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Scientific Publication

Immunoreactive peptide maps of SARS-CoV-2

Mishra, N., Huang, X., Joshi, S. et al. Immunoreactive peptide maps of SARS-CoV-2. Commun Biol 4, 225 (2021). https://doi.org/10.1038/s42003-021-01743-9

Abstract

Serodiagnosis of SARS-CoV-2 infection is impeded by immunological cross-reactivity among the human coronaviruses (HCoVs): SARS-CoV-2, SARS-CoV-1, MERS-CoV, OC43, 229E, HKU1, and NL63. Here we report the identification of humoral immune responses to SARS-CoV-2 peptides that may enable discrimination between exposure to SARS-CoV-2 and other HCoVs. We used a high-density peptide microarray and plasma samples collected at two time points from 50 subjects with SARS-CoV-2 infection confirmed by qPCR, samples collected in 2004–2005 from 11 subjects with IgG antibodies to SARS-CoV-1, 11 subjects with IgG antibodies to other seasonal human coronaviruses (HCoV), and 10 healthy human subjects. Through statistical modeling with linear regression and multidimensional scaling we identified specific peptides that were reassembled to identify 29 linear SARS-CoV-2 epitopes that were immunoreactive with plasma from individuals who had asymptomatic, mild or severe SARS-CoV-2 infections. Larger studies will be required to determine whether these peptides may be useful in serodiagnostics.

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Nimble supported collaboration with Roche featured in Nature Communications focus on translational and clinical research.

Nimble’s collaboration with Roche, recently published in Nature Communications as “Protease-activation using anti-idiotypic masks enables tumor specificity of a folate receptor 1-T cell bispecific antibody” has been featured in a focus on translational and clinical research. A link to the articles featured as well as the publication can be found below:

Featured Articles: https://www.nature.com/collections/ecgchegdhe

Publication: https://www.nature.com/articles/s41467-020-16838-w

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Scientific Publication

Protease-activation using anti-idiotypic masks enables tumor specificity of a folate receptor 1-T cell bispecific antibody.

Geiger M, et al. (2020) “Protease-activation using anti-idiotypic masks enables tumor specificity of a folate receptor 1-T cell bispecific antibody.” Nat Commun. 11: 3196. doi: 10.1038/s41467-020-16838-w

Abstract

T-cell bispecific antibodies (TCBs) crosslink tumor and T-cells to induce tumor cell killing. While TCBs are very potent, on-target off-tumor toxicity remains a challenge when selecting targets. Here, we describe a protease-activated anti-folate receptor 1 TCB (Prot-FOLR1-TCB) equipped with an anti-idiotypic anti-CD3 mask connected to the anti-CD3 Fab through a tumor protease-cleavable linker. The potency of this Prot- FOLR1-TCB is recovered following protease-cleavage of the linker releasing the anti-idiotypic anti-CD3 scFv. In vivo, the Prot-FOLR1-TCB mediates antitumor efficacy comparable to the parental FOLR1-TCB whereas a noncleavable control Prot-FOLR1-TCB is inactive. In contrast, killing of bronchial epithelial and renal cortical cells with low FOLR1 expression is prevented compared to the parental FOLR1-TCB. The findings are confirmed for mesothelin as alternative tumor antigen. Thus, masking the anti-CD3 Fab fragment with an anti-idiotypic mask and cleavage of the mask by tumor-specific proteases can be applied to enhance specificity and safety of TCBs.

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High-Throughput Identification of MHC Class I Binding Peptides Using an Ultradense Peptide Array.

Haj AK, et al. (2020) “High-Throughput Identification of MHC Class I Binding Peptides Using an Ultradense Peptide Array.” J Immunol. 204(6): 1689-96. doi: 10.4049/jimmunol.1900889

Abstract

Rational vaccine development and evaluation requires identifying and measuring the magnitude of epitope-specific CD8 T cell responses. However, conventional CD8 T cell epitope discovery methods are labor intensive and do not scale well. In this study, we accelerate this process by using an ultradense peptide array as a high-throughput tool for screening peptides to identify putative novel epitopes. In a single experiment, we directly assess the binding of four common Indian rhesus macaque MHC class I molecules (Mamu-A1*001, -A1*002, -B*008, and -B*017) to ∼61,000 8-mer, 9-mer, and 10-mer peptides derived from the full proteomes of 82 SIV and simian HIV isolates. Many epitope-specific CD8 T cell responses restricted by these four MHC molecules have already been identified in SIVmac239, providing an ideal dataset for validating the array; up to 64% of these known epitopes are found in the top 192 SIVmac239 peptides with the most intense MHC binding signals in our experiment. To assess whether the peptide array identified putative novel CD8 T cell epitopes, we validated the method by IFN-γ ELISPOT assay and found three novel peptides that induced CD8 T cell responses in at least two Mamu-A1*001–positive animals; two of these were validated by ex vivo tetramer staining. This high-throughput identification of peptides that bind class I MHC will enable more efficient CD8 T cell response profiling for vaccine development, particularly for pathogens with complex proteomes for which few epitope-specific responses have been defined.

