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Article: A hybrid demultiplexing strategy that improves performance and robustness of cell hashing.

Li, Lei / Sun, Jiayi / Fu, Yanbin / Changrob, Siriruk / McGrath, Joshua J C / Wilson, Patrick C

bioRxiv : the preprint server for biology

2023

Abstract: Recent advances in single cell RNA sequencing allow users to pool multiple samples and demultiplex in downstream analysis, which greatly increase experimental efficiency and cost-effectiveness. Among all the demultiplexing methods, nucleotide barcode- ... ...

Abstract	Recent advances in single cell RNA sequencing allow users to pool multiple samples and demultiplex in downstream analysis, which greatly increase experimental efficiency and cost-effectiveness. Among all the demultiplexing methods, nucleotide barcode-based cell hashing has gained widespread popularity due to its compatibility and simplicity. Despite these advantages, certain issues of this technic remain to be solved, such as challenges in distinguishing true positive from background, high reagent cost for samples with large cell numbers, and unpredictable false negative and false doublet rates. Here, we propose a hybrid demultiplexing strategy that increases calling accuracy and cell recovery of cell hashing without adding experimental cost. In this approach, we computationally cluster all single cells based on their natural genetic variations and assign donor identity by finding the dominant hashtag in each genotype cluster. This hybrid strategy assigns donor identity to any cell that is identified as singlet by either genotype clustering or cell hashing, which allows us to demultiplex most majority of cells even if only a small fraction of cells are labeled with hashtags. When comparing its performance with cell hashing on multiple real-world datasets, this hybrid approach consistently generates reliable demultiplexing results with increased cell recovery and accuracy.
Language	English
Publishing date	2023-11-28
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.04.02.535299
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Article ; Online: Imprinting, immunodominance, and other impediments to generating broad influenza immunity.

Knight, Matthew / Changrob, Siriruk / Li, Lei / Wilson, Patrick C

Immunological reviews

2020 Volume 296, Issue 1, Page(s) 191–204

Abstract: Natural influenza virus infections and seasonal vaccinations often do not confer broadly neutralizing immunity across diverse influenza strains. In addition, the virus is capable of rapid antigenic drift in order to evade pre-existing immunity. The ... ...

Abstract	Natural influenza virus infections and seasonal vaccinations often do not confer broadly neutralizing immunity across diverse influenza strains. In addition, the virus is capable of rapid antigenic drift in order to evade pre-existing immunity. The surface glycoproteins, hemagglutinin, and neuraminidase can easily mutate their immunodominant epitopes without impacting fitness. Skewing human antibody repertoires to target more conserved epitopes is thus an expanding area of research: Many groups are attempting to produce universal influenza vaccines that can protect across a wide variety of strains. Achieving this goal will require a detailed understanding of how infection history impacts humoral responses. It will also require the ability to manipulate or enhance B cell selection in order to expand clones that can recognize subdominant but protective epitopes. In this review, we will discuss what immune imprinting means to immunologists and describe efforts to overcome or silence imprinting in order to improve vaccination efficiency.
MeSH term(s)	Animals ; Antigens, Viral/immunology ; Clonal Selection, Antigen-Mediated ; Host-Pathogen Interactions/immunology ; Humans ; Immunity, Cellular ; Immunity, Humoral ; Immunodominant Epitopes/immunology ; Influenza Vaccines/administration & dosage ; Influenza Vaccines/immunology ; Influenza, Human/immunology ; Influenza, Human/prevention & control ; Influenza, Human/virology ; Orthomyxoviridae/immunology ; Vaccination
Chemical Substances	Antigens, Viral ; Immunodominant Epitopes ; Influenza Vaccines
Language	English
Publishing date	2020-07-14
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Review
ZDB-ID	391796-4
ISSN	1600-065X ; 0105-2896
ISSN (online)	1600-065X
ISSN	0105-2896
DOI	10.1111/imr.12900
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Article ; Online: Cross-reactive inhibitory antibody and memory B cell responses to variant strains of Duffy binding protein II at post-Plasmodium vivax infection.

Thawornpan, Pongsakorn / Changrob, Siriruk / Kochayoo, Piyawan / Wangriatisak, Kittikorn / Ntumngia, Francis B / De, Sai Lata / Han, Eun-Taek / Adams, John H / Chootong, Patchanee

PloS one

2022 Volume 17, Issue 10, Page(s) e0276335

Abstract: Duffy binding protein region II (DBPII) is considered a strong potential vaccine candidate of blood-stage P. vivax. However, the highly polymorphic nature of this protein often misdirects immune responses, leading them to be strain-specific. Details of ... ...

