Leveraging the biotechnological promise of the hagfish variable lymphocyte receptors: tools for aquatic microbial diseases.

The jawless vertebrates (agnathans/cyclostomes) are ancestral animals comprising lampreys and hagfishes as the only extant representatives. They possess an alternative adaptive immune system (AIS) that uses leucine-rich repeats (LRR)-based variable lymphocyte receptors (VLRs) instead of the immunoglobulin (Ig)-based antigen receptors of jawed vertebrates (gnathostomes). The different VLR types are expressed on agnathan lymphocytes and functionally resemble gnathostome antigen receptors. In particular, VLRB is functionally similar to the B cell receptor and is expressed and secreted by B-like lymphocytes as VLRB antibodies that bind antigens with high affinity and specificity. The potential repertoire scale of VLR-based antigen receptors is believed to be at least comparable to that of Ig-based systems. VLR proteins inherently possess characteristics that render them excellent candidates for biotechnological development, including tractability to recombinant approaches. In recent years, scientists have explored the biotechnological development and utility of VLRB proteins as alternatives to conventional mammalian antibodies. The VLRB antibody platform represents a non-traditional approach to generating a highly diverse repertoire of unique antibodies. In this review, we first describe some aspects of the biology of the AIS of the jawless vertebrates, which recognizes antigens by means of unique receptors. We then summarize reports on the development of VLRB-based antibodies and their applications, particularly those from the inshore hagfish (Eptatretus burgeri) and their potential uses to address microbial diseases in aquaculture. Hagfish VLRB antibodies (we call Ccombodies) are being developed and improved, while obstacles to the advancement of the VLRB platform are being addressed to utilize VLRBs effectively as tools in immunology. VLRB antibodies for novel antigen targets are expected to emerge to provide new opportunities to tackle various scientific questions. We anticipate a greater interest in the agnathan AIS in general and particularly in the hagfish AIS for greater elucidation of the evolution of adaptive immunity and its applications to address microbial pathogens in farmed aquatic animals and beyond.

Characterization of Hagfish (Eptatretus burgeri) Variable Lymphocyte Receptor-Based Antibody and Its Potential Role in the Neutralization of Nervous Necrosis Virus.

The variable lymphocyte receptor (VLR) mediates the humoral immune response in jawless vertebrates, including lamprey (Petromyzon marinus) and hagfish (Eptatretus burgeri). Hagfish VLRBs are composed of leucine-rich repeat (LRR) modules, conjugated with a superhydrophobic C-terminal tail, which contributes to low levels of expression in recombinant protein technology. In this study, we screened Ag-specific VLRBs from hagfish immunized with nervous necrosis virus (NNV). The artificially multimerized form of VLRB was constructed using a mammalian expression system. To enhance the level of expression of the Ag-specific VLRB, mutagenesis of the VLRB was achieved in vitro through domain swapping of the LRR C-terminal cap and variable LRR module. The mutant VLRB obtained, with high expression and secretion levels, was able to specifically recognize purified and progeny NNV, and the Ag binding ability of this mutant was increased by at least 250-fold to that of the nonmutant VLRB. Furthermore, preincubation of the Ag-specific VLRB with NNV reduced the infectivity of NNV in E11 cells in vitro, and in vivo experiment. Our results suggest that the newly developed Ag-specific VLRB has the potential to be used as diagnostic and therapeutic reagents for NNV infections in fish.

Generation and characterization of hagfish variable lymphocyte receptor B against glycoprotein of viral hemorrhagic septicemia virus (VHSV).

Variable lymphocyte receptors B (VLRBs) are non-immunoglobulin components of the humoral immune system in jawless vertebrates including hagfish (Eptatretus burgeri) and lamprey (Petromyzon marinus). Hagfish VLRBs consist of leucine rich repeat (LRR) modules with a superhydrophobic C-terminal tail, the latter of which leads to extremely low expression levels in recombinant protein technology. Here, we present an artificially oligomerized VLRB (arVLRB) that conjugates via the C4bp oligomerization domain derived from human C4b-binding protein (hC4bp) rather than the superhydrophobic tail. The resulting arVLRB had a tightly multimerized form with seven monomeric VLRB arms and showed high expression and secretion levels in a mammalian expression system. To isolate antigen-specific arVLRB, we constructed large VLRB libraries from hagfish immunized with the fish pathogen, viral hemorrhagic septicemia virus (VHSV). The selected arVLRBs were found to recognize various types of antigens, including the recombinant target protein, purified viruses, and progeny viruses, with high antigen binding abilities and specificities. We also performed in vitro affinity maturation of the arVLRBs through LRRCT mutagenesis, and found that this enhanced their antigen-binding properties by at least 125-fold. Our epitope mapping analysis revealed that 37DWDTPL42, which is located in a region conserved among the glycoproteins of all VHSV isolates, is the recognition epitope of the arVLRBs. Thus, our newly developed arVLRB could prove useful in the development of universal diagnostic tools and/or therapeutic agents for the virus. Together, our novel findings provide valuable insights into hagfish VLRB and its potential use as a novel alternative to conventional antibodies for biotechnological applications.

