a poor prognostic factor in patients with activated B-cell like type of DLBCL. Molecular classification of DLBCL, such as germinal center B-cell (GCB) type and non-GCB type, was performed according to the Hans algorithm. Among 175 newly diagnosed DLBCL patients, 96 (54.9 %) patients had an elevated FLC. MG and PG were observed in 34 and 68 patients, respectively. The 2-year overall survival (OS) and event-free survival (EFS) rates were 79.0 % and 71.6 %, respectively. In multivariate analysis, high-intermediate/high International Prognostic Index score and elevated FLC were significant for the OS (P = 0.002, P = 0.005, respectively) and EFS (P < 0.002, P = 0.010, respectively). MG and PG were also associated with inferior OS (P = 0.002, P = 0.011, respectively) and EFS (P = 0.002, P = 0.013, respectively). Ninety-six patients from a total 133 evaluable patients were classified to the non-GCB type. Patients with PG showed inferior clinical outcome for OS and EFS in patients with the GCB type (P = 0.006, P = 0.035, respectively). MG was a significant poor prognostic factor for OS and EFS in patients with the non-GCB type (P = 0.017, P = 0.004, respectively). MG was a poor prognostic maker in patients with the non-GCB type and PG was a poor prognostic indicator for the GCB type of DLBCL who were treated with R-CHOP.

Evolutionary redefinition of immunoglobulin light chain isotypes in tetrapods using molecular markers.

The phylogenetic relationships of Ig light chain (IGL) genes are difficult to resolve, because these genes are short and evolve relatively fast. Here, we classify the IGL sequences from 12 tetrapod species into three distinct groups (kappa, lambda, and sigma isotypes) using conserved amino acid residues, recombination signal sequences, and genomic organization of IGL genes as cladistic markers. From the distribution of the markers we conclude that the earliest extant tetrapods, the amphibians, possess three IGL isotypes: kappa, lambda, and sigma. Of these, two (kappa and lambda) are also found in reptiles and some mammals. The lambda isotype is found in all tetrapods tested to date, whereas the kappa isotype seems to have been lost at least in some birds and in the microbat. Conservation of the cladistic molecular markers suggests that they are associated with functional specialization of the three IGL isotypes. The genomic maps of IGL loci reveal multiple gene rearrangements that occurred in the evolution of tetrapod species. These rearrangements have resulted in interspecific variation of the genomic lengths of the IGL loci and the number and order of IGL constituent genes, but the overall organization of the IGL loci has not changed.

Antibody elbow angles are influenced by their light chain class.

We have examined the elbow angles for 365 different Fab fragments, and observe that Fabs with lambda light chains have adopted a wider range of elbow angles than their kappa chain counterparts, and that the lambda light chain Fabs are frequently found with very large (>195 degrees ) elbow angles. This apparent hyperflexibility of lambda chain Fabs may be due to an insertion in their switch region, which is one residue longer than in kappa chains, with glycine occurring most frequently at the insertion position. A new, web-based computer program that was used to calculate the Fab elbow angles is described.

Mouse monoclonal antibodies against rabbit b6 allotype and their use for characterization of light-chain subpopulations.

Mouse anti-allotypic hybridomas directed against different antigenic determinants of the b6 rabbit allotype have been raised. The fine specificity of these monoclonal antibodies (mAbs) was determined by radioimmunoassay and it was possible to classify them into three groups, each directed against distinct epitopes of the b6 allotype. Hare "b6" IgG were tested with anti-b6 mAb and no reaction was found indicating that the number of allotopes present on b6 molecules is greater than the three detected by the mAb. Comparative analysis by precipitation in gel of IgG from homozygous b6/b6 rabbits using mouse mAb and rabbit anti-b6 antibodies suggested that at least two categories of molecules can be discriminated. The observation of b6 subpopulations was confirmed by isolation of a minor subpopulation of IgG on a mouse monoclonal immunoadsorbent.

Anti-acetylcholine receptor antibody: use of polyethylene glycol as an aid to precipitation of antibody-receptor complexes in determination of light chain and subclass.

Polyethylene glycol 6000 (PEG) was used as an aid to precipitation of antibody-acetylcholine receptor (AChR) complexes. In the absence of anti-human IgG, 8% PEG can be used to precipitate antibody-AChR complexes. In the presence of low titre specific antiserum, 3% PEG selectively precipitates anti-IgG-IgG-AChR complexes and allows analysis of light chain and subclass contribution to the anti-AChR. The specificity of the various antisera is presented and results in 16 patients described.

