Longitudinal Dynamics of Human B-Cell Response at the Single-Cell Level in Response to Tdap Vaccination

To mount an adequate immune response against pathogens, stepwise mutation and selection processes are crucial functions of the adaptive immune system. To better characterize a successful vaccination response, we performed longitudinal (days 0, 5, 7, 10, and 14 after Boostrix vaccination) analysis of the single-cell transcriptome as well as the B-cell receptor (BCR) repertoire (scBCR-rep) in plasma cells of an immunized donor and compared it with baseline B-cell characteristics as well as flow cytometry findings. Based on the flow cytometry knowledge and literature findings, we discriminated individual B-cell subsets in the transcriptomics data and traced over-time maturation of plasmablasts/plasma cells (PB/PCs) and identified the pathways associated with the plasma cell maturation. We observed that the repertoire in PB/PCs differed from the baseline B-cell repertoire e.g., regarding expansion of unique clones in post-vaccination visits, high usage of IGHG1 in expanded clones, increased class-switching events post-vaccination represented by clonotypes spanning multiple IGHC classes and positive selection of CDR3 sequences over time. Importantly, the Variable gene family-based clustering of BCRs represented a similar measure as the gene-based clustering, but certainly improved the clustering of BCRs, as BCRs from duplicated Variable gene families could be clustered together. Finally, we developed a query tool to dissect the immune response to the components of the Boostrix vaccine. Using this tool, we could identify the BCRs related to anti-tetanus and anti-pertussis toxoid BCRs. Collectively, we developed a bioinformatic workflow which allows description of the key features of an ongoing (longitudinal) immune response, such as activation of PB/PCs, Ig class switching, somatic hypermutation, and clonal expansion, all of which are hallmarks of antigen exposure, followed by mutation & selection processes.

Cotton Leafroll Dwarf Virus US Genomes Comprise Divergent Subpopulations and Harbor Extensive Variability

Cotton leafroll dwarf virus (CLRDV) was first reported in the United States (US) in 2017 from cotton plants in Alabama (AL) and has become widespread in cotton-growing states of the southern US. To investigate the genomic variability among CLRDV isolates in the US, complete genomes of the virus were obtained from infected cotton plants displaying mild to severe symptoms from AL, Florida, and Texas. Eight CLRDV genomes were determined, ranging in size from 5865 to 5867 bp, and shared highest nucleotide identity with other CLRDV isolates in the US, at 95.9–98.7%. Open reading frame (ORF) 0, encoding the P0 silencing suppressor, was the most variable gene, sharing 88.5–99.6% and 81.2–89.3% amino acid similarity with CLRDV isolates reported in cotton growing states in the US and in Argentina and Brazil in South America, respectively. Based on Bayesian analysis, the complete CLRDV genomes from cotton in the US formed a monophyletic group comprising three relatively divergent sister clades, whereas CLRDV genotypes from South America clustered as closely related sister-groups, separate from US isolates, patterns reminiscent of phylogeographical structuring. The CLRDV isolates exhibited a complex pattern of recombination, with most breakpoints evident in ORFs 2 and 3, and ORF5. Despite extensive nucleotide diversity among all available CLRDV genomes, purifying selection (dN/dS < 1) was implicated as the primary selective force acting on viral protein evolution.

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

Background Recent 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. Methods Our 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. Results In 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). Conclusion Certain 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

SpatialDE2: Fast and localized variance component analysis of spatial transcriptomics

Spatial transcriptomics is now a mature technology, allowing to assay gene expression changes in the histological context of complex tissues. A canonical analysis workflow starts with the identification of tissue zones that share similar expression profiles, followed by the detection of highly variable or spatially variable genes. Rapid increases in the scale and complexity of spatial transcriptomic datasets demand that these analysis steps are conducted in a consistent and integrated manner, a requirement that is not met by current methods. To address this, we here present SpatialDE2, which unifies the mapping of tissue zones and spatial variable gene detection as integrated software framework, while at the same time advancing current algorithms for both of these steps. Formulated in a Bayesian framework, the model accounts for the Poisson count noise, while simultaneously offering superior computational speed compared to previous methods. We validate SpatialDE2 using simulated data and illustrate its utility in the context of two real-world applications to the spatial transcriptomics profiles of the mouse brain and human endometrium.

