Isotype characterization of monoclonalantibodies. The isotype characterization (classes and subclasses) of the produced MAbs were determined by ELISA as described (Harlow & Lane 1988). Briely, after the addition of hybridoma supernatants to the plates (PoliSorp™, Nunc, Danmark), sensitized with sonicated antigen of C. fetus subsp. venerealis NCTC 10354 at a concentra- tion of 3 ng/μL, 50µL of of each speciic MAbs against mouse IgA, IgM, IgG 1 , IgG 2a , IgG 2b and IgG 3 (BD Biosciences Pharmingen, EUA) (2 µL/µL) were added per well and the plates were incubated at room temperature for 1h. The plates were then washed three times with PBS + 0.5% Tween 20, and 50µL of anti-rat IgG pe- roxidase conjugate (Sigma, USA), diluted 1:10,000 in incubation buffer, were added to each well, followed by incubation for 1 h at 37°C. Reactions were detected with OPD as previously described.
in patients. From just transient anti-drug antibodies (ADA) without any clinical relevance to serious effects including anaphylaxis and premature drug clearance interfering in the drug PK properties (Chirmule, Jawa, Meibohm, 2012), the immunological effects of mAbs may be caused by several factors, either patient’s or drug related: genetic background, previous immunity, drug schedule, manufacturing process, formulation and molecular characteristics (Jefferis, 2016). Fully human monoclonalantibodies are conceptually less immunogenic than chimeric or humanized. However, adalimumab, e.g., is known to induce ADA in up to 30% of the patients (Bartelds et al., 2007; Goupille, 2016). In this case, these responses can be T-cells dependent, i.e. small linear sequences (9-mer) in the therapeutic protein may bind to the major compatibility complex activating the presentation to the compatible cells thus eliciting the immunogenic response. In this context, there are many approaches to identify and suppress CD4+ T cell epitopes within the protein sequence. According to a search on the online tool PubMed, from January 2012 to November 2017 using the keywords “T-cell epitope prediction” 209 articles were found from vaccine design to therapeutic proteins deimmunization. In silico processes are based in computer algorithms able to analyze the protein sequences mapping the MHC-I and II restricted T cell epitopes (Weber et al., 2009; Delluc, Ravot, Maillere, 2011). Ex vivo identification of CD4+ T epitopes has been successfully applied to identify the immunogenic sequences in infliximab and rituximab (Hamze et al., 2017) both chimeric monoclonalantibodies. Once identified, epitopes may be engineered as a less immunogenic molecule.
Efforts to initiate a specific approach to immunotherapy of cancer started with the attachment of chemotherapeutic drugs by a weak covalent link to antitumor antibodies, at that stage still polyclonal [1–3]. Spacers such as dextran or polyglutamic acid were used to allow high drug load. At a later stage the anti- bodies were biotinylated, whereas the drug was attached to avidin, allowing a two stage drug targeting to the tumor . During these experiments we noted that there was no need to covalently attach the drug to the antibody, which by itself had some antitumor activity. For example, when monoclonalantibodies (mAbs) to epidermal growth factor receptor (EGFR) were injected together with cisplatin  they exerted a strong synergistic effect on the ability to reduce the size of tumors (KB human epidermal carcinoma). This early observation, of a synergistic effect on cancer between an antibody and a chemotherapeutic drug, has paved the way for an extensively used clinical protocol  .
intraperitoneally and intravenously, 3 days before fusion. Hybridomas were expanded and cultured in the peritoneal cavities of Pristane-primed mice to obtain ascitic fluid. All procedures with animals were in accordance with the principles of the Brazilian Code for the Use of Laboratory Animals. The anti-class 5 MAbs selected for this study were: 3E6-2 (5.1), 3BH4-C7 (5.3), 1BG11-C7 (5.4), 3DH-F5G9 (5.5), and 5F1F4-T3(5c), and the 2 new monoclonalantibodies C14F10Br2 (5.8) and 7F11B5Br3 (5.9). Except for the 2 novel ones (5.8) and (5.9), the remaining anti-class 5 monoclonalantibodies were prepared in the laboratory of one of the authors (WZ). The specificities of the 5.8 and 5.9 MAbs were determined by dot ELISA. The class and subclass of the 2 new MAb were determined by ELISA using conjugated anti-mouse immunoglobulins as described by the manufacturer (mouse hybridoma subtyping kits, Boehringer, Mannheim).
