SARS-CoV-2 spike-reactive naïve B cells and pre-existing memory B cells contribute to antibody responses in unexposed individuals after vaccination.

Introduction: Since December 2019, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) has presented considerable public health challenges. Multiple vaccines have been used to induce neutralizing antibodies (nAbs) and memory B-cell responses against the viral spike (S) glycoprotein, and many essential epitopes have been defined. Previous reports have identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-reactive naïve B cells and preexisting memory B cells in unexposed individuals. However, the role of these spike-reactive B cells in vaccine-induced immunity remains unknown. Methods: To elucidate the characteristics of preexisting SARS-CoV-2 S-reactive B cells as well as their maturation after antigen encounter, we assessed the relationship of spike-reactive B cells before and after vaccination in unexposed human individuals. We further characterized the sequence identity, targeting domain, broad-spectrum binding activity and neutralizing activity of these SARS-CoV-2 S-reactive B cells by isolating monoclonal antibodies (mAbs) from these B cells. Results: The frequencies of both spike-reactive naïve B cells and preexisting memory B cells before vaccination correlated with the frequencies of spike-reactive memory B cells after vaccination. Isolated mAbs from spike-reactive naïve B cells before vaccination had fewer somatic hypermutations (SHMs) than mAbs isolated from spike-reactive memory B cells before and after vaccination, but bound SARS-CoV-2 spike in vitro. Intriguingly, these germline-like mAbs possessed broad binding profiles for SARS-CoV-2 and its variants, although with low or no neutralizing capacity. According to tracking of the evolution of IGHV4-4/IGKV3-20 lineage antibodies from a single donor, the lineage underwent SHMs and developed increased binding activity after vaccination. Discussion: Our findings suggest that spike-reactive naïve B cells can be expanded and matured by vaccination and cocontribute to vaccine-elicited antibody responses with preexisting memory B cells. Selectively and precisely targeting spike-reactive B cells by rational antigen design may provide a novel strategy for next-generation SARS-CoV-2 vaccine development.

TREG cells and CXCR3+ circulating TFH cells concordantly shape the neutralizing antibody responses in individuals who have recovered from mild COVID-19.

Regulatory T (TREG) cells are involved in the antiviral immune response in patients with COVID-19; however, whether TREG cells are involved in the neutralizing antibody (nAb) response remains unclear. Here, we found that individuals who recovered from mild but not severe COVID-19 had significantly greater frequencies of TREG cells and lower frequencies of CXCR3+ circulating TFH (cTFH) cells than healthy controls. Furthermore, TREG and CXCR3+ cTFH cells were negatively and positively correlated with the nAb responses, respectively, and TREG cells was inversely associated with CXCR3+ cTFH cells in individuals who recovered from mild COVID-19 but not in those with severe disease. Mechanistically, TREG cells inhibited memory B-cell differentiation and antibody production by limiting the activation and proliferation of cTFH cells, especially CXCR3+ cTFH cells, and functional molecule expression. This study provides novel insight showing that mild COVID-19 elicits a concerted nAb responses which are shaped by both TREG and TFH cells.

SARS-CoV-2 spike-specific TFH cells exhibit unique responses in infected and vaccinated individuals.

Long-term humoral immunity to SARS-CoV-2 is essential for preventing reinfection. The production of neutralizing antibody (nAb) and B cell differentiation are tightly regulated by T follicular help (TFH) cells. However, the longevity and functional role of TFH cell subsets in COVID-19 convalescents and vaccine recipients remain poorly defined. Here, we show that SARS-CoV-2 infection and inactivated vaccine elicited both spike-specific CXCR3+ TFH cell and CXCR3- TFH cell responses, which showed distinct response patterns. Spike-specific CXCR3+ TFH cells exhibit a dominant and more durable response than CXCR3- TFH cells that positively correlated with antibody responses. A third booster dose preferentially expands the spike-specific CXCR3+ TFH cell subset induced by two doses of inactivated vaccine, contributing to antibody maturation and potency. Functionally, spike-specific CXCR3+ TFH cells have a greater ability to induce spike-specific antibody secreting cells (ASCs) differentiation compared to spike-specific CXCR3- TFH cells. In conclusion, the persistent and functional role of spike-specific CXCR3+ TFH cells following SARS-CoV-2 infection and vaccination may play an important role in antibody maintenance and recall response, thereby conferring long-term protection. The findings from this study will inform the development of SARS-CoV-2 vaccines aiming to induce long-term protective immune memory.

