SARS-CoV-2 nucleocapsid: Biological functions and implication for disease diagnosis and vaccine design.

The coronavirus disease 2019 (COVID-19) pandemic is transmitted by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has affected millions of people all around the world, leading to more than 6.5 million deaths. The nucleocapsid (N) phosphoprotein plays important roles in modulating viral replication and transcription, virus-infected cell cycle progression, apoptosis, and regulation of host innate immunity. As an immunodominant protein, N protein induces strong humoral and cellular immune responses in COVID-19 patients, making it a key marker for studying N-specific B cell and T cell responses and the development of diagnostic serological assays and efficient vaccines. In this review, we focus on the structural and functional features and the kinetic and epitope mapping of B cell and T cell responses against SARS-CoV-2 N protein to extend our understanding on the development of sensitive and specific diagnostic immunological tests and effective vaccines.

Discovery of a novel marker for human granulocytes and tissue macrophages: RTL1 revisited.

Here, retrotransposon-like 1 (RTL1) is introduced as a marker for circulating and tissue neutrophils, tissue macrophages, and tumor-associated macrophages (TAM) and neutrophils (TAN). Anti-RTL1 polyclonal and monoclonal antibodies were produced, and their reactivity was examined by Western blotting (WB), ELISA, and immunostaining of human normal and cancer tissues. The reactivity of the anti-RTL1 antibodies with peripheral blood leukocytes and a panel of hematopoietic cell lines was examined. The generated antibodies specifically detected RTL1 in the WB of the placenta and U937 cells. The polyclonal antibody showed excellent reactivity with tissue-resident macrophages, Hofbauer cells, alveolar and splenic macrophages, Kupffer cells, and inflammatory cells in the tonsil, appendix, and gallbladder. In vitro GM-CSF-differentiated macrophages also showed a high level of intracellular RTL1 expression. TAM and TAN also showed excellent reactivity with this antibody. Almost all circulating granulocytes but not lymphocytes or monocytes expressed RTL1 at their surface. Serial sections of the appendix stained with CD15 and RTL1 and placenta stained with CD68 and RTL1 showed a considerable overlap in RTL1 expression in CD15+ granulocytes and CD68+ macrophages. A small percentage of myelomonocytic cell lines was positive for surface RTL1, while promyelocytic, monocytic, megaloblastic, and lymphoblastic cell lines were negative. Endothelial cells of normal and cancer tissues highly expressed RTL1. RTL1 could be considered a new marker for different normal tissue macrophages, TAM, circulating and tissue neutrophils, and TAN.

Diagnostic performance of a novel antigen-capture ELISA for the detection of SARS-CoV-2.

BACKGROUND AND AIMS: The coronavirus disease 2019 (COVID-19) pandemic is a serious health problem worldwide. Early virus detection is essential for disease control and management. Viral antigen detection by ELISA is a cost-effective, rapid, and accurate antigen diagnostic assay which could facilitate early viral detection. METHOD: An antigen-capture sandwich ELISA was developed using novel nucleocapsid (NP)-specific mouse monoclonal antibodies (MAbs). The clinical performance of the assay was assessed using 403 positive and 150 negative respiratory samples collected during different SARS-CoV-2 variants outbreaks in Iran. RESULTS: The limit of detection of our ELISA assay was found to be 43.3 pg/ml for recombinant NP. The overall sensitivity and specificity of this assay were 70.72% (95% CI: 66.01-75.12) and 100% (95% CI: 97.57-100), respectively, regardless of Ct values and SARS-CoV-2 variants. There was no significant difference in our assay sensitivity for the detection of Omicron subvariants compared to Delta variant. Assay sensitivity for the BA.5 Omicron subvariant was calculated as 91.89% (95% CI: 85.17-96.23) for samples with Ct values < 25 and 82.70% (95% CI: 75.19-88.71) for samples with Ct values < 30. CONCLUSION: Our newly developed ELISA method is reasonably sensitive and highly specific for detection of SARS-CoV-2 regardless of the variants and subvariants of the virus.