convulsions, paddling, and inevitably death (2,4,5). Like other α-herpesviruses, BHV-5 establishes a lifelong latent infection in sen- sory nerve ganglia after primary infection (5,6). Viral reactivation and shedding may occur under certain induced or naturally oc- curring stimuli and provide favorable condi- tions for viral transmission and spread (5,7). BHV-5 is genetically and antigenically closely related to the widespread respiratory and genital bovine herpesvirus type 1 (BHV- 1), so that until recently it was classified as a BHV-1 subtype (6,8). In fact, the recently determined nucleotide sequence of the en- tire DNA genome of a Brazilian BHV-5 isolate has revealed a nearly identical ge- nome structure and organization and a strik- ingly high sequence homology with BHV-1 (9). Although some antigenic differences have been detected between the envelope glycoproteins of BHV-1 and BHV-5, the major differences between these viruses are concentrated in the genes encoding the enve- lope glycoprotein C (9-13). Glycoprotein C is the most abundant envelope glycoprotein, it is involved in the initial interactions of virions with the cell surface, and represents a major target for antibodies with virus-neu- tralizing activity (14,15). In spite of the dif- ferences in the envelope glycoproteins, the traditional serological techniques and the majority of BHV-1 and BHV-5 monoclonalantibodies (mAbs) are unable to distinguish between these two viruses (16,17). Like- wise, extensive serological cross-reactivity has been demonstrated between BHV-1 and BHV-5 (17-19). Nevertheless, the occurrence of cross-protection between BHV-1 and BHV- 5 is still controversial (8,17,19-22). A possible cross-protection between these viruses would be of obvious interest for immunization strat- egies and vaccine production since only BHV-1 vaccines are available to date (21).
and quantify the immunoglobulins, which are the main components of the system that detects invaders in solutions of biological fluids or associated cells of this same medium. The bovine immunoglobulins consist of four classes: IgM, IgG, IgA, and IgE. The IgGs contain three subclasses: IgG1, IgG2, and IgG3 as well as two light ג (lambda) and K (Kappa) chains (Knight et al., 1988; Symons et al., 1989; Kacskovics et al., 1996; Rabbani et al., 1997). The three genes that codify the heavy chains of the IgGs (γ1, γ2, γ3) are located in the chromosome 21q24 (Gu et al., 1992). The production of monoclonalantibodies allows the development of specific laboratory tests for each class as well as each subclass. So far, no protein or mRNA has been identified for the IgD class. For the light chains, more than 90% are ג (lambda); however, as no specific reagents to either kappa or lambda exist, future studies would have to be carried out in order to establish the relationship between ג and Ќ chains (Pastoret, 1998).
Burkholderia pseudomallei is a Gram-negative bacillus that is the causative agent of melioidosis. The bacterium is inherently resistant to many antibiotics and mortality rates remain high in endemic areas. The lipopolysaccharide (LPS) and capsular polysaccharide (CPS) are two surface-associated antigens that contribute to pathogenesis. We previously developed two monoclonalantibodies (mAbs) specific to the CPS and LPS; the CPS mAb was shown to identify antigen in serum and urine from melioidosis patients. The goal of this study was to determine if passive immunization with CPS and LPS mAbs alone and in combination would protect mice from a lethal challenge with B. pseudomallei. Intranasal (i.n.) challenge experiments were performed with B. pseudomallei strains 1026b and K96423. Both mAbs provided significant protection when administered alone. A combination of mAbs was protective when low doses were administered. In addition, combination therapy provided a significant reduction in spleen colony forming units (cfu) compared to results when either the CPS or LPS mAbs were administered alone.