The antigenicity of SARS-CoV-2 Delta variants aggregated 10 high-frequency mutations in RBD has not changed sufficiently to replace the current vaccine strain.

Emerging SARS-CoV-2 variants are the most serious problem for COVID-19 prophylaxis and treatment. To determine whether the SARS-CoV-2 vaccine strain should be updated following variant emergence like seasonal flu vaccine, the changed degree on antigenicity of SARS-CoV-2 variants and H3N2 flu vaccine strains was compared. The neutralization activities of Alpha, Beta and Gamma variants' spike protein-immunized sera were analysed against the eight current epidemic variants and 20 possible variants combining the top 10 prevalent RBD mutations based on the Delta variant, which were constructed using pseudotyped viruses. Meanwhile, the neutralization activities of convalescent sera and current inactivated and recombinant protein vaccine-elicited sera were also examined against all possible Delta variants. Eight HA protein-expressing DNAs elicited-animal sera were also tested against eight pseudotyped viruses of H3N2 flu vaccine strains from 2011-2019. Our results indicate that the antigenicity changes of possible Delta variants were mostly within four folds, whereas the antigenicity changes among different H3N2 vaccine strains were approximately 10-100-fold. Structural analysis of the antigenic characterization of the SARS-CoV-2 and H3N2 mutations supports the neutralization results. This study indicates that the antigenicity changes of the current SARS-CoV-2 may not be sufficient to require replacement of the current vaccine strain.

Aggregation of high-frequency RBD mutations of SARS-CoV-2 with three VOCs did not cause significant antigenic drift.

Variants of SARS-CoV-2 continue to emerge, posing great challenges in outbreak prevention and control. It is important to understand in advance the impact of possible variants of concern (VOCs) on infectivity and antigenicity. Here, we constructed one or more of the 15 high-frequency naturally occurring amino acid changes in the receptor-binding domain (RBD) of Alpha, Beta, and Gamma variants. A single mutant of A520S, V367F, and S494P in the above three VOCs enhanced infectivity in ACE2-overexpressing 293T cells of different species, LLC-MK2 and Vero cells. Aggregation of multiple RBD mutations significantly reduces the infectivity of the possible three VOCs. Regarding neutralization, it is noteworthy that E484K, N501Y, K417N, and N439K predispose to monoclonal antibodies (mAbs) protection failure in the 15 high-frequency mutations. Most importantly, almost all possible VOCs (single RBD mutation or aggregation of multiple mutations) showed no more than a fourfold decrease in neutralizing activity with convalescent sera, vaccine sera, and immune sera of guinea pigs with different immunogens, and no significant antigenic drift was formed. In conclusion, our pseudovirus results could reduce the concern that the aggregation of multiple high-frequency mutations in the RBD of the spike protein of the three VOCs would lead to severe antigenic drift, and this would provide value for vaccine development strategies.

Reduced sensitivity of the SARS-CoV-2 Lambda variant to monoclonal antibodies and neutralizing antibodies induced by infection and vaccination.

Severe acute respiratory syndrome coronavirus 2 variants have continued to emerge in diverse geographic locations with a temporal distribution. The Lambda variant containing multiple mutations in the spike protein, has thus far appeared mainly in South America. The variant harbours two mutations in the receptor binding domain, L452Q and F490S, which may change its infectivity and antigenicity to neutralizing antibodies. In this study, we constructed 10 pseudoviruses to study the Lambda variant and each individual amino acid mutation's effect on viral function, and used eight cell lines to study variant infectivity. In total, 12 monoclonal antibodies, 14 convalescent sera, and 23 immunized sera induced by mRNA vaccines, inactivated vaccine, and adenovirus type 5 vector vaccine were used to study the antigenicity of the Lambda variant. We found that compared with the D614G reference strain, Lambda demonstrated enhanced infectivity of Calu-3 and LLC-MK2 cells by 3.3-fold and 1.6-fold, respectively. Notably, the sensitivity of the Lambda variant to 5 of 12 neutralizing monoclonal antibodies, 9G11, AM180, R126, X593, and AbG3, was substantially diminished. Furthermore, convalescent- and vaccine-immunized sera showed on average 1.3-2.5-fold lower neutralizing titres against the Lambda variant. Single mutation analysis revealed that this reduction in neutralization was caused by L452Q and F490S mutations. Collectively, the reduced neutralization ability of the Lambda variant suggests that the efficacy of monoclonal antibodies and vaccines may be compromised during the current pandemic.

