Recall of B cell memory depends on relative locations of prime and boost

Re-entry of memory B cells to recall germinal centers (GCs) is essential for updating their B-cell antigen receptors (BCRs). Using single B-cell culture and fate-mapping, we have characterized BCR repertoires in recall GCs following boost immunizations at sites local or distal to the priming. Local boosts with homologous antigen recruit to recall GCs progeny of primary GC B cells more efficiently than do distal boosts. Recall GCs following local boosts contain significantly more B cells with elevated levels of Ig mutations and higher avidity BCRs. This local preference is unaffected by blockade of CD40:CD154 interaction that terminate active, primary GC responses. Local boosts with heterologous antigens elicit secondary GCs with B-cell populations enriched for cross-reactivity to the priming and boosting antigens; in contrast, cross-reactive GC B cells are rare following distal boosts. Our findings indicate the importance of locality in humoral immunity and inform serial vaccination strategies for evolving viruses.

Liposomal Formulation of ChimeraT, a Multiple T-Cell Epitope-Containing Recombinant Protein, Is a Candidate Vaccine for Human Visceral Leishmaniasis

Background: Leishmaniases are neglected diseases caused by infection with Leishmania parasites and there are no human vaccines in use routinely. The purpose of this study was to examine the immunogenicity of ChimeraT, a novel synthetic recombinant vaccine against visceral leishmaniasis (VL), incorporated into a human-compatible liposome formulation. Methods: BALB/c mice were immunized subcutaneously with ChimeraT/liposome vaccine, ChimeraT/saponin adjuvant, or ChimeraT/saline and immune responses examined in vitro and in vivo. Results: Immunization with the ChimeraT/liposome formulation induced a polarized Th1-type response and significant protection against L. infantum infection. ChimeraT/liposome vaccine stimulated significantly high levels of interferon (IFN)-γ, interleukin (IL)-12, and granulocyte macrophage-colony stimulating factor (GM-CSF) cytokines by both CD4 and CD8 T-cells, with correspondingly lower levels of IL-4 and IL-10 cytokines. Induced antibodies were predominantly IgG2a isotype, and homologous antigen-stimulated spleen cells produced significant nitrite as a proxy for nitric oxide (NO). Furthermore, we examined a small number of treated VL patients and found higher levels of circulating anti-ChimeraT protein IgG2 antibodies, compared to IgG1 levels. Conclusions: Overall, the liposomal formulation of ChimeraT induced a protective Th1-type immune response and thus could be considered in future studies as a vaccine candidate against human VL.

Cross-Reaction, Enhancement, and Neutralization Activity of Dengue Virus Antibodies against Zika Virus: A Study in the Mexican Population

Zika virus (ZIKV), an emerging mosquito-borne flavivirus, has quickly spread in many regions around the world where dengue virus (DENV) is endemic. This represents a major health concern, given the high homology between these two viruses, which can result in cross-reactivity. The aim of this study was to determine the cross-reacting antibody response of the IgM and IgG classes against the recombinant envelope protein of ZIKV (rE-ZIKV) in sera from patients with acute-phase infection of different clinical forms of dengue, i.e., dengue fever (DF) and dengue hemorrhagic fever (DHF) (before the arrival of ZIKV in Mexico 2010), as well as acute-phase sera of ZIKV patients, together with the implications in neutralization and antibody-dependent enhancement. Differences in IgM responses were observed in a number of DF and DHF patients whose sera cross-reacted with the rE-ZIK antigen, with 42% recognition between acute-phase DHF and ZIKV but 27% recognition between DF and ZIKV. Regarding IgG antibodies, 71.5% from the DF group showed cross-reactivity to rE-ZIKV in contrast with 50% and only 25% of DHF and ZIKV serum samples, respectively, which specifically recognized the homologous antigen. The DHF group showed more enhancement of ZIKV infection of FCRγ-expressing cells compared to the DF group. Furthermore, the DHF group also showed a higher cross-neutralizing ability than that of DF. This is the first report where DF and DHF serum samples were evaluated for cross-reactivity against Zika protein and ZIKV. Furthermore, DENV serum samples cross-protect against ZIKV through neutralizing antibodies but at the same time mediate antibody-dependent enhancement in the sequential ZIKV infection.

