Virus-specific Monoclonal Antibodies Research Articles

A study on viruses and bacteria with particular interest on Mycoplasma pneumoniae in children with exacerbation of asthma from a tertiary care hospital in Sri Lanka.

Asthma is a significant public health concern, particularly in children with severe symptoms. Exacerbation of asthma (EOA) is life-threatening, and respiratory infections (RIs) play a crucial role. Though viruses play a significant role in EOA, patients are empirically treated with antibiotics, contributing to antibiotic resistance development. Although there are widely reported associations of EOA with viral or Mycoplasma pneumoniae infections, there are no published data for Sri Lanka. The present study aimed to identify the association of common respiratory viruses, typical respiratory bacterial pathogens and M. pneumoniae in children with EOA and relate them with the compatibility of antimicrobial use. A case-control study was conducted in the paediatric unit of North Colombo Teaching Hospital, Sri Lanka, involving two groups of children between 5 and 15 years of age. Group 1 is children with EOA and Group 2 is children with stable asthma (SA). Each group consisted of 100 children. Sputum/throat swabs were tested for common respiratory viruses using virus-specific fluorescein isothiocyanate-labelled monoclonal antibodies (MAbs), bacteria by routine culture, and M. pneumoniae by real-time polymerase chain reaction. Macrolide resistance in M. pneumoniae was detected using conventional PCR and sequencing specific genetic mutations in the 23S rRNA gene. M. pneumoniae was genotyped using nested multilocus sequence typing, which targeted eight housekeeping genes (ppa, pgm, gyrB, gmk, glyA, atpA, arcC and adk). There was no significant difference in age, gender, demographic or geographical location between the two groups. In children with EOA, antibiotics were used in 66 % (66/100) and macrolides in 42 % (42/100). Samples comprised 78 % (78/100) sputum and 22 % (22/100) throat swabs. Adenovirus was the most common virus identified, and it was significantly higher in children with EOA compared to those with SA. Still, the two groups had no significant difference in typical bacteria findings. M. pneumoniae was detected in one patient with EOA, but none was detected in the SA group. The M. pneumoniae was macrolide-sensitive and ST14 by multilocus sequence typing. This study showed that the empiric use of antibiotics in children with asthma might be better targeted with prior pathogen screening to inform appropriate treatment to minimize antibiotic resistance.

Production of rabies virus-specific monoclonal antibodies and evaluation of their neutralizing and protective potentials

Production of rabies virus-specific monoclonal antibodies and evaluation of their neutralizing and protective potentials

Characterization of a Panel of Monoclonal Antibodies Targeting the F-Protein of the Respiratory Syncytial Virus (RSV) for the Typing of Contemporary Circulating Strains.

Respiratory syncytial virus (RSV) is the most common cause of upper and lower respiratory tract infections in infants and young children. Virus-specific monoclonal antibodies (mAbs) can be used for diagnosis, prophylaxis, and research of RSV pathogenesis. A panel of 16 anti-RSV mAbs was obtained from mice immunized by RSV strain Long. Half of them had virus-neutralizing activity. According to Western blot all of these mAbs effectively bound native oligomeric (homodimeric and homotrimeric) forms of the RSV fusion (F) protein. Only five of the mAbs interacted with the monomeric form, and only one of these possessed neutralizing activity. None of these mAbs, nor the commercial humanized neutralizing mAb palivizumab, reacted with the denaturated F protein. Thus, interaction of all these mAbs with F protein had clear conformational dependence. Competitive ELISA and neutralization assays allowed the identification of nine antigenic target sites for the interaction of mAb with the F protein. Five partially overlapping sites may represent a complex spatial structure of one antigenic determinant, including one neutralizing and four non-neutralizing epitopes. Four sites (three neutralizing and one non-neutralizing) were found to be distinct. As a result of virus cultivation RSV-A, strain Long, in the presence of a large amount of one of the neutralizing mAbs, an escape mutant with a substitution, N240S, in the F protein, was obtained. Thus, it was shown for the first time that position 240 is critical for the protective effect of an anti-RSV antibody. To assess the ability of these mAbs to interact with modern RSV strains circulating in St. Petersburg (Russia) between 2014 and 2022, 73 RSV-A and 22 RSV-B isolates were analyzed. Six mAbs were directed to conserved epitopes of the F protein as they interacted most efficiently with both RSV subtypes in a fixed cell-ELISA and could be used for diagnostic assays detecting RSV.