Convergent evolution of the adaptive immune response in jawed vertebrates and cyclostomes: An evolutionary biology approach based study.

Two different adaptive immune systems (AIS) are present in the two phyla of vertebrates (jawed vertebrates and cyclostomes). The jawed vertebrate system is based on IG/TCR/RAG/MHC while the cyclostome system is based on VLRCs and AID-like enzymes both systems using homologous Cell types (B-cell and B-cell Like, T-cell and T-cell like). We will present our current view of the evolution of these two AISs and present alternative hypotheses that could explain the apparent convergent evolution of the two systems. We will also discuss why comparative immunology analyses should be based on evolutionary biology approaches and not on the scale of progress one.

Investigation of variable lymphocyte receptors in the alternative adaptive immune response of hagfish.

Jawless vertebrates have an alternative adaptive immune system mediated by variable lymphocyte receptors (VLRs), VLRA, VLRC and VLRB. In investigation on the adaptive immunity of hagfish, avian influenza virus hemagglutinin (H9N2-HA1) was used as a model antigen, with mRNA expression levels of VLRA, VLRC and Ikaros were up-regulated in the first week post-immunization. CD45 was up-regulated after the first week; and expression of VLRB progressively increased over the course of the trial. The transcriptional/translational activation of VLRB in blood was verified. The VLRBs cloned from these transcripts showed diversity in their leucine-rich repeats (LRRs). The production of specific VLRB increased in a time- and dose-dependent manner, detected by an anti-VLRB antibody (11G5). The plasma VLRB could distinguish H9N2-HA1 from unrelated proteins, but not from other HA1 subtypes. Together, our findings show that VLRs play a major role in the alternative adaptive immune system of hagfish by responding to specific foreign substances, such as H9N2-HA1.

Variable Lymphocyte Receptors: A Current Overview.

Jawless vertebrates represented by lampreys and hagfish mount antigen-specific immune responses using variable lymphocyte receptors. These receptors generate diversity comparable to that of T-cell and B-cell receptors by assembling multiple leucine-rich repeat modules with highly variable sequences. Although it is true that jawed and jawless vertebrates have structurally unrelated antigen receptors, their adaptive immune systems have much in common. Most notable is the conservation of lymphocyte lineages. It appears that specialized lymphocyte lineages emerged in a common vertebrate ancestor and that jawed and jawless vertebrates co-opted different antigen receptors within the context of such lymphocyte lineages.

Evolution of two prototypic T cell lineages.

Jawless vertebrates, which occupy a unique position in chordate phylogeny, employ leucine-rich repeat (LRR)-based variable lymphocyte receptors (VLR) for antigen recognition. During the assembly of the VLR genes (VLRA, VLRB and VLRC), donor LRR-encoding sequences are copied in a step-wise manner into the incomplete germ-line genes. The assembled VLR genes are differentially expressed by discrete lymphocyte lineages: VLRA- and VLRC-producing cells are T-cell like, whereas VLRB-producing cells are B-cell like. VLRA(+) and VLRC(+) lymphocytes resemble the two principal T-cell lineages of jawed vertebrates that express the αß or γδ T-cell receptors (TCR). Reminiscent of the interspersed nature of the TCRα/TCRδ locus in jawed vertebrates, the close proximity of the VLRA and VLRC loci facilitates sharing of donor LRR sequences during VLRA and VLRC assembly. Here we discuss the insight these findings provide into vertebrate T- and B-cell evolution, and the alternative types of anticipatory receptors they use for adaptive immunity.

Genomic donor cassette sharing during VLRA and VLRC assembly in jawless vertebrates.