Cross talk between hepatitis B virus and innate immunity of hepatocytes.

Innate immunity plays a major role in controlling viral infections. Recent exploration of sodium taurocholate co-transporting polypeptide receptor as specific hepatitis B virus (HBV) receptor in human hepatocytes has provided appropriate cell culture tools to study the innate immunity of hepatocytes and its cross talk with HBV. In this review, we give a brief update on interaction between HBV and innate immunity using the currently available in vitro cellular models that support the complete life cycle of HBV. We will discuss how HBV can act as a 'stealth' virus to counteract the innate immune responses mediated by the pathogen recognition receptors of hepatocytes and escape the first line of surveillance of the host immune system. We give an overview of the cellular components of innate immunity that present in these in vitro models and discuss how activating these innate immunity components may contribute to the eradication of HBV infection.

Epitope mapping of neutralising anti-SARS-CoV-2 monoclonal antibodies: Implications for immunotherapy and vaccine design.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. This disease has currently affected more than 346 million people and resulted in more than 5.5 million deaths in many countries. Neutralising monoclonal antibodies (MAbs) against the SARS-CoV-2 virus could serve as prophylactic/therapeutic agents in COVID-19 infection by providing passive protection against the virus in individuals. Until now, no Food and Drug Administration/European Medicines Agency-approved neutralising MAb against SARS-CoV-2 virus exists in the market, though a number of MAbs have been authorised for emergency use. Therefore, there is an urgent need for development of efficient anti-SARS-CoV-2 neutralising MAbs for use in the clinic. Moreover, neutralising anti-SARS-CoV-2 MAbs could be used as beneficial tools for designing epitope-based vaccines against the virus. Given that the target epitope of a MAb is a crucial feature influencing its neutralising potency, target epitopes of neutralising anti-SARS-CoV-2 MAbs already reported in the literature and reactivity of these MAbs with SARS-CoV-2 variants are reviewed herein.

Cloning, expression and characterization of a peptibody to deplete myeloid derived suppressor cells in a murine mammary carcinoma model.

BACKGROUND: Myeloid derived suppressor cells (MDSCs) are an immature heterogeneous population of myeloid lineage that attenuate the anti-tumor immune responses. Depletion of MDSCs has been shown to improve efficacy of cancer immunotherapeutic approaches. Here, we expressed and characterized a peptibody which had previously been defined by phage display technique capable of recognizing and depleting murine MDSCs. MATERIALS AND METHODS: Using splicing by overlap extension (SOE) PCR, the coding sequence of the MDSC binding peptide and linker were synthesized and then ligated into a home-made expression plasmid containing mouse IgG2a Fc. The peptibody construct was transfected into CHO-K1 cells by lipofectamine 3000 reagent and the resulting fusion protein was purified with protein G column and subsequently characterized by ELISA, SDS-PAGE and immunoblotting. The binding profile of the peptibody to splenic MDSCs and its MDSC depletion ability were then tested by flow cytometry. RESULTS: The purified peptibody appeared as a 70 KDa band in Western blot. It could bind to 98.8% of splenic CD11b+/Gr-1+ MDSCs. In addition, the intratumoral MDSCs were significantly depleted after peptibody treatment compared to their PBS-treated negative control counterparts (P < 0.05). CONCLUSION: In this study, a peptibody capable of depleting intratumoral MDSCs, was successfully expressed and purified. Our results imply that it could be considered as a potential tool for research on cancer immunotherapy.

T-cell engager antibodies enable T cells to control HBV infection and to target HBsAg-positive hepatoma in mice.

