Split Antigen Research Articles

Inactivated Split MERS-CoV Antigen Prevents Lethal Middle East Respiratory Syndrome Coronavirus Infections in Mice.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes fatal infections, with about 36% mortality in humans, and is endemic to the Middle East. MERS-CoV uses human dipeptidyl peptidase 4 (hDPP4) as a receptor for infection. Despite continued research efforts, no licensed vaccine is available for protection against this disease in humans. Therefore, this study sought to develop an inactivated fragmented MERS-CoV vaccine grown in Vero cells in an hDPP4-transgenic mouse model. Two-dose immunisation in mice with 15, 20, or 25 μg of spike proteins of inactivated split MERS-CoV antigens induced neutralising antibodies, with titres ranging from NT 80 to 1280. In addition, all immunised mice were completely protected, with no virus detection in tissues, weight loss, or mortality. The immunised splenocytes produced more cytokines that stimulate immune response (IFN-γ and TNF-α) than those that regulate it (IL-4 and IL-10). Taken together, the inactivated fragmented MERS-CoV vaccine is effective for the protection of mice against lethal MERS-CoV. Thus, the inactivated fragmented MERS-CoV vaccine warrants further testing in other hosts.

Characterization of the Efficacy of a Split Swine Influenza A Virus Nasal Vaccine Formulated with a Nanoparticle/STING Agonist Combination Adjuvant in Conventional Pigs.

Swine influenza A viruses (SwIAVs) are pathogens of both veterinary and medical significance. Intranasal (IN) vaccination has the potential to reduce flu infection. We investigated the efficacy of split SwIAV H1N2 antigens adsorbed with a plant origin nanoparticle adjuvant [Nano11-SwIAV] or in combination with a STING agonist ADU-S100 [NanoS100-SwIAV]. Conventional pigs were vaccinated via IN and challenged with a heterologous SwIAV H1N1-OH7 or 2009 H1N1 pandemic virus. Immunologically, in NanoS100-SwIAV vaccinates, we observed enhanced frequencies of activated monocytes in the blood of the pandemic virus challenged animals and in tracheobronchial lymph nodes (TBLN) of H1N1-OH7 challenged animals. In both groups of the virus challenged pigs, increased frequencies of IL-17A+ and CD49d+IL-17A+ cytotoxic lymphocytes were observed in Nano11-SwIAV vaccinates in the draining TBLN. Enhanced frequency of CD49d+IFNγ+ CTLs in the TBLN and blood of both the Nano11-based SwIAV vaccinates was observed. Animals vaccinated with both Nano11-based vaccines had upregulated cross-reactive secretory IgA in the lungs and serum IgG against heterologous and heterosubtypic viruses. However, in NanoS100-SwIAV vaccinates, a slight early reduction in the H1N1 pandemic virus and a late reduction in the SwIAV H1N1-OH7 load in the nasal passages were detected. Hence, despite vast genetic differences between the vaccine and both the challenge viruses, IN vaccination with NanoS100-SwIAV induced antigen-specific moderate levels of cross-protective immune responses.

Development of a calculated panel reactive antibody calculator for the United Arab Emirates: a proof of concept study

Calculated panel reactive antibody (CPRA) is used to help increase sensitized patient’s access to transplantation. United Arab Emirates (UAE) has a diverse resident population hence we developed a UAE–CPRA calculator based on HLA antigen frequencies of the different ethnic groups that represent the UAE population. HLA antigen frequencies at serological split antigen level for HLA-A, -B, -C, -DRB1 and -DQB1 of 1002 healthy unrelated donors were performed. We subsequently compared the performance of the UAE CPRA calculator with the Organ Procurement and Transplantation Network (OPTN) and the Canadian CPRA calculators in 110 Kidney Transplant waitlist patients from January 2016 to December 2018. Lin’s concordance correlation coefficient showed a moderate agreement between the UAE and OPTN calculator (Rc = 0.949, 95% CI 0.929–0.963) and the UAE and Canadian calculators (Rc = 0.952, 95% CI 0.932–0.965). While there continued to be a moderate agreement (Rc = 0.937, UAE versus OPTN calculator) in the lower sensitized group, a poor agreement (Rc = 0.555, UAE versus OPTN calculator) was observed in the higher sensitized group. In this study, we provide a template for countries to develop their own population-specific CPRA calculator. Implementation of the CPRA algorithm based on HLA frequencies of the multi-ethnic UAE population will be more fitting to increase access to transplantation and improve transplant outcomes. Our study demonstrates that the CPRA calculators developed using the data from the western population had poor correlation in our higher sensitized patients disadvantaging them in potential organ allocations systems. We plan to further refine this calculator by using high resolution HLA typing to address the problem of a genetically diverse population.

