Reshaping antibody diversity.

Some species mount a robust antibody response despite having limited genome-encoded combinatorial diversity potential. Cows are unusual in having exceptionally long CDR H3 loops and few V regions, but the mechanism for creating diversity is not understood. Deep sequencing reveals that ultralong CDR H3s contain a remarkable complexity of cysteines, suggesting that disulfide-bonded minidomains may arise during repertoire development. Indeed, crystal structures of two cow antibodies reveal that these CDR H3s form a very unusual architecture composed of a ß strand "stalk" that supports a structurally diverse, disulfide-bonded "knob" domain. Diversity arises from somatic hypermutation of an ultralong DH with a severe codon bias toward mutation to cysteine. These unusual antibodies can be elicited to recognize defined antigens through the knob domain. Thus, the bovine immune system produces an antibody repertoire composed of ultralong CDR H3s that fold into a diversity of minidomains generated through combinations of somatically generated disulfides.

Evolution of immunogenetic components encoding ultralong CDR H3.

The genomes of most vertebrates contain many V, D, and J gene segments within their Ig loci to construct highly variable CDR3 sequences through combinatorial diversity. This nucleotide variability translates into an antibody population containing extensive paratope diversity. Cattle have relatively few functional VDJ gene segments, requiring innovative approaches for generating diversity like the use of ultralong-encoding IGHV and IGHD gene segments that yield dramatically elongated CDR H3. Unique knob and stalk microdomains create protracted paratopes, where the antigen-binding knob sits atop a long stalk, allowing the antibody to bind both surface and recessed antigen epitopes. We examined genomes of twelve species of Bovidae to determine when ultralong-encoding IGHV and IGHD gene segments evolved. We located the 8-bp duplication encoding the unique TTVHQ motif in ultralong IGHV segments in six Bovid species (cattle, zebu, wild yak, domestic yak, American bison, and domestic gayal), but we did not find evidence of the duplication in species beyond the Bos and Bison genera. Additionally, we analyzed mRNA from bison spleen and identified a rich repertoire of expressed ultralong CDR H3 antibody mRNA, suggesting that bison use ultralong IGHV transcripts in their host defense. We found ultralong-encoding IGHD gene segments in all the same species except domestic yak, but again not beyond the Bos and Bison clade. Thus, the duplication event leading to this ultralong-encoding IGHV gene segment and the emergence of the ultralong-encoding IGHD gene segment appears to have evolved in a common ancestor of the Bos and Bison genera 5-10 million years ago.

Rapid elicitation of broadly neutralizing antibodies to HIV by immunization in cows.

No immunogen to date has reliably elicited broadly neutralizing antibodies to HIV in humans or animal models. Advances in the design of immunogens that antigenically mimic the HIV envelope glycoprotein (Env), such as the soluble cleaved trimer BG505 SOSIP, have improved the elicitation of potent isolate-specific antibody responses in rabbits and macaques, but so far failed to induce broadly neutralizing antibodies. One possible reason for this failure is that the relevant antibody repertoires are poorly suited to target the conserved epitope regions on Env, which are somewhat occluded relative to the exposed variable epitopes. Here, to test this hypothesis, we immunized four cows with BG505 SOSIP. The antibody repertoire of cows contains long third heavy chain complementary determining regions (HCDR3) with an ultralong subset that can reach more than 70 amino acids in length. Remarkably, BG505 SOSIP immunization resulted in rapid elicitation of broad and potent serum antibody responses in all four cows. Longitudinal serum analysis for one cow showed the development of neutralization breadth (20%, n = 117 cross-clade isolates) in 42 days and 96% breadth (n = 117) at 381 days. A monoclonal antibody isolated from this cow harboured an ultralong HCDR3 of 60 amino acids and neutralized 72% of cross-clade isolates (n = 117) with a potent median IC50 of 0.028 µg ml-1. Breadth was elicited with a single trimer immunogen and did not require additional envelope diversity. Immunization of cows may provide an avenue to rapidly generate antibody prophylactics and therapeutics to address disease agents that have evolved to avoid human antibody responses.

Nurse shark T-cell receptors employ somatic hypermutation preferentially to alter alpha/delta variable segments associated with alpha constant region.