BACKGROUND & AIMS: Current antiviral therapies control but rarely eliminate HBV, leaving chronic HBV carriers at risk of developing hepatocellular carcinoma (HCC). Lacking or dysfunctional virus-specific adaptive immunity prevents control of HBV and allows the virus to persist. Restoring antiviral T-cell immunity could lead to HBV elimination and cure of chronically infected patients. METHODS: We constructed bispecific T-cell engager antibodies that are designed to induce antiviral immunity through simultaneous binding of HBV envelope proteins (HBVenv) on infected hepatocytes and CD3 or CD28 on T cells. T-cell engager antibodies were employed in co-cultures with healthy donor lymphocytes and HBV-infected target cells. Activation of the T-cell response was determined by detection of pro-inflammatory cytokines, effector function (by cytotoxicity) and antiviral effects. To study in vivo efficacy, immune-deficient mice were transplanted with HBVenv-positive and -negative hepatoma cells. RESULTS: The 2 T-cell engager antibodies synergistically activated T cells to become polyfunctional effectors that in turn elicited potent antiviral effects by killing infected cells and in addition controlled HBV via non-cytolytic, cytokine-mediated antiviral mechanisms. In vivo in mice, the antibodies attracted T cells specifically to the tumors expressing HBVenv resulting in T-cell activation, tumor infiltration and reduction of tumor burden. CONCLUSION: This study demonstrates that the administration of HBVenv-targeting T-cell engager antibodies facilitates a robust T-cell redirection towards HBV-positive target cells and provides a feasible and promising approach for the treatment of chronic viral hepatitis and HBV-associated HCC. LAY SUMMARY: T-cell engager antibodies are an interesting, novel therapeutic tool to restore immunity in patients with chronic hepatitis B. As bispecific antibodies, they bind envelope proteins on the surface of the hepatitis B virus (HBV) and CD3 or CD28 on T cells. This way, they induce a potent antiviral and cytotoxic T-cell response that leads to the elimination of HBV-positive cells. These bispecific T-cell engager antibodies are exciting therapeutic candidates for chronic hepatitis B and HBV-associated hepatocellular carcinoma.

Illuminating the in vitro effects of Epstein-Barr virus and vitamin D on immune response in multiple sclerosis patients.

Given the complexity of immune complex diseases including multiple sclerosis (MS) and the plausible interactions between different risk factors, delineating the interplay between them would be imperative. The current study aimed to evaluate the in vitro effects of Epstein-Barr virus (EBV) and vitamin D on immune response in MS patients and healthy controls. The status of vitamin D and EBV load was evaluated using multiple techniques. In vitro EBV-infected peripheral blood mononuclear cells (PBMCs), in the presence or absence of vitamin D, were checked for IL-10, IFN-γ, and vitamin D receptor. MS patients showed significantly higher plasma levels of 1,25-(OH)2D but not 25-OHD, increased EBV load, and lower levels of vitamin D receptor (VDR) expression compared with healthy controls. Interestingly, an inverse correlation was observed between VDR expression and EBV load in PBMCs. Indeed, the levels of IFN-γ and IL-10 production were significantly higher in supernatant collected from in vitro EBV-infected PBMCs in MS patients compared with controls. While all vitamin D-treated PBMCs showed reduced levels of IFN-γ production, in vitro treatment of vitamin D showed no influence in IL-10 production. EBV and vitamin D were found to exert opposite in vitro effects on immune dysregulation in these patients. Our results highlight the complex interactions of different risk factors with immune system.

Potent synergistic anti-tumor activity of a novel humanized anti-HER2 antibody hersintuzumab in combination with trastuzumab in xenograft models.