Content and specificity of the Anti-SARS-CoV-2 antibodies in solutions for immunoglobulin replacement therapy

BackgroundSpecific antibodies are important for post-vaccination and post-infection immune responses against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The role of antibodies in preventing and treating Coronavirus disease 2019 (COVID-19) in high-risk populations has been highlighted through the use of virus-specific monoclonal antibodies, which has raised the question of immunoglobulin replacement therapy (IRT) used in immunocompromised patients. MethodsVirus-specific anti-receptor-binding domain (RBD) and anti-nucleocapsid protein (NCAP) antibodies (assessed using a chemiluminescence assay and virus-neutralizing antibodies (virus neutralization test against Delta and Omicron variants)) were analyzed in 20 batches of 10 % (100 mg/mL) immunoglobulin solutions for intravenous IRT from two commercially available producers between January 2022 and March 2023 for clinical use. ResultsAnti-RBD and anti-NCAP antibodies were detected in all 20 batches of assessed IRT solutions (mean concentrations of 2817 IU/mL and 2380 IU/mL, respectively). Notably, the concentration of the virus-specific antibodies increased continuously during the follow-up period (from 822.5 IU/mL to 4066.4 IU/mL and 102 IU/mL to 3455.9 IU/mL). These antibodies demonstrated high virus-neutralizing activity against the Delta variant (mean titers of 436 and 325) but were limited to the Omicron variant (mean titers 78 and 70). The differences observed between the two brands were not statistically significant. ConclusionIRT solutions contain high concentrations of anti-SARS-CoV-2 specific antibodies, which may prevent COVID-19; however, the efficacy can be influenced by variable virus-neutralizing activities against different viral strains. Therefore, appropriate IRT should be combined with other approaches, such as vaccination or pre- and post-exposure prophylaxis. Passively transmitted specific antibodies may also lead to false-positive serological test results.

Somatic Hypermutation and Framework Mutations of Variable Region Contribute to Anti-Zika Virus-Specific Monoclonal Antibody Binding and Function.

ABSTRACTZika virus (ZIKV) is a global public health concern due to its ability to cause congenital Zika syndrome and lack of approved vaccine, therapeutic, or other control measures. We discovered eight novel rabbit monoclonal antibodies (MAbs) that bind to distinct ZIKV envelope protein epitopes. The majority of the MAbs were ZIKV specific and targeted the lateral ridge of the envelope (E) protein domain III, while the MAb with the highest neutralizing activity recognized a putative quaternary epitope spanning E protein domains I and III. One of the non-neutralizing MAbs specifically recognized ZIKV precursor membrane protein (prM). Somatic hypermutation of immunoglobulin variable regions increases antibody affinity maturation and triggers antibody class switching. Negative correlations were observed between the somatic hypermutation rate of the immunoglobulin heavy-chain variable region and antibody binding parameters such as equilibrium dissociation constant, dissociation constant, and half-maximal effective concentration value of MAb binding to ZIKV virus-like particles. Complementarity-determining regions recognize the antigen epitopes and are scaffolded by canonical framework regions. Reversion of framework region amino acids to the rabbit germ line sequence decreased anti-ZIKV MAb binding activity of some MAbs. Thus, antibody affinity maturation, including somatic hypermutation and framework region mutations, contributed to the binding and function of these anti-ZIKV MAbs.IMPORTANCE ZIKV is a global health concern against which no vaccine or therapeutics are available. We characterized eight novel rabbit monoclonal antibodies recognizing ZIKV envelope and prM proteins and studied the relationship between somatic hypermutation of complementarity-determining regions, framework regions, mutations, antibody specificity, binding, and neutralizing activity. The results contribute to understanding structural features and somatic mutation pathways by which potent Zika virus-neutralizing antibodies can evolve, including the role of antibody framework regions.

Antiviral Cell Products against COVID-19: Learning Lessons from Previous Research in Anti-Infective Cell-Based Agents.

