Enhanced antibody responses in fully vaccinated individuals against pan-SARS-CoV-2 variants following Omicron breakthrough infection.

Omicron has become the globally dominant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant, creating additional challenges due to its ability to evade neutralization. Here, we report that neutralizing antibodies against Omicron variants are undetected following COVID-19 infection with ancestral or past SARS-CoV-2 variant viruses or after two-dose mRNA vaccination. Compared with two-dose vaccination, a three-dose vaccination course induces broad neutralizing antibody responses with improved durability against different SARS-CoV-2 variants, although neutralizing antibody titers against Omicron remain low. Intriguingly, among individuals with three-dose vaccination, Omicron breakthrough infection substantially augments serum neutralizing activity against a broad spectrum of SARS-CoV-2 variants, including Omicron variants BA.1, BA.2, and BA.5. Additionally, after Omicron breakthrough infection, memory T cells respond to the spike proteins of both ancestral and Omicron SARS-CoV-2 by producing cytokines with polyfunctionality. These results suggest that Omicron breakthrough infection following three-dose mRNA vaccination induces pan-SARS-CoV-2 immunity that may protect against emerging SARS-CoV-2 variants of concern.

Development and evaluation of an anti-rabies virus phosphoprotein-specific monoclonal antibody for detection of rabies neutralizing antibodies using RFFIT.

BACKGROUND: Rabies is a major public health problem with a fatality rate close to 100%; however, complete prevention can be achieved through pre- or post-exposure prophylaxis. The rapid fluorescent focus inhibition test (RFFIT) is one of the recommended testing methods to determine the production of neutralizing antibodies after vaccination. Here, we report the development of a new monoclonal antibody (mAb) designed to react specifically with Rabies virus (RABV) phosphoprotein (P protein), and the evaluation of its applicability to the RFFIT and its effectiveness as a diagnostic reagent for human rabies. METHODOLOGY/PRINCIPAL FINDINGS: The mAb KGH P 16B8 was produced to target the P protein of the Korean KGH RABV strain. An indirect immunofluorescence assay (IFA) was conducted to detect various strains of RABV in various cell lines. Alexa-conjugated KGH P 16B8 (16B8-Alexa) was developed for the RFFIT. The IFA test could detect RABV up to a 1:2,500 dilution, with a detection limit comparable to that of a commercial diagnostic reagent. The sensitivity, specificity, positive predictive value, and negative predictive value of the RFFIT using 16B8-Alexa in 414 clinical specimens were 98.67%, 99.47%, 99.55%, and 98.42%, respectively. The results of the RFFIT with 16B8-Alexa were strongly correlated with those obtained using an existing commercial diagnostic reagent (r = 0.995, p<0.001). CONCLUSIONS/SIGNIFICANCE: The mAb developed in this study shows high sensitivity and specificity, confirming its clinical utility with the RFFIT to measure the rabies neutralizing antibody titer and establish a diagnosis in human. Thus, 16B8-Alexa is expected to serve as an alternative diagnostic reagent that is widely accessible, with potentially broad applications beyond those of the RFFIT in Korea. Further studies with 16B8-Alexa should provide insight into the immunological mechanism of the P protein of Korean RABV.

Molecular characterization of KGH, the first human isolate of rabies virus in Korea.