Abstract	Duffy binding protein region II (DBPII) is considered a strong potential vaccine candidate of blood-stage P. vivax. However, the highly polymorphic nature of this protein often misdirects immune responses, leading them to be strain-specific. Details of cross-reactive humoral immunity to DBPII variants have therefore become an important focus for the development of broadly protective vaccines. Here, cross-reactive humoral immunity against a panel of Thai DBPII variants (DBL-THs) was demonstrated in immunized BALB/c mice and P. vivax patients, by in vitro erythrocyte-binding inhibition assay. Sera from immunized animals showed both strain-transcending (anti-DBL-TH2 and -TH4) and strain-specific (anti-DBL-TH5, -TH6 and -TH9) binding to DBL-TH variants. Using anti-DBL-TH sera at 50% inhibitory concentration (IC50) of the homologous strain, anti-DBL-TH2 sera showed cross inhibition to heterologous DBL-TH strains, whereas anti-DBL-TH5 sera exhibited only strain-specific inhibition. In P. vivax patients, 6 of 15 subjects produced and maintained cross-reactive anti-DBL-TH inhibitory antibodies through the 1-year post-infection timepoint. Cross-reactive memory B cell (MBC) responses to DBL-TH variants were analyzed in subjects recovered from P. vivax infection (RC). The plasma samples from 5 RC subjects showed broad inhibition. However, MBC-derived antibodies of these patients did not reveal cross-inhibition. Altogether, broadly anti-DBP variant inhibitory antibodies developed and persisted in P. vivax infections. However, the presence of cross-reactive anti-DBL-TH inhibitory function post-infection was not related with MBC responses to these variants. More detailed investigation of long-lasting, broadly protective antibodies to DBPII will guide the design of vivax malaria vaccines.
MeSH term(s)	Mice ; Animals ; Plasmodium vivax ; Malaria Vaccines ; Antigens, Protozoan ; Antibodies, Protozoan ; Carrier Proteins ; Memory B Cells ; Protozoan Proteins ; Receptors, Cell Surface ; Malaria, Vivax ; Antibodies, Blocking ; Mice, Inbred BALB C
Chemical Substances	Malaria Vaccines ; Antigens, Protozoan ; Antibodies, Protozoan ; Carrier Proteins ; Protozoan Proteins ; Receptors, Cell Surface ; Antibodies, Blocking
Language	English
Publishing date	2022-10-18
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2267670-3
ISSN	1932-6203 ; 1932-6203
ISSN (online)	1932-6203
ISSN	1932-6203
DOI	10.1371/journal.pone.0276335
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Article ; Online: Interferon-γ signal drives differentiation of T-bet

Kochayoo, Piyawan / Thawornpan, Pongsakorn / Wangriatisak, Kittikorn / Changrob, Siriruk / Leepiyasakulchai, Chaniya / Khowawisetsut, Ladawan / Adams, John H / Chootong, Patchanee

Scientific reports

2022 Volume 12, Issue 1, Page(s) 4842

Abstract: For development of a long-lasting protective malaria vaccine, it is crucial to understand whether Plasmodium-induced memory B cells (MBCs) or plasma cells develop and stably contribute to protective immunity, or on the contrary the parasite suppresses ... ...

Abstract	For development of a long-lasting protective malaria vaccine, it is crucial to understand whether Plasmodium-induced memory B cells (MBCs) or plasma cells develop and stably contribute to protective immunity, or on the contrary the parasite suppresses antibody responses by inducing MBC dysfunction. The expansion of T-bet
MeSH term(s)	B-Lymphocytes ; Humans ; Immunoglobulin G ; Immunologic Memory ; Interferon-gamma ; Malaria ; Malaria, Vivax ; Memory B Cells ; Plasma Cells ; Plasmodium vivax
Chemical Substances	IFNG protein, human ; Immunoglobulin G ; Interferon-gamma (82115-62-6)
Language	English
Publishing date	2022-03-22
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-022-08976-6
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Article ; Online: Epitopes recognition of SARS-CoV-2 nucleocapsid RNA binding domain by human monoclonal antibodies.

Kim, Youngchang / Maltseva, Natalia / Tesar, Christine / Jedrzejczak, Robert / Endres, Michael / Ma, Heng / Dugan, Haley L / Stamper, Christopher T / Chang, Changsoo / Li, Lei / Changrob, Siriruk / Zheng, Nai-Ying / Huang, Min / Ramanathan, Arvind / Wilson, Patrick / Michalska, Karolina / Joachimiak, Andrzej

iScience

2024 Volume 27, Issue 2, Page(s) 108976

Abstract: Coronavirus nucleocapsid protein (NP) of SARS-CoV-2 plays a central role in many functions important for virus proliferation including packaging and protecting genomic RNA. The protein shares sequence, structure, and architecture with nucleocapsid ... ...

