Strong Neutralizing Activity Research Articles

Critical role for cross-linking of trimeric lectin domains of surfactant protein D in antiviral activity against influenza A virus

Collectins are multimeric host defence lectins with trimeric CRDs (carbohydrate-recognition domains) and collagen and N-terminal domains that form higher-order structures composed of four or more trimers. Recombinant trimers composed of only the CRD and adjacent neck domain (termed NCRD) retain binding activity for some ligands and mediate some functional activities. The lung collectin SP-D (surfactant protein D) has strong neutralizing activity for IAVs (influenza A viruses) in vitro and in vivo, however, the NCRD derived from SP-D has weak viral-binding ability and lacks neutralizing activity. Using a panel of mAbs (monoclonal antibodies) directed against the NCRD in the present study we show that mAbs binding near the lectin site inhibit antiviral activity of full-length SP-D, but mAbs which bind other sites on the CRD do not. Two of the non-blocking mAbs significantly increased binding and antiviral activity of NCRDs as assessed by haemagglutination and neuraminidase inhibition and by viral neutralization. mAb-mediated cross-linking also enabled NCRDs to induce viral aggregation and to increase viral uptake by neutrophils and virus-induced respiratory burst responses by these cells. These results show that antiviral activities of SP-D can be reproduced without the N-terminal and collagen domains and that cross-linking of NCRDs is essential for antiviral activity of SP-D with respect to IAV.

Efficient neutralizing activity of cocktailed recombinant human antibodies against hepatitis a virus infection in vitro and in vivo

Hepatitis A virus (HAV) is the major pathogen responsible for acute infectious hepatitis A, a disease that is prevalent worldwide. Although HAV immunization effectively prevents infection, primary immunizations must be administered at least 2 weeks prior to HAV exposure. In contrast, passive immunization with pooled human immunoglobulin (Ig) can provide immediate and rapid protection from HAV infection. Because the use of human sera-derived Igs carries the risk of contamination, we sought to develop recombinant HAV-neutralizing human antibodies. We prepared a combinatorial phage display library of recombinant human anti-HAV antibodies from RNA extracted from the blood lymphocytes of a convalescent hepatitis A patient. Two recombinant human IgG antibodies, HAIgG16 and HAIgG78, were screened from the antibody library by their ability to bind with high affinity to purified, inactivated HAV virions. These antibodies recognized different epitopes of the HAV virion capsid, and competed with both patient sera and well-characterized neutralizing mouse monoclonal antibodies. A cocktailed mixture of HAIgG16 and HAIgG78 at a 3:1 ratio was prepared to compare its combined biological activity with that conferred by each antibody individually. The cocktailed antibodies displayed a stronger neutralizing activity in vitro than that observed with either HAIgG16 and HAIgG78 alone. To determine the in vivo neutralizing abilities of these antibodies, rhesus monkeys were inoculated with cocktailed antibodies and challenged with HAV. Whereas control animals developed hepatitis A and seroconverted to the HAV antibody, animals receiving cocktailed antibodies were protected either from viral infection or from developing clinical hepatitis. These results demonstrate that recombinant human antibody preparations could be used to prevent or treat early-stage HAV infection.

Development of neutralization antibodies against highly pathogenic H5N1 avian influenza virus using ostrich (Struthio camelus) yolk

The rapid outbreak of the highly pathogenic H5N1 avian influenza virus and its transmission to humans have induced world-wide fears of a new influenza pandemic. The most effective method for the reduction of the impact of such a pandemic would be prophylaxis with a safe and effective vaccine, as well as anti-viral materials. In this study, we generated the specific antibodies 'immunoglobulin yolk (IgY)' from ostrich eggs immunized with a full-length glycosylated recombinant H5 protein of the strain H5N1/Vietnam/1203/2004. Using this simple method, abundant specific antibody (about 200 g) against H5 was successfully produced by one female ostrich in a year. The IgY from the immunized ostrich eggs had strong reactivity to the H5N1 virus as well as to H5 proteins. Furthermore, the antibodies strongly inhibited cytopathic effects in MDCK cells and prevented the death of an embryonated chick after a viral inoculation, indicating strong neutralization activity against H5N1 infections. These findings suggest that the neutralization antibody produced by the H5-immunized ostrich is suitable for industrial purposes, such as the development of antibody-binding filters, which can be applied to a mask or to air-conditioners to prevent the influenza pandemic through antigen-antibody reactions. Of note, the mortality rate of chicks inoculated with the H5N1 virus was dramatically decreased with antibody injection. This indicates that ostrich IgY is a potentially effective therapeutic modality for H5N1 infection.

