Protective neutralizing antibodies from human survivors of Crimean-Congo hemorrhagic fever.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a World Health Organization priority pathogen. CCHFV infections cause a highly lethal hemorrhagic fever for which specific treatments and vaccines are urgently needed. Here, we characterize the human immune response to natural CCHFV infection to identify potent neutralizing monoclonal antibodies (nAbs) targeting the viral glycoprotein. Competition experiments showed that these nAbs bind six distinct antigenic sites in the Gc subunit. These sites were further delineated through mutagenesis and mapped onto a prefusion model of Gc. Pairwise screening identified combinations of non-competing nAbs that afford synergistic neutralization. Further enhancements in neutralization breadth and potency were attained by physically linking variable domains of synergistic nAb pairs through bispecific antibody (bsAb) engineering. Although multiple nAbs protected mice from lethal CCHFV challenge in pre- or post-exposure prophylactic settings, only a single bsAb, DVD-121-801, afforded therapeutic protection. DVD-121-801 is a promising candidate suitable for clinical development as a CCHFV therapeutic.

Protective human monoclonal antibodies target conserved sites of vulnerability on the underside of influenza virus neuraminidase.

Continuously evolving influenza viruses cause seasonal epidemics and pose global pandemic threats. Although viral neuraminidase (NA) is an effective drug and vaccine target, our understanding of the NA antigenic landscape still remains incomplete. Here, we describe NA-specific human antibodies that target the underside of the NA globular head domain, inhibit viral propagation of a wide range of human H3N2, swine-origin variant H3N2, and H2N2 viruses, and confer both pre- and post-exposure protection against lethal H3N2 infection in mice. Cryo-EM structures of two such antibodies in complex with NA reveal non-overlapping epitopes covering the underside of the NA head. These sites are highly conserved among N2 NAs yet inaccessible unless the NA head tilts or dissociates. Our findings help guide the development of effective countermeasures against ever-changing influenza viruses by identifying hidden conserved sites of vulnerability on the NA underside.

Structural analyses of the GI.4 norovirus by cryo-electron microscopy and X-ray crystallography revealing binding sites for human monoclonal antibodies.

Noroviruses are major causative agents of acute nonbacterial gastroenteritis in humans. There are neither antiviral therapeutic agents nor vaccines for noroviruses at this time. To evaluate the potential usefulness of two previously isolated human monoclonal antibody fragments, CV-1A1 and CV-2F5, we first conducted a single-particle analysis to determine the cryo-electron microscopy structure of virus-like particles (VLPs) from the genogroup I genotype 4 (GI.4) Chiba strain uniformly coated with CV-1A1 fragments. The results revealed that the GI.4-specific CV-1A1 antibody bound to the P2 subdomain, in which amino acids are less conserved and variable. Interestingly, a part of the CV-1A1 intrudes into the histo-blood group antigen-binding site, suggesting that this antibody might exert neutralizing activity. Next, we determined the crystal structure of the protruding (P) domain of the capsid protein in the complex form with the CV-2F5 antibody fragment. Consistent with the cross-reactivity, the CV-2F5 bound to the P1 subdomain, which is rich in amino acids conserved among the GI strains, and moreover induced a disruption of Chiba VLPs. These results suggest that the broadly reactive CV-2F5 antibody can be used as both a universal detection reagent and an antiviral drug for GI noroviruses. IMPORTANCE: We conducted the structural analyses of the VP1 protein from the GI.4 Chiba norovirus to identify the binding sites of the previously isolated human monoclonal antibodies CV-1A1 and CV-2F5. The cryo-electron microscopy of the Chiba virus-like particles (VLPs) complexed with the Fv-clasp forms of GI.4-specific CV-1A1 revealed that this antibody binds to the highly variable P2 subdomain, suggesting that this antibody may have neutralizing ability against the GI.4 strains. X-ray crystallography revealed that the CV-2F5 antibody bound to the P1 subdomain, which is rich in conserved amino acids. This result is consistent with the ability of the CV-2F5 antibody to react with a wide variety of GI norovirus strains. It is also found that the CV-2F5 antibody caused a disruption of VLPs. Our findings, together with previous reports on the structures of VP1 proteins and VLPs, are expected to open a path for the structure-based development of antivirals and vaccines against norovirus disease.