The significant immune escape of pseudotyped SARS-CoV-2 variant Omicron.

The emergence of Omicron/BA.1 has brought new challenges to fight against SARS-CoV-2. A large number of mutations in the Spike protein suggest that its susceptibility to immune protection elicited by the existing COVID-19 infection and vaccines may be altered. In this study, we constructed the pseudotyped SARS-CoV-2 variant Omicron. The sensitivity of 28 serum samples from COVID-19 convalescent patients infected with SARS-CoV-2 original strain was tested against pseudotyped Omicron as well as the other variants of concern (VOCs, Alpha, Beta, Gamma, Delta) and variants of interest (VOIs, Lambda, Mu). Our results indicated that the mean neutralization ED50 of these sera against Omicron decreased to 66, which is about 8.4-folds compared to the D614G reference strain (ED50 = 556), whereas the neutralization activity of other VOC and VOI pseudotyped viruses decreased only about 1.2-4.5-folds. The finding from our in vitro assay suggest that Omicron variant may lead to more significant escape from immune protection elicited by previous SARS-CoV-2 infection and perhaps even by existing COVID-19 vaccines.

Monoclonal antibodies constructed from COVID-19 convalescent memory B cells exhibit potent binding activity to MERS-CoV spike S2 subunit and other human coronaviruses.

Introduction: The Middle East respiratory syndrome coronavirus (MERS-CoV) and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are two highly contagious coronaviruses causing MERS and COVID-19, respectively, without an effective antiviral drug and a long-lasting vaccine. Approaches for diagnosis, therapeutics, prevention, etc., particularly for SARS-CoV-2 that is continually spreading and evolving, are urgently needed. Our previous study discovered that >60% of sera from convalescent COVID-19 individuals, but <8% from general population, showed binding activity against the MERS-CoV spike protein, indicating that SARS-CoV-2 infection boosted antibodies cross-reactive with MERS-CoV. Methods: To generate antibodies specific to both SARS-CoV-2 and MERS-CoV, here we screened 60 COVID-19 convalescent sera against MERS-CoV spike extracellular domain and S1 and S2 subunits. We constructed and characterized monoclonal antibodies (mAbs) from COVID-19 convalescent memory B cells and examined their binding and neutralizing activities against human coronaviruses. Results and Discussion: Of 60 convalescent serum samples, 34 showed binding activity against MERS-CoV S2, with endpoint titers positively correlated with the titers to SARS-CoV-2 S2. By sorting single memory B cells from COVID-19 convalescents, we constructed 38 mAbs and found that 11 mAbs showed binding activity with MERS-CoV S2, of which 9 mAbs showed potent cross-reactivity with all or a proportion of spike proteins of alphacoronaviruses (229E and NL63) and betacoronaviruses (SARS-CoV-1, SARS-CoV-2, OC43, and HKU1). Moreover, 5 mAbs also showed weak neutralization efficiency against MERS-CoV spike pseudovirus. Epitope analysis revealed that 3 and 8 mAbs bound to linear and conformational epitopes in MERS-CoV S2, respectively. In summary, we have constructed a panel of antibodies with broad-spectrum reactivity against all seven human coronaviruses, thus facilitating the development of diagnosis methods and vaccine design for multiple coronaviruses.

The Antigenicity of Epidemic SARS-CoV-2 Variants in the United Kingdom.