Potent Human Immunodeficiency Virus-Neutralizing and Complement Lysis Activities of Antibodies Are Not Obligatorily Linked

To evaluate the contribution of complement-mediated lysis to the in vivo activities of neutralizing antibodies, we analyzed the influence of complement activation on treatment success in a recent passive immunization trial with the neutralizing monoclonal antibodies 2G12, 2F5, and 4E10. Administration of monoclonal antibodies led to an immediate, high activation of the complement system even in the absence of viremia in the 14 participating human immunodeficiency virus-infected individuals. Lysis activity measured in patient plasma increased during passive immunization; however, the increases were modest and only partially attributable to the administration of antibodies. We found that unlike neutralization activity, lysis activity was not associated with treatment success in this trial. Compared to complement lysis mounted by the polyclonal antibody response in vivo, monoclonal antibodies were weak inducers of this activity, suggesting that polyclonal responses are more effective in reaching the required threshold of complement activation. Importantly, strong neutralization activity of the monoclonal antibodies did not predict complement lysis activity against patient and reference viruses, suggesting that these activities are not linked. In summary, our data support the notion that the in vivo activities of 2G12, 2F5, and 4E10 are likely due to direct neutralization or Fc receptor-mediated mechanisms such as phagocytosis and antibody-dependent cellular cytotoxicity.

Endogenous Cytotoxic T-Cell Response Contributes to the Long-Term Antiretroviral Protection Induced by a Short Period of Antibody-Based Immunotherapy of Neonatally Infected Mice

Neutralizing monoclonal antibodies (MAbs) are increasingly being considered for blunting human viral infections. However, whether they can also exert indirect effects on endogenous antiviral immune responses has been essentially overlooked. We have recently shown that a short (several-day) period of immunotherapy with the neutralizing 667 MAb of mouse neonates shortly after infection with the lethal FrCas(E) retrovirus not only has an immediate effect on the viral load but also permits an endogenous antiviral immunity to emerge. Even though passive immunotherapy was administered during the particular period of immunocompetence acquisition, the endogenous response eventually arising was protective and persisted long (>1 year) after the MAb has disappeared. As very high levels of anti-FrCas(E) antibodies, predominantly of the immunoglobulin G2a (IgG2a) isotype and showing strong neutralization activity, were found in the sera of MAb-treated mice, it was necessary to address whether this humoral immunity was sufficient on its own to confer full protection against FrCas(E) or whether a cytotoxic T-lymphocyte (CTL) response was also necessary. Using a variety of in vivo assays in young and adult animals previously infected by FrCas(E) and treated by 667, we show here that transient 667 immunotherapy is associated with the emergence of a CTL response against virus-infected cells. This cytotoxic activity is indispensable for long-term antiviral protective immunity, as high neutralizing antibody titers, even enhanced in in vivo CD8(+) cell depletion experiments, cannot prevent the FrCas(E)-induced death of infected/treated mice. Our work may have important therapeutic consequences, as it indicates that a short period of MAb-based immunotherapy conducted at a stage where the immune system is still developing can be associated with the mounting of a functional Th1-type immune response characterized by both CTL and IgG2a-type humoral contributions, the cooperation of which is known to be essential for the containment of chronic infections by a variety of viruses.

Generation of a recombinant Oka varicella vaccine expressing mumps virus hemagglutinin-neuraminidase protein as a polyvalent live vaccine

We constructed a recombinant varicella-zoster virus (VZV) Oka vaccine strain (vOka) that contained the mumps virus (MuV) hemagglutinin-neuraminidase (HN) gene, inserted into the site of the ORF 13 gene by using the bacterial artificial chromosome (BAC) system in Escherichia coli. Insertion of the HN gene into the VZV genome was confirmed by PCR and Southern blot. The infectious virus reconstituted from the vOka-HN genome (rvOka-HN) had a growth curve similar to the original recombinant vOka without the HN gene. The mumps virus HN protein expressed in rvOka-HN infected cells was expressed diffusely in the cytoplasm, and modification of the protein was similar to that seen in MuV-infected cells. Electron microscopic examination of infected cells revealed that HN was expressed on the plasma membrane of the cells but not in the viral envelope, suggesting that the tropism of rvOka-HN would be unchanged from that of the original vOka strain. Immunization of guinea pigs with rvOka-HN-induced VZV- and HN-specific antibodies. Interestingly, the induced antibodies had a strong neutralizing activity against virus-cell infections of both MuV and VZV. Therefore, the novel varicella vaccine expressing MuV HN protein is suitable as a polyvalent live attenuated vaccine against VZV and MuV infections.

