A Heterologous Challenge Rescues the Attenuated Immunogenicity of SARS-CoV-2 Omicron BA.1 Variant in Syrian Hamster Model.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant is becoming a dominant circulator and has several mutations in the spike glycoprotein, which may cause shifts of immunogenicity, so as to result in immune escape and breakthrough infection among the already infected or vaccinated populations. It is unclear whether infection with Omicron could generate adequate cross-variant protection. To investigate this possibility, we used Syrian hamsters as an animal model for infection of SARS-CoV-2. The serum from Omicron BA.1 variant-infected hamsters showed a significantly lower neutralization effect against infection of the same or different SARS-CoV-2 variants than the serum from Beta variant-infected hamsters. Furthermore, the serum from Omicron BA.1 variant-infected hamsters were insufficient to protect against rechallenge of SARS-CoV-2 Prototype, Beta and Delta variants and itself. Importantly, we found that rechallenge with different SARS-CoV-2 lineages elevated cross-variant serum neutralization titers. Overall, our findings indicate a weakened immunogenicity feature of Omicron BA.1 variant that can be overcome by rechallenge of a different SARS-CoV-2 lineages. Our results may lead to a new guideline in generation and use of the vaccinations to combat the pandemic of SARS-CoV-2 Omicron variant and possible new variants. IMPORTANCE The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant causes breakthrough infections among convalescent patients and vaccinated populations. However, Omicron does not generate robust cross-protective responses. Here, we investigate whether heterologous SARS-CoV-2 challenge is able to enhance antibody response in a sensitive animal model, namely, Syrian hamster. Of note, a heterologous challenge of Beta and Omicron BA.1 variant significantly broadens the breadth of SARS-CoV-2 neutralizing responses against the prototype, Beta, Delta, and Omicron BA.1 variants. Our findings confirm that vaccination strategy with heterologous antigens might be a good option to protect against the evolving SARS-CoV-2.

Genetic engineering of porcine endothelial cell lines for evaluation of human-to-pig xenoreactive immune responses.

Xenotransplantation (cross-species transplantation) using genetically-engineered pig organs offers a potential solution to address persistent organ shortage. Current evaluation of porcine genetic modifications is to monitor the nonhuman primate immune response and survival after pig organ xenotransplantation. This measure is an essential step before clinical xenotransplantation trials, but it is time-consuming, costly, and inefficient with many variables. We developed an efficient approach to quickly examine human-to-pig xeno-immune responses in vitro. A porcine endothelial cell was characterized and immortalized for genetic modification. Five genes including GGTA1, CMAH, ß4galNT2, SLA-I α chain, and ß2-microglobulin that are responsible for the production of major xenoantigens (αGal, Neu5Gc, Sda, and SLA-I) were sequentially disrupted in immortalized porcine endothelial cells using CRISPR/Cas9 technology. The elimination of αGal, Neu5Gc, Sda, and SLA-I dramatically reduced the antigenicity of the porcine cells, though the cells still retained their ability to provoke human natural killer cell activation. In summary, evaluation of human immune responses to genetically modified porcine cells in vitro provides an efficient method to identify ideal combinations of genetic modifications for improving pig-to-human compatibility, which should accelerate the application of xenotransplantation to humans.

Recombinant Lactobacillus plantarum NC8 strain expressing porcine rotavirus VP7 induces specific antibodies in BALB/c mice.

The major etiologic agent that causes acute gastroenteritis worldwide in young animals and children is Group A rotavirus. Currently, commercially available vaccines do not often prevent porcine rotavirus (PRV) infection. In this study, we evaluated the efficacy of oral recombinant Lactobacillus vaccine against PRV in a mouse model. Lactobacillus plantarum NC8 was used as the host strain, and bacterial vectors were constructed, because the NC8 isolated has shown the capability to survive gastric transit and to colonize the intestinal tract of humans and other mammals. To explore the immunological mechanisms, lactic acid bacterial vectors were used to express VP7 antigen from PRV. We constructed an L. plantarum strain with surface-displayed VP7, named NC8-pSIP409-pgsA-VP7-DCpep. The expressed recombinant protein had a molecular weight of ∼37 kDa. The strain was used to immunize BALB/c mice to evaluate their immunomodulatory characteristics. Mice were orally immunized with recombinant L. plantarum NC8-pSIP409-pgsA-VP7-DCpep at a dose of 2 × 109 colony forming units/200 µl. The results showed that NC8-pSIP409-pgsA-VP7-DCpep significantly stimulated the differentiation of dendritic cells (DCs) in Peyer's patches (PPs) and increased the serum levels of IL-4 and IFN-γ, as measured by enzyme-linked immunosorbent assay in mice treated with NC8-pSIP409-pgsA-VP7-DCpep. Compared to the empty vector group, NC8-pSIP409-pgsA-VP7-DCpep significantly increased the production of B220+ B cells in mesenteric lymph nodes (MLNs) and PPs and also increased the titer levels of the VP7-specific antibodies, including IgG and sIgA. The administration of NC8-pSIP409-pgsA-VP7-DCpep mediated relatively broad cellular responses. This study reveals that clear alternatives exist for PRV control strategies and provides information on PRV infection.

