The major outer membrane protein of Chlamydia trachomatis: critical binding site and conformation determine the specificity of antibody binding to viable chlamydiae

The major outer membrane protein (MOMP) is the prime candidate for the development of a chlamydial vaccine. Antibodies to the subspecies-specific epitope neutralize chlamydial infection. Monoclonal antibodies (MAbs) to this epitope were prepared either by immunization with whole chlamydiae or with a 16 amino acid synthetic peptide. The critical binding site on the subspecies epitope for these MAbs was determined to single amino acid resolution using several hundred solid-phase peptides. A frame shift of just one amino acid in critical binding site completely prevented antibody binding to viable chlamydiae. A single MAb to whole organisms was capable of spanning both the surface-exposed, conformation-dependent, subspecies epitope and a buried, conformation-independent species epitope some 10 A distant. Immunization with peptide generated an MAb with reduced binding constraints which permitted the antibody to bind with broadened species-specificity at the subspecies binding site. The results show for the first time the importance of both critical binding site and conformation at the subspecies epitope. We suggest that the conformational flexibility of short, epitopic peptide vaccines may in some cases be advantageous, giving rise to extended specificity not attained with the natural protein.     

Analysis and simulation of a neutralizing epitope of transmissible gastroenteritis virus.

The amino acid sequences recognized by monoclonal antibodies (MAbs) specific for the antigenic site IV of the spike protein S of transmissible gastroenteritis virus were analyzed by PEPSCAN. All MAbs of group IV recognized peptides from the S region consisting of residues 378 to 390. In addition, the neutralizing MAbs (subgroup IV-A) also bound to peptides from the region consisting of residues 1173 to 1184 and to several other peptides with a related amino acid composition. The contribution of the individual residues of both sequences to the binding of a MAb was determined by varying the length of the peptide and by a consecutive deletion or replacement of parental residues by the 19 other amino acids. The sequence consisting of residues 326 to 558, tested as part of a cro-beta-galactosidase hybrid protein, was antigenic, but the sequence consisting of residues 1150 to 1239 was not. Furthermore, antibodies raised in rabbits against the peptide SDSSFFSYGEIPFGN (residues 377 to 391), but not those raised against the peptide VRASRQLAKDKVNEC (residues 1171 to 1185), recognized the virus and had neutralizing activity. We infer that the epitope of the neutralizing MAbs is composite and consists of the linear sequence SFFSYGEI (residues 380 to 387) with contributions of A, D, K, N, Q, or V residues from other parts of the S molecule. The complex epitope was simulated by synthesizing peptides in which the sequences consisting of residues 380 to 387 and 1176 to 1184 were combined. MAbs of subgroup IV-A recognized the combination peptides two to six times better than the individual sequences. These results may offer prospects for the development of an experimental vaccine.     

Topology of Na+,K+-ATPase. Identification of the extra- and intracellular hydrophilic loops of the catalytic subunit by specific antibodies

To study the topology of Na+,K+-ATPase monoclonal antibodies (MAbs) specific for membrane-bound enzyme were produced. Using immunofluorescence staining of viable cells or smears of a pig kidney embryonic (PKE) cell line, two groups of MAbs were selected, namely those binding to extra- or intracellular portions of the alpha-subunit. The extracellular location of peptide loop 804-841 linking the Vth and VIth intramembrane hydrophobic segments was proved using MAb VG2. Another MAb, IIC9, interacting with PKE cells only after membrane perforation (4% formaldehyde and 0.1% Tween-20), was shown to bind to the hydrophilic loop 868-945. The antigenic determinants recognized by MAb IIC9 and VG2 are located in peptides 887-904 and 810-825, respectively. The C-terminus of the alpha-subunit molecule was positioned on the outer side of the cytoplasmic membrane utilizing affinity-purified antibodies to the synthetic peptide corresponding to fragment 999-1008.     

Characterization of murine polyclonal antisera and monoclonal antibodies generated against intact and denatured human papillomavirus type 1 virions.