COVID-19 is a real challenge for the protective immunity. Some people do not respond to vaccination by acquiring an appropriate immunological memory. The risk groups for this particular infection such as the elderly and people with compromised immunity (cancer patients, pregnant women, etc.) have the most serious problems in developing an adequate immune response. Therefore, dendritic cell (DC) vaccines that are loaded ex vivo with SARS-CoV-2 antigens in the optimal conditions are promising for immunization. Lymphocyte effector cells with chimeric antigen receptor (CAR lymphocytes) are currently used mainly as anti-tumor treatment. Before 2020, few studies on the antiviral CAR lymphocytes were reported, but since the outbreak of SARS-CoV-2 the number of such studies has increased. The basis for CARs against SARS-CoV-2 were several virus-specific neutralizing monoclonal antibodies. We propose a similar, but basically novel and more universal approach. The extracellular domain of the immunoglobulin G receptors will be used as the CAR receptor domain. The specificity of the CAR will be determined by the antibodies, which it has bound. Therefore, such CAR lymphocytes are highly universal and have functional activity against any infectious agents that have protective antibodies binding to a foreign surface antigen on the infected cells.

Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans

Pigs are natural hosts for the same subtypes of influenza A viruses as humans and integrally involved in virus evolution with frequent interspecies transmissions in both directions. The emergence of the 2009 pandemic H1N1 virus illustrates the importance of pigs in evolution of zoonotic strains. Here we generated pig influenza-specific monoclonal antibodies (mAbs) from H1N1pdm09 infected pigs. The mAbs recognized the same two major immunodominant haemagglutinin (HA) epitopes targeted by humans, one of which is not recognized by post-infection ferret antisera that are commonly used to monitor virus evolution. Neutralizing activity of the pig mAbs was comparable to that of potent human anti-HA mAbs. Further, prophylactic administration of a selected porcine mAb to pigs abolished lung viral load and greatly reduced lung pathology but did not eliminate nasal shedding of virus after H1N1pdm09 challenge. Hence mAbs from pigs, which target HA can significantly reduce disease severity. These results, together with the comparable sizes of pigs and humans, indicate that the pig is a valuable model for understanding how best to apply mAbs as therapy in humans and for monitoring antigenic drift of influenza viruses in humans, thereby providing information highly relevant to making influenza vaccine recommendations.

Generation and characterization of genetically stable heterohybridomas producing foot-and-mouth disease virus-specific porcine monoclonal antibodies

This report covers the methodology for generation of stable heterohybridoma clones producing Foot-and-mouth disease virus (FMDV) reactive porcine monoclonal antibodies (mAbs). Swine received five inoculations of an inactivated O1 Manisa FMDV vaccine prior to the harvest of splenocytes. Due to the lack of a species-specific hybridoma fusion partner, the Sp2/0 murine myeloma cell line was utilized for the formation of porcine-murine heterohybridoma clones. Twenty-nine FMDV-reactive parental clones were generated. Following sub-cloning and monitoring of reactivity over 20 serial passages, eleven subclones derived from unique parental origins were characterized and are reported herein.This methodology demonstrated the production of porcine mAbs by fusion of porcine splenocytes from immunized pigs with murine myeloma cells to generate heterohybridomas. The porcine immune response may differ from the murine immune response in relation to recognized epitopes. Therefore, application of this methodology may provide valuable resources for swine immunology and enhance the understanding of the mechanisms for antibody based protection from diseases in swine.

Potential Antiviral Options against SARS-CoV-2 Infection.

As of June 2020, the number of people infected with severe acute respiratory coronavirus 2 (SARS-CoV-2) continues to skyrocket, with more than 6.7 million cases worldwide. Both the World Health Organization (WHO) and United Nations (UN) has highlighted the need for better control of SARS-CoV-2 infections. However, developing novel virus-specific vaccines, monoclonal antibodies and antiviral drugs against SARS-CoV-2 can be time-consuming and costly. Convalescent sera and safe-in-man broad-spectrum antivirals (BSAAs) are readily available treatment options. Here, we developed a neutralization assay using SARS-CoV-2 strain and Vero-E6 cells. We identified the most potent sera from recovered patients for the treatment of SARS-CoV-2-infected patients. We also screened 136 safe-in-man broad-spectrum antivirals against the SARS-CoV-2 infection in Vero-E6 cells and identified nelfinavir, salinomycin, amodiaquine, obatoclax, emetine and homoharringtonine. We found that a combination of orally available virus-directed nelfinavir and host-directed amodiaquine exhibited the highest synergy. Finally, we developed a website to disseminate the knowledge on available and emerging treatments of COVID-19.