Epitopes of an antibody that neutralizes a wide range of SARS-CoV-2 variants in a conserved subdomain 1 of the spike protein.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued, enabling the virus to escape from host immunity by changing its spike antigen, while biased toward the receptor-binding domain and N-terminal domain. Here, we isolated a novel pan-SARS-CoV-2 neutralizing antibody (which we named MO11) for even the recent dominators XBB.1.16 and EG.5.1, from a convalescent patient who had received three doses of an original mRNA COVID-19 vaccination. A cryo-electron microscopy analysis of the spike-MO11 complex at 2.3 Å atomic resolution revealed that it recognizes a conserved epitope hidden behind a glycan shield at N331 on subdomain 1 (SD1), holding both the N- and C-terminal segments comprising SD1. Our identification of MO11 unveiled the functional importance of SD1 for the spike's function, and we discuss the potential availability of a novel common epitope among the SARS-CoV-2 variants.IMPORTANCENovel severe acute respiratory syndrome coronavirus 2 variants with immune evasion ability are still repeatedly emerging, nonetheless, a part of immunity developed in responding to the antigen of earlier variants retains efficacy against recent variants irrespective of the numerous mutations. In exploration for the broadly effective antibodies, we identified a cross-neutralizing antibody, named MO11, from the B cells of the convalescent patient. MO11 targets a novel epitope in subdomain 1 (SD1) and was effective against all emerging variants including XBB.1.16 and EG.5.1. The neutralizing activity covering from D614G to EG.5.1 variants was explained by the conservation of the epitope, and it revealed the importance of the subdomain on regulating the function of the antigen for viral infection. Demonstrated identification of the neutralizing antibody that recognizes a conserved epitope implies basal contribution of such group of antibodies for prophylaxis against COVID-19.

Identification and Analysis of Monoclonal Antibodies with Neutralizing Activity against Diverse SARS-CoV-2 Variants.

We identified neutralizing monoclonal antibodies against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) variants (including Omicron variants BA.5 and BA.2.75) from individuals who received two doses of mRNA vaccination after they had been infected with the D614G virus. We named them MO1, MO2, and MO3. Among them, MO1 showed particularly high neutralizing activity against authentic variants: D614G, Delta, BA.1, BA.1.1, BA.2, BA.2.75, and BA.5. Furthermore, MO1 suppressed BA.5 infection in hamsters. A structural analysis revealed that MO1 binds to the conserved epitope of seven variants, including Omicron variants BA.5 and BA.2.75, in the receptor-binding domain of the spike protein. MO1 targets an epitope conserved among Omicron variants BA.1, BA.2, and BA.5 in a unique binding mode. Our findings confirm that D614G-derived vaccination can induce neutralizing antibodies that recognize the epitopes conserved among the SARS-CoV-2 variants. IMPORTANCE Omicron variants of SARS-CoV-2 acquired escape ability from host immunity and authorized antibody therapeutics and thereby have been spreading worldwide. We reported that patients infected with an early SARS-CoV-2 variant, D614G, and who received subsequent two-dose mRNA vaccination have high neutralizing antibody titer against Omicron lineages. It was speculated that the patients have neutralizing antibodies broadly effective against SARS-CoV-2 variants by targeting common epitopes. Here, we explored human monoclonal antibodies from B cells of the patients. One of the monoclonal antibodies, named MO1, showed high potency against broad SARS-CoV-2 variants including BA.2.75 and BA.5 variants. The results prove that monoclonal antibodies that have common neutralizing epitopes among several Omicrons were produced in patients infected with D614G and who received mRNA vaccination.

Human cerebrospinal fluid monoclonal CASPR2 autoantibodies induce changes in electrophysiology, functional MRI, and behavior in rodent models.

