Increasing human monoclonal antibody cloning efficiency with a whole-cell modified immunoglobulin-capture assay (mICA)

Expression cloning of fully human monoclonal antibodies (hmAbs) is seeing powerful utility in the field of vaccinology, especially for elucidating vaccine-induced B-cell responses and novel vaccine candidate antigen discovery. Precision of the hmAb cloning process relies on efficient isolation of hmAb-producing plasmablasts of interest. Previously, a novel immunoglobulin-capture assay (ICA) was developed, using single protein vaccine antigens, to enhance the pathogen-specific hmAb cloning output. Here, we report a novel modification of this single-antigen ICA using formalin-treated, fluorescently stained whole cell suspensions of the human bacterial invasive pathogens, Streptococcus pneumoniae and Neisseria meningitidis. Sequestration of IgG secreted by individual vaccine antigen-specific plasmablasts was achieved by the formation of an anti-CD45-streptavidin and biotin anti-IgG scaffold. Suspensions containing heterologous pneumococcal and meningococcal strains were then used to enrich for polysaccharide- and protein antigen-specific plasmablasts, respectively, during single cell sorting. Following application of the modified whole-cell ICA (mICA), ~61% (19/31) of anti-pneumococcal polysaccharide hmAbs were cloned compared to 14% (8/59) obtained using standard (non-mICA) methods – representing a ~4.4-fold increase in hmAb cloning precision. A more modest ~1.7-fold difference was obtained for anti-meningococcal vaccine hmAb cloning; ~88% of hmAbs cloned via mICA versus ~53% cloned via the standard method were specific for a meningococcal surface protein. VDJ sequencing revealed that cloned hmAbs reflected an anamnestic response to both pneumococcal and meningococcal vaccines; diversification within hmAb clones occurred by positive selection for replacement mutations. Thus, we have shown successful utilization of whole bacterial cells in the ICA protocol enabling isolation of hmAbs targeting multiple disparate epitopes, thereby increasing the power of approaches such as reverse vaccinology 2.0 (RV 2.0) for bacterial vaccine antigen discovery

PROCALCITONIN AS A BIOMARKER OF BACTERIAL INFECTION IN SICKLE CELL VASO-OCCLUSIVE CRISIS.

Bacterial infection is an important trigger of vaso-occlusive crisis (VOC) in sickle cell anaemia (SCA). SCA Patients with VOC have signs of inflammation and it is difficult to diagnose bacterial infection in them. This study was undertaken to evaluate serum procalcitonin (PCT) as a biomarker of bacterial infection in acute sickle cell vaso-occlusive crisis. Hundred SCA patients were studied at Sickle Cell Clinic and Molecular Biology Laboratory, V.S.S. Medical College, Burla, Odisha, India. SCA was diagnosed by haemoglobin electrophoresis, HPLC and molecular analysis. Patients were divided into 3categories namely Category-A (VOC/ACS with fever but without evidence of bacterial infection-66 patients); Category-B (VOC with fever and documentedbacterial infection-24 patients); and Category-C (Patients in steady statewithout VOC/ACS or fever-10 patients). Investigations like complete blood count, C-reactive protein estimation and PCT measurement was done in all the cases. There was no significant difference in total leucocytes count and C-reactiveprotein values between category A and B. In category A the PCT level was 0.5ng/mL with 87.5% of cases having >2ng/mL. In category C, PCT value was 2ng/mL is indicative of bacterial infection necessitating antimicrobial therapy. Patients with indeterminate PCT value of0.5-2ng/mL, need a repeat PCT estimation or an empirical antibiotic therapyawaiting the availability of microbiological report as deemed necessary

Study of seminal fluid parameters and fertility of male sickle-cell disease patients and potential impact of hydroxyurea treatment

Context: Male sickle-cell disease (SCD) patients often have moderate to severe hypogonadism resulting in abnormal seminal fluid parameters. Hydroxyurea (HU) used in SCD patients has been found to further aggravate the testicular dysfunction. Aims: To study the seminal fluid parameters and fertility of male SCD patients and to evaluate the potential impact of HU treatment on the above factors. Settings and Design: This was a prospective study of 100 male SCD patients of age group 18 to 45 years. Materials and Methods: Seminal fluid analysis was done before starting low dose of HU (10 mg/kg/day) and every 3 months after initiation of HU. Patients with abnormal seminal parameters before HU therapy were not given HU therapy. In SCD patients with HU therapy, who developed abnormal seminal fluid parameters, HU was stopped for 3 months and seminal fluid parameters were re-evaluated. Results: Among SCD patients without HU therapy, nine (18%) patients developed oligospermia and two (4%) developed azoospermia. Among SCD patients with HU therapy, 10 (20%) patients developed oligospermia and five (10%) developed azoospermia. Seminal fluid parameters reverted to normal after stoppage of HU for 3 months in 73% of patients. Conclusions: Alteration of sperm parameters is seen in a significant number of SCD patients. Also, alterations of seminal fluid parameters are exacerbated by HU treatment even with low dose

Comparative study of clinical presentation and hematological indices in hospitalized sickle cell patients with severe Plasmodium falciparum malaria

Background: Sickle-cell-gene has a high frequency in malaria endemic regions. In India, though the prevalence of both sickle-cell-gene and malaria are high, no study has been carried out. This study aims to find out the possible differences in hematological and clinical parameters in severe falciparum malaria with respect to sickle cell genotypes. Methods: Five hundred fourteen adults with severe falciparum malaria hospitalized in Department of Medicine, Veer Surendra Sai Institute of Medical Sciences and Research, Burla, between August, 2010 to December, 2014 were included and categorized on the basis of sickle cell genotypes. The hematological parameters were compared by one-way-analysis-of-variance and incidence of sub-phenotypes of severe malaria was compared by χ2 test across the groups. Results: Patients with sickle cell anemia (HbSS) and severe falciparum malaria had lower hemoglobin level compared to patients with normal β-globin genotype (HbAA) and sickle cell trait (HbAS). Most of the hematological parameters were homogeneous in patients with HbAA and HbAS and different from patients with HbSS. Incidence of acute renal failure was low (χ2, 9.91; p, 0.002) and jaundice was high (χ2, 5.20; p, 0.022) in patients with HbSS. No clinical difference was observed in patients with HbAA and HbAS. The mortality was low (χ2, 4.33; p, 0.037) and high (χ2, 10.48; p, 0.001) in patients with HbAS and HbSS respectively compared to patients with HbAA. Conclusion: Though sickle-cell-gene protects against falciparum infections, the hematological parameters and sub-phenotypes of severe malaria remain unchanged when the infection progresses to a severe form in patients with HbAA and HbAS. Presence of hemolytic anemia in patients with HbSS shows diverse hematological and clinical phenotypes as compared to others. High mortality in patients with HbSS emphasizes the need for a better preventive approach to save valuable lives. Keywords: Plasmodium falciparum, Severe malaria, Sickle cell gene, Anemia, Mortalit