Molecular cloning, expression, and purification, along with in silico epitope analysis of recombinant enolase proteins (a potential vaccine candidate) from Candida albicans and Candida auris.

Candida albicans is the predominant cause of systemic candidiasis, although other non albicans Candida species are progressively becoming more widespread nowadays. Candida auris has emerged as a deadly multidrug-resistant fungal pathogen, posing a significant threat to global public health. In the absence of effective antifungal therapies, the development of a vaccine against C. auris infections is imperative. Enolase, a key glycolytic enzyme, has emerged as a promising vaccine candidate due to its immunogenic properties and essential role in fungal virulence. Herein, full-length Enolase gene sequences from C. albicans and C. auris were cloned into suitable expression vector and transformed into Escherichia coli expression hosts. Recombinant Enolase proteins were successfully expressed and purified using affinity chromatography under native conditions, followed by SDS-PAGE characterization and Western blot analysis. CD spectroscopy verified the existence of expressed proteins in soluble native conformation. Preliminary in silico studies verified the immunogenicity of recombinant Enolase proteins isolated from both C. albicans and C. auris. Furthermore, bioinformatics analysis revealed conserved B-cell and T-cell epitopes across C. albicans and C. auris Enolase proteins, suggesting potential cross-reactivity and broad-spectrum vaccine efficacy. Our findings are anticipated to play a role in advancing therapeutic as well as diagnostic strategies against systemic candidiasis.

Recent Advances in the Development of Monoclonal Antibodies and Next-Generation Antibodies.

mAbs are highly indispensable tools for diagnostic, prophylactic, and therapeutic applications. The first technique, hybridoma technology, was based on fusion of B lymphocytes with myeloma cells, which resulted in generation of single mAbs against a specific Ag. Along with hybridoma technology, several novel and alternative methods have been developed to improve mAb generation, ranging from electrofusion to the discovery of completely novel technologies such as B cell immortalization; phage, yeast, bacterial, ribosome, and mammalian display systems; DNA/RNA encoded Abs; single B cell technology; transgenic animals; and artificial intelligence/machine learning. This commentary outlines the evolution, methodology, advantages, and limitations of various mAb production techniques. Furthermore, with the advent of next-generation Ab technologies such as single-chain variable fragments, nanobodies, bispecific Abs, Fc-engineered Abs, Ab biosimilars, Ab mimetics, and Ab-drug conjugates, the healthcare and pharmaceutical sectors have become resourceful to develop highly specific mAb treatments against various diseases such as cancer and autoimmune and infectious diseases.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms.

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less. It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm. The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

COVID-19-Associated Mucormycosis: A Matter of Concern Amid the SARS-CoV-2 Pandemic.

Mucormycosis is an invasive fungal infection caused by fungi belonging to order Mucorales. Recently, with the increase in COVID-19 infections, mucormycosis infections have become a matter of concern globally, because of the high morbidity and mortality rates associated with them. Due to the association of mucormycosis with COVID-19 disease, it has been termed COVID-19-associated mucormycosis (CAM). In the present review, we focus on mucormycosis incidence, pathophysiology, risk factors, immune dysfunction, interactions of Mucorales with endothelial cells, and the possible role of iron in Mucorales growth. We review the limitations associated with current diagnostic procedures and the requirement for more specific, cost-effective, convenient, and sensitive assays, such as PCR-based assays and monoclonal antibody-based assays for the effective diagnosis of mucormycosis. We discuss the current treatment options involving antifungal drug therapies, adjunctive therapy, surgical treatment, and their limitations. We also review the importance of nutraceuticals-based therapy for the prevention as well as treatment of mucormycosis. Our review also highlights the need to explore the potential of novel immunotherapeutics, which include antibody-based therapy, cytokine-based therapy, and combination/synergistic antifungal therapy, as treatment options for mucormycosis. In summary, this review provides a complete overview of COVID-19-associated mucormycosis, addressing the current research gaps and future developments required in the field.

Synthesis of vildagliptin loaded acrylamide-g-psyllium/alginate-based core-shell nanoparticles for diabetes treatment.

