Publications by authors named "Amin Addetia"

26 Publications

  • Page 1 of 1

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

PLoS Negl Trop Dis 2021 Sep 7;15(9):e0009753. Epub 2021 Sep 7.

Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pntd.0009753DOI Listing
September 2021

Broad sarbecovirus neutralization by a human monoclonal antibody.

Nature 2021 09 19;597(7874):103-108. Epub 2021 Jul 19.

Humabs Biomed SA, a subsidiary of Vir Biotechnology, Bellinzona, Switzerland.

The recent emergence of SARS-CoV-2 variants of concern and the recurrent spillovers of coronaviruses into the human population highlight the need for broadly neutralizing antibodies that are not affected by the ongoing antigenic drift and that can prevent or treat future zoonotic infections. Here we describe a human monoclonal antibody designated S2X259, which recognizes a highly conserved cryptic epitope of the receptor-binding domain and cross-reacts with spikes from all clades of sarbecovirus. S2X259 broadly neutralizes spike-mediated cell entry of SARS-CoV-2, including variants of concern (B.1.1.7, B.1.351, P.1, and B.1.427/B.1.429), as well as a wide spectrum of human and potentially zoonotic sarbecoviruses through inhibition of angiotensin-converting enzyme 2 (ACE2) binding to the receptor-binding domain. Furthermore, deep-mutational scanning and in vitro escape selection experiments demonstrate that S2X259 possesses an escape profile that is limited to a single substitution, G504D. We show that prophylactic and therapeutic administration of S2X259 protects Syrian hamsters (Mesocricetus auratus) against challenge with the prototypic SARS-CoV-2 and the B.1.351 variant of concern, which suggests that this monoclonal antibody is a promising candidate for the prevention and treatment of emergent variants and zoonotic infections. Our data reveal a key antigenic site that is targeted by broadly neutralizing antibodies and will guide the design of vaccines that are effective against all sarbecoviruses.
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http://dx.doi.org/10.1038/s41586-021-03817-4DOI Listing
September 2021

Multivalent designed proteins protect against SARS-CoV-2 variants of concern.

bioRxiv 2021 Jul 7. Epub 2021 Jul 7.

Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness.

One-sentence Summary: We designed, developed, and characterized potent, trivalent miniprotein binders that provide prophylactic and therapeutic protection against emerging SARS-CoV-2 variants of concern.
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http://dx.doi.org/10.1101/2021.07.07.451375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8282097PMC
July 2021

SARS-CoV-2 RBD antibodies that maximize breadth and resistance to escape.

Nature 2021 09 14;597(7874):97-102. Epub 2021 Jul 14.

Humabs BioMed SA, a subsidiary of Vir Biotechnology, Bellinzona, Switzerland.

An ideal therapeutic anti-SARS-CoV-2 antibody would resist viral escape, have activity against diverse sarbecoviruses, and be highly protective through viral neutralization and effector functions. Understanding how these properties relate to each other and vary across epitopes would aid the development of therapeutic antibodies and guide vaccine design. Here we comprehensively characterize escape, breadth and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD). Despite a trade-off between in vitro neutralization potency and breadth of sarbecovirus binding, we identify neutralizing antibodies with exceptional sarbecovirus breadth and a corresponding resistance to SARS-CoV-2 escape. One of these antibodies, S2H97, binds with high affinity across all sarbecovirus clades to a cryptic epitope and prophylactically protects hamsters from viral challenge. Antibodies that target the angiotensin-converting enzyme 2 (ACE2) receptor-binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency. Nevertheless, we also characterize a potent RBM antibody (S2E12) with breadth across sarbecoviruses related to SARS-CoV-2 and a high barrier to viral escape. These data highlight principles underlying variation in escape, breadth and potency among antibodies that target the RBD, and identify epitopes and features to prioritize for therapeutic development against the current and potential future pandemics.
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http://dx.doi.org/10.1038/s41586-021-03807-6DOI Listing
September 2021

Molecular Features of the Measles Virus Viral Fusion Complex That Favor Infection and Spread in the Brain.

mBio 2021 06 1;12(3):e0079921. Epub 2021 Jun 1.

Center for Host-Pathogen Interaction, Columbia University Vagelos College of Physicians and Surgeons, New York, New York, USA.

Measles virus (MeV) bearing a single amino acid change in the fusion protein (F)-L454W-was isolated from two patients who died of MeV central nervous system (CNS) infection. This mutation in F confers an advantage over wild-type virus in the CNS, contributing to disease in these patients. Using murine organotypic brain cultures and human induced pluripotent stem cell-derived brain organoids, we show that CNS adaptive mutations in F enhance the spread of virus . The spread of virus in human brain organoids is blocked by an inhibitory peptide that targets F, confirming that dissemination in the brain tissue is attributable to F. A single mutation in MeV F thus alters the fusion complex to render MeV more neuropathogenic. Measles virus (MeV) infection can cause serious complications in immunocompromised individuals, including measles inclusion body encephalitis (MIBE). In some cases, MeV persistence and subacute sclerosing panencephalitis (SSPE), another severe central nervous system (CNS) complication, develop even in the face of a systemic immune response. Both MIBE and SSPE are relatively rare but lethal. It is unclear how MeV causes CNS infection. We introduced specific mutations that are found in MIBE or SSPE cases into the MeV fusion protein to test the hypothesis that dysregulation of the viral fusion complex-comprising F and the receptor binding protein, H-allows virus to spread in the CNS. Using metagenomic, structural, and biochemical approaches, we demonstrate that altered fusion properties of the MeV H-F fusion complex permit MeV to spread in brain tissue.
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http://dx.doi.org/10.1128/mBio.00799-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8263006PMC
June 2021

