Jason T. Ladner

Introduction: Vaccination aims to prevent or mitigate disease by priming the immune system prior to infection. While historical vaccine development relied mostly on trial-and-error, modern approaches have become more directed. By leveraging our growing understanding of pathogen biology and immune correlates of protection, we can design vaccines in ways that promote protective responses. However, the complexity of many pathogens (e.g., bacteria and fungi), as well as our immune responses against them, continue to present important challenges for vaccine development.Aim: Here, we demonstrate the utility of the PepSeq platform for highly multiplexed serology to both broadly and finely characterize antibody responses against complex pathogens, using the bacterium, Burkholderia pseudomallei, as a case study.Methods: We designed and synthesized three diverse pools of DNA-barcoded peptides (i.e., PepSeq libraries) and used them to characterize antibodies against a variety of B. pseudomallei proteins.Results: Epitope-resolved antibody binding profiles were generated for 85 individuals with culture-confirmed melioidosis, 89 US blood bank controls, and 6 monoclonal antibodies. Using these data, we identify novel B cell antigens/epitopes and finely characterize the epitopes of three monoclonal antibodies against the B. pseudomallei GroEL protein.Conclusion: Highly multiplexed serology platforms, like PepSeq, enable more comprehensive characterization of antibodies, both polyclonal and monoclonal, which can aid in the development of vaccines, diagnostics and therapeutics, even for pathogens with large, complex genomes.

Despite advancements in medical interventions, the disease burden caused by viral pathogens remains large and highly diverse. This burden includes the wide range of signs and symptoms associated with active viral replication as well as a variety of clinical sequelae of infection. Moreover, there is growing evidence supporting the existence of sex- and ethnicity-based health disparities linked to viral infections and their associated diseases. Despite several well-documented disparities in viral infection rates, our current understanding of virus-associated health disparities remains incomplete. This knowledge gap can be attributed, in part, to limitations of the most commonly used viral detection methodologies, which lack the breadth needed to characterize exposures across the entire virome. Additionally, virus-related health disparities are dynamic and often differ considerably through space and time. In this study, we utilize PepSeq, an approach for highly multiplexed serology, to broadly assess an individual’s history of viral exposures, and we demonstrate the effectiveness of this approach for detecting infection disparities through a pilot study of 400 adults aged 30–60 in Phoenix, AZ. Using a human virome PepSeq library, we observed expected seroprevalence rates for several common viruses and detected both expected and previously undocumented differences in inferred rates of infection between our male/female and Hispanic/non-Hispanic White individuals. IMPORTANCE: Our understanding of population-level virus infection rates and associated health disparities is incomplete. In part, this is because of the high diversity of human-infecting viruses and the limited breadth and sensitivity of traditional approaches for detecting infection events. Here, we demonstrate the potential for modern, highly multiplexed antibody detection methods to greatly increase our understanding of disparities in rates of infection across subpopulations (e.g., different sexes or ethnic groups). The use of antibodies as biomarkers allows us to detect evidence of past infections over an extended period, and our approach for highly multiplexed serology (PepSeq) allows us to measure antibody responses against hundreds of viruses in an efficient and cost-effective manner.

Despite advancements in medical interventions, the disease burden caused by viral pathogens remains large and highly diverse. This burden includes the wide range of signs and symptoms associated with active viral replication as well as a variety of clinical sequelae of infection. Moreover, there is growing evidence supporting the existence of sex– and ethnicity-based health disparities linked to viral infections and their associated diseases. Despite several well-documented disparities in viral infection rates, our current understanding of virus-associated health disparities remains incomplete. This knowledge gap can be attributed, in part, to limitations of the most commonly used viral detection methodologies, which lack the breadth needed to characterize exposures across the entire virome. Additionally, virus-related health disparities are dynamic and often differ considerably through space and time. In this study, we utilize PepSeq, an approach for highly-multiplexed serology, to broadly assess an individual’s history of viral exposures, and we demonstrate the effectiveness of this approach for detecting infection disparities through a pilot study of 400 adults aged 30-60 in Phoenix, AZ. Using a human virome PepSeq library, we observed expected seroprevalence rates for several common viruses and detected both expected and previously undocumented differences in inferred rates of infection between our Hispanic White and non-Hispanic White individuals. Importance Our understanding of population-level virus infection rates and associated health disparities is incomplete. In part, this is because of the high diversity of human-infecting viruses and the limited breadth and sensitivity of traditional approaches for detecting infection events. Here, we demonstrate the potential for modern, highly-multiplexed antibody detection methods to greatly increase our understanding of disparities in rates of infection across subpopulations (e.g., different sexes or ethnic groups). The use of antibodies as biomarkers allows us to detect evidence of past infections over an extended period of time, and our approach for highly-multiplexed serology (PepSeq) allows us to measure antibody responses against 100s of viruses in an efficient and cost-effective manner.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages of the Omicron variant rapidly became dominant in early 2022 and frequently cause human infections despite vaccination or prior infection with other variants. In addition to antibody-evading mutations in the receptor-binding domain, Omicron features amino acid mutations elsewhere in the Spike protein; however, their effects generally remain ill defined. The Spike D796Y substitution is present in all Omicron sub-variants and occurs at the same site as a mutation (D796H) selected during viral evolution in a chronically infected patient. Here, we map antibody reactivity to a linear epitope in the Spike protein overlapping position 796. We show that antibodies binding this region arise in pre-Omicron SARS-CoV-2 convalescent and vaccinated subjects but that both D796Y and D796H abrogate their binding. These results suggest that D796Y contributes to the fitness of Omicron in hosts with pre-existing immunity to other variants of SARS-CoV-2 by evading antibodies targeting this site. IMPORTANCE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved substantially through the coronavirus disease 2019 (COVID-19) pandemic: understanding the drivers and consequences of this evolution is essential for projecting the course of the pandemic and developing new countermeasures. Here, we study the immunological effects of a particular mutation present in the Spike protein of all Omicron strains and find that it prevents the efficient binding of a class of antibodies raised by pre-Omicron vaccination and infection. These findings reveal a novel consequence of a poorly understood Omicron mutation and shed light on the drivers and effects of SARS-CoV-2 evolution.

