The canonical theory of immunology stating that "Immunoglobulin (Ig) is produced by B lymphocytes and exerts antibody activity" has been established since the 1970s. However, the discovery of non B cell-derived Igs (non B-Igs), which can exert multiple biological activities in addition to their antibody activities, necessitates a reevaluation of the classic concept of Ig. This has been documented with a number of characteristics related to their structure, modification, genetic regulation as well as the functions associated with clinical conditions, particularly multiple cancers. The discovery of non B-Ig provides us with a new perspective to better understand not only basic immunology, but also various Ig-related clinical manifestations including autoimmune diseases, chronic inflammation, and anaphylaxis. Notably, non B-Ig can directly promote the occurrence of malignant tumours.

Fungal infections usually occur in immunocompromised patients. Intravenous immunoglobulin (IVIG) has been used as therapeutic interventions for many infectious diseases, but seldom applied in mycosis due to unknown antifungal specificity. This study aims to determine the presence of antifungal antibodies in IVIG. Binding reactivity of IVIG with crude and recombinant antigens of Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans and Talaromyces marneffei were observed in a dose-dependent manner, similar with mixed normal human sera. The antifungal specificity was further confirmed by competitive enzyme-linked immunosorbent assays (ELISA) inhibited by rabbit specific antifungal polyclonal antibodies (PAbs) and homogenous crude antigens with inhibitions of 65.5-87.2% and 73.1-94.2%, respectively. Moreover, IVIG also reacted with fungal glycoproteins (Csa2, Cpl1 and Mp1p) in a dose-dependent way, which was inhibited by specific rabbit PAbs and homogenous antigens with different inhibitions and pulled down 72.8-83.8% of specific antibodies if preabsorption IVIG with Dynabeads® coupled with homogenous glycoproteins. These results furthermore verified the antifungal specificity of IVIG. Among four brands of IVIG, there was different antifungal IgG against C. albicans (P < 0.05) and C. neoformans (P < 0.05), while no difference for A. fumigatus (P = 0.086) and T. marneffei (P = 0.057). IVIG contained a significantly higher level of specific IgG for C. albicans than other three fungi (P <0.001). In conclusion, we proved antifungal IgG against C. albicans, A. fumigatus, C. neoformans and T. marneffei present in IVIG, which might be expected to provide a possible immunoregulation choice for mycosis and an evaluation to humoral immunity against fungi.

Background: Antibodies induced by viral infection can not only prevent subsequent virus infection, but can also mediate pathological injury following infection. Therefore, understanding the B-cell receptor (BCR) repertoire of either specific neutralizing or pathological antibodies from patients convalescing from Coronavirus disease 2019 (COVID-19) infection is of benefit for the preparation of therapeutic or preventive antibodies, and may provide insight into the mechanisms of COVID-19 pathological injury. Methods: In this study, we used a molecular approach of combining 5’ Rapid Amplification of cDNA Ends (5’-RACE) with PacBio sequencing to analyze the BCR repertoire of all 5 IgH and 2 IgL genes in B-cells harvested from 35 convalescent patients after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Results: We observed numerous BCR clonotypes within most COVID-19 patients, but not in healthy controls, which validates the association of the disease with a prototypical immune response. In addition, many clonotypes were found to be frequently shared between different patients or different classes of antibodies. Conclusions: These convergent clonotypes provide a resource to identify potential therapeutic/prophylactic antibodies, or identify antibodies associated with pathological effects following infection with SARS-CoV-2.

Historically, immunoglobulin (Ig) has been known as an antibody and is expressed only in B lineage cells; importantly, Ig light chains are conjugated to heavy chains to form intact Igs. However, in this study, we found a free Igκ light chain with a unique Vκ4-1/Jκ3 rearrangement (Vκ4-1/Jκ3-FLC) that was widely expressed in different non-B lineages and was overexpressed in cancer cells. Further study indicated that Vκ4-1/Jκ3-FLC was hydrophobic, formed obvious insoluble deposits in the extracellular matrix (ECM) and existed in free form. Functional analyses demonstrated that Vκ4-1/Jκ3-FLC promoted the proliferation, migration and metastasis of colon cancer cells in vitro and in vivo. Mechanistically, Vκ4-1/Jκ3-FLC bound to integrin β1 and activated the FAK and Src pathways. More importantly, specific antibodies against the variable region of Vκ4-1/Jκ3-FLC significantly inhibited the growth of colon cancer tumors. Our findings suggested that Vκ4-1/Jκ3-FLC is a novel ECM protein and integrin β1 ligand and that it is involved in cancer progression and is a potential therapeutic target in cancer, particularly colon cancer.