Abstract	Coronavirus nucleocapsid protein (NP) of SARS-CoV-2 plays a central role in many functions important for virus proliferation including packaging and protecting genomic RNA. The protein shares sequence, structure, and architecture with nucleocapsid proteins from betacoronaviruses. The N-terminal domain (NP
Language	English
Publishing date	2024-01-19
Publishing country	United States
Document type	Journal Article
ISSN	2589-0042
ISSN (online)	2589-0042
DOI	10.1016/j.isci.2024.108976
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Article ; Online: The persistence of naturally acquired antibodies and memory B cells specific to rhoptry proteins of Plasmodium vivax in patients from areas of low malaria transmission.

Kochayoo, Piyawan / Changrob, Siriruk / Wangriatisak, Kittikorn / Lee, Seong Kyun / Chootong, Patchanee / Han, Eun-Taek

Malaria journal

2019 Volume 18, Issue 1, Page(s) 382

Abstract: Background: Rhoptries are the large, paired, secretory organelles located at the apical tip of the malaria merozoite that are considered important for parasite invasion processes. Plasmodium vivax rhoptry proteins have been shown to induce humoral ... ...

Abstract	Background: Rhoptries are the large, paired, secretory organelles located at the apical tip of the malaria merozoite that are considered important for parasite invasion processes. Plasmodium vivax rhoptry proteins have been shown to induce humoral immunity during natural infections. Therefore, these proteins may be potential novel vaccine candidates. However, there is a lack of data on the duration of antibody and memory B cell (MBC) responses. Here, the longitudinal analysis of antibody and MBC responses to the P. vivax rhoptry proteins PvRALP1-Ecto and PvRhopH2 were monitored and analysed in individuals to determine their persistence. Methods: Thirty-nine samples from P. vivax-infected subjects (age 18-60 years) were recruited to explore the frequency and persistence of antibody and MBC responses against rhoptry proteins (PvRALP1-Ecto and PvRhopH2) using both cross-sectional and longitudinal cohort study designs. Antibody levels were determined by ELISA during clinical malaria, and at 3, 9 and 12 months post-infection. The frequency of MBC sub-sets and presence of rhoptry-specific MBCs in subjects 18 months after treatment were detected by flow cytometry and ELISPOT assay. Results: The seroprevalence of antibodies against PvRALP1-Ecto and PvRhopH2 proteins was found to be high during acute infection, with IgG1, IgG2 and IgG3 sub-classes predominant. However, these anti-rhoptry responses were short-lived and significantly decreased at 9 months post-infection. To relate the durability of these antibody responses to MBC persistence at post-infection, 18-month post-infection peripheral blood mononuclear cells (PBMCs) samples were taken to detect rhoptry-specific MBCs and frequency of MBC sub-sets, and correlate with antibody responses. These late post-infection samples revealed that rhoptry-specific MBCs were present in about 70% of total subjects. However, the persistence of specific MBCs was not correlated with antibody responses as the majority of malaria subjects who were positive for PvRALP1-Ecto- or PvRhopH2-specific MBCs were seronegative for the rhoptry antigens. The frequencies of classical MBCs were increased after infection, whereas those of activated and atypical MBCs were decreased, indicating that MBC responses could switch from activated or atypical MBCs to classical MBCs after parasite clearance, and were maintained in blood circulating at post-infection. Conclusion: The study showed that rhoptry antigens induced the development and persistence of MBC responses in P. vivax-infected subjects who lived in a region of low malaria transmission, which were not related to the longevity of antibody responses.
MeSH term(s)	Adult ; Antibodies, Protozoan/blood ; Cross-Sectional Studies ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Longitudinal Studies ; Malaria, Vivax/immunology ; Malaria, Vivax/parasitology ; Male ; Middle Aged ; Plasmodium vivax/physiology ; Protozoan Proteins/immunology ; Seroepidemiologic Studies ; Thailand ; Time Factors ; Young Adult
Chemical Substances	Antibodies, Protozoan ; Protozoan Proteins
Language	English
Publishing date	2019-11-29
Publishing country	England
Document type	Journal Article
ISSN	1475-2875
ISSN (online)	1475-2875
DOI	10.1186/s12936-019-3009-2
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Article ; Online: Severe COVID-19 induces autoantibodies against angiotensin II that correlate with blood pressure dysregulation and disease severity.

