Publications

Class II HLA-DRB4 is a predictive biomarker for survival following immunotherapy in metastatic non-small cell lung cancer

Updated on Scientific Reports, 2024

Immune checkpoint inhibitors (ICI) are important treatment options for metastatic non-small cell lung cancer (mNSCLC). However, not all patients benefit from ICIs and can experience immune-related adverse events (irAEs). Limited understanding exists for germline determinants of ICI efficacy and toxicity, but Human Leukocyte Antigen (HLA) genes have emerged as a potential predictive biomarker. We performed HLA typing on 85 patients with mNSCLC, on ICI therapy and analyzed the impact of HLA Class II genotype on progression free survival (PFS), overall survival (OS), and irAEs. Most patients received pembrolizumab (83.5%). HLA-DRB4 genotype was seen in 34/85 (40%) and its presence correlated with improved OS in both univariate (p = 0.022; 26.3 months vs 10.2 months) and multivariate analysis (p = 0.011, HR 0.49, 95% CI [0.29, 0.85]). PFS did not reach significance (univariate, p = 0.12, 8.2 months vs 5.1 months). Eleven patients developed endocrine irAEs. HLA-DRB4 was the predominant genotype among these patients (9/11, 81.8%). Cumulative incidence of endocrine irAEs was higher in patients with HLA-DRB4 (p = 0.0139). Our study is the first to suggest that patients with metastatic NSCLC patients on ICI therapy with HLA-DRB4 genotype experience improved survival outcomes. Patients with HLA-DRB4 had the longest median OS (26.3 months). Additionally, we found a correlation between HLA-DRB4 and the occurrence of endocrine irAEs.

Recommended citation: Jiang, C.Y., Zhao, L., Green, M.D. et al. Class II HLA-DRB4 is a predictive biomarker for survival following immunotherapy in metastatic non-small cell lung cancer. Sci Rep 14, 345 (2024). https://doi.org/10.1038/s41598-023-48546-y https://www.nature.com/articles/s41598-023-48546-y

The first complete genome of the simian malaria parasite Plasmodium brasilianum

Updated on Scientific Reports, 2022

Naturally occurring human infections by zoonotic Plasmodium species have been documented for P. knowlesi, P. cynomolgi, P. simium, P. simiovale, P. inui, P. inui-like, P. coatneyi, and P. brasilianum. Accurate detection of each species is complicated by their morphological similarities with other Plasmodium species. PCR-based assays offer a solution but require prior knowledge of adequate genomic targets that can distinguish the species. While whole genomes have been published for P. knowlesi, P. cynomolgi, P. simium, and P. inui, no complete genome for P. brasilianum has been available. Previously, we reported a draft genome for P. brasilianum, and here we report the completed genome for P. brasilianum. The genome is 31.4 Mb in size and comprises 14 chromosomes, the mitochondrial genome, the apicoplast genome, and 29 unplaced contigs. The chromosomes consist of 98.4% nucleotide sites that are identical to the P. malariae genome, the closest evolutionarily related species hypothesized to be the same species as P. brasilianum, with 41,125 non-synonymous SNPs (0.0722% of genome) identified between the two genomes. Furthermore, P. brasilianum had 4864 (82.1%) genes that share 80% or higher sequence similarity with 4970 (75.5%) P. malariae genes. This was demonstrated by the nearly identical genomic organization and multiple sequence alignments for the merozoite surface proteins msp3 and msp7. We observed a distinction in the repeat lengths of the circumsporozoite protein (CSP) gene sequences between P. brasilianum and P. malariae. Our results demonstrate a 97.3% pairwise identity between the P. brasilianum and the P. malariae genomes. These findings highlight the phylogenetic proximity of these two species, suggesting that P. malariae and P. brasilianum are strains of the same species, but this could not be fully evaluated with only a single genomic sequence for each species.

Recommended citation: Bajic, M., Ravishankar, S., Sheth, M. et al. The first complete genome of the simian malaria parasite Plasmodium brasilianum. Sci Rep 12, 19802 (2022). https://doi.org/10.1038/s41598-022-20706-6 https://www.nature.com/articles/s41598-022-20706-6

Serial Analysis of the T-Cell Receptor β-Chain Repertoire in People Living With HIV Reveals Incomplete Recovery After Long-Term Antiretroviral Therapy

