A high-dose inoculum size results in persistent viral infection and arthritis in mice infected with chikungunya virus.

Chikungunya virus (CHIKV) is an emerging mosquito-transmitted alphavirus that leads to acute fever and chronic debilitating polyarthralgia. To date, the mechanism underlying chronic recurrent arthralgia is unknown. In the present study, newborn wild-type C57BL/6 mice were infected with CHIKV, and the virological and pathological features of CHIKV infection were analyzed over a period of 50 days. Acute viral infection was readily established by footpad inoculation of CHIKV at doses ranging from 10 plaque forming unit (PFU) to 106 PFU, during which inoculation dose-dependent viral RNA and skeletal muscle damage were detected in the foot tissues. However, persistent CHIKV was observed only when the mice were infected with a high dose of 106 PFU of CHIKV, in which low copy numbers (103-104) of viral positive strand RNA were continuously detectable in the feet from 29 to 50 dpi, along with a low level and progressive reduction in virus-specific CD8+ T cell responses. In contrast, viral negative strand RNA was detected at 50 dpi but not at 29 dpi and was accompanied by significant local skeletal muscle damage at 50 dpi when mild synovial hyperplasia appeared in the foot joints, although the damage was briefly repaired at 29 dpi. These results demonstrated that a high viral inoculation dose leads to viral persistence and progression to chronic tissue damage after recovery from acute infection. Taken together, these results provide a useful tool for elucidating the pathogenesis of persistent CHIKV infection and viral relapse-associated chronic arthritis.

Rab11-FIP1 and Rab11-FIP5 Regulate pIgR/pIgA Transcytosis through TRIM21-Mediated Polyubiquitination.

Polymeric immunoglobulin receptor (pIgR)-mediated polymeric immunoglobulin A (pIgA) transcytosis across mucosal epithelial cells plays an essential role in mucosal immunity. The general trafficking process has been well investigated, yet the elaborate regulatory mechanisms remain enigmatic. We identified a new pIgR interacting protein, the Rab11 effector Rab11-FIP1. Rab11-FIP1 and Rab11-FIP5 knockdown additively impaired pIgA transcytosis in both polarized and incompletely polarized cells. Moreover, Rab11-FIP1 and Rab11-FIP5 knockdown exhibited more significant inhibitory effects on pIgA transcytosis in incompletely polarized cells than in polarized cells. Interestingly, the trafficking process of pIgA in incompletely polarized cells is distinct from that in polarized cells. In incompletely polarized cells, the endocytic pIgR/pIgA was first transported from the basolateral plasma membrane to the vicinity of the centrosome where Rab11-FIP1 and Rab11-FIP5 bound to it, before the Rab11a-positive endosomes containing pIgR/pIgA, Rab11-FIP1 and Rab11-FIP5 were further transported to the apical plasma membrane via Golgi apparatus. During the trafficking process, TRIM21 mediated the K11-linked polyubiquitination of Rab11-FIP1 and the K6-linked polyubiquitination of Rab11-FIP5 to promote their activation and pIgA transcytosis. This study indicates that polyubiquitinated Rab11-FIP1 and Rab11-FIP5 mediated by TRIM21 cooperatively facilitate pIgA transcytosis and provides new insights into the intracellular trafficking process of pIgA in incompletely polarized cells.

A safe and effective mucosal RSV vaccine in mice consisting of RSV phosphoprotein and flagellin variant.

Respiratory syncytial virus (RSV) is a major cause of serious acute lower respiratory tract infection in infants and the elderly. The lack of a licensed RSV vaccine calls for the development of vaccines with other targets and vaccination strategies. Here, we construct a recombinant protein, designated P-KFD1, comprising RSV phosphoprotein (P) and the E.-coli-K12-strain-derived flagellin variant KFD1. Intranasal immunization with P-KFD1 inhibits RSV replication in the upper and lower respiratory tract and protects mice against lung disease without vaccine-enhanced disease (VED). The P-specific CD4+ T cells provoked by P-KFD1 intranasal (i.n.) immunization either reside in or migrate to the respiratory tract and mediate protection against RSV infection. Single-cell RNA sequencing (scRNA-seq) and carboxyfluorescein succinimidyl ester (CFSE)-labeled cell transfer further characterize the Th1 and Th17 responses induced by P-KFD1. Finally, we find that anti-viral protection depends on either interferon-γ (IFN-γ) or interleukin-17A (IL-17A). Collectively, P-KFD1 is a promising safe and effective mucosal vaccine candidate for the prevention of RSV infection.

