Variable Region Sequences Research Articles

1008. Presence of Antibody Dependent Cell Cytotoxicity (ADCC) Functional Antibodies that Target a Complex Gp41 Epitope Correlates with Long-term Non-progression and ADCC is Maintained with Mutants Using Germline Heavy Chain Variable Gene Sequence of VH1-02 Gene

BackgroundRecent data supports that improved qualitative antibody responses correlate with elite controllers (EC) of HIV. As ADCC has been associated with protection in vaccine studies, thorough exploration of antibodies that facilitate ADCC is warranted. In studies on monoclonal antibodies from long-term non-progressors (LTNPs), our laboratory has previously described highly mutated antibodies against a complex conformational epitope with contributions from both gp41 heptad repeat regions. Despite using the VH1-02 gene segment, known to contribute to some of the broadest neutralizing antibodies against HIV, members of these antibodies, termed group 76C antibodies, did not exhibit broad neutralization. MethodsOur goal was to characterize the non-neutralizing functions of antibodies of group 76C, to assess targeting of the epitope in various clinical presentations, and to assess the development of these antibodies by comparison to their predicted common ancestor. Serum samples were obtained from HIV+ clinical groups: EC, LTNP, stable CD4 counts on therapy, and those off therapy.ResultsIn antibody/serum competition assays, comparison to VRC01 which also uses VH1-02, showed that antibodies targeting the 76C group epitope were enriched in LTNPs. We then show recombinant antibodies of 76C members 6F5 and 6F11 both have robust ADCC activity, despite their sequence disparity. Sequence analysis predicted the common ancestor of this clonal group would utilize the germline non-mutated variable gene. We produced a recombinant ancestor Ab (76Canc) with a heavy chain utilizing the germline variable gene sequence paired to the 6F5 light chain. 76Canc binds HIV envelope constructs near the original group C epitope. 76Canc also shows comparable ADCC to 6F5 and 6F11 on both clade B and C constructs.Common ancestor antibodies maintain function and these types of antibodies correlate to a non-progressive clinical state. (A) Serum from long-term non-progressors (LTNPs) compared to serum from a group of HIV infected with lower CD4 levels as a control for viral load were used to compete against biotinylated CD4 binding site (VRC01) and 76C Gp41 conformational epitope (6F11) targeting antibodies. Serum dilutions were chosen to align means near 50%. Means with 95% confidence intervals are shown. (B) Monoclonal antibody 76Canc was created using the germline sequence of the heavy chain variable region with the CDR3 and light chain of 76C member. Antibody dependent cell cytotoxicity flow cytometric based assays were performed using gp41 proteins from clade B (MN) and clade C (ZA1197).ConclusionCertain antibodies present early on in infection may contribute to overall clinical course. Variable gene germline sequences that support functional activity against HIV could be targeted in vaccine regimens.Disclosures All Authors: No reported disclosures

Sequence variations, flanking region mutations, and allele frequency at 31 autosomal STRs in the central Indian population by next generation sequencing (NGS)

Capillary electrophoresis-based analysis does not reflect the exact allele number variation at the STR loci due to the non-availability of the data on sequence variation in the repeat region and the SNPs in flanking regions. Herein, this study reports the length-based and sequence-based allelic data of 138 central Indian individuals at 31 autosomal STR loci by NGS. The sequence data at each allele was compared to the reference hg19 sequence. The length-based allelic results were found in concordance with the CE-based results. 20 out of 31 autosomal STR loci showed an increase in the number of alleles by the presence of sequence variation and/or SNPs in the flanking regions. The highest gain in the heterozygosity and allele numbers was observed in D5S2800, D1S1656, D16S539, D5S818, and vWA. rs25768 (A/G) at D5S818 was found to be the most frequent SNP in the studied population. Allele no. 15 of D3S1358, allele no. 19 of D2S1338, and allele no. 22 of D12S391 showed 5 isoalleles each with the same size and with different intervening sequences. Length-based determination of the alleles showed Penta E to be the most useful marker in the central Indian population among 31 STRs studied; however, sequence-based analysis advocated D2S1338 to be the most useful marker in terms of various forensic parameters. Population genetics analysis showed a shared genetic ancestry of the studied population with other Indian populations. This first-ever study to the best of our knowledge on sequence-based STR analysis in the central Indian population is expected to prove the use of NGS in forensic case-work and in forensic DNA laboratories.

