Emerging trends and therapeutic applications of monoclonal antibodies.

Monoclonal antibodies (mAbs) are being used to prevent, detect, and treat a broad spectrum of malignancies and infectious and autoimmune diseases. Over the past few years, the market for mAbs has grown exponentially. They have become a significant part of many pharmaceutical product lines, and more than 250 therapeutic mAbs are undergoing clinical trials. Ever since the advent of hybridoma technology, antibody-based therapeutics were realized using murine antibodies which further progressed into humanized and fully human antibodies, reducing the risk of immunogenicity. Some of the benefits of using mAbs over conventional drugs include a drastic reduction in the chances of adverse reactions, interactions between drugs, and targeting specific proteins. While antibodies are very efficient, their higher production costs impede the process of commercialization. However, their cost factor has been improved by developing biosimilar antibodies, which are affordable versions of therapeutic antibodies. Along with biosimilars, innovations in antibody engineering have helped to design bio-better antibodies with improved efficacy than the conventional ones. These novel mAb-based therapeutics are set to revolutionize existing drug therapies targeting a wide spectrum of diseases, thereby meeting several unmet medical needs. In the future, mAbs generated by applying next-generation sequencing (NGS) are expected to become a powerful tool in clinical therapeutics. This article describes the methods of mAb production, pre-clinical and clinical development of mAbs, approved indications targeted by mAbs, and novel developments in the field of mAb research.

Mapping of conserved immunodominant epitope peptides in the outer membrane porin (Omp) L of prominent Enterobacteriaceae pathogens associated with gastrointestinal infections.

BACKGROUND: Members of Enterobacteriaceae such as Escherichia coli O 157:H7, Salmonella sp., Shigella sp., Klebsiella sp., and Citrobacter freundii are responsible for the outbreak of serious foodborne illness and other mucosal infections across the globe. The outer membrane proteins (OMPs) of Enterobacteriaceae are highly immunogenic in eliciting immune responses against pathogens. Moreover, the OMPs are highly conserved in the Enterobacteriaceae family. Sequence homology in the OMPs will ensure the presence of conserved immunodominant regions with predominant epitopes. The OmpL is such an immunogen that is highly conserved among the Enterobacteriaceae pathogens. In this study, we performed computational analysis on the outer membrane porin (Omp) L of prominent Enterobacteriaceae pathogens. RESULTS: Multiple sequence and structural alignment analysis have revealed that the OmpL protein is highly conserved among the selected Enterobacteriaceae pathogens. This amount of sequence and structural homology uncovered the conserved antibody binding B-cell epitopes in the OmpL protein. The B-cell epitopes predicted in the OmpL of Salmonella typhimurium are highly conserved among the other Enterobacteriaceae pathogens. CONCLUSION: In conclusion, these conserved B-cell epitopes will vouch for the generation of heterologous humoral immune response in conferring cross protection against the Enterobacteriaceae pathogens and control their outbreaks across the globe.

Phenotypic and genetic screening of Klebsiella pneumoniae isolates from human UTI patients for beta-lactamases and their genetic diversity analysis by ERIC and REP PCRs.

Klebsiella pneumoniae is one of the major nosocomial pathogens responsible for pneumoniae, septicaemia, liver abscesses, and urinary tract infections. Coordinated efforts by antibiotic stewardship and clinicians are underway to curtail the emergence of antibiotic-resistant strains. The objective of the present study is to characterize K. pneumoniae strains through antibiotic resistance screening for production of beta-lactamases (ß-lactamases) such as extended spectrum beta lactamases (ESBLs), AmpC ß-lactamases, and carbapenemases by phenotypic and genotypic methods and genetic fingerprinting by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and repetitive element palindromic PCR (REP-PCR). A total of 85 K. pneumoniae strains isolated from 504 human urinary tract infections (UTI) were used in this study. Only 76 isolates showed positive in phenotypic screening test (PST), while combination disc method (CDM) as phenotypic confirmatory test (PCT) confirmed 72 isolates as ESBL producers. One or more ß-lactamase genes were detected by PCR in 66 isolates (91.66%, 66/72) with blaTEM gene being the most predominant (75.75%, 50/66). AmpC genes could be detected in 21 isolates (31.8%, 21/66) with FOX gene being the predominant (24.24%, 16/66), whereas NDM-I was detected in a single strain (1.51%, 1/66). Genetic fingerprinting using ERIC-PCR and REP-PCR revealed wide heterogeneity among ß-lactamase producing isolates with discriminatory power of 0.9995 and 1, respectively.