In addition to canonical TCR and BCR, cartilaginous fish assemble noncanonical TCR that employ various B-cell components. For example, shark T cells associate alpha (TCR-α) or delta (TCR-δ) constant (C) regions with Ig heavy chain (H) variable (V) segments or TCR-associated Ig-like V (TAILV) segments to form chimeric IgV-TCR, and combine TCRδC with both Ig-like and TCR-like V segments to form the doubly rearranging NAR-TCR. Activation-induced (cytidine) deaminase-catalyzed somatic hypermutation (SHM), typically used for B-cell affinity maturation, also is used by TCR-α during selection in the shark thymus presumably to salvage failing receptors. Here, we found that the use of SHM by nurse shark TCR varies depending on the particular V segment or C region used. First, SHM significantly alters alpha/delta V (TCRαδV) segments using TCR αC but not δC. Second, mutation to IgHV segments associated with TCR δC was reduced compared to mutation to TCR αδV associated with TCR αC. Mutation was present but limited in V segments of all other TCR chains including NAR-TCR. Unexpectedly, we found preferential rearrangement of the noncanonical IgHV-TCRδC over canonical TCR αδV-TCRδC receptors. The differential use of SHM may reveal how activation-induced (cytidine) deaminase targets V regions.

Lost structural and functional inter-relationships between Ig and TCR loci in mammals revealed in sharks.

Immunoglobulins and T cell receptors (TCR) have obvious structural similarities as well as similar immunogenetic diversification and selection mechanisms. Nevertheless, the two receptor systems and the loci that encode them are distinct in humans and classical murine models, and the gene segments comprising each repertoire are mutually exclusive. Additionally, while both B and T cells employ recombination-activating genes (RAG) for primary diversification, immunoglobulins are afforded a supplementary set of activation-induced cytidine deaminase (AID)-mediated diversification tools. As the oldest-emerging vertebrates sharing the same adaptive B and T cell receptor systems as humans, extant cartilaginous fishes allow a potential view of the ancestral immune system. In this review, we discuss breakthroughs we have made in studies of nurse shark (Ginglymostoma cirratum) T cell receptors demonstrating substantial integration of loci and diversification mechanisms in primordial B and T cell repertoires. We survey these findings in this shark model where they were first described, while noting corroborating examples in other vertebrate groups. We also consider other examples where the gnathostome common ancestry of the B and T cell receptor systems have allowed dovetailing of genomic elements and AID-based diversification approaches for the TCR. The cartilaginous fish seem to have retained this T/B cell plasticity to a greater extent than more derived vertebrate groups, but representatives in all vertebrate taxa except bony fish and placental mammals show such plasticity.

Analysis of shark NCR3 family genes reveals primordial features of vertebrate NKp30.

Natural killer (NK) cells play major roles in innate immunity against viruses and cancer. Natural killer receptors (NKR) expressed by NK cells recognize foreign- or self-ligands on infected and transformed cells as well as healthy cells. NKR genes are the most rapidly evolving loci in vertebrates, and it is generally difficult to detect orthologues in different taxa. The unique exception is NKp30, an activating NKR in mammals that binds to the self-ligand B7H6. The NKp30-encoding gene, NCR3, has been found in most vertebrates including sharks, the oldest vertebrates with human-type adaptive immunity. NCR3 has a special, non-rearranging VJ-type immunoglobulin superfamily (IgSF) domain that predates the emergence of the rearranging antigen receptors. Herein we show that NCR3 loci are linked to the shark major histocompatibility complex (MHC), proving NCR3's primordial association with the MHC. We identified eight subtypes of differentially expressed highly divergent shark NCR3 family genes. Using in situ hybridization, we detected one subtype, NS344823, to be expressed by predominantly single cells outside of splenic B cell zones. The expression by non-B cells was also confirmed by PCR in peripheral blood lymphocytes. Surprisingly, high expression of NS344823 was detected in the thymic cortex, demonstrating NS344823 expression in developing T cells. Finally, we show for the first time that shark T cells are found as single cells or in small clusters in the splenic red pulp, also unassociated with the large B cell follicles we previously identified.

Ancient Use of Ig Variable Domains Contributes Significantly to the TCRδ Repertoire.