Immunotherapy of HER2-overexpressing cancers by FDA approved monoclonal antibodies (mAbs) such as trastuzumab and pertuzumab has shown promising results. We have recently produced a novel humanized anti-HER2 mAb, hersintuzumab, which did not sterically inhibit binding of trastuzumab and pertuzumab to HER2, thus recognizing a distinct epitope on subdomain I + II of HER2. In this study, we assessed the in vitro and in vivo anti-tumor activity of this mAb individually and in combination with trastuzumab. Different HER2-overexpressing human cancer cell lines, including SKOV3, NCI-N87 HCC1954 and BT-474 were cultured and binding reactivity of Hersintuzumab to these cell lines was analyzed by flow cytometry. In addition, the inhibitory effect of different concentrations of hersintuzumab, trastuzumab and their combination on tumor cells growth was assessed by XTT assay. For Assessment of tumor growth inhibition in xenograft model, Balb/c athymic nude mice were subcutaneously injected with NCI-N87 and SKOV3 tumor cells and then treated intravenously with these mAbs. Our results showed that hersintuzumab could bind to all HER2-overexpressing cell lines similar to trastuzumab. In vitro experiments showed that both hersintuzumab and trastuzumab individually and in combination inhibited growth of all cell lines with the exception of HCC-1954.Inhibitory effect of the combination of mAbs was significantly higher than that of each mAb alone. Similar results were obtained in the gastric (NCI-N87) and ovarian (SKOV-3) tumor xenograft models. Hersintuzumab in combination with trastuzumab induces synergic anti-tumor effects on HER2-overexpressing cells in vitro and in vivo and is potentially a therapeutic tool for treatment of HER2-overexpressing cancers.

The interplay between vitamin D and viral infections.

The pleiotropic role of vitamin D has been explored over the past decades and there is compelling evidence for an epidemiological association between poor vitamin D status and a variety of diseases. While the potential anti-viral effect of vitamin D has recently been described, the underlying mechanisms by which vitamin D deficiency could contribute to viral disease development remain poorly understood. The possible interactions between viral infections and vitamin D appear to be more complex than previously thought. Recent findings indicate a complex interplay between viral infections and vitamin D, including the induction of anti-viral state, functional immunoregulatory features, interaction with cellular and viral factors, induction of autophagy and apoptosis, and genetic and epigenetic alterations. While crosstalk between vitamin D and intracellular signalling pathways may provide an essential modulatory effect on viral gene transcription, the immunomodulatory effect of vitamin D on viral infections appears to be transient. The interplay between viral infections and vitamin D remains an intriguing concept, and the global imprint that vitamin D can have on the immune signature in the context of viral infections is an area of growing interest.

Immune function of plasmacytoid dendritic cells, natural killer cells, and their crosstalk in HBV infection.

Chronic hepatitis B virus infection is a major health problem, with over 245 million chronic carriers worldwide. This persistent infection is thought to be associated with inefficient innate and adaptive immune responses. Natural killer cells (NK cells) and plasmacytoid dendritic cells (pDCs) are the major innate immune cells which respond to viral infection at the early phase and are considered major components of the antiviral immune response. In this review, we summarize recent findings regarding the role of NK cells, pDCs, and their cross-talk in HBV infection and its chronicity. Although the data regarding the biological function of pDCs and NK cells in HBV infection is still controversial, many studies show that in chronic HBV infection, the cytotoxicity of NK cells is retained, while their capacity to secrete cytokines is strongly impaired. In addition, interferon-α production by pDCs is impaired during chronic HBV infection, and the virus interferes with pDC-NK cell interaction.

Hersintuzumab: A novel humanized anti-HER2 monoclonal antibody induces potent tumor growth inhibition.

Humanized monoclonal antibodies (mAbs) against HER2 including trastuzumab and pertuzumab are widely used to treat HER2 overexpressing metastatic breast cancers. These two mAbs recognize distinct epitopes on HER2 and their combination induces a more potent blockade of HER2 signaling than trastuzumab alone. Recently, we have reported characterization of a new chimeric mAb (c-1T0) which binds to an epitope different from that recognized by trastuzumab and significantly inhibits proliferation of HER2 overexpressing tumor cells. Here, we describe humanization of this mAb by grafting all six complementarity determining regions (CDRs) onto human variable germline genes. Humanized VH and VL sequences were synthesized and ligated to human γ1 and κ constant region genes using splice overlap extension (SOE) PCR. Subsequently, the humanized antibody designated hersintuzumab was expressed and characterized by ELISA, Western blot and flow cytometry. The purified humanized mAb binds to recombinant HER2 and HER2-overexpressing tumor cells with an affinity comparable with the chimeric and parental mouse mAbs. It recognizes an epitope distinct from those recognized by trastuzumab and pertuzumab. Binding of hersintuzumab to HER2 overexpressing tumor cells induces G1 cell cycle arrest, inhibition of ERK and AKT signaling pathways and growth inhibition. Moreover, hersintuzumab could induce antibody-dependent cell cytotoxicity (ADCC) on BT-474 cells. This new humanized mAb is a potentially valuable tool for single or combination breast cancer therapy.