Optimization and evaluation of a multiplex PCR assay for detection of Staphylococcus aureus and its major virulence genes for assessing food safety.

Staphylococcus aureus is a natural commensal microflora of humans which causes opportunistic infections due to its large arsenal of exotoxins, invasion, immune evasion, and antibiotic resistance mechanisms. The primary goal of this study is to develop a multiplex PCR (mPCR) assay for simultaneous detection of Staphylococcus aureus (nuc) and its virulence genes coding for prominent exotoxins namely alpha hemolysin (hla), enterotoxins A (sea), enterotoxin B (seb), toxic shock syndrome toxin (tsst-1), and the gene coding for methicillin resistance (mecA). A competitive internal amplification control (IAC) was included in the assay to exclude the false negative outcomes. Highly specific primer pairs were designed for the target genes using in silico resources. At the outset, monoplex PCRs were standardized using reference S. aureus strains. Primer specificity to the target genes was authenticated through restriction digestion analysis of amplified PCR products. Multiplex PCR was optimized in increments of one gene starting with nuc and IAC amplified simultaneously using one pair of primers (nuc) in a competitive manner. The mPCR assay was found to be highly sensitive with a detection limit of ~10 CFUs per reaction for pure cultures. Multiplex PCR assay was further evaluated on the retail and processed food samples to test the prevalence of S. aureus and study their exotoxin profiles. Of the 57 samples examined, 13 samples (22.80%) were found to be contaminated with S. aureus whose DNA was extracted after a 6-h enrichment period. Among these, a high percentage of hemolytic and enterotoxin A positive strains were encountered. The mPCR assay developed in this study would be a useful tool for rapid and reliable monitoring of S. aureus for food quality testing and from clinical infections.

Enhancement of effector functions of anti-CD20 monoclonal antibody by increased afucosylation in CHO cell line through cell culture medium optimization.

BACKGROUND: Recombinant therapeutic anti-CD20 monoclonal antibody (mAb) is used for the treatment of non-Hodgkin's lymphoma, a common B cell lymphoma constituting 80% of all lymphomas. Anti-CD20 mAb contains an Fc-linked biantennary glycan. Although, anti-CD20 monoclonal antibodies are being increasingly used for immunotherapy, their efficacy is limited in a section of patients with drug resistance to immunotherapy. There is a need to improve the efficacy by increasing the effector functions, such as the antibody-dependent cellular cytotoxicity (ADCC) activity of anti-CD20 monoclonal antibodies. RESULTS: We developed a simple and cost-effective approach to enhance ADCC effector activity in an in-house developed clone of anti-CD20 monoclonal antibody by increasing afucosylation in a new clone of Chinese Hamster Ovary (CHO) cells using 8X uridine, manganese, and galactose (UMG) to modulate the osmolality of the medium. The purified anti-CD20 monoclonal antibody from 8X UMG-containing medium showed a 2-fold increase in afucose content and 203% ADCC activity in comparison to control antibody. CONCLUSIONS: Our study reports enhanced ADCC activity by modulating afucosylation using osmolality by altering simple feed additives in the culture medium.

Immunoinformatics analysis and evaluation of recombinant chimeric triple antigen toxoid (r-HAB) against Staphylococcus aureus toxaemia in mouse model.