The loci encoding B and T cell Ag receptors are generally distinct in commonly studied mammals, with each receptor's gene segments limited to intralocus, cis chromosomal rearrangements. The nurse shark (Ginglymostoma cirratum) represents the oldest vertebrate class, the cartilaginous fish, with adaptive immunity provided via Ig and TCR lineages, and is one species among a growing number of taxa employing Ig-TCRδ rearrangements that blend these distinct lineages. Analysis of the nurse shark Ig-TCRδ repertoire found that these rearrangements possess CDR3 characteristics highly similar to canonical TCRδ rearrangements. Furthermore, the Ig-TCRδ rearrangements are expressed with TCRγ, canonically found in the TCRδ heterodimer. We also quantified BCR and TCR transcripts in the thymus for BCR (IgHV-IgHC), chimeric (IgHV-TCRδC), and canonical (TCRδV-TCRδC) transcripts, finding equivalent expression levels in both thymus and spleen. We also characterized the nurse shark TCRαδ locus with a targeted bacterial artifical chromosome sequencing approach and found that the TCRδ locus houses a complex of V segments from multiple lineages. An IgH-like V segment, nestled within the nurse shark TCRδ translocus, grouped with IgHV-like rearrangements we found expressed with TCRδ (but not IgH) rearrangements in our phylogenetic analysis. This distinct lineage of TCRδ-associated IgH-like V segments was termed "TAILVs." Our data illustrate a dynamic TCRδ repertoire employing TCRδVs, NARTCRVs, bona fide trans-rearrangements from shark IgH clusters, and a novel lineage in the TCRδ-associated Ig-like V segments.

TLR4 and TLR8 variability in Amazonian and West Indian manatee species from Brazil.

Amazonian (Trichechus inunguis) and West Indian (Trichechus manatus) manatees are aquatic mammals vulnerable to extinction found in the Amazon basin and the coastal western Atlantic. Toll-like receptors (TLR) play a key role in recognizing pathogen-associated molecular patterns using leucine-rich repeats (LRRs). We described the diversity of TLR4 and TLR8 genes in these two species of manatee. Amazonian manatee showed seven SNPs in TLR4 and the eight in TLR8, while West Indian manatee shared four and six of those SNPs, respectively. In our analysis, TLR4 showed one non-conservative amino acid replacement substitution in LRR7 and LRR8, on the other hand, TLR8 was less variable and showed only conserved amino acid substitutions. Selection analysis showed that only one TLR4 site was subjected to positive selection and none in TLR8. TLR4 in manatees did not show any evidence of convergent evolution compared to species of the cetacean lineage. Differences in TLR4 and TLR8 polymorphism may be related to distinct selection by pathogens, population reduction of West Indian manatees, or an expected consequence of population expansion in Amazonian manatees. Future studies combining pathogen association and TLR polymorphism may clarify possible roles of these genes and be used for conservation purposes of manatee species.

Comparative study of cartilaginous fish divulges insights into the early evolution of primary, secondary and mucosal lymphoid tissue architecture.

Cartilaginous fish are located at a pivotal point in phylogeny where the adaptive immune system begins to resemble that of other, more-derived jawed vertebrates, including mammals. For this reason, sharks and other cartilaginous fish are ideal models for studying the natural history of immunity. Insights from such studies may include distinguishing the (evolutionarily conserved) fundamental aspects of adaptive immunity from the (more recent) accessory. Some lymphoid tissues of sharks, including the thymus and spleen, resemble those of mammals in both appearance and function. The cartilaginous skeleton of sharks has no bone marrow, which is also absent in bony fish despite calcified bone, but cartilaginous fish have other Leydig's and epigonal organs that function to provide hematopoiesis analogous to mammalian bone marrow. Conserved across all vertebrate phylogeny in some form is gut-associated lymphoid tissues, or GALT, which is seen from agnathans to mammals. Though it takes many forms, from typhlosole in lamprey to Peyer's patches in mammals, the GALT serves as a site of antigen concentration and exposure to lymphocytes in the digestive tract. Though more complex lymphoid organs are not present in agnathans, they have several primitive tissues, such as the thymoid and supraneural body, that appear to serve their variable lymphocyte receptor-based adaptive immune system. There are several similarities between the adaptive immune structures in cartilaginous and bony fish, such as the thymus and spleen, but there are mechanisms employed in bony fish that in some instances bridge their adaptive immune systems to that of tetrapods. This review summarizes what we know of lymphoid tissues in cartilaginous fishes and uses these data to compare primary and secondary tissues in jawless, cartilaginous, and bony fishes to contextualize the early natural history of vertebrate mucosal immune tissues.

Haptoglobin Is a Divergent MASP Family Member That Neofunctionalized To Recycle Hemoglobin via CD163 in Mammals.

