Determination Of Groups Research Articles

Immunoinformatics Design of Multi-Epitope Peptide-Based Vaccine Against Cyprinid Herpesvirus-3 (CyHV-3) Targeting Thymidine Kinase Proteins

The common carp Cyprinus carpio is a freshwater teleost and is among the most economically significant fishes in aquaculture throughout the world. Taxonomically, C. carpio are a complex of species including subspecies Cyprinus carpio carpio. C. carpio are now threatened by Cyprinid Herpesvirus-3 (CyHV-3), the causative agent of Koi Herpesvirus Disease (KHVD), which causes severe morbidity and mortality in ornamental koi and common carp and can infect or be transmitted by other species. Despite these devastating circumstances, effective vaccinations or other medications for the control of KHVD are not readily available. For this reason, the aim of the current study was to formulate a multi-epitope vaccine against Cyprinid Herpesvirus-3 (CyHV-3) using an immunoinformatics approach. To assess the immunodominant T- and B-cell epitopes, the CyHV-3 proteomes were employed. Following a thorough evaluation, we constructed a strategy for vaccination employing four possible epitopes selected from among each of the three relevant epitope groups: cytotoxic T-lymphocyte, helper T-lymphocyte and linear B-lymphocyte. Important qualities used in the evaluation of the resultant vaccine are that it will be highly soluble, antigenic, immunogenic and non-allergenic. Among acceptable physicochemical qualities, the anticipated structure of the vaccine bears a close resemblance to that of the original protein. Additional considerations include a robust and sustained predicted binding between the vaccine and the Toll-Like Receptor (TLR9). Simulations of molecular dynamics confirm the likelihood of a strong binding stability and structural tightness. Moreover, the computer-generated immunological simulation revealed that the vaccine, when administered to fish, should induce immune responses comparable to those in real life. Finally, codon optimization based on Escherichia coli K12 produced favorable indications of GC content and acceptably high CAI value, as applicable to the cloning vector pET28+ (a). Overall, these results show that the proposed peptide vaccine is a promising option for CyHV-3 prophylaxis.

HLA molecular mismatches and induced donor-specific tolerance in combined living donor kidney and hematopoietic stem cell transplantation.

We investigated the potential role of HLA molecular mismatches (MM) in achieving stable chimerism, allowing for donor-specific tolerance in patients undergoing combined living donor kidney and hematopoietic stem cell transplantation (HSCT). All patients with available DNA samples (N=32) who participated in a phase 2 clinical trial (NCT00498160) where they received an HLA mismatched co-transplantation of living donor kidney and facilitating cell-enriched HSCT were included in this study. High-resolution HLA genotyping data were used to calculate HLA amino acid mismatches (AAMM), Eplet MM, three-dimensional electrostatic mismatch scores (EMS-3D), PIRCHE scores, HLA-DPB1 T-cell epitope group MM, HLA-B leader sequence MM, and KIR ligands MM between the donor and recipient in both directions. HLA MM were analyzed to test for correlation with the development of chimerism, graft vs. host disease (GvHD), de novo DSA, and graft rejection. Follow-up time of this cohort was 6-13.5 years. Of the 32 patients, 26 developed high-level donor or mixed stable chimerism, followed by complete withdrawal of immunosuppression (IS) in 25 patients. The remaining six of the 32 patients had transient chimerism or no engraftment and were maintained on IS (On-IS). In host versus graft direction, a trend toward higher median number of HLA-DRB1 MM scores was seen in patients On-IS compared to patients with high-level donor/mixed chimerism, using any of the HLA MM modalities; however, initial statistical significance was observed only for the EMS-3D score (0.45 [IQR, 0.30-0.61] vs. 0.24 [IQR, 0.18-0.36], respectively; p=0.036), which was lost when applying the Bonferroni correction. No statistically significant differences between the two groups were observed for AAMM, EMS-3D, Eplet MM, and PIRCHE-II scores calculated in graft versus host direction. No associations were found between development of chimerism and GvHD and non-permissive HLA-DPB1 T-cell epitope group MM, HLA-B leader sequence, and KIR ligands MM. Our results suggest an association between HLA-DRB1 molecular mismatches and achieving stable chimerism, particularly when electrostatic quality of the mismatch is considered. The non-permissive HLA-DPB1 T-cell epitope group, HLA-B leader sequence, and KIR ligands MM do not predict chimerism and GvHD in this combined kidney/HSCT transplant patient cohort. Further work is needed to validate our findings. https://clinicaltrials.gov/study/NCT00498160, identifier NCT00498160.