Human papillomavirus type 1 (HPV1) virions, both as intact virion particles (IVP) and as detergent-denatured virions (DDV), were used to prepare polyclonal antisera and monoclonal antibodies (MAbs) in BALB/c mice. Anti-IVP antiserum contained type-specific HPV1 L2-reactive antibodies and no detectable HPV1 L1-reactive antibodies. Anti-IVP MAbs recognized a linear epitope between L2 amino acids 102 and 108 (PIDVVDP). Anti-DDV antiserum contained type-specific HPV1 L1-reactive and HPV1 L2-reactive antibodies. An anti-DDV MAb recognized a linear epitope between L1 amino acids 127 and 133 (AENPTNY). HPV1a L1- and L2-encoded polypeptides expressed in Saccharomyces cerevisiae and by in vitro translation were equivalent in size to the major and minor virion capsid proteins, respectively.     

Recognition by human monoclonal antibodies of free and complexed peptides representing the prefusogenic and fusogenic forms of human immunodeficiency virus type 1 gp41

Human immunodeficiency virus type 1 (HIV-1) entry into target cells appears to be triggered when two heptad repeat regions in the ectodomain of gp41 associate, converting the prefusogenic form of gp41 to a fusogenic form. Peptides from these two heptad repeat regions, designated N51 and C43, form a coiled coil consisting of an alpha-helical trimer of heterodimers which approximates the core of the fusogenic form of gp41. To understand the antigenic structures of gp41 in these two configurations, and to examine the specificity of anti-gp41 antibodies produced by HIV-1-infected individuals, human anti-gp41 monoclonal antibodies (MAbs) were tested for their reactivity against N51, C43, and the complex formed by these peptides. Of 11 MAbs, 7 reacted with the complex but with neither of the parent peptides. These MAbs reacted optimally with the N51-C43 complex prepared at a 1:1 ratio and appeared to recognize the fusogenic form of gp41 in which the two heptad repeat regions are associated to form the coiled coil. The existence of antibodies from HIV-infected humans that exclusively recognize the N51-C43 complex constitutes the first proof that the coiled-coil conformation of gp41 exists in vivo and is immunogenic. Two of the 11 MAbs were specific for the hydrophilic loop region of gp41 and failed to react with either peptide alone or with the peptide complex, while the remaining 2 MAbs reacted with peptide C43. One of these two latter MAbs, 98-6, also reacted well with the equimolar N51-C43 complex, while reactivity with C43 by the other MAb, 2F5, was inhibited by even small amounts of N51, suggesting that the interaction of these peptides occludes or disrupts the epitope recognized by MAb 2F5. MAbs 98-6 and 2F5 are also unusual among the MAbs tested in their ability to neutralize multiple primary HIV isolates, although 2F5 displays more broad and potent activity. The data suggest that anti-gp41 neutralizing activity is associated with specificity for a region in C43 which participates in complex formation with N51.     

Sites in Myelin Basic Protein that React with Monoclonal Antibodies

The epitopes (antigenic sites) for seven monoclonal antibodies (MAbs) evoked in rats or mice by guinea pig or monkey myelin basic protein (BP) have been located in four different sequences of the BPs extracted from various species. Six of the MAbs were evoked by guinea pig BP. (1) One epitope, possibly a pair, is included within residues 1-14 of all BPs tested and reacts with two rat IgG MAbs. (2) A definite pair of overlapping epitopes includes the central Phe91-Phe92 sequence. One epitope is contained entirely within sequence 90-99 and reacts with a rat IgG MAb. The substitution of Ser in chicken BP for Thr97 destroys this epitope. The other epitope appears to include residues on the amino side of Phe44 and even of His32 and suggests some tertiary structure in BP. This epitope reacts with a mouse IgM MAb that does not recognize the chicken substitution. (3) The third epitope lies within residues 114-121, specifically including Trp118, and reacts with a rat IgG MAb. A cross-reacting epitope probably includes residues 44-45 in certain species (guinea pig and bovine but not rabbit). (4) Another pair of epitopes is located within residues 131-140 but is severely species-restricted. This region in guinea pig BP evoked a species-specific mouse IgM MAb. The same region in monkey BP evoked the seventh MAb, a mouse IgG, which reacts with human, chimpanzee, monkey, bovine, and rat-18.5 kDa BPs and to a lesser extent rabbit BP but not with guinea pig, pig, or chicken BPs. Some tertiary structure in guinea pig BP is also suggested by the reactivities with the IgM MAb. All of the MAbs react with myelin in histologic preparations, but the optimum method of preparation of the tissue varies with each.     