Anti-contactin associated protein receptor 2 (CASPR2) encephalitis is a severe autoimmune encephalitis with a variable clinical phenotype including behavioral abnormalities, cognitive decline, epileptic seizures, peripheral nerve hyperexcitability and neuropathic pain. The detailed mechanisms of how CASPR2 autoantibodies lead to synaptic dysfunction and clinical symptoms are largely unknown. Aiming for analyses from the molecular to the clinical level, we isolated antibody-secreting cells from the cerebrospinal fluid of two patients with CASPR2 encephalitis. From these we cloned four anti-CASPR2 human monoclonal autoantibodies (mAbs) with strong binding to brain and peripheral nerves. All were highly hypermutated and mainly of the IgG4 subclass. Mutagenesis studies determined selective binding to the discoidin domain of CASPR2. Surface plasmon resonance revealed affinities with dissociation constants KD in the pico- to nanomolar range. CASPR2 mAbs interrupted the interaction of CASPR2 with its binding partner contactin 2 in vitro and were internalized after binding to CASPR2-expressing cells. Electrophysiological recordings of rat hippocampal slices after stereotactic injection of CASPR2 mAbs showed characteristic afterpotentials following electrical stimulation. In vivo experiments with intracerebroventricular administration of human CASPR2 mAbs into mice and rats showed EEG-recorded brain hyperexcitability but no spontaneous recurrent seizures. Behavioral assessment of infused mice showed a subtle clinical phenotype, mainly affecting sociability. Mouse brain MRI exhibited markedly reduced resting-state functional connectivity without short-term structural changes. Together, the experimental data support the direct pathogenicity of CASPR2 autoantibodies. The minimally invasive EEG and MRI techniques applied here may serve as novel objective, quantifiable tools for improved animal models, in particular for subtle neuropsychiatric phenotypes or repeated measurements.

Usefulness of Belimumab in Adult Patients With Systemic Lupus Erythematosus Evaluated Using Single Indexes: A Meta-Analysis and Systematic Review.

Background: Belimumab is the first antibody drug approved for systemic lupus erythematosus (SLE), and is a fully human monoclonal antibody that inhibits soluble B lymphocyte stimulator protein. In clinical trials, a composite index was used to assess efficacy of belimumab. However, clinical guidelines on SLE treatment currently use single efficacy indexes. Objective: The main objective of this study was to perform a meta-analysis to evaluate the efficacy of belimumab utilizing single indexes used in routine clinical practice, rather than the composite efficacy index used in clinical trials during the development phase. As a secondary endpoint, safety was also evaluated. Methods: Several databases were searched to identify reports published up to December 1, 2021 on randomized controlled trials examining the efficacy of belimumab in adult patients with SLE. From the clinical trial data, efficacy was evaluated using single indexes including the SLE Disease Activity Index (SLEDAI), British Isles Lupus Assessment Group Index, and Physician Global Assessment. Safety was also assessed. Data were synthesized and analyzed using Review Manager 5.4. This study protocol was registered in the UMIN Clinical Trials Registry (Registration number: UMIN000052846). Results: The search identified 12 reports that met the inclusion criteria. Five reports were included in efficacy evaluation and 9 in safety evaluation. The primary endpoint was SLEDAI. Significantly more belimumab-treated patients achieved a ≥4-point reduction in SLEDAI (relative risk 1.28; 95% confidence interval, 1.16-1.40; P < 0.00001) compared with placebo. Other efficacy endpoints were also improved significantly in the belimumab group. No difference in safety was found between belimumab and placebo. Conclusions: The present meta-analysis evaluating clinical trial data using various single indexes recommended by clinical guidelines for SLE verifies that addition of belimumab to standard of care is efficacious for moderate-to-severe SLE.

Human monoclonal antibodies against Staphylococcus aureus A protein identified by high-throughput single-cell sequencing of phase I clinical volunteers' B cells.

Methicillin-resistant Staphylococcus aureus, poses a significant threat through infections in both community and hospital settings. To address this challenge, we conducted a phase I clinical trial study involving a recombinant Staphylococcus aureus vaccine. Utilizing peripheral blood lymphocytes from 64 subjects, we isolated antigen-specific memory B cells for subsequent single-cell sequencing. Among the 676 identified antigen-binding IgG1+ clones, we selected the top 10 antibody strains for construction within expression vectors. Successful expression and purification of these monoclonal antibodies led to the discovery of a highly expressed human antibody, designated as IgG-6. This antibody specifically targets the pentameric form of the Staphylococcus aureus protein A (SpA5). In vivo assessments revealed that IgG-6 provided prophylactic protection against MRSA252 infection. This study underscores the potential of human antibodies as an innovative strategy against Staphylococcus aureus infections, offering a promising avenue for further research and clinical development.