Historically, immunoglobulin (Ig) has been known as an antibody and is expressed only in B lineage cells; importantly, Ig light chains are conjugated to heavy chains to form intact Igs. However, in this study, we found a free Ig kappa light chain with a unique V kappa 4-1/J kappa 3 rearrangement (V kappa 4-1/J kappa 3-FLC) that was widely expressed in different non-B lineages and was overexpressed in cancer cells. Further study indicated that V kappa 4-1/J kappa 3-FLC was hydrophobic, formed obvious insoluble deposits in the extracellular matrix (ECM) and existed in free form. Functional analyses demonstrated that V kappa 4-1/J kappa 3-FLC promoted the proliferation, migration and metastasis of colon cancer cells in vitro and in vivo. Mechanistically, V kappa 4-1/J kappa 3-FLC bound to integrin beta 1 and activated the FAK and Src pathways. More importantly, specific antibodies against the variable region of V kappa 4-1/J kappa 3-FLC significantly inhibited the growth of colon cancer tumors. Our findings suggested that V kappa 4-1/J kappa 3-FLC is a novel ECM protein and integrin beta 1 ligand and that it is involved in cancer progression and is a potential therapeutic target in cancer, particularly colon cancer.

Pam3CSK4 is a synthetic tripalmitoylated lipopeptide that acts as a ligand of TLR1/TLR2 by mimicking the acetylated amino terminus of bacterial lipoproteins. Here we found that pretreatment of Pam3CSK4 protected mice from systemic infection of methicillin-resistant Staphylococcus aureus (MRSA), and enhanced the bacterial clearance in bacteremia model. Pro-inflammatory cytokines, such as TNF-α, IL-6, MCP-1 and IFN-γ were significantly decreased in serum from Pam3CSK4-treated mice. Besides, upon PamCSK4 treatment, the TLR2 expression was down-regulated, IRAK1 phosphorylation was inhibited, and the expression of IRAK-M and Tollip, two negative regulators of NF-κB pathway, was up-regulated. All of these indicated that Pam3CSK4 attenuated inflammation via inhibiting TLR1/TLR2 and the downstream NF-κB pathways, and suggested that Pam3CSK4 could be a potential immune modulator for MRSA systemic infection.

To understand why vaccine-activated tumor-specific T cells often fail to generate antitumor effects, we have focused on studying the intrinsic attributes of two alpha fetoprotein-specific CD8 T cells (Tet212 and Tet499). We found that while Tet499 survived in vitro re-stimulation and maintained their functions, Tet212 underwent exhaustion and apoptosis. In vivo, Tet499 generated more expansion than Tet212 and stronger antitumor effects. The different antigen responsiveness and antitumor effects of Tet212 and Tet499 correlated to their activation and differentiation state. Compared to Tet212, Tet499 were in a lower activation state and contained more stem-like memory T cells (Tscm) that could undergo more expansion in vivo. Mechanistically, Tet499 TCR was more downregulated than Tet212 TCR, which may be responsible for the lower activation and differentiation state of Tet499. In contrast to the notion that TCR downregulation dampens T cell antigen reactivity, our data show that it may help T cells maintain their antigen reactivity by halting their activation and differentiation at Tscm stage and by protecting T effector cells from antigen-induced exhaustion and apoptosis. These findings indicate that the focus of cancer vaccination should be on eliciting tumor-specific T cells at earlier differentiation stage including Tscm for better antigen reactivity.