Living Donor Liver Transplantation in Patients with Preformed Donor-Specific Anti-Human Leukocyte Antigen Antibodies Using Preoperative Desensitization Therapy According to Intensity of Donor-Specific Antibodies: A Single-Center Study.

BACKGROUND In liver transplantation (LT), preoperative desensitization therapy is considered necessary in patients positive for donor-specific anti-human leukocyte antigen antibodies (DSAs). However, the relationship between DSA intensity and the necessary desensitization therapy is unclear. MATERIAL AND METHODS A total of 37 adult living donor (LD) LTs performed between January 2016 and March 2022 were examined. Mycophenolate mofetil (MMF) was administered preoperatively in DSA-positive cases with positive lymphocyte cross-matching who underwent LDLT. In those with strongly positive DSA (mean fluorescence intensity 10 000), rituximab was administered 2 weeks before LDLT in addition to MMF. Cross-reactive epitope group antigen (CREG)-alone-positive cases were also treated with preoperative MMF when lymphocyte cross-matching was positive. RESULTS Of the 37 patients, 9 were DSA-positive, 7 were CREG-alone-positive, and the others were double-negative. Of 9 DSA-positive cases, desensitization therapy was performed in 7, among which rituximab administration was performed in 3 strongly DSA-positive cases. Of 7 CREG-alone-positive cases, 2 were lymphocyte cross-match-positive and underwent desensitization therapy. The 1-year survival rate was 100% in both DSA- and CREG-alone-positive cases. The frequency of T-cell mediated rejection in DSA-positive, CREG-alone-positive, and double-negative cases was 22%, 43%, and 29%, respectively, with no significant difference. Antibody-mediated rejection occurred in only 1 patient, who was strongly DSA-positive and blood-group incompatible. There was also no significant difference among the 3 groups in terms of the frequency of biliary complications or 90-day mortality. CONCLUSIONS Satisfactory LDLT results were achieved in DSA- and CREG-alone-positive cases following desensitization therapy.

Separate Gut Plasma Cell Populations Produce Auto-Antibodies against Transglutaminase 2 and Transglutaminase 3 in Dermatitis Herpetiformis.

Dermatitis herpetiformis (DH) is an inflammatory skin disorder often considered as an extra intestinal manifestation of celiac disease (CeD). Hallmarks of CeD and DH are auto-antibodies to transglutaminase 2 (TG2) and transglutaminase 3 (TG3), respectively. DH patients have auto-antibodies reactive with both transglutaminase enzymes. Here it is reported that in DH both gut plasma cells and serum auto-antibodies are specific for either TG2 or TG3 with no TG2-TG3 cross reactivity. By generating monoclonal antibodies from TG3-specific duodenal plasma cells of DH patients, three conformational epitope groups are defined. Both TG2-specific and TG3-specific gut plasma cells have few immunoglobulin (Ig) mutations, and the two transglutaminase-reactive populations show distinct selection of certain heavy and light chain V-genes. Mass spectrometry analysis of TG3-specific serum IgA corroborates preferential usage of IGHV2-5 in combination with IGKV4-1. Collectively, these results demonstrate parallel induction of anti-TG2 and anti-TG3 auto-antibody responses involving separate B-cell populations in DH patients.

Rhesus isoimmunization: An underappreciated reproductive risk

In sub-Saharan Africa, pregnancy-related complications are a significant contributor to morbidity, mortality, and rising healthcare costs (1). The membrane of human red blood cells (RBCs) may contain the Rhesus (Rh) antigen. There are several types of Rh antigens but most important is the RhD antigen. Along the whole RhD protein, a group of conformation-dependent epitopes make up the D antigen. The most clinically significant blood group antigens that determine whether fetomaternal blood compatibility exists are the RhD and ABO systems (2). While the D-negative phenotype is linked to a deletion of RHD in most Caucasians, it is uncertain why the D antigen is not expressed other populations like the Japanese and Africans where it is linked to a grossly normal RHD (3). Accordingly, if a mother is Rh D-negative and the fetus is Rh D positive, she may produce antibodies through a process called RhD sensitization if she is exposed to fetal antigens. Sensitization is unlikely to damage the index fetus in normal conditions, but it may result in recurrent pregnancy losses, an infant with sensitized RBCs or to severe types of hemolytic disease of the newborn presenting with jaundice, anemia, developmental problems, or stillbirth (4).
 Rhesus isoimmunization can be prevented, but most Sub-Saharan African countries have not fully utilized these opportunities. As a result, many women are at risk due to poor medical records following potentially sensitizing events and a lack of access to crucial diagnostic and therapeutic commodities. These have significantly increased the difficulty of managing Rh-negative pregnancies (5).
 There has been a contentious suggestion for the universal prophylactic use of anti-D immunoglobulin in all pregnancies, including those following termination or ectopic pregnancy (6). We know that only a few at-risk women will profit from this strategy, which will unquestionably be highly expensive. However, there is no question that the Rh-negative pregnant population of Africa needs to have access to anti-D immunoglobulin on a global basis (7)(8). It follows that appropriate strategies should be instituted to effectively identify at-risk women and treat them accordingly (9).
 Leaders in obstetrics practice from various African countries met towards the end of last year to discuss the unmet need to address the burden of Rhesus disease. They noted that, despite being entirely preventable, Rhesus disease was identified as one of the roadblocks to achieving maternal universal health coverage. The team recommended all stakeholders to facilitate access to anti-D immunoglobulin and screening tools for widespread screening and treatment.

