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Rheumatoid arthritis (RA) patients often develop rheumatoid factors (RFs), antibodies that bind IgG Fc, and anti-modified protein antibodies (AMPAs), multireactive autoantibodies that commonly bind citrullinated, homocitrullinated, and acetylated antigens. Recently, antibodies that bind citrulline-containing IgG epitopes were discovered in RA, suggesting that additional undiscovered IgG epitopes could exist and that IgG could be a shared antigen for RFs and AMPAs. This study was undertaken to reveal new IgG epitopes in rheumatic disease and to determine if multireactive AMPAs bind IgG.Using sera from patients with RA, systemic lupus erythematosus, Sjögren's disease (SjD), or spondyloarthropathy, IgG binding to native, citrulline-containing, and homocitrulline-containing linear epitopes of the IgG constant region was evaluated by peptide array, with highly bound epitopes further evaluated by enzyme-linked immunosorbent assay (ELISA). Binding of monoclonal AMPAs to IgG-derived peptides and IgG Fc was also evaluated by ELISA.Seropositive RA sera showed high IgG binding to multiple citrulline- and homocitrulline-containing IgG-derived peptides, whereas anti-SSA+ sera from SjD patients showed consistent binding to a single linear native epitope of IgG in the hinge region. Monoclonal AMPAs bound citrulline- and homocitrulline-containing IgG peptides and modified IgG Fc.The repertoire of epitopes bound by AMPAs includes modified IgG epitopes, positioning IgG as a common antigen that connects the otherwise divergent reactivities of RFs and AMPAs.

The peptidylarginine deiminases (PADs) and the citrullinated proteins that they generate have key roles in innate immunity and rheumatoid arthritis, an inflammatory arthritis with antibodies that target citrullinated proteins. However, the importance of PADs, particularly PAD2, in the adaptive immune response, both normal and pathogenic, is newly emerging. In this study, we evaluated a requirement for PAD2 in the antibody response in collagen-induced arthritis (CIA), a T and B cell-driven murine model of rheumatoid arthritis, and in the protective antibody response to murine influenza infection. Using PAD2-/- and PAD2+/+ mice on the DBA/1J background, we found that PAD2 is required for maximal anti-collagen antibody levels, but not collagen-specific plasma cell numbers, T cell activation or polarization, or arthritis severity in CIA. Also, we found that PAD2 is required not just for normal levels of persistent hemagglutination inhibiting antibodies but also for full protection from lethal influenza rechallenge. Together, these data provide evidence for a novel modest requirement for PAD2 in a normal antiviral antibody response and in an abnormal autoantibody response in inflammatory arthritis.

Serological population surveillance can elucidate immunity landscapes against SARS-CoV-2 variants and are critical in monitoring infectious disease spread, evolution, and outbreak risks. However, current serological tests fall short of capturing complex humoral immune responses from different communities. Here, we report a machine-learning (ML)-aided nanobiosensor that can simultaneously quantify antibodies against the ancestral strain and Omicron variants of SARS-CoV-2 with epitope resolution. Our approach is based on a multiplexed, rapid, and label-free nanoplasmonic biosensor, which can detect past infection and vaccination status and is sensitive to SARS-CoV-2 variants. After training an ML model with antigen-specific antibody datasets from four COVID-19 immunity groups (naïve, convalescent, vaccinated, and convalescent-vaccinated), we tested our approach on 100 blind blood samples collected in Dane County, WI. Our results are consistent with public epidemiological data, demonstrating that our user-friendly and field-deployable nanobiosensor can capture community-representative public health trends and help manage COVID-19 and future outbreaks.

Lasting immunity will be critical for overcoming COVID-19. However, the factors associated with the development of high titers of anti–SARS-CoV-2 Abs and how long those Abs persist remain incompletely defined. In particular, an understanding of the relationship between COVID-19 symptoms and anti–SARS-CoV-2 Abs is limited. To address these unknowns, we quantified serum anti–SARS- CoV-2 Abs in clinically diverse COVID-19 convalescent human subjects 5 wk (n = 113) and 3 mo (n = 79) after symptom resolution with three methods: a novel multiplex assay to quantify IgG against four SARS-CoV-2 Ags, a new SARS-CoV-2 receptor binding domain-angiotensin converting enzyme 2 inhibition assay, and a SARS-CoV-2 neutralizing assay. We then identified clinical and demographic factors, including never-before-assessed COVID-19 symptoms, that consistently correlate with high anti–SARS-CoV-2 Ab levels. We detected anti–SARS-CoV-2 Abs in 98% of COVID-19 convalescent subjects 5 wk after symptom resolution, and Ab levels did not decline at 3 mo. Greater disease severity, older age, male sex, higher body mass index, and higher Charlson Comorbidity Index score correlated with increased anti–SARS-CoV-2 Ab levels. Moreover, we report for the first time (to our knowledge) that COVID-19 symptoms, most consistently fever, body aches, and low appetite, correlate with higher anti–SARS-CoV-2 Ab levels. Our results provide robust and new insights into the development and persistence of anti–SARS-CoV-2 Abs.