Broad phenotypic alterations and potential dysfunction of lymphocytes in individuals clinically recovered from COVID-19.

Although millions of patients have clinically recovered from COVID-19, little is known about the immune status of lymphocytes in these individuals. In this study, the peripheral blood mononuclear cells of a clinically recovered (CR) cohort were comparatively analyzed with those of an age- and sex-matched healthy donor cohort. We found that CD8+ T cells in the CR cohort had higher numbers of effector T cells and effector memory T cells but lower Tc1 (IFN-γ+), Tc2 (IL-4+), and Tc17 (IL-17A+) cell frequencies. The CD4+ T cells of the CR cohort were decreased in frequency, especially the central memory T cell subset. Moreover, CD4+ T cells in the CR cohort showed lower programmed cell death protein 1 (PD-1) expression and had lower frequencies of Th1 (IFN-γ+), Th2 (IL-4+), Th17 (IL-17A+), and circulating follicular helper T (CXCR5+PD-1+) cells. Accordingly, the proportion of isotype-switched memory B cells (IgM-CD20hi) among B cells in the CR cohort showed a significantly lower proportion, although the level of the activation marker CD71 was elevated. For CD3-HLA-DR- lymphocytes in the CR cohort, in addition to lower levels of IFN-γ, granzyme B and T-bet, the correlation between T-bet and IFN-γ was not observed. Additionally, by taking into account the number of days after discharge, all the phenotypes associated with reduced function did not show a tendency toward recovery within 4‒11 weeks. The remarkable phenotypic alterations in lymphocytes in the CR cohort suggest that  severe acute respiratory syndrome coronavirus 2 infection profoundly affects lymphocytes and potentially results in dysfunction even after clinical recovery.

Characteristics of T-cell responses in COVID-19 patients with prolonged SARS-CoV-2 positivity - a cohort study.

OBJECTIVE: SARS-CoV-2 has caused a worldwide pandemic of COVID-19. The existence of prolonged SARS-CoV-2 positivity (PP) has further increased the burden on the health system. Since T cells are vital for viral control, we aimed to evaluate the characteristics of T-cell responses associated with PP. METHODS: We established a PP cohort and two age- and sex-matched control cohorts: a regular clinical recovery (CR) cohort and a healthy donor (HD) cohort. The mean time for RNA negativity conversion in the PP cohort was markedly longer than that in the CR cohort (66.2 vs 25.3 days), while the time from illness onset to sampling was not significantly different. T-cell responses in the PP cohort were assayed, analysed and compared with those in the CR and HD cohorts by flow cytometry and ELISpot analysis of peripheral blood mononuclear cells. RESULTS: Compared with the CR cohort, the proliferation, activation and functional potential of CD8+ and CD4+ T cells in the PP cohort were not significantly different. However, the frequencies and counts of Teff and Tem in CD8+ but not in CD4+ T cells of the PP cohort were prominently lower. Moreover, a weaker SARS-CoV-2 N protein-specific IFN-γ+ T-cell response and a higher frequency of Tregs were detected in the PP cohort. CONCLUSION: Suppressed CD8+ T-cell differentiation is associated with PP and may be an indicator for the prediction of prolonged SARS-CoV-2 positivity in COVID-19 patients. The association between suppressed CD8+ T-cell differentiation and elevated Tregs warrants studies in the future.

Alterations in Phenotypes and Responses of T Cells Within 6 Months of Recovery from COVID-19: A Cohort Study.

