Variable Lymphocyte Receptor B Research Articles

High throughput long-read sequencing of circulating lymphocytes of the evolutionarily distant sea lamprey reveals diversity and common elements of the variable lymphocyte receptor B (VLRB) repertoire.

The leucine-rich repeat-based variable lymphocyte receptor B (VLRB) antibody system of jawless vertebrates is capable of generating an antibody repertoire equal to or exceeding the diversity of antibody repertoires of jawed vertebrates. Unlike immunoglobulin-based immune repertoires, the VLRB repertoire diversity is characterized by variable lengths of VLRB encoding transcripts, rendering conventional immunoreceptor repertoire sequencing approaches unsuitable for VLRB repertoire sequencing. Here we demonstrate that long-read single-molecule real-time (SMRT) sequencing (PacBio) approaches permit the efficient large-scale assessment of the VLRB repertoire. We present a computational pipeline for sequence data processing and provide the first repertoire-based analysis of VLRB protein characteristics including properties of its subunits and regions of diversity within each structural leucine-rich repeat subunit. Our study provides a template to explore changes in the VLRB repertoire during immune responses and to establish large scale VLRB repertoire databases for computational approaches aimed at isolating monoclonal VLRB reagents for biomedical research and clinical applications.

Passive Immunization with Recombinant Antibody VLRB-PirAvp/PirBvp-Enriched Feeds against Vibrio parahaemolyticus Infection in Litopenaeus vannamei Shrimp.

The causative agent of acute hepatopancreatic necrosis disease (AHPND) is the bacterium, Vibrio parahaemolyticus, which secretes toxins into the gastrointestinal tract of its host. Vibrio parahaemolyticus toxins A and B (PirAvp/PirBvp) have been implicated in the pathogenesis of this disease, and are, therefore, the focus of studies developing treatments for AHPND. We previously produced recombinant antibodies based on the hagfish variable lymphocyte receptor B (VLRB) capable of neutralizing some viruses, suggesting that this type of antibody may have a potential application for treatment of AHPND. Here, recombinant PirAvp/PirBvp, produced using a bacterial expression system, were used as antigens to screen a hagfish VLRB cDNA library to obtain PirAvp/PirBvp-specific antibodies. A cell line secreting these antibodies was established by screening and cloning the DNA extracted from hagfish B cells. Supernatants collected from cells secreting the PirAvp/PirBvp antibodies were collected and concentrated, and used to passively immunize shrimp to neutralize the toxins PirAvp or PirBvp associated with AHPND. Briefly, 10 μg of PirAvp and PirBvp antibodies, 7C12 and 9G10, respectively, were mixed with the shrimp feed, and fed to shrimp for three days consecutive days prior to experimentally infecting the shrimp with V. parahaemolyticus (containing toxins A and B), and resulting mortalities recorded for six days. Results showed significantly higher level of survival in shrimp fed with the PirBvp-9G10 antibody (60%) compared to the group fed the PirAvp-7C12 antibody (3%) and the control group (0%). This suggests that VLRB antibodies may be a suitable alternative to immunoglobulin-based antibodies, as passive immunization treatments for effective management of AHPND outbreaks within shrimp farms.

Complement component C1q plays a critical role in VLRA/VLRC-mediated immune response

In jawless vertebrates, the lamprey complement component C1q (LC1q) acts as a lectin and activates lamprey complement component C3 (LC3) in association with mannose-binding lectin (MBL)-associated serine protease (MASP) via the lectin pathway. Furthermore, LC1q may interact with variable lymphocyte receptor B (VLRB) in a complex with antigens and mediate the activation of LC3, leading to cytolysis. In the present study, we found, for the first time, that LC1q plays a critical role in VLRA/VLRC-mediated immune response. Escherichia coli, Shigella flexneri, Aeromonas hydrophila, Pseudomonas plecoglossicida, Aeromonas allosaccharophila, P. luteola, Brevundimonas diminuta, and Bacillus cereus were isolated from infected Lampetra morii in our laboratory and identified using the 16s rRNA method. A. hydrophila was confirmed as a rapidly spreading lethal pathogen in the larvae of L. morii and was used in subsequent immune stimulation experiments. The results of real-time quantitative polymerase chain reaction (Q-PCR) and immunofluorescence analyses indicated that the RNA and protein expression levels of LC1q were upregulated following exposure to 107 cfu/mL of A. hydrophila, compared to the levels of the naïve group. We obtained LC1q morphants (LC1q MO) of lamprey larvae by morpholino-mediated knockdowns. We found that LC1q played key roles in the embryonic development of lamprey. The median lethal time (LT50) of LC1q MO larvae was 2 d after being exposed to the pathogens, whereas the LT50 of control MO was 5 d. The drastic decrease in LT50 values after LC1q knockdown implies that LC1q plays a critical role in lamprey immune response. Gene expression profiles of LC1q-deficient A. hydrophila, control MO A. hydrophila, wild type A. hydrophila, and naive 1-month-old ammocoetes larvae were compared by examining the expression levels of a selected panel of orthologous genes. It is worth mentioning that LC1q MO affected the VLRA+/VLRC + population genes but did not affect the VLRB + populations. Immunohistochemical data indicated that LC1q deficiency also affected VLRA and VLRC but not VLRB. Thus, LC1q plays a critical role in VLRA/VLRC-mediated immune response in lamprey.

