Mechanism of HIV-1 Neutralization by Antibodies Targeting a Membrane-Proximal Region of gp41

Induction of broadly neutralizing antibodies (bNAbs) is an important goal for HIV-1 vaccine development. Two autoreactive bNAbs, 2F5 and 4E10, recognize a conserved region on the HIV-1 envelope glycoprotein gp41 adjacent to the viral membrane known as the membrane-proximal external region (MPER). They block viral infection by targeting a fusion-intermediate conformation of gp41, assisted by an additional interaction with the viral membrane. Another MPER-specific antibody, 10E8, has recently been reported to neutralize HIV-1 with potency and breadth much greater than those of 2F5 or 4E10, but it appeared not to bind phospholipids and might target the untriggered envelope spikes, raising the hope that the MPER could be harnessed for vaccine design without major immunological concerns. Here, we show by three independent approaches that 10E8 indeed binds lipid bilayers through two hydrophobic residues in its CDR H3 (third heavy-chain complementarity-determining region). Its weak affinity for membranes in general and preference for cholesterol-rich membranes may account for its great neutralization potency, as it is less likely than other MPER-specific antibodies to bind cellular membranes nonspecifically. 10E8 binds with high affinity to a construct mimicking the fusion intermediate of gp41 but fails to recognize the envelope trimers representing the untriggered conformation. Moreover, we can improve the potency of 4E10 without affecting its binding to gp41 by a modification of its lipid-interacting CDR H3. These results reveal a general mechanism of HIV-1 neutralization by MPER-specific antibodies that involves interactions with viral lipids.     

Variable Loop Glycan Dependency of the Broad and Potent HIV-1-Neutralizing Antibodies PG9 and PG16