The immunological characterization of human antibodies to factor VIII isolated by immuno-affinity chromatography.

Nine human antibodies to factor VIII were isolated from haemophilic plasmas by affinity chromatography and gel filtration and six were subsequently subjected to immunological characterization. Three partially purified preparations were similarly characterized. Eight of the antibodies were characterized as being exclusively IgG and one preparation was found to contain IgM. Seven of the antibodies contained only a single light chain type, four being of type lambda and three of type kappa. Two antibody preparations contained both kappa and lambda light chains. In four of the preparations, only a single heavy chain sub-class could be demonstrated, three of IgG3 and one of IgG4. Of the remainder, three were a mixture of IgG3 and IgG4 sub-classes and one contained both IgG2 and IgG4. IgG sub-classification could not be achieved with the IgM-containing preparation. These results demonstrate a restricted heterogeneity of light and heavy chains in human antibodies to factor VIII.

Light-chain profiles in sera from patients with subacute sclerosing panencephalitis.

Sera from 2 subacute sclerosing panencephalitis (SSPE) patients were absorbed with a concentrated preparation of measles virus. Measles-specific IgG was eluted from the precipitates containing measles antigen-antibody complex. These IgGs, when subjected to immunofixation after isoelectric focusing showed a number of oligoclonal bands with one type of light-(L) chain. In urea polyacrylamide gel electrophoresis, the reduced and alkylated measles-specific IgG showed 1-3 homogeneous L-chain bands, whereas IgG isolated from unabsorbed sera and IgG isolated from supernatants of SSPE sera after absorption with measles virus showed a diffuse L-chain band. It can be concluded that in SSPE, measles virus is responsible for the synthesis of L-chain with restricted heterogeneity.

Fish lymphocytes differ in the expression of surface immunoglobulin.

Catfish peripheral blood and splenic lymphocytes were assayed for surface immunoglobulin using fifteen different mouse hybridoma antibodies to catfish immunoglobulin (Ig). These studied showed that this battery of monoclonal antibodies did not detect significant amounts of Ig on all lymphocytes. Unlike polyclonal antisera which demonstrated nearly 100% surface Ig+ cells, the monoclonal antibodies detected approximately 40% surface Ig+ cells. Furthermore, the percentage of Ig+ cells reactive with two of these monoclonals, tentatively shown to react with two different types of catfish light chains, was found to be nearly additive when the two antibodies were mixed. Thus it seems that fish lymphocytes, like their mammalian counterparts, have two different populations of lymphocytes; one which contains abundant surface Ig and one which does not. Whether these two types of cells represent the fish equivalents of B and T cells remains to be determined.

Ig light chain variability in DNP(494)-KLH immunised sea bass (Dicentrarchus labrax L.): evidence for intra-molecular induced suppression.

The coding sequence of the sea bass light chain was obtained by sequential anchored PCR on a head kidney cDNA library of a DNP(494)-KLH immunised sea bass. The cDNA sequence obtained codes for a leader peptide of 21aa and a mature IgL chain of 223aa. Both the amino acid sequence comparisons and neighbour-joining trees showed that the IgL chain of sea bass obtained is of the L1/G type. To study the variability of the light chain, additional PCRs on the cDNA library and cDNA from pooled head kidneys were performed. Multiple alignment of unique sequences (N=17) could be performed without introducing gaps, and showed extremely low variability in CDR1, and no variability in CDR2 or CDR3. A possible explanation for this low variability of the IgL1 chain might be the enhanced expression of monospecific anti-DNP antibodies. The isolation and characterisation of partial genomic and cDNA IgL sequences, which showed normal variability, corroborate this explanation.

Characterisation of immunoglobulin light chain cDNAs of the Atlantic salmon, Salmo salar L.; evidence for three IgL isotypes.

By screening a cDNA library and analysis of DNA produced by a combined 3'RACE/5'-anchored PCR, we have isolated three isotypes of IgL in the Atlantic salmon. Two of the isotypes were homologous to rainbow trout IgL1 and L2 sequences, while the third represents a previously uncharacterised salmonid IgL. The novel type 3 CL region is homologous to spotted wolffish c1 and yellowtail sequences, while the VL region is more similar to channel catfish F class than to any other fish VL sequences. Southern analysis indicates that the gene segments of all three isotypes are organised in multiple clusters. In addition, the VL gene segments of type 3 are arranged in opposite orientation relative to the JL and CL segments, while gene segments in type 2 clusters are all in the same orientation. Although transcripts of type 1 and 3 were readily found in the spleen and head kidney, only minute amounts of type 2 transcripts were seen. The majority of type 3 messages were truncated, suggesting that spliced and full-length transcripts of this isotype probably are present at a low level compared to type 1 transcripts. The uniqueness of the type 3 VLJL sequences suggests that this isotype offers additional diversity to the antigen-binding site of Atlantic salmon immunoglobulins.