Aspects of histocompatibility testing in xenotransplantation.

Xenotransplantation, using genetically-modified pigs for clinical organ transplantation, is a solution to the organ shortage. The biggest barrier to clinical implementation is the antigenicity of pig cells. Humans possess preformed antibody to pig cells that initiate antibody-mediated rejection of pig organs in primates. Advances in genetic engineering have led to the development of a pig lacking the three known glycan xenoantigens (triple-knockout [TKO] pigs). A significant number of human sera demonstrate no antibody binding to TKO pig cells. As a result of the TKO pig's low antigen expression, survival of life-supporting pig organs in immunosuppressed nonhuman primates has significantly increased, and hope has been renewed for clinical trials of xenotransplantation. It is important to understand the context in which xenotransplantation's predecessor, allotransplantation, has been successful, and the steps needed for the success of xenotransplantation. Successful allotransplantation has been based on two main immunological approaches - (i) adequate immunosuppressive therapy, and (ii) careful histocompatibility matching. In vivo studies suggest that the available immunosuppressive regimens are adequate to suppress the human anti-pig cellular response. Methods to evaluate and screen patients for the first clinical xenotransplantation trial are the next challenge. The goal of this review is to summarize the history of histocompatibility testing, and the available tools that can be utilized to determine xenograft histocompatibility.

The Role of SLAs in Xenotransplantation.

Advances in genetic engineering, particularly CRISPR/Cas9, have resulted in the development of a triple glycan-knockout (TKO) pig. There is minimal human antipig antibody binding to TKO pig cells. The TKO background has decreased antibody binding to a sufficiently low level that any additional xenoantigens expressed on the cells can now be more easily detected. One of these xenoantigens is the swine major histocompatibility complex, termed swine leukocyte antigens (SLA). SLA are the homolog to HLAs, a protein complex expressed on human tissue capable of stimulating the development of new antibodies in allotransplantation. These antibodies can result in graft failure through hyperacute, acute, or chronic rejection. Our knowledge of SLA, particularly in the last 5 years, has grown considerably. The presence, cause, and methods to detect anti-SLA antibodies will need to be carefully considered for the first clinical trial of xenotransplantation. The focus of this review is to summarize the role of SLA in xenotransplantation and consider whether it will prove to be a major barrier. Techniques are now available to mutate target SLA amino acids to ensure that cross-reactive anti-HLA antibodies no longer bind to SLA on the cells of the organ-source pigs. While deletion of SLA expression is possible, it would render the pig at risk for infectious complications. The ideal organ-source pig for HLA highly sensitized recipients may therefore be 1 with site-specific mutations to eliminate cross-reactive binding.

Mycobacterium tuberculosis infection is associated with increased B cell responses to unrelated pathogens.

Antigens from Mycobacterium tuberculosis (M.tb), have been shown to stimulate human B cell responses to unrelated recall antigens in vitro. However, it is not known whether natural M.tb infection or whether vaccination with, Mycobacterium bovis BCG, has a similar effect. This study investigated the effects of M.tb infection and BCG vaccination on B cell responses to heterologous pathogen recall antigens. Antibodies against several bacterial and viral pathogens were quantified by ELISA in 68 uninfected controls, 62 individuals with latent TB infection (LTBI) and 107 active pulmonary TB (APTB) cases, and 24 recently BCG-vaccinated adolescents and naive controls. Antibody avidity was investigated using surface plasmon resonance and B cell ELISPOTs were used to measure plasmablast and memory B cell responses (MBC) in APTB cases and healthy donor controls. APTB was associated with higher levels of antibodies to respiratory syncytial virus and measles virus, compared to uninfected controls. BCG vaccination did not alter levels of antibodies against heterologous pathogens. Tetanus toxoid (TT)-specific antibody avidity was increased in APTB cases in comparison to uninfected individuals and the ratio of TT-specific plasmablasts to MBCs in the APTB cases was 7:1. M.tb infection is associated with increased antibody responses to heterologous pathogens in human subjects.