Production and Characterization of Monoclonal Antibodies Specific for a Conserved Epitope within Hepatitis C Virus Hypervariable Region 1

Frequent mutations in hypervariable region 1 (HVR1) of the main envelope protein of hepatitis C virus (HCV) is a major mechanism of persistence by escaping the host immune recognition. HVR1 contains an epitope eliciting neutralizing antibodies. This study was aimed to prepare broadly cross-reacting, high-affinity, monoclonal antibodies (MAb) to the HVR1 C terminus of HCV with potential therapeutic neutralizing capacity. A conserved amino residue group of glycine (G) at position 23 and glutamic acid (Q) at position 26 in HVR1 was confirmed as a key epitope against which two MAbs were selected and characterized. MAbs 2P24 and 15H4 were immunoglobulin G1 kappa chain [IgG1(kappa)], cross-reacted with 32 and 30 of 39 random C-terminal HVR1 peptides, respectively, and did not react with other HCV peptides. The V(H) of 2P24 and 15H4 heavy chains originated from Igh germ line v gene family 1 and 8, respectively. In contrast, the V(L) kappa sequences were highly homologous. The affinity (K(d)) of 2P24 and 15H4 (10(-9) or 10(-8) M with two immunizing peptides and 10(-8) M with two nonimmunizing HVR1 peptides) paralleled the reactivity obtained with peptide enzyme immunoassay. MAbs 2P24 and 15H4 captured 25 of 31 (81%) HCV in unselected patients' plasmas. These antibodies also blocked HCV binding to Molt-4 cells in a dose-dependent fashion. The data presented suggest that broadly cross-reactive MAbs to a conserved epitope within HCV HVR1 can be produced. Clinical application for passive immunization in HCV-related chronic liver disease and after liver transplantation is considered.     

Multiple epitopes in a dodecapeptide of myelin basic protein determined by monoclonal antibodies

Three custom synthesized myelin basic protein (MBP) peptides, bovine peptide 79-88, human peptide 80-89, and human peptide 82-91, were used to produce four murine monoclonal antibodies (MAb) that were selected on the basis of reaction in a solid phase radioimmunoassay (SRIA) with human MBP. The MAb were compared with respect to antigen specificity against intact MBP and 10 overlapping MBP peptides. One MAb recognized an epitope near the amino-terminus of bovine MBP peptide 79-88. A second MAb was directed towards an epitope that is more reactive in human MBP peptide 45-89 than in intact MBP, but is not recognized in any of the small MBP peptides examined. The third MAb detected an epitope near the middle of human MBP peptide 80-89, whereas the fourth MAb reacted with the carboxyl-terminal portion of human MBP peptide 82-91. Epitopes recognized in SRIA were sometimes not detected by the same MAb in a fluid phase double antibody radioimmunoassay. These results demonstrate the multiplicity of potential epitopes in a dodecapeptide of MBP and do not support the concept of a single, dominant epitope in the region of MBP peptide 80-89.     

Rapid and precise epitope mapping of monoclonal antibodies against Plasmodium falciparum AMA1 by combined phage display of fragments and random peptides.

We describe an approach for the rapid mapping of epitopes within a malaria antigen using a combination of phage display techniques. Phage display of antigen fragments identifies the location of the epitopes, then random peptide libraries displayed on phage are employed to identify accurately amino acids involved in the epitope. Finally, phage display of mutant fragments confirms the role of each residue in the epitope. This approach was applied to the apical membrane antigen-1 (AMA1), which is a leading candidate for inclusion in a vaccine directed against the asexual blood stages of Plasmodium falciparum. As part of the effort both to understand the function of AMA1 in the parasite life cycle and to define the specificity of protective immune responses, a panel of monoclonal antibodies (MAbs) was generated to obtain binding reagents to the various domains within the molecule. There is a pressing need to determine rapidly the regions recognized by these antibodies and the structural requirements required within AMA1 for high affinity binding of the MAbs. Using phage displaying random AMA1 fragments, it was shown that MAb5G8 recognizes a short linear epitope within the pro-domain of AMA1 whereas the epitope recognized by MAb 1F9 is reduction sensitive and resides within a disulphide-bonded 57 amino acid sub-domain of domain-1. Phage displaying random peptide libraries and mutant AMA1 fragments were employed for fine mapping of the MAb5G8 core epitope to a three-residue sequence in the AMA1 prodomain.     