Supplementary Figure from The Antitumor Effects of Vaccine-Activated CD8+ T Cells Associate with Weak TCR Signaling and Induction of Stem-Like Memory T Cells

Objective:To ascertain whether the immune complexes (ICs) formed by Dengue virus 1 non-structure protein 1 (DENV1 NS1)and its IgG antibodies could mediate passive systemic anaphylaxis (PSA) and to explain the pathogenesis of Dengue hemorrhagic fever or Dengue shock syndrome (DHF/DSS).Methods:The monoclonal antibodies (mAbs) or mAb cocktails from 20 IgG mAbs of DENV1 NS1 prepared in this lab were screened to initiate PSA and passive cutaneous anaphylaxis (PCA) in mice.Meanwhile, the effects of GdCl3 and platelet activating factor ( PAF) antagonist CV-3988 on PSA induced by the NS1-IgG ICs were observed.Results:Two groups of monoclonal antibody cocktails with purified NS 1 were proved to be capable of provoking PCA and PSA in mice,whereas the other mAbs or mAb cocktails could be not .The murine PSA initiated by NS1-IgG(5D25+3B1) ICs could be sig-nificantly inhibited by in vivo treatment with GdCl3 or PAF antagonist CV-3988.Conclusion: The NS1-IgG ICs formed with DENV1 NS1 and IgG mAb cocktails can mediate PSA and PCA ,but not all of ICs formed by DENV 1 NS1 mAbs or mAb cocktails with DENV 1 NS1 can induce PSA ,indicating that it may be related to the special epitopes of DENV 1 NS1.The monocyte/macrophages and PAF may be as major effector cells and the major mediator for PSA induced by NS 1-IgG ICs,respectively.

Objective To study the viral etiologic characteristics of acute respiratory viruses associated with adults influenza like illness (ILI) case.Methods ILI cases and non-ILI cases of nose swabs specimen were collected from Zhujiang Hospital of Southern Medical University from Nov 2009 to Oct 2014.qRT-PCR was used to test for 18 respiratory viruses.Results 201 virus positive responses were detected in samples from 420 (47.9％) patients with influenza like illness,while only 3 cases (2.6％) were found to be infected with virus in 115 non-ILI cases.The most common virus identified was influenza virus (IFV) in frequency of 51.2％ (103/201),followed by human rhinovirus (RHV) and coronaviruses (Coy).There was no significant difference among each age group in virus detection rate,however,there were obvious differences in viruses detected of several age groups,which showed that the main inflect crowd of influenza was 18 to 30 age group,the main inflect crowd of human coronavirus was 51-77 age group,and the main inflect crowd of human rhinovirus was 51-90 age group.Conclusion ILI cases in adult mosdy were caused by infection with several kinds of respiratory virus,and the dominant virus for ILI was proved to be the seasonal influenza viruses.

Cancer-associated antigen 215 (CA215) is an immunoglobulin molecule expressed by numerous tumor types. Membrane‑bound and soluble CA215 have been detected in the majority of cancer cells and rarely identified in normal tissues. In addition, CA215C is a carbohydrate‑associated epitope in the variable region of CA215, which is specifically recognized by the monoclonal antibody, RP215. However, CA215C is not a suitable vaccine candidate as it is a thymus‑independent antigen. In the present study, RP215 was used as a target to screen short peptide mimics of CA215C from a phage display peptide library. Following three rounds of screening, 30 positive phage clones that specifically bound to RP215 were identified and sequenced. The result of amino‑acid sequence analysis revealed five conserved sequence groups for seventeen of the positive phage clones. The sequences of phage clones 2, 13 and 42 were selected for peptide synthesis and binding analysis. The synthetic peptides R2 and R42 specifically bound RP215. Antisera from mice immunized with R2‑BSA or R42‑BSA bound purified CA215C and innate CA215C expressed on human hepatic and rectal carcinoma tissues, as demonstrated by immunohistochemistry. Furthermore, R2‑BSA and R42‑BSA antisera inhibited RP215 binding to cancer tissues. These results revealed that R2‑BSA and R42‑BSA antisera had similar characteristics to RP215 and that the synthetic peptides R2 and R42 may mimic the CA215C epitope. R2 and R42 peptides may therefore have potential for development into a tumor vaccine.