Diabetes mellitus has become a major public health concern all over the world. Vildagliptin is one of the antidiabeticdrug that can overcome the existing problem of this prevalent disease. Present study aims to synthesize and investigate the role of vildagliptin-loaded core-shell nanoparticle of grafted psyllium and alginate (VG@P/A-NPs) in anti-diabetes application. FTIR, SEM, XRD, 13CNMR and zeta analyzer were used for characterization of the core-shell nanoparticles (VG@P/A-NPs). The synthesized acrylamide-grafted-psyllium was also optimized through varying grafting parameters such as acrylamide and ceric ammonium nitrate (CAN) concentration, time and temperature to obtain the maximum yield of acrylamide-grafted-psyllium. Rheological analysis of pure psyllium, grafted psyllium and alginate were also performed. For biological studies, the first cytotoxicity of grafted psyllium and VG@P/A-NPs were examined on human lung adenocarcinoma cell line A549 in which it was observed that VG@P/A-NPs did not exhibited any toxicity. The antidiabetic potential of VG@P/A-NPs was investigated by glucose uptake assay, using TNF-α induced insulin resistance skeletal cell model using mouse muscle L6 cell line. The insulin signaling impaired cell line displayed a highly significant (p < 0.0001) dose-dependent increase in glucose uptake after treatment with increasing doses of VG@P/A-NPs.The drug release behavior of VG@P/A-NPs was examined at various pH and the highest drug release (98 %) was obtained at pH (7.4). The drug release kinetic data was following the Higuchi (R2 = 0.9848) kinetic model, suggesting the release of drug from vildagliptin-loaded grafted psyllium-alginate core-shell nanoparticles (VG@P/A-NPs) as a square root of time-dependent process and diffusion controlled. This study provides an economical and environment-friendly approach towards the synthesis of VG@P/A-NPs with antidiabetes applications.

Expression, Purification, and Refolding of Chikungunya Virus Full-Length Envelope E2 Protein along with B-Cell and T-Cell Epitope Analyses Using Immuno-Informatics Approaches.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus, which causes severe illness in humans and is responsible for epidemic outbreaks in Africa, Asia, North and South America, and Europe. Despite its increased global prevalence, no licensed vaccines are available to date for treating or preventing CHIKV infection. The envelope E2 protein is one of the promising subunit vaccine candidates against CHIKV. In this study, we describe successful cloning, expression, and purification of CHIKV E2 full-length (E2-FL) and truncated (E2-ΔC and E2-ΔNC) proteins in the Escherichia coli expression system. The recombinant E2 proteins were purified from inclusion bodies using Ni-NTA chromatography. Further, we describe a detailed refolding procedure for obtaining the CHIKV E2-FL protein in native conformation, which was confirmed using circular dichroism and Fourier transform infrared spectroscopy. BALB/c mice immunized with the three different E2 proteins exhibited increased E2-specific antibody titers compared to sham-immunized controls, suggesting induction of strong humoral immune response. On analyzing the E2-specific antibody response generated in immunized mice, the CHIKV E2-FL protein was observed to be the most immunogenic among the three different CHIKV E2 antigens used in the study. Our B-cell and T-cell epitope mapping results indicate that the presence of specific immunogenic peptides located in the N-terminal and C-terminal regions of the CHIKV E2-FL protein may contribute to its increased immunogenicity, compared to truncated CHIKV E2 proteins. In summary, our study provides a detailed protocol for expressing, purifying, and refolding of the CHIKV E2-FL protein and provides an understanding of its immunogenic epitopes, which can be exploited for the development of novel multiepitope-based anti-CHIKV vaccine strategies.

Host-parasite interactions during Plasmodium infection: Implications for immunotherapies.

Malaria is a global infectious disease that remains a leading cause of morbidity and mortality in the developing world. Multiple environmental and host and parasite factors govern the clinical outcomes of malaria. The host immune response against the Plasmodium parasite is heterogenous and stage-specific both in the human host and mosquito vector. The Plasmodium parasite virulence is predominantly associated with its ability to evade the host's immune response. Despite the availability of drug-based therapies, Plasmodium parasites can acquire drug resistance due to high antigenic variations and allelic polymorphisms. The lack of licensed vaccines against Plasmodium infection necessitates the development of effective, safe and successful therapeutics. To design an effective vaccine, it is important to study the immune evasion strategies and stage-specific Plasmodium proteins, which are targets of the host immune response. This review provides an overview of the host immune defense mechanisms and parasite immune evasion strategies during Plasmodium infection. Furthermore, we also summarize and discuss the current progress in various anti-malarial vaccine approaches, along with antibody-based therapy involving monoclonal antibodies, and research advancements in host-directed therapy, which can together open new avenues for developing novel immunotherapies against malaria infection and transmission.