Updated on Frontiers in Immunology, 2022

Long-term antiretroviral therapy (ART) in people living with HIV (PLHIV) is associated with sustained increases in CD4+ T-cell count, but its effect on the peripheral blood T-cell repertoire has not been comprehensively evaluated. In this study, we performed serial profiling of the composition and diversity of the T-cell receptor β-chain (TRB) repertoire in 30 adults with HIV infection before and after the initiation of ART to define its long-term impact on the TRB repertoire. Serially acquired blood samples from 30 adults with HIV infection collected over a mean of 6 years (range, 1-12) years, with 1-4 samples collected before and 2-8 samples collected after the initiation of ART, were available for analysis. TRB repertoires were characterized via high-throughput sequencing of the TRB variable region performed on genomic DNA extracted from unsorted peripheral blood mononuclear cells. Additional laboratory and clinical metadata including serial measurements of HIV viral load and CD4+ T-cell count were available for all individuals in the cohort. A previously published control group of 189 TRB repertoires from peripheral blood samples of adult bone marrow transplant donors was evaluated for comparison. ART initiation in PLHIV was associated with a sustained reduction in viral load and a significant increase in TRB repertoire diversity. However, repertoire diversity in PLHIV remained significantly lower than in the control group even after long-term ART. The composition of TRB repertoires of PLHIV after ART also remained perturbed compared to the control cohort, as evidenced by large persistent private clonal expansions, reduced efficiency in the generation of TRB CDR3 amino acid sequences, and a narrower range of CDR3 lengths. Network analysis revealed an antigen-experienced structure in the TRB repertoire of PLHIV both before and after ART initiation that was quite distinct from the structure of control repertoires, with a slight shift toward a more naïve structure observed after ART initiation. Though we observe significant improvement in TRB repertoire diversity with durable viral suppression in PLHIV on long-term ART, the composition and structure of these repertoires remain significantly perturbed compared to the control cohort of adult bone marrow transplant donors.

Recommended citation: Towlerton AMH, Ravishankar S, Coffey DG, Puronen CE and Warren EH (2022) Serial Analysis of the T-Cell Receptor β-Chain Repertoire in People Living With HIV Reveals Incomplete Recovery After Long-Term Antiretroviral Therapy. Front. Immunol. 13:879190. doi: 10.3389/fimmu.2022.879190 https://www.frontiersin.org/articles/10.3389/fimmu.2022.879190/full

RNA helicase, DDX3X, is actively recruited to sites of DNA damage in live cells

Updated on DNA Repair, 2021

Recent studies have suggested that human RNA helicase, DDX3X, is important for DNA repair, but little is known about the nuclear activity of this protein. In vitro analysis of nuclear DDX3X interactions and localization with DNA damage pointed to a direct role for DDX3X in the DNA damage response. We aimed to investigate whether DDX3X plays a direct role in the DNA damage response in live cells. In order to track nuclear DDX3X, we generated a nuclear-export deficient DDX3X mutant construct and performed microirradiation in live cells. We found that DDX3X accumulates at sites of microirradiation shortly after DNA damage induction. We further found DDX3X recruitment to be mediated by its intrinsically disordered domains, similar to other RNA binding proteins that are recruited to sites of DNA damage. Inhibition of liquid-liquid phase separation also reduced DDX3X recruitment. CRISPR/Cas9-mediated knockout of PARP1 ablated DDX3X recruitment, which was restored upon transgenic expression of wild-type PARP1 but not catalytically inactive PARP1, suggesting that DDX3X recruitment is PARP1-dependent.

Recommended citation: Cargill MJ, Morales A, Ravishankar S, Warren EH. RNA helicase, DDX3X, is actively recruited to sites of DNA damage in live cells. DNA repair. 2021 Jul 1;103:103137. https://www.sciencedirect.com/science/article/pii/S1568786421000938

Targeted deep amplicon sequencing of antimalarial resistance markers in Plasmodium falciparum isolates from Cameroon

Updated on International Journal of Infectious Diseases, 2021

Recent studies showed the first emergence of the R561H artemisinin-associated resistance marker in Africa, which highlights the importance of continued molecular surveillance to assess the selection and spread of this and other drug resistance markers in the region. In this study, we used targeted amplicon deep sequencing of 116 isolates collected in two areas of Cameroon to genotype the major drug resistance genes, k13, crt, mdr1, dhfr, and dhps, and the cytochrome b gene (cytb) in Plasmodium falciparum. No confirmed or associated artemisinin resistance markers were observed in Pfk13. In comparison, both major and minor alleles associated with drug resistance were found in Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps. Notably, a high frequency of other nonsynonymous mutations was observed across all the genes, except for Pfcytb, suggesting continued selection pressure. The results from this study supported the continued use of artemisinin-based combination therapy and administration of sulfadoxine-pyrimethamine for intermittent preventive therapy in pregnant women, and for seasonal chemoprevention in these study sites in Cameroon.