To determine whether the neutralization activity of monoclonal antibodies, convalescent sera and vaccine-elicited sera was affected by the top five epidemic SARS-CoV-2 variants in the UK, including D614G+L18F+A222V, D614G+A222V, D614G+S477N, VOC-202012/01(B.1.1.7) and D614G+69-70del+N439K, a pseudovirus-neutralization assay was performed to evaluate the relative neutralization titers against the five SARS-CoV-2 variants and 12 single deconvolution mutants based on the variants. In this study, 18 monoclonal antibodies, 10 sera from convalescent COVID-19 patients, 10 inactivated-virus vaccine-elicited sera, 14 mRNA vaccine-elicited sera, nine RBD-immunized mouse sera, four RBD-immunized horse sera, and four spike-encoding DNA-immunized guinea pig sera were tested and analyzed. The N501Y, N439K, and S477N mutations caused immune escape from nine of 18 mAbs. However, the convalescent sera, inactivated virus vaccine-elicited sera, mRNA vaccine-elicited sera, spike DNA-elicited sera, and recombinant RBD protein-elicited sera could still neutralize these variants (within three-fold changes compared to the reference D614G variant). The neutralizing antibody responses to different types of vaccines were different, whereby the response to inactivated-virus vaccine was similar to the convalescent sera.

SARS-CoV-2 501Y.V2 variants lack higher infectivity but do have immune escape.

The 501Y.V2 variants of SARS-CoV-2 containing multiple mutations in spike are now dominant in South Africa and are rapidly spreading to other countries. Here, experiments with 18 pseudotyped viruses showed that the 501Y.V2 variants do not confer increased infectivity in multiple cell types except for murine ACE2-overexpressing cells, where a substantial increase in infectivity was observed. Notably, the susceptibility of the 501Y.V2 variants to 12 of 17 neutralizing monoclonal antibodies was substantially diminished, and the neutralization ability of the sera from convalescent patients and immunized mice was also reduced for these variants. The neutralization resistance was mainly caused by E484K and N501Y mutations in the receptor-binding domain of spike. The enhanced infectivity in murine ACE2-overexpressing cells suggests the possibility of spillover of the 501Y.V2 variants to mice. Moreover, the neutralization resistance we detected for the 501Y.V2 variants suggests the potential for compromised efficacy of monoclonal antibodies and vaccines.

Spike-specific circulating T follicular helper cell and cross-neutralizing antibody responses in COVID-19-convalescent individuals.

Coronavirus disease 2019 (COVID-19) is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1-3 and individuals with COVID-19 have symptoms that can be asymptomatic, mild, moderate or severe4,5. In the early phase of infection, T- and B-cell counts are substantially decreased6,7; however, IgM8-11 and IgG12-14 are detectable within 14 d after symptom onset. In COVID-19-convalescent individuals, spike-specific neutralizing antibodies are variable3,15,16. No specific drug or vaccine is available for COVID-19 at the time of writing; however, patients benefit from treatment with serum from COVID-19-convalescent individuals17,18. Nevertheless, antibody responses and cross-reactivity with other coronaviruses in COVID-19-convalescent individuals are largely unknown. Here, we show that the majority of COVID-19-convalescent individuals maintained SARS-CoV-2 spike S1- and S2-specific antibodies with neutralizing activity against the SARS-CoV-2 pseudotyped virus, and that some of the antibodies cross-neutralized SARS-CoV, Middle East respiratory syndrome coronavirus or both pseudotyped viruses. Convalescent individuals who experienced severe COVID-19 showed higher neutralizing antibody titres, a faster increase in lymphocyte counts and a higher frequency of CXCR3+ T follicular help (TFH) cells compared with COVID-19-convalescent individuals who experienced non-severe disease. Circulating TFH cells were spike specific and functional, and the frequencies of CXCR3+ TFH cells were positively associated with neutralizing antibody titres in COVID-19-convalescent individuals. No individuals had detectable autoantibodies. These findings provide insights into neutralizing antibody responses in COVID-19-convalescent individuals and facilitate the treatment and vaccine development for SARS-CoV-2 infection.

Circulating CXCR3+ Tfh cells positively correlate with neutralizing antibody responses in HCV-infected patients.