Staphylococcus aureus is a serious pathogen unleashing its virulence through several classes of exotoxins such as hemolysins and enterotoxins. In this study, we designed a novel multi-antigen subunit vaccine which can induce innate, humoral and cellular immune responses. Alpha hemolysin, enterotoxins A and B were selected as protective antigens for combining into a triple antigen chimeric protein (HAB). Immunoinformatics analysis predicted HAB protein as a suitable vaccine candidate for inducing both humoral and cellular immune responses. Tertiary structure of the HAB protein was predicted and validated through computational approaches. Docking studies were performed between the HAB protein and mice TLR2 receptor. Furthermore, we constructed and generated recombinant HAB (r-HAB) protein in E. coli and studied its toxicity, immunogenicity and protective efficacy in a mouse model. Triple antigen chimeric protein (r-HAB) was found to be highly immunogenic in mouse as the anti-r-HAB hyperimmune serum was strongly reactive to all three native exotoxins on Western blot. In vitro toxin neutralization assay using anti-r-HAB antibodies demonstrated > 75% neutralization of toxins on RAW 264.7 cell line. Active immunization with r-HAB toxoid gave ~ 83% protection against 2 × lethal dosage of secreted exotoxins. The protection was mediated by induction of strong antibody responses that neutralized the toxins. Passive immunization with anti-r-HAB antibodies gave ~ 50% protection from lethal challenge. In conclusion, in vitro and in vivo testing of r-HAB found the molecule to be nontoxic, highly immunogenic and induced excellent protection towards native toxins in actively immunized and partial protection to passively immunized mice groups. KEY POINTS: • HAB protein was computationally designed to induce humoral and cellular responses. • r-HAB protein was found to be nontoxic, immunogenic and protective in mouse model. • r-HAB conferred protection against lethal challenge in active and passive immunization.

Application of IgY antibodies against staphylococcal protein A (SpA) of Staphylococcus aureus for detection and prophylactic functions.

In the present study, immunoglobulin Y (IgY) antibodies were raised in hens against the surface staphylococcal protein A (SpA) of Staphylococcus aureus. Anti-SpA IgY were tested in vitro for diagnostic applications, bacteriostatic, and biofilm inhibition effects. A specific and sensitive immunocapture PCR (IPCR) was developed to detect S. aureus from food, clinical, and environmental samples. Anti-SpA IgY were used for capturing S. aureus cells from different matrices. Chicken antibodies were chosen over mammalian antibodies based on its inertness to immunoglobulin (Ig)-binding property of SpA protein. No cross-reactivity was encountered with closely related Gram-positive and Gram-negative food pathogens. Inter-assay variation is < 10%. The assay was found suitable for testing on solid and liquid food samples, skin, and nasal swabs. The assay showed limit of detection of ≥ 102 CFU/mL from broth cultures and 102 to 103 CFU/ml from diverse natural samples. This assay overcomes the false positives commonly encountered while using mammalian immunoglobulins (IgG). Anti-SpA IgY antibodies were tested for their bacteriostatic effect on the growth of S. aureus. IgY antibodies at a concentration of 150 µg/ml inhibited the growth of S. aureus completely indicating the potential of IgY antibodies in neutralization of infectious pathogens. Similarly, anti-SpA IgY at MIC50 concentration reduced biofilm formation by ~ 45%. In view of advantages offered by IgY antibodies for specific detection of S. aureus in immunocapture PCR (IPCR) assay and in vitro neutralization potential of S. aureus, we recommend using IgY over conventional IgG of mammals involving S. aureus and its antigens. KEY POINTS: • IPCR with anti-SpA IgY for S. aureus was specific and sensitive for natural samples. • Anti-SpA IgY at 150 ug/ml displayed growth inhibition of S. aureus strains temporarily. • Anti-SpA IgY at MIC50 concentrations inhibited the biofilm formation partially.

Functional characterization of a broad and potent neutralizing monoclonal antibody directed against outer membrane protein (OMP) of Salmonella typhimurium.