The HIV-1-specific antibodies PG9 and PG16 show marked cross-isolate neutralization breadth and potency. Antibody neutralization has been shown to be dependent on the presence of N-linked glycosylation at position 160 in gp120. We show here that (i) the loss of several key glycosylation sites in the V1, V2, and V3 loops; (ii) the generation of pseudoviruses in the presence of various glycosidase inhibitors; and (iii) the growth of pseudoviruses in a mutant cell line (GnT1^−/−) that alters envelope glycosylation patterns all have significant effects on the sensitivity of virus to neutralization by PG9 and PG16. However, the interaction of antibody is not inhibited by sugar monosaccharides corresponding to those found in glycans on the HIV surface. We show that some of the glycosylation effects described are isolate dependent and others are universal and can be used as diagnostic for the presence of PG9 and PG16-like antibodies in the sera of HIV-1-infected patients. The results suggest that PG9 and PG16 recognize a conformational epitope that is dependent on glycosylation at specific variable loop N-linked sites. This information may be valuable for the design of immunogens to elicit PG9 and PG16-like antibodies, as well as constructs for cocrystallization studies.It is argued that an effective HIV vaccine should include a component that induces a broadly neutralizing antibody response (2, 3, 21, 25, 32, 37, 39, 54). The key target for broadly neutralizing HIV antibodies is the envelope spike, which consists of a compact, metastable heterodimeric trimer of the glycoproteins gp120 and gp41 (43, 62).gp120 is one of the most heavily glycosylated proteins known, with up to 50% of its mass arising from carbohydrates attached to roughly 25 N-linked glycosylation sites (31) determined by the NXT/S consensus sequence (where X can be any amino acid except Pro) (1). Glycosylation significantly impacts the folding and conformation of envelope spikes, thus affecting antigenicity and immunogenicity (30, 35). Carbohydrates are generally poorly immunogenic, and the dense covering of glycans is often referred to as the “silent face” or “glycan shield” (58). The glycans have also been suggested to have an important role in viral transmission through interaction with lectins, in particular the C-type lectin DC-SIGN, which is found on the surfaces of dendritic cells and is thought to aid the transport of virus to anatomical sites rich in CD4⁺ T cells, such as lymph nodes (8, 16).Although the positioning of N-linked protein glycosylation is encoded by the protein sequence (1), the type of glycan displayed (high mannose, hybrid, or complex) is not under direct genetic control but is determined by the three-dimensional structure of a protein and its interaction with the biosynthetic cellular environment, including accessibility to glycan-processing enzymes (50). For example, highly clustered glycans prevent access of the processing enzymes, leading to high-mannose-type glycans being displayed (6, 23). Therefore, the glycosylation of recombinant HIV envelope proteins can vary significantly depending on the protein sequence, structure, and the cell in which they are expressed (50). Although the positions of many glycans are relatively conserved between isolates and clades (60), there can be variation in the occupancy and precise nature of the glycans displayed at these positions on recombinant envelope (7, 17-19, 61). However, we have recently observed major differences between the glycosylation of recombinant envelope proteins and envelope expressed on the virion surface, with the latter being dominated by Man_5-9GlcNAc₂ oligomannose glycans (9). Nevertheless, significant glycan heterogeneity remains on the virion surface.Recently, two new neutralizing antibodies, PG9 and PG16, were isolated from an African clade A-infected donor and shown to be both broad and potent (56). From a panel of 162 viruses, PG9 neutralized 127 and PG16 neutralized 119 viruses at a median potency that exceeded that of the broadly neutralizing antibodies—2G12, b12, 2F5, and 4E10—by about an order of magnitude. In a TZM-bl neutralization assay, PG9 has been shown to neutralize 87% of a panel of 82 viruses (M. Seaman, unpublished data). Both PG9 and PG16 show preferential trimer binding and interact with an epitope formed from conserved regions of the V1/V2 and V3 variable loops. Mutation of N160, an N-linked glycosylation site in the V2 loop, completely abolishes PG9 and PG16 neutralization, suggesting the N160 glycan is important in forming the PG9 and PG16 epitope. Further, PG9 shows significant binding to monomeric gp120 DU422 and treatment of the glycoprotein with Endo H (removing high-mannose glycans) results in significant reduction in antibody binding. Occasionally, neutralization of some pseudoviruses by PG16 in particular has revealed an unusual neutralization profile with a shallow slope and plateaus at <100%. We hypothesized that this unusual neutralization profile may be related to antibody sensitivity to glycosylation and, more specifically, could be due to glycan profile or partial glycosylation at critical sites.We show here that loss of any one of several glycosylation sites in the V1, V2, and V3 loops has significant effects on the sensitivity of pseudovirus to neutralization by PG9 and PG16. Generating pseudovirus in the presence of various glycosidase inhibitors also has notable effects on antibody neutralization. We show that some of these effects are isolate dependent and others are universal and can be used to help identify the presence of PG9 and PG16-like antibodies in the serum of HIV-1-infected patients (57). For some isolates displaying aberrant neutralization profiles as described above, we found that changing the glycan profile on the HIV-1 trimer using glycosidase inhibitors or a mutant cell line resulted in higher neutralization plateaus and neutralization profiles with the more usual sigmoidal shape. Changes in sensitivity to neutralization were also observed for some but not all isolates. The antibody-gp120 interaction was not inhibited by sugar monosaccharides found in glycans on the HIV envelope. The results suggest PG9 and PG16 recognize a conformational epitope that is dependent on the glycosylation at specific variable loop N-linked glycosylation sites. This information may be valuable for the design of immunogens to elicit PG9 and PG16-like antibodies, as well as constructs for cocrystallization studies.     

Viral Escape from HIV-1 Neutralizing Antibodies Drives Increased Plasma Neutralization Breadth through Sequential Recognition of Multiple Epitopes and Immunotypes

Identifying the targets of broadly neutralizing antibodies to HIV-1 and understanding how these antibodies develop remain important goals in the quest to rationally develop an HIV-1 vaccine. We previously identified a participant in the CAPRISA Acute Infection Cohort (CAP257) whose plasma neutralized 84% of heterologous viruses. In this study we showed that breadth in CAP257 was largely due to the sequential, transient appearance of three distinct broadly neutralizing antibody specificities spanning the first 4.5 years of infection. The first specificity targeted an epitope in the V2 region of gp120 that was also recognized by strain-specific antibodies 7 weeks earlier. Specificity for the autologous virus was determined largely by a rare N167 antigenic variant of V2, with viral escape to the more common D167 immunotype coinciding with the development of the first wave of broadly neutralizing antibodies. Escape from these broadly neutralizing V2 antibodies through deletion of the glycan at N160 was associated with exposure of an epitope in the CD4 binding site that became the target for a second wave of broadly neutralizing antibodies. Neutralization by these CD4 binding site antibodies was almost entirely dependent on the glycan at position N276. Early viral escape mutations in the CD4 binding site drove an increase in wave two neutralization breadth, as this second wave of heterologous neutralization matured to recognize multiple immunotypes within this site. The third wave targeted a quaternary epitope that did not overlap any of the four known sites of vulnerability on the HIV-1 envelope and remains undefined. Altogether this study showed that the human immune system is capable of generating multiple broadly neutralizing antibodies in response to a constantly evolving viral population that exposes new targets as a consequence of escape from earlier neutralizing antibodies.     