Effect of Promoter Region Sequence Variations in Relation to Antibiotic Resistance of Methicillin- Resistant Staphylococcus Aureus

Multidrug resistant bacterial strains are one of the concerns of healthcare sector. Among these, Methicillin- resistant Staphylococcus aureus (MRSA) is one of the major hospitals acquired multidrug resistant strains. MRSA shows it is resistant against methicillin and other penicillin like antibiotics by producing PBP2a (penicillin binding protein 2a) which is encoded by its mecA gene. In this process mecR1 and mecI gene act as regulator of mecA gene. The aim of this study is to identify sequence variance located in mecA, mecR1 and mecI promoter region and the effect of those changes in relation to pathogenicity of these MRSA strains. In this current research, work wound infections samples were collected from 90 patients who were infected during post-surgical mamagement. Samples were collected from Rajshahi Medical college hospital, Bangladesh. In antibiotic sensitivity test, MRSA was found 100% sensitive against only Choramphenicol and Bacitracin. It also shows partial resistance against Amikacin, Impenem, Doxycycline, Gentamycin and Neomycin. In HRM, analysis six types of genotypes (TT, AA, CC, GG, GA and CT) were identified in mecA, mecR1 and mecI genes. Two types of mutation (T>C and G>A) were found in current study. This HRM analysis was further correlate with antibiotic sensitivity test in terms of antibiotic resistance and sequence variance.

Immunotherapeutic Targeting of Mesothelin Positive Pediatric AML Using Bispecific T Cell Engaging Antibodies.

Simple SummaryImmunotherapy development in pediatric AML has been slow due to the paucity of validated AML-specific targets. We recently identified mesothelin (MSLN) as a therapeutic target in pediatric AML. Mice receiving T cell engaging bispecific antibodies (BsAbs) targeting MSLN and CD3 achieved complete remission and durable responses in two MSLN-positive patient-derived xenograft (PDX) models. This is a first report showing MSLN-targeting BsAbs are a viable immunotherapy for MSLN-positive pediatric AML.Advances in the treatment of pediatric AML have been modest over the past four decades. Despite maximally intensive therapy, approximately 40% of patients will relapse. Novel targeted therapies are needed to improve outcomes. We identified mesothelin (MSLN), a well-validated target overexpressed in some adult malignancies, to be highly expressed on the leukemic cell surface in a subset of pediatric AML patients. The lack of expression on normal bone marrow cells makes MSLN a viable target for immunotherapies such as T-cell engaging bispecific antibodies (BsAbs) that combine two distinct antibody-variable regions into a single molecule targeting a cancer-specific antigen and the T-cell co-receptor CD3. Using antibody single-chain variable region (scFv) sequences derived from amatuximab-recognizing MSLN, and from either blinatumomab or AMG330 targeting CD3, we engineered and expressed two MSLN/CD3-targeting BsAbs: MSLNAMA-CD3L2K and MSLNAMA-CD3AMG, respectively. Both BsAbs promoted T-cell activation and reduced leukemic burden in MV4;11:MSLN xenografted mice, but not in those transplanted with MSLN-negative parental MV4;11 cells. MSLNAMA-CD3AMG induced complete remission in NTPL-146 and DF-5 patient-derived xenograft models. These data validate the in vivo efficacy and specificity of MSLN-targeting BsAbs. Because prior MSLN-directed therapies appeared safe in humans, MSLN-targeting BsAbs could be ideal immunotherapies for MSLN-positive pediatric AML patients.

First report of mitochondrial COI in foraminifera and implications for DNA barcoding

Foraminifera are a species-rich phylum of rhizarian protists that are highly abundant in many marine environments and play a major role in global carbon cycling. Species recognition in Foraminifera is mainly based on morphological characters and nuclear 18S ribosomal RNA barcoding. The 18S rRNA contains variable sequence regions that allow for the identification of most foraminiferal species. Still, some species show limited variability, while others contain high levels of intragenomic polymorphisms, thereby complicating species identification. The use of additional, easily obtainable molecular markers other than 18S rRNA will enable more detailed investigation of evolutionary history, population genetics and speciation in Foraminifera. Here we present the first mitochondrial cytochrome c oxidase subunit 1 (COI) gene sequences (“barcodes”) of Foraminifera. We applied shotgun sequencing to single foraminiferal specimens, assembled COI, and developed primers that allow amplification of COI in a wide range of foraminiferal species. We obtained COI sequences of 49 specimens from 17 species from the orders Rotaliida and Miliolida. Phylogenetic analysis showed that the COI tree is largely congruent with previously published 18S rRNA phylogenies. Furthermore, species delimitation with ASAP and ABGD algorithms showed that foraminiferal species can be identified based on COI barcodes.

