Efficient encoding of large antigenic spaces by epitope prioritization with Dolphyn.

We investigate a relatively underexplored component of the gut-immune axis by profiling the antibody response to gut phages using Phage Immunoprecipitation Sequencing (PhIP-Seq). To cover large antigenic spaces, we develop Dolphyn, a method that uses machine learning to select peptides from protein sets and compresses the proteome through epitope-stitching. Dolphyn compresses the size of a peptide library by 78% compared to traditional tiling, increasing the antibody-reactive peptides from 10% to 31%. We find that the immune system develops antibodies to human gut bacteria-infecting viruses, particularly E.coli-infecting Myoviridae. Cost-effective PhIP-Seq libraries designed with Dolphyn enable the assessment of a wider range of proteins in a single experiment, thus facilitating the study of the gut-immune axis.

Epitope Mapping of SARS-CoV-2 Spike Antibodies in Vaccinated Kidney Transplant Recipients Reveals Poor Spike Coverage Compared to Healthy Controls.

Kidney transplant recipients (KTRs) develop decreased antibody titers to SARS-CoV-2 vaccination compared to healthy controls (HCs), but whether KTRs generate antibodies against key epitopes associated with neutralization is unknown. Plasma from 78 KTRs from a clinical trial of third doses of SARS-CoV-2 vaccines and 12 HCs underwent phage display immunoprecipitation and sequencing (PhIP-Seq) to map antibody responses against SARS-CoV-2. KTRs had lower antibody reactivity to SARS-CoV-2 than HCs, but KTRs and HCs recognized similar epitopes associated with neutralization. Thus, epitope gaps in antibody breadth of KTRs are unlikely responsible for decreased efficacy of SARS-CoV-2 vaccines in this immunosuppressed population.

A robust pipeline for high-content, high-throughput immunophenotyping reveals age- and genetics-dependent changes in blood leukocytes.

High-dimensional flow cytometry is the gold standard to study the human immune system in large cohorts. However, large sample sizes increase inter-experimental variation because of technical and experimental inaccuracies introduced by batch variability. Our high-throughput sample processing pipeline in combination with 28-color flow cytometry focuses on increased throughput (192 samples/experiment) and high reproducibility. We implemented quality control checkpoints to reduce technical and experimental variation. Finally, we integrated FlowSOM clustering to facilitate automated data analysis and demonstrate the reproducibility of our pipeline in a study with 3,357 samples. We reveal age-associated immune dynamics in 2,300 individuals, signified by decreasing T and B cell subsets with age. In addition, by combining genetic analyses, our approach revealed unique immune signatures associated with a single nucleotide polymorphism (SNP) that abrogates CD45 isoform splicing. In summary, we provide a versatile and reliable high-throughput, flow cytometry-based pipeline for immune discovery and exploration in large cohorts.

Applications of VirScan to broad serological profiling of bat reservoirs for emerging zoonoses.

Introduction: Bats are important providers of ecosystem services such as pollination, seed dispersal, and insect control but also act as natural reservoirs for virulent zoonotic viruses. Bats host multiple viruses that cause life-threatening pathology in other animals and humans but, themselves, experience limited pathological disease from infection. Despite bats' importance as reservoirs for several zoonotic viruses, we know little about the broader viral diversity that they host. Bat virus surveillance efforts are challenged by difficulties of field capture and the limited scope of targeted PCR- or ELISA-based molecular and serological detection. Additionally, virus shedding is often transient, thus also limiting insights gained from nucleic acid testing of field specimens. Phage ImmunoPrecipitation Sequencing (PhIP-Seq), a broad serological tool used previously to comprehensively profile viral exposure history in humans, offers an exciting prospect for viral surveillance efforts in wildlife, including bats. Methods: Here, for the first time, we apply PhIP-Seq technology to bat serum, using a viral peptide library originally designed to simultaneously assay exposures to the entire human virome. Results: Using VirScan, we identified past exposures to 57 viral genera-including betacoronaviruses, henipaviruses, lyssaviruses, and filoviruses-in semi-captive Pteropus alecto and to nine viral genera in captive Eonycteris spelaea. Consistent with results from humans, we find that both total peptide hits (the number of enriched viral peptides in our library) and the corresponding number of inferred past virus exposures in bat hosts were correlated with poor bat body condition scores and increased with age. High and low body condition scores were associated with either seropositive or seronegative status for different viruses, though in general, virus-specific age-seroprevalence curves defied assumptions of lifelong immunizing infection, suggesting that many bat viruses may circulate via complex transmission dynamics. Discussion: Overall, our work emphasizes the utility of applying biomedical tools, like PhIP-Seq, first developed for humans to viral surveillance efforts in wildlife, while highlighting opportunities for taxon-specific improvements.