Correction to: Blocking of opioid receptors in experimental formaline-inactivated respiratory syncytial virus (FI-RSV) immunopathogenesis: from beneficial to harmful impacts.

In the original publication, seventh author's name was incorrectly published as 'Maryam Golaram'.

Blocking of opioid receptors in experimental formaline-inactivated respiratory syncytial virus (FI-RSV) immunopathogenesis: from beneficial to harmful impacts.

Opioid system plays a significant role in pathophysiological processes, such as immune response and impacts on disease severity. Here, we investigated the effect of opioid system on the immunopathogenesis of respiratory syncytial virus (RSV) vaccine (FI-RSV)-mediated illness in a widely used mouse model. Female Balb/c mice were immunized at days 0 and 21 with FI-RSV (2 × 106 pfu, i.m.) and challenged with RSV-A2 (3 × 106 pfu, i.n.) at day 42. Nalmefene as a universal opioid receptors blocker administered at a dose of 1 mg/kg in combination with FI-RSV (FI-RSV + NL), and daily after live virus challenge (RSV + NL). Mice were sacrificed at day 5 after challenge and bronchoalveolar lavage (BAL) fluid and lungs were harvested to measure airway immune cells influx, T lymphocyte subtypes, cytokines/chemokines secretion, lung histopathology, and viral load. Administration of nalmefene in combination with FI-RSV (FI-RSV + NL-RSV) resulted in the reduction of the immune cells infiltration to the BAL fluid, the ratio of CD4/CD8 T lymphocyte, the level of IL-5, IL-10, MIP-1α, lung pathology, and restored weight loss after RSV infection. Blocking of opioid receptors during RSV infection in vaccinated mice (FI-RSV-RSV + NL) had no significant effects on RSV immunopathogenesis. Moreover, administration of nalmefene in combination with FI-RSV and blocking opioid receptors during RSV infection (FI-RSV + NL-RSV + NL) resulted in an increased influx of the immune cells to the BAL fluid, increases the level of IFN-γ, lung pathology, and weight loss in compared to control condition. Although nalmefene administration within FI-RSV vaccine decreases vaccine-enhanced infection during subsequent exposure to the virus, opioid receptor blocking during RSV infection aggravates the host inflammatory response to RSV infection. Thus, caution is required due to beneficial/harmful functions of opioid systems while targeting as potentially therapies.

All-trans retinoic acid in combination with sodium butyrate enhances specific monoclonal antibody productivity in recombinant CHO cell line.

The effects of all-trans retinoic acid (RA) and sodium butyrate (NaBu) on growth, viability and antibody production of two types of transfected Chinese hamster ovary cell lines (CHO-K1 and CHO-S) were investigated using a batch mode cell culture. By adding 0.5 mM NaBu in the CHO-K1 cell culture, the cell specific productivity (Qp) and antibody concentration increased by five- and threefold, respectively. The optimal concentration of RA was 100 nM which resulted in twofold increase in antibody production. In a combination model, RA applied at early growth phase of CHO-K1 cells followed by addition of NaBu with lowering culture temperature at the end of stationary phase resulted in two- and threefold increase in Qp and final antibody concentration, respectively. The latter strategy was also applied on suspended CHO-S cells with enhanced Qp and antibody concentration, but to a lesser extent than the CHO-K1 cells. In conclusion, our results demonstrate that the addition of RA and NaBu along with lowering the culture temperature can increase cell culture period as well as Qp and the final concentration of recombinant monoclonal antibody in both CHO-K1 and CHO-S cells without any significant change in binding affinity of the mAb.

