Chimeric synthetic peptides from the envelope (gp46) and the transmembrane (gp21) glycoproteins for the detection of antibodies to human T-cell leukemia virus type II.

Two chimeric synthetic peptides incorporating immunodominant sequences from HTLV-II virus were synthesized. Monomeric peptides P2 and P3 represent sequences from transmembrane protein (gp21) and envelope protein (gp46) of the virus. The peptide P2 is a gp21 (370-396) sequence and the peptide P3 is a gp46 (178-205) sequence. Those peptides were arranged in a way that permits one to obtain different combinations of chimeric peptides (P2-GG-P3 and P3-GG-P2), separated by two glycine residues as spacer arms. The antigenic activity of these peptides was evaluated by UltramicroEnzyme-linked immunosorbent assay (UMELISA) by using panels anti-HTLV-II-positive sera (n = 11), anti-HTLV-I/II-positive sera (n = 2), HTLV-positive (untypeable) serum samples (n = 2), and anti-HTLV-I-positive sera (n = 22), while specificity was evaluated with anti-HIV-positive samples (n = 19) and samples from healthy blood donors (n = 30). The efficacy of the chimeric peptides in solid-phase immunoassays was compared with the monomeric peptides and a mixture of the monomeric peptides. Higher sensitivity was observed for chimeric peptide Q5 assay. Those results may be related to a higher peptide adsorption capacity to the solid surface and for epitope accessibility to the antibodies. This chimeric peptide would be very useful for HTLV-II diagnostic.     

Chimeric synthetic peptides containing two immunodominant epitopes from the envelope gp46 and the transmembrane gp21 glycoproteins of HTLV-I virus.

Two chimeric synthetic peptides incorporating immunodominant sequences from HTLV-I virus were synthesized. Monomeric peptides P7 and P8 represent sequences from transmembrane protein (gp21) and envelope protein (gp46) of the virus. The peptide P7 is a gp21 (374-400) sequence and the peptide P8 is a gp46 (190-207) sequence. Those peptides were arranged in a way that permits one to obtain different combinations of chimeric peptides (P7-GG-P8 and P8-GG-P7), separated by two glycine residues as spacer arms. The antigenic activity of these peptides were evaluated by UltramicroEnzyme-linked immunosorbent assay (UMELISA) by using panels of anti-HTLV-I-positive sera (n = 22), anti-HTLV-I/II-positive sera (n = 2), HTLV-positive (untypeable) serum samples (n = 2), and anti-HTLV-II-positive sera (n = 11), while specificity was evaluated with anti-HIV-positive samples (n = 19) and samples from healthy blood donors (n = 30). The efficacy of the chimeric peptides in solid-phase immunoassays was compared with the monomeric peptides and monomeric peptides together. The chimeric peptide P7-GG-P8 proved to be the most reactive with anti-HTLV-I-positive sera. These results may be related to a higher peptide adsorption capacity to the solid surface and for epitope accessibility to the antibodies. This chimeric peptide would be very useful for HTLV-I diagnostics.     

Study of the peptide length and amino acid specific substitution in the antigenic activity of the chimeric synthetic peptides, containing the p19 core and gp46 envelope proteins of the HTLV-I virus

Four chimeric synthetic peptides (Q5, Q6, Q7(multiply sign in circle), and Q8(multiply sign in circle)), incorporating immunodominant epitopes of the core p19 (105-124 a.a.) and envelope gp46 proteins (175-205 a.a.), of HTLV-I were obtained. Also, two gp46 monomeric peptides M4 and M5(multiply sign in circle) (Ser at position 192) were synthesized. The analysis of the influence of the peptide lengths and the proline to serine substitution on the chimeric and monomeric peptides' antigenicity, with regard to the chimeric peptides Q1, Q2, Q3(multiply sign in circle), and Q4(multiply sign in circle), reported previously, for HTLV-I was carried out. The peptides' antigenicity was evaluated in an ultramicroenzyme-linked immunosorbent assay (UMELISA) using sera of HTLV-I/II. The peptides' antigenicity was affected appreciably by the change of the peptide length and amino acid substitutions into the immunodominant sequence of gp46 peptide.     