Single B Cell Immunoglobulin Sequencing Identifies Distinct Features of Monoclonal Antibodies in Patients with Immmune Thrombotic Thrombocytoepenic Purpura

Introduction. Immune thrombotic thrombocytopenic purpura (iTTP) is a potentially fatal blood disorder, resulting from autoantibodies against ADAMTS13, a plasma metalloprotease that cleaves von Willebrand factor. However, the structural feature, binding epitope, and the mechanism of action of these autoantibodies in patients with acute iTTP are not fully understood.Methods. To further understand the pathogenesis of iTTP, single B cell immunoglobulin (Ig) sequencing using 10xChromium in 4 patients experiencing an acute episode of iTTP was performed; the expression and preliminary functional characterizations of selected clones were also carried out.Results. Approximately 2,631 viable and fluoresceinated ADAMTS13 labeled B cells (e.g., 7AAD -CD19 +CD20 +ADAMTS13 +) were sorted out from peripheral blood mononuclear cells of four patients with acute iTTP. These enriched ADAMTS13 antibody-producing B cells were then used for single cell analysis using 10xGenomics 5'-VDJ kit following the manufacturer's instruction. The single-cell gene expression libraries and VDJ libraries were constructed and sequenced by Hiseq at 20,000 reads/cell for gene expression and 5,000 reads/cell for VDJ sequences. Sequencing FASTQ files were mapped and counted by running through the Cell Ranger pipeline, and the final data were then further analyzed by the Loupe browser.We showed for the first time that the most frequent VJ combinations in the anti-ADAMTS13 IgG were: IGHV4-39:ILGJ4, IGHV3-48:ILGJ4, IGLV1-44:ILGLJ2, GLV5-45:ILGLJ3, IGLV2-14:ILGJ2, and IGLV3-21:ILGJ3 as shown in Figure 1. Of the top ten clones, the most frequently observed CDR3 (complementarity-determining region-3) sequences of these antibodies were CARDQLGISETQGSDLW on the heavy chain and CVIWHNSAWVF on the light chain as shown in Figure 2 and Table 1.The variable region sequences from the heavy and the light chains of Ig molecules were cloned into a human IgHG1 and a human IgL vector, respectively, which was then cotransfected in HEK293 cells. Western blotting, ELISA, immunoprecipitation, and functional assays were used to determine the expression and the function of human monoclonal IgG antibodies. Our preliminary results demonstrated the human monoclonal IgG antibodies bound and/or inhibited plasma ADAMTS13 activity.Conclusions. We conclude that there is clonal expansion of ADAMTS13 antibody producing B cells in acute iTTP and the cloned human monoclonal antibodies using the single B cell sequencing approach are functional. Our ongoing analysis on the structural and functional relationship of a large number of isolated human monoclonal antibodies may shed new light on the pathogenesis of iTTP. These antibodies may be useful to explore structural elements required for allosteric regulation of ADAMTS13 activity. [Display omitted] DisclosuresZheng: AJMC: Honoraria; Clotsolution: Other: Co-founder; Takeda: Consultancy, Honoraria; Sanofi-Genzyme: Honoraria, Speakers Bureau; Alexion: Speakers Bureau.

T Cell Engaging Bispecific Antibodies Produce Durable Response in Mesothelin-Positive Patient-Derived Xenograft Models of Pediatric AML