Plant derived Nano-11 particle adsorbed with stimulator of interferon genes adjuvant and split influenza virus antigens elicits the cross-protective immunity in pigs

Abstract We evaluated the performance of split swine influenza A virus (SwIAV) H1N2 antigens with a plant-derived nanoparticle adjuvant (Nano-11) or its combination with a synthetic STING agonist ADU-S100 (NanoS100). Specific pathogen free pigs were vaccinated twice via intramuscular (IM) and intradermal (ID) routes and challenged with a virulent heterologous H1N1. Animals vaccinated IM or ID with H1N2-ADU-S100-Nano-11 had significantly increased cross-reactive IgG and IgA titers in serum, nasal secretion and bronchoalveolar lavage fluid. There was a significant increase of IFNγ+ effector memory T-helper/memory cells and late effector cytotoxic T cells (CTLs), IL-17A+ central and effector memory T-helper/memory cells, IL-17A+ early effector CTLs, and CXCL10+ plasmacytoid dendritic cells in the PBMCs of the ID injected H1N2-ADU-S100-Nano-11 compared to H1N2-Nano-11 group. The frequency of IFNγ+ late effector CTLs and effector memory T-helper/memory cells and IL-17A+ late effectorCTLs, as well as in lungs CXCL10+ plasmacytoid dendritic cells was increased in PBMCs of IM injected H1N2-ADUS100-Nano-11 pigs. Increased expression of IL-4 and IL-6 mRNA was observed in tracheobronchial lymph nodes of IM Nano-11 based vaccinates following challenge. Furthermore, the challenge virus load in the lungs and nasal passage was undetectable in IM H1N2+ADU-S100-Nano-11 vaccinates by day 6 post infection along with reduced macroscopic lung lesions. In conclusion, despite vast genetic difference (77% HA gene identity) between the H1N2 and H1N1 SwIAV, the NanoS100 adjuvanted vaccine elicited cross protective immune responses, suggesting the potential role of this combination adjuvant in inducing cross-protective immunity in pigs. Supported by USDA National Institute of Food and Agriculture, AFRI project 2019-67015-29814

Novel Adapter Chimeric Antigen Receptor (aCAR) T Cells for Temporally Controllable Targeting of Single and Multiple Tumor Antigens