Evaluation of Monoclonal Antibody (IgG2bMAb) for Detection of Coproantigen from Experimentally Infected Rats with Strongyloides ratti

Background and Aim: Highly sensitive and specific diagnostic assay for the detection of Strongyloides is needed due to the intermittent and low concentration of eggs, larvae and adult worms that can be found in a faecal specimen. In some cases, repeated sampling of the faecal specimen is required to obtain satisfactory and reliable results. The aim of the study is to develop and evaluates monoclonal antibody-based Sandwich ELISA for the detection of coproantigen associated with Strongyloides infection using S. ratti as a model. Place and Duration of Study: Department of Medical Microbiology and Parasitology, Faculty of Medicine and Health Sciences, University Malaysia, Between September 2018 and March 2019. Methodology: The monoclonal antibody was raised against a soluble antigen of the infective filariform larvae (iL3) of S. ratti. The monoclonal antibody produced (IgG2bMAb) was evaluated for cross-reactivity against homologous and heterologous helminth antigens such as excretory-secretory (ES), infective larvae (iL3) and coproantigen of S. ratti, adult worms of A. caninum, A. suum, T. canis and T. cati. Results: An IgG2bMAb was observed to react with 30 kDa proteins associated with all homologous antigen from iL3, ES and coproantigen of S. ratti and cross-reacted with one heterologous antigen from adult worm of A. caninum at the same molecular weight. There was no cross-reaction observed with other heterologous antigens from adult worms of T. canis, T. cati and A. suum. The sensitivity of IgG2bMAb for the detection of S. ratti was 85% in Sandwich ELISA. Cross- reaction was observed with hookworm antigen that caused by A. caninum in Western immunoblotting. Conclusion: The results indicated that IgG2b have an immunodiagnostic property as IgG2bMAb and was able to detect antigens from coproantigen related to S. ratti with 85% sensitivity based on Sandwich ELISA) even though cross-reaction was observed with A. caninum. These findings will be very useful to tackle many cases of multiple worms’ infections such as both strongyloidiasis and hookworm. Therefore, we recommend that further evaluation and study in the human area where multiple infections can be common should be carried out.

Modulated Zika virus NS1 conjugate offers advantages for accurate detection of Zika virus specific antibody in double antigen binding and Ig capture enzyme immunoassays

The accurate diagnosis and seroprevalence investigations of Zika virus (ZKV) infections remain complex due to cross reactivity with other flaviviruses. Two assay formats, both using labelled Zika virus NS1 antigen as a revealing agent (a double antigen binding assay, DABA, and an immunoglobulin Ig capture assay, IgG capture) were initially developed and compared with the indirect EuroimmunZ assay for the detection of anti-Zika antibody. Of 147 pre-Zika period serum samples, 39 (27%) were reactive in the EuroimmunZ or the DABA assays, 28 sera concordantly so. Such false reactivity was influenced by the serotype of Dengue virus (DV) to which individuals had been exposed to. Thus, of sera from patients undergoing secondary Dengue virus infection of known serotype, 91%, 45% and 28% of Dengue virus serotype 2, 3 and 4 respectively were reactive in one or more of the three assays. A novel method of quenching false sero-reactivity was therefore developed for the DABA and IgG capture assays. Initial addition of a single homologous Dengue virus serotype 3 NS1Ag quench significantly ablated false reactivities in the pre-Zika period sera. An equipotent quadrivalent quench comprising homologous Dengue virus serotypes 1 to 4 NS1Ag was shown to be optimum yet retained sensitivity for the detection of specific anti-Zika antibody. Comparing DABA and IgG capture assays using quenched and unquenched conjugates in comparison with EuroimmunZ early in the course of PCR-confirmed infection indicated that a significant component of the apparent early anti-ZIKA antibody response is likely to be due to a Zika virus-driven anamnestic anti-Dengue virus response. The increased specificity provided by homologous antigen quenching is likely to provide a significant improvement in sero-diagnostics and to be of clinical value.