Circulating T follicular helper (cTfh) cells have been identified as counterparts of germinal center Tfh (GC Tfh) cells in humans and can support T-dependent B cell maturation and antibody production in vitro. However, the role of cTfh cells in neutralizing antibody (nAb) responses in HCV infection remains unclear. Here, we characterized the phenotype and function of cTfh cells and demonstrated the associations of cTfh cells and their subsets with nAb responses in HCV infection. A total of 38 HCV-infected individuals and 28 healthy controls were enrolled from a pool of injection drug users. The frequency and function of blood Tfh cells were analyzed by flow cytometry. The titers and breadths of serum nAbs were measured using HCV pseudo-particle neutralization assays. Herein, we report several key observations. First, HCV infection skewed cTfh toward CXCR3+ cTfh cell differentiation. Second, the frequency of CXCR3+ cTfh cells positively correlated with HCV nAb titers and breadths. Third, CXCR3+ cTfh cells showed higher expression of Tfh-associated molecules (PD-1, ICOS, IL-21, Bcl-6) compared with CXCR3- cTfh cells from individuals with HCV infection. Coculture of cTfh cells and autologous memory B cells in vitro indicated that CXCR3+ cTfh cells show a superior ability to support HCV E2-specific B cell expansion compared with CXCR3- cTfh cells from individuals with HCV infection. HCV infection skews cTfh cells toward CXCR3-biased Tfh cell differentiation, which positively correlates with the magnitude and breadth of the HCV nAb response. It is our hope that these findings will provide insights for the rational design of a nAb-based HCV vaccine.

Elevated LAG-3 on CD4+ T cells negatively correlates with neutralizing antibody response during HCV infection.

Lymphocyte activation gene-3 (LAG-3), an inhibitory molecule, which has been shown co-expressed with multiple inhibitory receptors on CD8+ T and natural killer (NK) cells and negatively regulates T and NK cell responses during hepatitis C virus (HCV) infection. However, whether LAG-3 is involved in the regulation of the antibody response remains unclear. This study aims to investigate the relationship of LAG-3 with neutralizing antibody (nAb) response during HCV infection. A total of 66 HCV-infected individuals and 36 sex- and age-matched healthy controls from a population of intravenous drug users were recruited. Circulating follicular helper T (cTfh) cells and LAG-3-expressing CD4+ T cells, type 1 regulatory T (Tr1) cells, and regulatory T (Treg) cells were characterized by flow cytometry. Serum nAb response of HCV-infected individuals was determined using pseudoparticle neutralization assays. We found that HCV infection enhanced LAG-3 expression on CD4+ T cells and exhibited regulatory T cell-like phenotype and inversely associated with the HCV nAb response. Further analysis showed that frequency of CXCR3+ cTfh cells positively correlated with nAb response, however LAG-3+ CD4+ T cells inversely associated with CXCR3+ cTfh cells. This study observed that LAG-3+ CD4+ T cells exhibit a regulatory cell phenotype and negatively associate with the HCV nAb response, implying that LAG-3 may be involved in the negative regulation of humoral immunity during HCV infection.

Infection of human pegivirus 2 (HPgV-2) is associated with hepatitis C virus but not hepatitis B virus infection in people who inject drugs.

We evaluated the association between human pegivirus-2 (HPgV-2) infection and hepatitis C virus (HCV)/hepatitis B virus (HBV) co-infection in 745 plasma samples collected from HCV-positive but human immunodeficiency virus type one (HIV-1)-negative people who inject drugs in Hunan, China. The prevalence of anti-HPgV-2 was 4.43  % (33/745) and, within this, the HCV 6a genotype showed significantly higher prevalence as compared with the HCV non-6a genotypes, 6.29  % (18/286) vs. 1.69  % (4/236), respectively (P=0.009). HPgV-2 RNA was detected in 2.15  % (16/745), and was not significantly different between the HCV 6a and non-6a genotypes, 2.45  % (7/286) vs. 2.54  % (6/236), respectively (P =0.945). HBV single infection did not increase the risk of HPgV-2 infection. Compared with HCV single infection, HCV/HBV co-infection increased the risk of HPgV-2 infection by about three-fold: odds ratio (OR)=3.24 [95  % confidence interval (CI) 1.34-7.82, P=0.014] according to anti-HPgV-2 positivity or OR=3.51 (95  % CI 1.15-10.74, P=0.051) according to HPgV-2 viraemia. HPgV-2 infection did not increase the levels of liver-specific enzymes. Our study provides new findings regarding the association between HPgV-2 and HCV genotypes as well as HCV/HBV co-infection.

