Immunogenicity and reactogenicity after booster dose with AZD1222 via intradermal route among adult who had received CoronaVac.

BACKGROUND: Currently, booster dose is needed after 2 doses of non-live COVID-19 vaccine. With limited resources and shortage of COVID-19 vaccines, intradermal(ID) administration might be a potential dose-sparing strategy. OBJECTIVE: To determine immunologic response and reactogenicity of ID ChAdOx1 nCoV-19 vaccine (AZD1222,Oxford/AstraZeneca) as a booster dose after completion of 2-dose CoronaVac(SV) in healthy adult. METHODS: This is a prospective cohort study of adult aged 18-59 years who received 2-dose SV at 14-35 days apart for more than 2 months. Participants received ID AZD1222 at fractional low dose(1×1010 viral particles,0.1 ml). Antibody responses were evaluated by surrogate virus neutralization test(sVNT) against delta variant and wild type, and anti-spike-receptor-binding-domain immunoglobulin G(anti-S-RBD IgG) at prior, day14, 28, 90, and 180 post booster. Solicited reactogenicity was collected for 7 days post-booster. Primary endpoint was the differences of sVNT against delta strain ≥ 80% inhibition at day14 and 90 compared with the parallel cohort study of 0.5-ml intramuscular(IM) route. RESULTS: From August2021, 100 adults with median age of 46 years(IQR 41-52) participated. Prior to booster, geometric mean(GM) of sVNT against delta strain was 22.4% inhibition(95 %CI 18.7-26.9) and of anti-S-RBD IgG was 109.3 BAU/ml(95.4-125.1). Post ID booster, GMs of sVNT against delta strain were 95.5% inhibition (95%CI 94.2-96.8) at day14, 73.1% inhibition (66.7-80.2) at day90, and 22.7% inhibition (14.9-34.6) at day180. The differences of proportion of participants achieving sVNT against delta strain ≥ 80% inhibition in ID recipients versus IM were + 4.2% (95 %CI -2.0to10.5) at day14, and -37.3%(-54.2to-20.3) at day90. Anti-S-RBD IgG GMs were 2037.1 BAU/ml (95%CI 1770.9-2343.2) at day14 and 744.6 BAU/ml(650.1-852.9) at day90, respectively. Geometric mean ratios(GMRs) of anti-S-RBD IgG were 0.99(0.83-1.20) at day14, and 0.82(0.66-1.02) at day90. Only 18% reported feverish, compared with 37% of IM (p = 0.003). Common reactogenicity was erythema at injection site(53%) while 7% reported blister. CONCLUSION: Low-dose ID AZD1222 booster enhanced lower neutralizing antibodies at 3 months compared with IM route. Less systemic reactogenicity occurred, but higher local reactogenicity.

Nucleocapsid proteins from other swine enteric coronaviruses differentially modulate PEDV replication.

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV) share tropism for swine intestinal epithelial cells. Whether mixing of viral components during co-infection alters pathogenic outcomes or viral replication is not known. In this study, we investigated how different coronavirus nucleocapsid (CoV N) proteins interact and affect PEDV replication. We found that PDCoV N and TGEV N can competitively interact with PEDV N. However, the presence of PDCoV or TGEV N led to very different outcomes on PEDV replication. While PDCoV N significantly suppresses PEDV replication, overexpression of TGEV N, like that of PEDV N, increases production of PEDV RNA and virions. Despite partial interchangeability in nucleocapsid oligomerization and viral RNA synthesis, endogenous PEDV N cannot be replaced in the production of infectious PEDV particles. Results from this study give insights into functional compatibilities and evolutionary relationship between CoV viral proteins during viral co-infection and co-evolution.

Porcine Epidemic Diarrhea Virus 3C-Like Protease-Mediated Nucleocapsid Processing: Possible Link to Viral Cell Culture Adaptability.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high mortality rates in newborn piglets, leading to massive losses to the swine industry worldwide during recent epidemics. Intense research efforts are now focusing on defining viral characteristics that confer a growth advantage, pathogenicity, or cell adaptability in order to better understand the PEDV life cycle and identify suitable targets for antiviral or vaccine development. Here, we report a unique phenomenon of PEDV nucleocapsid (N) cleavage by the PEDV-encoded 3C-like protease (3Cpro) during infection. The identification of the 3Cpro cleavage site at the C terminus of N supported previous observations that PEDV 3Cpro showed a substrate requirement slightly different from that of severe acute respiratory syndrome coronavirus (SARS-CoV) 3Cpro and revealed a greater flexibility in its substrate recognition site. This cleavage motif is present in the majority of cell culture-adapted PEDV strains but is missing in emerging field isolates. Remarkably, reverse-genetics-derived cell culture-adapted PEDVAVCT12 harboring uncleavable N displayed growth retardation in Vero E6-APN cells compared to the wild-type virus. These observations altogether shed new light on the investigation and characterization of the PEDV nucleocapsid protein and its possible link to cell culture adaptation. IMPORTANCE: Recurrent PEDV outbreaks have resulted in enormous economic losses to swine industries worldwide. To gain the upper hand in combating this disease, it is necessary to understand how this virus replicates and evades host immunity. Characterization of viral proteins provides important clues to mechanisms by which viruses survive and spread. Here, we characterized an intriguing phenomenon in which the nucleocapsids of some PEDV strains are proteolytically processed by the virally encoded main protease. Growth retardation in recombinant PEDV carrying uncleavable N suggests a replication advantage provided by the cleavage event, at least in the cell culture system. These findings may direct us to a more complete understanding of PEDV replication and pathogenicity.