Multiresistant nosocomial pathogens often cause life-threatening infections that are some- times untreatable with currently available antibiotics. Staphylococci and enterococci are the predominant Gram-positive species associated with hospital-acquired infections. These in- fections often lead to extended hospital stay and excess mortality. In this study, a panel of fully human monoclonalantibodies was isolated from a healthy individual by selection of B- cells producing antibodies with high opsonic killing against E. faecalis 12030. Variable do- mains (VH and VL) of these immunoglobulin genes were amplified by PCR and cloned into an eukaryotic expression vector containing the constant domains of a human IgG1 mole- cule and the human lambda constant domain. These constructs were transfected into CHO cells and culture supernatants were collected and tested by opsonophagocytic assay against E. faecalis and S. aureus strains (including MRSA). At concentrations of 600 pg/ml, opsonic killing was between 40% and 70% against all strains tested. Monoclonalantibodies were also evaluated in a mouse sepsis model (using S. aureus LAC and E. faecium), a mouse peritonitis model (using S. aureus Newman and LAC) and a rat endocarditis model (using E. faecalis 12030) and were shown to provide protection in all models at a concentra- tion of 4 μg/kg per animal. Here we present a method to produce fully human IgG1 monoclo- nal antibodies that are opsonic in vitro and protective in vivo against several multiresistant Gram-positive bacteria. The monoclonalantibodies presented in this study are significantly more effective compared to another monoclonal antibody currently in clinical trials.
We have raised monoclonalantibodies (mAbs) directed towards amastigote forms of Trypanosoma cruzi, and shown that mAbs 1D9 and 4B9 are carbohydrate while mAb 4B5 activity is resistant to periodate oxidation of the antigen. Here we used an ELISA to quanti- tate and compare the expression of surface epitopes on fixed parasites among different parasite isolates. The expression of markers varied among T. cruzi amastigotes isolated from infected cells or after extracellular differentiation of trypomastigotes. Moreover, we also observed an extensive polymorphic expression of these epitopes among amastigotes derived from different strains and clones. For instance, mAb 2C2 strongly and evenly reacted with 9 strains and clones (G, Y, CL, Tulahuen, MD, and F, and clones Sylvio X-10/4, D11, and CL.B), with absorbance at 492 nm (A 492 nm) from 0.6 to 0.8. By contrast, mAb 4B5 had a higher expression in Tulahuen amasti- gotes (around 0.9 at 492 nm) whereas its reactivity with amastigotes from clones CL.B, Sylvio X-10/4 and D11 was much lower (around 0.4). mAb 1D9 displayed an interesting pattern of reactivity with amastigotes of the different strains and clones (A 492 nm of
Clostridium difficile (C. difficile) infection (CDI) is the main cause of nosocomial antibiotic- associated colitis and increased incidence of community-associated diarrhea in industrial- ized countries. At present, the primary treatment of CDI is antibiotic administration, which is effective but often associated with recurrence, especially in the elderly. Pathogenic strains produce enterotoxin, toxin A (TcdA), and cytotoxin, toxin B (TcdB), which are necessary for C. difficile induced diarrhea and gut pathological changes. Administration of anti-toxin anti- bodies provides an alternative approach to treat CDI, and has shown promising results in preclinical and clinical studies. In the current study, several humanized anti-TcdA and anti- TcdB monoclonalantibodies were generated and their protective potency was character- ized in a hamster infection model. The humanized anti-TcdA (CANmAbA4) and anti-TcdB (CANmAbB4 and CANmAbB1) antibodies showed broad spectrum in vitro neutralization of toxins from clinical strains and neutralization in a mouse toxin challenge model. Moreover, co-administration of humanized antibodies (CANmAbA4 and CANmAbB4 cocktail) pro- vided a high level of protection in a dose dependent manner (85% versus 57% survival at day 22 for 50 mg/kg and 20 mg/kg doses, respectively) in a hamster gastrointestinal infec- tion (GI) model. This study describes the protective effects conferred by novel neutralizing anti-toxin monoclonalantibodies against C. difficile toxins and their potential as therapeutic agents in treating CDI.
About 20% of proteins produced by the human genome contain single amino acid repeats, among which histidine (His) repeats are uncommonly seen with only 86 proteins bearing stretches of five or more consecutive histidine residues [3, 5, 6]. The functional studies on His- repeats have been very limited so far. Some studies have described His-repeats as nuclear speckle-targeting signals [1,2,3]. In this study, we confirmed the nuclear speckle localization of both human and mouse FAM76B by immunocytochemistry (data not shown for mouse FAM76B). Other studies showed that proteins with histidine stretches had functions related to RNA synthesis , and played a major role in development of the nervous system [3, 5]. How- ever, until now, study of poly(His)-containing proteins and their functions has been hampered by a lack of sensitive and specific monoclonalantibodies. The anti-hFAM76B MAbs produced in this study fill this need, and as tools, can facilitate the study the biological functions of FAM76B.