Differentiation of Cytopathic Effects (CPE) induced by influenza virus infection using deep Convolutional Neural Networks (CNN).

Cell culture remains as the golden standard for primary isolation of viruses in clinical specimens. In the current practice, researchers have to recognize the cytopathic effects (CPE) induced by virus infection and subsequently use virus-specific monoclonal antibody to confirm the presence of virus. Considering the broad applications of neural network in various fields, we aimed to utilize convolutional neural networks (CNN) to shorten the timing required for CPE identification and to improve the assay sensitivity. Based on the characteristics of influenza-induced CPE, a CNN model with larger sizes of filters and max-pooling kernels was constructed in the absence of transfer learning. A total of 601 images from mock-infected and influenza-infected MDCK cells were used to train the model. The performance of the model was tested by using extra 400 images and the percentage of correct recognition was 99.75%. To further examine the limit of our model in evaluating the changes of CPE overtime, additional 1190 images from a new experiment were used and the recognition rates at 16 hour (hr), 28 hr, and 40 hr post virus infection were 71.80%, 98.25%, and 87.46%, respectively. The specificity of our model, examined by images of MDCK cells infected by six other non-influenza viruses, was 100%. Hence, a simple CNN model was established to enhance the identification of influenza virus in clinical practice.

Contribution of antibody-dependent enhancement to the pathogenesis of coronavirus infections

Since the emergence of the SARS-CoV-2 virus in late 2019, vaccines against the COVID-19 infection have been under development using different approaches. At present, protective immunity factors against COVID-19 infection are not completely characterized. Of the four structural proteins of coronavirus, the spike protein (S) and the nucleocapsid protein (N) are most widely expressed in viral infections and elicit the antibody response. Antibody-dependent enhancement (ADE) presents a problem for developing a vaccine against SARS-CoV. It was shown in animal studies that SARS-CoV-1 vaccines containing recombinant S-protein or DNA-vaccine expressed S-protein led to pulmonary immunopathology after infection with SARS virus. Antibodies to the coronavirus S-protein produced by the human immune system in response to infection may contribute to the penetration of SARS-CoV into monocytes and macrophages through the Fc-gamma receptor (FcγR) and may aggravate the course of infection. The demonstration of ADE with coronavirus infection raises fundamental questions regarding the development of vaccines against the SARS-CoV-2 virus and the use of passive prophylaxis or treatment with virus-specific monoclonal antibodies. Evaluation of the mechanisms of immunopathology, including the responses of immunoglobulins and cytokines to vaccines, and tests for antigen-antibody complexes after infection and vaccination can help address these issues.

No Evidence for Cross-reactivity of Virus-specific Antibodies With HLA Alloantigens.

Antibodies directed against HLA can develop through pregnancy, blood transfusions, or organ transplants. Anecdotal evidence suggests that virus-specific antibodies may have the capacity to cross-react with HLA, a phenomenon called heterologous immunity, which is well described for T-cell alloreactivity. To determine whether antibody cross-reactivity between viral antigens and HLA is common, we tested 51 virus-specific human monoclonal antibodies (mAbs) specific for human immunodeficiency virus, varicella zoster virus, cytomegalovirus, and parvovirus, for reactivity against HLA class I and class II in single-antigen bead assays. In addition, we tested the reactivity of 41 HLA-specific human mAbs against common viral antigens of cytomegalovirus, varicella zoster virus, human immunodeficiency virus, Epstein-Barr virus, and BK polyomavirus. No cross-reactivity of any of the virus-specific mAbs with either HLA class I or class II molecules, as well as no cross-reactivity of any of the HLA-specific mAbs with any of the viral antigens was observed. These findings indicate that the frequency of cross-reactivity on the antibody level between viral antigens and HLA, if present at all, is low. The emergence of HLA antibodies upon viral infection or vaccination is therefore probably due to bystander activation of dormant HLA-specific memory B cells.