Viral genomes reveal patterns of the SARS-CoV-2 outbreak in Washington State.

Sci Transl Med 2021 05 3;13(595). Epub 2021 May 3.

Seattle Children's Research Institute, Seattle, WA 98101, USA.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has gravely affected societies around the world. Outbreaks in different parts of the globe have been shaped by repeated introductions of new viral lineages and subsequent local transmission of those lineages. Here, we sequenced 3940 SARS-CoV-2 viral genomes from Washington State (USA) to characterize how the spread of SARS-CoV-2 in Washington State in early 2020 was shaped by differences in timing of mitigation strategies across counties and by repeated introductions of viral lineages into the state. In addition, we show that the increase in frequency of a potentially more transmissible viral variant (614G) over time can potentially be explained by regional mobility differences and multiple introductions of 614G but not the other variant (614D) into the state. At an individual level, we observed evidence of higher viral loads in patients infected with the 614G variant. However, using clinical records data, we did not find any evidence that the 614G variant affects clinical severity or patient outcomes. Overall, this suggests that with regard to D614G, the behavior of individuals has been more important in shaping the course of the pandemic in Washington State than this variant of the virus.
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http://dx.doi.org/10.1126/scitranslmed.abf0202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8158963PMC
May 2021

Structural basis for broad sarbecovirus neutralization by a human monoclonal antibody.

bioRxiv 2021 Apr 8. Epub 2021 Apr 8.

Humabs Biomed SA, a subsidiary of Vir Biotechnology, 6500 Bellinzona, Switzerland.

The recent emergence of SARS-CoV-2 variants of concern (VOC) and the recurrent spillovers of coronaviruses in the human population highlight the need for broadly neutralizing antibodies that are not affected by the ongoing antigenic drift and that can prevent or treat future zoonotic infections. Here, we describe a human monoclonal antibody (mAb), designated S2×259, recognizing a highly conserved cryptic receptor-binding domain (RBD) epitope and cross-reacting with spikes from all sarbecovirus clades. S2×259 broadly neutralizes spike-mediated entry of SARS-CoV-2 including the B.1.1.7, B.1.351, P.1 and B.1.427/B.1.429 VOC, as well as a wide spectrum of human and zoonotic sarbecoviruses through inhibition of ACE2 binding to the RBD. Furthermore, deep-mutational scanning and escape selection experiments demonstrate that S2×259 possesses a remarkably high barrier to the emergence of resistance mutants. We show that prophylactic administration of S2×259 protects Syrian hamsters against challenges with the prototypic SARS-CoV-2 and the B.1.351 variant, suggesting this mAb is a promising candidate for the prevention and treatment of emergent VOC and zoonotic infections. Our data unveil a key antigenic site targeted by broadly-neutralizing antibodies and will guide the design of pan-sarbecovirus vaccines.
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http://dx.doi.org/10.1101/2021.04.07.438818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8043460PMC
April 2021

Antibodies to the SARS-CoV-2 receptor-binding domain that maximize breadth and resistance to viral escape.

bioRxiv 2021 Apr 8. Epub 2021 Apr 8.

Vir Biotechnology, San Francisco, CA 94158, USA.

An ideal anti-SARS-CoV-2 antibody would resist viral escape , have activity against diverse SARS-related coronaviruses , and be highly protective through viral neutralization and effector functions . Understanding how these properties relate to each other and vary across epitopes would aid development of antibody therapeutics and guide vaccine design. Here, we comprehensively characterize escape, breadth, and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD), including S309 , the parental antibody of the late-stage clinical antibody VIR-7831. We observe a tradeoff between SARS-CoV-2 neutralization potency and breadth of binding across SARS-related coronaviruses. Nevertheless, we identify several neutralizing antibodies with exceptional breadth and resistance to escape, including a new antibody (S2H97) that binds with high affinity to all SARS-related coronavirus clades via a unique RBD epitope centered on residue E516. S2H97 and other escape-resistant antibodies have high binding affinity and target functionally constrained RBD residues. We find that antibodies targeting the ACE2 receptor binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency, but we identify one potent RBM antibody (S2E12) with breadth across sarbecoviruses closely related to SARS-CoV-2 and with a high barrier to viral escape. These data highlight functional diversity among antibodies targeting the RBD and identify epitopes and features to prioritize for antibody and vaccine development against the current and potential future pandemics.
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http://dx.doi.org/10.1101/2021.04.06.438709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8043444PMC
April 2021

SARS-CoV-2 ORF6 Disrupts Bidirectional Nucleocytoplasmic Transport through Interactions with Rae1 and Nup98.

mBio 2021 04 13;12(2). Epub 2021 Apr 13.

Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA

RNA viruses that replicate in the cytoplasm often disrupt nucleocytoplasmic transport to preferentially translate their own transcripts and prevent host antiviral responses. The accessory protein ORF6 has previously been shown to be a major inhibitor of interferon production in both severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we show SARS-CoV-2-infected cells display an elevated level of nuclear mRNA accumulation compared to mock-infected cells. We demonstrate that ORF6 is responsible for this nuclear imprisonment of host mRNA, and using a cotransfected reporter assay, we show this nuclear retention of mRNA blocks expression of newly transcribed mRNAs. ORF6's nuclear entrapment of host mRNA is associated with its ability to copurify with the mRNA export factors, Rae1 and Nup98. These protein-protein interactions map to the C terminus of ORF6 and can be abolished by a single amino acid mutation in Met58. Overexpression of Rae1 restores reporter expression in the presence of SARS-CoV-2 ORF6. SARS-CoV ORF6 also interacts with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly copurifies with Rae1 and Nup98 and results in significantly reduced expression of reporter proteins compared to SARS-CoV ORF6, a potential mechanism for the delayed symptom onset and presymptomatic transmission uniquely associated with the SARS-CoV-2 pandemic. We also show that both SARS-CoV and SARS-CoV-2 ORF6 block nuclear import of a broad range of host proteins. Together, these data support a model in which ORF6 clogs the nuclear pore through its interactions with Rae1 and Nup98 to prevent both nuclear import and export, rendering host cells incapable of responding to SARS-CoV-2 infection. SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), is an RNA virus with a large genome that encodes multiple accessory proteins. While these accessory proteins are not required for growth , they can contribute to the pathogenicity of the virus. We demonstrate that SARS-CoV-2-infected cells accumulate poly(A) mRNA in the nucleus, which is attributed to the accessory protein ORF6. Nuclear entrapment of mRNA and reduced expression of newly transcribed reporter proteins are associated with ORF6's interactions with the mRNA export proteins Rae1 and Nup98. SARS-CoV ORF6 also shows the same interactions with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly represses reporter expression and copurifies with Rae1 and Nup98 compared to SARS-CoV ORF6. Both SARS-CoV ORF6 and SARS-CoV-2 ORF6 block nuclear import of a wide range of host factors through interactions with Rae1 and Nup98. Together, our results suggest ORF6's disruption of nucleocytoplasmic transport prevents infected cells from responding to the invading virus.
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http://dx.doi.org/10.1128/mBio.00065-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092196PMC
April 2021

Generation of BK and JC Polyomavirus Defective Viral Genomes in Human Urine Samples Associated with Higher Viral Loads.

J Virol 2021 05 24;95(12). Epub 2021 May 24.

Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA

Defective viral genomes (DVGs) are parasitic viral sequences containing point mutations, deletions, or duplications that might interfere with replication. DVGs are often associated with viral passage at high multiplicities of infection in culture systems but have been increasingly reported in clinical specimens. To date however, only RNA viruses have been shown to contain DVGs in clinical specimens. Here, using direct deep sequencing with multiple library preparation strategies and confirmatory digital droplet PCR (ddPCR) of urine samples taken from immunosuppressed individuals, we show that clinical BK polyomavirus (BKPyV) and JC polyomavirus (JCPyV) strains contain widespread genomic rearrangements across multiple loci that likely interfere with viral replication. BKPyV DVGs were derived from BKPyV genotypes Ia, Ib-1, and Ic. The presence of DVGs was associated with specimens containing higher viral loads but never reached clonality, consistent with a model of parasitized replication. These DVGs persisted during clinical infection as evidenced in two separate pairs of samples containing BK virus collected from the same individual up to 302 days apart. In a separate individual, we observed the generation of DVGs after a 57.5-fold increase in viral load. In summary, by extending the presence of DVGs in clinical specimens to DNA viruses, we demonstrate the ubiquity of DVGs in clinical virology. Defective viral genomes (DVGs) can have a significant impact on the production of infectious virus particles. DVGs have only been identified in cultured viruses passaged at high multiplicities of infection and RNA viruses collected from clinical specimens; no DNA virus in the wild has been shown to contain DVGs. Here, we identified BK and JC polyomavirus DVGs in clinical urine specimens and demonstrated that these DVGs are more frequently identified in samples with higher viral loads. The strains containing DVGs had rearrangements throughout their genomes, with the majority affecting genes required for viral replication. Longitudinal analysis showed that these DVGs can persist during an infection but do not reach clonality within the chronically infected host. Our identification of polyomavirus DVGs suggests that these parasitic sequences exist across the many classes of viruses capable of causing human disease.
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http://dx.doi.org/10.1128/JVI.00250-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8316075PMC
May 2021

Inactivation of genes in oxidative respiration and iron acquisition pathways in pediatric clinical isolates of Small colony variant Enterobacteriaceae.