Comprehensive profiling of pre-infection antibodies identifies HIV targets associated with viremic control and viral load.

Background: High HIV viral load (VL) is associated with increased transmission risk and faster disease progression. HIV controllers achieve viral suppression without antiretroviral (ARV) treatment. We evaluated viremic control in a community-randomized trial with >48,000 participants. Methods: A massively multiplexed antibody profiling system, VirScan, was used to quantify pre- and post-infection antibody reactivity to HIV peptides in 664 samples from 429 participants (13 controllers, 135 viremic non-controllers, 64 other non-controllers, 217 uninfected persons). Controllers had VLs <2,000 copies/mL with no ARV drugs detected at the first HIV-positive visit and one year later. Viremic non-controllers had VLs 2,000 copies/mL with no ARV drugs detected at the first HIV-positive visit. Other non-controllers had either ARV drugs detected at the first HIV-positive visit (n=47) or VLs <2,000 copies/mL with no ARV drugs detected at only one HIV-positive visit (n=17). Results: We identified pre-infection HIV antibody reactivities that correlated with post-infection VL. Pre-infection reactivity to an epitope in the HR2 domain of gp41 was associated with controller status and lower VL. Pre-infection reactivity to an epitope in the C2 domain of gp120 was associated with non-controller status and higher VL. Different patterns of antibody reactivity were observed over time for these two epitopes. Conclusion: These studies suggest that pre-infection HIV antibodies are associated with controller status and modulation of HIV VL. These findings may inform research on antibody-based interventions for HIV treatment.

Barcoding intracellular reverse transcription enables high-throughput phenotype-coupled T cell receptor analyses.

Assays linking cellular phenotypes with T cell or B cell antigen receptor sequences are crucial for characterizing adaptive immune responses. Existing methodologies are limited by low sample throughput and high cost. Here, we present INtraCEllular Reverse Transcription with Sorting and sequencing (INCERTS), an approach that combines molecular indexing of receptor repertoires within intact cells and fluorescence-activated cell sorting (FACS). We demonstrate that INCERTS enables efficient processing of millions of cells from pooled human peripheral blood mononuclear cell (PBMC) samples while retaining robust association between T cell receptor (TCR) sequences and cellular phenotypes. We used INCERTS to discover antigen-specific TCRs from patients with cancer immunized with a novel mutant KRAS peptide vaccine. After ex vivo stimulation, 28 uniquely barcoded samples were pooled prior to FACS into peptide-reactive and non-reactive CD4+ and CD8+ populations. Combining complementary patient-matched single-cell RNA sequencing (scRNA-seq) data enabled retrieval of full-length, paired TCR alpha and beta chain sequences for future validation of therapeutic utility.

Transplantation elicits a clonally diverse CD8+ T cell response that is comprised of potent CD43+ effectors.

CD8+ T cells mediate acute rejection of allografts, which threatens the long-term survival of transplanted organs. Using MHC class I tetramers, we find that allogeneic CD8+ T cells are present at an elevated naive precursor frequency relative to other epitopes, only modestly increase in number after grafting, and maintain high T cell receptor diversity throughout the immune response. While antigen-specific effector CD8+ T cells poorly express the canonical effector marker KLRG-1, expression of the activated glycoform of CD43 defines potent effectors after transplantation. Activated CD43+ effector T cells maintain high expression of the coreceptor induced T cell costimulator (ICOS) in the presence of CTLA-4 immunoglobulin (Ig), and dual CTLA-4 Ig/anti-ICOS treatment prolongs graft survival. These data demonstrate that graft-specific CD8+ T cells have a distinct response profile relative to anti-pathogen CD8+ T cells and that CD43 and ICOS are critical surface receptors that define potent effector CD8+ T cell populations that form after transplantation.