Identification of a linear peptide recognized by monoclonal antibody 2D7 capable of generating CCR5-specific antibodies with human immunodeficiency virus-neutralizing activity

CCR5 is the major coreceptor for human immunodeficiency virus (HIV) infection. The murine monoclonal antibody (MAb) 2D7, which recognizes a conformation-dependent epitope in the second extracellular loop of CCR5, is one of the most potent inhibitors of R5 virus cell entry. However, attempts to humanize 2D7 for in vivo human use have been unsuccessful so far. A filamentous phage library expressing random peptides was used to identify a peptide mimitope that is recognized by MAb 2D7. A synthetic peptide containing this sequence (2D7-2SK) bound to MAb 2D7 with high affinity and reversed its HIV type 1 (HIV-1) fusion inhibitory activity. The peptide contains sequence homologies to two distal regions of the second extracellular loop of human CCR5, both of which are required for MAb 2D7 binding. Rabbit anti-2D7-mimitope antibodies competed with MAb 2D7 for binding to the 2D7-2SK peptide in Biacore biosensor testing. Importantly, the rabbit anti-2D7-2SK antibodies bound to CCR5 on cells and specifically inhibited R5 (but not X4) envelope-mediated syncytium formation. These antibodies also neutralized infection of human peripheral blood mononuclear cells with R5 HIV isolates comparably to MAb 2D7. In summary, we have identified a novel peptide that closely mimics the MAb 2D7 epitope on CCR5. This peptide could be included as a potential vaccine candidate or to isolate 2D7-like human antibodies as entry inhibitors for R5 viruses.     

Neutralizing monoclonal antibodies against human immunodeficiency virus type 2 gp120.

Monoclonal antibodies (MAbs) were obtained by immunizing mice with synthetic peptides corresponding to the third variable (V3) or the third conserved (C3) domain of the external envelope protein (gp120) of human immunodeficiency virus type 2 (HIV-2ROD). One MAb, designated B2C, which was raised against V3 peptide NKI26, bound to the surface of HIV-2-infected cells but not to their uninfected counterparts. B2C was capable of neutralizing cell-free and cell-associated virus infection in an isolate-specific fashion. The antibody-binding epitope was mapped to a 6-amino-acid peptide in the V3 variable domain which had the core sequence His-Tyr-Gln. Two MAbs, 2H1B and 2F19C, which were raised against the C3 peptide TND27 reacted with gp120 of HIV-2ROD in a Western immunoblot assay. The C3 epitopes recognized by these two MAbs appeared inaccessible because of their poor reactivity in a surface immunofluorescence assay. Although partial inhibition of syncytium formation was observed in the presence of the anti-C3 MAbs, their neutralizing activity appeared weak. Finally, the effects of these MAbs against CD4-gp120 binding were assessed. Partial inhibition of CD4-gp120 binding was observed in the presence of high concentrations of B2C. On the other hand, no inhibition of CD4-gp120 binding was observed in the presence of anti-C3 MAbs. Since complete neutralization could be achieved at a concentration corresponding to that of partial binding inhibition by B2C, some different mechanisms may be involved in the B2C-mediated neutralization. These results, taken together, indicated that analogous to the function of the V3 region of HIV-1, the V3 region of HIV-2ROD contained at least a type-specific fusion-inhibiting neutralizing epitope. In this respect, the V3 sequence of HIV-2 may be a useful target in an animal model for HIV vaccine development.     