Sci Rep 2021 Apr 2;11(1):7457. Epub 2021 Apr 2.

Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA.

Isolation of bacterial small colony variants (SCVs) from clinical specimens is not uncommon and can fundamentally change the outcome of the associated infections. Bacterial SCVs often emerge with their normal colony phenotype (NCV) co-isolates in the same sample. The basis of SCV emergence in vivo is not well understood in Gram-negative bacteria. In this study, we interrogated the causal genetic lesions of SCV growth in three pairs of NCV and SCV co-isolates of Escherichia coli, Citrobacter freundii, and Enterobacter hormaechei. We confirmed SCV emergence was attributed to limited genomic mutations: 4 single nucleotide variants in the E. coli SCV, 5 in C. freundii, and 8 in E. hormaechei. In addition, a 10.2 kb chromosomal segment containing 11 genes was deleted in the E. hormaechei SCV isolate. Each SCV had at least one coding change in a gene associated with bacterial oxidative respiration and another involved in iron capture. Chemical and genetic rescue confirmed defects in heme biosynthesis for E. coli and C. freundii and lipoic acid biosynthesis in E. hormaachei were responsible for the SCV phenotype. Prototrophic growth in all 3 SCV Enterobacteriaceae species was unaffected under anaerobic culture conditions in vitro, illustrating how SCVs may persist in vivo.
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http://dx.doi.org/10.1038/s41598-021-86764-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018945PMC
April 2021

Prospective mapping of viral mutations that escape antibodies used to treat COVID-19.

Science 2021 02 25;371(6531):850-854. Epub 2021 Jan 25.

Basic Sciences and Computational Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

Antibodies are a potential therapy for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the risk of the virus evolving to escape them remains unclear. Here we map how all mutations to the receptor binding domain (RBD) of SARS-CoV-2 affect binding by the antibodies in the REGN-COV2 cocktail and the antibody LY-CoV016. These complete maps uncover a single amino acid mutation that fully escapes the REGN-COV2 cocktail, which consists of two antibodies, REGN10933 and REGN10987, targeting distinct structural epitopes. The maps also identify viral mutations that are selected in a persistently infected patient treated with REGN-COV2 and during in vitro viral escape selections. Finally, the maps reveal that mutations escaping the individual antibodies are already present in circulating SARS-CoV-2 strains. These complete escape maps enable interpretation of the consequences of mutations observed during viral surveillance.
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http://dx.doi.org/10.1126/science.abf9302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7963219PMC
February 2021

Prospective mapping of viral mutations that escape antibodies used to treat COVID-19.

bioRxiv 2020 Dec 1. Epub 2020 Dec 1.

Basic Sciences and Computational Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109.

Antibodies are becoming a frontline therapy for SARS-CoV-2, but the risk of viral evolutionary escape remains unclear. Here we map how all mutations to SARS-CoV-2's receptor-binding domain (RBD) affect binding by the antibodies in Regeneron's REGN-COV2 cocktail and Eli Lilly's LY-CoV016. These complete maps uncover a single amino-acid mutation that fully escapes the REGN-COV2 cocktail, which consists of two antibodies targeting distinct structural epitopes. The maps also identify viral mutations that are selected in a persistently infected patient treated with REGN-COV2, as well as in lab viral escape selections. Finally, the maps reveal that mutations escaping each individual antibody are already present in circulating SARS-CoV-2 strains. Overall, these complete escape maps enable immediate interpretation of the consequences of mutations observed during viral surveillance.
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http://dx.doi.org/10.1101/2020.11.30.405472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724661PMC
December 2020

Estimation of Full-Length TprK Diversity in Treponema pallidum subsp. .

mBio 2020 10 27;11(5). Epub 2020 Oct 27.

Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA

Immune evasion and disease progression of subsp. are associated with sequence diversity in the hypervariable outer membrane protein TprK. Previous attempts to study variation within TprK have sequenced at depths insufficient to fully appreciate the hypervariable nature of the protein, failed to establish linkage between the protein's seven variable regions, or were conducted on isolates passed through rabbits. As a consequence, a complete profile of during infection in the human host is still lacking. Furthermore, prior studies examining how subsp. uses its repertoire of genomic donor sites to generate diversity within the variable regions of the have yielded a partial understanding of this process due to the limited number of alleles examined. In this study, we used short- and long-read deep sequencing to directly characterize full-length alleles from subsp. collected from early lesions of patients attending two sexually transmitted infection clinics in Italy. We demonstrate that strains collected from cases of secondary syphilis contain significantly more unique variable region sequences and full-length TprK sequences than those from cases of primary syphilis. Our data, combined with recent data available on Chinese subsp. specimens, show the near-complete absence of overlap in TprK sequences among the 41 specimens profiled to date. We further estimate that the potential antigenic variability carried by TprK rivals that of current estimates of the human adaptive immune system. These data underscore the immunoevasive ability of TprK that allows subsp. to establish lifelong infection. Syphilis continues to be a significant public health issue in both low- and high-income countries, including the United States where the rate of syphilis infection has increased over the past 5 years. subsp. , the causative agent of syphilis, carries the outer membrane protein TprK that undergoes segmental gene conversion to constantly create new sequences. We performed full-length deep sequencing of TprK to examine TprK diversity in clinical subsp. strains. We then combined our results with data from all samples for which TprK deep sequencing results were available. We found almost no overlap in TprK sequences between different patients. Moreover, our data allowed us to estimate the total number of TprK variants that subsp. can potentially generate. Our results support how the subsp. TprK antigenic variation system is an equal adversary of the human immune system leading to pathogen persistence in the host.
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http://dx.doi.org/10.1128/mBio.02726-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593977PMC
October 2020