The molecular basis of antibody-mediated neutralization of West Nile virus

The study of the interaction between the West Nile virus envelope protein and monoclonal antibodies has provided insight into the molecular mechanisms of neutralization. Structural studies have identified an epitope on the lateral ridge of domain III of the West Nile virus E protein that is recognized by antibodies with the strongest neutralizing activity in vitro and in vivo. Antibodies that bind to this epitope are particularly inhibitory because they block infection at a post-attachment step and at concentrations that result in a low occupancy of the available sites on the virion.

The Neutralization Properties of a HIV-Specific Antibody Are Markedly Altered by Glycosylation Events Outside the Antigen-Binding Domain

Neutralizing Abs constitute a pivotal mechanism of the adaptive immune response against HIV-1 infection. Yet, most of the Abs that appear in the circulation during HIV infection are nonneutralizing. In this study, we report a dramatic change of the neutralizing properties of a human Ab reactive with the nonneutralizing epitope termed cluster I on the HIV-1 transmembrane protein gp41 when the Ab was produced in Chinese hamster ovary (CHO)-K1 cells. Our laboratory has previously reported that the Ab F240, when produced in a hybridoma, is nonneutralizing as assessed by standard neutralization assays. The F240 IgG1 Ab expressed in CHO cells acquired a strong neutralization activity against a broad range of HIV isolates without a change in immunoreactivity. Sequencing of the F240 mRNAs produced in the parental hybridoma and CHO cells revealed identical sequences, suggesting that acquired neutralization resulted from cell-specific posttranslational modifications. We found that the Ab produced by CHO cells is glycosylated to a greater extent than the parental Ab produced by the hybridoma. Moreover, treatment with peptide N-glycosidase F abrogated F240 neutralization, in an isolate-specific manner, but not Ab b12 neutralization. Interestingly, the F240 isotype-switched variants IgG3 and IgG4, also expressed in CHO cells, exhibited identical immunoreactivity to IgG1 isotypes but had clear differences in viral neutralization. These results suggest that structural features of the Ig molecule other than the primary sequence of the variable regions play a more prominent role in HIV neutralization than anticipated.

Isolation of therapeutic human monoclonal antibodies for varicella‐zoster virus and the effect of light chains on the neutralizing activity

Therapeutic antibodies against varicella-zoster virus (VZV) were isolated from a combinatorial library of human antibodies using a phage-display system. Purified gH:gL was used to screen the library, and approximately 300 clones were isolated. Eight kinds of Fab-cp3-fused molecules (clones 10, 24, 36, 60, 94, 120, 192, and 431) neutralized viral infectivity. After conversion of Fab-cp3 antibodies to the Fab-protein A form, the concentrations of antibodies showing 50% inhibition of plaque formation ranged from 0.12 to 400 nM. Clones 10, 24, 94, 120 and 431 neutralized wild strains without showing strain specificity and were further converted to human IgG(1). Two clones (24 and 94) were confirmed to react with gH:gL and VZV-infected cells. IgG of clone 94 prevented spreading of infected cells. Thus these antibodies exhibited the typical phenotype of anti-gH antibody. Next the contribution of light (L) chains to neutralizing activity was analyzed by comparing the effect of L chain of clones 10, 120, and 192 with the identical heavy chain on their neutralizing activity. The L chain in the Fab form of clone 94 was replaced by L chains of clones 10, 24, 36, and 60 and the neutralizing activity of these replaced antibodies was weaker than that of the prototype clone 94. When the kappa-L chain of clone 94 was replaced by the lambda-L chain of clone 24, this antibody possessed neutralizing activity despite the kappa-lambda class change. Thus, human antibody library against VZV-gH has been established and characterized the role of the L chain in VZV-neutralizing activity to engineering further an antibody with stronger neutralizing activity.