SARS-CoV-2 lineages of the Omicron variant rapidly became dominant in early 2022 and frequently cause human infections despite vaccination or prior infection with other variants. In addition to antibody-evading mutations in the Receptor Binding Domain, Omicron features amino acid mutations elsewhere in the Spike protein, however their effects generally remain ill-defined. The Spike D796Y substitution is present in all Omicron sub-variants and occurs at the same site as a mutation (D796H) selected during viral evolution in a chronically-infected patient. Here we map antibody reactivity to a linear epitope in the Spike protein overlapping position 796. We show that antibodies binding this region arise in pre-Omicron SARS-CoV-2 convalescent and vaccinated subjects, but that both D796Y and D796H abrogate their binding. These results suggest that D796Y contributes to the fitness of Omicron in hosts with pre-existing immunity to other variants of SARS-CoV-2 by evading antibodies targeting this site.

Pathogen genome sequencing has become a routine part of our response to active outbreaks of infectious disease and should be an important part of our preparations for future epidemics. In this Essay, we discuss the innovations that have enabled routine pathogen genome sequencing, as well as how genome sequences can be used to understand and control the spread of infectious disease. We also explore the impact of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic on the field of pathogen genomics and outline the challenges we must address to further improve the utility of pathogen genome sequencing in the future.

Current methods for detecting infections either require a sample collected from an actively infected site, are limited in the number of agents they can query, and/or yield no information on the immune response. Here we present an approach that uses temporally coordinated changes in highly-multiplexed antibody measurements from longitudinal blood samples to monitor infection events at sub-species resolution across the human virome. In a longitudinally-sampled cohort of South African adolescents representing >100 person-years, we identify >650 events across 48 virus species and observe strong epidemic effects, including high-incidence waves ofAichivirus Aand the D68 subtype ofEnterovirus Dearlier than their widespread circulation was appreciated. In separate cohorts of adults who were sampled at higher frequency using self-collected dried blood spots, we show that such events temporally correlate with symptoms and transient inflammatory biomarker elevations, and observe the responding antibodies to persist for periods ranging from ≤1 week to >5 years. Our approach generates a rich view of viral/host dynamics, supporting novel studies in immunology and epidemiology.

PepSeq is an in vitro platform for building and conducting highly multiplexed proteomic assays against customizable targets by using DNA-barcoded peptides. Starting with a pool of DNA oligonucleotides encoding peptides of interest, this protocol outlines a fully in vitro and massively parallel procedure for synthesizing the encoded peptides and covalently linking each to a corresponding cDNA tag. The resulting libraries of peptide/DNA conjugates can be used for highly multiplexed assays that leverage high-throughput sequencing to profile the binding or enzymatic specificities of proteins of interest. Here, we describe the implementation of PepSeq for fast and cost-effective epitope-level analysis of antibody reactivity across hundreds of thousands of peptides from <1 µl of serum or plasma input. This protocol includes the design of the DNA oligonucleotide library, synthesis of DNA-barcoded peptide constructs, binding of constructs to sample, preparation for sequencing and data analysis. Implemented in this way, PepSeq can be used for a number of applications, including fine-scale mapping of antibody epitopes and determining a subject’s pathogen exposure history. The protocol is divided into two main sections: (i) design and synthesis of DNA-barcoded peptide libraries and (ii) use of libraries for highly multiplexed serology. Once oligonucleotide templates are in hand, library synthesis takes 1–2 weeks and can provide enough material for hundreds to thousands of assays. Serological assays can be conducted in 96-well plates and generate sequencing data within a further ~4 d. A suite of software tools, including the PepSIRF package, are made available to facilitate the design of PepSeq libraries and analysis of assay data.

Antibodies are generated in response to infections with viruses and other pathogens, and they help protect against future exposures. Mature antibodies are long lived, are highly specific, and can bind to their protein targets with high affinity.