Human monoclonal antibodies to influenza virus: IgG subclass and light chain distribution.

Three adults and three children were immunized with inactivated or live attenuated influenza vaccines and 98 IgG monoclonal antibodies derived from EBV immortalization of their blood lymphocytes were studied. All antibodies reacted specifically with influenza A H3N2 or H1N1 whole virus and 73 of 74 tested reacted with the purified HA glycoproteins. The majority (76%) of 77 monoclonal antibodies adequately tested by ELISA or solid phase RIA contained lambda light chains. ELISA analysis of the IgG subclass of the six persons' postimmunization anti-influenza serum activity and of monoclonal antibodies derived from them showed a similar predominance of IgG1.

Isoelectric focusing of the human IgG light chains: a classification of the fractions and their normal values.

The method of isoelectrofocusing was employed to obtain a reproducible pattern of human IgG light chain bands. The map of IF spectra, which contained 13 main fractions and some subfractions, was delineated. Isoelectric points of these fractions and their quantitative relations in 10 healthy individuals were determined. Initial studies performed in 3 patients with monoclonal immunoglobulins and in 1 patient with gastric ulcer, negative for monoclonal protein, showed significant deviations from normal values in certain fractions of the IF spectrum.

Reactive lymphoid hyperplasia with single class (monoclonal) surface immunoglobulin.

Lymphoid tissues from 12 patients were diagnosed as reactive lymphoid hyperplasia, but surface immunoglobulin studies revealed monoclonal (single class) immunoglobulin staining patterns. Infectious, autoimmune, and immunodeficient conditions were diagnosed on the basis of histology and clinical features. Such surface immunoglobulin restriction has been used as an indicator of a neoplastic lymphoid proliferation, but the cases of these patients, in whom the histologic diagnosis was benign, emphasize the importance of a multiparameter approach to diagnosis. Although at the time of this report none of the patients still available to follow-up study have developed known lymphoid neoplasms, the possibility that monoclonal SIg patterns are a harbinger of neoplastic disease makes continuing follow-up of such patients important.

Production of monoclonal antibodies with haemagglutination-inhibition activity to the Skalica strain from the tick-borne encephalitis complex.

Hybridomas secreting monoclonal antibodies with haemagglutination-inhibition (HI) activity to the Skalica strain of tick-borne encephalitis (TBE) complex were prepared by the fusion of P3-NS1-Ag4-1 myeloma cell line with spleen cells of BALB/c mice immunized with the purified Skalica strain. The highest titres of monoclonal antibodies obtained from the hybridomas S-9, S-15 and S-16 ranged from 512 to 10,240, respectively; the ascitic fluid contained as many as 4.6 mg/ml of monoclonal antibodies. Its analysis by Ouchterlony's double immunodiffusion, agarose electrophoresis, and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of monoclonal antibodies with mu isotype of the heavy and kappa isotype of the light chain. The specificity of the monoclonal antibodies was proved using 11 different antigens from family Togaviridae in the HI test.

[Certain characteristics of isolation of human myelomic IgG of different subclasses and their distribution according to the allotypes, subclasses and types of light chain]. / Nekotorye osobennosti vydeleniia mielomnykh IgG cheloveka razlichnykh subklassov i ikh raspredelenie po allotipam, subklassam i tipam legkikh tsepei.

A combination of the methods of preparative electrophoresis in agar gel and of the ion-exchange chromatography on DE-32 cellulose permitted to obtain 32 immunochemically pure human myelomic IgG. The proteins of the first three subclasses were obtained by elution in the 0.01 phosphate buffer at pH 7.6. IgG4 was eluted with the increase of the gradient to 1 M NaCl in the phosphate buffer. Of the 32 human myelomic IgG 26 represented IgG1,4--IgG2, 1--IgG3, and 1--IgG4. Among the 26 IgG1 11 were of the Gm(a) allotype, and 15 proteins had the Gm(f) determinant; one IgG2 protein was Gm(n+) and 3--Gm(n-). One IgG3 protein was referred to the Gm(b) variant. The majority of the IgG proteins of the subclass I had chi-type of the L-chains, and the chi: lambda ratio constituted 2.71.