The consequences of past COVID-19 infection for personal health and long-term population immunity are only starting to be revealed. Unfortunately, detecting past infection is currently a challenge, limiting clinical and research endeavors. Widely available anti-SARS-CoV-2 antibody tests cannot differentiate between past infection and vaccination given vaccine-induced anti-spike antibodies and the rapid loss of infection-induced anti-nucleocapsid antibodies. Anti-membrane antibodies develop after COVID-19, but their long-term persistence is unknown. Here, we demonstrate that anti-membrane IgG is a sensitive and specific marker of past COVID-19 infection and persists at least one year. We also confirm that anti-receptor binding domain (RBD) Ig is a long-lasting, sensitive, and specific marker of past infection and vaccination, while anti-nucleocapsid IgG lacks specificity and quickly declines after COVID-19. Thus, a combination of anti-membrane and anti-RBD antibodies can accurately differentiate between distant COVID-19 infection, vaccination, and naïve states to advance public health, individual healthcare, and research goals.

Lasting immunity will be critical for overcoming COVID-19. However, the factors associated with the development of high titers of anti-SARS-CoV-2 antibodies and how long those antibodies persist remain incompletely defined. In particular, an understanding of the relationship between COVID-19 symptoms and anti-SARS-CoV-2 antibodies is limited. To address these unknowns, we quantified serum anti-SARS-CoV-2 antibodies in clinically diverse COVID-19 convalescent human subjects five weeks (n=113) and three months (n=79) after symptom resolution with three methods: a novel multiplex assay to quantify IgG against four SARS-CoV-2 antigens, a new SARS-CoV-2 receptor binding domain-angiotensin converting enzyme 2 inhibition assay, and a SARS-CoV-2 neutralizing assay. We then identified clinical and demographic factors, including never before assessed COVID-19 symptoms, that consistently correlate with high anti-SARS-CoV-2 antibody levels. We detected anti-SARS-CoV-2 antibodies in 98% of COVID-19 convalescent subjects five weeks after symptom resolution and antibody levels did not decline at three months. Greater disease severity, older age, male sex, higher body mass index, and higher Charlson Comorbidity Index score correlated with increased anti-SARS-CoV-2 antibody levels. Moreover, we report for the first time that COVID-19 symptoms, most consistently fever, body aches, and low appetite, correlate with higher anti-SARS-CoV-2 antibody levels. Our results provide robust and new insights into the development and persistence of anti-SARS-CoV-2 antibodies.

Holometabolous insects have been able to radiate to vast ecological niches as adults through the evolution of adult-specific structures such as wings, antennae and eyes. These structures arise from imaginal discs that show regenerative capacity when damaged. During imaginal disc regeneration, development has been shown to be delayed in the fruit fly Drosophila melanogaster, but how conserved the delay-inducing mechanisms are across holometabolous insects has not been assessed. The goal of this research was to develop the hornworm Manduca sexta as an alternative model organism to study such damage-induced mechanisms, with the advantage of a larger hemolymph volume enabling access to the hormonal responses to imaginal disc damage. Upon whole-body X-ray exposure, we noted that the imaginal discs were selectively damaged, as assessed by TUNEL and Acridine Orange stains. Moreover, development was delayed, predominantly at the pupal-to-adult transition, with a concomitant delay in the prepupal ecdysteroid peak. The delays to eclosion were dose dependent, with some ability for repair of damaged tissues. We noted a shift in critical weight, as assessed by the point at which starvation no longer impacted developmental timing, without a change in growth rate, which was uncoupled from juvenile hormone clearance in the body. The developmental profile was different from that of D. melanogaster, which suggests species differences may exist in the mechanisms delaying development.

Holometabolous insects have been able to radiate to vast ecological niches as adults through the evolution of adult-specific structures such as wings, antennae and eyes. These structures arise from imaginal discs that show regenerative capacity when damaged. During imaginal disc regeneration, development has been shown to be delayed in the fruit fly Drosophila melanogaster, but how conserved the delay-inducing mechanisms are across holometabolous insects has not been assessed. The goal of this research was to develop the hornworm, Manduca sexta, as an alternative model organism to study such damage-induced mechanisms, with the advantage of a larger hemolymph volume enabling access to the hormonal responses to imaginal disc damage. Upon whole-body x-ray exposure, we noted that the imaginal discs were selectively damaged, as assessed by TUNEL and acridine orange stains. Moreover, development was delayed, predominantly at the pupal-to-adult transition, with a concomitant delay in the prepupal ecdysteroid peak. The delays to eclosion were dose-dependent, with some ability for repair of damaged tissues. We noted a shift in critical weight, as assessed by the point at which starvation no longer impacted developmental timing, without a change in growth rate, which was uncoupled from juvenile hormone clearance in the body. The developmental profile was different from Drosophila melanogaster, which suggests species differences may exist in the mechanisms delaying development.