Briquez, Priscilla S / Rouhani, Sherin J / Yu, Jovian / Pyzer, Athalia R / Trujillo, Jonathan / Dugan, Haley L / Stamper, Christopher T / Changrob, Siriruk / Sperling, Anne I / Wilson, Patrick C / Gajewski, Thomas F / Hubbell, Jeffrey A / Swartz, Melody A

Science advances

2022 Volume 8, Issue 40, Page(s) eabn3777

Abstract: Patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can experience life-threatening respiratory distress, blood pressure dysregulation, and thrombosis. This is thought to be associated with an impaired activity of ... ...

Abstract	Patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can experience life-threatening respiratory distress, blood pressure dysregulation, and thrombosis. This is thought to be associated with an impaired activity of angiotensin-converting enzyme 2 (ACE2), which is the main entry receptor of SARS-CoV-2 and which also tightly regulates blood pressure by converting the vasoconstrictive peptide angiotensin II (AngII) to a vasopressor peptide. Here, we show that a significant proportion of hospitalized patients with COVID-19 developed autoantibodies against AngII, whose presence correlates with lower blood oxygenation, blood pressure dysregulation, and overall higher disease severity. Anti-AngII antibodies can develop upon specific immune reaction to the SARS-CoV-2 proteins Spike or receptor-binding domain (RBD), to which they can cross-bind, suggesting some epitope mimicry between AngII and Spike/RBD. These results provide important insights on how an immune reaction against SARS-CoV-2 can impair blood pressure regulation.
MeSH term(s)	Angiotensin II ; Angiotensin-Converting Enzyme 2 ; Autoantibodies ; Blood Pressure ; COVID-19 ; Epitopes/metabolism ; Humans ; Peptidyl-Dipeptidase A/metabolism ; Protein Binding ; SARS-CoV-2 ; Severity of Illness Index ; Spike Glycoprotein, Coronavirus
Chemical Substances	Autoantibodies ; Epitopes ; Spike Glycoprotein, Coronavirus ; spike protein, SARS-CoV-2 ; Angiotensin II (11128-99-7) ; Peptidyl-Dipeptidase A (EC 3.4.15.1) ; Angiotensin-Converting Enzyme 2 (EC 3.4.17.23)
Language	English
Publishing date	2022-10-07
Publishing country	United States
Document type	Journal Article
ZDB-ID	2810933-8
ISSN	2375-2548 ; 2375-2548
ISSN (online)	2375-2548
ISSN	2375-2548
DOI	10.1126/sciadv.abn3777
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Article ; Online: Librator: a platform for the optimized analysis, design, and expression of mutable influenza viral antigens.

Li, Lei / Changrob, Siriruk / Fu, Yanbin / Stovicek, Olivia / Guthmiller, Jenna J / McGrath, Joshua J C / Dugan, Haley L / Stamper, Christopher T / Zheng, Nai-Ying / Huang, Min / Wilson, Patrick C

Briefings in bioinformatics

2022 Volume 23, Issue 2

Abstract: Artificial mutagenesis and protein engineering have laid the foundation for antigenic characterization and universal vaccine design for influenza viruses. However, many methods used in this process require manual sequence editing and protein expression, ... ...

Abstract	Artificial mutagenesis and protein engineering have laid the foundation for antigenic characterization and universal vaccine design for influenza viruses. However, many methods used in this process require manual sequence editing and protein expression, limiting their efficiency and utility in high-throughput applications. More streamlined in silico tools allowing researchers to properly analyze and visualize influenza viral protein sequences with accurate nomenclature are necessary to improve antigen design and productivity. To address this need, we developed Librator, a system for analyzing and designing custom protein sequences of influenza virus hemagglutinin (HA) and neuraminidase (NA) glycoproteins. Within Librator's graphical interface, users can easily interrogate viral sequences and phylogenies, visualize antigen structures and conservation, mutate target residues and design custom antigens. Librator also provides optimized fragment design for Gibson Assembly of HA and NA expression constructs based on peptide conservation of all historical HA and NA sequences, ensuring fragments are reusable and compatible across related subtypes, thereby promoting reagent savings. Finally, the program facilitates single-cell immune profiling, epitope mapping of monoclonal antibodies and mosaic protein design. Using Librator-based antigen construction, we demonstrate that antigenicity can be readily transferred between HA molecules of H3, but not H1, lineage viruses. Altogether, Librator is a valuable tool for analyzing influenza virus HA and NA proteins and provides an efficient resource for optimizing recombinant influenza antigen synthesis.
MeSH term(s)	Antibodies, Viral ; Antigens, Viral/genetics ; Hemagglutinin Glycoproteins, Influenza Virus/genetics ; Humans ; Influenza Vaccines ; Influenza, Human ; Neuraminidase/genetics ; Orthomyxoviridae/genetics
Chemical Substances	Antibodies, Viral ; Antigens, Viral ; Hemagglutinin Glycoproteins, Influenza Virus ; Influenza Vaccines ; Neuraminidase (EC 3.2.1.18)
Language	English
Publishing date	2022-02-20
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	2068142-2
ISSN	1477-4054 ; 1467-5463
ISSN (online)	1477-4054
ISSN	1467-5463
DOI	10.1093/bib/bbac028
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Article ; Online: Mutability and hypermutation antagonize immunoglobulin codon optimality