The envelope domain III (EDIII) of the dengue virus (DENV) has been confirmed to be involved in receptor binding. It is the target of specific neutralizing antibodies, and is considered to be a promising subunit dengue vaccine candidate. However, several recent studies have shown that anti‑EDIII antibodies contribute little to the neutralizing or enhancing ability of human DENV‑infected serum. The present study involved an analysis of the neutralization and antibody‑dependent enhancement (ADE) activities of EDIII‑reactive antibodies in human convalescent sera from patients with primary DENV‑1 infection and rabbit antiserum immunized with recombinant DENV‑1 EDIII protein. The results indicated that serum neutralization was not associated with titres of EDIII‑binding antibodies in the human DENV‑1‑infected sera. The depletion of anti‑EDIII antibodies from these serum samples revealed that the anti‑EDIII antibodies of the patients contributed little to neutralization and ADE. However, the EDIII‑reactive antibodies from the rabbit antiserum exhibited protective abilities of neutralization at a high dilution (~1:50,000) and ADE at a low dilution (~1:5,000) for the homotypic DENV infection. Notably, the rabbit antiserum displayed ADE activity only at a dilution of 1:40 for the heterotypic virus infection, which suggests that EDIII‑reactive antibodies may be safe in secondary infection with heterotypic viruses. These results suggest that DENV EDIII is not the predominant antigen of the DENV infection process; however, purified or recombinant DENV EDIII may be used as a subunit vaccine to provoke an effective and safe antibody response.

Human adenoviruses (HAdVs) are highly contagious pathogens causing acute respiratory disease (ARD), such as community-acquired pneumonia. HAdV-7d, a re-emergent genomic variant, has been recently reported in Asia and the United States after a several-decade absence. However, whether HAdV-7d is associated with higher severity than other types is currently unclear. In this study, the clinical and epidemiological investigation showed that fever, cough, and sore throat were the three most common respiratory symptoms of HAdV infections. HAdV-7 caused longer duration of fever, higher morbidity of tachypnea/dyspnea, pleural effusion, diarrhea, hepatosplenomegaly, consciousness alteration, as well as higher rates of pneumonia, mechanical ventilation and higher fatality rate (28.6%) than other types, particularly HAdV-3 and HAdV-2. The genomes of seven HAdV-7d isolates from mild, severe, and fatal cases were sequenced and highly similar with each other. Surprisingly, two isolates (2011, 2012) had 100% identical genomes with an earlier strain from a fatal ARD outbreak in China (2009), which elucidates the virus origin and confirms the unexpected HAdV genomic conservation and stability. Phylogenetic analysis indicated that L1 52/55-kDa DNA packaging protein may be associated with the higher severity of illness and fatality rate of HAdV-7. Clinicians need to be aware of HAdVs in children with ARD.

Due to the enormous capacity of Staphylococcus aureus to acquire antibiotic resistance, it becomes imperative to develop vaccines for decreasing the risk of its life-threatening infections. Peptidoglycan (PGN) is a conserved and major component of S. aureus cell wall. However, it has not been used as a vaccine candidate since it is a thymus-independent antigen. In this study, we synthesized a multiple antigenic peptide, named MAP27, which comprised four copies of a peptide that mimics the epitope of PGN. After immunization with MAP27 five times and boosting with heat-inactivated bacterium one time, anti-MAP27 serum bound directly to S. aureus or PGN. Immunization with MAP27 decreased the bacterial burden in organs of BALB/c mice and significantly prolonged their survival time after S. aureus lethal-challenge. The percentage of IFN-γ(+)CD3(+) T cells and IL-17(+)CD4(+) T cells in spleen, as well as the levels of IFN-γ, IL-17A/F and CCL3 in spleen and lung, significantly increased in the MAP27-immunized mice after infection. Moreover, in vitro incubation of heat-inactivated S. aureus with splenocytes isolated from MAP27-immunized mice stimulated the production of IFN-γ and IL-17A/F. Our findings demonstrated that MAP27, as a thymus-dependent antigen, is efficient at eliciting T cell-mediated responses to protect mice from S. aureus infection. This study sheds light on a possible strategy to design vaccines against S. aureus.