Drift of the HIV-1 Envelope Glycoprotein gp120 toward Increased Neutralization Resistance over the Course of the Epidemic: a Comprehensive Study Using the Most Potent and Broadly Neutralizing Monoclonal Antibodies

Extending our previous analyses to the most recently described monoclonal broadly neutralizing antibodies (bNAbs), we confirmed a drift of HIV-1 clade B variants over 2 decades toward higher resistance to bNAbs targeting almost all the identified gp120-neutralizing epitopes. In contrast, the sensitivity to bNAbs targeting the gp41 membrane-proximal external region remained stable, suggesting a selective pressure on gp120 preferentially. Despite this evolution, selected combinations of bNAbs remain capable of neutralizing efficiently most of the circulating variants.     

A Combination of Broadly Neutralizing HIV-1 Monoclonal Antibodies Targeting Distinct Epitopes Effectively Neutralizes Variants Found in Early Infection

Neutralizing antibody protection against HIV-1 may require broad and potent antibodies targeting multiple epitopes. We tested 7 monoclonal antibodies (MAbs) against 45 viruses of diverse subtypes from early infection. The CD4 binding site MAb NIH45-46W was most broad and potent (91% coverage; geometric mean 50% inhibitory concentration [IC(50)], 0.09 μg/ml). Combining NIH45-46W and a V3-specific MAb, PGT128, neutralized 96% of viruses, while PGT121, another V3-specific MAb, neutralized the remainder. Thus, 2 or 3 antibody specificities may prevent infection by most HIV-1 variants.     

Computational Prediction of Broadly Neutralizing HIV-1 Antibody Epitopes from Neutralization Activity Data

Broadly neutralizing monoclonal antibodies effective against the majority of circulating isolates of HIV-1 have been isolated from a small number of infected individuals. Definition of the conformational epitopes on the HIV spike to which these antibodies bind is of great value in defining targets for vaccine and drug design. Drawing on techniques from compressed sensing and information theory, we developed a computational methodology to predict key residues constituting the conformational epitopes on the viral spike from cross-clade neutralization activity data. Our approach does not require the availability of structural information for either the antibody or antigen. Predictions of the conformational epitopes of ten broadly neutralizing HIV-1 antibodies are shown to be in good agreement with new and existing experimental data. Our findings suggest that our approach offers a means to accelerate epitope identification for diverse pathogenic antigens.     

Rationally Targeted Mutations at the V1V2 Domain of the HIV-1 Envelope to Augment Virus Neutralization by Anti-V1V2 Monoclonal Antibodies

HIV-1 envelope glycoproteins (Env) are the only viral antigens present on the virus surface and serve as the key targets for virus-neutralizing antibodies. However, HIV-1 deploys multiple strategies to shield the vulnerable sites on its Env from neutralizing antibodies. The V1V2 domain located at the apex of the HIV-1 Env spike is known to encompass highly variable loops, but V1V2 also contains immunogenic conserved elements recognized by cross-reactive antibodies. This study evaluates human monoclonal antibodies (mAbs) against V2 epitopes which overlap with the conserved integrin α4β7-binding LDV/I motif, designated as the V2i (integrin) epitopes. We postulate that the V2i Abs have weak or no neutralizing activities because the V2i epitopes are often occluded from antibody recognition. To gain insights into the mechanisms of the V2i occlusion, we evaluated three elements at the distal end of the V1V2 domain shown in the structure of V2i epitope complexed with mAb 830A to be important for antibody recognition of the V2i epitope. Amino-acid substitutions at position 179 that restore the LDV/I motif had minimal effects on virus sensitivity to neutralization by most V2i mAbs. However, a charge change at position 153 in the V1 region significantly increased sensitivity of subtype C virus ZM109 to most V2i mAbs. Separately, a disulfide bond introduced to stabilize the hypervariable region of V2 loop also enhanced virus neutralization by some V2i mAbs, but the effects varied depending on the virus. These data demonstrate that multiple elements within the V1V2 domain act independently and in a virus-dependent fashion to govern the antibody recognition and accessibility of V2i epitopes, suggesting the need for multi-pronged strategies to counter the escape and the shielding mechanisms obstructing the V2i Abs from neutralizing HIV-1.     