Chimeric synthetic peptide as antigen for immunodiagnosis of HIV-1 infection

One chimeric peptide incorporating antigenic sequences from the gp41 transmembrane region (peptide H-18) and the gp120 envelope region (peptide H-15) corresponding to amino acids (587-617) on gp41 and (495-516) on gp120 of human immunodeficiency virus (HIV 1) was synthesized. Both sequences were separated by two glycine residues. This peptide was evaluated as antigen in an ultramicro-enzyme-linked immunosorbent assay (UMELISA) with samples derived from HIV-1 (n = 30) with different titers of antibodies and healthy blood donors (n = 30). The results were compared to plates coated with monomeric peptides and to plates coated with two monomeric peptides together. Results demonstrated that monomeric peptides gp41 (H-18) and gp120 (H-15) were good as antigens with samples that present antibodies to these regions. The chimeric peptide was the most antigenic. Those results may be related to the peptide structure, adsorption to the solid surface, and epitope accessibility to the antibodies. This chimeric peptide would be very useful for HIV-1 diagnostics.     

Use of a chimeric synthetic peptide from the core p19 protein and the envelope gp46 glycoprotein in the immunodiagnosis of HTLV-II virus infection

A chimeric synthetic peptide incorporating immunodominant epitope of the p19 gag protein (116-134) and the gp46 env protein (178-200) of HTLV-II virus, separated by two glycine residues, was synthesized by conventional solid-phase peptide synthesis. The antigenic activity of this peptide was evaluated by Ultramicro Enzyme-linked immunosorbent assay (UMELISA) by using panels of anti-HTLV-II positive sera (n = 9), anti-HTLV-I/II positive sera (n = 2), HTLV-positive (untypeable) serum samples (n = 1),and anti-HTLV-I positive sera (n = 14), while specificity was evaluated with samples from healthy blood donors (n = 20). The efficacy of the chimeric peptide in solid-phase immunoassays was compared with the monomeric peptides. Data demonstrated that the chimeric peptide was the most reactive because it detected antibodies to virus efficiently. This may be related to peptide adsorption to the solid surface and epitope accessibility to the antibodies. The results indicate that chimeric peptide as coating antigen is very useful for the immunodiagnosis of HTLV-II infection.     

Antigenic activity of three chimeric synthetic peptides of the transmembrane (gp41) and the envelope (gp120) glycoproteins of HIV-1 virus

The antigenicity of three chimeric synthetic peptides (Qm, Qm-16, and Qm-17) incorporating an immunodominant epitope of the gp41 transmembrane protein (587-617) and the different epitopes of the gp120 envelope protein (495-516), (301-335), (502-516) of human immunodeficiency virus (HIV-1), separated by two glycine residues, was evaluated by UltramicroEnzyme-linked immunosorbent assay (UMELISA) by using panels of anti-HIV-1 positive sera (n = 47). The specificity was evaluated with samples from healthy blood donors (n = 20) and anti-HIV-2 positive samples (n = 10). The results indicate that the chimeric peptide, Qm, was the most reactive one because it detected antibodies to virus efficiently. This may be related to peptide adsorption onto the solid surface, the C-terminal region of HIV-1 gp120 (495-516) combined with gp41 (587-617) in the chimera, and the epitope accessibility to the antibodies. This study showed the usefulness of the chimeric peptides as antigen to detect antibodies to HIV-1 virus.     

Chimeric synthetic peptides as antigens for detection of antibodies to Trypanosoma cruzi

Six chimeric synthetic peptides (QCha-1, QCha-2, QCha-3, QCha-4, QCha-5, and QCha-6) incorporating antigenic sequences of two immunodominant repeat B-cell epitopes of Trypanosoma cruzi were synthesized by conventional solid-phase peptide synthesis. The antigenic activity of these peptides was evaluated by UltramicroEnzyme-linked immunosorbent assay (UMELISA) by using panels of positive Chagasic sera (n=82), while specificity was evaluated with samples from healthy blood donors (n=44) and patients with other infectious diseases (n=86). The antigenicity of the chimeric peptides in solid-phase immunoassays was compared with that of the monomeric peptides. Data demonstrated that the chimeric peptide QCha-5 was the most reactive because it detected antibodies to parasite efficiently. The results indicate that chimeric peptide as coating antigen is very useful for the immunodiagnosis of Chagas' disease.     