Recommended citation: L’Episcopia M, Kelley J, Dongho BG, Patel D, Schmedes S, Ravishankar S, Perrotti E, Modiano D, Lucchi NW, Russo G, Talundzic E. Targeted deep amplicon sequencing of antimalarial resistance markers in Plasmodium falciparum isolates from Cameroon. International Journal of Infectious Diseases. 2021 Jun 1;107:234-41. https://www.sciencedirect.com/science/article/pii/S1201971221003933

Evolution and Genetic Diversity of the k13 Gene Associated with Artemisinin Delayed Parasite Clearance in Plasmodium falciparum

Updated on Antimicrobial Agents and Chemotherapy, 2019

Mutations in the Plasmodium falciparum k13 (Pfk13) gene are linked to delayed parasite clearance in response to artemisinin-based combination therapies (ACTs) in Southeast Asia. To explore the evolutionary rate and constraints acting on this gene, k13 orthologs from species sharing a recent common ancestor with P. falciparum and Plasmodium vivax were analyzed. These comparative studies were followed by genetic polymorphism analyses within P. falciparum using 982 complete Pfk13 sequences from public databases and new data obtained by next-generation sequencing from African and Haitian isolates. Although k13 orthologs evolve at heterogeneous rates, the gene was conserved across the genus, with only synonymous substitutions being found at residues where mutations linked to the delayed parasite clearance phenotype have been reported. This suggests that those residues were under constraint from undergoing nonsynonymous changes during evolution of the genus. No fixed nonsynonymous differences were found between Pfk13 and its orthologs in closely related species found in African apes. This indicates that all nonsynonymous substitutions currently found in Pfk13 are younger than the time of divergence between P. falciparum and its closely related species. At the population level, no mutations linked to delayed parasite clearance were found in our samples from Africa and Haiti. However, there is a high number of single Pfk13 mutations segregating in P. falciparum populations, and two predominant alleles are distributed worldwide. This pattern is discussed in terms of how changes in the efficacy of natural selection, affected by population expansion, may have allowed for the emergence of mutations tolerant to ACTs.

Recommended citation: Pacheco MA, Kadakia ER, Chaudhary Z, Perkins DJ, Kelley J, Ravishankar S, Cranfield M, Talundzic E, Udhayakumar V, Escalante AA. Evolution and genetic diversity of the k13 gene associated with artemisinin delayed parasite clearance in Plasmodium falciparum. Antimicrobial Agents and Chemotherapy. 2019 Aug;63(8):10-128. https://journals.asm.org/doi/full/10.1128/aac.02550-18

Next-generation Sequence-analysis Toolkit (NeST): A standardized bioinformatics framework for analyzing Single Nucleotide Polymorphisms in next-generation sequencing data

Updated on Antimicrobial Agents and Chemotherapy, 2018

Rapid advancements in next-generation sequencing (NGS) technologies have led to the development of numerous bioinformatics tools and pipelines. As these tools vary in their output function and complexity and some are not well-standardized, it is harder to choose a suitable pipeline to identify variants in NGS data. Here, we present NeST (NGS-analysis Toolkit), a modular consensus-based variant calling framework. NeST uses a combination of variant callers to overcome potential biases of an individual method used alone. NeST consists of four modules, that integrate open-source bioinformatics tools, a custom Variant Calling Format (VCF) parser and a summarization utility, that generate high-quality consensus variant calls. NeST was validated using targeted-amplicon deep sequencing data from 245 Plasmodium falciparum isolates to identify single-nucleotide polymorphisms conferring drug resistance. The results were verified using Sanger sequencing data for the same dataset in a supporting publication. NeST offers a user-friendly pipeline for variant calling with standardized outputs and minimal computational demands for easy deployment for use with various organisms and applications.

Recommended citation: Ravishankar S, Schmedes SE, Patel DS, Plucinski M, Udhayakumar V, Talundzic E, Vannberg F. Next-generation Sequence-analysis Toolkit (NeST): A standardized bioinformatics framework for analyzing Single Nucleotide Polymorphisms in next-generation sequencing data. bioRxiv. 2018 May 16:323535. https://www.biorxiv.org/content/10.1101/323535v3.abstract

Mapping-free variant calling using haplotype reconstruction from k-mer frequencies

Updated on Oxford Bioinformatics, 2018

The standard protocol for detecting variation in DNA is to map millions of short sequence reads to a known reference and find loci that differ. While this approach works well, it cannot be applied where the sample contains dense variants or is too distant from known references. De novo assembly or hybrid methods can recover genomic variation, but the cost of computation is often much higher. We developed a novel k-mer algorithm and software implementation, Kestrel, capable of characterizing densely packed SNPs and large indels without mapping, assembly or de Bruijn graphs. When applied to mosaic penicillin binding protein (PBP) genes in Streptococcus pneumoniae, we found near perfect concordance with assembled contigs at a fraction of the CPU time. Multilocus sequence typing (MLST) with this approach was able to bypass de novo assemblies. Kestrel has a very low false-positive rate when applied to the whole genome, and while Kestrel identified many variants missed by other methods, limitations of a purely k-mer based approach affect overall sensitivity. Source code and documentation for a Java implementation of Kestrel can be found at https://github.com/paudano/kestrel. All test code for this publication is located at https://github.com/paudano/kescases.