In the present study, we have generated a murine monoclonal antibody (mAb) named Sal-06 by using the crude outer membrane protein preparation of Salmonella enteric subsp. enterica serovar Typhimurium ATCC 14028 strain as antigen. Sal-06mAb belonging to IgG1 isotype demonstrated broad cross-reactivity to standard and isolated strains of genus Salmonella and others such as Escherichia coli, Klebsiella pneumonia, and Proteus mirabilis. Cross-reactivity across several bacterial genera indicated that the epitopes reactive to Sal-06mAb are conserved among these members. Neutralizing effects of Sal-06mAb on Salmonella growth and survival was evaluated in vitro using bacteriostatic and bactericidal activity with and without complement and bacterial invasion inhibition assay. Sal-06mAb demonstrated a bacteriostatic effect on the growth of S. typhimurium ATCC 14028 strain which is both time and concentration (of mAb) dependent. It was also found that the bacterial growth inhibition was complement independent. When the bacterial cells were preincubated with Sal-06mAb, it reduced the adherence and invasion of bacterial cells into A549 epithelial cell line. This was confirmed by CFU count analysis, phase contrast, and fluorescence microscopy. Scanning electron microscope (SEM) imaging confirmed the antimicrobial effects of Sal-06mAb on S. typhimurium ATCC 14028. The development of broadly reactive and cross protective Sal-06mAb opens new possibilities for immunotherapy of sepsis caused by Gram-negative Enterobacteriaceae members.

A novel tandem repeat cloning technique for creation of multiple short peptide repeats to differentiate closely related antigens.

Antibody cross-reactivity is a problem often associated with closely related antigens. This study was aimed to develop a method enabling differentiation of closely related toxins based on antigen designing strategy. The method involves identification of disparate amino acids (AA) confined to target antigen in comparison with two or more closely related antigens, their assembly into a DNA oligomer and further cloning as six tandem repeats (TR) using restriction and ligation strategy into a desired vector and finally generation of antigen specific antibodies. The practical utility of this method was demonstrated by generating and testing the specificity of polyclonal antibodies against staphylococcal enterotoxin C (SEC). Cross-reactivity is a problem often associated with SEC in immunoassays due to its amino acid sequence identity with staphylococcal enterotoxin B (SEB) (40-60%). To circumvent the same, the above-mentioned strategy was applied. Unique AA of SEC (36 AA) in comparison to SEB were selected, reassembled and with deduced corresponding nucleotides, an oligomer of 117 bases was designed. Using primers with restriction overhangs, three constructs were created each with two repeats using a common restriction site. The resulting three constructs were sequentially cloned into alternating restriction sites of pRSET A vector in directional orientation, expressed in E. coli for rTR/SEC protein which was used to generate specific polyclonal antibodies against SEC. Specificity was compared with antibody raised against whole SEC recombinant protein using Western blot and dot blot assays. High specificity was achieved through the developed strategy signifying its possible application to address cross-reactivity problem associated with closely related antigens.

Recent Advances in Probiotics as Live Biotherapeutics Against Gastrointestinal Diseases.

BACKGROUND: Gastrointestinal (GI) diseases are a major cause of emergency department visits requiring hospitalizations leading to considerable burden on global economy. Several factors contribute to the onset of gastrointestinal diseases such as pathogens (parasites, bacteria, virus, toxins etc.), autoimmune disorders and severe inflammation of intestine. OBJECTIVE: One common feature among all these diseases is the dysentery and alteration of gut microbiota composition (gut dysbiosis). Apart from conventional therapies such as antibiotics and ORS supplementation, gut microbiota modulation with probiotic supplementation has emerged as a successful and healthy alternative in mitigating GI diseases. In this review our goal is to discuss the causes of gastrointestinal diseases and the present state of various therapeutic strategies such as probiotics as live biotherapeutics and Fecal Microbial Transplants (FMT's). CONCLUSION: Several reports and clinical trials point out to the beneficial effects of probiotics in modulating the gut microbiota and improving the side effects of gastrointestinal diseases. Live biotherapeutics and FMT's could be suitable and successful alternatives to conventional therapies in mitigating the gastrointestinal pathogens.