Leveraging T-cell receptor - epitope recognition models to disentangle unique and cross-reactive T-cell response to SARS-CoV-2 during COVID-19 progression/resolution.

Despite the general agreement on the significance of T cells during SARS-CoV-2 infection, the clinical impact of specific and cross-reactive T-cell responses remains uncertain. Understanding this aspect could provide insights for adjusting vaccines and maintaining robust long-term protection against continuously emerging variants. To characterize CD8+ T-cell response to SARS-CoV-2 epitopes unique to the virus (SC2-unique) or shared with other coronaviruses (CoV-common), we trained a large number of T-cell receptor (TCR) - epitope recognition models for MHC-I-presented SARS-CoV-2 epitopes from publicly available data. These models were then applied to longitudinal CD8+ TCR repertoires from critical and non-critical COVID-19 patients. In spite of comparable initial CoV-common TCR repertoire depth and CD8+ T-cell depletion, the temporal dynamics of SC2-unique TCRs differed depending on the disease severity. Specifically, while non-critical patients demonstrated a large and diverse SC2-unique TCR repertoire by the second week of the disease, critical patients did not. Furthermore, only non-critical patients exhibited redundancy in the CD8+ T-cell response to both groups of epitopes, SC2-unique and CoV-common. These findings indicate a valuable contribution of the SC2-unique CD8+ TCR repertoires. Therefore, a combination of specific and cross-reactive CD8+ T-cell responses may offer a stronger clinical advantage. Besides tracking the specific and cross-reactive SARS-CoV-2 CD8+ T cells in any TCR repertoire, our analytical framework can be expanded to more epitopes and assist in the assessment and monitoring of CD8+ T-cell response to other infections.

SENGKETA KEPUTUSAN TATA USAHA NEGARA PADA PENGADAAN BARANG/JASA PEMERINTAH DI KEMENTERIAN PERHUBUNGAN REPUBLIK INDONESIA

The tender process at the Ministry of Transportation uses an Electronic Procurement System (SPSE) and a supporting system where the final results in the application tendered by the Selection Working Group are only in the form of Minutes of Election Results (BAHP), which are then reported to the Commitment Making Officer (PPK). to be followed up in the form of a Letter of Appointment of Goods/Services Provider (SPPBJ) whether to accept the results of the Working Group or reject it. In the process of determining this, it turned out that there was a legal debate which resulted in legal uncertainty where the Selection Working Group's determination was in the form of BAHP as the result which was used as the object of the lawsuit. Meanwhile, the opinion of the other panel of judges is inversely proportional to the BAHP issue as the object of this dispute. Apart from that, administrative efforts before filing a lawsuit were also a problem where some of the Panel of Judges passed the dismissal stage and some did not.
 From the results of the study it was found that the determination of the object of dispute state administrative decisions on the procurement of government goods/services, especially at the Ministry of Transportation of the Republic of Indonesia, was normatively in the basic regulations regarding Government Procurement of Goods/Services, showing the decision to determine the winner of the Working Group in the form of BAHP even though it required approval with PPK can be used as th e object of dispute in disputes over state administrative decisions. In addition, the results also show that the explanations and decisions at the State Administrative Court, the administrative e arrangements for the procurement of goods/services are objections and appeals and a guarantee of 1% (one percent).
 
 Keywords: Procurement, goods/services, administrative decision.

Screening of linear B-cell epitopes and its proinflammatory activities of Haemophilus parasuis outer membrane protein P2.