Efficient encoding of large antigenic spaces by epitope prioritization with Dolphyn.

We investigated a relatively underexplored component of the gut-immune axis by profiling the antibody response to gut phages using Phage Immunoprecipitation Sequencing (PhIP-Seq). To enhance this approach, we developed Dolphyn, a novel method that uses machine learning to select peptides from protein sets and compresses the proteome through epitope-stitching. Dolphyn improves the fraction of gut phage library peptides bound by antibodies from 10% to 31% in healthy individuals, while also reducing the number of synthesized peptides by 78%. In our study on gut phages, we discovered that the immune system develops antibodies to bacteria-infecting viruses in the human gut, particularly E.coli-infecting Myoviridae. Cost-effective PhIP-Seq libraries designed with Dolphyn enable the assessment of a wider range of proteins in a single experiment, thus facilitating the study of the gut-immune axis.

Coexisting autoantibodies against transcription factor Sp4 are associated with decreased cancer risk in patients with dermatomyositis with anti-TIF1γ autoantibodies.

OBJECTIVES: In dermatomyositis (DM), autoantibodies are associated with unique clinical phenotypes. For example, anti-TIF1γ autoantibodies are associated with an increased risk of cancer. The purpose of this study was to discover novel DM autoantibodies. METHODS: Phage ImmunoPrecipitation Sequencing using sera from 43 patients with DM suggested that transcription factor Sp4 is a novel autoantigen; this was confirmed by showing that patient sera immunoprecipitated full-length Sp4 protein. Sera from 371 Johns Hopkins patients with myositis (255 with DM, 28 with antisynthetase syndrome, 40 with immune-mediated necrotising myopathy, 29 with inclusion body myositis and 19 with polymyositis), 80 rheumatological disease controls (25 with Sjogren's syndrome, 25 with systemic lupus erythematosus and 30 with rheumatoid arthritis (RA)) and 200 healthy comparators were screened for anti-SP4 autoantibodies by ELISA. A validation cohort of 46 anti-TIF1γ-positive patient sera from the University of Pittsburgh was also screened for anti-Sp4 autoantibodies. RESULTS: Anti-Sp4 autoantibodies were present in 27 (10.5%) patients with DM and 1 (3.3%) patient with RA but not in other clinical groups. In patients with DM, 96.3% of anti-Sp4 autoantibodies were detected in those with anti-TIF1γ autoantibodies. Among 26 TIF1γ-positive patients with anti-Sp4 autoantibodies, none (0%) had cancer. In contrast, among 35 TIF1γ-positive patients without anti-Sp4 autoantibodies, 5 (14%, p=0.04) had cancer. In the validation cohort, among 15 TIF1γ-positive patients with anti-Sp4 autoantibodies, 2 (13.3%) had cancer. By comparison, among 31 TIF1γ-positive patients without anti-Sp4 autoantibodies, 21 (67.7%, p<0.001) had cancer. CONCLUSIONS: Anti-Sp4 autoantibodies appear to identify a subgroup of anti-TIF1γ-positive DM patients with lower cancer risk.

Comprehensive profiling of antibody responses to the human anellome using programmable phage display.

Anelloviruses represent a major constituent of the commensal human virome; however, little is known about their immunobiology. Here, we present "AnelloScan," a T7 phage library representing the open reading frame 1 (ORF1), ORF2, ORF3, and torque teno virus (TTV)-derived apoptosis-inducing protein (TAIP) sequences of more than 800 human anelloviruses and profile the antibody reactivities of serum samples from a cross-sectional cohort of 156 subjects by using phage-immunoprecipitation sequencing (PhIP-Seq). A majority of anellovirus peptides are not reactive in any of the subjects tested (n = ∼28,000; ∼85% of the library). Antibody-reactive peptides are largely restricted to the C-terminal region of the capsid protein ORF1. Moreover, using a longitudinal cohort of matched blood-transfusion donors and recipients, we find that most transmitted anelloviruses do not elicit a detectable antibody reactivity in the recipient and that the remainder elicit delayed responses appearing ∼100-150 days after transfusion.