Expression and Purification along with Evaluation of Serological Response and Diagnostic Potential of Recombinant Sap2 Protein from C. parapsilosis for Use in Systemic Candidiasis.

Systemic candidiasis is the fourth most common bloodstream infection in ICU patients worldwide. Although C. albicans is a predominant species causing systemic candidiasis, infections caused by non-albicans Candida (NAC) species are increasingly becoming more prevalent globally along with the emergence of drug resistance. The diagnosis of systemic candidiasis is difficult due to the absence of significant clinical symptoms in patients. We investigated the diagnostic potential of recombinant secreted aspartyl proteinase 2 (rSap2) from C. parapsilosis for the detection of Candida infection. The rSap2 protein was successfully cloned, expressed and purified using Ni-NTA chromatography under denaturing conditions using an E. coli-based prokaryotic expression system, and refolded using a multi-step dialysis procedure. Structural analysis by CD and FTIR spectroscopy revealed the refolded protein to be in its near native conformation. Immunogenicity analysis demonstrated the rSap2 protein to be highly immunogenic as evident from significantly high titers of Sap2-specific antibodies in antigen immunized Balb/c mice, compared to sham-immunized controls. The diagnostic potential of rSap2 protein was evaluated using immunoblotting and ELISA assays using proven candidiasis patient serum and controls. Immunoblotting results indicate that reactivity to rSap2 was specific to candidiasis patient sera with no cross reactivity observed in healthy controls. Increased levels of anti-Sap2-specific Ig, IgG and IgM antibodies were observed in candidiasis patients compared to controls and was similar in sensitivity obtained when whole Candida was used as coating antigen. In summary, the rSap2 protein from C. parapsilosis has the potential to be used in the diagnosis of systemic candidiasis, providing a rapid, convenient, accurate and cost-effective strategy.

The Role of B-Cells and Antibodies against Candida Vaccine Antigens in Invasive Candidiasis.

Systemic candidiasis is an invasive fungal infection caused by members of the genus Candida. The recent emergence of antifungal drug resistance and increased incidences of infections caused by non-albicans Candida species merit the need for developing immune therapies against Candida infections. Although the role of cellular immune responses in anti-Candida immunity is well established, less is known about the role of humoral immunity against systemic candidiasis. This review summarizes currently available information on humoral immune responses induced by several promising Candida vaccine candidates, which have been identified in the past few decades. The protective antibody and B-cell responses generated by polysaccharide antigens such as mannan, ß-glucan, and laminarin, as well as protein antigens like agglutinin-like sequence gene (Als3), secreted aspartyl proteinase (Sap2), heat shock protein (Hsp90), hyphally-regulated protein (Hyr1), hyphal wall protein (Hwp1), enolase (Eno), phospholipase (PLB), pyruvate kinase (Pk), fructose bisphosphate aldolase (Fba1), superoxide dismutase gene (Sod5) and malate dehydrogenase (Mdh1), are outlined. As per studies reviewed, antibodies induced in response to leading Candida vaccine candidates contribute to protection against systemic candidiasis by utilizing a variety of mechanisms such as opsonization, complement fixation, neutralization, biofilm inhibition, direct candidacidal activity, etc. The contributions of B-cells in controlling fungal infections are also discussed. Promising results using anti-Candida monoclonal antibodies for passive antibody therapy reinforces the need for developing antibody-based therapeutics including anti-idiotypic antibodies, single-chain variable fragments, peptide mimotopes, and antibody-derived peptides. Future research involving combinatorial immunotherapies using humanized monoclonal antibodies along with antifungal drugs/cytokines may prove beneficial for treating invasive fungal infections.

Synthesis of rifaximin loaded chitosan-alginate core-shell nanoparticles (Rif@CS/Alg-NPs) for antibacterial applications.