In mammals, haptoglobin (Hp) is an acute-phase plasma protein that binds with high affinity to hemoglobin (Hb) released by intravascular hemolysis. The resultant Hp-Hb complexes are bound and cleared by the scavenger receptor CD163, limiting Hb-induced oxidative damage. In this study, we show that Hp is a divergent member of the complement-initiating MASP family of proteins, which emerged in the ancestor of jawed vertebrates. We demonstrate that Hp has been independently lost from multiple vertebrate lineages, that characterized Hb-interacting residues of mammals are poorly conserved in nonmammalian species maintaining Hp, and that the extended loop 3 region of Hp, which mediates CD163 binding, is present only in mammals. We show that the Hb-binding ability of cartilaginous fish (nurse shark, Ginglymostoma cirratum; small-spotted catshark, Scyliorhinus canicula; and thornback ray, Raja clavata) and teleost fish (rainbow trout, Oncorhynchus mykiss) Hp is species specific, and where binding does occur it is likely mediated through a different structural mechanism to mammalian Hp. The continued, high-level expression of Hp in cartilaginous fishes in which Hb binding is not evident signals that Hp has (an)other, yet unstudied, role(s) in these species. Previous work indicates that mammalian Hp also has secondary, immunomodulatory functions that are independent of Hb binding; our work suggests these may be remnants of evolutionary more ancient functions, retained after Hb removal became the primary role of Hp in mammals.

Post-Translational Protein Deimination Signatures in Serum and Serum-Extracellular Vesicles of Bos taurus Reveal Immune, Anti-Pathogenic, Anti-Viral, Metabolic and Cancer-Related Pathways for Deimination.

The bovine immune system is known for its unusual traits relating to immunoglobulin and antiviral responses. Peptidylarginine deiminases (PADs) are phylogenetically conserved enzymes that cause post-translational deimination, contributing to protein moonlighting in health and disease. PADs also regulate extracellular vesicle (EV) release, forming a critical part of cellular communication. As PAD-mediated mechanisms in bovine immunology and physiology remain to be investigated, this study profiled deimination signatures in serum and serum-EVs in Bos taurus. Bos EVs were poly-dispersed in a 70-500 nm size range and showed differences in deiminated protein cargo, compared with whole sera. Key immune, metabolic and gene regulatory proteins were identified to be post-translationally deiminated with some overlapping hits in sera and EVs (e.g., immunoglobulins), while some were unique to either serum or serum-EVs (e.g., histones). Protein-protein interaction network analysis of deiminated proteins revealed KEGG pathways common for serum and serum-EVs, including complement and coagulation cascades, viral infection (enveloped viruses), viral myocarditis, bacterial and parasitic infections, autoimmune disease, immunodeficiency intestinal IgA production, B-cell receptor signalling, natural killer cell mediated cytotoxicity, platelet activation and hematopoiesis, alongside metabolic pathways including ferroptosis, vitamin digestion and absorption, cholesterol metabolism and mineral absorption. KEGG pathways specific to EVs related to HIF-1 signalling, oestrogen signalling and biosynthesis of amino acids. KEGG pathways specific for serum only, related to Epstein-Barr virus infection, transcription mis-regulation in cancer, bladder cancer, Rap1 signalling pathway, calcium signalling pathway and ECM-receptor interaction. This indicates differences in physiological and pathological pathways for deiminated proteins in serum-EVs, compared with serum. Our findings may shed light on pathways underlying a number of pathological and anti-pathogenic (viral, bacterial, parasitic) pathways, with putative translatable value to human pathologies, zoonotic diseases and development of therapies for infections, including anti-viral therapies.

"Double-duty" conventional dendritic cells in the amphibian Xenopus as the prototype for antigen presentation to B cells.

Two populations of dendritic cells (DCs) are found in mammals, one derived from hematopoietic precursors (conventional/cDC), and another derived from mesenchymal precursors, the follicular DC (FDC); the latter is specialized for antigen presentation to B cells, and has only been definitively demonstrated in mammals. Both cDC and FDC are necessary for induction of germinal centers (GC) and GC-dependent class switch recombination (CSR) and somatic hypermutation (SHM). We demonstrate that in Xenopus, an amphibian in which immunoglobulin CSR and SHM occur without GC formation, a single type of DC has properties of both cDC and FDC, including high expression of MHC class II for the former and display of native antigen at the cell surface for the latter. Our data confirm that the advent of FDC functionality preceded emergence of bona fide FDC, which was in turn crucial for the development of GC formation and efficient affinity maturation in mammals.

Deiminated proteins in extracellular vesicles and plasma of nurse shark (Ginglymostoma cirratum) - Novel insights into shark immunity.

