Comirnaty-Elicited and Convalescent Sera Recognize Different Spike Epitopes

Many of the approved SARS-CoV-2 vaccines are based on a stabilized variant of the spike protein. This raises the question of whether the immune response against the stabilized spike is identical to the immune response that is elicited by the native spike in the case of a SARS-CoV-2 infection. Using a peptide array-based approach, we analysed the binding of antibodies from Comirnaty-elicited, convalescent, and control sera to the peptides covering the spike protein. A total of 37 linear epitopes were identified. A total of 26 of these epitopes were almost exclusively recognized by the convalescent sera. Mapping these epitopes to the spike structures revealed that most of these 26 epitopes are masked in the pre-fusion structure. In particular, in the conserved central helix, three epitopes that are only exposed in the post-fusion conformation were identified. This indicates a higher spike-specific antibody diversity in convalescent sera. These differences could be relevant for the breadth of spike-specific immune response.

Butyrate and Forskolin Augment Host Defense, Barrier Function, and Disease Resistance Without Eliciting Inflammation

Host defense peptides (HDPs) are an integral part of the innate immune system with both antimicrobial and immunomodulatory activities. Induction of endogenous HDP synthesis is being actively explored as an antibiotic-alternative approach to disease control and prevention. Butyrate, a short-chain fatty acid, and forskolin, a phytochemical, have been shown separately to induce HDP gene expression in human cells. Here, we investigated the ability of butyrate and forskolin to induce the expressions of chicken HDP genes and the genes involved in barrier function such as mucin 2 and claudin 1 both in vitro and in vivo. We further evaluated their efficacy in protecting chickens from Clostridium perfringens-induced necrotic enteritis. Additionally, we profiled the transcriptome and global phosphorylation of chicken HD11 macrophage cells in response to butyrate and forskolin using RNA sequencing and a kinome peptide array, respectively. Our results showed a strong synergy between butyrate and forskolin in inducing the expressions of several, but not all, HDP genes. Importantly, dietary supplementation of butyrate and a forskolin-containing plant extract resulted in significant alleviation of intestinal lesions and the C. perfringens colonization in a synergistic manner in a chicken model of necrotic enteritis. RNA sequencing revealed a preferential increase in HDP and barrier function genes with no induction of proinflammatory cytokines in response to butyrate and forskolin. The antiinflammatory and barrier protective properties of butyrate and forskolin were further confirmed by the kinome peptide array. Moreover, we demonstrated an involvement of inducible cAMP early repressor (ICER)-mediated negative feedback in HDP induction by butyrate and forskolin. Overall, these results highlight a potential for developing butyrate and forskolin, two natural products, as novel antibiotic alternatives to enhance intestinal health and disease resistance in poultry and other animals.

Identification and Mapping of HBsAg Loss-Related B-Cell Linear Epitopes in Chronic HBV Patients by Peptide Array

Identification of immunogenic targets against hepatitis B virus (HBV)-encoded proteins will provide crucial advances in developing potential antibody therapies. In this study, 63 treatment-naïve patients with chronic HBV infection and 46 patients who achieved hepatitis B surface antigen loss (sAg loss) following antiviral treatment were recruited. Moreover, six patients who transitioned from the hepatitis B e antigen-positive chronic infection phase (eAg+CInf) to the hepatitis phase (eAg+CHep) were enrolled from real-life clinical practice. Additionally, telbivudine-treated eAg+CHep patients and relapsers or responders from an off-treatment cohort were longitudinally studied. The frequencies and function of B cells were assessed by flow cytometry. We devised a peptide array composed of 15-mer overlapping peptides of HBV-encoded surface (S), core (C), and polymerase (P) proteins and performed a screening on B-cell linear epitopes with sera. Naïve B cells and plasmablasts were increased, whereas total memory, activated memory (AM), and atypical memory (AtM) B cells were reduced in sAg- patients compared with sAg+ patients. Importantly, longitudinal observations found that AtM B cells were associated with successful treatment withdrawal. Interestingly, we identified six S-specific dominant epitopes (S33, S34, S45, S76, S78, and S89) and one C-specific dominant epitope (C37) that reacted with the majority of sera from sAg- patients. Of note, more B-cell linear epitopes were detected in CHep patients with alanine aminotransferase (ALT) flares than in nonflare CInf patients, and five B-cell linear epitopes (S4, S5, S10, S11, and S68) were overwhelmingly recognized by ALT flare patients. The recognition rates of epitopes on C and P proteins were significantly increased in CHep patients relative to CInf patients. Strikingly, a statistically significant elevation in the number of positive epitopes was observed when ALT nonflare patients shifted into the flare phase. Moreover, S76 identified at baseline was confirmed to be associated with a complete response after 48 weeks of telbivudine therapy. Taken together, we identified several functional cure-related B-cell linear epitopes of chronic HBV infection, and these epitopes may serve as vaccine candidates to elicit neutralizing antibodies to treat HBV infection.