The present work aims to synthesize the rifaximin loaded chitosan-alginate core-shell nanoparticles (Rif@CS/Alg-NPs) for antibacterial applications. The core-shell nanoparticles (Rif@CS/Alg-NPs) were characterized by Fourier Transform Infrared (FT-IR) spectroscopy, Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), X-rays diffraction (XRD) and zeta analyzer. The antibacterial activities of Rif@CS/Alg-NPs were investigated against three species of bacteria namely Escherichia coli (E. coli), Pseudomonas aeruginosa (PA) and Bacillus haynesii (BH). Rif@CS/Alg-NPs exhibited outstanding antibacterial activities against E. coli, P. aeroginosa and Bacillus haynesii (BH) with 24 mm, 30 mm and 34 mm zone of inhibitions, respectively. Cytotoxicity of Rif@CS/Alg-NPs was also evaluated against human lung adenocarcinoma cell line A549 and found to be nontoxic. The drug release behavior of Rif@CS/Alg-NPs was investigated at different pH levels and maximum drug release (80%) was achieved at pH (7.2). The drug release kinetic data followed the Higuchi (R2 = 0.9963) kinetic model, indicating the drug release from Rif@CS/Alg-NPs as a square root of time-dependent process and diffusion controlled. Current research provides a cost-effective and green approach toward the synthesis of Rif@CS/Alg-NPs for its antibacterial applications.

Vaccination with Secreted Aspartyl Proteinase 2 Protein from Candida parapsilosis Can Enhance Survival of Mice during C. tropicalis-Mediated Systemic Candidiasis.

The rising incidence of non-albicans Candida species globally, along with the emergence of drug resistance, is a cause for concern. This study investigated the protective efficacy of secreted aspartyl proteinase 2 (Sap2) in systemic C. tropicalis infection. Vaccination with recombinant Sap2 (rSap2) protein from C. parapsilosis enhanced survival of mice compared to rSap2 vaccinations from C. albicans (P = 0.02), C. tropicalis (P = 0.06), and sham immunization (P = 0.04). Compared to sham-immunized mice, the fungal CFU number was significantly reduced in organs of Sap2-parapsilosis-immunized mice. Histopathologically, increased neutrophilic recruitment was observed in Sap2-parapsilosis- and Sap2-tropicalis-immunized mice. Among different rSap2 proteins, Sap2-parapsilosis vaccination induced increased titers of Sap2-specific Ig, IgG, and IgM antibodies, which could bind whole fungus. Between different groups, sera from Sap2-parapsilosis-vaccinated mice exhibited increased C. tropicalis biofilm inhibition ability in vitro and enhanced neutrophil-mediated fungal killing. Passive transfer of anti-Sap2-parapsilosis immune serum in naive mice significantly reduced fungal burdens compared to those in mice receiving anti-sham immune serum. Higher numbers of plasma cells and Candida-binding B cells in Sap2-vaccinated mice suggest a role of B cells during early stages of Sap2-mediated immune response. Additionally, increased levels of Th1/Th2/Th17 cytokines observed in Sap2-parapsilosis-vaccinated mice indicate immunomodulatory properties of Sap2. Epitope analysis performed using identified B-cell epitopes provides a basis to understand differences in immunogenicity observed among Sap2-antigens and can aid the development of a multivalent or multiepitope anti-Candida vaccine(s). In summary, our results suggest that Sap2-parapsilosis vaccination can improve mouse survival during C. tropicalis infection by inducing both humoral and cellular immunity, and higher titers of Sap2-induced antibodies are beneficial during systemic candidiasis.

Antibody and B Cell Subset Perturbations in Human Immunodeficiency Virus-Uninfected Patients With Cryptococcosis.

The importance of antibody immunity in protection against Cryptococcus neoformans remains unresolved. We measured serum C neoformans-specific and total antibody levels and peripheral blood B cell subsets of 12 previously healthy patients with cryptococcosis (cases) and 21 controls. Before and after adjustment for age, sex, and race, cryptococcal capsular polysaccharide immunoglobulin G was higher in cases than controls, whereas total B and memory B cell levels were lower. These associations parallel previous findings in patients with human immunodeficiency virus-associated cryptococcosis and suggest that B cell subset perturbations may also associate with disease in previously normal individuals with cryptococcosis.

Naïve B cells reduce fungal dissemination in Cryptococcus neoformans infected Rag1-/- mice.