T cells, engineered to express chimeric antigen receptors (CAR), are capable to generate an extremely potent and highly specific immune response against defined surface-expressed antigens. The very same features however hinder a more general clinical application, first because on-target off-tumor toxicity can cause life-threatening damage to vitally important healthy tissue and second because heterogeneity as well as plasticity of tumor tissue circumvent elimination through modulation or loss of target epitopes.One elegant way to achieve temporal (on/off) as well as qualitative (change and combine target antigens) control of CAR T cell function, is to split antigen recognition and effector cell activation using adapter molecules. Here, we report on the development of a novel adapter CAR (aCAR) system. By harnessing the unique properties of a novel single chain variable fragment (scFv), recognizing a neo-epitope comprising the endogenous vitamin biotin, conjugated to an adapter molecule e.g. a monoclonal antibody, in context of a special linker moiety, the technology can be used to specifically redirect CAR-T cells to a target structure referred to as linker-label epitope (LLE).CAR design was empirically tested using five different extracellular spacer formats combined with a second generation 4-1BB-CD3-ζ signaling backbone. aCAR expression was found to transmit potent T cell activation, cytokine secretion and target cell lysis exclusively in presence of biotinylated antibodies (b-mAbs) or antibody fragments targeting specific antigens at concentrations spanning several log units (10 pg/ml - 100 µg/ml). Importantly, no impairment or inhibition of signaling was detected in human serum or at supra-physiological concentrations of free biotin, providing evidence for specificity against the LLE. Short spacers (IgG4 or CD8 hinge) performed best, independent of the utilized biotinylated antibody format (full-size monoclonal antibodies (mAb), Fab2 or Fab fragments). aCAR T cells were tested in vitro in combination with over 30 different b-mAbs directed against antigens expressed on tumor cells of various cancer entities. With all tested antibodies, aCAR T cells were highly cytotoxic in the presence of a single antibody against an expressed antigen, thusverifying the specificity and applicability of the aCAR system. Further, we were able to demonstrate the simultaneous targeting of multiple antigens can prevent selection of intrinsically resistant antigen-loss variants. Importantly, we found that combining adapters against different antigens expressed on the same target cell mediates aCAR activation at adapter concentrations below the activation threshold for single antigen targeting, allowing target cell recognition and elimination based on multifactorial expression profiles. Finally, in vivo efficacy was demonstrated, targeting CD19 in a NALM-6 NSG mouse xenograft model.In conclusion, we demonstrate a novel aCAR system for maximal flexibility. aCAR T cell activation and cytotoxicity exclusively depends on the presence of biotinylated adapter molecules. Therefore, aCAR-T cell function can be stringently titrated by b-mAb infusion and withdrawal. This could add to an increased safety profile of CAR T cell therapy. Moreover, simultaneous targeting of multiple antigens can reduce selection pressure and the occurrence of resistance to therapy. Finally, since various mAbs have been FDA-approved or are close to enter the clinical setting and since the biotinylation of mAbs can be easily performed in a GMP-compliant manner, future clinical trials in leukemia and solid tumors can be envisioned based on our aCAR system. DisclosuresSeitz:Miltenyi Biotec GmbH: Patents & Royalties, Research Funding. Schlegel:Miltenyi Biotec GmbH: Patents & Royalties, Research Funding. Mittelstaet:Miltenyi Biotec GmbH: Employment. Lock:Miltenyi Biotec GmbH: Employment. Kaiser:Miltenyi Biotec GmbH: Employment, Patents & Royalties. Lang:Miltenyi Biotec GmbH: Patents & Royalties, Research Funding. Handgretinger:Miltenyi Biotec GmbH: Patents & Royalties, Research Funding.

Can PIRCHE-II Matching Outmatch Traditional HLA Matching?

We analyzed in a cohort of 68,606 first deceased donor kidney transplantations reported to the Collaborative Transplant Study whether an epitope-based matching of donor-recipient pairs using the Predicted Indirectly ReCognizable HLA Epitopes algorithm (PIRCHE-II) is superior to currently applied HLA antigen matching. PIRCHE-II scores were calculated based on split antigen HLA-A, -B, -DRB1 typing and adjusted to the 0–6 range of HLA mismatches. PIRCHE-II scores correlated strongly with the number of HLA mismatches (Spearman ρ = 0.65, P < 0.001). In multivariable analyses both parameters were found to be significant predictors of 5-year death-censored graft loss with high prognostic power [hazard ratio (HR) per adjusted PIRCHE-II score = 1.102, per HLA mismatch = 1.095; z-value PIRCHE-II: 9.8, HLA: 11.2; P < 0.001 for both]. When PIRCHE-II scores and HLA mismatches were analyzed simultaneously, their predictive power decreased but remained significant (PIRCHE-II: P = 0.002; HLA: P < 0.001). Influence of PIRCHE-II was especially strong in presensitized and influence of HLA mismatches in non-sensitized recipients. If the level of HLA-incompatibility was low (0–3 mismatches), PIRCHE-II scores showed a low impact on graft survival (HR = 1.031) and PIRCHE-II matching did not have additional significant benefit (P = 0.10). However, if the level of HLA-incompatibility was high (4–6 mismatches), PIRCHE-II improved the positive impact of matching compared to applying the traditional HLA matching alone (HR = 1.097, P = 0.005). Our results suggest that the PIRCHE-II score is useful and can be included into kidney allocation algorithms in addition to HLA matching; however, at the resolution level of HLA typing that is currently used for allocation it cannot fully replace traditional HLA matching.