Inhibition of Coronavirus Entry and by a Lipid-Conjugated Peptide Derived from the SARS-CoV-2 Spike Glycoprotein HRC Domain.

mBio 2020 10 20;11(5). Epub 2020 Oct 20.

Department of Pediatrics, Columbia University Medical Center, New York, New York, USA

The emergence of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the etiological agent of the 2019 coronavirus disease (COVID-19), has erupted into a global pandemic that has led to tens of millions of infections and hundreds of thousands of deaths worldwide. The development of therapeutics to treat infection or as prophylactics to halt viral transmission and spread is urgently needed. SARS-CoV-2 relies on structural rearrangements within a spike (S) glycoprotein to mediate fusion of the viral and host cell membranes. Here, we describe the development of a lipopeptide that is derived from the C-terminal heptad repeat (HRC) domain of SARS-CoV-2 S that potently inhibits infection by SARS-CoV-2. The lipopeptide inhibits cell-cell fusion mediated by SARS-CoV-2 S and blocks infection by live SARS-CoV-2 in Vero E6 cell monolayers more effectively than previously described lipopeptides. The SARS-CoV-2 lipopeptide exhibits broad-spectrum activity by inhibiting cell-cell fusion mediated by SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV) and blocking infection by live MERS-CoV in cell monolayers. We also show that the SARS-CoV-2 HRC-derived lipopeptide potently blocks the spread of SARS-CoV-2 in human airway epithelial (HAE) cultures, an model designed to mimic respiratory viral propagation in humans. While viral spread of SARS-CoV-2 infection was widespread in untreated airways, those treated with SARS-CoV-2 HRC lipopeptide showed no detectable evidence of viral spread. These data provide a framework for the development of peptide therapeutics for the treatment of or prophylaxis against SARS-CoV-2 as well as other coronaviruses. SARS-CoV-2, the causative agent of COVID-19, continues to spread globally, placing strain on health care systems and resulting in rapidly increasing numbers of cases and mortalities. Despite the growing need for medical intervention, no FDA-approved vaccines are yet available, and treatment has been limited to supportive therapy for the alleviation of symptoms. Entry inhibitors could fill the important role of preventing initial infection and preventing spread. Here, we describe the design, synthesis, and evaluation of a lipopeptide that is derived from the HRC domain of the SARS-CoV-2 S glycoprotein that potently inhibits fusion mediated by SARS-CoV-2 S glycoprotein and blocks infection by live SARS-CoV-2 in both cell monolayers () and human airway tissues (). Our results highlight the SARS-CoV-2 HRC-derived lipopeptide as a promising therapeutic candidate for SARS-CoV-2 infections.
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http://dx.doi.org/10.1128/mBio.01935-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587434PMC
October 2020

Chlorhexidine gluconate bathing in children with cancer or those undergoing hematopoietic stem cell transplantation: A double-blinded randomized controlled trial from the Children's Oncology Group.

Cancer 2020 01 20;127(1):56-66. Epub 2020 Oct 20.

Division of Haematology/Oncology, Program in Child Health Evaluative Sciences, The Hospital for Sick Children, Toronto, Ontario, Canada.

Background: To the authors' knowledge, information regarding whether daily bathing with chlorhexidine gluconate (CHG) reduces central line-associated bloodstream infection (CLABSI) in pediatric oncology patients and those undergoing hematopoietic stem cell transplantation (HCT) is limited.

Methods: In the current multicenter, randomized, double-blind, placebo-controlled trial, patients aged ≥2 months and <22 years with cancer or those undergoing allogeneic HCT were randomized 1:1 to once-daily bathing with 2% CHG-impregnated cloths or control cloths for 90 days. The primary outcome was CLABSI. Secondary endpoints included total positive blood cultures, acquisition of resistant organisms, and acquisition of cutaneous staphylococcal isolates with an elevated CHG mean inhibitory concentration.