Identification and Functional Characterization of Protest SWI/SNF and ISWI Complexes

The thesis aims to identify and initiate functional characterization of the SWI/SNF and ISWI complexes in Tetrahymena thermophila. Through affinity purification of the conserved subunit Snf5 followed by mass spectrometry (AP-MS), I identified the first SWI/SNF complex in protists. One of the subunits I found is a small bromodomain containing protein named Ibd1. Through AP-MS of Ibd1 I found Ibd1 is versatile and interacts with several additional chromatin remodeling complexes. Bromodomains are known to have affinity for acetylated lysine residues within proteins such as histones. A peptide array experiment suggests that Ibd1 also has affinity for acetylated chromatin. Indirect immunofluorescence (IF) of Ibd1 hints at a role in transcription. My analysis of Tetrahymena Iswi1 shows expression during meiosis, vegetative growth and starvation. IF data shows its localization is consistent with Iswi1 function in mitosis/meiosis or maintenance of silent chromatin. AP-MS of ISW1 discovered several interacting proteins of unknown function.

Potent Neutralization Antibodies Induced by a Recombinant Trimeric Spike Protein Vaccine Candidate Containing PIKA Adjuvant for COVID-19

The structures of immunogens that elicit the most potent neutralization antibodies to prevent COVID-19 infection are still under investigation. In this study, we tested the efficacy of a recombinant trimeric Spike protein containing polyI:C (PIKA) adjuvant in mice immunized by a 0–7–14 day schedule. The results showed that a Spike protein-specific antibody was induced at Day 21 with titer of above 50,000 on average, as measured by direct binding. The neutralizing titer was above 1000 on average, as determined by a pseudo-virus using monoclonal antibodies (40592-MM57 and 40591-MM43) with IC50 at 1 μg/mL as standards. The protein/peptide array-identified receptor-binding domain (RBD) was considered as immunodominant. No linear epitopes were found in the RBD, although several linear epitopes were found in the C-terminal domain right after the RBD and heptad repeat regions. Our study supports the efficacy of a recombinant trimeric Spike protein vaccine candidate for COVID-19 that is safe and ready for storage and distribution in developing countries.

Identification of a Zika NS2B Epitope for Which Absence of IgG Response Is Associated with Severe Neurological Symptoms and the Design of a Biomarker Capable of Discriminatory Diagnostics Between Severe and Non4 Severe Clinical Phenotypes

<div> <div> <div><p><a></a>In this manuscript we describe the engineering of a biomarker for the diagnosis and prognosis of Zika-associated neurological disease. Although the causal association between congenital Zika virus (ZIKV) infection and neurological manifestations has been well documented in the recent years, biomarkers for proper diagnostic and disease outcome still remain to be defined. Combining high-density peptide array and multivariate analysis, we have identified an ZIKV epitope that is associated to a lack of IgG antibody response in patients with severe neurological symptoms. An engineered chimera was developed to discriminate between mild and severe clinical forms of the disease.</p></div> </div> </div>

Identification of a Zika NS2B Epitope for Which Absence of IgG Response Is Associated with Severe Neurological Symptoms and the Design of a Biomarker Capable of Discriminatory Diagnostics Between Severe and Non4 Severe Clinical Phenotypes

<div> <div> <div><p><a></a>In this manuscript we describe the engineering of a biomarker for the diagnosis and prognosis of Zika-associated neurological disease. Although the causal association between congenital Zika virus (ZIKV) infection and neurological manifestations has been well documented in the recent years, biomarkers for proper diagnostic and disease outcome still remain to be defined. Combining high-density peptide array and multivariate analysis, we have identified an ZIKV epitope that is associated to a lack of IgG antibody response in patients with severe neurological symptoms. An engineered chimera was developed to discriminate between mild and severe clinical forms of the disease.</p></div> </div> </div>