Development of anti-Aeromonas hydrophila serum from Rabbits

An experiment was conducted to prepare anti-Aeromonas hydrophila serum in two rabbits (Oryctolagus cuniculus). Formalin killed cell (FKC) and heat killed cell (HKC) were prepared by adding 0.5% formalin and 2.5 h heat treatment at 60°C of freshly cultured Aeromonas hydrophila (AQC810) isolated from naturally infected koi fish kidney respectively. Slide agglutination tests were performed for the determination of the presence of specific antibody against Aeromonas hydrophila in the serum collected from the blood of pre-immune rabbits or post immune rabbits. No agglutination of pre-immune serum with the homologous antigen proved that the rabbits had no previous Aeromonas contamination. Rabbits were injected subcutaneously with the FKC added with same amount of Freund’s complete adjuvant. Three booster doses were given without the adjuvant at weekly intervals after second week of first injection. Agglutination titration of anti-Aeromonas hydrophila rabbit serum against HKC and FKC of homologous bacteria showed that the titers increased with the post inoculation days which rose at their peak during the fifth week of immunization and after three consecutive booster doses at weekly intervals starting from second week of first immunization. Rabbits were sacrificed after six weeks of immunization, whole blood, collected and centrifuged to accumulate the serum that were complementinactivated by heating to 58ºC for 30 min and stored at -20°C in appendorph tubes, with the aim of rapid diagnosis of Aeromonas infections in fishes. Result of this study would initiate the way of vaccine development against MAS (motile Aeromonas septicemia) in the aquaculture fishes of Bangladesh.Res. Agric., Livest. Fish.5(1): 137-145, April 2018

Modulation of the Effects of Lung Immune Response on Bone Marrow by Oral Antigen Exposure

Allergic airway inflammation is attenuated by oral tolerization (oral exposure to allergen, followed by conventional sensitization and challenge with homologous antigen), which decreases airway allergen challenge-induced eosinophilic infiltration of the lungs and bone marrow eosinophilia. We examined its effects on bone marrow eosinophil and neutrophil production. Mice of wild type (BP-2, BALB/c, and C57BL/6) and mutant strains (lacking iNOS or CD95L) were given ovalbumin (OVA) or water (vehicle) orally and subsequently sensitized and challenged with OVA (OVA/OVA/OVA and H2O/OVA/OVA groups, resp.). Anti-OVA IgG and IgE, bone marrow eosinophil and neutrophil numbers, and eosinophil and neutrophil production ex vivo were evaluated. T lymphocytes from OVA/OVA/OVA or control H2O/OVA/OVA donors were transferred into naïve syngeneic recipients, which were subsequently sensitized/challenged with OVA. Alternatively, T lymphocytes were cocultured with bone marrow eosinophil precursors from histocompatible sensitized/challenged mice. OVA/OVA/OVA mice of the BP-2 and BALB/c strains showed, relative to H2O/OVA/OVA controls, significantly decreased bone marrow eosinophil counts and ex vivo eosinopoiesis/neutropoiesis. Full effectiveness in vivo required sequential oral/subcutaneous/intranasal exposures to the same allergen. Transfer of splenic T lymphocytes from OVA/OVA/OVA donors to naive recipients prevented bone marrow eosinophilia and eosinopoiesis in response to recipient sensitization/challenge and supressed eosinopoiesis upon coculture with syngeneic bone marrow precursors from sensitized/challenged donors.