Identification and mapping of functional domains on human T-cell lymphotropic virus type 1 envelope proteins by using synthetic peptides.

To identify the regions that are important in human T-cell leukemia virus type 1 (HTLV-1) envelope function, we synthesized 23 kinds of peptides covering the envelope proteins and examined the inhibitory effect of each peptide on syncytium formation induced by HTLV-1-bearing cells. Of the 23 synthetic peptides, 2, corresponding to amino acids 197 to 216 on gp46 and 400 to 429 on gp21, inhibited syncytium formation induced by HTLV-1-bearing cells but did not affect syncytium formation induced by human immunodeficiency virus type 1-producing cells. The peptide concentrations giving 50% inhibition of syncytium formation for gp46 197 to 216 and gp21 400 to 429 were 14.9 and 6.0 microM, respectively. A syncytium formation assay with overlapping synthetic peptides containing amino acids 175 to 236 and 391 to 448 of the envelope proteins showed that syncytium formation was inhibited by peptides that contained the amino acid sequences 197 to 205 (Asp-His-Ile-Leu-Glu-Pro-Ser-Ile-Pro) and 397 to 406 (Gln-Glu-Gln-Cys-Arg-Phe- Pro-Asn-Ile-Thr). These observations suggest that the two regions corresponding to amino acids 197 to 216 and 400 to 429 are involved] in HTLV-1 envelope function.     

Proline-rich tandem repeats of antibody complementarity-determining regions bind and neutralize human immunodeficiency virus type 1 particles.

The proline-rich tandem repeat domain of human mucin MUC1 forms an extended structure containing large repeating loops that are crested by a turn. We show that the repeating-loop structure of MUC1 can be replaced by an antibody complementarity-determining region loop of a human immunodeficiency virus type 1 (HIV-1)-specific neutralizing antibody to create a chimeric, multivalent, mucin-like, anti-HIV-1 compound. We used 8 residues of an antibody molecule to replace 8 of 20 residues of the MUC1 tandem-repeat sequence. The antiviral peptide discussed here contains three copies of a 20-residue tandem repeat, (IYYDYEEDPAPGSTAPPAHG)3, for a total of 60 residues. We demonstrate that the mucin-antibody chimera retains the binding specificity of the parent antibody (monoclonal antibody F58), GPGR of the HIV-1 gp120 V3 neutralizing epitope, and the ability to neutralize virus particles. In inhibition enzyme-linked immunosorbent assay, the mucin-antibody chimeric peptide could inhibit 71 to 84% of binding to a V3 loop peptide by monoclonal antibodies known to be specific for GPGR in the V3 loop. The mucin-antibody chimeric peptide could also inhibit monoclonal antibody binding to native gp120 captured from virus particles. In addition, the chimeric peptide neutralized the homologous HIV-IIIB virus in a standard neutralization assay. The methods of antiviral peptide design and construction presented here are general and theoretically limited only by the size of the antibody repertoire. This approach could be used to synthesize peptides for a variety of therapeutic applications.     

Structural and immunogenicity analysis of chimeric B-cell epitope constructs derived from the gp46 and gp21 subunits of the envelope glycoproteins of HTLV-1.

B-cell epitopes were selected from the gp21 and gp46 subunits of the envelope glycoprotein of human T-cell lymphotropic virus type 1 (HTLV-1) by computer-aided analyses of protein antigenicity. Molecular modeling was used to design and synthesize the epitopes as chimeric constructs with promiscuous T-helper epitopes derived either from the tetanus toxoid (amino acids 947-967) or measles virus fusion protein (amino acids 288-302). Circular dichroism measurements revealed that the peptides had a secondary structure that correlated well with the crystal structure data or predicted structure. The chimeric peptides were then evaluated for their immunogenicity in rabbits or mice. Antibodies against one of the epitopes derived from the gp21 subunit were found to be neutralizing in its ability to inhibit the formation of virus-induced syncytia. These studies underscore the importance of the gp21 transmembrane region for the development of vaccine candidates. The applicability of a chimeric approach is discussed in the context of recent findings regarding the role of gp21 transmembrane region in the viral fusion process.     