The Broad Neutralizing Antibody Responses after HIV-1 Superinfection Are Not Dominated by Antibodies Directed to Epitopes Common in Single Infection

HIV-1 vaccines designed to date have failed to elicit neutralizing antibodies (Nabs) that are capable of protecting against globally diverse HIV-1 subtypes. One relevant setting to study the development of a strong, cross-reactive Nab response is HIV-1 superinfection (SI), defined as sequential infections from different source partners. SI has previously been shown to lead to a broader and more potent Nab response when compared to single infection, but it is unclear whether SI also impacts epitope specificity and if the epitopes targeted after SI differ from those targeted after single infection. Here the post-SI Nab responses were examined from 21 Kenyan women collectively exposed to subtypes A, C, and D and superinfected after a median time of ~1.07 years following initial infection. Plasma samples chosen for analysis were collected at a median time point ~2.72 years post-SI. Because previous studies of singly infected populations with broad and potent Nab responses have shown that the majority of their neutralizing activity can be mapped to 4 main epitopes on the HIV-1 Envelope, we focused on these targets, which include the CD4-binding site, a V1/V2 glycan, the N332 supersite in V3, and the membrane proximal external region of gp41. Using standard epitope mapping techniques that were applied to the previous cohorts, the present study demonstrates that SI did not induce a dominant Nab response to any one of these epitopes in the 21 women. Computational sera delineation analyses also suggested that 20 of the 21 superinfected women’s Nab responses could not be ascribed a single specificity with high confidence. These data are consistent with a model in which SI with diverse subtypes promotes the development of a broad polyclonal Nab response, and thus would provide support for vaccine designs using multivalent HIV immunogens to elicit a diverse repertoire of Nabs.     

Structural Basis of Potent and Broad HIV-1 Fusion Inhibitor CP32M

CP32M is a newly designed peptide fusion inhibitor possessing potent anti-HIV activity, especially against T20-resistant HIV-1 strains. In this study, we show that CP32M can efficiently inhibit a large panel of diverse HIV-1 variants, including subtype B', CRF07_BC, and CRF01_AE recombinants and naturally occurring or induced T20-resistant viruses. To elucidate its mechanism of action, we determined the crystal structure of CP32M complexed with its target sequence. Differing from its parental peptide, CP621-652, the (621)VEWNEMT(627) motif of CP32M folds into two α-helix turns at the N terminus of the pocket-binding domain, forming a novel layer in the six-helix bundle structure. Prominently, the residue Asn-624 of the (621)VEWNEMT(627) motif is engaged in the polar interaction with a hydrophilic ridge that borders the hydrophobic pocket on the N-terminal coiled coil. The original inhibitor design of CP32M provides several intra- and salt bridge/hydrogen bond interactions favoring the stability of the helical conformation of CP32M and its interactions with N-terminal heptad repeat (NHR) targets. We identified a novel salt bridge between Arg-557 on the NHR and Glu-648 of CP32M that is critical for the binding of CP32M and resistance against the inhibitor. Therefore, our data present important information for developing novel HIV-1 fusion inhibitors for clinical use.     

人乳头瘤病毒中和表位及抗体中和作用机制的研究进展

人乳头瘤病毒(Human papillomavirus,HPV)是一类无包膜的小DNA病毒,其衣壳蛋白由主要衣壳蛋白L1和次要衣壳蛋白L2组成,持续感染HPV将引起宫颈癌和尖锐湿疣等多种疾病。HPV衣壳蛋白L1和L2中分布着大量中和表位,并具有较强的免疫原性,HPV疫苗可诱导机体产生高滴度的中和抗体并阻碍病毒感染,进而预防宫颈癌等疾病的发生。分析阐述HPV衣壳蛋白中和表位及抗体的中和作用机理,有助于阐明HPV疫苗预防病毒感染的作用机制,为今后设计新一代保护范围更广的HPV疫苗奠定良好的基础。本文就HPV衣壳蛋白中和表位及抗体的中和作用机制进行综述。     

Antibodies to the Buried N Terminus of Rhinovirus VP4 Exhibit Cross-Serotypic Neutralization