Evaluation of multi-assay algorithms for cross-sectional HIV incidence estimation in settings with universal antiretroviral treatment.

BACKGROUND: Multi-assay algorithms (MAAs) are used to estimate population-level HIV incidence and identify individuals with recent infection. Many MAAs use low viral load (VL) as a biomarker for long-term infection. This could impact incidence estimates in settings with high rates of early HIV treatment initiation. We evaluated the performance of two MAAs that do not include VL. METHODS: Samples were collected from 219 seroconverters (infected < 1 year) and 4376 non-seroconverters (infected > 1 year) in the HPTN 071 (PopART) trial; 28.8% of seroconverter samples and 73.2% of non-seroconverter samples had VLs ≤ 400 copies/mL. Samples were tested with the Limiting Antigen Avidity assay (LAg) and JHU BioRad-Avidity assays. Antibody reactivity to two HIV peptides was measured using the MSD U-PLEX assay. Two MAAs were evaluated that do not include VL: a MAA that includes the LAg-Avidity assay and BioRad-Avidity assay (LAg + BR) and a MAA that includes the LAg-Avidity assay and two peptide biomarkers (LAg + PepPair). Performance of these MAAs was compared to a widely used MAA that includes LAg and VL (LAg + VL). RESULTS: The incidence estimate for LAg + VL (1.29%, 95% CI: 0.97-1.62) was close to the observed longitudinal incidence (1.34% 95% CI: 1.17-1.53). The incidence estimates for the other two MAAs were higher (LAg + BR: 2.56%, 95% CI 2.01-3.11; LAg + PepPair: 2.84%, 95% CI: 1.36-4.32). LAg + BR and LAg + PepPair also misclassified more individuals infected > 2 years as recently infected than LAg + VL (1.2% [42/3483 and 1.5% [51/3483], respectively, vs. 0.2% [6/3483]). LAg + BR classified more seroconverters as recently infected than LAg + VL or LAg + PepPair (80 vs. 58 and 50, respectively) and identified ~ 25% of virally suppressed seroconverters as recently infected. CONCLUSIONS: The LAg + VL MAA produced a cross-sectional incidence estimate that was closer to the longitudinal estimate than two MAAs that did not include VL. The LAg + BR MAA classified the greatest number of individual seroconverters as recently infected but had a higher false recent rate.

Detecting antibody reactivities in Phage ImmunoPrecipitation Sequencing data.

Phage ImmunoPrecipitation Sequencing (PhIP-Seq) is a recently developed technology to assess antibody reactivity, quantifying antibody binding towards hundreds of thousands of candidate epitopes. The output from PhIP-Seq experiments are read count matrices, similar to RNA-Seq data; however some important differences do exist. In this manuscript we investigated whether the publicly available method edgeR (Robinson et al., Bioinformatics 26(1):139-140, 2010) for normalization and analysis of RNA-Seq data is also suitable for PhIP-Seq data. We find that edgeR is remarkably effective, but improvements can be made and introduce a Bayesian framework specifically tailored for data from PhIP-Seq experiments (Bayesian Enrichment Estimation in R, BEER).

PhIP-Seq Reveals Autoantibodies for Ubiquitously Expressed Antigens in Viral Myocarditis.