BackgroundImmunotherapy development in pediatric AML is lagging because of dearth of validated AML-specific targets. We showed recently that mesothelin (MSLN) is highly expressed on the leukemia cell surface in a subset of pediatric AML patients, and validated MSLN as a therapeutic target using antibody-drug conjugates directed against MSLN (Kaeding et al., Blood Adv, 5:2350-2361, 2021). Antibody single-chain variable region (scFv) sequences derived from amatuximab recognizing MSLN and from either blinatumomab or AMG330 targeting CD3 were used to engineer and express two MSLN/CD3-targeting BsAbs: MSLN AMA-CD3 L2K and MSLN AMA-CD3 AMG respectively. Both these antibodies demonstrated anti-leukemic activity in mice engrafted with MV4;11 cells engineered to overexpress MSLN, while they failed to show any effect in mice bearing MV4;11 cells without MSLN, confirming that these antibodies specifically targeted MSLN (Gopalakrishnapillai et al., Blood, 134:3925, 2019).MethodsMSLN cell surface expression was quantitated using BD Quantibrite PE Phycoreythrin Fluorescence Quantitation kit. 3x10 6 NTPL-146 cells were injected in NSG-B2m mice and 2x10 6 DF-5 cells were injected in NSG-SGM3 mice via the tail vein. Mice were randomly assigned to treatment groups when human cells were detectable in blood. The percentage of human chimerism in mouse peripheral blood was evaluated weekly by flow cytometry. Bipsecific antibodies were administered ip at 3 mg/Kg daily for six days. Human peripheral blood pan T cells from StemCell Technologies were injected iv (3x10 6 cells per mouse) to act as effector cells. Chemotherapy (DA) consisted of 3 doses of 1.5 mg/kg daunorubicin iv and 5 doses of 50 mg/kg cytarabine ip. Mice were monitored daily and euthanized when any of the experimental endpoints were met.ResultsIn this study, we evaluated the efficacy of two bispecific antibodies in two distinct patient-derived xenograft models of pediatric AML with endogenous MSLN expression quantitated at 6617 and 7414 MSLN antibodies bound per cell in NTPL-146 and DF-5 respectively. A Kaplan-Meier survival plot based on the time when each mouse reached experimental endpoint showed that 6/8 NTPL-146 engrafted mice receiving MSLN AMA-CD3 AMG and T cells survived disease-free until the end of the experiment at day 520 whereas all the mice in control groups had died by day 138 (Fig. 1a). The AML bone marrow load of MSLN AMA-CD3 AMG-treated mice was < 0.01% at 520 days, whereas the bone marrow load of mice from the other treatment groups was greater than 90% at the time of death, consistent with marrow failure as the proximal cause of death (Fig. 1b). These data show that treatment with MSLN AMA-CD3 AMG is curative in the vast majority of mice.Treatment with MSLN AMA-CD3 L2K and T cells increased the median survival by 109.5 days compared to untreated mice while treatment with MSLN AMA-CD3 AMG showed a complete remission in 6/8 mice (**P<0.001). Thus, the survival benefit of both MSLN AMA-CD3 L2K and MSLN AMA-CD3 AMG treated mice greatly exceeded the allogeneic effect T cells alone, which only showed a 22-day improvement in median survival. Either MSLN AMA-CD3 L2K or MSLN AMA-CD3 AMG treatment alone showed no improvement in survival compared to untreated mice.The efficacy of the potent BsAb MSLN AMA-CD3 AMG in comparison with chemotherapy (DA) was evaluated in DF-5. DA treatment, like T cell infusion, did not significantly change median survival compared to untreated mice, while BsAb MSLN AMA-CD3 AMG in the presence of human T cells was curative (Fig. 1c, **P<0.005). Mice treated with only T cells showed a rapid rise in AML cell percentage in peripheral blood until day 34 when they succumbed to disease, whereas the mice receiving T cells with BsAb had AML cell burden of less than 1% (Fig. 1d, *P<0.05). Mice treated with BsAbs showed greater expansion of allogeneic human T cells compared to mice receiving T cells alone (Fig. 1e, *P<0.05). Eight weeks post treatment initiation, the surviving mice were euthanized and AML burden in the bone marrow was evaluated. In contrast with mice receiving T cells alone, mice treated with T cells and BsAbs had no detectable AML cells (Fig. 1f, **P<0.005).ConclusionThese data validate the efficacy of MSLN-targeting BsAbs in PDX models with endogenous MSLN expression. Because prior MSLN-directed therapies appeared safe in humans, MSLN-targeting BsAbs could be ideal immunotherapies for MSLN-positive pediatric AML patients. [Display omitted] DisclosuresGopalakrishnapillai: Geron: Research Funding. Correnti: Link Immunotherapeutics: Current Employment. Kaeding: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Barwe: Prelude Therapeutics: Research Funding.

The Global Soil Mycobiome consortium dataset for boosting fungal diversity research

Fungi are highly important biotic components of terrestrial ecosystems, but we still have a very limited understanding about their diversity and distribution. This data article releases a global soil fungal dataset of the Global Soil Mycobiome consortium (GSMc) to boost further research in fungal diversity, biogeography and macroecology. The dataset comprises 722,682 fungal operational taxonomic units (OTUs) derived from PacBio sequencing of full-length ITS and 18S-V9 variable regions from 3200 plots in 108 countries on all continents. The plots are supplied with geographical and edaphic metadata. The OTUs are taxonomically and functionally assigned to guilds and other functional groups. The entire dataset has been corrected by excluding chimeras, index-switch artefacts and potential contamination. The dataset is more inclusive in terms of geographical breadth and phylogenetic diversity of fungi than previously published data. The GSMc dataset is available over the PlutoF repository.

Longitudinal TprK profiling of in vivo and in vitro-propagated Treponema pallidum subsp. pallidum reveals accumulation of antigenic variants in absence of immune pressure.