Immune Responses to Adjuvanted H7N9 Split Antigen in Aged Mice.

The avian influenza A H7N9 virus has caused severe infection and high mortality in humans. It can be extremely hazardous to the elderly since age might diminish the immune response, and poor immunogenicity of H7 hemagglutinin could diminish the vaccine efficacy in this population. To overcome this issue, adjuvants are used to induce a stronger immune response. In this study, we generated a recombinant H7N9 influenza virus using reverse genetic techniques, consisting of hemagglutinin and neuraminidase genes derived from a human H7N9 virus, with the remaining genes from H1N1 A/Puerto Rico/8/34 (PR8). To evaluate whether the adjuvant can improve immune responses in aged animals, the humoral and cellular immune responses of 18-month-old BALB/c mice to different doses of split avian influenza A H7N9 vaccine with and without the adjuvant MF59 were compared. Our data showed that aged mice immunized with MF59 elicited higher levels of hemagglutination inhibition and microneutralization antibodies and interferon-gamma-specific enzyme-linked immunospot assay (ELISPOT) responses when compared with antigens alone. It is suggested that the split avian influenza A H7N9 vaccine combined with MF59 may significantly improve immune responses to influenza vaccination in elderly humans.

CAN EPITOPE MATCHING OUTMATCH TRADITIONAL HLA MATCHING?

Collaborative Transplant Study. Introduction: Pre-transplant presence and post-transplant development of alloantibodies against highly polymorphic human leukocyte antigen (HLA) allele mismatches (mm) play a major role in rejection of kidney allografts. However, antibodies recognize small sequences on HLA alleles called epitopes and the same epitope can be present on different alleles, impairing the precision of matching. Using the Collaborative Transplant Study database, we analyzed whether a T-cell epitope-based matching of donor-recipient pairs using the PIRCHE-II algorithm is a better predictor of graft survival than the currently applied allele group-based HLA-A, -B, -DR matching. Methods: 68,606 European deceased donor kidney-only transplantations during 1996–2016 were analyzed. PIRCHE-II mismatch score was calculated based on split typing using the 2007 NMDP haplotype frequency database and the PIRCHE matching service. Multivariable Cox regression analyses were performed to calculate hazard ratios (HRs) for death-censored graft loss. Correlation was analyzed with Spearman’s rank correlation. In order to achieve comparable HRs, a log-transformed PIRCHE-II score was used, followed by a linear transformation to match the value range of 0–6 HLA A+B+DR mm. Linear transformations in Cox regression analysis change the HR per point without changing the P- and Z-values. Results and Discussion: The HLA mismatch-derived PIRCHE-II score correlated strongly with the number of HLA mm (correlation coefficient ρ=0.65, P<0.001). Cox regression analyses revealed that Pirche II score and HLA mm are predictive confounders for death-censored graft loss at year 5 with highly significant prognostic power (Z-value for Pirche II: 9.8; for HLA: 11.2; P<0.001) (Table 1).The full Cox model per transformed PIRCHE-II score without HLA mm resulted in a hazard ratio (HR) for graft loss of 1.102 (95% CI=1.081–1.123; P<0.001). When HLA mm were added to the model, the HR per PIRCHE-II score decreased to 1.042 but remained statistically significant (95% CI=1.015–1.071; P=0.002) (Table 1). Overall, considering the HLA loci currently used for typing at split antigen level, HLA mm still had a higher predictive value than the PIRCHE-II score (11.2 vs. 9.8) regarding graft survival. As illustrated in Figure 1, the variance in PIRCHE-II score increased with higher number of HLA mm.Therefore, the possible clinical benefit of PIRCHE-II score matching improved with higher number of HLA mismatches. As demonstrated in Table 1, the risk of graft loss increases with each additional PIRCHE-II score. Conclusion: Our results indicate that PIRCHE-II score is useful and can be included into matching algorithms. However, it cannot fully replace traditional HLA matching. There seems to be information on immunological differences PIRCHE-II score cannot cover in its present form using HLA typing data at split level. This needs to be analyzed also for HLA matching at 4-digit molecular typing level.