McGrath, Joshua J.C. / Park, Juyeon / Troxell, Chloe A. / Chervin, Jordan C. / Li, Lei / Kent, Johnathan R. / Changrob, Siriruk / Fu, Yanbin / Huang, Min / Zheng, Nai-Ying / Wilbanks, G. Dewey / Nelson, Sean A. / Sun, Jiayi / Inghirami, Giorgio / Madariaga, Maria Lucia L. / Georgiou, George / Wilson, Patrick C.

bioRxiv

Abstract: The efficacy of polyclonal antibody responses is inherently linked to paratope diversity, as generated through V(D)J recombination and somatic hypermutation (SHM). These processes arose in early jawed vertebrates; however, little is known about how ... ...

Abstract	The efficacy of polyclonal antibody responses is inherently linked to paratope diversity, as generated through V(D)J recombination and somatic hypermutation (SHM). These processes arose in early jawed vertebrates; however, little is known about how immunoglobulin diversity, mutability, and hypermutation have evolved in tandem with another more ubiquitous feature of protein-coding DNA - codon optimality. Here, we explore these relationships through analysis of germline IG genes, natural V(D)J repertoires, serum VH usage, and monoclonal antibody (mAb) expression, each through the lens of multiple optimality metrics. Strikingly, proteomic serum IgG sequencing showed that germline IGHV codon optimality positively correlated with VH representation after influenza vaccination, and in vitro, codon deoptimization of mAbs with synonymous amino acid sequences caused consistent expression loss. Germline V genes exhibit a range of codon optimality that is maintained by functionality, and inversely related to mutability. SHM caused a load-dependent deoptimization of IGH VDJ repertoires within human tonsils, bone marrow, and lymph nodes (including SARS-CoV-2-specific clones from mRNA vaccinees), influenza-infected mice, and zebrafish. Comparison of natural mutation profiles to true random suggests the presence of selective pressures that constrain deoptimization. These findings shed light on immunoglobulin evolution, providing unanticipated insights into the antagonistic relationship between variable region diversification, codon optimality, and antibody secretion; ultimately, the need for diversity takes precedence over that for the most efficient expression of the antibody repertoire.
Keywords	covid19
Language	English
Publishing date	2024-03-14
Publisher	Cold Spring Harbor Laboratory
Document type	Article ; Online
DOI	10.1101/2024.03.13.584690
Database	COVID19

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Article: SARS-CoV-2 infection induces cross-reactive autoantibodies against angiotensin II.

medRxiv : the preprint server for health sciences

2021

Abstract: Patients infected with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can experience life-threatening respiratory distress, blood pressure dysregulation and thrombosis. This is thought to be associated with an impaired activity of ... ...

Abstract	Patients infected with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can experience life-threatening respiratory distress, blood pressure dysregulation and thrombosis. This is thought to be associated with an impaired activity of angiotensin-converting enzyme-2 (ACE-2), which is the main entry receptor of SARS-CoV-2 and which also tightly regulates blood pressure by converting the vasoconstrictive peptide angiotensin II (AngII) to a vasopressor peptide. Here, we show that a significant proportion of hospitalized COVID-19 patients developed autoantibodies against AngII, whose presence correlates with lower blood oxygenation, blood pressure dysregulation, and overall higher disease severity. Anti-AngII antibodies can develop upon specific immune reaction to the SARS-CoV-2 proteins Spike or RBD, to which they can cross-bind, suggesting some epitope mimicry between AngII and Spike/RBD. These results provide important insights on how an immune reaction against SARS-CoV-2 can impair blood pressure regulation.
Language	English
Publishing date	2021-11-02
Publishing country	United States
Document type	Preprint
DOI	10.1101/2021.11.02.21265789
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