Immune evasion by Treponema pallidum subspecies pallidum (T. pallidum) has been attributed to antigenic variation of its putative outer-membrane protein TprK. In TprK, amino acid diversity is confined to seven variable (V) regions, and generation of sequence diversity within the V regions occurs via a non-reciprocal segmental gene conversion mechanism where donor cassettes recombine into the tprK expression site. Although previous studies have shown the significant role of immune selection in driving accumulation of TprK variants, the contribution of baseline gene conversion activity to variant diversity is less clear. Here, combining longitudinal tprK deep sequencing of near clonal Chicago C from immunocompetent and immunosuppressed rabbits along with the newly developed in vitro cultivation system for T. pallidum, we directly characterized TprK alleles in the presence and absence of immune selection. Our data confirm significantly greater sequence diversity over time within the V6 region during syphilis infection in immunocompetent rabbits compared to immunosuppressed rabbits, consistent with previous studies on the role of TprK in evasion of the host immune response. Compared to strains grown in immunocompetent rabbits, strains passaged in vitro displayed low level changes in allele frequencies of TprK variable region sequences similar to that of strains passaged in immunosuppressed rabbits. Notably, we found significantly increased rates of V6 allele generation relative to other variable regions in in vitro cultivated T, pallidum strains, illustrating that the diversity within these hypervariable regions occurs in the complete absence of immune selection. Together, our results demonstrate antigenic variation in T. pallidum can be studied in vitro and occurs even in the complete absence of immune pressure, allowing the T. pallidum population to continuously evade the immune system of the infected host.

Combining Fluorescent Cell Sorting and Single B Cell Amplification to Screen the Monoclonal Antibody Gene against Human Glypican-1 in Pancreatic Cancer.

In this report, one novel method has been developed to screen the monoclonal antibody against human pancreatic cancer biomarker glypican-1 (GPC1) through the combination of fluorescent cell sorting and single B cell amplification. GPC1-positive B cells were sorted out from the peripheral blood mononuclear cells (PBMCs) by fluorescent cell sorting after the GPC1 immunization to the New Zealand white rabbit. Then, total RNA was extracted and reversely transcribed into cDNA, which was used as the template, and the variable region sequences of both heavy and light chains were amplified from the same B cell. Next, their recombinant antibody was expressed and purified from the human 293T cell after the antibody gene amplification and expression vector construction. The enzyme-linked immunosorbent assay (ELISA) and flow cytometry assays were used to determine the antibody affinity. The antibody named GPC-12 that we screened and obtained was proven to have natural heavy-light chain pairing information, and it was highly specific to the GPC1 antigen, and the affinity could reach 1 × 10−7 M. Overall, an effective and novel method has been successfully developed to screen the antibody by combining the fluorescent cell sorting and single-cell amplifying technologies, which was proved to be workable in our setting.

Determination of variable region sequences from hybridoma immunoglobulins that target Mycobacterium tuberculosis virulence factors.

Mycobacterium tuberculosis (Mtb) infects one-quarter of the world's population. Mtb and HIV coinfections enhance the comorbidity of tuberculosis (TB) and AIDS, accounting for one-third of all AIDS-associated mortalities. Humoral antibody to Mtb correlates with TB susceptibility, and engineering of Mtb antibodies may lead to new diagnostics and therapeutics. The characterization and validation of functional immunoglobulin (Ig) variable chain (IgV) sequences provide a necessary first step towards developing therapeutic antibodies against pathogens. The virulence-associated Mtb antigens SodA (Superoxide Dismutase), KatG (Catalase), PhoS1/PstS1 (regulatory factor), and GroES (heat shock protein) are potential therapeutic targets but lacked IgV sequence characterization. Putative IgV sequences were identified from the mRNA of hybridomas targeting these antigens and isotype-switched into a common immunoglobulin fragment crystallizable region (Fc region) backbone, subclass IgG2aκ. Antibodies were validated by demonstrating recombinant Ig assembly and secretion, followed by the determination of antigen-binding specificity using ELISA and immunoblot assay.

Multiomic Approach to Analyze Infant Gut Microbiota: Experimental and Analytical Method Optimization.

Background: The human intestinal microbiome plays a central role in overall health status, especially in early life stages. 16S rRNA amplicon sequencing is used to profile its taxonomic composition; however, multiomic approaches have been proposed as the most accurate methods for study of the complexity of the gut microbiota. In this study, we propose an optimized method for bacterial diversity analysis that we validated and complemented with metabolomics by analyzing fecal samples. Methods: Forty-eight different analytical combinations regarding (1) 16S rRNA variable region sequencing, (2) a feature selection approach, and (3) taxonomy assignment methods were tested. A total of 18 infant fecal samples grouped depending on the type of feeding were analyzed by the proposed 16S rRNA workflow and by metabolomic analysis. Results: The results showed that the sole use of V4 region sequencing with ASV identification and VSEARCH for taxonomy assignment produced the most accurate results. The application of this workflow showed clear differences between fecal samples according to the type of feeding, which correlated with changes in the fecal metabolic profile. Conclusion: A multiomic approach using real fecal samples from 18 infants with different types of feeding demonstrated the effectiveness of the proposed 16S rRNA-amplicon sequencing workflow.