Bacillus anthracis, the causative agent of anthrax disease, could be used as a biothreat reagent. It is vital to develop a rapid, convenient method to detect B. anthracis. In the current study, three high affinity and specificity monoclonalantibodies (mAbs, designated 8G3, 10C6 and 12F6) have been obtained using fully washed B. anthracis spores as an immunogen. These mAbs, confirmed to direct against EA1 protein, can recognize the surface of B. anthracis spores and intact vegetative cells with high affinity and species-specificity. EA1 has been well known as a major S-layer component of B. anthracis vegetative cells, and it also persistently exists in the spore preparations and bind tightly to the spore surfaces even after rigorous washing. Therefore, these mAbs can be used to build a new and rapid immunoassay for detection of both life forms of B. anthracis, either vegetative cells or spores.
Unfortunately, the accuracy of SMM is far from ideal. The limited accuracy is the result of the imperfect epitope-defining approach, which only considers the side chain composition of an epitope but not the conformational preferences of its peptide backbone [13,14]. Suboptimal accuracy could limit the potential utility of SMM in analyzing circulating viruses or viral isolates from clinical trials, as well as narrow the applicability of the method in vaccine immunogen design. Accordingly, in this study we developed a more accurate high throughput computational method, the Method of Dynamic Epitopes (MDE), which predicts dynamic (both in sequence and in structure) epitopes of human anti-HIV-1 Abs. We optimized our method for two different anti- V3-loop monoclonalantibodies (mAbs), 2219  and 447-52D , and used this method in one of its useful applications: to obtain new calculated measurements of breadth of reactivity of these mAbs among major prevalent HIV-1 group M subtypes and circulating recombinant forms (CRFs).
The mosaic of tomato crops can be caused by a specific virus species classified in the Tobamovirus genus (14). The infected tomato plants show light and dark green mottled areas on the leaves and the fruits may be reduced in size and number with uneven ripening (12). The disease can be disseminated through infected tools and seeds, which control can be more difficult. It is very important in Europe and already do occur in Brazil (1,5). Three Tomato mosaic tobamovirus strains (ToMV) were reported and one have spread out in São Paulo plantations (1), where 30% of the tomato Brazilian production is harvested (6). The precise identification of ToMV is done by time- consuming differentiation by host plant tests and polyclonal antibody serology, which presents cross-reaction with tobacco mosaic tobamovirus (TMV), which also infects tomato plants. This could be avoided by using highly specific and uniform monoclonalantibodies (MAb) suitable for plant virus diagnosis (5,8,13). This work tested this approach aiming practical uses in selecting monoclonalantibodies to recognize specific ToMV epitopes.
Paracoccidioidomycosis (PCM) is a systemic granulomatous mycosis whose agent is Paracoccidioides bra- siliensis. In the culture supernatant, the fungus expresses glycoproteins of from 13 to 148 kDa. A cell surface glycoprotein of 43 kDa is the major antigenic component of P. brasiliensis. Another expressed glycoprotein, gp70, is recognized by 96% of sera from PCM patients and is able to induce lymphoproliferation. Since, little is known about this glycoprotein, we produced monoclonalantibodies (MAbs) against gp70 to isolate the molecule from total fungus extracts and to investigate its possible role in the pathogenesis of PCM. Using these MAbs, it was observed by confocal microscopy that gp70 is located mainly in the intracellular compartment of the fungus, although it was also detected in the culture supernatant. Based on observations showing that gp43 has a down-regulatory effect on mouse peritoneal macrophages, we tested the effects of gp70 on their phagocytic ability. Purified gp70 was able to inhibit the activity of macrophages through the mannose receptors and also through the Fc receptors; the latter effect was not observed with gp43. gp70 inhibits NO and H 2 O 2 liberation
tested; suggesting that these anti-idiotypic antibodies were directed to either the TARDY inser- tion in the CDRH3 region and/or the 6-amino acid changes in the CDR1 and CDR2. Impor- tantly, while macaques injected with PGT121 did not exhibit anti-PGT121 antibodies, plasma from animals injected twice with 10–1074 contained antibodies specific for both 10–1074 as well as the related family member PGT121. No monkey plasma showed significant binding to b12 or 10E8 (not shown) and all anti-idiotypic antibodies tested showed similar binding pat- terns to either mammalian- and plant-derived bnAbs. It is important to note that many ma- caques appeared to have pre-existing “anti-idiotypic” antibodies in their circulation prior to injection; presumably due to polyreactivity or environmental stimulation. Fourteen naïve ma- caques were tested for pre-existing reactivities to PGT121, VRC01, 10–1074 and NIH45–46 by ELISA. The results in Fig. 5B illustrate two points: (i) similar to the cross-reactivity previously observed (macaque #5193 in Fig. 3A), most monkeys tested had low to moderate levels of anti- bodies specific for NIH45–46 G54W and (ii) sera from some macaques e.g. #5191, 5194 and 5844 bound at varying levels to several of the bnAbs tested.