Haemophilus parasuis is a commensal organism of the upper respiratory tract of pigs, but virulent strains can cause Glässer's disease, resulting in significant economic losses to the swine industry. OmpP2 is an outer membrane protein of this organism that shows considerable heterogeneity between virulent and non-virulent strains, with classification into genotypes I and II. It also acts as a dominant antigen and is involved in the inflammatory response. In this study, 32 monoclonal antibodies (mAbs) against recombinant OmpP2 (rOmpP2) of different genotypes were tested for reactivity to a panel of OmpP2 peptides. Nine linear B cell epitopes were screened, including five common genotype epitopes (Pt1a, Pt7/Pt7a, Pt9a, Pt17, and Pt19/Pt19a) and two groups of genotype-specific epitopes (Pt5 and Pt5-II, Pt11/Pt11a, and Pt11a-II). In addition, we used positive sera from mice and pigs to screen for five linear B-cell epitopes (Pt4, Pt14, Pt15, Pt21, and Pt22). After porcine alveolar macrophages (PAMs) were stimulated with overlapping OmpP2 peptides, we found that the epitope peptides Pt1 and Pt9, and the loop peptide Pt20 which was adjacent epitopes could all significantly upregulated the mRNA expression levels of IL-1α, IL-1β, IL-6, IL-8, and TNF-α. Additionally, we identified epitope peptides Pt7, Pt11/Pt11a, Pt17, Pt19, and Pt21 and loop peptides Pt13 and Pt18 which adjacent epitopes could also upregulate the mRNA expression levels of most proinflammatory cytokines. This suggested that these peptides may be the virulence-related sites of the OmpP2 protein, with proinflammatory activity. Further study revealed differences in the mRNA expression levels of proinflammatory cytokines, including IL-1β and IL-6, between genotype-specific epitopes, which may be responsible for pathogenic differences between different genotype strains. Here, we profiled a linear B-cell epitope map of the OmpP2 protein and preliminarily analyzed the proinflammatory activities and effects of these epitopes on bacterial virulence, providing a reliable theoretical basis for establishing a method to distinguish strain pathogenicity and to screen candidate peptides for subunit vaccines.

Characterizing T cell responses to enzymatically modified beta cell neo-epitopes.

Previous studies verify the formation of enzymatically post-translationally modified (PTM) self-peptides and their preferred recognition by T cells in subjects with type 1 diabetes (T1D). However, questions remain about the relative prevalence of T cells that recognize PTM self-peptides derived from different antigens, their functional phenotypes, and whether their presence correlates with a specific disease endotype. To address this question, we identified a cohort of subjects with T1D who had diverse levels of residual beta cell function. Using previously developed HLA class II tetramer reagents, we enumerated T cells that recognize PTM GAD epitopes in the context of DRB1*04:01 or PTM IA2 epitopes in the context of DQB1*03:02 (DQ8). Consistent with prior studies, we observed higher overall frequencies and a greater proportion of memory T cells in subjects with T1D than in HLA matched controls. There were significantly higher numbers of GAD specific T cells than IA2 specific T cells in subjects with T1D. T cells specific for both groups of epitopes could be expanded from the peripheral blood of subjects with established T1D and at-risk subjects. Expanded neo-epitope specific T cells primarily produced interferon gamma in both groups, but a greater proportion of T cells were interferon gamma positive in subjects with T1D, including some poly-functional cells that also produced IL-4. Based on direct surface phenotyping, neo-epitope specific T cells exhibited diverse combinations of chemokine receptors. However, the largest proportion had markers associated with a Th1-like phenotype. Notably, DQ8 restricted responses to PTM IA2 were over-represented in subjects with lower residual beta cell function. Neo-epitope specific T cells were present in at-risk subjects, and those with multiple autoantibodies have higher interferon gamma to IL-4 ratios than those with single autoantibodies, suggesting a shift in polarization during progression. These results reinforce the relevance of PTM neo-epitopes in human disease and suggest that distinct responses to neo-antigens promote a more rapid decline in beta cell function.