Lampreys possess two T-like lymphocyte lineages that express either variable lymphocyte receptor (VLR) A or VLRC antigen receptors. VLRA(+) and VLRC(+) lymphocytes share many similarities with the two principal T-cell lineages of jawed vertebrates expressing the αß and γδ T-cell receptors (TCRs). During the assembly of VLR genes, several types of genomic cassettes are inserted, in step-wise fashion, into incomplete germ-line genes to generate the mature forms of antigen receptor genes. Unexpectedly, the structurally variable components of VLRA and VLRC receptors often possess partially identical sequences; this phenomenon of module sharing between these two VLR isotypes occurs in both lampreys and hagfishes. By contrast, VLRA and VLRC molecules typically do not share their building blocks with the structurally analogous VLRB receptors that are expressed by B-like lymphocytes. Our studies reveal that VLRA and VLRC germ-line genes are situated in close proximity to each other in the lamprey genome and indicate the interspersed arrangement of isotype-specific and shared genomic donor cassettes; these features may facilitate the shared cassette use. The genomic structure of the VLRA/VLRC locus in lampreys is reminiscent of the interspersed nature of the TCRA/TCRD locus in jawed vertebrates that also allows the sharing of some variable gene segments during the recombinatorial assembly of TCR genes.

Copy number and sequence variation of leucine-rich repeat modules suggests distinct functional constraints operating on variable lymphocyte receptors expressed by agnathan T cell-like and B cell-like lymphocytes.

Unlike jawed vertebrates that use T cell and B cell receptors for antigen recognition, jawless vertebrates represented by lampreys and hagfish use variable lymphocyte receptors (VLR) as antigen receptors. VLRs generate high levels of diversity by assembling variable leucine-rich repeat (LRR) modules. Of the three VLRs thus far identified, VLRB is expressed on B cell-like lymphocytes and functions as antibodies, whereas VLRA and VLRC are expressed on T cell-like lymphocytes and function as membrane-bound receptors. In the present study, we show that the copy number of LRRV modules in lamprey and hagfish VLRB transcripts follows a binominal distribution with the success rates of 15.5 and 22.4 %, respectively. By contrast, the copy number distribution of LRRV modules in VLRA and VLRC transcripts deviates from the binominal distribution mainly because transcripts with two or less LRRV modules occur infrequently. Notably, the second LRRV module shows distinctive sequence signatures in VLRA and VLRC, but not in VLRB transcripts. These observations suggest that distinct functional constraints operate on VLRs expressed by agnathan T cell-like and B cell-like lymphocytes.

Two forms of adaptive immunity in vertebrates: similarities and differences.

Unlike jawed vertebrates that use T-cell and B-cell receptors for antigen recognition, jawless vertebrates represented by lampreys and hagfish use variable lymphocyte receptors (VLRs) as antigen receptors. VLRs generate diversity comparable to that of gnathostome antigen receptors by assembling variable leucine-rich repeat modules. The discovery of VLR has revolutionized our understanding of how adaptive immunity emerged and highlighted the differences between the adaptive immune systems (AISs) of jawed and jawless vertebrates. However, emerging evidence also indicates that their AISs have much in common. Particularly striking is the conservation of lymphocyte lineages. The basic architecture of the AIS including the dichotomy of lymphocytes appears to have been established in a common ancestor of jawed and jawless vertebrates. We review here the current knowledge on the AIS of jawless vertebrates, emphasizing both the similarities to and differences from the AIS of jawed vertebrates.

Hagfish C1q: its unique binding property.

Hagfish C1q (HaC1q) was identified and characterized as a pattern-recognition molecule (PRM) in the hagfish complement system. The serum from hagfish, Eptatretus burgeri, was applied to a GlcNAc-agarose column and eluted sequentially with GlcNAc and EDTA. Four (31, 27, 26, and 19 kDa) and one (26 kDa) proteins were detected as bound molecules in the GlcNAc- and the EDTA-eluates, respectively. Among these, the 26 kDa protein from the EDTA eluate was found to be a homologue of mammalian C1q through cDNA analysis. HaC1q had an ability to bind to various microbes in a Ca(2+)-dependent manner and its target ligands on the microbes were lipopolysaccharide, lipoteichoic acid, and peptidoglycan. The binding of HaC1q to GlcNAc-agarose was not inhibited by an excess amount of monosaccharide such as GlcNAc. While HaC1q bound to Sepharose 6B with a matrix of GlcNAc-agarose (polymer of agarobiose), it did not bind to Sepharose 4B that contained lower concentration of agarobiose than Sepharose 6B. Therefore, the target of HaC1q on GlcNAc-agarose was concluded to be agarobiose and high density of the target moiety seemed to be required for the stable binding. This finding was in accordance with the known behavior of other lectins involved in the complement system. We have concluded that HaC1q recognizes agarobiose-like structures present on the surface of microbes and acts as a pattern-recognition molecule in the process for elimination of invading microbes.

Definition of a third VLR gene in hagfish.