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Microarray analyses reveal strain-specific antibody responses to Plasmodium falciparum apical membrane antigen 1 variants following natural infection and vaccination.

Bailey JA, et al. (2020) “Microarray analyses reveal strain-specific antibody responses to Plasmodium falciparum apical membrane antigen 1 variants following natural infection and vaccination.” Sci Rep. 10(1): 3952. doi: 10.1038/s41598-020-60551-z

Abstract

Vaccines based on Plasmodium falciparum apical membrane antigen 1 (AMA1) have failed due to extensive polymorphism in AMA1. To assess the strain-specificity of antibody responses to malaria infection and AMA1 vaccination, we designed protein and peptide microarrays representing hundreds of unique AMA1 variants. Following clinical malaria episodes, children had short-lived, sequence-independent increases in average whole-protein seroreactivity, as well as strain-specific responses to peptides representing diverse epitopes. Vaccination resulted in dramatically increased seroreactivity to all 263 AMA1 whole-protein variants. High-density peptide analysis revealed that vaccinated children had increases in seroreactivity to four distinct epitopes that exceeded responses to natural infection. A single amino acid change was critical to seroreactivity to peptides in a region of AMA1 associated with strain-specific vaccine efficacy. Antibody measurements using whole antigens may be biased towards conserved, immunodominant epitopes. Peptide microarrays may help to identify immunogenic epitopes, define correlates of vaccine protection, and measure strain-specific vaccine-induced antibodies.

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Identification of immunoreactive linear epitopes of Borrelia miyamotoi.

Tokarz R, et al. (2020) “Identification of immunoreactive linear epitopes of Borrelia miyamotoi.” Ticks Tick Borne Dis. 11(1): 101314. doi: 10.1016/j.ttbdis.2019.101314

Abstract

Borrelia miyamotoi is an emerging tick-borne spirochete transmitted by ixodid ticks. Current serologic assays for B. miyamotoi are impacted by genetic similarities to other Borrelia and limited understanding of optimal antigenic targets. In this study, we employed the TBD-Serochip, a peptide array platform, to identify new linear targets for serologic detection of B. miyamotoi. We examined a wide range of suspected B. miyamotoi antigens and identified 352 IgM and 91 IgG reactive peptides, with the majority mapping to variable membrane proteins. These included peptides within conserved fragments of variable membrane proteins that may have greater potential for differential diagnosis. We also identified reactive regions on FlaB, and demonstrate crossreactivity of B. burgdorferi s.l. C6 with a B. miyamotoi C6-like peptide. The panel of linear peptides identified in this study can be used to enhance serodiagnosis of B. miyamotoi.

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RNA Transcription and Splicing Errors as a Source of Cancer Frameshift Neoantigens for Vaccines.

Shen L, et al. (2019) “RNA Transcription and Splicing Errors as a Source of Cancer Frameshift Neoantigens for Vaccines.” Scientific Reports. 9(1): 14184. doi: 10.1038/s41598-019-50738-4

Abstract

The success of checkpoint inhibitors in cancer therapy is largely attributed to activating the patient’s immune response to their tumor’s neoantigens arising from DNA mutations. This realization has motivated the interest in personal cancer vaccines based on sequencing the patient’s tumor DNA to discover neoantigens. Here we propose an additional, unrecognized source of tumor neoantigens. We show that errors in transcription of microsatellites (MS) and mis-splicing of exons create highly immunogenic frameshift (FS) neoantigens in tumors. The sequence of these FS neoantigens are predictable, allowing creation of a peptide array representing all possible neoantigen FS peptides. This array can be used to detect the antibody response in a patient to the FS peptides. A survey of 5 types of cancers reveals peptides that are personally reactive for each patient. This source of neoantigens and the method to discover them may be useful in developing cancer vaccines.

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Comprehensive Profiling of Rheumatoid Arthritis Antibody Repertoire.

Lo KC, et al. (2020) “Comprehensive Profiling of Rheumatoid Arthritis Antibody Repertoire.” Arthritis Rheumatol. 72(2): 242-250. doi: 10.1002/art.41089

Abstract

Objective

Autoantibodies against citrullinated proteins are found in 64–89% of rheumatoid arthritis (RA) patients, with 88–99% specificity. This study was undertaken to create an unbiased, comprehensive profile of serum antibodies against the human proteome, including the citrullinome and the homocitrullinome, in RA patients, using a high‐density peptide array.