Results: The study was stopped early because of poor accrual. Among the 177 enrolled patients, 174 were considered as evaluable (88 were randomized to the CHG group and 86 were randomized to the control group). The rate of CLABSI per 1000 central line days in the CHG group was 5.44 versus 3.10 in the control group (risk difference, 2.37; 95% confidence interval, 0.05-4.69 [P = .049]). Post hoc conditional power analysis demonstrated a 0.2% chance that the results would have favored CHG had the study fully enrolled. The rate of total positive blood cultures did not differ between groups (risk difference, 2.37; 95% confidence interval, -0.41 to 5.14 [P = .078]). The number of patients demonstrating the new acquisition of resistant organisms did not differ between groups (P = .54). Patients in the CHG group were found to be more likely to acquire cutaneous staphylococcal isolates with an elevated CHG mean inhibitory concentration (P = .032).

Conclusions: The data from the current study do not support the use of routine CHG bathing in children with cancer or those undergoing allogeneic HCT.
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http://dx.doi.org/10.1002/cncr.33271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820990PMC
January 2020

Viral genomes reveal patterns of the SARS-CoV-2 outbreak in Washington State.

medRxiv 2020 Sep 30. Epub 2020 Sep 30.

University of Washington, Seattle, WA, USA.

The rapid spread of SARS-CoV-2 has gravely impacted societies around the world. Outbreaks in different parts of the globe are shaped by repeated introductions of new lineages and subsequent local transmission of those lineages. Here, we sequenced 3940 SARS-CoV-2 viral genomes from Washington State to characterize how the spread of SARS-CoV-2 in Washington State (USA) was shaped by differences in timing of mitigation strategies across counties, as well as by repeated introductions of viral lineages into the state. Additionally, we show that the increase in frequency of a potentially more transmissible viral variant (614G) over time can potentially be explained by regional mobility differences and multiple introductions of 614G, but not the other variant (614D) into the state. At an individual level, we see evidence of higher viral loads in patients infected with the 614G variant. However, using clinical records data, we do not find any evidence that the 614G variant impacts clinical severity or patient outcomes. Overall, this suggests that at least to date, the behavior of individuals has been more important in shaping the course of the pandemic than changes in the virus.
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http://dx.doi.org/10.1101/2020.09.30.20204230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536883PMC
September 2020

Neutralizing Antibodies Correlate with Protection from SARS-CoV-2 in Humans during a Fishery Vessel Outbreak with a High Attack Rate.

J Clin Microbiol 2020 10 21;58(11). Epub 2020 Oct 21.

Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, Washington, USA

The development of vaccines against SARS-CoV-2 would be greatly facilitated by the identification of immunological correlates of protection in humans. However, to date, studies on protective immunity have been performed only in animal models and correlates of protection have not been established in humans. Here, we describe an outbreak of SARS-CoV-2 on a fishing vessel associated with a high attack rate. Predeparture serological and viral reverse transcription-PCR (RT-PCR) testing along with repeat testing after return to shore was available for 120 of the 122 persons on board over a median follow-up of 32.5 days (range, 18.8 to 50.5 days). A total of 104 individuals had an RT-PCR-positive viral test with a cycle threshold ( ) of <35 or seroconverted during the follow-up period, yielding an attack rate on board of 85.2% (104/122 individuals). Metagenomic sequencing of 39 viral genomes suggested that the outbreak originated largely from a single viral clade. Only three crew members tested seropositive prior to the boat's departure in initial serological screening and also had neutralizing and spike-reactive antibodies in follow-up assays. None of the crew members with neutralizing antibody titers showed evidence of bona fide viral infection or experienced any symptoms during the viral outbreak. Therefore, the presence of neutralizing antibodies from prior infection was significantly associated with protection against reinfection (Fisher's exact test,  = 0.002).
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http://dx.doi.org/10.1128/JCM.02107-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587101PMC
October 2020

Neutralizing antibodies correlate with protection from SARS-CoV-2 in humans during a fishery vessel outbreak with high attack rate.

medRxiv 2020 Aug 14. Epub 2020 Aug 14.

Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, WA.

The development of vaccines against SARS-CoV-2 would be greatly facilitated by the identification of immunological correlates of protection in humans. However, to date, studies on protective immunity have only been performed in animal models and correlates of protection have not been established in humans. Here, we describe an outbreak of SARS-CoV-2 on a fishing vessel associated with a high attack rate. Predeparture serological and viral RT-PCR testing along with repeat testing after return to shore was available for 120 of the 122 persons on board over a median follow-up of 32.5 days (range 18.8 to 50.5 days). A total of 104 individuals had an RT-PCR positive viral test with Ct <35 or seroconverted during the follow-up period, yielding an attack rate on board of 85.2% (104/122 individuals). Metagenomic sequencing of 39 viral genomes suggested the outbreak originated largely from a single viral clade. Only three crewmembers tested seropositive prior to the boat's departure in initial serological screening and also had neutralizing and spike-reactive antibodies in follow-up assays. None of these crewmembers with neutralizing antibody titers showed evidence of bona fide viral infection or experienced any symptoms during the viral outbreak. Therefore, the presence of neutralizing antibodies from prior infection was significantly associated with protection against re-infection (Fisher's exact test, p=0.002).
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http://dx.doi.org/10.1101/2020.08.13.20173161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430625PMC
August 2020

Comparative genomics and full-length Tprk profiling of Treponema pallidum subsp. pallidum reinfection.