Jawless vertebrates (cyclostomes) have an alternative adaptive immune system in which lymphocytes somatically diversify their variable lymphocyte receptors (VLR) through recombinatorial use of leucine-rich repeat cassettes during VLR gene assembly. Three types of these anticipatory receptors in lampreys (VLRA, VLRB, and VLRC) are expressed by separate lymphocyte lineages. However, only two VLR genes (VLRA and VLRB) have been found in hagfish. Here we have identified a third hagfish VLR, which undergoes somatic assembly to generate sufficient diversity to encode a large repertoire of anticipatory receptors. Sequence analysis, structural comparison, and phylogenetic analysis indicate that the unique hagfish VLR is the counterpart of lamprey VLRA and the previously identified hagfish "VLRA" is the lamprey VLRC counterpart. The demonstration of three orthologous VLR genes in both lampreys and hagfish suggests that this anticipatory receptor system evolved in a common ancestor of the two cyclostome lineages around 480 Mya.

A major allogenic leukocyte antigen in the agnathan hagfish.

All vertebrates, from jawless fish to mammals, possess adaptive immune systems that can detect and inactivate non-self-antigens through a vast repertoire of antigen receptors. Unlike jawed vertebrates, the hagfish utilizes variable lymphocyte receptors (VLRs) that are unrelated to immunoglobulin molecules but are diversified by copy-choice gene conversion mechanism. Here, we report that hagfish VLRs react with allogenic leukocyte antigens but not with self-antigens. We found that a highly polymorphic membrane protein, NICIR3, is recognized by VLRs as an allogenic leukocyte antigen (ALA). In a serological cross-reactivity test, a close correlation was observed between the amino acid differences in the protein sequences and the VLR cross-reactivities. This leukocyte antigen was predominantly expressed in phagocytic leukocytes, where it was associated with phagocytosed protein antigens. These findings suggest that a polymorphic leukocyte antigen, NICIR3/ALA, plays a pivotal role in jawless vertebrate adaptive immunity.

Design principles of adaptive immune systems.

Both jawless vertebrates, such as lampreys and hagfish, and jawed vertebrates (encompassing species as diverse as sharks and humans) have an adaptive immune system that is based on somatically diversified and clonally expressed antigen receptors. Although the molecular nature of the antigen receptors and the mechanisms of their assembly are different, recent findings suggest that the general design principles underlying the two adaptive immune systems are surprisingly similar. The identification of such commonalities promises to further our understanding of the mammalian immune system and to inspire the development of new strategies for medical interventions targeting the consequences of faulty immune functions.

Alternative adaptive immunity in jawless vertebrates.

Jawless vertebrates use variable lymphocyte receptors (VLRs) that are generated by RAG-independent combinatorial assembly of leucine-rich repeat cassettes for Ag recognition, instead of the Ig-based Ag receptors used by jawed vertebrates. The VLR genes encode for crescent-shaped proteins that use variable beta-strands and a C-terminal loop to bind to Ags rather than the six CDR loops used by BCRs and TCRs. VLR mAbs have been isolated recently, which enabled the structure of VLR-Ag complexes to be defined. The jawless vertebrate adaptive immune system has many similarities to the Ig-based system of jawed vertebrates, including the compartmentalized development of B-like and T-like lymphocyte lineages that proliferate and differentiate into VLR-secreting plasmacytes and proinflammatory cytokine-producing cells in response to Ags. The definition of common features of the VLR-based and Ig-based systems offers fresh insight into the evolution of adaptive immunity.

Regulation of antigen-receptor gene assembly in hagfish.

Variable lymphocyte receptors (VLRs) are antigen receptors in the jawless vertebrates lamprey and hagfish. VLR genes are classified into VLRA and VLRB, and lymphocytes expressing VLRA are T-cell-like, whereas those expressing VLRB are B-cell-like in the sea lamprey. Diverse VLR genes are assembled somatically in lymphocytes; however, how the assembly is regulated is still largely unknown. Here, we analyse VLR gene assembly at the single-cell level in the inshore hagfish (Eptatretus burgeri). Each lymphocyte assembles and transcribes only one type of VLR gene, either VLRA or VLRB. In general, monoallelic assembly of VLR was observed, but diallelic assembly was found in some cases--in many of which, one allele was functional and the other was defective. In fact, all VLR-assembled lymphocytes contained at least one functional VLR gene. Together, these results indicate a feedback inhibition of VLR assembly and selection of VLR-positive lymphocytes.

Myxinidin, a novel antimicrobial peptide from the epidermal mucus of hagfish, Myxine glutinosa L.