A fully human monoclonal antibody possesses antibody-dependent cellular cytotoxicity (ADCC) activity against the H1 subtype of influenza A virus by targeting a conserved epitope at the HA1 protomer interface.

The DiversitabTM system produces target specific high titer fully human polyclonal IgG immunoglobulins from transchromosomic (Tc) bovines shown to be safe and effective against multiple virulent pathogens in animal studies and Phase 1, 2 and 3 human clinical trials. We describe the functional properties of a human monoclonal antibody (mAb), 38C2, identified from this platform, which recognizes recombinant H1 hemagglutinins (HAs) and induces appreciable antibody-dependent cellular cytotoxicity (ADCC) activity in vitro. Interestingly, 38C2 monoclonal antibody demonstrated no detectable neutralizing activity against H1N1 virus in both hemagglutination inhibition and virus neutralization assays. Nevertheless, this human monoclonal antibody induced appreciable ADCC against cells infected with multiple H1N1 strains. The HA-binding activity of 38C2 was also demonstrated in flow cytometry using Madin-Darby canine kidney cells infected with multiple influenza A H1N1 viruses. Through further investigation with the enzyme-linked immunosorbent assay involving the HA peptide array and 3-dimensional structural modeling, we demonstrated that 38C2 appears to target a conserved epitope located at the HA1 protomer interface of H1N1 influenza viruses. A novel mode of HA-binding and in vitro ADCC activity pave the way for further evaluation of 38C2 as a potential therapeutic agent to treat influenza virus infections in humans.

Development of a recombinant hepatitis B immunoglobulin derived from B cells collected from healthy individuals administered with hepatitis B virus vaccines: A feasibility study.

BACKGROUND: In Japan, plasma with a high concentration of Hepatitis B Virus (HBV) antibodies for hepatitis B immunoglobulin (HBIG) is almost entirely imported. We aimed to produce recombinant HBIG by isolating immunoglobulin cDNAs against the HBV surface antigen (HBsAg). STUDY DESIGN AND METHODS: B cells expressing HBsAg antibodies were obtained from blood center personnel who had been administered HB vaccine booster and then isolated by either an Epstein-Barr virus hybridoma or an antigen-specific memory B cell sorting method. Each cDNA of the heavy and light chains of the target antibody was cloned into an IgG1 expression vector and transfected into Expi293F cells to produce a recombinant monoclonal antibody (mAb), which was screened by ELISA and in vitro HBV neutralizing assays. The cross-reactivity of the mAbs to normal human molecules was evaluated by ELISA and immunohistochemistry. RESULTS: Antibody cDNAs were cloned from 11 hybridoma cell lines and 204 HBsAg-bound memory B cells. Three of the resulting recombinant mAbs showed stronger neutralizing activity in vitro than the currently used HBIG. All three bind to the conformational epitope(s) of HBsAg but not to human DNA or cells. DISCUSSION: We successfully isolated HBV-neutralizing monoclonal antibodies from B cells collected from healthy plasma donors boosted against the HBV. To obtain an alternative source for HBIG, HBV-neutralizing monoclonal antibodies from B cells collected from healthy plasma donors boosted against the HBV may be useful.

Generation and Next-Generation Sequencing-Based Characterization of a Large Human Combinatorial Antibody Library.

Antibody phage display is a key technology for the discovery and development of target-specific monoclonal antibodies (mAbs) for use in research, diagnostics, and therapy. The construction of a high-quality antibody library, with larger and more diverse antibody repertoires, is essential for the successful development of phage display-derived mAbs. In this study, a large human combinatorial single-chain variable fragment library (1.5 × 1011 colonies) was constructed from Epstein-Barr virus-infected human peripheral blood mononuclear cells stimulated with a combination of two of the activators of human B cells, the Toll-like receptor 7/8 agonist R848 and interleukin-2. Next-generation sequencing analysis with approximately 1.9 × 106 and 2.7 × 106 full-length sequences of heavy chain variable (VH) and κ light chain variable (Vκ) domains, respectively, revealed that the library consists of unique VH (approximately 94%) and Vκ (approximately 91%) sequences with greater diversity than germline sequences. Lastly, multiple unique mAbs with high affinity and broad cross-species reactivity could be isolated from the library against two therapeutically relevant target antigens, validating the library quality. These findings suggest that the novel antibody library we have developed may be useful for the rapid development of target-specific phage display-derived recombinant human mAbs for use in therapeutic and diagnostic applications.