Mechanistic study of intertypic nucleoprotein complex formation and its inhibitory effect toward influenza A virus.

Co-infection of influenza A and B viruses (IAV and IBV) results in marked decreases in IAV replication. Multiple mechanisms have been proposed for this phenomenon. Recently, we reported that IBV nucleoprotein (BNP) alone can suppress IAV replication and proposed an inhibition model in which BNP binds IAV nucleoprotein (ANP) and disrupts IAV polymerase complexes. Here, using mutagenesis and co-immunoprecipitation, we determined the protein motifs mediating the intertypic ANP-BNP complex and showed that it specifically interferes with ANP׳s interaction with the PB2 subunit of the IAV polymerase but not with the other subunit PB1. We further demonstrated that BNP only suppresses growth of IAVs but not other RNA viruses. However, different IAV strains display varied sensitivity toward the BNP׳s inhibitory effect. Together, our data provide mechanistic insights into intertypic nucleoprotein complex formation and highlight the role of BNP as a potential broad-spectrum anti-IAV agent.

Nucleoprotein of influenza B virus binds to its type A counterpart and disrupts influenza A viral polymerase complex formation.

Upon co-infection with influenza B virus (FluB), influenza A virus (FluA) replication is substantially impaired. Previously, we have shown that the nucleoprotein of FluB (BNP) can inhibit FluA polymerase machinery, retarding the growth of FluA. However, the molecular mechanism underlying this inhibitory action awaited further investigation. Here, we provide evidence that BNP hinders the proper formation of FluA polymerase complex by competitively binding to the nucleoprotein of FluA. To exert this inhibitory effect, BNP must be localized in the nucleus. The interaction does not require the presence of the viral RNA but needs an intact BNP RNA-binding motif. The results highlight the novel role of BNP as an anti-influenza A viral agent and provide insights into the mechanism of intertypic interference.

Inhibition of influenza A virus replication by influenza B virus nucleoprotein: an insight into interference between influenza A and B viruses.

Given that co-infection of cells with equivalent titers of influenza A and B viruses (FluA and FluB) has been shown to result in suppression of FluA growth, it is possible that FluB-specific proteins might hinder FluA polymerase activity and replication. We addressed this possibility by individually determining the effect of each gene of FluB on the FluA polymerase assay and found that the nucleoprotein of FluB (NP(FluB)) inhibits polymerase activity of FluA in a dose-dependent manner. Mutational analyses of NP(FluB) suggest that functional NP(FluB) is necessary for this inhibition. Slower growth of FluA was also observed in MDCK cells stably expressing NP(FluB). Further analysis of NP(FluB) indicated that it does not affect nuclear import of NP(FluA). Taken together, these findings suggest a novel role of NP(FluB) in inhibiting replication of FluA, providing more insights into the mechanism of interference between FluA and FluB and the lack of reassortants between them.

ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP subunit.

Membrane proteins impose enormous challenges to cellular protein homeostasis during their post-translational targeting, and they require chaperones to keep them soluble and translocation competent. Here we show that a novel targeting factor in the chloroplast signal recognition particle (cpSRP), cpSRP43, is a highly specific molecular chaperone that efficiently reverses the aggregation of its substrate proteins. In contrast to 'ATPases associated with various cellular activities' (AAA(+)) chaperones, cpSRP43 uses specific binding interactions with its substrate to mediate its 'disaggregase' activity. This disaggregase capability can allow targeting machineries to more effectively capture their protein substrates and emphasizes a close connection between protein folding and trafficking processes. Moreover, cpSRP43 provides the first example to our knowledge of an ATP-independent disaggregase and shows that efficient reversal of protein aggregation can be attained by specific binding interactions between a chaperone and its substrate.