Exploring the sequence features determining amyloidosis in human antibody light chains

The light chain (AL) amyloidosis is caused by the aggregation of light chain of antibodies into amyloid fibrils. There are plenty of computational resources available for the prediction of short aggregation-prone regions within proteins. However, it is still a challenging task to predict the amyloidogenic nature of the whole protein using sequence/structure information. In the case of antibody light chains, common architecture and known binding sites can provide vital information for the prediction of amyloidogenicity at physiological conditions. Here, in this work, we have compared classical sequence-based, aggregation-related features (such as hydrophobicity, presence of gatekeeper residues, disorderness, β-propensity, etc.) calculated for the CDR, FR or VL regions of amyloidogenic and non-amyloidogenic antibody light chains and implemented the insights gained in a machine learning-based webserver called “VLAmY-Pred” (https://web.iitm.ac.in/bioinfo2/vlamy-pred/). The model shows prediction accuracy of 79.7% (sensitivity: 78.7% and specificity: 79.9%) with a ROC value of 0.88 on a dataset of 1828 variable region sequences of the antibody light chains. This model will be helpful towards improved prognosis for patients that may likely suffer from diseases caused by light chain amyloidosis, understanding origins of aggregation in antibody-based biotherapeutics, large-scale in-silico analysis of antibody sequences generated by next generation sequencing, and finally towards rational engineering of aggregation resistant antibodies.

Detection of Minimal Residual Disease in Autograft Is Prognostic of Survival Following Stem Cell Transplant in Multiple Myeloma

▪IntroductionMultiple myeloma (MM) is an incurable disease with a variety of treatment options. In those capable of handling aggressive treatment, often high dose chemotherapy (HDT) and autologous stem cell transplant (ASCT) are used in first remission to improve progression free (PFS) and overall survival (OS). It has been theorized and demonstrated that propagation of the disease following HDT/ASCT is partially due to the return of the disease in the autograft. What is less clear is whether or not a threshold of disease in the autograft correlates with a higher risk of earlier recurrence. This study hypothesized that such a threshold exists.MethodsMM patients in this single institution study were prospectively observed following initial diagnosis and consent from 1998 to 2014. Basic patient demographics collected included age, sex, International Staging (ISS), type of induction therapy and response. Patients were observed for primarily time to progression following HDT/ASCT. OS was a secondary endpoint. At diagnosis, a bone marrow aspirate aliquot was used to create patient-specific primers for detection of their unique MM variable region sequence (VDJ). These primers were subsequently used during quantitative PCR on each patient's autograft at time of collection and reinfusion (i.e. post thawing). Cut-point analysis was used to determine a threshold for which detection of minimal residual disease in the autograft may translate into poorer survival. Cox proportional hazards multivariate analysis took into consideration age at transplant, sex, ISS, type of induction and response to induction.ResultsBetween 1998 and 2014, 89 patients were accrued to study. 77 patients are included here for analysis. 12 patients were not included due to an inability to properly sequence patient specific primers (10 of which harbored light chain disease). Basic demographics included median age at diagnosis of 58 years (range 36-71), 61 male, and a majority of patients having ISS stage 3 (n=31; n=24 for ISS 2). Given the time points involved, induction therapy was variable including bortezomib-containing (n=31), doxorubicin-based (n=33), melphalan (n=4), and dexamethasone (n=9). A threshold of detection at ≥ 1 in 100,000 circulating white blood cells in the autograft was associated with poorer PFS following HDT/ASCT (median 1.7 vs 2.6 years, hazard ratio [HR] 1.78, p=0.03). A trend to poorer OS was also seen in this group (median 5.6 vs. 15.8 years, HR 2.65, p=0.08). This detriment remained significant when taking into consideration age, sex and ISS staging. Inclusion of induction and response was not done due to the heterogeneity of treatment regimens and will require further study. Further, the aggregate number of malignant cells in the reinfused autograft was the same as the number prior to cryopreservation - indicating that essentially all malignant cells in the autograft survive the cryopreservation process and thawing.ConclusionsThis is the first study of which we are aware that demonstrates an association between the degree of autograft contamination with MM cells and survival. Whether this phenomenon continues to hold in the current era where more focused treatments continue to enter pre-ASCT remains to be seen. However, as HDT/ASCT remain standard treatment, our results may point toward a renewed focus on autograft purging as potential means of improving at least PFS. This study remains open and continues to accrue patients. DisclosuresBelch:Amgen, Celgene, Takeda: Honoraria. Venner:Celgene, Janssen: Research Funding; Celgene, Janssen, Takeda, Amgen, Merck: Honoraria.