Human anti-α-fucose antibodies are xenoreactive toward GGTA1/CMAH knockout pigs.

Progress has been made in overcoming antibody-mediated rejection of porcine xenografts by deleting pig genes that produce unique carbohydrate epitopes. Pigs deficient in galactose α-1,3 galactose (gene modified: GGTA1) and neu5Gc (gene modified: CMAH) have reduced levels of human antibody binding. Previously we identified α-fucose as a glycan that was expressed in high levels on cells of GGTA1/CMAH KO pigs. To validate the α-fucose phenotype observed previously we compared lectin affinity toward human and pig serum glycoproteins by dot blot analysis and confocal microscopy. Human anti-fucose antibody isolated by affinity chromatography was tested for specificity to L-fucose by custom macroarray. The affinity and cytotoxicity of the isolated human anti-fucose antibody toward human and GGTA1/CMAH KO pig PBMCs was determined by flow cytometry. Dot blot and confocal analysis support out previous findings that α-fucose is more highly expressed in pigs than humans. Pig kidney glomeruli and tubules contain abundant α-fucose and may represent focal sites for anti-α-fucose antibody binding. The Isolated human anti-fucose IgA, IgG and IgM bound to GGTA1/CMAH KO pig PBMC and were cytotoxic. Interestingly, the isolated human IgG cross reacted with the methyl pentose, L-rhamnose. Human anti-fucose antibody bound and was cytotoxic to GGTA1/CMAH KO pig peripheral blood monocytes. We have shown that α-fucose is an abundant target for cytotoxic human antibody in the organs of genetically modified pigs important to xenotransplantation.

Old World Monkeys are less than ideal transplantation models for testing pig organs lacking three carbohydrate antigens (Triple-Knockout).

Triple-knockout (TKO) pigs (with added protective human transgenes) are likely to be optimal sources of organs for clinical organ xenotransplantation because many humans have minimal or no natural antibody to TKO pig cells. However, Old World monkeys (OWMs) have naturally-existing antibodies directed to TKO cells. We measured anti-pig IgM/IgG binding, and complement-dependent cytotoxicity to wild-type (WT), α1,3-galactosyltransferase gene-knockout (GTKO), and TKO pig peripheral blood mononuclear cells (PBMCs) using sera from humans, several OWMs, and two New World monkeys (NWMs). Furthermore, we compared survival of GTKO (n = 5) and TKO (n = 3) pig kidneys in baboons. OWMs had significantly greater IgM binding and cytotoxicity to TKO PBMCs than humans or NWMs. Mean anti-TKO IgM was significantly higher in OWMs and significantly lower in NWMs than in humans. Cytotoxicity of OWM sera to TKO PBMCs was significantly greater than of human serum, but there was no significant difference between human and NWM sera. The median survival of TKO pig kidneys (4 days) in baboons was significantly shorter than that of GTKO kidneys (136 days) (p < 0.05). Even though considered ideal for clinical xenotransplantation, the presence of naturally-existing antibodies to TKO pig cells in OWMs complicates the transplantation of TKO pig kidneys in OWMs.

Identification by mass spectrometry and immunoblotting of xenogeneic antigens in the N- and O-glycomes of porcine, bovine and equine heart tissues.