Molecular dynamics simulations provide molecular insights into the role of HLA-B51 in Behçet's disease pathogenesis.

Behçet's disease is an inflammatory disorder of unknown etiology. Genetic tendency has an important role in its pathogenesis, and HLA-B51, a class I MHC antigen, has been recognized as the strongest susceptibility factor for Behçet's disease. Despite the confirmation of the association of HLA-B51 with Behçet's disease in different populations, its pathogenic mechanisms remain elusive. HLA-B51 differs in only two amino acids from HLA-B52, other split antigen of HLA-B5, which is not associated with Behçet's disease. These two amino acids are located in the B pocket of the antigen-binding groove, which occupies the second amino acids of the bound peptides. To understand the nature of the HLA-peptide interactions, differences in structure and dynamics of two HLA alleles were investigated by molecular dynamics simulations using YAYDGKDYI, LPRSTVINI, and IPYQDLPHL peptides. For HLA-B51, all bound peptides fluctuated to larger extent than HLA-B52. Free energy profiles of unbinding process for YAYDGKDYI by steered molecular dynamics simulations showed that unbinding from HLA-B52 results in greater free energy differences than HLA-B51. These results suggest the possibility of an instability of HLA-B51 associated with the repertoire of peptides, and this finding may provide significant insight to its pathogenic role in Behçet's disease.

Molecular‐level HLA mismatch is associated with rejection and worsened graft survival in heart transplant recipients – a retrospective study

The aim was to evaluate the association of molecular‐level human leukocyte antigen (HLA) mismatching with post‐transplant graft survival, rejection, and cardiac allograft vasculopathy (CAV). We retrospectively analyzed all primary cardiac transplant recipients between 01/1984‐06/2016. 1167 patients fulfilled inclusion criteria and had HLA typing information available. In 312 donor‐recipient pairs, typing at serological split antigen level was available. We used the Epitope MisMatch Algorithm to calculate the number of amino acid differences in antibody‐verified HLA eplets (amino acid mismatch load (AAMM)) between donor and recipient. Patients with a higher HLA‐DR AAMM load had inferior 1‐year graft survival (hazard ratio [HR], 1.14; 95% confidence interval [CI], 1.01–1.28). The HLA‐AB AAMM load showed no impact on graft survival. In the subgroup with available split‐level information, we observed an inferior graft survival for a higher HLA‐DR AAMM load 3 months after transplantation (HR, 1.22; 95% CI, 1.04–1.44) and a higher risk for rejection for an increasing HLA‐AB (HR, 1.70; 95% CI, 1.29–2.24) and HLA‐DR (HR, 1.32; 95% CI, 1.09–1.61) AAMM load. No impact on the development of CAV was found. Molecular‐level HLA mismatch analysis could serve as a tool for risk stratification after heart transplantation and might take us one step further into precision medicine.

Specific amino acid patterns define split specificities of HLA-B15 antigens enabling conversion from DNA-based typing to serological equivalents

The HLA-B15 typing by serological approaches defined the serological subgroups (or splits) B62, B63, B75, B76, B77 and B70 (B71 and B72). The scarcity of sera with specific anti-HLA antibodies makes the serological typing method difficult to discriminate a high variety of HLA antigens, especially between the B15 antigen subgroups. Advancements in DNA-based technologies have led to a switch from serological typing to high-resolution DNA typing methods. DNA sequencing techniques assign B15 specificity to all alleles in the HLA-B*15 allele group, without distinction of the serological split equivalents. However, the presence of antibodies in the patient defined as split B15 antigens urges the identification of HLA-B*15 allele subtypes of the donor, since the presence of donor-specific antibodies is an important contraindication for organ transplantation. Although the HLA dictionary comprises information regarding the serological subtypes of HLA alleles, there are currently 394 B15 antigens out of 516 in the IPD-IMGT/HLA database (3.38.0) without any assigned serological subtype. In this regard, we aimed to identify specific amino acid patterns for each B*15 serological split, in order to facilitate the assignment of B*15 alleles to serological equivalents after high-resolution molecular typing. As a result, serological specificities of 372/394 not yet assigned alleles could be predicted based on amino acid motifs. Furthermore, two new serological types were identified and added, B62-Bw4 and B71-Bw4.