Variation of Housekeeping Genes in Clinical Isolates and Vaccine Strains of Bordetella pertussis.

BACKGROUND: Bordetella pertussis causes serious contagious infections, primarily in childhood. A whole-cell vaccine, diphtheria-tetanus-whole cell pertussis (DTwP), has been used to protect against pertussis in children in Iran, but the pertussis cases have been increasing during recent years. We determined the allelic variation level of housekeeping genes in isolates recovered from pertussis patients and vaccine strains used in national vaccination program. METHODS: Five clinical isolates, 2 vaccine strains and a Tohama I strain were studied through multilocus sequence typing (MLST) of housekeeping genes. The relatedness between STs, the founder, single- and double-locus variants (SLVs, DLVs) was determined using eBURST algorithm. The concordance between the type assignments by MLST and PFGE was determined. RESULTS: In the 5 clinical isolates, 2 STs were identified, ST2 and ST79. The vaccine strains displayed two distinct allelic profiles assigned to ST1 and ST2. ST2 was predicted as founder and the remaining STs were SLVs of ST2. MLST and PFGE type assignments were 86.6% concordant. CONCLUSIONS: The clinical isolates of B. pertussis were different from vaccine strains used in the national vaccination program. This study confirms the low level of variation in housekeeping genes of B. pertussis. MLST of virulent antigenic genes needs to be applied as a complementary method for the characterization of new ST-harboring isolates that may predominate periodically. The combination of these data allows rapid and efficient surveillance of currently circulating isolates. These data might elucidate the future trends and considerations for vaccine formulation and design.

Antibody response to HER2 extracellular domain and subdomains in mouse following DNA immunization.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in 15-20 % of breast cancer patients and is an appropriate target for immunotherapy in these patients. Monoclonal antibodies (mAbs) specific to HER2 are currently applied to treat breast cancer patients with HER2 overexpression. Active immunization with HER2 DNA or protein has been considered as a suitable alternative. The aim of this study is to evaluate anti-HER2 antibody response in serum of mice immunized with DNA constructs containing full extracellular domain (fECD) or subdomains of human HER2. Four extracellular subdomains and also fECD of HER2 were cloned into pCMV6-Neo vector. Different groups of Balb/C mice were immunized with HER2 DNA constructs and boosted with HER2 recombinant protein. The anti-HER2 antibody was subsequently determined by ELISA, flow cytometry, and immunohistochemistry. Anti-HER2 antibody was detected only in serum of mice immunized with fECD DNA. None of HER2 extracellular subdomains induced appreciable levels of anti-HER2 antibody. However, boosting with fECD or extracellular subdomain III (DIII) recombinant protein resulted in enhanced anti-HER2 fECD as well as anti-HER2 subdomain antibody responses. In this regard, almost all (99 %) of HER2-overexpressing BT474 cells could be detected by serum antibody from mice immunized with HER2 subdomain DNA and boosted with recombinant HER2 protein by flow cytometry. Similarly, serum of mice immunized with DIII DNA construct and boosted with recombinant DIII protein could also recognize these cells, but to a lesser extent (50 %). Our findings suggest that combination of HER2 DNA and protein immunization could effectively induce anti-HER2 antibody response in Balb/C mice.

Downregulation of CD73 in 4T1 breast cancer cells through siRNA-loaded chitosan-lactate nanoparticles.