Monoclonal antibodies from B cells of patients with anti-MDA5 antibody-positive dermatomyositis directly stimulate interferon gamma production.

Anti-melanoma differentiation-associated gene 5 (MDA5) antibody (Ab) positive dermatomyositis (anti-MDA5 DM) is a rare entity associated with poor prognosis and multiple immunologic abnormalities. These include the presence of autoAbs and deleterious interferon (IFN)-gamma production in the severe form of the disease. Here, we show that the autoAbs profile differs between patients, depending on disease severity, and that autoAbs from B cells of patients directly stimulate IFN-gamma production by peripheral blood cells. Serum of 29 anti-MDA5 DM patients were analyzed by indirect immunofluorescence (IIF) on Hep-2 cells, to identify patterns associated with poor outcome. Seventeen (59%) serum gave a specific cytoplasmic MDA5 pattern on Hep-2 cells, while 12 (41%) gave an unspecific pattern. Specific MDA5 pattern was associated with a higher risk to develop interstitial lung disease (p = 0.003). Monoclonal autoAbs were generated from B cell clones of two patients with extreme clinical presentation, one who developed a lethal form of the disease, and the other with a favorable outcome. Supernatants of the autoreactive B cell clones that gave an IIF cytoplasmic pattern were tested for their abilities to stimulate IFN-gamma production by peripheral blood cells. Out of 120,000 B cell clones analyzed, 12 produced monoclonal Abs that triggered direct IFN-gamma secretion by peripheral blood cells, by a monocyte-dependent mechanism. None of them was directed against the MDA5 antigen. Altogether, these findings demonstrated that autoAbs other than the highly specific anti-MDA5 Ab are direct contributors of the IFN-gamma upregulation that is linked to the severity of the disease.

Design, synthesis, and evaluation of HIV-1 entry inhibitors based on broadly neutralizing antibody 447-52D and gp120 V3loop interactions.

The third variable loop region (V3 loop) on gp120 plays an important role in cellular entry of HIV-1. Its interaction with the cellular CD4 and coreceptors is an important hallmark in facilitating the bridging by gp41 and subsequent fusion of membranes for transfer of viral genetic material. Further, the virus phenotype determines the cell tropism via respective co- receptor binding. Thus, coreceptor binding motif of envelope is considered to be a potent anti-viral drug target for viral entry inhibition. However, its high variability in sequence is the major hurdle for developing inhibitors targeting the region. In this study, we have used an in silico Virtual Screening and "Fragment-based" method to design small molecules based on the gp120 V3 loop interactions with a potent broadly neutralizing human monoclonal antibody, 447-52D. From the in silico analysis a potent scaffold, 1,3,5-triazine was identified for further development. Derivatives of 1,3,5-triazine with specific functional groups were designed and synthesized keeping the interaction with co-receptor intact. Finally, preliminary evaluation of molecules for HIV-1 inhibition on two different virus strains (clade C, clade B) yielded IC50 < 5.0 µM. The approach used to design molecules based on broadly neutralizing antibody, was useful for development of target specific potent antiviral agents to prevent HIV entry. The study reported promising inhibitors that could be further developed and studied.

Impact of Host Immune Status on Discordant Anti-SARS-CoV-2 Circulating B Cell Frequencies and Antibody Levels.