Peptidylarginine deiminases (PADs) are phylogenetically conserved calcium-dependent enzymes which post-translationally convert arginine into citrulline in target proteins in an irreversible manner, causing functional and structural changes in target proteins. Protein deimination causes generation of neo-epitopes, affects gene regulation and also allows for protein moonlighting. Extracellular vesicles are found in most body fluids and participate in cellular communication via transfer of cargo proteins and genetic material. In this study, post-translationally deiminated proteins and extracellular vesicles (EVs) are described for the first time in shark plasma. We report a poly-dispersed population of shark plasma EVs, positive for phylogenetically conserved EV-specific markers and characterised by TEM. In plasma, 6 deiminated proteins, including complement and immunoglobulin, were identified, whereof 3 proteins were found to be exported in plasma-derived EVs. A PAD homologue was identified in shark plasma by Western blotting and detected an expected 70 kDa size. Deiminated histone H3, a marker of neutrophil extracellular trap formation, was also detected in nurse shark plasma. This is the first report of deiminated proteins in plasma and EVs, highlighting a hitherto unrecognized post-translational modification in key immune proteins of innate and adaptive immunity in shark.

Genomic organization of the zebrafish (Danio rerio) T cell receptor alpha/delta locus and analysis of expressed products.

In testing the hypothesis that all jawed vertebrate classes employ immunoglobulin heavy chain V (IgHV) gene segments in their T cell receptor (TCR)δ encoding loci, we found that some basic characterization was required of zebrafish TCRδ. We began by annotating and characterizing the TCRα/δ locus of Danio rerio based on the most recent genome assembly, GRCz10. We identified a total of 141 theoretically functional V segments which we grouped into 41 families based upon 70 % nucleotide identity. This number represents the second greatest count of apparently functional V genes thus far described in an antigen receptor locus with the exception of cattle TCRα/δ. Cloning, relative quantitative PCR, and deep sequencing results corroborate that zebrafish do express TCRδ, but these data suggest only at extremely low levels and in limited diversity in the spleens of the adult fish. While we found no evidence for IgH-TCRδ rearrangements in this fish, by determining the locus organization we were able to suggest how the evolution of the teleost α/δ locus could have lost IgHVs that exist in sharks and frogs. We also found evidence of surprisingly low TCRδ expression and repertoire diversity in this species.

Karyotypic stasis and swarming influenced the evolution of viral tolerance in a species-rich bat radiation.

The emergence of COVID-19 and severe acute respiratory syndrome (SARS) has prioritized understanding bats' viral tolerance. Myotis bats are exceptionally species rich and have evolved viral tolerance. They also exhibit swarming, a cryptic behavior where large, multi-species assemblages gather for mating, which has been hypothesized to promote interspecific hybridization. To resolve the coevolution of genome architecture and their unusual antiviral tolerance, we undertook a phylogenomic analysis of 60 Old World Myotis genomes. We demonstrate an extensive history of introgressive hybridization that has replaced the species phylogeny across 17%-93% of the genome except for pericentromeric regions of macrochromosomes. Introgression tracts were enriched on microchromosome regions containing key antiviral pathway genes overexpressed during viral challenge experiments. Together, these results suggest that the unusual Myotis karyotype may have evolved to selectively position immune-related genes in high recombining genomic regions prone to introgression of divergent alleles, including a diversity of interleukin loci responsible for the release of pro-inflammatory cytokines.

Impact of PEG sensitization on the efficacy of PEG hydrogel-mediated tissue engineering.

While poly(ethylene glycol) (PEG) hydrogels are generally regarded as biologically inert blank slates, concerns over PEG immunogenicity are growing, and the implications for tissue engineering are unknown. Here, we investigate these implications by immunizing mice against PEG to stimulate anti-PEG antibody production and evaluating bone defect regeneration after treatment with bone morphogenetic protein-2-loaded PEG hydrogels. Quantitative analysis reveals that PEG sensitization increases bone formation compared to naive controls, whereas histological analysis shows that PEG sensitization induces an abnormally porous bone morphology at the defect site, particularly in males. Furthermore, immune cell recruitment is higher in PEG-sensitized mice administered the PEG-based treatment than their naive counterparts. Interestingly, naive controls that were administered a PEG-based treatment also develop anti-PEG antibodies. Sex differences in bone formation and immune cell recruitment are also apparent. Overall, these findings indicate that anti-PEG immune responses can impact tissue engineering efficacy and highlight the need for further investigation.

Intramuscular but not nebulized administration of a mRNA vaccine against Rhodococcus equi stimulated humoral immune responses in neonatal foals.