Human antibodies for immunotherapy development generated via a human B‐cell hybridoma technology

Current strategies for the production of therapeutic MAbs include the use of mammalian cell systems to recombinantly produce antibodies derived from mice bearing human immunoglobulin transgenes, humanization of rodent antibodies, or phage libraries. We have previously reported the use of transient regulation of cellular DNA mismatch repair processes to enhance traits (e.g. affinity, titers) of MAb-producing cell lines, including hybridomas. We reasoned that this process, named morphogenics, could be utilized to improve suboptimal hybridoma cells generated via ex-vivo immunization and immortalization of antigen-specific human B-cells for therapeutic antibodies development. Here we present a platform process that combines hybridoma and morphogenics technologies for the generation of fully human MAbs specific for disease-associated human antigens. We were able to generate hybridoma lines secreting MAbs with high binding specificity and biological activity. One MAb with strong neutralizing activity against human GM-CSF was identified that is currently advanced to preclinical development for autoimmune disease indications. Moreover, these hybridoma cells have proven suitable for genetic optimization using the morphogenics process and have shown potential for large scale manufacturing.

Human antibodies for immunotherapy development generated via a human B cell hybridoma technology.

Current strategies for the production of therapeutic mAbs include the use of mammalian cell systems to recombinantly produce Abs derived from mice bearing human Ig transgenes, humanization of rodent Abs, or phage libraries. Generation of hybridomas secreting human mAbs has been previously reported; however, this approach has not been fully exploited for immunotherapy development. We previously reported the use of transient regulation of cellular DNA mismatch repair processes to enhance traits (e.g., affinity and titers) of mAb-producing cell lines, including hybridomas. We reasoned that this process, named morphogenics, could be used to improve suboptimal hybridoma cells generated by means of ex vivo immunization and immortalization of antigen-specific human B cells for therapeutic Ab development. Here we present a platform process that combines hybridoma and morphogenics technologies for the generation of fully human mAbs specific for disease-associated human antigens. We were able to generate hybridoma lines secreting mAbs with high binding specificity and biological activity. One mAb with strong neutralizing activity against human granulocyte-macrophage colony-stimulating factor was identified that is now considered for preclinical development for autoimmune disease indications. Moreover, these hybridoma cells have proven suitable for genetic optimization using the morphogenics process and have shown potential for large-scale manufacturing.

Production and Characterization of Neutralizing Monoclonal Antibodies Against Human Metapneumovirus F Protein

Human metapneumovirus (hMPV) F protein promotes fusion of viral and cell membranes, and is thought to be a major antigenic determinant that mediates effective neutralization and protection against hMPV infection. In this paper, the development of two mouse monoclonal antibodies (MAbs) by immunization with hMPVinfected cells is described. Immunofluorescence assay (IFA) using hMPV F protein-expressing cells indicated that two MAbs, designated 1G3 and 9B10, recognized hMPV F protein. Both MAbs were found to be reactive to two groups of hMPV by an IFA using two groups of hMPV-infected cells. The 9B10 mAb had strong neutralizing activity against both groups of hMPV, while the 1G3 MAb had only weak neutralization activity. These results indicate that the hMPV F protein is a major antigenic determinant that mediates extensive cross-lineage neutralization and protection. Production of MAbs to the hMPV F protein is critical for development of diagnostic techniques, vaccine research, and studies on viral pathogenesis.

Amino Acids 1055 to 1192 in the S2 Region of Severe Acute Respiratory Syndrome Coronavirus S Protein Induce Neutralizing Antibodies: Implications for the Development of Vaccines and Antiviral Agents

The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-SDelta10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.

A novel intranasal Protollin ™-based measles vaccine induces mucosal and systemic neutralizing antibody responses and cell-mediated immunity in mice

Protollin-MV is a vaccine produced by mixing split measles virus (MV) antigen with the novel adjuvant Protollin ™ ( Neisseria meningitidis outer membrane proteins non-covalently complexed with Shigella flexneri 2a lipopolysaccharide). Intranasal immunization of mice with two or three doses of Protollin-MV induces both serum IgG and mucosal IgA with strong neutralizing activity. There is a dose-dependent shift towards lower IgG 1:IgG 2a ratios and MV-specific IFNγ production in splenocytes. Intranasal Protollin-MV can therefore induce systemic and mucosal neutralizing antibody responses as well as elicit a balanced T H1/T H2-type response.