The polypeptide biophysics of proline/alanine‐rich sequences (PAS): Recombinant biopolymers with PEG‐like properties

PAS polypeptides comprise long repetitive sequences of the small L‐amino acids proline, alanine and/or serine that were developed to expand the hydrodynamic volume of conjugated pharmaceuticals and prolong their plasma half‐life by retarding kidney filtration. Here, we have characterized the polymer properties both of the free polypeptides and in fusion with the biopharmaceutical IL‐1Ra. Data from size exclusion chromatography, dynamic light scattering, circular dichroism spectroscopy and quantification of hydrodynamic and polar properties demonstrate that the biosynthetic PAS polypeptides exhibit random coil behavior in aqueous solution astonishingly similar to the chemical polymer poly‐ethylene glycol (PEG). The solvent‐exposed PAS peptide groups, in the absence of secondary structure, account for strong hydrophilicity, with negligible contribution by the Ser side chains. Notably, PAS polypeptides exceed PEG of comparable molecular mass in hydrophilicity and hydrodynamic volume while exhibiting lower viscosity. Their uniform monodisperse composition as genetically encoded polymers and their biological nature, offering biodegradability, render PAS polypeptides a promising PEG mimetic for biopharmaceutical applications.     

Identification and characterization of an immunogenic hybrid epitope formed by both HIV gp120 and human CD4 proteins

Certain antibodies from HIV-infected humans bind conserved transition state (CD4 induced [CD4i]) domains on the HIV envelope glycoprotein, gp120, and demonstrate extreme dependence on the formation of a gp120-human CD4 receptor complex. The epitopes recognized by these antibodies remain undefined although recent crystallographic studies of the anti-CD4i monoclonal antibody (MAb) 21c suggest that contacts with CD4 as well as gp120 might occur. Here, we explore the possibility of hybrid epitopes that demand the collaboration of both gp120 and CD4 residues to enable antibody reactivity. Analyses with a panel of human anti-CD4i MAbs and gp120-CD4 antigens with specific mutations in predicted binding domains revealed one putative hybrid epitope, defined by the human anti-CD4i MAb 19e. In virological and immunological tests, MAb 19e did not bind native or constrained gp120 except in the presence of CD4. This contrasted with other anti-CD4i MAbs, including MAb 21c, which bound unliganded, full-length gp120 held in a constrained conformation. Conversely, MAb 19e exhibited no specific reactivity with free human CD4. Computational modeling of MAb 19e interactions with gp120-CD4 complexes suggested a distinct binding profile involving antibody heavy chain interactions with CD4 and light chain interactions with gp120. In accordance, targeted mutations in CD4 based on this model specifically reduced MAb 19e interactions with stable gp120-CD4 complexes that retained reactivity with other anti-CD4i MAbs. These data represent a rare instance of an antibody response that is specific to a pathogen-host cell protein interaction and underscore the diversity of immunogenic CD4i epitope structures that exist during natural infection.     

Common Neutralization Epitope in Minor Capsid Protein L2 of Human Papillomavirus Types 16 and 6

Studies of virus neutralization by antibody are a prerequisite for development of a prophylactic vaccine strategy against human papillomaviruses (HPVs). Using HPV16 and -6 pseudovirions capable of inducing beta-galactosidase in infected monkey COS-1 cells, we examined the neutralizing activity of mouse monoclonal antibodies (MAbs) that recognize surface epitopes in HPV16 minor capsid protein L2. Two MAbs binding to a synthetic peptide with the HPV16 L2 sequence of amino acids (aa) 108 to 120 were found to inhibit pseudoinfections with HPV16 as well as HPV6. Antisera raised by immunizing BALB/c mice with the synthetic peptide had a cross-neutralizing activity similar to that of the MAb. The data indicate that HPV16 and -6 have a common cross-neutralization epitope (located within aa 108 to 120 of L2 in HPV16), suggesting that this epitope may be shared by other genital HPVs.     

Establishment of monoclonal antibodies against human acidic fibroblast growth factor.

Four kinds of hybridomas secreting monoclonal antibodies (MAbs) against human acidic fibroblast growth factor (haFGF) were established using recombinant haFGF as an immunogen. The recognition sites of four MAbs designated AF1-52, 81, 114 and 1C10 for the haFGF molecule were examined by binding studies with synthetic polypeptides and with amino-terminal truncated forms of haFGF. These experiments suggested that AF1-52, 114, and 1C10 MAbs recognize epitopes within the 1-5, 44-132 and 6-43 amino acid sequences, respectively. However, the epitope recognized by the AF1-81 MAb could not be determined. The sandwich EIA method constructed with these MAbs was sensitive to 1.5 pg/well of haFGF and had no cross-reactivity with human basic FGF, bovine aFGF or the hst-1 gene product.     