Toward a more accurate view of human B-cell repertoire by next-generation sequencing, unbiased repertoire capture and single-molecule barcoding.

B-cell repertoire analysis using next-generation sequencing has become a valuable tool for interrogating the genetic record of humoral response to infection. However, key obstacles such as low throughput, short read length, high error rate, and undetermined bias of multiplex PCR method have hindered broader application of this technology. In this study, we report several technical advances in antibody repertoire sequencing. We first demonstrated the ability to sequence antibody variable domains using the Ion Torrent PGM platform. As a test case, we analyzed the PGT121 class of antibodies from IAVI donor 17, an HIV-1-infected individual. We then obtained "unbiased" antibody repertoires by sequencing the 5'-RACE PCR products of B-cell transcripts from IAVI donor 17 and two HIV-1-uninfected individuals. We also quantified the bias of previously published gene-specific primers by comparing the repertoires generated by 5'-RACE PCR and multiplex PCR. We further developed a single-molecule barcoding strategy to reduce PCR-based amplification noise. Lastly, we evaluated several new PGM technologies in the context of antibody sequencing. We expect that, based upon long-read and high-fidelity next-generation sequencing technologies, the unbiased analysis will provide a more accurate view of the overall antibody repertoire while the barcoding strategy will facilitate high-resolution analysis of individual antibody families.

The immunoglobulin light chain in poikilothermic vertebrates.

The immunoglobulin light chains are classified as kappa or lambda in mammals and birds (homeothermic vertebrates), but the traditional criteria for this classification are not applicable to the light chains found in poikilothermic vertebrates. Still it is possible to find some relationships between Ig light chain sequences in these animals and in those of the homeothermic animals. It is generally accepted that the Ig light chains contribute to the antigen binding capacity of antibodies and the variability is approximately similar in all studied vertebrate species except the elasmobranchs. This might be explained by the organisation of the Ig light chain locus in these animals and the fact that the variable and joining DNA segments are joined in the genome. These conclusions are limited by the small number of species studied in this respect.

Relevance of class, molecular weight and isoelectric point in predicting human light chain amyloidogenicity.

The ability to predict the amyloidogenicity of certain light chains may facilitate an earlier diagnosis of AL amyloidosis and, possibly, lead to more effective treatment. Using current methods, available in clinical chemistry laboratories, we assessed the class, the relative molecular mass (Mr) and the isoelectric point of urinary monoclonal light chains from 35 patients with AL amyloidosis (A+) and 51 without amyloidosis (A-). The light chain class (LCC) was lambda in 77% and 45% of A+ and A- patients, respectively. Light chain fragments (LCF) with low Mr (12-18 x 10(3) were detected in the urine of 30/35 A+ patients and in 15/51 A- ones. The mean (SD) isoelectric point (pI) of A+ light chains was 4.8 (1.1) while in A- patients it was 6.2 (1.6). Univariate analysis showed significant differences between the two groups for the three parameters. Discriminant analysis gave a function which allowed a correct allocation of 81% of the cases between the two groups.

Characterization of immunoglobulin light chain isotypes in the common carp.

Two or three isotypes of immunoglobulin (Ig) light (L) chain have been demonstrated in several teleost species thus far. A reverse transcription-polymerase chain reaction (RT-PCR) strategy was used with the aim of isolating cDNA clones encoding IgL chain from the common carp ( Cyprinus carpio. L.) different from the previously isolated isotype, designated L1A. cDNA clones representing two novel isotypes, designated L1B and L3, were obtained in this study. For CL segments, L1B sequences were similar to type L1/G in various teleost species, as well as L1A, while L3 sequences were closely related with type L3/F in catfish and zebrafish. For VL segments, however, L1A sequences demonstrated higher similarity to L3 rather than L1B. These results were also supported by the phylogenetic study. The similarity found between L1A and L3 VL sequences was based on the very high degree of nucleotide identity in FR1, and/or FR2 to FR3, strongly suggesting the involvement of interlocus recombination between them. Southern blot analyses suggested that the locus of L1B has a cluster-like organization, but that those of L3 and L1A are currently unknown due to cross-hybridization between those VL probes. Northern blot analyses showed that CL mRNA of each isotype was expressed in lymphoid tissues examined, particularly strongly in pronephros, and was predominantly composed of approximately 1-kb and 0.6-kb transcripts, possibly corresponding to fully spliced (LVJC) and truncated (JC/J-C) forms, respectively.