An efficient method for integration of PCR fragments into adjacent or overlapping restriction sites during gene cloning.

In the present work, a simple and straightforward method was developed to clone any PCR-amplified products into restriction sites that are very close, adjacent or overlapping in the expression vector. The novelty of the methodology involves a crucial primer-designing step by adding appropriate overhangs to the 5' ends of primers based on the multiple cloning sites (MCS) (polylinker) region of expression vector. After PCR amplification, actual cloning is performed not in adjacent RE sites, but in sites that are little distant in the MCS. However, the sites lost during this cloning step are maintained intact since they are provided by the cloned PCR product (through the primer overhangs). Gene for green fluorescent protein (GFP) was cloned and expressed employing this strategy to demonstrate its simplicity. This method is highly useful for vector modification without losing the restriction sites present in the MCS.

An Update on Clinical Burden, Diagnostic Tools, and Therapeutic Options of Staphylococcus aureus.

Staphylococcus aureus is an important pathogen responsible for a variety of diseases ranging from mild skin and soft tissue infections, food poisoning to highly serious diseases such as osteomyelitis, endocarditis, and toxic shock syndrome. Proper diagnosis of pathogen and virulence factors is important for providing timely intervention in the therapy. Owing to the invasive nature of infections and the limited treatment options due to rampant spread of antibiotic-resistant strains, the trend for development of vaccines and antibody therapy is increasing at rapid rate than development of new antibiotics. In this article, we have discussed elaborately about the host-pathogen interactions, clinical burden due to S aureus infections, status of diagnostic tools, and treatment options in terms of prophylaxis and therapy.

Congenital Hypothyroidism: Facts, Facets & Therapy.

BACKGROUND: Thyroid hormone (T3) is essential for normal development of children enabling brain development and somatic growth. However, certain individuals are genetically predisposed with insufficient or no thyroid hormones. Such a condition is termed congenital hypothyroidism (CH). OBJECTIVE: In the present review, a brief back ground about congenital hypothyroidism, factors associated with CH leading to thyroid dysgenesis and thyroid dyshormonogenesis is elaborated. Additionally, the guidelines for available treatment options, management and follow-up required for patients diagnosed with CH are discussed. Treatment options in terms of treatment initiation and dosage of hormone replacement are discussed. CONCLUSION: Though CH is considered as the most common neonatal metabolic disorder, it is also easily treatable compared to other metabolic or hereditary diseases. The outcome of CH treatment depends on the compliance of parents early in life and by patients themselves during later part of life.

Evaluation of recombinant SEA-TSST fusion toxoid for protection against superantigen induced toxicity in mouse model.

Treatment of Staphylococcus aureus infections has become complicated owing to growing antibiotic resistance mechanisms and due to the multitude of virulence factors secreted by this organism. Failures with traditional monovalent vaccines or toxoids have brought a shift towards the use of multivalent formulas and neutralizing antibodies to combat and prevent range of staphylococcal infections. In this study, we evaluated the efficacy of a fusion protein (r-ET) comprising truncated regions of staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin (TSST-1) in generating neutralizing antibodies against superantigen induced toxicity in murine model. Serum antibodies showed specific reactivity to both SEA and TSST-1 native toxins. Hyperimmune serum from immunized animals protected cultured splenocytes from non-specific superantigen induced proliferation completely. Passive antibody administration prevented tissue damage from acute inflammation associated with superantigen challenge from S. aureus cell free culture supernatants. Approximately 80% and 50% of actively and passively immunized mice respectively were protected from lethal dose against S. aureus toxin challenge. This study revealed that r-ET protein is non-toxic and a strong immunogen which generated neutralizing antibodies and memory immune response against superantigen induced toxic effects in mice model.