Successful induction of protective antibody responses against Haemophilus influenzae type b and diphtheria after transcutaneous immunization with the glycoconjugate polyribosyl ribitol phosphate-cross-reacting material 197 vaccine.

We examined the antibody responses elicited in rats after transcutaneous immunization (TCI) with the Haemophilus influenzae type b (Hib)-cross-reacting material (CRM(197)) glycoconjugate vaccine coadministered with cholera toxin or mutants of heat-labile enterotoxin of Escherichia coli (LTK63 and LTR72) as adjuvants. The glycoconjugate vaccine was immunogenic, eliciting high antibody responses to the capsular polysaccharide of Hib and to diphtheria toxin. Passively transferred immune serum protected infant rats against challenge with the Hib Eagan strain and exhibited strong neutralizing activity against diphtheria toxin both in vitro and in vivo. The finding that TCI of rats can elicit antibody responses surpassing the minimum levels required for protective immunity against Hib and diphtheria suggests that this immunization strategy holds a lot of promise for future pediatric use. However, further studies are required to confirm the potential of TCI with glycoconjugate vaccines in humans.

Immunodominant domains of the Measles virus hemagglutinin protein eliciting a neutralizing human B cell response.

The most important neutralizing and protective antibodies against Measles virus (MeV) are directed against the hemagglutinin protein (MeV-H). To define the MeV binding domains recognized by human antibodies a set of 10 non-redundant MeV-H-specific monoclonal antibodies (mabs) was used to block their binding in a competition ELISA. Sera from both naturally infected and vaccinated individuals showed similar competition patterns. Two distinct domains were identified as the main target of human antibodies. One domain corresponded to the region of the previously described hemagglutinin noose epitope (HNE, aa 380-400) [35], which is recognized by hemagglutination-inhibiting, neutralizing and protective mabs. The second region is defined by a mab with strong neutralizing but weak hemagglutination-inhibiting activity. Mabs with a strong neutralizing capacity with respect to wild-type viruses seemed to displace more human antibodies than those with a weaker neutralizing activity. Human antibodies seem to react more weakly with the hemagglutinin regions that bind the CD46 and the fusion protein and more strongly with the putative CD150 binding site and the top loops of beta-sheet 2 and 3 of the hemagglutinin.

Efficient isolation of novel human monoclonal antibodies with neutralizing activity against HIV-1 from transgenic mice expressing human Ig loci.

Despite considerable interest in the isolation of mAbs with potent neutralization activity against primary HIV-1 isolates, both for identifying useful targets for vaccine development and for the development of therapeutically useful reagents against HIV-1 infection, a relatively limited number of such reagents have been isolated to date. Human mAbs (hu-mAbs) are preferable to rodent mAbs for treatment of humans, but isolation of hu-mAbs from HIV-infected subjects by standard methods of EBV transformation of B cells or phage display of Ig libraries is inefficient and limited by the inability to control or define the original immunogen. An alternative approach for the isolation of hu-mAbs has been provided by the development of transgenic mice that produce fully hu-mAbs. In this report, we show that immunizing the XenoMouse G2 strain with native recombinant gp120 derived from HIV(SF162) resulted in robust humoral Ab responses against gp120 and allowed the efficient isolation of hybridomas producing specific hu-mAbs directed against multiple regions and epitopes of gp120. hu-mAbs possessing strong neutralizing activity against the autologous HIV(SF162) strain were obtained. The epitopes recognized were located in three previously described neutralization domains, the V2-, V3- and CD4-binding domains, and in a novel neutralization domain, the highly variable C-terminal region of the V1 loop. This is the first report of neutralizing mAbs directed at targets in the V1 region. Furthermore, the V2 and V3 epitopes recognized by neutralizing hu-mAbs were distinct from those of previously described human and rodent mAbs and included an epitope requiring a full length V3 loop peptide for effective presentation. These results further our understanding of neutralization targets for primary, R5 HIV-1 viruses and demonstrate the utility of the XenoMouse system for identifying new and interesting epitopes on HIV-1.

Efficient generation of respiratory syncytial virus (RSV)-neutralizing human MoAbs via human peripheral blood lymphocyte (hu-PBL)-SCID mice and scFv phage display libraries.