Animal bioprosthetic heart valves (BHV) are used to replace defective valves in patients with valvular heart disease. Especially young BHV recipients may experience a structural valve deterioration caused by an immune reaction in which α-Gal and Neu5Gc are potential target antigens. The expression of these and other carbohydrate antigens in animal tissues used for production of BHV was explored. Protein lysates of porcine aortic and pulmonary valves, and porcine, bovine and equine pericardia were analyzed by Western blotting using anti-carbohydrate antibodies and lectins. N-glycans were released by PNGase F digestion and O-glycans by ß-elimination. Released oligosaccharides were analyzed by liquid chromatography - tandem mass spectrometry. In total, 102 N-glycans and 40 O-glycans were identified in animal heart tissue lysates. The N- and O-glycan patterns were different between species. α-Gal and Neu5Gc were identified on both N- and O-linked glycans, N,N´-diacetyllactosamine (LacdiNAc) on N-glycans only and sulfated O-glycans. The relative amounts of α-Gal-containing N-glycans were higher in bovine compared to equine and porcine pericardia. In contrast to the restricted number of proteins carrying α-Gal and LacdiNAc, the distribution of proteins carrying Neu5Gc-determinants varied between species and between different tissues of the same species. Porcine pericardium carried the highest level of Neu5Gc-sialylated O-glycans, and bovine pericardium the highest level of Neu5Gc-sialylated N-glycans. The identified N- and O-linked glycans, some of which may be immunogenic and remain in BHVs manufactured for clinical use, could direct future genetic engineering to prevent glycan expression rendering the donor tissues less immunogenic in humans.

Cell-Based Assays for Modeling Xenogeneic Immune Responses.

Research in xenotransplantation implies a high experimental complexity comprising molecular, cellular, and in vivo studies to investigate the mechanisms of xenograft immune rejection and functional failure, as well as the strategies to counteract them. After major advances associated with the identification of the carbohydrate xenoantigens and their elimination through genomic edition of the source pigs, the study of the cellular immune response against the xenograft is gaining particular attention. Xenogeneic cell-based assays that put together pig cells and human leukocytes such as monocytes, NK cells, and T cells are relevant to address this hurdle. Thus, we describe here coculture, co-stimulatory, and cytotoxicity assays for investigating the cellular and molecular mechanisms of xenograft rejection. These techniques allow elucidating the key pathways that take place during the xenogeneic immune response in a simplified setting. Treatment with either pro-inflammatory or anti-inflammatory cytokines can be used for studying the regulation of adhesion, co-stimulatory molecules, and receptors involved in triggering the immune response under various conditions. Furthermore, these assays can be used for the follow-up of the immune response of in vivo studies as well as for the development of tolerogenic approaches that promote xenograft survival.

Profiling natural serum antibodies of non-human primates with a carbohydrate antigen microarray.

BACKGROUND: Engineering of α-Galactosyltransferase gene-knockout pigs circumvented hyperacute rejection of pig organs after xenotransplantation in non-human primates. Overcoming this hurdle revealed the importance of non-α-Gal carbohydrate antigens in the immunobiology of acute humoral xenograft rejection. METHODS: This study analyzed serum from seven naïve cynomolgus monkeys (blood type O/B/AB = 3/2/2) for the intensity of natural IgM and IgG signals using carbohydrate antigen microarray, which included historically reported α-Gal and non-α-Gal carbohydrate antigens with various modifications. RESULTS: The median (range) of IgM and IgG signals were 12.71 (7.23-16.38) and 9.05 (7.23-15.90), respectively. The highest IgM and IgG signals with narrowest distribution were from mono- and disaccharides, followed by modified structures. Natural anti-α-Gal antibody signals were medium to high in IgM (11.2-15.9) and medium in IgG (8.5-11.6) spectra, and was highest with Lac core structure (Galα1-3Galß1-4Glc, iGb3) and lowest with LacNAc core structure (Galα1-3Galß1-4GlcNAc). Similar signal intensities (up to 15.8 in IgM and up to 11.8 in IgG) were observed for historically detected natural non-α-Gal antigens, which included Tn antigen, T antigen, GM2 glycolipid, and Sda antigen. The hierarchical clustering analysis revealed the presence of clusters of anti-A antibodies and was capable of distinguishing between the blood group B and AB non-human primates. CONCLUSIONS: The results presented here provide the most comprehensive evaluation of natural antibodies present in cynomolgus monkeys.

The pathology of solid organ xenotransplantation.