Data provided by the CARE-MS II trial seem to help in clarifying the profile of patients benefiting most of this treatment. However, safety and tolerability issues were raised in all these studies and need also to be highlighted. Acute infusion reactions (headache, rash, fever and nausea) were recorded in 90% of patients receiving alemtuzumab, but their frequency decreased in subsequent infusions of the drug. Nasopharyngitis, respiratory and urinary tract infections as well as herpes infections were more frequently observed in patients taking alemtuzumab than in those receiving interferon beta-1a. Both CARE-MS I and II raised the issue of autoimmune adverse events: 18% of alemtuzumab-treated patients in CARE-MS I and 16% in CARE-MS II developed autoimmune thyroid disorders, 1% developed immune thrombocytopenia requiring treatment, one patient developed glomerulonephritis with elevated anti-glomerular basement membrane antibodies during safety follow-up and another patient was diagnosed with presumed autoimmune pancytopenia 19 months after his last alemtuzumab course. 26,27
The classical routine of monoclonal antibody prepar- ation is time-consuming and laborious; the resulted mAbs are generally high specific. Protein-specific antibodies can be generated by immunization of animals with peptides, if the peptide is an effective epitope of the protein. Bioinfor- matics prediction followed by concrete experimental validation is both economical and effective. For epitope prediction, bioinformatics software can reduce the experi- mental workload by 95% and increase the efficiency of new epitope location by 10 to 20 folds . In this study, we used DNAStar Protean program to predict epitopes of saPLIγ by comprehensively analyzing many parameters such as hydrophilicity, surface accessibility, antigenic index, secondary structure and flexibility. Finally, we choose 151 CPVLRLSNRTHEANRNDLIKVA 172 as a hapten and obtained 18 IgG mAb cell strains. The resulted PLIγ mAb could recognize a broad range of snake sera including venomous and non-venomous snake spe- cies, because the epitope peptide is highly homologous among snake PLIγs.
Construction of BsAb employing Redox methodology The redox method was employed for the preparation of bispecific antibodies. A schematic representation of the redox method used for BsAb production is shown in Figure 1. Increasing concentrations (0–60 mM) of reducing agents, viz. b– mercaptoethanesulfonic acid sodium salt and b–mercaptoethanol were analyzed for efficient reduction of the disulphide bonds of both monoclonals as well as polyclonals antibodies (data not shown for b–mercaptoethanol). The monoclonalantibodies were reduced by adding an equal volume of two fold concentrated b- mercaptoethanesulphonic acid sodium salt solution (60 mM) in distilled water to a mixture of 1 mg of mouse monoclonal anti-L. monocytogenes LZH1 IgG1 and mouse monoclonal IgG2a raised against the human erythrocyte membrane protein (Protein4.2 (2G- 12)) in PBS and incubated at 37 uC for 25 minutes. Thereafter, monovalent arms were exposed to oxidizing conditions by dialysis against three buffer exchanges of phosphate buffered saline (PBS), pH 7.4 for 24 hours at 4 uC . The same set of protocol was followed for the construction of the in-house prepared bispecific antibody, employing polyclonal antibodies specific for human erythrocyte or L. monocytogenes cell surface antigens. Bispecific antibodies from both sources (monoclonal and polyclonal) were purified from parent antibody mixtures over sequential affinity columns (as discussed in detail later) to ensure that only pure and surface antigen specific BsAbs were isolated.