Prevention of influenza virus infection and transmission by intranasal administration of a porous maltodextrin nanoparticle-formulated vaccine.

Influenza vaccines administered intramuscularly exhibit poor mucosal immune responses in the respiratory tract which is the prime site of the infection. Intranasal vaccination is a potential route for vaccine delivery which has been demonstrated effective in inducing protective immune responses in both systemic and mucosal compartments. For this purpose, nanoparticles have been used as antigen delivery systems to improve antigen capture by immune cells. In this paper we demonstrate efficient delivery of viral antigens to airway epithelial cells, macrophages and dendritic cells, using polysaccharide nanoparticles (NPL), leading to a strong protection against influenza virus infection. A formulation combining split Udorn virus antigens with NPL and the mucosal protein adjuvant CTA1-DD was administered intranasally and resulted in an enhanced specific humoral immune response. Furthermore, NPL carrying split Udorn, with or without CTA1-DD, inhibited virus transmission from infected to uninfected naive mice. These results demonstrate that an intranasal delivery system combining NPL, mucosal adjuvant CTA1-DD and split virus antigens confers robust protection against influenza infection and inhibits virus transmission.

Novel calixarene-based surfactant enables low dose split inactivated vaccine protection against influenza infection

Influenza A viruses cause major morbidity and represent a severe global health problem. Current influenza vaccines are mainly egg-based products requiring the split of whole viruses using classical detergents such as Triton X-100, which implies certain limitations. Here, we report the use of the novel calixarene-based surfactant CALX133ACE as an alternative to classical detergents for influenza inactivated split vaccine preparation. We confirmed that CALX133ACE-based split HA antigens are fully functional and quantifiable by the “gold standard” method SRID. Additionally, as in the case of the Triton X-100-based split, the CALX133ACE-based split antigens are stable for at least 6 months at 4 °C. Moreover, immunization of mice with CALX133ACE-based split NYMC X-179A (H1N1) antigens harboring 10 to 30-fold less antigen than the commercialized trivalent inactivated vaccines Vaxigrip® or Fluviral® induced comparable efficient protection and neutralizing antibody responses against A(H1N1)pdm09 infection. Taken together, our results demonstrate for the first time the use of a calixarene-based detergent as an efficient splitting agent for the production of optimized influenza split antigens, paving the way for significant improvement in the vaccine manufacturing process, notably with regard to the current regulation on the prohibition of endocrine disruptors, such as Triton X-100.

Microprojection arrays applied to skin generate mechanical stress, induce an inflammatory transcriptome and cell death, and improve vaccine-induced immune responses

Chemical adjuvants are typically used to improve immune responses induced by immunisation with protein antigens. Here we demonstrate an approach to enhance immune responses that does not require chemical adjuvants. We applied microprojection arrays to the skin, producing a range of controlled mechanical energy to invoke localised inflammation, while administering influenza split virus protein antigen. We used validated computational modelling methods to identify links between mechanical stress and energy generated within the skin strata and resultant cell death. We compared induced immune responses to those induced by needle-based intradermal antigen delivery and used a systems biology approach to examine the nature of the induced inflammatory response, and correlated this with markers of cell stress and death. Increasing the microprojection array application energy and the addition of QS-21 adjuvant were each associated with enhanced antibody response to delivered antigen and with induction of gene transcriptions associated with TNF and NF-κB signalling pathways. We concluded that microprojection intradermal antigen delivery inducing controlled local cell death could potentially replace chemical adjuvants to enhance the immune response to protein antigen.