The COVID-19 pandemic, caused by the SARS-CoV-2 infection, is a global health crisis. While many patients have clinically recovered, little is known about long-term alterations in T cell responses of COVID-19 convalescents. In this study, T cell responses in peripheral blood mononuclear cells of a long-time COVID-19 clinically recovered (20-26 weeks) cohort (LCR) were measured via flow cytometry and ELISpot. The T cell responses of LCR were comparatively analyzed against an age and sex matched short-time clinically recovered (4-9 weeks) cohort (SCR) and a healthy donor cohort (HD). All volunteers were recruited from Wuhan Jinyintan Hospital, China. Phenotypic analysis showed that activation marker PD-1 expressing on CD4+ T cells of LCR was still significantly lower than that of HD. Functional analysis indicated that frequencies of Tc2, Th2 and Th17 in LCR were comparable to those of HD, but Tc17 was higher than that of HD. In LCR, compared to the HD, there were fewer IFN-γ producing T cells but more IL-2 secreting T cells. In addition, the circulating Tfh cells in LCR were still slightly lower compared to HD, though the subsets composition had recovered. Remarkably, SARS-CoV-2 specific T cell responses in LCR were comparable to that of SCR. Collectively, T cell responses experienced long-term alterations in phenotype and functional potential of LCR cohort. However, after clinical recovery, SARS-CoV-2 specific T cell responses could be sustained at least for six months, which may be helpful in resisting re-infection.

TLR5 activation in hepatocytes alleviates the functional suppression of intrahepatic CD8+ T cells.

The liver is an immune-privileged organ with a tolerogenic environment for maintaining liver homeostasis. This hepatic tolerance limits the intrahepatic CD8+ T-cell response for eliminating infections. The tolerant microenvironment in the liver is orchestrated by liver-specific immunoregulatory cells that can be functionally regulated by pathogen-associated molecular patterns (PAMPs). Here, we report that flagellin, a key PAMP of gut bacteria, modulates the intrahepatic CD8+ T-cell response by activating the TLR5 signalling pathway of hepatocytes. We found that mice treated with Salmonella-derived recombinant flagellin (SF) by hydrodynamic injection had a significantly elevated IFN-γ production by the intrahepatic lymphocytes in 7 days after injection. This was correlated with a reduced immune suppressive effect of primary mouse hepatocytes (PMHs) in comparison with that of PMHs from mock-injected control mice. In vitro co-culture of SF-treated PMHs with splenocytes revealed that hepatocyte-induced immune suppression is alleviated through activation of the TLR5 but not the NLRC4 signalling pathway, leading to improved activation and function of CD8+ T cells during anti-CD3 stimulation or antigen-specific activation. In an acute HBV replication mouse model established by co-administration of SF together with an HBV-replicating plasmid by hydrodynamic injection, SF significantly enhanced the intrahepatic HBV-specific CD8+ T-cell response against HBV surface antigen. Our results clearly showed that flagellin plays a role in modulating the intrahepatic CD8+ T-cell response by activating the TLR5 pathway in PMHs, which suggests a potential role for gut bacteria in regulating liver immunity.

Immunoglobulin A Targeting on the N-Terminal Moiety of Viral Phosphoprotein Prevents Measles Virus from Evading Interferon-ß Signaling.

Immunoglobulin A (IgA) can inhibit intracellular viral replication during its transport across the epithelial cells. We find a monoclonal IgA antibody 7F1-IgA against the N-terminal moiety of the phosphoprotein (PNT) of measles virus (MV), which inhibits the intracellular replication of MV in Caco-2 cells but not in interferon-deficient Vero-pIgR cells. Transcytosis of 7F1-IgA across the MV-infected Caco-2 cells enhances the production of interferon-ß (IFN-ß) and the expression of IFN-stimulated genes, rendering Caco-2 cells with higher antiviral immunity. 7F1-IgA specifically interacts with MV phosphoprotein inside the MV-infected Caco-2 cell and prevents MV phosphoprotein from inhibiting the phosphorylation of JAK1 and STAT1. The intraepithelial interaction between 7F1-IgA and the viral phosphoprotein results in an earlier and stronger phosphorylation of JAK1 and STAT1 and, consequently, a more efficient nuclear translocation of STAT1 for the activation of the type I interferon pathway. Thus, IgA against phosphoprotein prevents a virus from evading type I IFN signaling and confers host epithelial cells efficient innate antiviral immunity, which potentiates a new antiviral target and an antiviral strategy.