IgM and B-1 cell deficient mice exhibit early C. neoformans dissemination from lungs to brain, but a definitive role for B cells in conferring resistance to C. neoformans dissemination has not been established. To address this question, we developed an intranasal (i.n.) C. neoformans infection model in B and T cell deficient Rag1-/- mice and found they also exhibit earlier fungal dissemination and higher brain CFU than wild-type C57Bl/6 (wild-type) mice. To probe the effect of B cells on fungal dissemination, Rag1-/- mice were given splenic (intravenously) or peritoneal (intraperitoneally) B cells from wild-type mice and infected i.n. with C. neoformans 7 d later. Mice that received B cells had lung histopathology resembling wild type mice 14 d post-infection, and B-1, not B-2 or T cells in their lungs, and serum and lung IgM and IgG 21 d post-infection. Lung CFU were comparable in wild-type, Rag1-/-, and Rag1-/- mice that received B cells 21 d post-infection, but brain CFU were significantly lower in mice that received B cells than Rag1-/- mice that did not. To determine if natural antibody can promote immunity in our model, we measured alveolar macrophage phagocytosis of C. neoformans in Rag1-/- mice treated with naive wild-type IgM-sufficient or sIgM-/- IgM-deficient sera before infection. Compared to IgM-deficient sera, IgM-sufficient sera significantly increased phagocytosis. Our data establish B cells are able to reduce early C. neoformans dissemination in mice and suggest natural IgM may be a key mediator of early antifungal immunity in the lungs.

Host immunity to Cryptococcus neoformans.

Cryptococcosis is caused by the fungal genus Cryptococcus. Cryptococcosis, predominantly meningoencephalitis, emerged with the HIV pandemic, primarily afflicting HIV-infected patients with profound T-cell deficiency. Where in use, combination antiretroviral therapy has markedly reduced the incidence of and risk for disease, but cryptococcosis continues to afflict those without access to therapy, particularly in sub-Saharan Africa and Asia. However, cryptococcosis also occurs in solid organ transplant recipients and patients with other immunodeficiencies as well as those with no known immunodeficiency. This article reviews innate and adaptive immune responses to C. neoformans, with an emphasis on recent studies on the role of B cells, natural IgM and Fc gamma receptor polymorphisms in resistance to cryptococcosis.

Fc gamma receptor 3A polymorphism and risk for HIV-associated cryptococcal disease.

UNLABELLED: Cryptococcus neoformans is one of the most common causes of fungal disease in HIV-infected persons, but not all of those who are infected develop cryptococcal disease (CD). Although CD4(+) T cell deficiency is a risk factor for HIV-associated CD, polymorphisms of phagocytic Fc gamma receptors (FCGRs) have been linked to CD risk in HIV-uninfected persons. To investigate associations between FCGR2A 131 H/R and FCGR3A 158 F/V polymorphisms and CD risk in HIV-infected persons, we performed PCR-based genotyping on banked samples from 164 men enrolled in the Multicenter AIDS Cohort Study (MACS): 55 who were HIV infected and developed CD and a matched control group of 54 who were HIV infected and 55 who were HIV uninfected. Using additive and allelic statistical models for analysis, the high-affinity FCGR3A 158V allele was significantly associated with CD status after adjusting for race/ethnicity (odds ratio [OR], 2.1; P = 0.005), as was the FCGR3A 158 VV homozygous genotype after adjusting for race/ethnicity, rate of CD4(+) T cell decline, and nadir CD4(+) T cell count (OR, 21; P = 0.005). No associations between CD and FCGR2A 131 H/R polymorphism were identified. In binding studies, human IgG (hIgG)-C. neoformans complexes exhibited more binding to CHO-K1 cells expressing FCGR3A 158V than to those expressing FCGR3A 158F, and in cytotoxicity assays, natural killer (NK) cells expressing FCGR3A 158V induced more C. neoformans-infected monocyte cytotoxicity than those expressing FCGR3A 158F. Together, these results show an association between the FCGR3A 158V allele and risk for HIV-associated CD and suggest that this polymorphism could promote C. neoformans pathogenesis via increased binding of C. neoformans immune complexes, resulting in increased phagocyte cargo and/or immune activation. IMPORTANCE: HIV-associated CD4(+) T cell deficiency is a sine qua non for HIV-associated cryptococcal disease (CD), but not all patients with CD4(+) T cell deficiency develop CD despite serological evidence of previous infection. At present, there are no biomarkers that predict HIV-associated CD risk. The goal of our study was to understand whether Fc gamma receptor (FCGR) polymorphisms that have been shown to portend CD risk in HIV-uninfected people are associated with CD risk in HIV-infected people. Such biomarkers could identify those who would benefit most from targeted prophylaxis and/or earlier treatment, particularly in sub-Saharan Africa, where there are nearly a million cases of HIV-associated CD annually. A biomarker of risk could also identify potential candidates for immunization, should there be a vaccine for Cryptococcus neoformans.