Parainfluenza virus spread into the lower respiratory tract of immunocompromised hosts: Antibodies but not G-CSF reduce viral load and accelerate clearance

Abstract In immunocompromised patients, parainfluenza virus (PIV) infections have a higher potential of spread to the lower respiratory tract (LRT), increasing morbidity and mortality. Understanding the immunological defects that facilitate the spread of the infection to the LRT among high-risk populations will help developing better management protocols. In this study, we suppressed the immune system in mice using the widely used anti-leukemic drugs: dexamethasone and/ or cyclophosphamide. We monitored the spread of viral infection into the LRT by non-invasive bioluminescence imaging system using a reporter Sendai virus (murine PIV type 1). Our results show immunosuppression leads to a delay in clearance of infection and higher lung viral loads. A rebound in the peripheral neutrophils counts coincided with initiation of viral clearance. However, treatment of immunosuppressed mice with neutrophil-specific antibodies to delay neutrophil rebound did not delay clearance, and treatment with G-CSF to increase neutrophils counts did not accelerate clearance. On the other hand, treating immunocompromised mice with either Sendai virus-specific polysera or monoclonal antibodies reduced viral progression to the lung and accelerated viral clearance. This study demonstrates the strength of non-invasive bioluminescence in both studying respiratory viral progression and evaluating candidate intervention strategies. The findings challenge the potential benefit of using G-CSF in PIV pneumonia and suggest a therapeutic potential of virus-specific antibodies.

P218: Generation of hepatitis E virus‐specific monoclonal antibodies

Journal of Viral HepatitisVolume 22, Issue S2 p. 129-130 Poster PresentationFree Access P218: Generation of hepatitis E virus-specific monoclonal antibodies First published: 25 June 2015 https://doi.org/10.1111/jvh.199_12425AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume22, IssueS2Special Issue: 15th International Symposium on Viral Hepatitis and Liver Diseases (ISVHLD) held from June 26th to 28th, 2015, in Berlin, GermanyJune 2015Pages 129-130 RelatedInformation

The Clinical Potential of Passive Immunization with Therapeutic Antibodies: Focus on Highly Pathogenic Avian Influenza Virus Infection

ABSTRACT The H5N1 highly pathogenic avian influenza (HPAI) virus has been reported to infect humans and posing a significant pandemic threat. Although neuraminidase inhibitors are currently available for the treatment of human influenza, alternative strategies need to be developed for the treatment of H5N1 HPAI virus infection in humans due to the appearance of drug-resistant viruses. Recently, passive immunization with virus-specific monoclonal antibodies has been tested for H5N1 HPAI virus infection in animal models, providing evidence that antibody therapy may be a promising option for prophylaxis or treatment of this infectious disease. Here we discuss potential benefits and limitations of antibody therapy in clinical cases of H5N1 virus infection in humans.

Validation of cross-genotype neutralization by hepatitis B virus-specific monoclonal antibodies by in vitro and in vivo infection.

Vaccines based on hepatitis B virus (HBV) genotype A have been used worldwide for immunoprophylaxis and are thought to prevent infections by non-A HBV strains effectively, whereas, vaccines generated from genotype C have been used in several Asian countries, including Japan and Korea, where HBV genotype C is prevalent. However, acute hepatitis B caused by HBV genotype A infection has been increasing in Japan and little is known about the efficacy of immunization with genotype C-based vaccines against non-C infection. We have isolated human monoclonal antibodies (mAbs) from individuals who were immunized with the genotype C-based vaccine. In this study, the efficacies of these two mAbs, HB0116 and HB0478, were analyzed using in vivo and in vitro models of HBV infection. Intravenous inoculation of HBV genotype C into chimeric mice with human hepatocytes resulted in the establishment of HBV infection after five weeks, whereas preincubation of the inocula with HB0116 or HB0478 protected chimeric mice from genotype C infection completely. Interestingly, both HB0116 and HB0478 were found to block completely genotype A infection. Moreover, infection by a genotype C strain with an immune escape substitution of amino acid 145 in the hepatitis B surface protein was also completely inhibited by incubation with HB0478. Finally, in vitro analysis of dose dependency revealed that the amounts of HB0478 required for complete protection against genotype C and genotype A infection were 5.5 mIU and 55 mIU, respectively. These results suggested that genotype C-based vaccines have ability to induce cross-genotype immunity against HBV infection.