HLA Epitope Mismatching is Associated with Rejection and Worsened Graft Survival in Heart Transplant Recipients

Purpose At present the assessment of the heart transplant (HTx) recipient's personal alloimmune risk is still not precise enough. The aim of this study was to evaluate the impact of molecular-level human leukocyte antigen (HLA) matching on post-transplant graft survival, rejection and cardiac allograft vasculopathy (CAV). Methods We retrospectively analyzed all primary cardiac transplant recipients between 01/1984 - 06/2016. 1,167 patients fulfilled inclusion criteria with HLA typing information available for donor and recipient. In 312 pairs high resolution HLA typing at split antigen level was available for AB and DR. We used the HLAMatchmaker algorithm (version 02.1) to calculate amino acid differences in antibody verified HLA epitopes (epitope mismatch load (EpMM)) between donor and recipient. In order to group patients in high and low EpMM, we split the collective at the respective median. Kaplan Meier analyses with log-rank tests were used to compare outcomes between groups. A p-value of Results Patients with a DR EpMM load below the median ( Conclusion HLA matching at the epitope level can serve as tool for risk stratification after heart transplantation and could take us one step further into precision medicine in solid organ transplantation.

Cytokine production in whole-blood cultures following immunization with an influenza vaccine

ABSTRACTA clinical trial of a quadrivalent split influenza vaccine was performed in the 2014/15 season. Sixty-four subjects aged 6 months to 18 years were enrolled in order to investigate the relationship between cellular and humoral immune responses. Subjects were categorized into two groups by measuring neutralizing antibodies: non-primed naïve/primed or seroconverted/non-seroconverted groups. Whole-blood cultures were stimulated with the H1N1 split antigen before immunization and one month after the first and second immunizations for subjects < 13 years and before and one month after the first dose for those ≥ 13 years in order to investigate cytokine production. Significant amounts of IL-2, IL-12, IL-13, MCP-1, MIP-1β, and TNF-α were detected from one month after the first dose in the naïve group. In addition to these cytokines, the production of IL-1β, IL-4, IL-6, IL-8, IL-10, IL-17, G-CSF, and IFN-γ was enhanced one month after the second dose. No significant increase was noted in the primed group, except in the production of IL-10. In seroconverted subjects, the production of IL-2, IL-4, IL-8, IL-10, G-CSF, MCP-1, TNF-α, and IFN-γ increased one month after the first dose, which was earlier than in the naïve group, whereas no significant cytokine response was noted in subjects without seroconversion. Subjects ≥ 13 years were primed and the production of G-CSF, IL-4, and IL-1β increased in subjects with seroconversion. Whole-blood cultures were also stimulated with the H3N2 split antigen and similar cytokine profiles were obtained. Many cytokines and chemokines, including inflammatory cytokines, were produced in seroconverted, but not non-seroconverted subjects.

P200 High-resolution HLA typing of deceased donors enables accurate identification of allle_specific DSA

HLA typing of donors for solid organ transplantation is performed with the primary goal of avoiding hyperacute antibody-mediated rejection (AMR) caused by recipient antibodies against donor antigens. Given the need for rapid testing of deceased donors, SSP or SSO methods are used to enable timely HLA typing at the serologic split antigen level. However, these methods do not provide unambiguous allele-level genotyping. This ambiguity can be problematic in patients with allele-specific anti-HLA antibodies. We report a case of suspected AMR following deceased donor kidney transplantation as an example of high-resolution HLA genotyping enabling identification of donor-specific antibodies (DSA). The patient was transplanted in 2010 with a flow cytometric crossmatch negative deceased donor kidney. History was remarkable for biopsy-proven C4d positive acute AMR in 2012 successfully treated by IVIG and rituximab. In late 2016, the patient developed increased proteinuria. Biopsy demonstrated changes consistent with both active and chronic AMR, but without presence of C4d. Solid-phase anti-HLA antibody testing did not demonstrate identifiable DSA, though there was evidence of allele-specific antibodies (Table 1). To investigate whether these represented DSA, donor DNA was re-typed for HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1,and -DPB1 by next-generation DNA sequencing (NGS). High-resolution HLA typing identified DSA against DQA1∗05:05 and DRB3∗03:01. Reactivity against DR52 or DQA05 would not have been called based upon average group reactivity. Identification of DSA supported diagnosis of AMR and enabled tracking of DSA as an objective measure of the efficacy of therapy. The increasing frequency of allele-specific DSA has led us to implement routine re-testing of solid organ donors for high-resolution HLA typing to promote accurate and efficient evaluation of potential allele-specific DSA. Download : Download high-res image (123KB) Download : Download full-size image