Molecular characterization of the early B cell response to pulmonary Cryptococcus neoformans infection.

The role of B cells in host defense against fungi has been difficult to establish. We quantified and determined the molecular derivation of B-1a, B-1b, and B-2 B cell populations in C57BL/6 mice after pulmonary infection with Cryptococcus neoformans. Total B-1 and B-2 cell numbers increased in lungs and peritoneal cavity as early as day 1 postinfection, but lacked signs of clonal expansion. Labeled capsular (24067) and acapsular (Cap67) C. neoformans strains were used to identify C. neoformans-binding B cell subsets by flow cytometry. Peritoneal cavity B-1a B cells exhibited the most acapsular and capsular C. neoformans binding in C. neoformans-infected mice, and C. neoformans-selected B-1 B cells secreted laminarin- and C. neoformans-binding IgM. Single-cell PCR-based sequence analysis of B-1a, B-1b, and B-2 cell IgH V region H chain (V(H)) genes revealed increased usage of V(H)11 and V(H)12, respectively, in acapsular and capsular C. neoformans-selected B-1a cells. Germline V(H) segments were used, with capsular C. neoformans-selected cells having less junctional diversity than acapsular C. neoformans-selected cells. Further studies in B-1 B cell-depleted mice showed that these mice had higher brain and lung fungal burdens and less alveolar macrophage phagocytosis of C. neoformans than did control and B-1a B cell-reconstituted mice. Taken together, these results establish a mechanistic role for B-1 B cells in the innate B cell response to pulmonary infection with C. neoformans and reveal that IgM-producing B-1a cells, which express germline V(H) genes, bind C. neoformans and contribute to early fungal clearance. Thus, B-1a B cells provide a first line of defense during pulmonary C. neoformans infection in mice.

Eliminating expression of erucic acid-encoding loci allows the identification of "hidden" QTL contributing to oil quality fractions and oil content in Brassica juncea (Indian mustard).

Oil content and oil quality fractions (viz., oleic, linoleic and linolenic acid) are strongly influenced by the erucic acid pathway in oilseed Brassicas. Low levels of erucic acid in seed oil increases oleic acid content to nutritionally desirable levels, but also increases the linoleic and linolenic acid fractions and reduces oil content in Indian mustard (Brassica juncea). Analysis of phenotypic variability for oil quality fractions among a high-erucic Indian variety (Varuna), a low-erucic east-European variety (Heera) and a zero-erucic Indian variety (ZE-Varuna) developed by backcross breeding in this study indicated that lower levels of linoleic and linolenic acid in Varuna are due to substrate limitation caused by an active erucic acid pathway and not due to weaker alleles or enzyme limitation. To identify compensatory loci that could be used to increase oil content and maintain desirable levels of oil quality fractions under zero-erucic conditions, we performed Quantitative Trait Loci (QTL) mapping for the above traits on two independent F1 doubled haploid (F1DH) mapping populations developed from a cross between Varuna and Heera. One of the populations comprised plants segregating for erucic acid content (SE) and was used earlier for construction of a linkage map and QTL mapping of several yield-influencing traits in B. juncea. The second population consisted of zero-erucic acid individuals (ZE) for which, an Amplified Fragment Length Polymorphism (AFLP)-based framework linkage map was constructed in the present study. By QTL mapping for oil quality fractions and oil content in the ZE population, we detected novel loci contributing to the above traits. These loci did not co-localize with mapped locations of the fatty acid desaturase 2 (FAD2), fatty acid desaturase 3 (FAD3) or fatty acid elongase (FAE) genes unlike those of the SE population wherein major QTL were found to coincide with mapped locations of the FAE genes. Some of the new loci identified in the ZE population could be detected as 'weak' contributors (with LOD < 2.5) in the SE population in which their contribution to the traits was "masked" due to pleiotropic effects of erucic acid genes. The novel loci identified in this study could now be used to improve oil quality parameters and oil content in B. juncea under zero-erucic conditions.