OBJECTIVE: Design and evaluate immune responses of neonatal foals to a mRNA vaccine expressing the virulence-associated protein A (VapA) of Rhodococcus equi. ANIMALS: Cultured primary equine respiratory tract cells; Serum, bronchoalveolar lavage fluid (BALF), and peripheral blood mononuclear cells (PBMCs) from 30 healthy Quarter Horse foals. METHODS: VapA expression was evaluated by western immunoblot in cultured equine bronchial cells transfected with 4 mRNA constructs encoding VapA. The mRNA construct with greatest expression was used to immunize foals at ages 2 and 21 days in 5 groups: (1) 300 µg nebulized mRNA (n = 6); (2) 600 µg nebulized mRNA (n = 4); (3) 300 µg mRNA administered intramuscularly (IM) (n = 5); (4) 300 µg VapA IM (positive controls; n = 6); or (5) nebulized water (negative controls; n = 6). Serum, BALF, and PBMCs were collected at ages 3, 22, and 35 days and tested for relative anti-VapA IgG1, IgG4/7, and IgA activities using ELISA and cell-mediated immunity by ELISpot. RESULTS: As formulated, nebulized mRNA was not immunogenic. However, a significant increase in anti-VapA IgG4/7 activity (P < .05) was noted exclusively in foals immunized IM with VapA mRNA by age 35 days. The proportion of foals with anti-VapA IgG1 activity > 30% of positive control differed significantly (P = .0441) between negative controls (50%; 3/6), IM mRNA foals (100%; 5/5), and IM VapA (100%; 6/6) groups. Natural exposure to virulent R equi was immunogenic in some negative control foals. CLINICAL RELEVANCE: Further evaluation of the immunogenicity and efficacy of IM mRNA encoding VapA in foals is warranted.

Crystal structure of shrimp arginine kinase in binary complex with arginine-a molecular view of the phosphagen precursor binding to the enzyme.

Arginine kinase (AK) is a key enzyme for energetic balance in invertebrates. Although AK is a well-studied system that provides fast energy to invertebrates using the phosphagen phospho-arginine, the structural details on the AK-arginine binary complex interaction remain unclear. Herein, we determined two crystal structures of the Pacific whiteleg shrimp (Litopenaeus vannamei) arginine kinase, one in binary complex with arginine (LvAK-Arg) and a ternary transition state analog complex (TSAC). We found that the arginine guanidinium group makes ionic contacts with Glu225, Cys271 and a network of ordered water molecules. On the zwitterionic side of the amino acid, the backbone amide nitrogens of Gly64 and Val65 coordinate the arginine carboxylate. Glu314, one of proposed acid-base catalytic residues, did not interact with arginine in the binary complex. This residue is located in the flexible loop 310-320 that covers the active site and only stabilizes in the LvAK-TSAC. This is the first binary complex crystal structure of a guanidine kinase in complex with the guanidine substrate and could give insights into the nature of the early steps of phosphagen biosynthesis.

Immunonutrition: facilitating mucosal immune response in teleost intestine with amino acids through oxidant-antioxidant balance.

Comparative animal models generate fundamental scientific knowledge of immune responses. However, these studies typically are conducted in mammals because of their biochemical and physiological similarity to humans. Presently, there has been an interest in using teleost fish models to study intestinal immunology, particularly intestinal mucosa immune response. Instead of targeting the pathogen itself, a preferred approach for managing fish health is through nutrient supplementation, as it is noninvasive and less labor intensive than vaccine administrations while still modulating immune properties. Amino acids (AAs) regulate metabolic processes, oxidant-antioxidant balance, and physiological requirements to improve immune response. Thus, nutritionists can develop sustainable aquafeeds through AA supplementation to promote specific immune responses, including the intestinal mucosa immune system. We propose the use of dietary supplementation with functional AAs to improve immune response by discussing teleost fish immunology within the intestine and explore how oxidative burst is used as an immune defense mechanism. We evaluate immune components and immune responses in the intestine that use oxidant-antioxidant balance through potential selection of AAs and their metabolites to improve mucosal immune capacity and gut integrity. AAs are effective modulators of teleost gut immunity through oxidant-antioxidant balance. To incorporate nutrition as an immunoregulatory means in teleost, we must obtain more tools including genomic, proteomic, nutrition, immunology, and macrobiotic and metabonomic analyses, so that future studies can provide a more holistic understanding of the mucosal immune system in fish.