Individuals with pre-existing chronic systemic low-grade inflammation are prone to develop severe COVID-19 and stronger anti-SARS-CoV-2 antibody responses. Whether this phenomenon reflects a differential expansion of antiviral B cells or a failure to regulate antibody synthesis remains unknown. Here, we compared the antiviral B cell repertoire of convalescent healthcare personnel to that of hospitalized patients with pre-existing comorbidities. Out of 277,500 immortalized B cell clones, antiviral B cell frequencies were determined by indirect immunofluorescence screening on SARS-CoV-2 infected cells. Surprisingly, frequencies of SARS-CoV-2 specific clones from the two groups were not statistically different, despite higher antibody levels in hospitalized patients. Moreover, functional analyses revealed that several B cell clones from healthcare personnel with low antibody levels had neutralizing properties. This study reveals for the first time a key qualitative defect of antibody synthesis in severe patients and calls for caution regarding estimated protective immunity based only on circulating antiviral antibodies.

Anti-OspA DNA-Encoded Monoclonal Antibody Prevents Transmission of Spirochetes in Tick Challenge Providing Sterilizing Immunity in Mice.

We recently developed anti-OspA human immunoglobulin G1 monoclonal antibodies (HuMAbs) that are effective in preventing Borrelia transmission from ticks in a murine model. Here, we investigated a novel approach of DNA-mediated gene transfer of HuMAbs that provide protection against Lyme disease. Plasmid DNA-encoded anti-OspA HuMAbs inoculated in mice achieved a serum antibody concentration of >6 µg/mL. Among mice injected with DNA-encoded monoclonal antibodies, 75%-77% were protected against an acute challenge by Borrelia-infected ticks. Our results represent the first demonstration of employing DNA transfer as a delivery system for antibodies that block transmission of Borrelia in animal models.

A therapeutic human antibody against the domain 4 of the Bacillus anthracis protective antigen shows protective efficacy in a mouse model.

Since Bacillus anthracis is a high-risk pathogen and a potential tool for bioterrorism, numerous therapeutic methods including passive immunization have been actively developed. Using a human monoclonal antibody phage display library, we screened new therapeutic antibodies for anthrax infection against protective antigen (PA) of B. anthracis. Among 5 selected clones of antibodies based on enzyme-linked immunosorbent assay (ELISA) results, 7B1 showed neutralizing activity to anthrax lethal toxin (LT) by inhibiting binding of the domain 4 of PA (PD4) to its cellular receptors. Through light chain shuffling process, we improved the productivity of 7B1 up to 25 folds. The light chain shuffled 7B1 antibody showed protective activity against LT both in vitro and in vivo. Furthermore, the antibody also conferred protection of mice from 3 × LD50 challenges of fully virulent anthrax spores. Our result expands the possibility of developing a new therapeutic antibody for anthrax cure.

Human Neutralizing Monoclonal Antibody Inhibition of Middle East Respiratory Syndrome Coronavirus Replication in the Common Marmoset.

Middle East respiratory syndrome coronavirus (MERS-CoV) infection in humans is highly lethal, with a fatality rate of 35%. New prophylactic and therapeutic strategies to combat human infections are urgently needed. We isolated a fully human neutralizing antibody, MCA1, from a human survivor. The antibody recognizes the receptor-binding domain of MERS-CoV S glycoprotein and interferes with the interaction between viral S and the human cellular receptor human dipeptidyl peptidase 4 (DPP4). To our knowledge, this study is the first to report a human neutralizing monoclonal antibody that completely inhibits MERS-CoV replication in common marmosets. Monotherapy with MCA1 represents a potential alternative treatment for human infections with MERS-CoV worthy of evaluation in clinical settings.

Pre-exposure Prophylaxis With OspA-Specific Human Monoclonal Antibodies Protects Mice Against Tick Transmission of Lyme Disease Spirochetes.