Enteroviruses such as group B coxsackieviruses (CVB) are commonly suspected as causes of myocarditis that can lead to dilated cardiomyopathy (DCM), and the mouse model of CVB3 myocarditis is routinely used to understand DCM pathogenesis. Mechanistically, autoimmunity is suspected due to the presence of autoantibodies for select antigens. However, their role continues to be enigmatic, which also raises the question of whether the breadth of autoantibodies is sufficiently characterized. Here, we attempted to comprehensively analyze the autoantibody repertoire using Phage ImmunoPrecipitation Sequencing (PhIP-Seq), a versatile and high-throughput platform, in the mouse model of CVB3 myocarditis. First, PhIP-Seq analysis using the VirScan library revealed antibody reactivity only to CVB3 in the infected group but not in controls, thus validating the technique in this model. Second, using the mouse peptide library, we detected autoantibodies to 32 peptides from 25 proteins in infected animals that are ubiquitously expressed and have not been previously reported. Third, by using ELISA as a secondary assay, we confirmed antibody reactivity in sera from CVB3-infected animals to cytochrome c oxidase assembly factor 4 homolog (COA4) and phosphoinositide-3-kinase adaptor protein 1 (PIK3AP1), indicating the specificity of antibody detection by PhIP-Seq technology. Fourth, we noted similar antibody reactivity patterns in CVB3 and CVB4 infections, suggesting that the COA4- and PIK3AP1-reactive antibodies could be common to multiple CVB infections. The specificity of the autoantibodies was affirmed with influenza-infected animals that showed no reactivity to any of the antigens tested. Taken together, our data suggest that the autoantibodies identified by PhIP-Seq may have relevance to CVB pathogenesis, with a possibility that similar reactivity could be expected in human DCM patients.

Detecting and quantifying antibody reactivity in PhIP-Seq data with BEER.

SUMMARY: Because of their high abundance, easy accessibility in peripheral blood, and relative stability ex vivo, antibodies serve as excellent records of environmental exposures and immune responses. Phage Immuno-Precipitation Sequencing (PhIP-Seq) is the most efficient technique available for assessing antibody binding to hundreds of thousands of peptides at a cohort scale. PhIP-Seq is a high-throughput approach for assessing antibody reactivity to hundreds of thousands of candidate epitopes. Accurate detection of weakly reactive peptides is particularly important for characterizing the development and decline of antibody responses. Here, we present BEER (Bayesian Enrichment Estimation in R), a software package specifically developed for the quantification of peptide reactivity from PhIP-Seq experiments. BEER implements a hierarchical model and produces posterior probabilities for peptide reactivity and a fold change estimate to quantify the magnitude. BEER also offers functionality to infer peptide reactivity based on the edgeR package, though the improvement in speed is offset by slightly lower sensitivity compared to the Bayesian approach, specifically for weakly reactive peptides. AVAILABILITY AND IMPLEMENTATION: BEER is implemented in R and freely available from the Bioconductor repository at https://bioconductor.org/packages/release/bioc/html/beer.html.

Greater Breadth of Vaccine-Induced Immunity in Females than Males Is Mediated by Increased Antibody Diversity in Germinal Center B Cells.

Inactivated influenza vaccines induce greater antibody responses in females than males among both humans and mice. To test the breadth of protection, we used recombinant mouse-adapted A/California/2009 (maA/Cal/09) H1N1 viruses containing mutations at one (1M), two (2M), or three (3M) antigenic sites, in addition to a virus containing the 1M mutation and a substitution of the Ca2 antigenic site (Sub) with one derived from an H5 hemagglutinin (HA) to challenge mice of both sexes. Following maA/Cal/09 vaccination, females produced greater virus-specific, class-switched total IgG and IgG2c antibodies against the vaccine and all mutant viruses, and antibodies from females recognized a greater number of unique, linear HA epitopes than did antibodies from males. While females had greater neutralizing antibody titers against the vaccine virus, both sexes showed a lower neutralization capacity against mutant viruses. After virus challenge, vaccinated females had lower pulmonary virus titers and reduced morbidity than males for the 1M and 2M viruses, but not the Sub virus. Females generated greater numbers of germinal center (GC) B cells containing superior somatic hypermutation (SHM) frequencies than vaccinated males. Deletion of activation-induced cytidine deaminase (Aicda) eliminated female-biased immunity and protection against the 2M virus. Harnessing methods to improve GC B cell responses and frequencies of SHM, especially in males, should be considered in the development of universal influenza vaccines. IMPORTANCE Adult females develop greater antibody responses to influenza vaccines than males. We hypothesized that female-biased immunity and protection would be dependent on the extent of virus diversity as well as molecular mechanisms in B cells which constrain the breadth of epitope recognition. We developed a panel of mouse-adapted (ma) A/Cal/09 viruses that had mutations in the immunodominant hemagglutinin. Following vaccination against maA/Cal/09, females were better able to neutralize maA/Cal/09 than males, but neutralization of mutant maA/Cal/09 viruses was equally poor in both sexes, despite vaccinated females being better protected against these viruses. Vaccinated females benefited from the greater production of class-switched, somatically hypermutated antibodies generated in germinal center B cells, which increased recognition of more diverse maA/Cal/09 hemagglutinin antigen epitopes. Female-biased protection against influenza infection and disease after vaccination is driven by differential mechanisms in males versus females and should be considered in the design of novel vaccine platforms.