Identification of genes for variable regions of immunoglobulins that recognize sialylated glycans

We previously reported an antibody (clone ID: FR9, IgM-κ) that recognizes the sialyl oligosaccharide Neu5Acα2,6Galβ1,4GlcNAc as an epitope on glycoproteins and glycolipids. In the present study, we developed an antibody (clone ID: AFR45, IgM-κ) that recognizes Neu5Acα2,3Galβ1,4GlcNAc/Glc as an epitope on glycoproteins and glycolipids and compared the nucleotide and amino acid sequences of the immunoglobulin gene variable regions with those of FR9. The heavy chain variable (VH) regions of FR9 and AFR45 were encoded by different VH gene segments, each of which was composed of a characteristic D gene segment. The major differences between VH genes encoding various antibodies deposited in public databases and FR9 and AFR45 were identified in the D gene segment, indicating that D genes play a critical role in determining the epitope specificity of these antibodies. Surprisingly, although FR9 and AFR45 were obtained independently from different mice immunized with different immunogens, the light chain variable (VL) region nucleotide sequences were identical. The VL gene consisted of Igkv4-57 and Igkj4 gene segments (Igkv4-57j4), the sequences of which were identical to VL genes for a number of antibodies against meningococcal group C capsular polysaccharide deposited in public databases. As this polysaccharide is a sialic acid homopolymer, these results indicate that Igkv4-57j4 encodes a VL common to immunoglobulins that recognize sialylated glycans.

Microbiome recovery in adult females with uncomplicated urinary tract infections in a randomised phase 2A trial of the novel antibiotic gepotidacin (GSK2140944)

BackgroundWith increasing concerns about the impact of frequent antibiotic usage on the human microbiome, it is important to characterize the potential for such effects in early antibiotic drug development clinical trials. In a randomised Phase 2a clinical trial study that evaluated the pharmacokinetics of repeated oral doses of gepotidacin, a first-in-chemical-class triazaacenaphthylene antibiotic with a distinct mechanism of action, in adult females with uncomplicated urinary tract infections for gepotidacin (GSK2140944) we evaluated the potential changes in microbiome composition across multiple time points and body-sites (ClinicalTrials.gov: NCT03568942).ResultsSamples of gastrointestinal tract (GIT), pharyngeal cavity and vaginal microbiota were collected with consent from 22 patients at three time points relative to the gepotidacin dosing regimen; Day 1 (pre-dose), Day 5 (end of dosing) and Follow-up (Day 28 ± 3 days). Microbiota composition was determined by DNA sequencing of 16S rRNA gene variable region 4 amplicons. By Day 5, significant changes were observed in the microbiome diversity relative to pre-dose across the tested body-sites. However, by the Follow-up visit, microbiome diversity changes were reverted to compositions comparable to Day 1. The greatest range of microbiome changes by body-site were GIT followed by the pharyngeal cavity then vagina. In Follow-up visit samples we found no statistically significant occurrences of pathogenic taxa.ConclusionOur findings suggest that gepotidacin alteration of the human microbiome after 5 days of dosing is temporary and rebound to pre-dosing states is evident within the first month post-treatment. We recommend that future antibiotic drug trials include similar exploratory investigations into the duration and context of microbiome modification and recovery.Trial registrationNCT03568942. Registered 26 June 2018.

Evaluation of Mycobacterium tuberculosis ripA gene to detect antibiotic resistance

Mycobacterium tuberculosis infection has remained a public health threat in Indonesia. This infection is complicated by the antibiotic-resistant of M. tuberculosis strains. The common essential in resistance comes from a mutation in genomic DNA. ripA gene is one of the regions critical in the replication and persistence of M. tuberculosis in their resistance. This gene has responsible for cell wall polymer peptidoglycan. The objective of this research was to evaluate the ripA gene in antibiotic resistance. This is to investigate and compare the ripA gene sequences of M. tuberculosis at an unprecedented rate. A total of five specimens of M. tuberculosis were isolated from tuberculosis patients with rifampicin resistance. The ripA gene from M. tuberculosis was isolated and amplified using a design primer for ripA N-terminal domain of peptidoglycan hydrolase. Further, ripA gene was analyzed using the Sanger method sequencing. The data were analyzed and compared using M. tuberculosis H37Rv from the National Center for Biotechnology Information (NCBI). In addition, the sequence was analyzed with multiple sequence alignment (Clustal IW) to identify the mutation. Our result suggests that the evaluation of genes in M. tuberculosis isolates revealed sequence variation in ripA regions (Ala701Gly). Understanding these mutations implies an evaluation of antibiotic-resistant. Furthermore, this information implies local diagnostic and treatment guidelines to cell-wall targeting antibiotics.