目的 了解我国手足口病常见病毒感染患者血清中抗体交叉反应及保护性,为揭示手足口病二次感染保护机制提供依据.方法 收集2010-2014年珠江医院经荧光定量PCR确诊为手足口病患儿血清,用间接免疫荧光鉴定非EV71肠道病毒感染血清与EV71病毒感染细胞间结合,及EV71感染恢复期血清和抗EV71单克隆抗体与非EV71肠道病毒感染细胞的结合,对存在交叉结合反应的血清检测其交叉中和保护性.结果 共收集149例非EV71肠道病毒感染血清,经鉴定其中54份标本无EV71混合感染及既往感染,血清型分别为CA6、CA10、CA9、CA5、CA16、CB3、CB5及未分型肠道病毒.46份(85.2％)非EV71感染血清IgG抗体与EV71病毒感染细胞结合,具统计学意义(Z=-2.39,P=0.017),并对EV71病毒具弱中和活性.EV71感染血清及5株抗EV71单克隆抗体仅与CA16感染细胞结合.结论 初步确定多种非EV71肠道病毒感染血清与EV71病毒存在交叉反应,且具有弱中和活性;而EV71感染血清对其他肠道病毒无交叉保护作用.首次证明抗EV71 VP2、VP4单抗可结合CA16病毒.

This study performed to identify and characterize the epitopes recognized by cross-neutralizing antibody against envelope protein domain Ⅲ(ED Ⅲ) of dengue virus(DENV). Two strains of cross-neutralizing monoclonal antibody(m Ab) against ED Ⅲ of dengue virus were used to screen the epitopes from phage peptide library, and the selected phage clones were tested by sandwich enzyme-linked immunosorbent assay(ELISA), and then analyzed by sequencing and bioinformatics. Furthermore, the antigenicity of the mimotope to conservative epitope within EDⅢ was identified by murine antisera immunized with positive phage clones. Phage clones binding to m Ab 2B11A35 and 2D73A7 were obtained and proved as conformational epitopes. The bioinformatics analysis showed that m Ab 2B11A35 and 2D73A7 could recognize the epitope that located in the EF loop on EDⅢ protein.The antisera of mice immunized with 3 phage clones binding to m Ab 2B11A35 were capable of binding to dengue1, 3 and 4 ED Ⅲ proteins and DENV 2 infected cells. In conclusion, we identified a new conservative and protective epitope in EDⅢ protein recognized by two strains of cross-neutralizing m Abs, which could be used for dengue virus vaccine design.

Objective:To screen epitope mimics to lipoteichoic acid from a random 12-mer phage display peptide library and i-dentify the specificity of the mimotopes of LTA.Methods:The monoclonal antibody against LTA was used as a target to screen the 12-mer phage display peptide library and the specificity of phage clones were identified by sandwich ELISA.The amino acid sequences of positive phage clones were deduced from DNA sequencing.The specificity of synthetic peptide were identified by sandwich ELISA.Results:4 clones were obtained after 3 rounds of screening.Amino acid sequence analysis revealed four different types of mimotope sequence.A linear peptide (GHxDFRQxxQPS),named L2,which derived from positive sequence was synthesized.ELISA result indicates that L2 can bind to anti-LTA mAb specifically in a dose-dependent manner.Conclusion:The mimotopes of LTA were obtained by using the phage display technology.