Phage display of environmental protein toxins and virulence factors reveals the prevalence, persistence, and genetics of antibody responses.

Microbial exposures are crucial environmental factors that impact healthspan by sculpting the immune system and microbiota. Antibody profiling via Phage ImmunoPrecipitation Sequencing (PhIP-Seq) provides a high-throughput, cost-effective approach for detecting exposure and response to microbial protein products. We designed and constructed a library of 95,601 56-amino acid peptide tiles spanning 14,430 proteins with "toxin" or "virulence factor" keyword annotations. We used PhIP-Seq to profile the antibodies of ∼1,000 individuals against this "ToxScan" library. In addition to enumerating immunodominant antibody epitopes, we studied the age-dependent stability of the ToxScan profile and used a genome-wide association study to find that the MHC-II locus modulates bacterial epitope selection. We detected previously described anti-flagellin antibody responses in a Crohn's disease cohort and identified an association between anti-flagellin antibodies and juvenile dermatomyositis. PhIP-Seq with the ToxScan library is thus an effective tool for studying the environmental determinants of health and disease at cohort scale.

Loss of TDP-43 function and rimmed vacuoles persist after T cell depletion in a xenograft model of sporadic inclusion body myositis.

Sporadic inclusion body myositis (IBM) is the most common acquired muscle disease in adults over age 50, yet it remains unclear whether the disease is primarily driven by T cell­mediated autoimmunity. IBM muscle biopsies display nuclear clearance and cytoplasmic aggregation of TDP-43 in muscle cells, a pathologic finding observed initially in neurodegenerative diseases, where nuclear loss of TDP-43 in neurons causes aberrant RNA splicing. Here, we show that loss of TDP-43­mediated splicing repression, as determined by inclusion of cryptic exons, occurs in skeletal muscle of subjects with IBM. Of 119 muscle biopsies tested, RT-PCR­mediated detection of cryptic exon inclusion was able to diagnose IBM with 84% sensitivity and 99% specificity. To determine the role of T cells in pathogenesis, we generated a xenograft model by transplanting human IBM muscle into the hindlimb of immunodeficient mice. Xenografts from subjects with IBM displayed robust regeneration of human myofibers and recapitulated both inflammatory and degenerative features of the disease. Myofibers in IBM xenografts showed invasion by human, oligoclonal CD8+ T cells and exhibited MHC-I up-regulation, rimmed vacuoles, mitochondrial pathology, p62-positive inclusions, and nuclear clearance and cytoplasmic aggregation of TDP-43, associated with cryptic exon inclusion. Reduction of human T cells within IBM xenografts by treating mice intraperitoneally with anti-CD3 (OKT3) suppressed MHC-I up-regulation. However, rimmed vacuoles and loss of TDP-43 function persisted. These data suggest that T cell depletion does not alter muscle degenerative pathology in IBM.

Analysis of antibody binding specificities in twin and SNP-genotyped cohorts reveals that antiviral antibody epitope selection is a heritable trait.