The complete genome sequence of the KGH strain of the first human rabies virus, which was isolated from a skin biopsy of a patient with rabies, whose symptoms developed due to bites from a raccoon dog in 2001. The size of the KGH strain genome was determined to be 11,928 nucleotides (nt) with a leader sequence of 58 nt, nucleoprotein gene of 1,353 nt, phosphoprotein gene of 894 nt, matrix protein gene of 609 nt, glycoprotein gene of 1,575 nt, RNA-dependent RNA polymerase gene of 6,384 nt, and trailer region of 69 nt. Sequence similarity was compared with 39 fully sequenced rabies virus genomes currently available, and the result showed 70.6-91.6 % at the nucleotide level, and 82.8-97.9 % at the amino acid level. The deduced amino acids in the viral protein were compared with those of other rabies viruses, and various functional regions were investigated. As a result, we found that the KGH strain only had a unique amino acid substitution that was identified to be associated either with host immune response and pathogenicity in the N protein, or with a related region regulating STAT1 in the P protein, and related to pathogenicity in G protein. Based on phylogenetic analyses using the complete genome of 39 rabies viruses, the KGH strain was determined to be closely related with the NNV-RAB-H strain and transplant rabies virus serotype 1, which are Indian isolates, and was confirmed to belong to the Arctic-like 2 clade. The KGH strain was most closely related to the SKRRD0204HC and SKRRD0205HC strain when compared with Korean animal isolates, which was separated around the same time and place, and belonged to the Gangwon III subgroup.

The association between prion proteins and Aß1₋42 oligomers in cytotoxicity and apoptosis.

Misfolding of prion protein (PrP to PrPSc) can cause neurodegenerative prion diseases. As a glycosylphosphatidylinositol (GPI)-anchored membrane protein, the normal form of PrP (PrPC) can function as a receptor for ligands in the extracellular space. PrPC was suggested to be involved in memory, synaptic neuronal communication, and anti-oxidation as a neuroprotective agent. The recently identified interaction between PrPC and Aß(1-42) oligomers suggested another role for PrP as a receptor for Aß(1-42) oligomers, thereby influencing cytotoxicity and apoptosis. Here, the association between PrPC and Aß(1-42) oligomers was investigated by visualizing protein localization in neuronal cells by immunocytochemistry. Aß(1-42) oligomer-induced cytotoxicity was tested in respective expressions of PrPC by using mouse neuroblastoma-2a (N2a) cells, the prion protein overexpressed cells (L2-2B1), and a Prnp-null mouse hippocampal cell line (HpL 3-4). Moreover, apoptotic proteins such as caspase-8 were used to assess the effect of PrPC on Aß(1-42) oligomer-mediated apoptosis. In L2-2B1 and HpL 3-4 cells, the difference in the cytotoxicity of Aß(1-42) oligomers could be clearly distinguished. In addition, Aß(1-42) oligomers induced mitochondria dysfunction, reactive oxygen species (ROS) generation, and calcium influx PrPC-dependently. Apoptosis, related to mitochondria dysfunction, was further investigated to determine the cytotoxic pathway; the results suggest that PrPC could be involved in both the intrinsic and extrinsic apoptotic pathways. Finally, cells with abundant PrPC expression seemed to be more susceptible to Aß(1-42) oligomer toxicity, suggesting the importance of the level of PrPC expression in the induction of apoptosis.

Serum MicroRNA Expression Profiling in Mice Infected with Rabies Virus.

OBJECTIVES: Serum or plasma microRNAs (miRNAs) are potential biomarkers for the diagnosis for cancer and prenatal diseases. This study was conducted to investigate whether rabies virus causes a change in serum miRNA expression. METHODS: ICR mice were intramuscularly inoculated with rabies virus and were sacrificed weekly to collect serum and brain tissue for 4 weeks postinoculation. Mice were assigned to four groups based on the results of indirect immunofluorescent assays, enzyme-linked immunosorbent assay, and nested reverse transcription-polymerase chain reaction and the expression profiles of serum miRNAs were compared using a commercial mouse miRNA expression profiling assay. RESULTS: The expression levels of miRNAs changed significantly with the different stages of the disease. The expression level of 94 serum miRNAs in infected mice changed at least twofold. Seven microRNAs of them were significantly upregulated or downregulated in all infected mice regardless of disease status. The number of miRNAs with an expression level change decreased with the progression of the disease. In a hierarchical cluster analysis, infected mice clustered into a group separate from uninfected control mice. CONCLUSIONS: Based on the relationship of miRNAs to gene expression regulation, miRNAs may be candidates for the study of viral pathogenesis and could have potential as biomarkers.