Recommended citation: Peter A Audano, Shashidhar Ravishankar, Fredrik O Vannberg, Mapping-free variant calling using haplotype reconstruction from k-mer frequencies, Bioinformatics, Volume 34, Issue 10, May 2018, Pages 1659–1665, https://doi.org/10.1093/bioinformatics/btx753 https://academic.oup.com/bioinformatics/article/34/10/1659/4657072

Next-generation sequencing and bioinformatics protocol for malaria drug resistance marker surveillance

Updated on Antimicrobial Agents and Chemotherapy, 2018

The recent advances in next-generation sequencing technologies provide a new and effective way of tracking malaria drug-resistant parasites. To take advantage of this technology, an end-to-end Illumina targeted amplicon deep sequencing (TADS) and bioinformatics pipeline for molecular surveillance of drug resistance in P. falciparum, called malaria resistance surveillance (MaRS), was developed. TADS relies on PCR enriching genomic regions, specifically target genes of interest, prior to deep sequencing. MaRS enables researchers to simultaneously collect data on allele frequencies of multiple full-length P. falciparum drug resistance genes (crt, mdr1, k13, dhfr, dhps, and the cytochrome b gene), as well as the mitochondrial genome. Information is captured at the individual patient level for both known and potential new single nucleotide polymorphisms associated with drug resistance. The MaRS pipeline was validated using 245 imported malaria cases that were reported to the Centers for Disease Control and Prevention (CDC). The chloroquine resistance crt CVIET genotype (mutations underlined) was observed in 42% of samples, the highly pyrimethamine-resistant dhps IRN triple mutant in 92% of samples, and the sulfadoxine resistance dhps mutation SGEAA in 26% of samples. The mdr1 NFSND genotype was found in 40% of samples. With the exception of two cases imported from Cambodia, no artemisinin resistance k13 alleles were identified, and 99% of patients carried parasites susceptible to atovaquone-proguanil. Our goal is to implement MaRS at the CDC for routine surveillance of imported malaria cases in the United States and to aid in the adoption of this system at participating state public health laboratories, as well as by global partners.

Recommended citation: Talundzic E, Ravishankar S, Kelley J, Patel D, Plucinski M, Schmedes S, Ljolje D, Clemons B, Madison-Antenucci S, Arguin PM, Lucchi NW. Next-generation sequencing and bioinformatics protocol for malaria drug resistance marker surveillance. Antimicrobial Agents and Chemotherapy. 2018 Apr;62(4):10-128. https://journals.asm.org/doi/full/10.1128/aac.02474-17

TLR-exosomes exhibit distinct kinetics and effector function

Updated on Scientific Reports, 2017

The innate immune system is vital to rapidly responding to pathogens and Toll-like receptors (TLRs) are a critical component of this response. Nanovesicular exosomes play a role in immunity, but to date their exact contribution to the dissemination of the TLR response is unknown. Here we show that exosomes from TLR stimulated cells can largely recapitulate TLR activation in distal cells in vitro. We can abrogate the action-at-a-distance signaling of exosomes by UV irradiation, demonstrating that RNA is crucial for their effector function. We are the first to show that exosomes derived from poly(I:C) stimulated cells induce in vivo macrophage M1-like polarization within murine lymph nodes. These poly(I:C) exosomes demonstrate enhanced trafficking to the node and preferentially recruit neutrophils as compared to control exosomes. This work definitively establishes the differential effector function for exosomes in communicating the TLR activation state of the cell of origin.

Recommended citation: Srinivasan, S., Su, M., Ravishankar, S. et al. TLR-exosomes exhibit distinct kinetics and effector function. Sci Rep 7, 41623 (2017). https://doi.org/10.1038/srep41623 https://www.nature.com/articles/srep41623

First Full Draft Genome Sequence of Plasmodium brasilianum

Updated on Genome Announcements, 2017

Plasmodium malariae is a protozoan parasite that can cause human malaria. The simian parasite Plasmodium brasilianum infects New World monkeys from Latin America and is morphologically indistinguishable from P. malariae. Here, we report the first full draft genome sequence for P. brasilianum.

Recommended citation: Talundzic E, Ravishankar S, Nayak V, Patel DS, Olsen C, Sheth M, Batra D, Loparev V, Vannberg FO, Udhayakumar V, Barnwell JW. First full draft genome sequence of Plasmodium brasilianum. Genome Announcements. 2017 Feb 9;5(6):10-128. https://journals.asm.org/doi/abs/10.1128/genomea.01566-16

Updated on , 2024