HBV coinfection with HCV alters circulating Tfh cell distribution and impairs HCV neutralizing antibody responses.

Hepatitis C virus (HCV) and hepatitis B virus (HBV) coinfection reciprocally influences viral replication and host defence responses. This study aimed to investigate the impact of HBV coinfection on circulating T follicular helper cell (cTfh) distribution and the HCV neutralizing antibody (nAb) response. HCV neutralizing antibody responses were measured in individuals with HCV monoinfection (n = 83) and HBV/HCV coinfection (n = 78) using the HCV pseudoparticle neutralization assay. The frequencies of cTfh cells and their subsets in HCV monoinfection (n = 34) and HBV/HCV coinfection (n = 30) were analysed by flow cytometry. The correlations of clinical parameters, cTfh cells and neutralizing antibody responses were analysed. Compared with HCV monoinfection, the HBV coinfection group showed significantly lower HCV neutralizing antibody responses (P < 0.001) and a decreased frequency of circulating Th1-like Tfh cells (Tfh1) (P = 0.004). In HCV monoinfection, the frequency of the Tfh1 subset was positively correlated with HCV neutralizing antibody responses (R = 0.378, P = 0.03), but this correlation was lost under HBV/HCV coinfection (R = 0.115, P = 0.551). In contrast, the frequency of circulating Th2-like Tfh cells (Tfh2) was negatively correlated with the HCV neutralizing antibody responses (R = 0.404, P = 0.003). Further analysis showed that HBV coinfection enhanced the Tfh2 subset composition within cTfh cells (P < 0.001), which was associated with serum HBsAg in HBV/HCV coinfection (R = 0.521, P = 0.003). As expected, HBsAg also exhibited an inverse association with HCV neutralizing antibody responses in HBV/HCV coinfection (R = 0.59, P < 0.001). In contrast to HCV monoinfection, HBV/HCV coinfection leads to altered cTfh cell distribution and impaired HCV neutralizing antibody responses, which are associated with HBsAg. These findings will be helpful for better understanding the immunopathogenesis of HBV/HCV coinfection.

Altered regulatory cytokine profiles in cases of pediatric respiratory syncytial virus infection.

OBJECTIVES: Regulatory cytokines are associated with viral infection. The objective of this study was to evaluate the relation between serum regulatory cytokines concentrations and respiratory syncytial virus (RSV) disease. METHODS: We enrolled 325 children aged < 24 months who were diagnosed with acute respiratory tract infection. Twenty age-matched healthy children were enrolled as controls. Nasopharyngeal swabs were analyzed to identify virus by reverse transcription polymerase chain reaction, and blood samples were taken to quantify the regulatory cytokine concentrations, including interleukin (IL)-35, IL-10 and transforming growth factor (TGF)-ß1 using the Bio-Plex immunoassay or enzyme-linked immunosorbent assay. RESULTS: RSV disease was associated with a great regulatory cytokine response than healthy children, among 89 RSV-infected patients, serum IL-35 (P = .0001) and IL-10 (P = .006) was significantly elevated in comparison with healthy controls. Young children (0< age ≤6 months) with RSV infection had significantly lower IL-35 and IL-10 expression but needed more oxygen therapy and more severe disease comparing with older children (12< age <24 months). Comparing with mild group, the expression levels of IL-10 were significantly lower in children with moderate and severe disease (P = .012 and P = .005, respectively). And levels of IL-10 was inversely associated with total duration of RSV infection symptoms (r = - 0.311, P = .019). CONCLUSION: Children with RSV infected had increased serum regulatory cytokine IL-10 and IL-35 concentrations. Elevated expression of IL-10 and IL-35 were contributed to protect hypoxia and reduce the severity of disease.

Dengue virus and Japanese encephalitis virus infection of the central nervous system share similar profiles of cytokine accumulation in cerebrospinal fluid.