Molecular dissection of antibody responses against pneumococcal surface protein A: evidence for diverse DH-less heavy chain gene usage and avidity maturation.

Immunization of human volunteers with a single dose of pneumococcal surface protein A (PspA) stimulates broad cross-reactive Abs to heterologous PspA molecules that, when transferred, protect mice from fatal infection with Streptococcus pneumoniae. In this study, we report the molecular characterization of 36 mouse mAbs generated against the extracellular domain of PspA (PspA(3-286)) from strain R36A. Abs to PspA(3-286) were encoded by diverse V(H) and V(kappa) families/genes. The H chain CDR3 and L chain CDR3 lengths were 3-13 (7.8 +/- 0.5) and 8-9 (8.7 +/- 0.2) codons, respectively. Unexpectedly, seven hybridomas expressed H chains that lack D(H) gene-derived amino acids. Nontemplate-encoded addition(s) were observed in the H chain expressed in six of these seven hybridomas; Palindromic addition(s) were absent. Absence of D(H) gene-derived amino acids did not prevent anti-PspA(3-286) mAbs from attaining average relative avidity. Avidity maturation occurred during primary IgG anti-PspA(3-286) polyclonal Ab response in PspA(3-286)- and R36A-immunized mice. Compared with PspA(3-286)-immunized mice, the relative avidity of the primary polyclonal IgG Abs was higher in R36A immunized mice on days 72, 86, and 100. Two pairs of clonally related hybridomas were observed. D(H) genes expressed in the majority (75.9%) of the hybridomas used reading frame 3. Analysis of replacement/silent mutation ratio in the CDR and framework regions provided evidence for Ag-driven selection in 11 mAbs. Based on epitope localization experiments, the mAbs were classified into 12 independent groups. ELISA additivity assay indicated that members within a group recognized topographically related epitopes. This study provides molecular insights into the biology of D(H)-less Abs.

Systematic design and testing of nested (RT-)PCR primers for specific amplification of mouse rearranged/expressed immunoglobulin variable region genes from small number of B cells.

The aim of this study was to develop a highly specific and sensitive (RT-)PCR capable of potentially amplifying the rearranged/expressed VH and VL gene belonging to any mouse immunoglobulin V gene family from a single or a small number of B cells. A database of germline immunoglobulin sequences was used to design 112 primers for a nested (RT-)PCR based strategy to cover all VH, VL, JH, JL, CH and CL gene families/genes from C57BL/6 and BALB/c mice. 93.7% of the primers had 4-fold or less, while 71.4% had no degeneracy. The proportions of germline V genes to which the primers bind with no, up to 1 and up to 2 mismatches are 59.7%, 84.1% and 94.9%, respectively. Most but not all V gene family specific primers designed allow amplification of full-length V genes. The nested primers permit PCR amplification of rearranged V genes belonging to all VH and VL gene families from splenocyte genomic DNA. The V gene family-specific nature of the primers was experimentally confirmed for randomly selected 6 VH and 6 Vkappa families, and all Vlambda genes. The broad V gene family coverage of our primer set was experimentally validated by amplifying the rearranged/expressed VH and VL genes from splenocytes and a panel of 38 hybridomas under conditions where primer mixes and genomic DNA or total RNA was used as starting template. We observed no or low-level cross-family priming. Pooled constant region specific primers allowed efficient RT-PCR amplification of H and L chain isotypes. The expressed VH and VL genes belonging to different V gene families RT-PCR amplified from a mixture of hybridomas in a representative manner. We successfully amplified the expressed VH and Vkappa gene from a single hybridoma cell by RT-PCR and from 10-15 microdissected B cells by genomic PCR. This, first of its kind, comprehensive set of highly sensitive and specific nested primers that provide broad V gene family coverage will open up new avenues and opportunities to study various aspects of mouse B cell biology.