IgA targeting on the α-molecular recognition element (α-MoRE) of viral phosphoprotein inhibits measles virus replication by interrupting formation and function of P-N complex intracellularly.

Secretory IgA (SIgA) antibody is unique for its capability to transit through epithelial cells by transcytosis and thus has opportunities and probabilities to interact with all viral components during viral replication which may result in the inhibition of viral replication intracellularly. Here, we report a novel IgA mAb 1D11-IgA against phosphoprotein (P) of measles virus (MV), which is able to interact specifically with P in MV infected Vero-pIgR cells grown in a two-chamber transwell system. The binding epitope of 1D11-IgA involves a key residue proline 23 in P protein, which is among the α-molecular recognition element (α-MoRE) of P and critical for N0-P complex. The antibody appears to block P to interact with N in P-N complex and thus may inhibit the function of viral RdRp complex, which results in decreased synthesis of viral genome RNA and mRNA. Our data together demonstrate that IgA is able to interact with viral phosphoprotein intraepithelial cells and neutralize viral replication by interrupting formation of P-N complex and function of RdRp. The findings highlight that IgA has a unique anti-viral activity by targeting viral conserved components critical for viral replication, which serves as a proof-of-concept assessment of the druggability of mononegavirales P-N interfaces.

Improved immune response against HIV-1 Env antigen by enhancing EEV production via a K151E mutation in the A34R gene of replication-competent vaccinia virus Tiantan.

The development of an effective HIV-1 vaccine is still a global priority. In recent years, vaccinia virus (VV) has been widely used as an HIV-1 vaccine vector, but its immune efficacy against HIV-1 antigens needs to be optimized. The extracellular enveloped virus (EEV) of VV is capable of faster entry, earlier release, and long-range dissemination. We hypothesized that an improvement in EEV formation by the manipulation of VV genes involved in the EEV release would consequently cause an improved expression of the VV carrying HIV-1 Env antigen and a subsequent enhanced immune response. To this end, an A34R K151E mutant (rVTT-A34Rmut) from VV Tiantan strain (VTT) with robustly increased EEV release was selected to serve as an optimized vaccine vector. The results were consistent with our hypothesis: the A34R mutant-based HIV-1 vaccine candidate rVTT-A34Rmut-Env produced more HIV-1 Env antigen in vitro and in vivo, and thus led to an improved HIV-1 Env-specific T cell immune response, binding antibody, and even the neutralizing antibody response in mice without increased virulence. Meanwhile, the application of the A34R mutation on another VV-based HIV-1 vaccine candidate, VTKgpe, also exhibited a similar immune enhancement effect with no enhanced virulence. The results in this study suggested that rVTT-A34Rmut is a potentially improved vaccine vector candidate for human application. In addition, the improvement of the EEV formation via the A34R gene mutation may also be potent in other poxvirus vector-based vaccines against HIV-1 or other pathogens and even cancer in the future.

Second-generation Flagellin-rPAc Fusion Protein, KFD2-rPAc, Shows High Protective Efficacy against Dental Caries with Low Potential Side Effects.

Dental caries is one of the most common global chronic diseases affecting all ages of the population; thus a vaccine against caries is urgently needed. Our previous studies demonstrated that a fusion protein, KF-rPAc, in which rPAc of S. mutans is directly fused to the C-terminal of E. coli-derived flagellin (KF), could confer high prophylactic and therapeutic efficiency against caries. However, possible side effects, including the high antigenicity of flagellin and possible inflammatory injury induced by flagellin, may restrict its clinical usage. Here, we produced a second-generation flagellin-rPAc fusion protein, KFD2-rPAc, by replacing the main antigenicity region domains D2 and D3 of KF with rPAc. Compared with KF-rPAc, KFD2-rPAc has lower TLR5 agonist efficacy and induces fewer systemic inflammatory responses in mice. After intranasal immunization, KFD2-rPAc induces significantly lower flagellin-specific antibody responses but a comparable level of rPAc-specific antibody responses in mice. More importantly, in rat challenge models, KFD2-rPAc induces a robust rPAc-specific IgA response, and confers efficient prophylactic and therapeutic efficiency against caries as does KF-rPAc, while the flagellin-specific antibody responses are highly reduced. In conclusion, low side effects and high protective efficiency against caries makes the second-generation flagellin-rPAc fusion protein, KFD2-rPAc, a promising vaccine candidate against caries.