Presentation of native epitopes in the V1/V2 and V3 regions of human immunodeficiency virus type 1 gp120 by fusion glycoproteins containing isolated gp120 domains.

The immune response to viral glycoproteins is often directed against conformation- and/or glycosylation-dependent structures; synthetic peptides and bacterially expressed proteins are inadequate probes for the mapping of such epitopes. This report describes a retroviral vector system that presents such native epitopes on chimeric glycoproteins in which protein fragments of interest are fused to the C terminus of the N-terminal domain of the murine leukemia virus surface protein, gp70. The system was used to express two disulfide-bonded domains from gp120, the surface protein of human immunodeficiency virus type 1 (HIV-1), that include potent neutralization epitopes. The resulting fusion glycoproteins were synthesized at high levels and were efficiently transported and secreted. A fusion protein containing the HXB2 V1/V2 domain was recognized by an HIVIIIB-infected patient serum as well as by 17 of 36 HIV-1 seropositive hemophiliac, homosexual male and intravenous drug user patient sera. Many of these HIV+ human sera reacted with V1/V2 domains from several HIV-1 clones expressed in fusion glycoproteins, indicating the presence of cross-reactive antibodies against epitopes in the V1/V2 domain. Recognition of gp(1-263):V1/V2HXB2 by the HIVIIIB-infected human patient serum was largely blocked by synthetic peptides matching V1 but not V2 sequences, while recognition of this construct by a broadly cross-reactive hemophiliac patient serum was not blocked by individual V1 or V2 peptides or by mixtures of these peptides. A construct containing the V3 domain of the IIIB strain of HIV-1, gp(1-263):V3HXB2, was recognized by sera from a human and a chimpanzee that had been infected by HIVIIIB but not by sera from hemophiliac patients who had been infected with HIV-1 of MN-like V3 serotype. The reactive sera had significantly higher titers when assayed against gp(1-263):V3HXB2 than when assayed against matching V3 peptides. Immunoprecipitation of this fusion glycoprotein by the human serum was only partially blocked by V3 peptide, indicating that this infected individual produced antibodies against epitopes in V3 that were expressed on the fusion glycoprotein but not by synthetic peptides. These data demonstrated that the chimeric glycoproteins described here effectively present native epitopes present in the V1/V2 and V3 domains of gp120 and provide efficient methods for detection of antibodies directed against native epitopes in these regions and for characterization of such epitopes.     

Interaction of fibrin(ogen) with leukocyte receptor alpha M beta 2 (Mac-1): further characterization and identification of a novel binding region within the central domain of the fibrinogen gamma-module

The fibrinogen gamma-module sequences, gamma190-202 or P1, and gamma377-395 or P2, were implicated in interaction with the alpha(M)I-domain of the leukocyte receptor alpha(M)beta(2). P1 is an integral part of the gamma-module central domain, while P2 is inserted into this domain forming an antiparallel beta-strand with P1. We hypothesized earlier that separation of P2 from P1 may regulate interaction of fibrin(ogen) with leukocytes during the inflammatory response. To test the relative contributions of these sequences to the interaction and the effect of their separation, we prepared the recombinant gamma-module (gamma148-411) and its halves, gamma148-286 and gamma287-411 fragments containing P1 and P2, respectively, and evaluated their affinities for the recombinant alpha(M)I-domain. In a solid-phase binding assay, the immobilized gamma-module exhibited high affinity for alpha(M)I (K(d) = 22 nM), while the affinities of the isolated gamma148-286 and gamma287-411 halves were much lower (K(d)'s = 521 and 194 nM, respectively), indicating that both halves contribute to the interaction in a synergistic manner. This is consistent with the above hypothesis. Further, we prepared the recombinant gamma148-191 and gamma192-286 fragments corresponding to the NH(2)-terminal and central domains, respectively, as well as gamma148-226 containing P1, and tested their interaction with alpha(M)I. The immobilized gamma192-286 fragment bound to alpha(M)I with K(d) = 559 nM, while both gamma148-191 and gamma148-226 failed to bind suggesting that P1 does not contribute substantially to the binding and that the binding occurs mainly through the gamma227-286 region. To further localize a putative binding sequence, we cleaved gamma192-286 and analyzed the resulting peptides. The only alpha(M)I-binding activity was associated with the gamma228-253 peptide, indicating that this region of the central domain contains a novel alpha(M)beta(2)-binding sequence.     