Human immune responses to viral infections are highly variable, but the genetic factors that contribute to this variability are not well characterized. We used VirScan, a high-throughput epitope scanning technology, to analyze pan-viral antibody reactivity profiles of twins and SNP-genotyped individuals. Using these data, we determined the heritability and genomic loci associated with antibody epitope selection, response breadth, and control of Epstein-Barr virus (EBV) viral load. 107 EBV peptide reactivities were heritable and at least two Epstein-Barr nuclear antigen 2 (EBNA-2) reactivities were associated with variants in the MHC class II locus. We identified an EBV serosignature that predicted viral load in peripheral blood mononuclear cells and was associated with variants in the MHC class I locus. Our study illustrates the utility of epitope profiling to investigate the genetics of pathogen immunity, reports heritable features of the antibody response to viruses, and identifies specific HLA loci important for EBV epitope selection.

Deconvoluting virome-wide antibody epitope reactivity profiles.

BACKGROUND: Comprehensive characterization of exposures and immune responses to viral infections is critical to a basic understanding of human health and disease. We previously developed the VirScan system, a programmable phage-display technology for profiling antibody binding to a library of peptides designed to span the human virome. Previous VirScan analytical approaches did not carefully account for antibody cross-reactivity among sequences shared by related viruses or for the disproportionate representation of individual viruses in the library. METHODS: Here we present the AntiViral Antibody Response Deconvolution Algorithm (AVARDA), a multi-module software package for analyzing VirScan datasets. AVARDA provides a probabilistic assessment of infection with species-level resolution by considering sequence alignment of all library peptides to each other and to all human viruses. We employed AVARDA to analyze VirScan data from a cohort of encephalitis patients with either known viral infections or undiagnosed etiologies. We further assessed AVARDA's utility in associating viral infection with type 1 diabetes and lupus. FINDINGS: By comparing acute and convalescent sera, AVARDA successfully confirmed or detected encephalitis-associated responses to human herpesviruses 1, 3, 4, 5, and 6, improving the rate of diagnosing viral encephalitis in this cohort by 44%. AVARDA analyses of VirScan data from the type 1 diabetes and lupus cohorts implicated enterovirus and herpesvirus infections, respectively. INTERPRETATION: AVARDA, in combination with VirScan and other pan-pathogen serological techniques, is likely to find broad utility in the epidemiology and diagnosis of infectious diseases. FUNDING: This work was made possible by support from the National Institutes of Health (NIH), the US Army Research Office, the Singapore Infectious Diseases Initiative (SIDI), the Singapore Ministry of Health's National Medical Research Council (NMRC) and the Singapore National Research Foundation (NRF).

HIV Antibody Profiles in HIV Controllers and Persons With Treatment-Induced Viral Suppression.

Introduction: Low HIV viral load is associated with delayed disease progression and reduced HIV transmission. HIV controllers suppress viral load to low levels in the absence of antiretroviral treatment (ART). We used an antibody profiling system, VirScan, to compare antibody reactivity and specificity in HIV controllers, non-controllers with treatment-induced viral suppression, and viremic non-controllers. Methods: The VirScan library contains 3,384 phage-displayed peptides spanning the HIV proteome. Antibody reactivity to these peptides was measured in plasma from a Discovery Cohort that included 13 elite controllers, 27 viremic controllers, 12 viremic non-controllers, and 21 non-controllers who were virally suppressed on ART. Antibody reactivity to selected peptides was also assessed in an independent cohort of 29 elite controllers and 37 non-controllers who were virally suppressed on ART (Validation Cohort) and in a longitudinal cohort of non-controllers. Results: In the Discovery Cohort, 62 peptides were preferentially targeted in HIV controllers compared to non-controllers who were virally suppressed on ART. These specificities were not significantly different when comparing controllers versus viremic non-controllers. Aggregate reactivity to these peptides was also high in elite controllers from the independent Validation Cohort. The 62 peptides formed seven clusters of homologous epitopes in env, gag, integrase, and vpu. Reactivity to one of these clusters located in gag p17 was inversely correlated with viral load set point in an independent cohort of non-controllers. Conclusions: Antibody reactivity was low in non-controllers suppressed on ART, but remained high in viremic controllers despite viral suppression. Antibodies in controllers and viremic non-controllers were directed against epitopes in diverse HIV proteins; higher reactivity against p17 peptides was associated with lower viral load set point. Further studies are needed to determine if these antibodies play a role in regulation of HIV viral load.