PURPOSE OF REVIEW: The use of genetically modified pigs has resulted in prolonged xenograft organ survival, overcoming the initial barriers that lead to hyperacute rejection and immediate loss of the graft. The purpose of the present review is to revisit the xenogeneic response and the pathologic changes in the xenograft organ in the context of recent publications of large animal studies that highlight existing challenges. RECENT FINDINGS: Transgenic modifications that have included complement regulatory proteins and coagulation regulatory proteins have prolonged xenograft survival in pig to nonhuman primate kidneys, livers, and hearts. Modifications of immunosuppressive regimens such as the addition of mTOR inhibition and costimulatory blockade have also led to better outcomes. Antibody-mediated rejection and thrombotic microangiopathy persist as primary challenges to the field and require further systematic exploration. SUMMARY: The efforts to overcome the natural antibody response to xenoantigens are largely sufficient. There is great opportunity for designing immunosuppression protocols and for detecting early coagulopathies, complement activation, and donor-specific antibody response. With graft survival prolongation, there is also a greater need to understand mechanisms and to enhance diagnostic tools for pathologic evaluation.

Bacterial Protein Toll-Like-Receptor Agonists: A Novel Perspective on Vaccine Adjuvants.

Adjuvants have been used in vaccines for over a century, however, the search for safe and effective vaccine adjuvants continues. In recent decades toll-like-receptor (TLR) agonists have been investigated as potential vaccine adjuvants. In this regard, the majority of the currently investigated TLR agonists are non-protein microbial components such as lipopolysaccharides, oligonucleotides, and lipopeptides. On the other hand, a growing number of studies reveal that TLR signaling and immune responses can be activated by numerous bacterial proteins. However, their potential roles as adjuvants have been somewhat overlooked. Herein, we discuss several such bacterial proteins which exhibit adjuvant properties, including the activation of TLR signaling, antigen presenting cell maturation, pro-inflammatory cytokine production and adaptive immune response. The protein nature of these TLR agonists presents several unique features not shared by non-protein TLR agonists. These properties include the amenability for modifying the structure and function as necessary for optimal immunogenicity and minimal toxicity. Protein adjuvants can be genetically fused to protein antigens which ensure the co-delivery of adjuvant-antigen not only into the same cell but also in the same endocytic cargo, leading to more effective activation of innate and adaptive immune response.

Xenoantigen-Dependent Complement-Mediated Neutralization of Lymphocytic Choriomeningitis Virus Glycoprotein-Pseudotyped Vesicular Stomatitis Virus in Human Serum.

Neutralization by antibodies and complement limits the effective dose and thus the therapeutic efficacy of oncolytic viruses after systemic application. We and others previously showed that pseudotyping of oncolytic rhabdoviruses such as maraba virus and vesicular stomatitis virus (VSV) with the lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) results in only a weak induction of neutralizing antibodies. Moreover, LCMV-GP-pseudotyped VSV (VSV-GP) was significantly more stable in normal human serum (NHS) than VSV. Here, we demonstrate that depending on the cell line used for virus production, VSV-GP showed different complement sensitivities in nonimmune NHS. The NHS-mediated titer reduction of VSV-GP was dependent on activation of the classical complement pathway, mainly by natural IgM antibodies against xenoantigens such as galactose-α-(1,3)-galactose (α-Gal) or N-glycolylneuraminic acid (Neu5Gc) expressed on nonhuman production cell lines. VSV-GP produced on human cell lines was stable in NHS. However, VSV-GP generated in transduced human cells expressing α-Gal became sensitive to NHS. Furthermore, GP-specific antibodies induced complement-mediated neutralization of VSV-GP independently of the producer cell line, suggesting that complement regulatory proteins potentially acquired by the virus during the budding process are not sufficient to rescue the virus from antibody-dependent complement-mediated lysis. Thus, our study points to the importance of a careful selection of cell lines for viral vector production for clinical use.IMPORTANCE Systemic application aims to deliver oncolytic viruses to tumors as well as to metastatic lesions. However, we found that xenoantigens incorporated onto the viral surface from nonhuman production cell lines are recognized by natural antibodies in human serum and that the virus is thereby inactivated by complement lysis. Hence, to maximize the effective dose, careful selection of cell lines for virus production is crucial.

Role of human and porcine MHC DRB1 alleles in determining the intensity of individual human anti-pig T-cell responses.