RSV is one of the major causes of pneumonia and bronchiolitis in infants and young children and is associated with high mortality. RSV neutralizing human antibody (hu-Ab) is known to mediate resistance to viral infection as well as to be an effective treatment for severe lower respiratory tract RSV infection. We have previously demonstrated that human primary and secondary immune responses can be established in severe combined immunodeficient mice engrafted with human peripheral blood lymphocytes (hu-PBL-SCID). By combining this animal model with the single-chain Fv antibody (scFv) phage display library technique, we were able to investigate further its clinical potential by generating a panel of human scFvs that exhibit both high F glycoprotein (RSV-F) binding affinities ( approximately 108 M(-1)) and strong neutralizing activities against RSV infection in vitro. Sequencing analysis of the randomly isolated anti-RSV-F scFv clones revealed that they were derived from different VH families with mutations in the complementarity-determining region 1 (CDR1). The results suggest that: (i) RSV-F-specific human immune responses and affinity maturation can be induced in hu-PBL-SCID mice; and (ii) this approach can be applied to generate large numbers of human scFvs with therapeutic potential. Despite the fact that hu-PBL-SCID mouse and human scFv phage display library have individually been established, our approach contributes a simple and significant step toward the generalization of antigen-specific human monoclonal antibody (hu-MoAb) production and their clinical applications.

Analysis of the functional domains of Arcanobacterium pyogenes pyolysin using monoclonal antibodies

Pyolysin (PLO), secreted by Arcanobacterium pyogenes, is a novel member of the thiol-activated cytolysin (TACY) family of bacterial toxins. Four monoclonal antibodies (mAbs) to PLO were prepared for the analysis of functional domains of this toxin. Two (mAbs S and H) of these markedly inhibited the hemolytic activity of PLO, but the inhibiting activity of the other two antibodies (mAbs C and G) was weaker. Subsequently, nine truncated PLOs were derived from recombinant Escherichia coli by various deletions from the N-terminus. Strong hemolytic activity was recognized in truncates of PLO following the deletion of 30 or 55 amino acids, but not in the truncate with deletion of 74 residues. Truncated PLOs were used in immunoblotting experiments to locate the epitopes for the mAbs. The epitope for mAbs C and G lies within the undecapeptide region (amino acids 487–505) of the C-terminus of PLO, which seems to be the binding site to erythrocytes. In contrast, the epitopes for mAbs S and H, which showed strong neutralizing activity, were found to lie in the N-terminal regions of the PLO ranging from 55 to 73 and 123 to 166 amino acids, respectively. From these results, it seems that the N-terminal region of PLO, in particular, the region of amino acids 55–74 is important for hemolytic activity.

Neutralization of feline infectious peritonitis virus: preparation of monoclonal antibody that shows cell tropism in neutralizing activity after viral absorption into the cells.

. Feline infectious peritonitis virus (FIPV) infection of feline macro-phages is enhanced by mouse anti-FIPV monoclonal antibody (MAb). This anti-body-dependent enhancement (ADE) of FIPV infection is dependent on mouse MAb subclass, and MAb of IgG2a subclass has a strong ADE activity. Furthermore, MAb showing strong neutralizing activity in Felis catus whole fetus (fcwf-4) cells and Crandell feline kidney (CrFK) cells shows strong enhancing activity in feline macrophages, indicating that the neutralizing epitope and the enhancing epitope are closely related. In this study, we prepared MAb FK50-4 that showed a strong neutralizing activity in feline macrophages, despite the fact that the MAb belonged to the IgG2a subclass. However, MAb FK50-4 did not exhibit neutralizing activity in CrFK cells or fcwf-4 cells, thus showing a very unusual property. MAb FK50-4 recognized FIPV small integral membrane glycoprotein (M protein). Even when feline macrophages were pretreated with MAb FK50-4 prior to FIPV inoculation, this antibody prevented FIPV infection. This reaction disappeared after treatment of FK50-4 with protein A. The neutralizing activity of FK50-4 was also effective on feline macrophages after the cells were inoculated with FIPV. These findings indicated that the FIPV replication mechanism differs between feline macrophages and CrFK/fcwf-4 cells and that a neutralizing epitope that can prevent FIPV infection of feline macrophages after viral absorption is present on M protein.