Development of a vaccine for the common cold has been thwarted by the fact that there are more than 100 serotypes of human rhinovirus (HRV). We previously demonstrated that the HRV14 capsid is dynamic and transiently displays the buried N termini of viral protein 1 (VP1) and VP4. Here, further evidence for this “breathing” phenomenon is presented, using antibodies to several peptides representing the N terminus of VP4. The antibodies form stable complexes with intact HRV14 virions and neutralize infectivity. Since this region of VP4 is highly conserved among all of the rhinoviruses, antiviral activity by these anti-VP4 antibodies is cross-serotypic. The antibodies inhibit HRV16 infectivity in a temperature- and time-dependent manner consistent with the breathing behavior. Monoclonal and polyclonal antibodies raised against the 30-residue peptide do not react with peptides shorter than 24 residues, suggesting that these peptides are adopting three-dimensional conformations that are highly dependent upon the length of the peptide. Furthermore, there is evidence that the N termini of VP4 are interacting with each other upon extrusion from the capsid. A Ser5Cys mutation in VP4 yields an infectious virus that forms cysteine cross-links in VP4 when the virus is incubated at room temperature but not at 4°C. The fact that all of the VP4s are involved in this cross-linking process strongly suggests that VP4 forms specific oligomers upon extrusion. Together these results suggest that it may be possible to develop a pan-serotypic peptide vaccine to HRV, but its design will likely require details about the oligomeric structure of the exposed termini.Rhinoviruses are the major causative agents of the common cold and cost the United States economy approximately $40 billion per year (6). Therefore, it is of great interest to prevent or ameliorate the symptoms of the common cold. The rhinovirus genus is a member of the picornavirus family and is characterized by nonenveloped capsid with a diameter of ∼300 Å containing a single-stranded, plus-sense RNA genome (19). Other members of the picornavirus family include foot-and-mouth disease virus, poliovirus, encephalomyocarditis virus, and hepatitis A virus. The capsids exhibit pseudo T = 3 icosahedral symmetry and are composed of 60 copies of the four capsid proteins VP1, VP2, VP3, and VP4. VP1, VP2, and VP3 have an eight-stranded antiparallel beta-barrel motif structure and form the outer surface of the capsid, while VP4 lies at the interface between the capsid and the interior genomic RNA (22). VP4 is approximately 70 amino acids in length and is myristoylated at the N terminus (3, 14).Antibodies are the major line of defense against picornavirus infections. In the case of human rhinovirus 14 (HRV14), a number of studies have been performed to detail the antibody recognition and neutralization processes (25). While it had been long suggested that antibodies neutralize viral infectivity by inducing large conformational changes in the capsid, both cryo-transmission electron microscopy (cryo-TEM) (2, 28) and crystallographic analysis (27) clearly demonstrated that this was not the case. Further, it was shown that antibody recognition is more plastic than previously thought in that it is able to bind into the relatively narrow receptor-binding region of the canyon (27). These results suggested that the major in vivo role of antibodies is to bind to virion and work synergistically with other immune system components (26). This hypothesis has gained further support from studies of other pathogens (1) and implies that vaccines need only to elicit antibodies that bind to the authentic pathogen with high affinity.While these results simplified the goal of creating a synthetic vaccine by focusing on capsid recognition rather than possible antibody-induced conformational changes, developing synthetic vaccines against all 100 serotypes of HRV remains a daunting task. As shown in the structures of HRV14/antibody complexes, the antibodies make extensive contacts with the surface of the capsid that is not limited to a single antigenic loop (2, 27). Further evidence for this extensive contact is that antibodies to peptides corresponding to antigenic NIm loops fail to neutralize the virions (17, 29), and antibodies raised against intact capsids do not bind effectively to peptides corresponding to NIm-IA loop (T. J. Smith, unpublished results). One notable exception is the case of HRV2, where there is cross-reactivity between the NIm-II site of the virion and a synthetic peptide (30). Nevertheless, developing a repertoire of peptides representing the entire antigenic ensemble of HRVs is not only impractical but also unlikely to elicit neutralizing antibodies.All of the studies described above were performed with the antibodies that were raised against intact particles or to peptides representing epitopes that reside on the outer surface of the capsid. In the case of poliovirus, however, antibodies were raised against VP4 and the N termini of VP1 of poliovirus serotype I (15, 21). It was shown that these antibodies are capable of neutralizing the virion despite the fact that those portions of the capsid protein are buried in the interior of the capsid at the capsid-RNA interface (8). These results suggested that the poliovirus capsid was more dynamic than indicated by the crystal structure and that these termini are presented to the exterior of the virion in a temperature-dependent and reversible manner. While the role of capsid dynamics in the viral life cycle was not clear, it was suggested that the N termini of VP1 and VP4 might facilitate cell membrane attachment and subsequent entry of the virus into the host cell (3, 4).More recently, evidence for capsid dynamics has been found in other viruses as well. In the cases of swine vesicular disease virus (10) and coxsackievirus A9 (18), antibodies were raised against the whole virus in pigs and rabbits, respectively. These polyclonal antibodies demonstrated a strong reaction to the peptides corresponding to the N termini of VP1 and VP3 of swine vesicular disease virus and coxsackievirus A9, respectively. In a similar study, antibodies from the plasma of patients suffering from type I diabetes were found to target VP4 protein of coxsackievirus B3, again suggesting the exposure of VP4 peptide during coxsackievirus infection (23). These results imply that capsid “breathing” may be a phenomenon common to many proteinaceous capsids.Using a very different approach, the dynamic nature of HRV14 was analyzed using limited proteolysis and mass spectrometry (matrix-assisted laser desorption ionization [MALDI]) analyses (14). In these experiments, the virus was treated with both matrix-bound and soluble forms of trypsin for various periods of time, and the resulting proteolytic fragments were identified by MALDI. Surprisingly, the N termini of VP4 and VP1 were found to be the most proteolytically sensitive portions of the capsid in spite of being buried inside the viral capsid. As an additional control, the antiviral “WIN” compounds, which had been previously shown to stabilize the virions against thermal and acid denaturation, were added during digestion. While these WIN compounds did not affect the intrinsic proteolytic activity of trypsin, they nearly completely protected the VP1 and VP4 termini from proteolysis for an extended period. Together, these results suggested that HRV14 is transiently exposing these termini in a “breathing” process and that the empty hydrophobic drug-binding region apparently plays an important role in facilitating these dynamics.In this study we further examined HRV14 capsid dynamics by raising polyclonal antibodies against several peptides representing the N termini of VP1 and VP4. In these experiments, only the antibodies against the VP4 N terminus were found to successfully neutralize viral infectivity in vitro. Further, we demonstrate that the HRV14 VP4 antiserum cross-reacts with other serotypes of rhinovirus (HRV16, and HRV29), which is likely due to the high degree of conservation of VP4. Antibody neutralization closely parallels the MALDI analysis in that antibody neutralization and proteolysis are enhanced at 37°C in the case of HRV16 whereas the elevated temperatures are not required for either phenomenon in the cases of HRV14 and HRV29. Epitope mapping of the N-terminal 30 residues of VP4 suggests that it adopts a nonlinear conformation, and this is further substantiated by results showing that all of the copies of VP4 in the Ser5Cys HRV14 mutant at room temperature form cysteine cross-linked dimers. This cysteine cross-link does not form at 4°C, suggesting that capsid breathing is essential for VP4 exposure and interactions. Since VP4 dimerization does not affect viral infectivity, it seems likely that VP4 extrusion is a normal part of the cell attachment and entry process of rhinovirus. Together, these results suggest that VP4 might be useful as a pan-serotypic rhinovirus vaccine, but it seems likely that better understanding of the VP4 oligomeric structure will be necessary for further optimization.     