Inhibition of Cell-Free Human T-Cell Leukemia Virus Type 1 Infection at a Postbinding Step by the Synthetic Peptide Derived from an Ectodomain of the gp21 Transmembrane Glycoprotein

To investigate the roles of human T-cell leukemia virus type 1 (HTLV-1) envelope (Env) proteins gp46 and gp21 in the early steps of infection, the effects of the 23 synthetic peptides covering the entire Env proteins on transmission of cell-free HTLV-1 were examined by PCR and by the plaque assay using a pseudotype of vesicular stomatis virus (VSV) bearing the Env of HTLV-1 [VSV(HTLV-1)]. The synthetic peptide corresponding to amino acids 400 to 429 of the gp21 Env protein (gp21 peptide 400-429, Cys-Arg-Phe-Pro-Asn-Ile-Thr-Asn-Ser-His-Val-Pro-Ile-Leu-Gln-Glu-Arg-P ro-Pro-Leu-Glu-Asn-Arg-Val-Leu-Thr-Gly-Trp-Gly-Leu) strongly inhibited infection of cell-free HTLV-1. By using the mutant peptide, Asn407, Ser408, and Leu413, -419, -424, and -429 were confirmed to be important amino acids for neutralizing activity of the gp21 peptide 400-429. Addition of this peptide before or during adsorption of HTLV-1 at 4 degrees C did not affect its entry. However, HTLV-1 infection was inhibited about 60% when the gp21 peptide 400-429 was added even 30 min after adsorption of HTLV-1 to cells, indicating that the amino acid sequence 400 to 429 on the gp21 Env protein plays an important role at the postbinding step of HTLV-1 infection. In contrast, a monoclonal antibody reported to recognize the gp46 191-196 peptide inhibited the infection of HTLV-1 at the binding step.     

Peptide affinity and concentration affect the sensitivity of M3-restricted CTLs induced in vitro.

In vitro stimulation of mouse splenocytes with hemagglutinin (HA) 173-190, a peptide derived from influenza virus hemagglutinin (A/JAP/305/57, H2N2), induces CTLs that are directed to the MHC class Ib molecule, H2-M3. M3 preferably binds peptides bearing an N-terminal formylmethionine. In this study, we show that several related nonformylated peptides can induce anti-HA CTLs in vitro: MLIIW (the minimal epitope), derived from HA186-190 at the C-terminal end of HA173-190; MLIIWG; MLIIWGV; and MLIIWGI, as well as formylated MLIIW. The heptamer peptides correspond to a polymorphism of HA192 in H2 strains of influenza; they have the highest relative affinities for M3 of the nonformylated peptides and higher affinities than some formylated mitochondrial peptides. Depending on the affinity of the peptide, a range of concentrations can be used to induce CTLs. One nanomolar of the high affinity f-MLIIW peptide can induce anti-HA CTLs, whereas 100-fold more of the lower affinity MLIIW peptide is needed. Lines induced with high concentrations (1 microM or greater) of f-MLIIW recognize Ag poorly, and the most efficient CTLs are induced with the lowest concentrations of peptide. Analysis with a panel of anti-TCRVbeta Abs shows that different T cells respond to high vs low peptide; the repertoire of cells responding to higher concentrations is more diverse, consistent with the expansion of more, but less efficient, clones. Thus, peptide affinity and concentration should be considered together for generating efficient antipeptide CTLs in vitro.     

Sensitivity of human immunodeficiency virus type 1 to fusion inhibitors targeted to the gp41 first heptad repeat involves distinct regions of gp41 and is consistently modulated by gp120 interactions with the coreceptor

T-20 is a synthetic peptide that corresponds to 36 amino acids within the C-terminal heptad repeat region (HR2) of human immunodeficiency virus type 1 (HIV-1) gp41. T-20 has been shown to potently inhibit viral replication of HIV-1 both in vitro and in vivo and is currently being evaluated in a Phase III clinical trial. T-649 is an inhibitory peptide that also corresponds to 36 amino acids within HR2. This sequence overlaps the T-20 sequence but is shifted 10 residues toward the N terminus of gp41. Both inhibitors are thought to exert their antiviral activity by interfering with the conformational changes that occur within gp41 to promote membrane fusion following gp120 interactions with CD4 and coreceptor molecules. We have shown previously that coreceptor specificity defined by the V3 loop of gp120 modulates sensitivity to T-20 and that a critical region within the N-terminal heptad repeat (HR1) of gp41 is the major determinant of sensitivity (C. A. Derdeyn et al., J. Virol. 74:8358-8367, 2000). This report shows that (i) regions within gp41 distinct from those associated with T-20 sensitivity govern the baseline sensitivity to T-649 and (ii) T-649 sensitivity of chimeric viruses that contain sequences derived from CXCR4- and CCR5-specific envelopes is also modulated by coreceptor specificity. Moreover, the pattern of sensitivity of CCR5-specific chimeras with only minor differences in their V3 loop was consistent for both inhibitors, suggesting that the individual affinity for coreceptor may influence accessibility of these inhibitors to their target sequence. Finally, an analysis of the sensitivity of 55 primary, inhibitor-naive HIV-1 isolates found that higher concentrations of T-20 (P < 0.001) and T-649 (P = 0.016) were required to inhibit CCR5-specific viruses compared to viruses that utilize CXCR4. The results presented here implicate gp120-coreceptor interactions in driving the complex conformational changes that occur in gp41 to promote fusion and entry and suggest that sensitivity to different HR1-directed fusion inhibitors is governed by distinct regions of gp41 but is consistently modulated by coreceptor specificity.     

Stabilization of the biologically active conformation of the principal neutralizing determinant of HIV-1(IIIB) containing a cis-proline surrogate: 1H NMR and molecular modeling study

The V3 loop is part of the gp120 glycoprotein, an extracellular protein located on the membrane of the human immunodeficiency virus (HIV-1). This loop is significantly important in many biological processes of the virus and contains the principal neutralizing determinant (PND). The PND is one of the most variable regions of the envelope, and this is probably related to the ability of the HIV virus to escape the immunologic defenses of the target host. Particular attention has been paid to the central part of the V3 loop which contains a highly conserved GPGR/GPGQ sequence and represents the binding site for antibodies. Many attempts have been made to design synthetic peptides as mimics of the V3 loop capable of eliciting immune response. However, this strategy suffers from the great conformational flexibility small peptides have in solution, which together with bioavailability represents the most important limitation to the usefulness of synthetic peptides as drugs and as synthetic immunogens. The use of conformationally constrained peptides can alleviate this problem. Early works using NMR studies have shown that a V3(IIIB) loop-derived peptide is conformationally heterogeneous when free in water. Upon complexation with 0.5beta, a monoclonal neutralizing antibody specific for the HIV-1(IIIB) strain, it adopts a beta-hairpin conformation with the central proline forming a type VIb beta-turn. In this study, we report the design and characterization of a conformationally restricted peptide with a sequence identical to that previously described, but with thiazolidine derivatives replacing the proline. The affinity of the 2,2-dimethylthiazolidine derivative for 0.5beta demonstrates that this moiety can successfully be used to mimic the proline in a cis conformation. This peptide not only displays a high propensity to adopt a beta-hairpin conformation but also retains the type VIb RGPG beta-turn similar to that found in the native complex. These compounds could help in elaborating more efficient immunogens for HIV-1 synthetic vaccine development.     

Chimeric influenza virus induces neutralizing antibodies and cytotoxic T cells against human immunodeficiency virus type 1.