Immunochemical Properties of Recombinant Polypeptides Mimicking Domains I and II of West Nile Virus Glycoprotein E

Complementary DNA fragments (nucleotides 935–1475, 1091–1310, and 935–1193) encoding the N-terminal portion of glycoprotein E of West Nile virus (WNV), strain LEIV-Vlg99-27889-human, were cloned. Recombinant polypeptides of glycoprotein E (E_1–180, E_53–126, and E_1–86) of the WNV having amino acid sequences corresponding to the cloned cDNA fragments and mimicking the main functional regions of domains I and II of surface glycoprotein E were purified by affinity chromatography. According to ELISA and Western blotting, 12 types of monoclonal antibodies (MAbs) raised in our laboratory against recombinant polypeptide E_1–180 recognized the WNV glycoprotein E. This is indicative of similarity between the antigenic structures of the short recombinant polypeptides and corresponding regions of the glycoprotein. Analysis of interactions of the MAbs with short recombinant polypeptides and protein E of tick-borne encephalitis virus revealed at least six epitopes within domains I and II of the WNV protein E. We found at least seven MAb types against the region between amino acid residues (aa) 86 and 126 of domain II, which contains the peptide responsible for fusion of the virus and cell membranes (residues 98–110). The epitope for antireceptor MAbs 10H10 was mapped within the 53–86 aa region of domain II of WNV protein E, which is evidence for the spatial proximity of the fusion peptide and the coreceptor of protein E (residues 53–86) for cellular laminin-binding protein (LBP). The X-ray pattern of protein E suggests that the bc loop (residues 73–89) of domain II interacts with LBP and, together with the cd loop (fusion peptide), determines the initial stages of flavivirus penetration into the cell.     

Molecular and biological characterization of human monoclonal antibodies binding to the spike and nucleocapsid proteins of severe acute respiratory syndrome coronavirus

Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein. One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease.     

Location of the Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin

We analyzed the binding site on Cry1Aa toxin for the Cry1Aa receptor in Bombyx mori, 115-kDa aminopeptidase N type 1 (BmAPN1) (K. Nakanishi, K. Yaoi, Y. Nagino, H. Hara, M. Kitami, S. Atsumi, N. Miura, and R. Sato, FEBS Lett. 519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking molecule N-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of 508STLRVN513 and 582VFTLSAHV589. These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding.     

The catalytic subunit of the DNA polymerase of herpes simplex virus type 1 interacts specifically with the C terminus of the UL8 component of the viral helicase-primase complex.

The herpes simplex virus type 1 (HSV-1) UL8 DNA replication protein is a component of a trimeric helicase-primase complex. Sixteen UL8-specific monoclonal antibodies (MAbs) were isolated and characterized. In initial immunoprecipitation experiments, one of these, MAb 804, was shown to coprecipitate POL, the catalytic subunit of the HSV-1 DNA polymerase, from extracts of insect cells infected with recombinant baculoviruses expressing the POL and UL8 proteins. Coprecipitation of POL was dependent on the presence of UL8 protein. Rapid enzyme-linked immunosorbent assays (ELISAs), in which one protein was bound to microtiter wells and binding of the other protein was detected with a UL8- or POL-specific MAb, were developed to investigate further the interaction between the two proteins. When tested in the ELISAs, five of the UL8-specific MAbs consistently inhibited the interaction, raising the possibility that these antibodies act by binding to epitopes at or near a site(s) on UL8 involved in its interaction with POL. The epitopes recognized by four of the inhibitory MAbs were approximately located by using a series of truncated UL8 proteins expressed in mammalian cells. Three of these MAbs recognized an epitope near the C terminus of UL8, which was subjected to fine mapping with a series of overlapping peptides. The C-terminal peptides were then tested in the ELISA for their ability to inhibit the POL-UL8 interaction: the most potent exhibited a 50% inhibitory concentration of approximately 5 microM. Our findings suggest that the UL8 protein may be involved in recruiting HSV-1 DNA polymerase into the viral DNA replication complex and also identify a potential new target for antiviral therapy.