1470. Human monoclonal antibodies for the treatment and prevention of Crimean Congo Hemorrhagic Fever

Abstract Background Crimean-Congo hemorrhagic fever (CCHF) is the most widely spread viral hemorrhagic disease, found in Europe, Asia, and Africa. The disease symptoms start and progress rapidly and can lead to bruising and bleeding, with high fatality rates of up to 50% in hospitalized patients. Currently there are no approved vaccines or therapeutics available, and treatment is supportive. The CCHFV glycoprotein is made up of two major structural glycoproteins, GC and GN, which are required for the virus attachment and entry. An additional protein, GP38, is a secreted byproduct derived from a glycoproptein precursor of GN and whose role remains unknown. Recently, the non-neutralizing mouse mAb 13G8 (anti-GP38) has shown 60-90% protection prophylactically or post-challenge in a mouse model when challenged with various CCHFV strains. This is noteworthy because neutralizing mouse mAbs to GC have not shown consistent protection in mouse models. Because most of the work has been on mice up till now, we decided to isolate and study mAbs from human survivors of CCHF. Methods The antibody response to GC and GP38 were measured in CCHF patients from Turkey ∼ 6 months after infection, and one high titer sample was selected for mAb isolation. For this purpose, a combined proteomics/genomics approach was performed; plasma Abs were isolated using sequential affinity chromatography, peptides subjected to Mass spectrometry, and then matched to single cell VDJ sequence libraries constructed from PBMCs. mAbs were tested for binding and neutralization, and one candidate used for in-vivo evaluation with Ifnar1- mice (lacking IFN) challenged with CCHF virus. Results 11 anti-GP38 and 8 anti-Gc antibodies were isolated. All anti-Gc (and none of the GP38) mAbs exhibited neutralization. Anti-GP38 antibodies were segregated into 6 epitope groups based on competition assays (including 3 not described before). CC5-17, the human mAb sharing the same epitope as 13G8, showed 50% protection two separate experiments in Ifnar1- mice (when given 30 minutes prior to challenge, or 1 and 4 days after challenge). Conclusion The non-neutralizing gp38 antibodies have proven to afford protection before or after exposure to CCHFV. Human mAbs have the potential for protection and/or prevention of CCHF. Disclosures All Authors: No reported disclosures.

Contriving multi-epitope vaccine ensemble for monkeypox disease using an immunoinformatics approach.

The current global outbreak of monkeypox (MPX) disease, caused by Monkeypox virus (MPXV), has resulted in 16 thousand infection cases, five deaths, and has been declared a global health emergency of international concern by the World Health Organization. Given current challenges in the safety of existing vaccines, a vaccine to prevent MPX infection and/or onset of symptoms would significantly advance disease management. In this context, a multi-epitope-based vaccine could be a well-suited approach. Herein, we searched a publicly accessible database (Virus Pathogen Database and Analysis Resource) for MPXV immune epitopes from various antigens. We prioritized a group of epitopes (10 CD8+ T cells and four B-cell epitopes) using a computer-aided technique based on desirable immunological and physicochemical properties, sequence conservation criteria, and non-human homology. Three multi-epitope vaccines were constructed (MPXV-1–3) by fusing finalized epitopes with the aid of appropriate linkers and adjuvant (beta-defensin 3, 50S ribosomal protein L7/L12, and Heparin-binding hemagglutinin). Codon optimization and in silico cloning in the pET28a (+) expression vector ensure the optimal expression of each construct in the Escherichia Coli system. Two and three-dimensional structures of the constructed vaccines were predicted and refined. The optimal binding mode of the construct with immune receptors [Toll-like receptors (TLR2, TLR3, and TLR4)] was explored by molecular docking, which revealed high docking energies of MPXV-1–TLR3 (–99.09 kcal/mol), MPXV-2–TLR3 (–98.68 kcal/mol), and MPXV-3–TLR2 (–85.22 kcal/mol). Conformational stability and energetically favourable binding of the vaccine-TLR2/3 complexes were assessed by performing molecular dynamics simulations and free energy calculations (Molecular Mechanics/Generalized Born Surface Area method). In silico immune simulation suggested that innate, adaptive, and humoral responses will be elicited upon administration of such potent multi-epitope vaccine constructs. The vaccine constructs are antigenic, non-allergen, non-toxic, soluble, topographically exposed, and possess favourable physicochemical characteristics. These results may help experimental vaccinologists design a potent MPX vaccine.

Potent neutralizing anti-SARS-CoV-2 human antibodies cure infection with SARS-CoV-2 variants in hamster model

SummaryTreatment with neutralizing monoclonal antibodies (mAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contributes to COVID-19 management. Unfortunately, SARS-CoV-2 variants escape several of these recently approved mAbs, highlighting the need for additional discovery and development. In a convalescent patient with COVID-19, we identified six mAbs, classified in four epitope groups, that potently neutralized SARS-CoV-2 D614G, beta, gamma, and delta infection in vitro, with three mAbs neutralizing omicron as well. In hamsters, mAbs 3E6 and 3B8 potently cured infection with SARS-CoV-2 Wuhan, beta, and delta when administered post-viral infection at 5 mg/kg. Even at 0.2 mg/kg, 3B8 still reduced viral titers. Intramuscular delivery of DNA-encoded 3B8 resulted in in vivo mAb production of median serum levels up to 90 μg/mL, and protected hamsters against delta infection. Overall, our data mark 3B8 as a promising candidate against COVID-19, and highlight advances in both the identification and gene-based delivery of potent human mAbs.