BACKGROUND: Tick transmission of Borrelia spirochetes to humans results in significant morbidity from Lyme disease worldwide. Serum concentrations of antibodies against outer surface protein A (OspA) were shown to correlate with protection from infection with Borrelia burgdorferi, the primary cause of Lyme disease in the United States. METHODS: Mice transgenic for human immunoglobulin genes were immunized with OspA from B. burgdorferi to generate human monoclonal antibodies (HuMabs) against OspA. HuMabs were generated and tested in in vitro borreliacidal assays and animal protection assays. RESULTS: Nearly 100 unique OspA-specific HuMabs were generated, and 4 HuMabs (221-7, 857-2, 319-44, and 212-55) were selected as lead candidates on the basis of borreliacidal activity. HuMabs 319-44, 857-2, and 212-55 were borreliacidal against 1 or 2 Borrelia genospecies, whereas 221-7 was borreliacidal (half maximal inhibitory concentration, < 1 nM) against B. burgdorferi, Borrelia afzelii, and Borrelia garinii, the 3 main genospecies endemic in the United States, Europe, and Asia. All 4 HuMabs completely protected mice from infection at 10 mg/kg in a murine model of tick-mediated transmission of B. burgdorferi CONCLUSIONS: Our study indicates that OspA-specific HuMabs can prevent the transmission of Borrelia and that administration of these antibodies could be employed as preexposure prophylaxis for Lyme disease.

RNA recognition by a human antibody against brain cytoplasmic 200 RNA.

Diverse functional RNAs participate in a wide range of cellular processes. The RNA structure is critical for function, either on its own or as a complex form with proteins and other ligands. Therefore, analysis of the RNA conformation in cells is essential for understanding their functional mechanisms. However, no appropriate methods have been established as yet. Here, we developed an efficient strategy for panning and affinity maturation of anti-RNA human monoclonal antibodies from a naïve antigen binding fragment (Fab) combinatorial phage library. Brain cytoplasmic 200 (BC200) RNA, which is also highly expressed in some tumors, was used as an RNA antigen. We identified MabBC200-A3 as the optimal binding antibody. Mutagenesis and SELEX experiments showed that the antibody recognized a domain of BC200 in a structure- and sequence-dependent manner. Various breast cancer cell lines were further examined for BC200 RNA expression using conventional hybridization and immunoanalysis with MabBC200-A3 to see whether the antibody specifically recognizes BC200 RNA among the total purified RNAs. The amounts of antibody-recognizable BC200 RNA were consistent with hybridization signals among the cell lines. Furthermore, the antibody was able to discriminate BC200 RNA from other RNAs, supporting the utility of this antibody as a specific RNA structure-recognizing probe. Intriguingly, however, when permeabilized cells were subjected to immunoanalysis instead of purified total RNA, the amount of antibody-recognizable RNA was not correlated with the cellular level of BC200 RNA, indicating that BC200 RNA exists as two distinct forms (antibody-recognizable and nonrecognizable) in breast cancer cells and that their distribution depends on the cell type. Our results clearly demonstrate that anti-RNA antibodies provide an effective novel tool for detecting and analyzing RNA conformation.

Characterization of a new human monoclonal antibody directed against the Vel antigen.

BACKGROUND AND OBJECTIVES: The Vel blood group antigen is a poorly characterized high-prevalence antigen. Until now, anti-Vel antibodies have been observed in only alloimmunized Vel-negative individuals. In this study, we aimed to establish a human hybridoma cell line secreting the first anti-Vel monoclonal antibody (mAb), clone SpG213Dc. MATERIALS AND METHODS: Peripheral blood lymphocytes from a French Vel-negative woman with anti-Vel in her plasma were transformed with Epstein-Barr virus and then hybridized with the myeloma cell line Sp2/O-Ag14 using the polyethylene glycol (PEG) method. A specific anti-Vel mAb was successfully produced and was extensively characterized by serological, flow cytometry and Western blot analyses. RESULTS: One human anti-Vel-secreting clone was produced and the secreted anti-Vel mAb (SpG213Dc) was examined. The specificity of the SpG213Dc mAb was assessed by its reactivity against a panel of nine genotyped RBCs including, respectively, three Vel-negative and six Vel-positive (three wild-type homozygous and three heterozygous) samples using flow cytometry method. Vel-positive RBCs were specifically stained and were subsequently used to perform Western blot and immunoprecipitation analysis of the Vel antigen. CONCLUSION: Serological characterization of the new monoclonal anti-Vel SpG213Dc showed a heterogeneous level of expression of the Vel antigen on the different RBCs. Our results suggest that the mAb SpG213Dc can be reliably used as a blood grouping reagent, thus allowing the mass-scale phenotyping of blood donors to strengthen rare blood banks with Vel-negative RBC units.