Sensitivity and specificity of monoclonal antibody Mn-Cs1 for detection and determination of Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato

Monoclonal antibody Mn-Cs1 with a high level of sensitivity and specificity for detection and determination of Clavibacter michiganensis subsp. sepedonicus was prepared. Strain C. m. subsp. sepedonicus NCPPB 3467 (as whole cell antigen and extracellular polysaccharides) was used for immunisation of four mice Balb/c. After cloning and verifying, two stable hybridoma clones were gained. One monoclonal antibody, designated Mn-Cs1, was used in all tests. It reacted intensely with extracellular polysaccharides from homologous antigen (> 0.5 mg/ml), weakly with proteins from cell walls (> 200 μg/ml) and with whole homologous antigen (concentration 104–103 cfu/ml) in DAS-ELISA. Monoclonal antibody Mn-Cs1 showed a high level of specificity. It reacted neither with bacterial strains of closely related subspecies of Clavibacter michiganensis (C. m. subsp. michiganensis and C. m. subsp. insidiosus) nor with the saprophytic bacteria Pseudomonas fluorescens and Pantoea agglomerans.

Use of elisa employing homologous and heterologous antigens for the detection of IgG and subclasses (IgG1 and IgG2) in the diagnosis of Canine visceral leishmaniasis

Indirect immunofluorescence is the method recommended for the diagnosis of visceral leishmanisis in dogs, however, the accuracy of this technique is low and its use on a large scale is limited. Since ELISA does not present these limitations, this technique might be an option for the detection of IgG or specific IgG1 and IgG2 subclasses. Canine ehrlichiosis is an important differential diagnosis of American Visceral Leishmaniasis (AVL). The present study compared ELISA using Leishmania chagasi and Leishmania braziliensis antigen for the detection of anti-Leishmania IgG and subclasses in serum samples from 37 dogs naturally infected with L. chagasi (AVL) and in samples from four dogs co-infected with L. braziliensis and L. chagasi (CI). The occurrence of cross-reactivity was investigated in control serum samples of 17 healthy dogs (HC) and 35 infected with Ehrlichia canis (EC). The mean optical density obtained for the detection of IgG was significantly higher when L. chagasi antigen was used, and was also higher in subgroup VLs (symptomatic) compared to subgroup Vla (asymptomatic). The correlation between IgG and IgG1 was low. The present results suggest that IgG ELISA using homologous antigen yields the best results, permitting the diagnosis of asymptomatic L. chagasi infection and the discrimination between cases of AVL and ehrlichiosis in dogs.

Hydrophobic fraction of Taenia saginata metacestodes, rather than hydrophilic fraction, contains immunodominant markers for diagnosing human neurocysticercosis

INTRODUCTION: Considering that alternative antigens for diagnosing neurocysticercosis continue to be a challenge because of the increasing difficulty in obtaining parasites from naturally infected pigs for preparation of Taenia solium homologous antigen, the aim of the present study was to evaluate the detergent (D) and aqueous (A) fractions from saline extract of Taenia saginata metacestodes for diagnosing neurocysticercosis. METHODS: Taenia saginata was obtained from naturally infected bovines in the Triângulo Mineiro region, State of Minas Gerais, Brazil. The carcasses came from cold storage units and had been slaughtered in accordance with the inspection technique recommended by the Federal Inspection Service. The D and A fractions were obtained by using Triton X-114 (TX-114). Serum samples were obtained from 40 patients with a diagnosis of neurocysticercosis, 45 with other parasitic diseases and 30 from apparently normal individuals. IgG antibody levels were evaluated using the ELISA and immunoblotting assays. RESULTS: The ELISA sensitivity and specificity were 95% and 73.3%, when using saline extract; 95% and 82.6% for the D fraction; and 65% and 61.3% for the A fraction, respectively. The immunoblotting assay confirmed the ELISA results, such that the D fraction was more efficient than the other extracts, and the 70-68kDa component was immunodominant among neurocysticercosis patients. CONCLUSIONS: These results demonstrated that the D fraction from Taenia saginata metacestodes obtained using TX-114 can be used as a heterologous antigenic fraction in the immunoblotting assay for serologically diagnosing human neurocysticercosis, given its ability to select immunodominant antigens.