Objective To compare the detection efficiency between multiplex RT-PCR method and liquichip technology for screening the viral etiological agents of diarrhea.Methods The development of the multiplex RT-PCR method.A total of 107 feces samples from patients who suffered from diarrhea and attended to Zhujiang Hospital of Southern University from September 2013 to February 2014 were collected and tested in parallel by both multiplex RT-PCR and xTAG Gastrointestinal Pathogen Panel ( xTAG GPP) for Adenovirus, Norovirus genogroupⅠandⅡ, as well as by both multiplex RT-PCR and monoplex RT-PCR for Astrovirus and Sapovirus.To evaluate the sensitivity and specificity of multiplex RT-PCR, xTAG GPP and monoplex RT-PCR were used as reference.Kappa coefficient test was used to evaluate the consistency among the methods.The detection limit and accuracy of multiplex RT-PCR were evaluated by detection of serial dilution of positive plasmids and products sequencing for the five viral agents.Results The multiplex RT-PCR showed high consistency with xTAG GPP and monoplex RT-PCR, in which Kappa value was 0.885 and 1.000 respectively( P=0.000 ).Compared to xTAG GPP, the sensitivity and specificity of the multiplex RT-PCR were at average of 80.8%( 21/26 ) and 100%( 295/295 ) respectively.The detection limit and accuracy of multiplex RT-PCR were 104 copies /μl-106 copies/μl.Conclusion The high consistency indicated that both the multiplex RT-PCR and xTAG GPP are useful as a special,sensitive, high throughput and rapid diagnostic tools for the detection of the major viral pathogens related to diarrhea in clinical laboratory.

IgG-induced passive systemic anaphylaxis (PSA), a serious adverse effect of passive immune therapy using therapeutic monoclonal antibodies, has been greatly emphasized. However, controversy exists regarding the type of effector cells involved in IgG-induced anaphylaxis as a result of the induction of PSA by different IgG subtypes or the use of mice with varying genetic backgrounds. To clarify the effector cells for PSA, the PSA model with serious hypothermia was established by IgG monoclonal antibody (mAb) against natural protein or complete antigen, not hapten conjugate, in BALB/c and C57BL/6 mice. The results indicated that PSA could be remarkably inhibited by the depletion of macrophages but not by the depletion of whole leukocytes, basophils, neutrophils or monocytes. We further confirmed that macrophages are indispensable for the PSA induced by all six IgG-natural antigen complexes in both strains of mice. Additionally, platelet-activating factor (PAF) was found to be the major effector mediator for IgG-induced anaphylaxis. Moreover, gene knock-out of the third component of complement (C3) did not affect PSA-related hypothermia in C57BL/6 mice. These results indicate that macrophages and PAF act as dominant effector cells and mediator molecules, respectively, and are indispensable components in the induction of IgG-mediated PSA induced by IgG mAb and natural protein antigen. Based on the above results, we hypothesize that inconsistencies in effector cells for PSA may be associated with different features of the mAb-antigen system that might affect the magnitude of FcγRs cross-linking on effector cells.

The early diagnosis of West Nile virus (WNV) infection is important for successful clinical management and epidemiological control. The non-structural protein 1 (NS1) of flavivirus, a highly conserved and secreted glycoprotein, is abundant in the serum of flavivirus-infected patients and represents a useful early diagnostic marker. We developed a WNV-specific NS1 antigen-capture ELISA using two mouse monoclonal antibodies (MAbs) that recognised distinct epitopes of the NS1 protein of WNV as capture and detection antibodies. The antigen-capture ELISA displayed exclusive specificity to WNV without cross-reaction with other related members of the flavivirus family, including the dengue virus, yellow fever virus, Japanese encephalitis virus, and tick-borne encephalitis virus. Additionally, the specificity was presented as no false positive in normal (0/1003) and DENV-infected (0/107) human serum specimens. The detection limit of the antigen-capture ELISA was as low as 15 pg/ml of recombinant WNV NS1 protein (rWNV-NS1) and 6.1 plaque-forming units (PFU)/0.1 ml of WNV-infected culture supernatant. In mice infected with WNV, the NS1 protein was readily detected in serum as early as one day after WNV infection, prior to the development of clinical signs of the disease. The sensitivity of the NS1 capture ELISA (93.7%) was significantly higher (79.4%) than that of real-time reverse transcription polymerase chain reaction in 63 serum samples from WNV-infected mice (p = 0.035). This newly developed NS1 antigen-capture ELISA with high sensitivity and specificity could be used as an efficient method for the early diagnosis of WNV infection in animals or humans.