P053 Evaluation of discrepant deceased donor HLA typings reported in UNET

Aim The recent implementation of the new Kidney Allocation System (KAS) has greatly increased the amount of deceased donor (DD) organ sharing across the country, especially for patients with 99–100% cPRA. This has led to an increased reliance on virtual crossmatching (vXM) for allocation and acceptance of these organs. In order to perform an accurate vXM the donor HLA typing must be correct. To assess the accuracy of HLA typings used for DD allocation, the OPTN Histocompatibility Committee investigated the frequency of and reasons for discrepant HLA typing. Methods All DD typings entered into UNet SM from 2015 to 2016 that included at least two separate entries (Match Run, Donor/Recipient Histocompatibility Forms) were analyzed using OPTN data. Each individual discrepancy was classified based on the type of error and whether the discrepant typing may have impacted organ allocation. Results Initial analysis of all DD typing showed many discrepancies among the data entered. While many of these entry errors were in the Donor or Recipient Histocompatibility forms, ∼2% of DDs had discrepancies that could have affected the match run and potentially organ allocation. The primary errors included testing or reporting an incorrect HLA type (wrong donor), clerical, technical and interpretive errors leading to incorrect data entry and the use of parent antigen nomenclature instead of the defined split antigens. The recent requirement for DPB1 typing of DDs was also a source of many discrepant results. Conclusions This analysis shows that there are a significant number of DD typings with discrepant results. A small proportion of these may have potentially affected organ allocation and appear to mainly be a result of data entry errors. Such errors can lead to a false negative vXM and potentially harmful transplant outcomes. The Committee continues to work with UNOS to reduce errors and has proposed several solutions aimed at decreasing the number of discrepant typings.

Low hemagglutinin antigen dose influenza vaccines adjuvanted with AS03 alter the long-term immune responses in BALB/c mice

ABSTRACTWe investigated the long-term immune profiles of dose-sparing, AS03-adjuvanted vaccines compared to a traditional high-dose, unadjuvated influenza vaccine formulation. BALB/c mice received 2 IM injections of influenza A/Uruguay/716/2007 (H3N2) split vaccine antigen: high-dose (HD) (3 µg hemagglutinin (HA)/dose) or low-dose (LD) formulations (0.03 µg or 0.003 µg HA) with AS03 and were followed to 34 weeks post-boost (pb). We examined serologic responses, spleen and bone marrow (BM) HA-specific antibody-secreting cells (ASCs) by ELISpot, influenza-specific cytokine/chemokine production in re-stimulated splenocytes by multiplex ELISA, and antigen-specific CD4+ T cells that express cytokines (IL-2, IFNγ, TNFα and IL-5) by flow cytometry. All formulations elicited robust serum antibody titers that persisted for at least 34 weeks. The number of antigen-specific ASCs in the spleen and BM were higher in the 2 LD +AS03 groups, but despite having fewer ASCs, the average spot size in the HD-unadjuvanted group was larger at later time-points, suggesting greater antibody production per cell. Striking differences in the long-term profiles induced by the different vaccine formulations may contribute to these different ASC profiles. The HD-unadjuvanted vaccine elicited strong Th2 cytokines during the first 6 weeks pb but LD+AS03 groups generated broader, more durable responses at later timepoints. Finally, the 0.03 µg HA+AS03 group generated the greatest number of antigen-specific CD4+ T cells and the highest percentage of poly-functional cells that expressed 2 or more cytokines. Although all of the tested vaccines induced durable antibody responses, we show that different vaccine formulations (dose-sparing, adjuvant) generate distinct long-term immune profiles. Furthermore, our data suggest that the different profiles may be generated through unique mechanisms.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature