Identification of new epitopes recognized by human monoclonal antibodies with neutralizing and antibody-dependent cellular cytotoxicity activities specific for human T cell leukemia virus type 1.

We have generated a number of EBV-transformed B cell lines producing human mAb against human T cell leukemia virus type 1 (HTLV-1) from the peripheral blood B lymphocytes obtained from patients with HTLV-1-associated myelopathy/tropical spastic paraparesis. Various synthetic peptides corresponding to antigenic regions of HTLV-1 gag and env proteins were used for the screening of antibodies in ELISA. In our study, four IgG mAb to the gag p19 amino acids 100 to 130, and 5 IgG mAb to the env p46 amino acids 175 to 199 were characterized. An immunofluorescence assay showed that all of these mAb specifically bound to the surface of HTLV-1-bearing cell lines. Among these mAb, one anti-gp46 mAb, designated KE36-11, neutralized the infectivity of HTLV-1 as determined by both the inhibition of HTLV-1-induced syncytium formation and transformation assays in vitro. An antibody-binding assay using overlapping oligopeptides revealed that KE36-11 recognized a new epitope locating between the gp46 amino acid sequence 187-193 (Ala-Pro-Pro-Leu-Leu-Pro-His). Another anti-gp46 mAb, designated KE36-7, showed antibody-dependent cellular cytotoxicity against HTLV-1-bearing cell line. KE36-7 bound strongly to the 10-mer peptide-gp46 187-196, and weakly to peptides containing the gp46 amino acid sequence 191-196 (Leu-Pro-His-Ser-Asn-Leu). These two epitopes, which are associated with HTLV-1 neutralization and antibody-dependent cellular cytotoxicity, are thus the first epitopes identified in human HTLV-1 infection. It is possible that passive immunization of humans with these two human mAb are effective on the protection of HTLV-1 infection in vivo.     

Identification of a linear epitope of interferon-alpha2b recognized by neutralizing monoclonal antibodies.

Four monoclonal antibodies (mAbs) directed against the recombinant human interferon-alpha2b (IFN-alpha2b) were used as probes to study the interaction of the IFN molecule to its receptors. The [125I]IFN-alpha2b binding to immobilized mAbs was completely inhibited by IFN-alpha2b and IFN-alpha2a but neither IFNbeta nor IFNgamma showed any effect. Gel-filtration HPLC of the immune complexes formed by incubating [125I]IFN-alpha2b with paired mAbs revealed the lack of simultaneous binding of two different antibodies to the tracer, suggesting that all mAbs recognize the same IFN antigenic domain. Furthermore, the mAbs were also able to neutralize the IFN-alpha2b anti-viral and anti-proliferative activities as well as [125I]IFN-alpha2b binding to WISH cell-membranes. As [125I]mAbs did not recognize IFN exposed epitopes in the IFN:receptor complexes, mAb induction of a conformational change in the IFN binding domain impairing its binding to receptors was considered unlikely. In order to identify the IFN region recognized by mAbs, IFN-alpha2b was digested with different proteolytic enzymes. Immunoreactivity of the resulting peptides was examined by Western blot and their sequences were established by Edman degradation after blotting to poly(vinylidene difluoride) membranes. Data obtained indicated that the smallest immunoreactive region recognized by mAbs consisted of residues 107-132 or 107-146. As this zone includes the sequence 123-140, which has been involved in the binding to receptors, and our mAbs did not show an allosteric behaviour, it is concluded that they are directed to overlapping epitopes located close to or even included in the IFN binding domain.     

Antigenic and functional organization of human parainfluenza virus type 3 fusion glycoprotein.

Twenty-six monoclonal antibodies (MAbs) (14 neutralizing and 12 nonneutralizing) were used to examine the antigenic structure, biological properties, and natural variation of the fusion (F) glycoprotein of human type 3 parainfluenza virus (PIV3). Analysis of laboratory-selected antigenic variants and of PIV3 clinical isolates indicated that the panel of MAbs recognizes at least 20 epitopes, 14 of which participate in neutralization. Competitive binding assays indicated that the 14 neutralization epitopes are organized into three nonoverlapping antigenic sites (A, B, and C) and one bridge site (AB) and that the 6 nonneutralization epitopes form four sites (D, E, F, and G). Most of the neutralizing MAbs were involved in nonreciprocal competitive binding reactions, suggesting that they induce conformational changes in other neutralization epitopes. Fusion-inhibition and complemented-enhanced neutralization assays indicated that antigenic sites AB, B, and C may correspond to functional domains of the F molecule. Our results indicated that antibody binding alone is not sufficient for virus neutralization and that many anti-F MAbs neutralize by mechanisms not involving fusion-inhibition. The degree of antigenic variation in the F epitopes of clinical strains was examined by binding and neutralization tests. It appears that PIV3 frequently develops mutations that produce F epitopes which efficiently bind antibodies, but are completely resistant to neutralization by these antibodies.     

Identification by phage display and characterization of two neutralizing chimpanzee monoclonal antibodies to the hepatitis E virus capsid protein

Two monoclonal antibodies (MAbs) against the ORF2 protein of the SAR-55 strain of hepatitis E virus (HEV) were isolated by phage display from a cDNA library of chimpanzee (Pan troglodytes) gamma1/kappa antibody genes. Both MAbs, HEV#4 and HEV#31, bound to reduced, denatured open reading frame 2 (ORF2) protein in a Western blot, suggesting that they recognize linear epitopes. The affinities (equilibrium dissociation constants, K(d)) for the SAR-55 ORF2 protein were 1.7 nM for HEV#4 and 5.4 nM for HEV#31. The two MAbs also reacted in an enzyme-linked immunosorbent assay with recombinant ORF2 protein from a heterologous HEV, the Meng strain. Each MAb blocked the subsequent binding of the other MAb to homologous ORF2 protein in indirect competition assays, suggesting that they recognize the same or overlapping epitopes. Radioimmunoprecipitation assays suggested that at least part of the linear epitope(s) recognized by the two MAbs is located between amino acids 578 and 607. MAbs were mixed with homologous HEV in vitro and then inoculated into rhesus monkeys (Macaca mulatta) to determine their neutralizing ability. Whereas all control animals developed hepatitis (elevated liver enzyme levels in serum) and seroconverted to HEV, those receiving an inoculum incubated with either HEV#4 or HEV#31 were not infected. Therefore, each MAb neutralized the SAR-55 strain of HEV in vitro.     

Effects of hemagglutinin-neuraminidase protein mutations on cell-cell fusion mediated by human parainfluenza type 2 virus

The monoclonal antibody M1-1A, specific for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 2 virus (HPIV2), blocks virus-induced cell-cell fusion without affecting the hemagglutinating and neuraminidase activities. F13 is a neutralization escape variant selected with M1-1A and contains amino acid mutations N83Y and M186I in the HN protein, with no mutation in the fusion protein. Intriguingly, F13 exhibits reduced ability to induce cell-cell fusion despite its multistep replication. To investigate the potential role of HPIV2 HN protein in the regulation of cell-cell fusion, we introduced these mutations individually or in combination to the HN protein in the context of recombinant HPIV2. Following infection at a low multiplicity, Vero cells infected with the mutant virus H-83/186, which carried both the N83Y and M186I mutations, remained as nonfused single cells at least for 24 h, whereas most of the cells infected with wild-type virus mediated prominent cell-cell fusion within 24 h. On the other hand, the cells infected with the mutant virus, carrying either the H-83 or H-186 mutation, mediated cell-cell fusion but less efficiently than those infected with wild-type virus. Irrespective of the ability to cause cell-cell fusion, however, every virus could infect all the cells in the culture within 48 h after the initial infection. These results indicated that both the N83Y and M186I mutations in the HN protein are involved in the regulation of cell-cell fusion. Notably, the limited cell-cell fusion by H-83/186 virus was greatly promoted by lysophosphatidic acid, a stimulator of the Ras and Rho family GTPases.     

Characterization of a discontinuous human immunodeficiency virus type 1 gp120 epitope recognized by a broadly reactive neutralizing human monoclonal antibody.

While one hypervariable, linear neutralizing determinant on the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein has been well characterized, little is known about the conserved, discontinuous gp120 epitopes recognized by neutralizing antibodies in infected individuals. Here, the epitope recognized by a broadly reactive neutralizing monoclonal antibody (F105) derived from an HIV-1-infected patient was characterized by examining the effects of changes in conserved gp120 amino acids on antibody reactivity. The F105 epitope was disrupted by changes in gp120 amino acids 256 and 257, 368 to 370, 421, and 470 to 484, which is consistent with the discontinuous nature of the epitope. Three of these regions are proximal to those previously shown to be important for CD4 binding, which is consistent with the ability of the F105 antibody to block gp120-CD4 interaction. Since F105 recognition was more sensitive to amino acid changes in each of the four identified gp120 regions than was envelope glycoprotein function, replication-competent mutant viruses that escaped neutralization by the F105 antibody were identified. These studies identify a conserved, functional HIV-1 gp120 epitope that is immunogenic in man and may serve as a target for therapeutic or prophylactic intervention.     

Isolation and characterization of neutralizing monoclonal antibodies to vaccinia virus.

Cells producing neutralizing monoclonal antibodies (mAbs) to UV-inactivated vaccinia virus strain WR were derived by fusion of hyperimmunized mouse spleen cells with mouse myeloma cells. Three mAbs that reacted strongly with purified virus envelopes as determined by enzyme-linked immunosorbent assay were studied. The three mAbs recognized a 14,000-molecular-weight (14K) envelope protein of vaccinia virus and were shown to be immunoglobulin G2b (mAbC3 and mAbB11) and immunoglobulin M (mAbF11). By using ascites, one of the antibodies, mAbC3, neutralized (50%) virus infectivity with a titer of about 10(-4), whereas the others exhibited lower neutralization titers of 10(-2) to 10(-3). The binding of the mAbs to vaccinia virus did not alter virus attachment to cells. However, virus uncoating was extensively blocked by mAbC3, whereas mAbB11 and mAbF11 had little or no effect. The three mAbs recognized a similar 14K protein in cowpox, rabbitpox, and vaccinia Elstree strains, indicating a high degree of protein conservation among orthopoxviruses. Based on the binding of mAbs to V-8 protease cleavage products of the 14K protein, the extent of protein recognition for other poxviruses, and differences in the degree of virus neutralization and of virus uncoating into cells, we suggest that the three mAbs recognize different domains of vaccinia 14K viral envelope protein. Furthermore, our findings indicate that the 14K protein may play a role in virus penetration.     

Generation and characterization of neutralizing human monoclonal antibodies against human immunodeficiency virus type 1 Tat antigen

The human immunodeficiency virus Tat regulatory protein is essential for virus replication and pathogenesis. From human peripheral blood mononuclear cells of three Tat toxoid-immunized volunteers, we isolated five Tat-specific human monoclonal antibodies (HMAbs): two full-length immunoglobulin G (IgG) antibodies and three single-chain fragment-variable (scFv) antibodies. The two IgGs were mapped to distinct epitopes within the basic region of Tat, and the three scFvs were mapped to the N-terminal domain of Tat. The three scFvs were highly reactive with recombinant Tat in Western blotting or immunoprecipitation, but results were in contrast to those for the two IgGs, which are sensitive to a particular folding of the protein. In transactivation assays, scFvs were able to inhibit both active recombinant Tat and native Tat secreted by a transfected CEM cell line while IgGs neutralized only native Tat. These HMAbs were able to reduce viral p24 production in human immunodeficiency virus type 1 strain IIIB chronically infected cell lines in a dose-dependent manner.     

Dissection of human immunodeficiency virus type 1 entry with neutralizing antibodies to gp41 fusion intermediates

Human immunodeficiency virus type 1 (HIV-1) entry requires conformational changes in the transmembrane subunit (gp41) of the envelope glycoprotein (Env) involving transient fusion intermediates that contain exposed coiled-coil (prehairpin) and six-helix bundle structures. We investigated the HIV-1 entry mechanism and the potential of antibodies targeting fusion intermediates to block Env-mediated membrane fusion. Suboptimal temperature (31.5 degrees C) was used to prolong fusion intermediates as monitored by confocal microscopy. After transfer to 37 degrees C, these fusion intermediates progressed to syncytium formation with enhanced kinetics compared with effector-target (E/T) cell mixtures that were incubated only at 37 degrees C. gp41 peptides DP-178, DP-107, and IQN17 blocked fusion more efficiently (5- to 10-fold-lower 50% inhibitory dose values) when added to E/T cells at the suboptimal temperature prior to transfer to 37 degrees C. Rabbit antibodies against peptides modeling the N-heptad repeat or the six-helix bundle of gp41 blocked fusion and viral infection at 37 degrees C only if preincubated with E/T cells at the suboptimal temperature. Similar fusion inhibition was observed with human six-helix bundle-specific monoclonal antibodies. Our data demonstrate that antibodies targeting gp41 fusion intermediates are able to bind to gp41 and arrest fusion. They also indicate that six-helix bundles can form prior to fusion and that the lag time before fusion occurs may include the time needed to accumulate preformed six-helix bundles at the fusion site.     

Proteins associated with human parainfluenza virus type 3.

The polypeptides associated with human parainfluenza virus type 3 were identified. Five proteins were present in detergent- and salt-resistant viral cores. Of these, three proteins designated NP0, NP1, and NP2 of 68,000, 58,000, and 52,000 daltons, respectively, were stably associated with 50S RNA in CsCl gradient-purified nucleocapsids. The amounts of NP1 and NP2 were variable, and these proteins were shown to be structurally related to the major nucleocapsid protein (NP0) by partial Staphylococcus aureus V8 protease mapping. The other core proteins included a 240K protein designated L (candidate for the viral polymerase) and an 84K protein designated as the phosphoprotein (P) on the basis of a predominant incorporation of Pi. The viral envelope had four prominent proteins (72, 53, 40, and 12K) under reducing conditions of electrophoresis. The 72 and 53K proteins were specifically labeled with [3H]glucosamine and [3H]mannose. When sulfhydryl reagents were removed, a new 62K protein was visualized in place of the 72, 53, and 12K proteins. The 53 and 12K proteins were interpreted to be the two subunits (F1 and F2) of the fusion protein, and the 72K protein was designated as the HN (hemagglutinin-neuraminidase) glycoprotein. The unglycosylated 40K protein represented the viral matrix protein (M). Immunoprecipitation of infected cell lysates with rabbit hyperimmune antiserum against purified virus confirmed the viral origin of these polypeptides.     

Characterization of murine monoclonal antibodies to the tat protein from human immunodeficiency virus type 1.

A panel of murine monoclonal antibodies (MAbs) to the human immunodeficiency virus type 1 trans-activator tat protein were characterized. The anti-tat MAbs were mapped to the different domains of the tat protein by Western blot (immunoblot) and Pepscan analyses. One-half of the MAbs tested mapped to the amino-terminal proline-rich region, and one-third of the MAbs tested mapped to the lysine-arginine-rich region of tat. The individual MAbs were tested for inhibition of tat-mediated trans activation, using a cell-based in vitro assay system. MAbs which mapped to the amino-terminal region of the tat protein demonstrated the highest degree of inhibition, whereas MAbs reactive to other portions of the molecule exhibited a less pronounced effect on tat function.     

Identification of the principal neutralizing determinant of human immunodeficiency virus type 1 as a fusion domain.

The V3 loop, located near the middle of the surface envelope glycoprotein gp120, is the major neutralizing domain of human immunodeficiency virus type 1 (HIV-1). Although the majority of the V3 loop is highly variable between different strains of HIV-1, a Gly-Pro-Gly-Arg motif at the tip of the loop is highly conserved. To determine whether this region plays a role in fusion mediated by the HIV-1 envelope glycoproteins, we introduced seven single-amino-acid changes in the V3 loop. The mutant envelope glycoproteins were expressed from an HIV-1 envelope expression vector and analyzed for their ability to induce cell fusion in the absence of virus replication. Our results indicated that single-amino-acid changes in the V3 loop were capable of completely abolishing or greatly reducing the ability of the HIV-1 envelope glycoproteins to induce cell fusion, thereby identifying the V3 loop as a fusion domain of HIV-1. Mutations in the highly conserved tip of the loop or in a nonconserved region flanking the highly conserved tip had no effect on envelope glycoprotein synthesis, processing, transport, or binding to the CD4 receptor molecule. Mutation of the putative disulfide bridge-forming Cys at residue 336 blocked gp160 cleavage and CD4 binding.     

Induction of human immunodeficiency virus neutralizing antibodies using fusion complexes

Human immunodeficiency virus-1 (HIV-1) infects cells by membrane fusion that is mediated by the envelope proteins gp120/gp41 and the cellular receptors CD4 and CCR5. During this process, some conserved viral epitopes are temporarily exposed and may induce a neutralizing antibody response when fixed in the fusogenic conformation. These transient structures are conserved and may be effective antigens for use in an anti-HIV-1 vaccine. In this study we tested different conditions of preparation of fusion complexes inducing neutralizing antibodies against both R5 and X4 tropic HIV-1 strains. Cell lines expressing HIV-1 gp120/gp41 and CD4-CCR5 were prepared and conditions for producing fusion complexes were tested. Complexes produced at different temperature and fixative combinations were used to immunize mice. Results indicated that (a) fusion complexes prepared at either 21 degrees C, 30 degrees C or 37 degrees C were immunogenic and induced neutralizing antibodies against both R5 and X4 HIV-1 heterologous isolates; (b) after extensive purification of antibodies there was no cytotoxic effect; (c) complexes prepared at 37 degrees C were more immunogenic and induced higher titers of neutralizing antibodies than complexes prepared at either 21 degrees C or 30 degrees C; (d) the fixative used did not affect the titer of neutralizing antibodies except for glutaraldehyde which was ineffective; (e) the neutralizing activity was retained after CD4-CCR5 antibody removal. The production of higher titers of neutralizing antibody with fusion complexes prepared at 37 degrees C, as compared to lower temperatures, may be related to the induction of antibodies against many different conformation intermediates that subsequently act synergistically at different steps in the fusion process.     

Localization of the HIV-1 gp120 conformational epitope recognized by virus neutralizing monoclonal antibodies 2G12

A phage peptide library was used to select peptides interacting with virus-neutralizing monoclonal antibodies (mAb) 2G12 which recognize a discontinuous surface epitope of HIV-1 gp120. With the published X-ray data, gp120 regions involved in the antigenic determinant were predicted. Binding with mAb 2G12 was ascribed to Trh-297, Phe-383, Tyr-384, Arg-419, Ile-420, Thr-415, Leu-416, Pro-417, Lys-421, and Trp-112. Though distant in the gp120 sequence, these residues are close in space and form the 2G12 epitope on the gp120 surface.     

Cell receptors for baboon endogenous virus recognized by monoclonal antibodies.

Hybridomas from mice immunized with baboon endogenous virus (BaEV) from A204(M7) cells produced several antiviral monoclonal antibodies and, in addition, antibodies D-12 and E-4, which appeared to be virus specific because they reacted with BaEV but not with Mason-Pfizer virus or RD-114 virus. However, they also bound to human virus-free cells, and they did not recognize BaEV from bat or canine host cells. Cell membrane targets for these antibodies comigrated with an 18,000-dalton protein, which may contain specific determinants of BaEV receptors since antibody masking of these cell sites prevented BaEV but not Mason-Pfizer virus or RD-114 virus adsorption. However, RD-114 virus interfered with BaEV adsorption. Thus, the two viral receptors must be adjacent, but the antibody D-12 and E-4 targets are not within the active site of RD-114 virus receptor. Conversely, cell coating with BaEV from bat or canine hosts inhibited antibody D-12 binding. Noncultivated human lymphocytes and cells from fetal organs bound much less antibody D-12 than did cells from established cell lines, with a correlation between amounts of antibody D-12 acceptor sites and BaEV receptors. Thus, in vivo, BaEV infection of human cells may be inefficient. In vitro, antibody D-12 treatment of chronically infected A204(M7) cells caused intracellular accumulation of viral proteins and decreased virus release, with no such effect on RD-114 virus-producing cells. Canine cells bound antibody D-12 only if coated with BaEV from A204(M7) cells, indicating that the human determinant coadsorbed with the virions to animal cells. Possibly, determinants of cell receptors participate in BaEV maturation and become associated with the virions.     

Expression of the surface glycoproteins of human parainfluenza virus type 3 by bovine parainfluenza virus type 3, a novel attenuated virus vaccine vector

Bovine parainfluenza virus type 3 (bPIV3) is being evaluated as an intranasal vaccine for protection against human PIV3 (hPIV3). In young infants, the bPIV3 vaccine appears to be infectious, attenuated, immunogenic, and genetically stable, which are desirable characteristics for an RNA virus vector. To test the potential of the bPIV3 vaccine strain as a vector, an infectious DNA clone of bPIV3 was assembled and recombinant bPIV3 (r-bPIV3) was rescued. r-bPIV3 displayed a temperature-sensitive phenotype for growth in tissue culture at 39 degrees C and was attenuated in the lungs of Syrian golden hamsters. In order to test whether r-bPIV3 could serve as a vector, the fusion and hemagglutinin-neuraminidase genes of bPIV3 were replaced with those of hPIV3. The resulting bovine/human PIV3 was temperature sensitive for growth in Vero cells at 37 degrees C. The replication of bovine/human PIV3 was also restricted in the lungs of hamsters, albeit not as severely as was observed for r-bPIV3. Despite the attenuation phenotypes observed for r-bPIV3 and bovine/human PIV3, both of these viruses protected hamsters completely upon challenge with hPIV3. In summary, bPIV3 was shown to function as a virus vector that may be especially suitable for vaccination of infants and children against PIV3 and other viruses.