Escape from neutralization by the respiratory syncytial virus-specific neutralizing monoclonal antibody palivizumab is driven by changes in on-rate of binding to the fusion protein

The role of binding kinetics in determining neutralizing potency for antiviral antibodies is poorly understood. While it is believed that increased steady-state affinity correlates positively with increased virus-neutralizing activity, the relationship between association or dissociation rate and neutralization potency is unclear. We investigated the effect of naturally-occurring antibody resistance mutations in the RSV F protein on the kinetics of binding to palivizumab. Escape from palivizumab-mediated neutralization of RSV occurred with reduced association rate (Kon) for binding to RSV F protein, while alteration of dissociation rate (Koff) did not significantly affect neutralizing activity. Interestingly, linkage of reduced Kon with reduced potency mirrored the effect of increased Kon found in a high-affinity enhanced potency palivizumab variant (motavizumab). These data suggest that association rate is the dominant factor driving neutralization potency for antibodies to RSV F protein antigenic site A and determines the potency of antibody somatic variants or efficiency of escape of viral glycoprotein variants.

Generation of recombinant porcine parvovirus virus-like particles in Saccharomyces cerevisiae and development of virus-specific monoclonal antibodies.

Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the self-assembly of virus-like particles (VLPs). Nine monoclonal antibodies (MAbs) against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeast S. cerevisiae represents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance.

The potent and broadly neutralizing human dengue virus-specific monoclonal antibody 1C19 reveals a unique cross-reactive epitope on the bc loop of domain II of the envelope protein.

ABSTRACTFollowing natural dengue virus (DENV) infection, humans produce some antibodies that recognize only the serotype of infection (type specific) and others that cross-react with all four serotypes (cross-reactive). Recent studies with human antibodies indicate that type-specific antibodies at high concentrations are often strongly neutralizing in vitro and protective in animal models. In general, cross-reactive antibodies are poorly neutralizing and can enhance the ability of DENV to infect Fc receptor-bearing cells under some conditions. Type-specific antibodies at low concentrations also may enhance infection. There is an urgent need to determine whether there are conserved antigenic sites that can be recognized by cross-reactive potently neutralizing antibodies. Here, we describe the isolation of a large panel of naturally occurring human monoclonal antibodies (MAbs) directed to the DENV domain II fusion loop (FL) envelope protein region from subjects following vaccination or natural infection. Most of the FL-specific antibodies exhibited a conventional phenotype, characterized by low-potency neutralizing function and antibody-dependent enhancing activity. One clone, however, recognized the bc loop of domain II adjacent to the FL and exhibited a unique phenotype of ultrahigh potency, neutralizing all four serotypes better than any other previously described MAb recognizing this region. This antibody not only neutralized DENV effectively but also competed for binding against the more prevalent poor-quality antibodies whose binding was focused on the FL. The 1C19 human antibody could be a promising component of a preventative or therapeutic intervention. Furthermore, the unique epitope revealed by 1C19 suggests a focus for rational vaccine design based on novel immunogens presenting cross-reactive neutralizing determinants.

Development of Norwalk Virus-Specific Monoclonal Antibodies with Therapeutic Potential for the Treatment of Norwalk Virus Gastroenteritis

Passive immunoprophylaxis or immunotherapy with norovirus-neutralizing monoclonal antibodies (MAbs) could be a useful treatment for high-risk populations, including infants and young children, the elderly, and certain patients who are debilitated or immunocompromised. In order to obtain antinorovirus MAbs with therapeutic potential, we stimulated a strong adaptive immune response in chimpanzees to the prototype norovirus strain Norwalk virus (NV) (genogroup I.1). A combinatorial phage Fab display library derived from mRNA of the chimpanzees' bone marrow was prepared, and four distinct Fabs reactive with Norwalk recombinant virus-like particles (rVLPs) were recovered, with estimated binding affinities in the subnanomolar range. Mapping studies showed that the four Fabs recognized three different conformational epitopes in the protruding (P) domain of NV VP1, the major capsid protein. The epitope of one of the Fabs, G4, was further mapped to a specific site involving a key amino acid residue, Gly365. One additional specific Fab (F11) was recovered months later from immortalized memory B cells and partially characterized. The anti-NV Fabs were converted into full-length IgG (MAbs) with human γ1 heavy chain constant regions. The anti-NV MAbs were tested in the two available surrogate assays for Norwalk virus neutralization, which showed that the MAbs could block carbohydrate binding and inhibit hemagglutination by NV rVLP. By mixing a single MAb with live Norwalk virus prior to challenge, MAbs D8 and B7 neutralized the virus and prevented infection in a chimpanzee. Because chimpanzee immunoglobulins are virtually identical to human immunoglobulins, these chimpanzee anticapsid MAbs may have a clinical application.