Dengue virus (DENV) and Japanese encephalitis virus (JEV) are two important pathogenic viruses that can cause severe encephalitis, which is accompanied by inflammatory cytokines. However, the inflammatory cytokine content of cerebrospinal fluid (CSF) in DENV and JEV infection of central nervous system are not sufficiently studied. To investigate cytokine levels in serum and CSF of hospitalised children with DENV and JEV infection of the central nervous system, a total of 183 hospitalised children with viral encephalitis-like syndrome were enrolled between May 2014 and April 2015 at the Children's Hospital of Chenzhou, Hunan, China. DENV and JEV infection was diagnosed by ELISA. Cytokine levels in the serum and CSF were measured by commercial ELISA kits. Twenty-nine (15.85%) and 26 (14.21%) DENV and JEV infections were detected in 183 patients with viral encephalitis-like syndrome, respectively. Higher granulocyte-macrophage colony-stimulating factor (GM-CSF) levels were detected in the serum of JEV infected patients than in DNEV patients (p < 0.05) or in healthy controls (p < 0.001), and levels of GM-CSF, interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) were higher in the CSF than serum in both DENV and JEV infection. Both DENV and JEV infection induced similar cytokine accumulation profiles in the CSF, which probably contributed to DENV- and JEV-induced immunopathogenesis.

Modulation of Hepatitis C Virus-Specific CD8 Effector T-Cell Function with Antiviral Effect in Infectious Hepatitis C Virus Coculture Model.

The antiviral effects of hepatitis C virus (HCV)-specific CD8 T cells have been shown in an HCV replicon system but not in an authentic infectious HCV cell culture (HCVcc) system. Here, we developed tools to examine the antigenicity of HCV-infected HLA-A2-positive Huh7.5 hepatoma cells (Huh7.5A2 cells) in activating HCV-specific CD8 T cells and the downstream antiviral effects. Infectious HCV epitope mutants encoding the well-defined genotype 1a-derived HLA-A2-restricted HCV NS3-1073 or NS5-2594 epitope were generated from a genotype 2a-derived HCV clone (Jc1Gluc2A) by site-directed mutagenesis. CD8 T-cell lines specific for NS3-1073 and NS5-2594 were expanded from HCV-seropositive persons by peptide stimulation in vitro or engineered from HCV-seronegative donor T cells by transduction of a lentiviral vector expressing HCV-specific T-cell receptors. HCV-specific CD8 T cells were cocultured with Huh7.5 cells that were pulsed with titrating doses of HCV epitope peptides or infected with HCV epitope mutants. HCV-specific CD8 T-cell activation (CD107a, gamma interferon, macrophage inflammatory protein 1ß, tumor necrosis factor alpha) was dependent on the peptide concentrations and the relative percentages of HCV-infected Huh7.5A2 cells. HCV-infected Huh7.5A2 cells activated HCV-specific CD8 T cells at levels comparable to those achieved with 0.1 to 2 µM pulsed peptides, providing a novel estimate of the level at which endogenously processed HCV epitopes are presented on HCV-infected cells. While HCV-specific CD8 T-cell activation with cytolytic and antiviral effects was blunted by PD-L1 expression on HCV-infected Huh7.5A2 cells, resulting in the improved viability of Huh7.5A2 cells, PD-1 blockade reversed this effect, producing enhanced cytolytic elimination of HCV-infected Huh7.5A2 cells. Our findings, obtained using an infectious HCVcc system, show that the HCV-specific CD8 T-cell function is modulated by antigen expression levels, the percentage of HCV-infected cells, and the PD-1/PD-L1 pathways and has antiviral and cytotoxic effects.IMPORTANCE We developed several novel molecular and immunological tools to study the interactions among HCV, HCV-infected hepatocytes, and HCV-specific CD8 T cells. Using these tools, we show the level at which HCV-infected hepatoma cells present endogenously processed HCV epitopes to HCV-specific CD8 T cells with antiviral and cytotoxic effects. We also show the marked protective effect of PD-L1 expression on HCV-infected hepatoma cells against HCV-specific CD8 T cells.