VSV-Displayed HIV-1 Envelope Identifies Broadly Neutralizing Antibodies Class-Switched to IgG and IgA

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Networks of HIV-1 Envelope Glycans Maintain Antibody Epitopes in the Face of Glycan Additions and Deletions

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Enhancement and Neutralization of Feline Infectious Peritonitis Virus Infection in Feline Macrophages by Neutralizing Monoclonal Antibodies Recognizing Different Epitopes

The interaction between the enhancing and neutralizing activities of three monoclonal antibodies (MAbs) (5-6-2, 6-4-2 and 7-4-1) to the spike protein of feline infectious peritonitis virus (FIPV) strain 79-1146 was determined using feline macrophages. At a high MAb concentration, all of the three MAbs completely inhibited the FIPV infection at 37 C. However, two of them (6-4-2 and 7-4-1) enhanced FIPV infection when either the MAb concentration or reaction temperature was lowered. These MAbs also exerted an immediate infectivity-enhancing activity for up to 10 min of reaction and by 20 min, neutralizing activities were observed. Only MAb 5-6-2 consistently showed neutralizing activity regardless of the reaction conditions. Competition with sera from cats experimentally infected with FIPV strain 79-1146 or feline enteric coronavirus strain 79-1683 showed that the two epitopes recognized by MAb 5-6-2 and MAb 6-4-2, respectively, are also recognized by the natural host.