Methods

Our high‐density peptide array, consisting of >4.6 million peptides, contained the entire annotated human proteome. The 20,246 proteins were represented as overlapping 16‐mer peptides. In addition to native peptides, citrullinated and homocitrullinated peptides were included, as substitutions for arginine and lysine, and provided a comprehensive screen against all possible epitopes. Twenty‐six serum samples (from 8 controls and 18 RA patients) were profiled on the high‐density peptide array. Using RA‐specific epitopes, we constructed an 8‐epitope diagnostic biomarker on a Gyrolab xPlore instrument with a cohort of 92 serum samples (from 29 controls and 63 RA patients). The diagnostic biomarker was further validated with an independent cohort of 181 serum samples (from 54 controls and 127 RA patients).

Results

In the initial cohort the diagnostic performance of the 8‐epitope biomarker yielded 96.6% specificity and 92.1% sensitivity. The overall diagnostic performance in the validation cohort was 94.4% specificity and 85% sensitivity. In both cohorts, the performance of the 8‐epitope diagnostic biomarker compared favorably against the Abnova cyclic citrullinated peptide 2 (CCP2) assay. Using data from the peptide array, we identified novel RA‐specific epitopes and formed the basis of a new RA diagnostic assay.

Conclusion

Comprehensive antibody profiling using a high‐density peptide array not only identified novel RA‐specific epitopes but also allowed us to construct a novel diagnostic biomarker that is as specific as and more sensitive than the Abnova CCP2 assay.

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Antibodies to Enteroviruses in Cerebrospinal Fluid of Patients with Acute Flaccid Myelitis.

Mishra N, et al. (2019) “Antibodies to Enteroviruses in Cerebrospinal Fluid of Patients with Acute Flaccid Myelitis.” mBio. 10(4). pii: e01903-19. doi: 10.1128/mBio.01903-19

ABSTRACT

Acute flaccid myelitis (AFM) has caused motor paralysis in >560 children in the United States since 2014. The temporal association of enterovirus (EV) outbreaks with increases in AFM cases and reports of fever, respiratory, or gastrointestinal illness prior to AFM in >90% of cases suggest a role for infectious agents. Cerebrospinal fluid (CSF) from 14 AFM and 5 non-AFM patients with central nervous system (CNS) diseases in 2018 were investigated by viral-capture high-throughput sequencing (VirCapSeq-VERT system). These CSF and serum samples, as well as multiple controls, were tested for antibodies to human EVs using peptide microarrays. EV RNA was confirmed in CSF from only 1 adult AFM case and 1 non-AFM case. In contrast, antibodies to EV peptides were present in CSF of 11 of 14 AFM patients (79%), significantly higher than controls, including non-AFM patients (1/5 [20%]), children with Kawasaki disease (0/10), and adults with non-AFM CNS diseases (2/11 [18%]) (P = 0.023, 0.0001, and 0.0028, respectively). Six of 14 CSF samples (43%) and 8 of 11 sera (73%) from AFM patients were immunoreactive to an EV-D68-specific peptide, whereas the three control groups were not immunoreactive in either CSF (0/5, 0/10, and 0/11; P = 0.008, 0.0003, and 0.035, respectively) or sera (0/2, 0/8, and 0/5; P = 0.139, 0.002, and 0.009, respectively).

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Whole genome-derived tiled peptide arrays detect pre-diagnostic autoantibody signatures in non-small cell lung cancer.

Yan Y, et al. (2019) “Whole genome-derived tiled peptide arrays detect pre-diagnostic autoantibody signatures in non-small cell lung cancer.” Cancer Res. 79(7): 1549-1557. doi: 10.1158/0008-5472.CAN-18-1536

Abstract

The majority of non–small-cell lung cancer (NSCLC) cases are diagnosed at advanced stages, primarily because earlier stages of the disease are either asymptomatic or may be attributed to other causes such as infection or long-term effects from smoking. Therefore, early detection of NSCLC would likely increase response and survival rates due to timely intervention. Here, we utilize a novel approach based on whole genome–derived tiled peptide arrays to identify epitopes associated with autoantibody reactivity in NSCLC as a potential means for early detection. Arrays consisted of 2,781,902 tiled peptides representing 20,193 proteins encoded in the human genome. Analysis of 86 prediagnostic samples and 86 matched normal controls from a high-risk cohort revealed 48 proteins with three or more reactive epitopes in NSCLC samples relative to controls. Independent mass spectrometry analysis identified 40 of the 48 proteins in prediagnostic sera from NSCLC samples, of which, 21 occurred in the immunoglobulin-bound fraction. In addition, 63 and 34 proteins encompassed three or more epitopes that were distinct for squamous cell lung cancer and lung adenocarcinoma, respectively. Collectively, these data show that tiled peptide arrays provide a means to delineate epitopes encoded across the genome that trigger an autoantibody response associated with tumor development.

Significance: This study provides a modality for early diagnosis of NSCLC for precision oncology that can be applied to other cancer types.