Antibody Variable Sequences Have a Pronounced Effect on Cellular Uptake, Transport and Plasma Half-Life

Monoclonal IgG antibodies are the fastest growing class of biologics, but large differences exist in their plasma half-life in humans. Thus, to design IgG antibodies with favourable pharmacokinetics, it is crucial to identify the determinants of such differences. Here, we demonstrate that the variable region sequences of IgG antibodies greatly affect cellular uptake and subsequent recycling and rescue from intracellular degradation by endothelial cells. When the variable sequences are masked by the cognate antigen, it influences both their transport behaviour and binding to the neonatal Fc receptor (FcRn), a key regulator of IgG plasma half-life. Furthermore, we show how charge patch differences in the variable domains modulate both binding and transport properties, and that a short plasma half-life, due to unfavourable charge patches, may partly be overcome by Fc-engineering for improved FcRn binding.

Functional and in silico Characterization of Neutralizing Interactions Between Antibodies and the Foot-and-Mouth Disease Virus Immunodominant Antigenic Site.

Molecular knowledge of virus–antibody interactions is essential for the development of better vaccines and for a timely assessment of the spread and severity of epidemics. For foot-and-mouth disease virus (FMDV) research, in particular, computational methods for antigen–antibody (Ag–Ab) interaction, and cross-antigenicity characterization and prediction are critical to design engineered vaccines with robust, long-lasting, and wider response against different strains. We integrated existing structural modeling and prediction algorithms to study the surface properties of FMDV Ags and Abs and their interaction. First, we explored four modeling and two Ag–Ab docking methods and implemented a computational pipeline based on a reference Ag–Ab structure for FMDV of serotype C, to be used as a source protocol for the study of unknown interaction pairs of Ag–Ab. Next, we obtained the variable region sequence of two monoclonal IgM and IgG antibodies that recognize and neutralize antigenic site A (AgSA) epitopes from South America serotype A FMDV and developed two peptide ELISAs for their fine epitope mapping. Then, we applied the previous Ag–Ab molecular structure modeling and docking protocol further scored by functional peptide ELISA data. This work highlights a possible different behavior in the immune response of IgG and IgM Ab isotypes. The present method yielded reliable Ab models with differential paratopes and Ag interaction topologies in concordance with their isotype classes. Moreover, it demonstrates the applicability of computational prediction techniques to the interaction phenomena between the FMDV immunodominant AgSA and Abs, and points out their potential utility as a metric for virus-related, massive Ab repertoire analysis or as a starting point for recombinant vaccine design.

Light chain skewing in autoantibodies and B-cell receptors of the citrullinated antigen-binding B-cell response in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting 1% of the world population. RA is associated with the presence of autoantibodies, of which anti-citrullinated protein antibodies (ACPA) are most prominent. ACPA are produced by citrullinated antigen-binding B cells that have presumably survived tolerance checkpoints. So far, it is unclear how and when such autoreactive B cells emerge. Light chain (LC) rearrangement and mutation rates can be informative with regard to selection steps during B-cell development. Therefore, we studied LC characteristics of ACPA-expressing B cells and secreted ACPA with the aim to better understand the development of this disease-specific, autoreactive B-cell response. Paired ACPA-IgG and ACPA-depleted IgG were isolated from serum (n = 87) and synovial fluid (SF, n = 21) of patients with established RA. We determined the LC composition for each fraction by ELISA using kappa(Igκ)- and lambda(Igλ) LC-specific antibodies. Cellular LC expression was determined using flow cytometry. In addition, we used a B-cell receptor (BCR)-specific PCR to obtain LC variable region sequences of citrullinated antigen- and tetanus toxoid (TT)-binding B cells. In serum, we observed an increased frequency of lambda LC in ACPA-IgG (1.64:1) compared to control IgG (2.03:1) and to the κ/λ ratio reported for healthy individuals (2:1). A similar trend towards higher frequencies of lambda LCs was observed for ACPA-IgG in SF (1.84:1). Additionally, the percentage of Igλ-expressing B cells was higher for citrullinated antigen-binding B cells (51%) compared to TT-specific (43%) and total CD19+CD20+ B cells (36%). Moreover, an increased Igλ percentage was observed in BCR-sequences derived from ACPA-expressing (49%) compared to TT-specific B cells (34%). Taken together, we report an enhanced frequency of lambda LCs in the secreted ACPA-IgG repertoire and, on the cellular level, in BCR sequences of ACPA-expressing B cells compared to control. This skewing in the autoreactive B-cell repertoire could reflect a process of active selection.