Characterization of anti-porcine CXCL10 monoclonal antibodies

Abstract C-X-C motif chemokine ligand 10 (CXCL10) facilitates chemoattraction of important immune cells to tissues. As part of the USDA-NIFA Swine Immune Toolkit Initiative, our goal is to provide the veterinary community with new commercial immune reagents and standardized assays for future research efforts. For the chemokine target CXCL10, we used yeast expressed, recombinant porcine CXCL10 (rPoCXCL10) to generate a panel of α-CXCL10 monoclonal antibodies (mAbs). Each of the α-PoCXCL10 mAbs was assessed by ELISA using cross-inhibition analyses of biotinylated mAbs, and direct binding to orthologous yeast expressed CXCL10 proteins. As a result, we assigned six distinct epitope groups for the nine generated α-CXCL10 mAbs. Subsequently, we screened AF647-tagged α-CXCL10 mAbs for intracellular staining of pig immune cells using different stimulation conditions. Of the nine mAbs only two detected intracellular CXCL10 expression in PMA/ionomycin or rPoIFNγ-stimulated porcine cells. Further, cell characterization assays verified CXCL10+ cells as CD3-CD4-CD172+, with occasional CD4+ subsets. Overall, we have determined that α-CXCL10-1.4 mAb is the best mAb clone for intracellular staining and recommend it for use in analysis of intracellular signaling to detect interactions that regulate cell migration. A sandwich ELISA was also developed to quantitate CXCL10 protein expression; it verified reactivity with native porcine CXCL10. Overall, these tools will be useful for evaluating immunity against swine infectious diseases and host-responses to vaccines. New reagents identified by the Swine Immune Toolkit Initiative will undoubtedly advance future swine research efforts. Supported by USDA-NIFA AFRI grant # 2019-67015-29815. Supported by USDA-ARS project # 8042?32000?102 and USDA-NIFA AFRI grant # 2019-67015-29815

Antibodies binding diverse pertactin epitopes protect mice from Bordetella pertussis infection

Infection by the bacterium Bordetella pertussis continues to cause considerable morbidity and mortality worldwide. Many current acellular pertussis vaccines include the antigen pertactin, which has presumptive adhesive and immunomodulatory activities, but is rapidly lost from clinical isolates after the introduction of these vaccines. To better understand the contributions of pertactin antibodies to protection and pertactin's role in pathogenesis, we isolated and characterized recombinant antibodies binding four distinct epitopes on pertactin. We demonstrate that four of these antibodies bind epitopes that are conserved across all three classical Bordetella strains, and competition assays further showed that antibodies binding these epitopes are also elicited by B. pertussis infection of baboons. Surprisingly, we found that representative antibodies binding each epitope protected mice against experimental B. pertussis infection. A cocktail of antibodies from each epitope group protected mice against a subsequent lethal dose of B. pertussis and greatly reduced lung colonization levels after sublethal challenge. Each antibody reduced B. pertussis lung colonization levels up to 100-fold when administered individually, which was significantly reduced when antibody effector functions were impaired, with no antibody mediating antibody-dependent complement-induced lysis. These data suggest that antibodies binding multiple pertactin epitopes protect primarily by the same bactericidal mechanism, which overshadows contributions from blockade of other pertactin functions. These antibodies expand the available tools to further dissect pertactin's role in infection and understand the impact of antipertactin antibodies on bacterial fitness.

Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies

The SARS-CoV-2 B.1.1.529 (Omicron) variant contains 15 mutations of the receptor-binding domain (RBD). How Omicron evades RBD-targeted neutralizing antibodies requires immediate investigation. Here we use high-throughput yeast display screening1,2 to determine the profiles of RBD escaping mutations for 247 human anti-RBD neutralizing antibodies and show that the neutralizing antibodies can be classified by unsupervised clustering into six epitope groups (A–F)—a grouping that is highly concordant with knowledge-based structural classifications3–5. Various single mutations of Omicron can impair neutralizing antibodies of different epitope groups. Specifically, neutralizing antibodies in groups A–D, the epitopes of which overlap with the ACE2-binding motif, are largely escaped by K417N, G446S, E484A and Q493R. Antibodies in group E (for example, S309)6 and group F (for example, CR3022)7, which often exhibit broad sarbecovirus neutralizing activity, are less affected by Omicron, but a subset of neutralizing antibodies are still escaped by G339D, N440K and S371L. Furthermore, Omicron pseudovirus neutralization showed that neutralizing antibodies that sustained single mutations could also be escaped, owing to multiple synergetic mutations on their epitopes. In total, over 85% of the tested neutralizing antibodies were escaped by Omicron. With regard to neutralizing-antibody-based drugs, the neutralization potency of LY-CoV016, LY-CoV555, REGN10933, REGN10987, AZD1061, AZD8895 and BRII-196 was greatly undermined by Omicron, whereas VIR-7831 and DXP-604 still functioned at a reduced efficacy. Together, our data suggest that infection with Omicron would result in considerable humoral immune evasion, and that neutralizing antibodies targeting the sarbecovirus conserved region will remain most effective. Our results inform the development of antibody-based drugs and vaccines against Omicron and future variants.

Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies

The SARS-CoV-2 B.1.1.529 (Omicron) variant contains 15 mutations of the receptor-binding domain (RBD). How Omicron evades RBD-targeted neutralizing antibodies requires immediate investigation. Here we use high-throughput yeast display screening1,2 to determine the profiles of RBD escaping mutations for 247 human anti-RBD neutralizing antibodies and show that the neutralizing antibodies can be classified by unsupervised clustering into six epitope groups (A–F)—a grouping that is highly concordant with knowledge-based structural classifications3,4,5. Various single mutations of Omicron can impair neutralizing antibodies of different epitope groups. Specifically, neutralizing antibodies in groups A–D, the epitopes of which overlap with the ACE2-binding motif, are largely escaped by K417N, G446S, E484A and Q493R. Antibodies in group E (for example, S309)6 and group F (for example, CR3022)7, which often exhibit broad sarbecovirus neutralizing activity, are less affected by Omicron, but a subset of neutralizing antibodies are still escaped by G339D, N440K and S371L. Furthermore, Omicron pseudovirus neutralization showed that neutralizing antibodies that sustained single mutations could also be escaped, owing to multiple synergetic mutations on their epitopes. In total, over 85% of the tested neutralizing antibodies were escaped by Omicron. With regard to neutralizing-antibody-based drugs, the neutralization potency of LY-CoV016, LY-CoV555, REGN10933, REGN10987, AZD1061, AZD8895 and BRII-196 was greatly undermined by Omicron, whereas VIR-7831 and DXP-604 still functioned at a reduced efficacy. Together, our data suggest that infection with Omicron would result in considerable humoral immune evasion, and that neutralizing antibodies targeting the sarbecovirus conserved region will remain most effective. Our results inform the development of antibody-based drugs and vaccines against Omicron and future variants.

Cross-neutralization of RBD mutant strains of SARS-CoV-2 by convalescent patient derived antibodies.

BackgroundThe emergence of COVID‐19 pandemic resulted in an urgent need for the development of therapeutic interventions. Of which, neutralizing antibodies play a crucial role in the prevention and resolution of viral infection.MethodsWe generated antibody libraries from 18 different COVID‐19 recovered patients and screened neutralizing antibodies to SARS‐CoV‐2 and its mutants. After 3 rounds of panning, 456 positive phage clones were obtained with high affinity to RBD (receptor binding domain). Clones were then reconstituted into whole human IgG for epitope binning assay and all 19 IgG were classified into 6 different epitope groups or Bins.ResultsAlthough all antibodies were found to bind RBD, the antibodies in Bin2 had superior inhibitory ability of the interaction between spike protein and angiotensin converting enzyme 2 receptor (ACE2). Most importantly, the antibodies from Bin2 showed stronger binding affinity or ability to mutant RBDs (N501Y, W463R, R408I, N354D, V367F, and N354D/D364Y) derived from different SARS‐CoV‐2 strains as well, suggesting the great potential of these antibodies in preventing infection of SARS‐CoV‐2 and its mutations. Furthermore, such neutralizing antibodies strongly restricted the binding of RBD to hACE2 overexpressed 293T cells. Consistently, these antibodies effectively neutralized wildtype and more transmissible mutant pseudovirus entry into hACE2 overexpressed 293T cells. In Vero‐E6 cells, one of these antibodies can even block the entry of live SARS‐CoV‐2 into cells at 12.5 nM.ConclusionsThese results indicate that the neutralizing human antibodies from the patient‐derived antibody libraries have the potential to fight SARS‐CoV‐2 and its mutants in this global pandemic.