Detection of Human CD38 Using Variable Lymphocyte Receptor (VLR) Tetramers.

CD38 is a multifunctional cell surface receptor expressed on multiple cell lineages of hematopoietic origin with high levels of expression on human plasma cells. Previously, we isolated the monoclonal variable lymphocyte receptor B (VLRB) MM3 antibody from the evolutionarily distant sea lamprey, which recognized the CD38 ectoenzyme exclusively on human plasma cells in a manner that correlated with CD38 enzymatic activity. The plasma cell-specific binding of VLRB MM3 contrasts with the broad pattern of expression of CD38-determined conventional antibodies specific for this antigen. In an effort to facilitate the application of this unique reagent in combination with conventional antibody panels, we explored a strategy to generate VLRB MM3 tetramers. The resulting reagent maintained the threshold-based recognition of CD38. Increased sensitivity achieved with VLRB MM3 tetramers also showed preferential recognition of germinal center centroblasts over centrocytes. VLRB MM3 tetramers thus provided a unique and versatile single-step staining reagent for the detection of human CD38 that is readily incorporated into multi-color flow cytometry panels.

Development of a modified yeast display system for screening antigen-specific variable lymphocyte receptor B in hagfish (Eptatretus burgeri)

The variable lymphocyte receptor B (VLRB) of jawless vertebrates has a similar function to the antibodies produced by jawed vertebrates, and has been considered as an alternative source to mammalian antibodies for use in biological research. We developed a modified yeast display vector system (pYD8) to display recombinant hagfish VLRB proteins on the extracellular surface of yeast for the isolation of antigen-specific VLRBs. After observing an up-regulation in the VLRB response in hagfish immunized with hemagglutinin 1 of avian influenza virus H9N2 subtype (H9N2-HA1), the antigen-specific VLRBs decorated on the yeast's surface were selected by quantitative library screening through magnetic-activated cell sorting (MACS) and fluorescent-activated cell sorting (FACS). We also demonstrated a strong specificity of the antigen-specific VLRBs, when expressed as a secreted protein using a mammalian expression system. Together, our findings suggest that the pYD8 vector system could be useful for screening antigen-specific hagfish VLRBs, and the specificity of secreted VLRB may have potential for a variety of biological applications.

A novel CD81 homolog identified in lamprey, Lampetra japonica, with roles in the immune response of lamprey VLRB+ lymphocytes.

The cluster of differentiation 81 (CD81), a member of the transmembrane 4 superfamily, is primarily found to be expressed in a wide variety of cells including T and B cells of vertebrates as a critical modulator. In the present study, the open reading frame of a CD81 gene homolog (Lja-CD81) was cloned in lamprey, Lampetra japonica, which is 702 bp long and encodes a protein of 233-amino acids. Although Lja-CD81 seems to be close to CD9 molecules in their full-length sequences, Lja-CD81 possesses higher identity to vertebrates' CD81 than to CD9 (including a lamprey CD9) molecules in their large extracellular loops. In addition, it also possesses a myristoylation site (Met-Gly-Val-Glu-Gly-Cys-Leu-Lys) in its N-terminal region which is identical to the N-terminal regions of CD81 molecules. These data suggest that CD9 and CD81 molecules diverged no later than the emergence of jawless vertebrates. The mRNA levels of Lja-CD81 in lymphocytes and supraneural myeloid bodies were up-regulated significantly after stimulation with mixed antigens, and a similar expressional pattern of Lja-CD81 at protein level was also confirmed. Furthermore, Lja-CD81 was found to be co-localized with variable lymphocyte receptor B (VLRB) evenly on the cell membrane of peripheral blood lymphocytes isolated from control group, but they were found to aggregate on one side of the membrane of peripheral blood VLRB+ lymphocytes after stimulation with mixed antigens. All these results indicate that the Lja-CD81 identified in lamprey may play an important role in the immune response of lamprey VLRB+ lymphocytes.