Fish epidermal mucus contains innate immune components that provide a first line of defense against various infectious pathogens. This study reports the bioassay-guided fractionation and characterization of a novel antimicrobial peptide, myxinidin, from the acidic epidermal mucus extract of hagfish (Myxine glutinosa L.). Edman sequencing and mass spectrometry revealed that myxinidin consists of 12 amino acids and has a molecular mass of 1,327.68 Da. Myxinidin showed activity against a broad range of bacteria and yeast pathogens at minimum bactericidal concentration (MBC) ranging from 1.0 to 10.0 microg/mL. Screened pathogens, Salmonella enterica serovar Typhimurium C610, Escherichia coli D31, Aeromonas salmonicida A449, Yersinia ruckeri 96-4, and Listonella anguillarum 02-11 were found to be highly sensitive to myxinidin at the MBC of 1.0-2.5 microg/mL; Staphylococcus epidermis C621 and yeast (Candida albicans C627) had an MBC of 10.0 microg/mL. The antimicrobial activity of myxinidin was found to be two to 16 times more active than a potent fish-derived antimicrobial peptide, pleurocidin (NRC-17), against most of the screened pathogens. The microbicidal activity of myxinidin was retained in the presence of sodium chloride (NaCl) at concentrations up to 0.3 M and had no hemolytic activity against mammalian red blood cells. These results suggest that myxinidin may have potential applications in fish and human therapeutics.

Comparison of the biochemical composition of normal epidermal mucus and extruded slime of hagfish (Myxine glutinosa L.).

Hagfish (Myxine glutinosa) secrete normal epidermal mucus and extruded slime. The epidermal mucus is produced continuously to prevent pathogen adherence while the extruded slime is observed predominantly during feeding, provocation or stress. To date little is known about the involvement of extruded slime in the physiological functions of hagfish. In this preliminary study, innate immune enzymes and the protein composition of hagfish normal epidermal mucus and extruded slime were analysed and compared. The lysozyme specific activity of hagfish was observed approximately two-fold higher in extruded slime than that of epidermal mucus. The extruded slime had approximately 3.5-5.0 fold increased levels of alkaline phosphatase, cathepsin B and proteases in comparison to epidermal mucus. Protease characterization using specific inhibitors showed that the extruded slime had higher levels of serine trypsin-like proteases compared to metalloproteases whereas epidermal mucus showed equal proportion of both serine and metalloproteases. SDS-PAGE analysis showed high levels of three proteins with molecular masses in the range of 13-16kDa in the extruded slime. The LC/MS/MS analysis of protein bands 1, 2 and 3 showed closest matches to hemoglobulin-3, histone H3 and H2B proteins, respectively. The observation of elevated levels of innate immune parameters in the extruded slime suggested that the extruded slime has a significant role in innate immunity of hagfish against infectious pathogens.

Two types of antigen receptor systems in vertebrates.

Extant jawless vertebrates, represented by lampreys and hagfishes, have innate immune receptors with variable domains structurally resembling those of T/B-cell receptors. However, they appear to lack cardinal elements of adaptive immunity shared by all jawed vertebrates: major histocompatibility complex molecules and T/B-cell receptors. Thus, it was widely believed that adaptive immunity is unique to jawed vertebrates. Recently, this belief was overturned by the discovery of agnathan antigen receptors named variable lymphocyte receptors. These receptors generate diversity in their antigen-binding sites through assembling highly diverse leucine-rich repeat modules. The crystal structures of hagfish variable lymphocyte receptor monomers indicate that they adopt a horseshoe-shaped structure and likely bind antigens through the hypervariable concave surface. Secreted variable lymphocyte receptors form pentamers or tetramers of dimers and bind antigens with high specificity and avidity. The fact that variable lymphocyte receptors are structurally unrelated to T/B-cell receptors indicates that jawed and jawless vertebrates have developed antigen receptors independently.

Two variable lymphocyte receptor genes of the inshore hagfish are located far apart on the same chromosome.

Variable lymphocyte receptors (VLR) generate enormous diversity through assembling highly diverse leucine-rich repeat (LRR) modules and presumably function as antigen receptors in jawless vertebrates. The hagfish, which constitute major extant members of jawless vertebrates along with lampreys, have two VLR genes designated VLRA and VLRB, whereas only a single VLR gene has been identified in the lamprey. In the present study, we show by fluorescence in situ hybridization (FISH) that hagfish VLRA and VLRB are located on the same chromosome, but are far apart from each other. Analysis of available inshore hagfish complementary DNA sequences indicates that VLRA and VLRB do not share a LRR module with an identical nucleotide sequence. Physical separation of VLRA and VLRB is consistent with this observation and indicates that the two VLR genes function as separate units. The FISH protocol developed in this study should be useful for the analysis of the agnathan genome.