BACKGROUND: Differences in quality and strength of immune responses between individuals are mainly due to polymorphisms in major histocompatibility complex (MHC) molecules. Focusing on MHC class-II, we asked whether the intensity of human anti-pig T-cell responses is influenced by genetic variability in the human HLA-DRB1 and/or the porcine SLA-DRB1 locus. METHODS: ELISpot assays were performed using peripheral blood mononuclear cells (PBMCs) from 62 HLA-DRB1-typed blood donors as responder and the porcine B cell line L23 as stimulator cells. Based on the frequency of IFN-γ-secreting cells, groups of weak, medium, and strong responder individuals were defined. Mixed lymphocyte reaction (MLR) assays were performed to study the stimulatory capacity of porcine PBMCs expressing different SLA-DRB1 alleles. RESULTS: Concerning the MHC class-II configuration of human cells, we found a significant overrepresentation of HLA-DRB1*01 alleles in the medium/strong responder group as compared to individuals showing weak responses to stimulation with L23 cells. Evaluation of the role of MHC class-II variability in porcine stimulators revealed that cells expressing SLA-DRB1*06 alleles triggered strong proliferation in approximately 70% of humans. Comparison of amino acid sequences indicated that strong human anti-pig reactivity may be associated with a high rate of similarity between human and pig HLA/SLA-DRB1 alleles. CONCLUSION: Variability in human and porcine MHC determines the intensity of individual human anti-pig T-cell responses. MHC typing and cross-matching of prospective recipients of xenografts and donor pigs could be relevant to select for donor-recipient combinations with minimal anti-porcine immunity.

Generation of cattle knockout for galactose-α1,3-galactose and N-glycolylneuraminic acid antigens.

Two well-characterized carbohydrate epitopes are absent in humans but present in other mammals. These are galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc) which are introduced by the activities of two enzymes including α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP-Neu5Gc hydroxylase (encoded by the CMAH gene) that are inactive in humans but present in cattle. Hence, bovine-derived products are antigenic in humans who receive bioprosthetic heart valves (BHVs) or those that suffer from red meat syndrome. Using programmable nucleases, we disrupted (knockout, KO) GGTA1 and CMAH genes encoding for the enzymes that catalyse the synthesis of αGal and Neu5Gc, respectively, in both male and female bovine fibroblasts. The KO in clonally selected fibroblasts was detected by polymerase chain reaction (PCR) and confirmed by Sanger sequencing. Selected fibroblasts colonies were used for somatic cell nuclear transfer (SCNT) to produce cloned embryos that were implanted in surrogate recipient heifers. Fifty-three embryos were implanted in 33 recipients heifers; 3 pregnancies were carried to term and delivered 3 live calves. Primary cell cultures were established from the 3 calves and following molecular analyses confirmed the genetic deletions. FACS analysis showed the double-KO phenotype for both antigens confirming the mutated genotypes. Availability of such cattle double-KO model lacking both αGal and Neu5Gc offers a unique opportunity to study the functionality of BHV manufactured with tissues of potentially lower immunogenicity, as well as a possible new clinical approaches to help patients with red meat allergy syndrome due to the presence of these xenoantigens in the diet.

Localization Analysis of Heterophilic Antigen Epitopes of H1N1 Influenza Virus Hemagglutinin.

Previous studies have indicated that two monoclonal antibodies (mAbs; A1-10 and H1-84) of the hemagglutinin (HA) antigen on the H1N1 influenza virus cross-react with human brain tissue. It has been proposed that there are heterophilic epitopes between the HA protein and human brain tissue (Guo et al. in Immunobiology 220:941-946, 2015). However, characterisation of the two mAbs recognising the heterophilic epitope on HA has not yet been performed. In the present study, the common antigens of influenza virus HA were confirmed using indirect enzyme-linked immunosorbent assays and analysed with DNAMAN software. The epitopes were localized to nine peptides in the influenza virus HA sequence and the distribution of the peptides in the three-dimensional structure of HA was determined using PyMOL software. Key amino acids and variable sequences of the antibodies were identified using abYsis software. The results demonstrated that there were a number of common antigens among the five influenza viruses studied that were recognised by the mAbs. One of the peptides, P2 (LVLWGIHHP191-199), bound both of the mAbs and was located in the head region of HA. The key amino acids of this epitope and the variable regions in the heavy and light chain sequences of the mAbs that recognised the epitope are described. A heterophilic epitope on H1N1 influenza virus HA was also introduced. The existence of this epitope provides a novel perspective for the occurrence of nervous system diseases that could be caused by influenza virus infection, which might aid in influenza prevention and control.