The immunosuppressive factors in tumor microenvironment enhance tumor growth and suppress anti-tumor immune responses. Adenosine is an important immunosuppressive factor which can be secreted by both tumor and immune cells trough action of two cell surface ecto-nucleotidase molecules CD39 and CD73. Blocking the adenosine generating molecules has emerged as an effective immunotherapeutic approach for treatment of cancer. In this study, CD73-siRNA encapsulated into chitosan-lactate (ChLa) nanoparticles (NPs) was employed to suppress the expression of CD73 molecule on 4T1 breast tumor cells, in vitro. ChLa NPs were generated through ionic gelation of ChLa by tripolyphosphate (TPP). Small interfering RNA (SiRNA)-loaded NPs had about 100 nm size with a polydispersive index below 0.3 and a zeta potential about 13. Our results showed that ChLa NPs with Ch 50 kDa exhibit the best physicochemical features with the high siRNA encapsulation capacity. Synthesized NPs were able to fully bind with siRNA, protect them against serum and heparin degradation, and promote the transfection process. While the NPs exhibited low toxicity during 72 h cell culture, the transfection of Ch-plasmid expressing green fluorescent protein (pEGFP) NPs was efficient in 4T1 cells with a transfection rate of 53.6 % as detected by flow cytometry. In addition, CD73-siRNA-loaded ChLa NPs could efficiently suppress the expression of CD73 as assayed by real-time polymerase chain reaction and flow cytometry. As a conclusion, CD73-siRNA-loaded ChLa NPs may be considered as a promising therapeutic tool for cancer therapy; however, further in vivo investigations are necessary.

EBV and vitamin D status in relapsing-remitting multiple sclerosis patients with a unique cytokine signature.

Multiple sclerosis, a debilitating autoimmune and inflammatory disease of the central nervous system, is associated with both infectious and non-infectious factors. We investigated the role of EBV infection, vitamin D level, and cytokine signature in MS patients. Molecular and serological assays were used to investigate immune biomarkers, vitamin D level, and EBV status in 83 patients with relapsing-remitting multiple sclerosis and 62 healthy controls. In total, 98.8 % of MS patients showed a history of EBV exposure compared to 88.6 % in the healthy group (p = 0.005). EBV DNA load was significantly higher in MS patients than healthy subjects (p < 0.0001). Using a panel of biomarkers, we found a distinct transcriptional signature in MS patients compared to the healthy group with mRNA levels of CD73, IL-6, IL-23, IFN-γ, TNF-α, IL-15, IL-28, and IL-17 significantly elevated in MS patients (p < 0.0001). In contrast, the mRNA levels for TGF-ß, IDO, S1PR1, IL-10, and CCL-3 were significantly lower in MS patients compared to healthy controls (p < 0.0001). No significant differences were found with the mRNA levels of IL-13, CCL-5, and FOXP3. Interestingly, in MS patients we found an inverse correlation between vitamin D concentration and EBV load, but not EBNA-1 IgG antibody levels. Our data highlight biomarker correlates in MS patients together with a complex interplay between EBV replication and vitamin D levels.

Localization of immunodominant epitopes within the "a" determinant of hepatitis B surface antigen using monoclonal antibodies.

The common "a" determinant is the major immunodominant region of hepatitis B surface antigen (HBsAg) shared by all serotypes and genotypes of hepatitis B virus (HBV). Antibodies against this region are thought to confer protection against HBV and are essential for viral clearance. Mutations within the "a" determinant may lead to conformational changes in this region, which can affect the binding of neutralizing antibodies. There is an increasing concern about identification and control of mutant viruses which is possible by comprehensive structural investigation of the epitopes located within this region. Anti-HBs monoclonal antibodies (mAbs) against different epitopes of HBsAg are a promising tool to meet this goal. In the present study, 19 anti-HBs mAbs were employed to map epitopes localized within the "a" determinant, using a panel of recombinant mutant HBsAgs. The topology of the epitopes was analyzed by competitive enzyme-linked immunosorbent assay (ELISA). Our results indicate that all of the mAbs seem to recognize epitopes within or in the vicinity of the "a" determinant of HBsAg. Different patterns of binding with mutant forms were observed with different mAbs. Amino acid substitutions at positions 123, 126, 129, 144, and 145 dramatically reduced the reactivity of antibodies with HBsAg. The T123N mutation had the largest impact on antibody binding to HBsAg. The reactivity pattern of our panel of mAbs with mutant forms of HBsAg could have important clinical implications for immunoscreening, diagnosis of HBV infection, design of a new generation of recombinant HB vaccines, and immunoprophylaxis of HBV infection as an alternative to therapy with hepatitis B immune globulin (HBIG).