Potent neutralizing RBD-specific antibody cocktail against SARS-CoV-2 and its mutant.

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and its variants has posed a serious global public health emergency. Therapeutic interventions or vaccines are urgently needed to treat and prevent the further dissemination of this contagious virus. This study described the identification of neutralizing receptor‐binding domain (RBD)‐specific antibodies from mice through vaccination with a recombinant SARS‐CoV‐2 RBD. RBD‐targeted monoclonal antibodies (mAbs) with distinct function and epitope recognition were selected to understand SARS‐CoV‐2 neutralization. High‐affinity RBD‐specific antibodies exhibited high potency in neutralizing both live and pseudotype SARS‐CoV‐2 viruses and the SARS‐CoV‐2 pseudovirus particle containing the spike protein S‐RBDV367F mutant (SARS‐CoV‐2(V367F)). These results demonstrated that these antibodies recognize four distinct groups (I–IV) of epitopes on the RBD and that mAbs targeting group I epitope can be used in combination with mAbs recognizing groups II and/or IV epitope to make mAb cocktails against SARS‐CoV‐2 and its mutants. Moreover, structural characterization reveals that groups I, III, and IV epitopes are closely located to an RBD hotspot. The identification of RBD‐specific antibodies and cocktails may provide an effective therapeutic and prophylactic intervention against SARS‐CoV‐2 and its isolates.

Development and Characterization of Nanobodies Targeting the Kupffer Cell.

Nanobodies that are derived from single-chain antibodies of camelids have served as powerful tools in diagnostics, therapeutics and investigation of membrane receptors' structure and function. In this study, we developed a series of nanobodies by a phage display screening building from lymphocytes isolated from an alpaca immunized with recombinant mouse Kupffer cell receptor Clec4F, which is involved in pathogen recognition by binding to galactose and N-acetylgalactosamine. Bio-panning selections retrieved 14 different nanobodies against Clec4F with an affinity ranging from 0.2 to 2 nM as determined by SPR. Those nanobodies mainly recognize 4 different epitopes as analyzed via competitive epitope binning. By analysis of the radioactivity in each organ after injection of 99mTc labeled Clec4F nanobodies in naïve mice, we found that these nanobodies are targeting the liver. Furthermore, we performed a structural characterization at atomic resolution of two of the Clec4F nanobodies from different epitope groups, which revealed distinct features within the CDR2 and CDR3 regions. Taken together, we developed a series of nanobodies targeting multiple distinct recognition epitopes of the Kupffer cell-specific receptor Clec4F which may be useful for its structural and functional investigation as well as for use as molecular imaging and therapeutic agents.

Post-transplant de novo non donor-specific HLA antibodies are not associated with poor graft outcome in non-sensitized pediatric recipients of kidney transplantation

While de novo donor-specific HLA antibodies (dnDSAs) have a detrimental impact on kidney graft outcome, the clinical significance of de novo non donor-specific antibodies (dnNDSAs) is more controversial. We retrospectively evaluated for Ab development and characteristics of dnNDSAs serially collected post-transplant sera and, when available, graft biopsy eluates, from 144 non-sensitized, primary pediatric kidney recipients, consecutively transplanted at a single center between 2003 and 2017, using HLA class I and class II single-antigen flow-bead assays (SAB). The results were compared with clinical-pathologic data from HLA antibody negative and HLA dnDSA-positive patients.Forty-five out of 144 patients developed dnNDSAs (31%). Among the dnNDSA-positive patients, 86% displayed one or more class I/II antibodies recognizing antigens included in the CREG/shared epitope groups that also comprise the mismatched donor HLA antigens. Despite potential pathogenicity, as suggested by their occasional presence within the graft, dnNDSAs displayed significantly lower MFI, and limited complement binding and graft homing properties, when compared with dnDSAs. In parallel, the graft survival probability was significantly lower in patients with dnDSA than in those with dnNDSA or without HLA antibodies (p < 0.005). Indeed, the dnNDSA-positive patients remaining dnDSA-negative throughout the posttransplant period did not develop clinical antibody mediated rejection and graft loss, and maintained good graft function at a median follow-up of 9 years. The biological characteristics of dnNDSAs may account for the low graft damaging capability when compared to dnDSAs.