PLoS Negl Trop Dis 2020 04 6;14(4):e0007921. Epub 2020 Apr 6.

Department of Laboratory Medicine, University of Washington, Seattle, Washington, United States of America.

Developing a vaccine against Treponema pallidum subspecies pallidum, the causative agent of syphilis, remains a public health priority. Syphilis vaccine design efforts have been complicated by lack of an in vitro T. pallidum culture system, prolific antigenic variation in outer membrane protein TprK, and lack of functional annotation for nearly half of the genes. Understanding the genetic basis of T. pallidum reinfection can provide insights into variation among strains that escape cross-protective immunity. Here, we present comparative genomic sequencing and deep, full-length tprK profiling of two T. pallidum isolates from blood from the same patient that were collected six years apart. Notably, this patient was diagnosed with syphilis four times, with two of these episodes meeting the definition of neurosyphilis, during this interval. Outside of the highly variable tprK gene, we identified 14 coding changes in 13 genes. Nine of these genes putatively localized to the periplasmic or outer membrane spaces, consistent with a potential role in serological immunoevasion. Using a newly developed full-length tprK deep sequencing protocol, we profiled the diversity of this gene that far outpaces the rest of the genome. Intriguingly, we found that the reinfecting isolate demonstrated less diversity across each tprK variable region compared to the isolate from the first infection. Notably, the two isolates did not share any full-length TprK sequences. Our results are consistent with an immunodominant-evasion model in which the diversity of TprK explains the ability of T. pallidum to successfully reinfect individuals, even when they have been infected with the organism multiple times.
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http://dx.doi.org/10.1371/journal.pntd.0007921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7162541PMC
April 2020

Transfer of skin microbiota between two dissimilar autologous microenvironments: A pilot study.

PLoS One 2019 30;14(12):e0226857. Epub 2019 Dec 30.

Seattle Children's Hospital, Seattle, WA, United States of America.

Dysbiosis of skin microbiota is associated with several inflammatory skin conditions, including atopic dermatitis, acne, and hidradenitis suppurativa. There is a surge of interest by clinicians and the lay public to explore targeted bacteriotherapy to treat these dermatologic conditions. To date, skin microbiota transplantation studies have focused on moving single, enriched strains of bacteria to target sites rather than a whole community. In this prospective pilot study, we examined the feasibility of transferring unenriched skin microbiota communities between two anatomical sites of the same host. We enrolled four healthy volunteers (median age: 28 [range: 24, 36] years; 2 [50%] female) who underwent collection and transfer of skin microbiota from the forearm to the back unidirectionally. Using culture methods and 16S rRNA V1-V3 deep sequencing, we compared baseline and mixed ("transplant") communities, at T = 0 and T = 24 hours. Our ability to detect movement from one site to the other relied on the inherent diversity of the microenvironment of the antecubital fossa relative to the less diverse back. Comparing bacterial species present in the arm and mixed ("transplant") communities that were absent from the baseline back, we saw evidence of transfer of a partial DNA signature; our methods limit conclusions regarding the viability of transferred organisms. We conclude that unenriched transfer of whole cutaneous microbiota is challenging, but our simple technique, intended to move viable skin organisms from one site to another, is worthy of further investigation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0226857PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936845PMC
April 2020

International Spread of Multidrug-Resistant Campylobacter coli in Men Who Have Sex With Men in Washington State and Québec, 2015-2018.

Clin Infect Dis 2020 11;71(8):1896-1904

Departments of Laboratory Medicine and Microbiology, University of Washington School of Medicine, Seattle, Washington, USA.

Background: Campylobacter species are among the most common causes of enteric bacterial infections worldwide. Men who have sex with men (MSM) are at increased risk for sexually transmitted enteric infections, including globally distributed strains of multidrug-resistant Shigella species.

Methods: This was a retrospective study of MSM-associated Campylobacter in Seattle, Washington and Montréal, Québec with phenotypic antimicrobial resistance profiles and whole genome sequencing (WGS).

Results: We report the isolation of 2 clonal lineages of multidrug-resistant Campylobacter coli from MSM in Seattle and Montréal. WGS revealed nearly identical strains obtained from the 2 regions over a 4-year period. Comparison with the National Center for Biotechnology Information's Pathogen Detection database revealed extensive Campylobacter species clusters carrying multiple drug resistance genes that segregated with these isolates. Examination of the genetic basis of antimicrobial resistance revealed multiple macrolide resistance determinants including a novel ribosomal RNA methyltransferase situated in a CRISPR (clustered regularly interspaced short palindromic repeats) array locus in a C. coli isolate.