Longitudinal dynamics of human B-cell response at single-cell level in response to Tdap vaccination

Adaptation of the immune system to mount an adequate immune response against pathogens is a crucial function of the adaptive immune system. To better characterize a successful vaccination response, we performed longitudinal (days 0, 5, 7, 10, and 14 after Boostrix vaccination) analysis of the single-cell transcriptome as well as the B-cell receptor (BCR) repertoire (scBCR-rep) in plasma cells of an immunized donor and compared it with baseline B cell characteristics as well as flow cytometry findings. Based on the flow cytometry knowledge and literature findings, we discriminated individual B cell subsets in the transcriptomics data and traced over-time maturation of plasmablasts/plasma cells (PB/PCs) and identified the pathways associated with the plasma cell maturation. We observed that the repertoire in PB/PCs differed from the baseline B cell repertoire e.g. regarding the expansion of unique clones in post-vaccination visits, high usage of IGHG1 in expanded clones, increased class switching events post-vaccination represented by clonotypes spanning multiple IGHC classes and positive selection of CDR3 sequences over time. Importantly, the Variable gene family-based clustering of BCRs represented a similar measure as the gene-based clustering, however, certainly improved the clustering of BCRs, as BCRs from duplicated Variable gene families could be clustered together. Finally, we developed a query tool to dissect the immune response to the components of the Boostrix vaccine. Using this tool, we could identify the BCRs related to anti-tetanus and anti-pertussis toxoids. Collectively, we developed a workflow that allows the description of key features of an ongoing immune response, such as activation of PB/PCs, Ig class switching, somatic hypermutation, and clonal expansion, all of which are hallmarks of antigen exposure.

The unmutated common ancestor of Gp41 conformational epitope targeting variable heavy chain 1-02 utilizing antibodies maintains antibody dependent cell cytotoxicity

In studies on monoclonal Abs (mAbs) from long-term non-progressors (LTNPs), our laboratory has previously described highly mutated Abs 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 Abs against HIV, members of these Abs, termed group 76C Abs, did not exhibit broad neutralization.<br />Because of the excessive mutations and use of VH1-02, our goal was to characterize the non-neutralizing functions of Abs of group 76C, to assess targeting of the epitope in various clinical presentations, and to assess the development of these Abs by comparison to their predicted common ancestor. Serum competition assays showed group 76C Abs were enriched in LTNPs, in comparison to VRC-01. Specific group 76C clones 6F5 and 6F11, expressed as recombinant Abs, 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. Competition with group 76C recombinant Ab 6F5 confirms 76Canc binds HIV envelope constructs near the original group C epitope. 76Canc demonstrates comparable ADCC to 6F5 and 6F11 when targeting both clade B and C HIV constructs. The functional capability of Abs utilizing germline VH1-02 has implications for disease control and vaccine development.

Nanopore sequencing approach for immunoglobulin gene analysis in chronic lymphocytic leukemia

The evaluation of the somatic hypermutation of the clonotypic immunoglobulin heavy variable gene has become essential in the therapeutic management in chronic lymphocytic leukemia patients. European Research Initiative on Chronic Lymphocytic Leukemia promotes good practices and standardized approaches to this assay but often they are labor-intensive, technically complex, with limited in scalability. The use of next-generation sequencing in this analysis has been widely tested, showing comparable accuracy and distinct advantages. However, the adoption of the next generation sequencing requires a high sample number (run batching) to be economically convenient, which could lead to a longer turnaround time. Here we present data from nanopore sequencing for the somatic hypermutation evaluation compared to the standard method. Our results show that nanopore sequencing is suitable for immunoglobulin heavy variable gene mutational analysis in terms of sensitivity, accuracy, simplicity of analysis and is less time-consuming. Moreover, our work showed that the development of an appropriate data analysis pipeline could lower the nanopore sequencing error rate attitude.

PEG3 controls lipogenesis through ACLY

Peg3 (Paternally expressed gene 3) is an imprinted gene encoding a DNA-binding protein that is a well-known transcriptional repressor. Previous studies have shown that the mutant phenotypes of Peg3 are associated with the over-expression of genes involved in lipid metabolism. In the current study, we investigated four potential downstream genes of Peg3, which were identified through ChIP-seq data: Acly, Fasn, Idh1, and Hmgcr. In vivo binding of PEG3 to the promoter region of these key genes involved in lipogenesis was subsequently confirmed through individual ChIP experiments. We observed the opposite response of Acly expression levels against the variable gene dosages of Peg3, involving 0x, 1x, and 2x Peg3. This suggests the transcriptional repressor role of Peg3 in the expression levels of Acly. Another set of analyses showed a sex-biased response in the expression levels of Acly, Fasn, and Idh1 against 0x Peg3 with higher levels in female and lower levels in male mammary glands. These results overall highlight that Peg3 may be involved in regulating the expression levels of several key genes in adipogenesis.