Frontline Science: Nasal epithelial GM-CSF contributes to TLR5-mediated modulation of airway dendritic cells and subsequent IgA response.

Flagellin, as a TLR5 agonist, is an established mucosal adjuvant for enhancing mucosal IgA responses by i.n. immunization. Nasal epithelial cells (NECs) are the first sentinel cells to be exposed to antigen and adjuvant in i.n. immunization, and it is suggested that they play an important role in the mucosal adjuvant activity of flagellin. However, the molecular mechanism leading to modulation and the response by flagellin-activated NECs remain unknown. We aimed to identify the soluble mediator(s) from flagellin-activated NECs that modulate the functions of airway dendritic cells (DCs) and enhance subsequent IgA response. In vitro studies showed that compared with the TLR4 agonist LPS, flagellin directly triggered slight up-regulation of CD80 on airway DCs but was insufficient to affect CD86 expression and DC-mediated IgA response. With the use of an in vitro system for culturing mouse primary NECs (mNECs), we demonstrated that flagellin-activated mNECs could functionally modulate airway DCs, which manifested as significant up-regulation of CD80/CD86 and enhancement of IgA production. The functional modulation of airway DCs was dependent on TLR5 activation of mNECs rather than direct TLR5 activation of airway DCs. With the use of cytokine array and antibody-blocking assays, we further identified that GM-CSF, a cytokine secreted from TLR5-activated mNECs, contributes to the activation of mNECs to airway DCs and subsequent IgA enhancement. In vivo blocking experiments confirmed that GM-CSF is an important factor in recombinant flagellin derived from Salmonella typhi (FliC)-induced airway DC activation and antigen-specific IgA enhancement. Our data directly demonstrate that nasal epithelial GM-CSF contributes to TLR5-mediated modulation of airway DCs and a subsequent IgA response.

Flagellin-rPAc vaccine inhibits biofilm formation but not proliferation of S. mutans.

As the main etiologic bacterium of dental caries, Streptococcus mutans (S. mutans) has been considered as the primary object of vaccine research. We previously constructed a recombinant flagellin-rPAc fusion protein (KF-rPAc) that consists of an alanine-rich region to proline-rich region fragment of PAc (rPAc) from S. mutans and flagellin KF from E.coli K12 strain. Intranasal (i.n) immunization of KF-rPAc could induce high level of rPAc-specific antibody responses and offer robust protection against dental caries. In caries development, biofilm formation was considered as the necessary process involved. As PAc possesses other activities besides affecting adherence of S. mutans to salivary glycoproteins, we wondered whether rPAc-specific antibody responses induced by KF-rPAc could inhibit biofilm formation. Hence, in the present study, a simple and convenient in vitro biofilm model of S. mutans was constructed without saliva pre-coated. Both serum and saliva from KF-rPAc immunized rats significantly inhibited biofilm formation. Moreover, with the presence of serum or saliva, the biofilm formation is negatively correlated with the level of rPAc-specific antibody, and positively correlated with caries scores in rat. Moreover, in immunized mice, the level of rPAc-specific antibody also negatively correlated with the biofilm formation. Unlike ampicillin, serum of KF-rPAc immunized mice only inhibited biofilm formation but not proliferation. All together, we discovered that besides the well known blocking adherence of S. mutans to salivary glycoproteins by rPAc-specific antibody, flagellin-rPAc vaccine could also protects tooth from caries by inhibiting biofilm structure formation in between bacteria.