Conclusions: As previously reported for Shigella, specific multidrug-resistant strains of Campylobacter are circulating by sexual transmission in MSM populations across diverse geographic locations, suggesting a need to incorporate sexual behavior in the investigation of clusters of foodborne pathogens revealed by WGS data.
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http://dx.doi.org/10.1093/cid/ciz1060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7643735PMC
November 2020

A Novel, Widespread Allele Results in Reduced Chlorhexidine Susceptibility in .

Antimicrob Agents Chemother 2019 06 24;63(6). Epub 2019 May 24.

Seattle Children's Research Institute, Seattle, Washington, USA

Chlorhexidine gluconate (CHG) is a topical antiseptic widely used in health care settings. In spp., the pump QacA effluxes CHG, while the closely related QacB cannot due to a single amino acid substitution. We characterized 1,050 cutaneous isolates obtained from 173 pediatric oncology patients enrolled in a multicenter CHG bathing trial. CHG susceptibility testing revealed that 63 (6%) of these isolates had elevated CHG MICs (≥4 μg/ml). Screening of all 1,050 isolates for the gene (the same gene with A or B allele) by restriction fragment length polymorphism (RFLP) yielded 56 isolates with a novel RFLP pattern, The CHG MIC was significantly higher for -positive isolates (MIC, 4 μg/ml; MIC range, 0.5 to 4 μg/ml) than for other groups: -positive isolates ( = 559; MIC, 1 μg/ml; MIC range, 0.5 to 4 μg/ml), -positive isolates ( = 17; MIC, 1 μg/ml; MIC range, 0.25 to 2 μg/ml), and -negative isolates ( = 418, MIC, 1 μg/ml; MIC range, 0.125 to 2 μg/ml) ( = 0.001). A high proportion of the -positive isolates also displayed methicillin resistance (96.4%) compared to the other groups (24.9 to 61.7%) ( = 0.001). Whole-genome sequencing revealed that -positive isolates encoded a variant of QacA with 2 amino acid substitutions. This new allele, named , was carried on the novel plasmid pAQZ1. The -carrying isolates belonged to the highly resistant sequence type 2 clone. By searching available sequence data sets, we identified 39 additional -carrying strains from 5 countries. Curing an isolate of resulted in a 4-fold decrease in the CHG MIC, confirming the role of in the elevated CHG MIC. Our results highlight the importance of further studying and its functional role in clinical staphylococci.
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http://dx.doi.org/10.1128/AAC.02607-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6535555PMC
June 2019

Heterogeneous Antimicrobial Susceptibility Characteristics in Isolates from Cystic Fibrosis Patients.

mSphere 2018 Mar-Apr;3(2). Epub 2018 Mar 14.

Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA.

Clinical isolates of from patients with cystic fibrosis (CF) are known to differ from those associated with non-CF hosts by colony morphology, drug susceptibility patterns, and genomic hypermutability. isolates from CF patients have long been recognized for their overall reduced rate of antimicrobial susceptibility, but their intraclonal MIC heterogeneity has long been overlooked. Using two distinct cohorts of clinical strains ( = 224 from 56 CF patients, = 130 from 68 non-CF patients) isolated in 2013, we demonstrated profound Etest MIC heterogeneity in CF isolates in comparison to non-CF isolates. On the basis of whole-genome sequencing of 19 CF isolates from 9 patients with heterogeneous MICs, the core genome phylogenetic tree confirmed the within-patient CF clonal lineage along with considerable coding sequence variability. No extrachromosomal DNA elements or previously characterized antibiotic resistance mutations could account for the wide divergence in antimicrobial MICs between coisolates, though many heterogeneous mutations in efflux and porin genes and their regulators were present. A unique OprD sequence was conserved among the majority of isolates of CF analyzed, suggesting a pseudomonal response to selective pressure that is common to the isolates. Genomic sequence data also suggested that CF pseudomonal hypermutability was not entirely due to mutations in , , and . We conclude that the net effect of hundreds of adaptive mutations, both shared between clonally related isolate pairs and unshared, accounts for their highly heterogeneous MIC variances. We hypothesize that this heterogeneity is indicative of the pseudomonal syntrophic-like lifestyle under conditions of being "locked" inside a host focal airway environment for prolonged periods. Patients with cystic fibrosis endure "chronic focal infections" with a variety of microorganisms. One microorganism, , adapts to the host and develops resistance to a wide range of antimicrobials. Interestingly, as the infection progresses, multiple isogenic strains of emerge and coexist within the airways of these patients. Despite a common parental origin, the multiple strains of develop vastly different susceptibility patterns to actively used antimicrobial agents-a phenomenon we define as "heterogeneous MICs." By sequencing pairs of isolates displaying heterogeneous MICs, we observed widespread isogenic gene lesions in drug transporters, DNA mismatch repair machinery, and many other structural or cellular functions. Coupled with the heterogeneous MICs, these genetic lesions demonstrated a symbiotic response to host selection and suggested evolution of a multicellular syntrophic bacterial lifestyle. Current laboratory standard interpretive criteria do not address the emergence of heterogeneous growth and susceptibilities with treatment implications.
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http://dx.doi.org/10.1128/mSphere.00615-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5853491PMC
March 2018
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