The desirable donor pig to eliminate all xenoreactive antigens.

The humoral barrier has been the limiting factor in moving xenotransplantation towards the clinic. Improvements in somatic cell nuclear transfer and genome editing, particularly CRISPR-Cas9, have made it possible to create pigs with multiple glycan xenoantigen deletions for the purposes of reducing xenoreactive antibody binding to the xenografted organ. Recent studies have also considered the aetiology and existence of antibodies directed at the swine leucocyte antigen (SLA) complex, and potential genetic engineering strategies to avoid these antibodies. Evaluation of xenoreactive antibody binding is very important for the advancement of xenotransplantation, because if patients do not have any detectable xenoreactive antibody, then it is reasonable to expect that cellular rejection and not antibody-mediated rejection (AMR) will be the next hurdle to clinical application.

Cross-presentation of Exogenous Antigens.

Presentation of exogenous antigens loaded on major histocompatibility complex class I molecules by antigen presenting cells, termed cross-presentation, is essential for the induction of CD8+ T cells and is performed mainly by specialized dendritic cell subsets. Research into this field has described two main mechanisms of cross-presentation, the cytosolic pathway and the vacuolar pathway. As the first step in cross-presentation, surface receptors relating to cross-presentation are required in the recognition and uptake of Ags, which include C-type lectin receptors, immunoglobulin γ Fc region receptor, chemokine receptor, scavenger receptor etc. After uptake by the cells, there are also many molecules that enable Ags to participate in cross-presentation pathways. By this approach, exogenous Ags can induce CD8+ T cells into cytotoxic T lymphocytes, which is of great significance to induce antitumor and antiviral immune responses, and the molecular mechanism would facilitate the development of related adjuvants. However, the detailed mechanisms of cross-presentation still remain unknown. In this paper, some latest researches, including two major pathways, DC surface receptors and application prospects are summarized.

Adoptive transfer of xenoantigen­stimulated T cell receptor Vß­restricted human regulatory T cells prevents porcine islet xenograft rejection in humanized mice.

Polyclonal expansion of human regulatory T cells (Tregs) prevents xenogeneic rejection by suppressing effector T cell responses in vitro and in vivo. However, a major limitation to using polyclonally expanded Tregs is that they may cause pan­immunosuppressive effects. The present study was conducted to compare the ability of ex vivo expanded human xenoantigen­stimulated Tregs (Xeno­Treg) and polyclonal Tregs (Poly­Treg) to protect islet xenografts from rejection in NOD­SCID interleukin (IL)­2 receptor (IL2r)γ­/­ mice. Human cluster of differentiation (CD)4+CD25+CD127lo Tregs, expanded either by stimulating with porcine peripheral blood mononuclear cells (PBMCs) or anti­CD3/CD28 beads, were characterized by immune cell phenotyping, T cell receptor (TCR) Vß CDR3 spectratyping and performing suppressive activity assays in vitro. The efficiency of adoptively transferred ex vivo human Tregs was evaluated in vivo using neonatal porcine islet cell clusters (NICC) transplanted into NOD­SCID IL­2rγ­/­ mice, which received human PBMCs with or without Xeno­Treg or Poly­Treg. Xeno­Treg, which expressed increased levels of human leukocyte antigen­DR and secreted higher levels of IL­10, demonstrated enhanced suppressive capacity in a pig­human mixed lymphocyte reaction. Spectratypes of TCR Vß4, Vß10, Vß18 and Vß20 in Xeno­Treg showed restriction and expanded clones at sizes of 205, 441, 332 and 196 respectively, compared to those of Poly­Treg. Reconstitution of mice with human PBMCs and Poly­Treg resulted in NICC xenograft rejection at 63 days. Adoptive transfer with human PBMCs and Xeno­Treg prolonged islet xenograft survival beyond 84 days, with grafts containing intact insulin­secreting cells surrounded by a small number of human CD45+ cells. This study demonstrated that adoptive transfer of ex vivo expanded human Xeno­Treg may potently prevent islet xenograft rejection in humanized NOD­SCID IL2rγ­/­ mice compared with Poly­Treg. These findings suggested that adoptive Treg therapy may be used for immunomodulation in islet xenotransplantation by minimizing systemic immunosuppression.