Expression and characterization of monomeric variable lymphocyte receptor B specific to the glycoprotein of viral hemorrhagic septicemia virus (VHSV)

Monomeric variable lymphocyte receptor B (VLRB) is one of the smallest binding scaffold (20–25 kDa) from jawless vertebrates, hagfish and lamprey. This relatively new class of binding scaffold has various advantages: i) it has a single peptide composition, amenable to molecular engineering for enhancing its stability and affinity; ii) it has a small size, contributing better tissue penetration and easier production using microorganism expression system. Monomeric arVLRB142, which can specifically bind to the glycoprotein of viral hemorrhagic septicemia virus (VHSV), was expressed in Pichia pastoris. High quantity recombinant monomeric arVLRB142 (rVLR142mono) was purified from 100 ml of culture with a resulting yield of 2.6 ±1.3 mg of target protein. Functional studies revealed that the purified rVLR142mono can specifically recognize low levels of the target antigen (recombinant glycoprotein) (i.e. as low as 0.1 nM), but also the native glycoprotein of VHSV. The expressed rVLR142mono exhibited high levels of stability and it retained it binding capacity over broad temperature (4 °C ~ 60 °C) and pH ranges (pH 1.5–12.5). We developed an effective expression system for mass production of monomeric VLRB based on P. pastoris. The recombinant protein that was obtained offers promising binding avidity and biophysical stability and its potential use in various biotechnological applications.

Development of a novel therapeutic vaccine carrier that sustains high antibody titers against several targets simultaneously.

With the aim to improve the efficacy of therapeutic vaccines that target self-antigens, we have developed a novel fusion protein vaccine on the basis of the C-terminal multimerizing end of the variable lymphocyte receptor B (VLRB), the Ig equivalent in jawless fishes. Recombinant vaccines were produced in Escherichia coli by fusing the VLRB sequence to 4 different cancer-associated target molecules. The anti-self-immune response generated in mice that were vaccinated with VLRB vaccines was compared with the response in mice that received vaccines that contained bacterial thioredoxin (TRX), previously identified as an efficient carrier. The anti-self-Abs were analyzed with respect to titers, binding properties, and duration of response. VLRB-vaccinated mice displayed a 2- to 10-fold increase in anti-self-Ab titers and a substantial decrease in Abs against the foreign part of the fusion protein compared with the response in TRX-vaccinated mice (P < 0.01). VLRB-generated Ab response had duration similar to the corresponding TRX-generated Abs, but displayed a higher diversity in binding characteristics. Of importance, VLRB vaccines could sustain an immune response against several targets simultaneously. VLRB vaccines fulfill several key criteria for an efficient therapeutic vaccine that targets self-antigens as a result of its small size, its multimerizing capacity, and nonexposed foreign sequences in the fusion protein.-Saupe, F., Reichel, M., Huijbers, E. J. M., Femel, J., Markgren, P.-O., Andersson, C. E., Deindl, S., Danielson, U. H., Hellman, L. T., Olsson, A.-K. Development of a novel therapeutic vaccine carrier that sustains high antibody titers against several targets simultaneously.

Identification and characterisation of the immune response properties of Lampetra japonica BLNK.

B cell linker protein (BLNK) is a central linker protein involved in B cell signal transduction in jawed vertebrates. In a previous study, we have reported the identification of a BLNK homolog named Lj-BLNK in lampreys. In this study, a 336 bp cDNA fragment encoding the Lj-BLNK Src homology 2 (SH2) domain was cloned into the vector pET-28a(+) and overexpressed in Escherichia coli BL21. The recombinant fragment of Lj-BLNK (rLj-BLNK) was purifiedby His-Bind affinity chromatography, and polyclonal antibodies against rLj-BLNK were raised in male New Zealand rabbits. Fluorescenceactivated cell sorting (FACS) analysisrevealed that Lj-BLNK was expressed in approximately 48% of the lymphocyte-like cells of control lampreys, and a significant increase in Lj-BLNK expression was observed in lampreys stimulated with lipopolysaccharide (LPS). Western blotting analysis showed that variable lymphocyte receptor B (VLRB) and Lj-BLNKwere distributed in the same immune-relevant tissues, and the levels of both were upregulated in supraneural myeloid bodies and lymphocyte-like cells after LPS stimulation. Immunofluorescence demonstrated that Lj-BLNK was localized in VLRB+ lymphocyte-like cells. These results indicate that the Lj-BLNK protein identified in lampreys might play an important role in the VLRB-mediated adaptive immune response.

Lamprey VLRB response to influenza virus supports universal rules of immunogenicity and antigenicity.

Immunoglobulins (Igs) are a crown jewel of jawed vertebrate evolution. Through recombination and mutation of small numbers of genes, Igs can specifically recognize a vast variety of natural and man-made organic molecules. Jawless vertebrates evolved a parallel system of humoral immunity, which recognizes antigens not with Ig, but with a structurally unrelated receptor called the variable lymphocyte receptor B (VLRB). We exploited the convergent evolution of Ig and VLRB antibodies (Abs) to investigate if intrinsic chemical features of foreign proteins determine their antigenicity and immunogenicity. Surprisingly, we find lamprey VLRB and mouse Ig responses to influenza A virus are extremely similar. Each focuses ~80% of the response on hemagglutinin (HA), mainly through recognition of the major antigenic sites in the HA globular head domain. Our findings predict basic conservation of Ab responses to protein antigens, strongly supporting the use of animal models for understanding human Ab responses to viruses and protein immunogens.