Expression vectors based on DNA or plus-stranded RNA viruses are being developed as vaccine carriers directed against various pathogens. Less is known about the use of negative-stranded RNA viruses, whose genomes have been refractory to direct genetic manipulation. Using a recently described reverse genetics method, we investigated whether influenza virus is able to present antigenic structures from other infectious agents. We engineered a chimeric influenza virus which expresses a 12-amino-acid peptide derived from the V3 loop of gp120 of human immunodeficiency virus type 1 (HIV-1) MN. This peptide was inserted into the loop of antigenic site B of the influenza A/WSN/33 virus hemagglutinin (HA). The resulting chimeric virus was recognized by specific anti-V3 peptide antibodies and a human anti-gp120 monoclonal antibody in both hemagglutination inhibition and neutralization assays. Mice immunized with the chimeric influenza virus produced anti-HIV antibodies which were able to bind to synthetic V3 peptide, to precipitate gp120, and to neutralize MN virus in human T-cell culture system. In addition, the chimeric virus was also capable of inducing cytotoxic T cells which specifically recognize the HIV sequence. These results suggest that influenza virus can be used as an expression vector for inducing both B- and T-cell-mediated immunity against other infectious agents.     

Identification and synthesis of the epitope for a human monoclonal antibody which can neutralize human T-cell leukemia/lymphotropic virus type I

A monoclonal antibody (mAb), designated 0.5 alpha, derived from a patient with adult T-cell leukemia was found previously to neutralize the human T-cell leukemia/lymphotropic type I (HTLV-I) virus in in vitro assays and bind to the major envelope glycoprotein (gp46) of HTLV-I (Matsushita, S., Guroff, M.R., Trepel, J., Crossman, J., Mitsuya, H., and Broder, S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2671-2676). We have designed experiments to determine the epitope for this mAb. Using simultaneous multiple peptide synthesis, we synthesized 481 overlapping octapeptides which corresponded to the sequence of gp46. We mapped the epitope for mAb 0.5 alpha to lie between residues 186 and 195 of gp46. This result was confirmed by independently synthesizing a peptide containing this epitope which bound specifically to mAb 0.5 alpha with an approximate Ka = 4 x 10(7) M-1. In addition, the peptide inhibited mAb 0.5 alpha binding to gp46 derived from T-cells infected with HTLV-I. This epitope containing peptide may facilitate understanding HTLV-1 infection of T-cells.     

Mapping of B-neutralizing and T-helper cell epitopes on the bovine leukemia virus external glycoprotein gp51.

A battery of 19 synthetic peptides was used to characterize efficient neutralizing and helper T-cell epitopes on the bovine leukemia virus (BLV) external envelope glycoprotein gp51. Four of the antipeptide antisera raised in rabbits inhibited the formation of BLV-induced syncytia; these antisera are directed against peptides 64-73, 98-117, and 177-192. Only antisera directed against the 177-192 region also neutralized vesicular stomatitis virus-BLV pseudotypes. This study clearly demonstrates that neutralizing properties can be observed with antibodies raised to regions undescribed so far and included in both the amino-terminal and central parts of the antigen. In addition, some helper T-cell determinants were defined from gp51-immunized mice and from BLV-infected cattle. Although none of the peptides tested behaved as a universal helper T-cell epitope, peptide 98-117 stimulated T-cell proliferation from BALB/c mice and from three infected cows, while peptide 169-188 strongly stimulated T-cell proliferation from one infected cow. Further experiments performed with three peptides overlapping the 169-188 region (177-192, 179-192, 181-192) demonstrated the particular relevance of residue(s) P-177 and/or D-178 in the helper T-cell epitope. These data should assist in the design of an efficient subunit vaccine against BLV infection that contains peptides possessing both B-neutralizing and helper T-cell determinants.     

Immunization with gp96 from Listeria monocytogenes-infected mice is due to N-formylated listerial peptides.

N-Formylated (N-f-met) peptides derived from proteins of the intracellular bacterium Listeria monocytogenes generate a protective, H2-M3-restricted CD8 T cell response in C57BL/6 mice. N-f-met peptide-specific CTL were generated in vitro when mice previously immunized with gp96 isolated from donor mice infected with L. monocytogenes were stimulated with these peptides. No significant peptide-specific CTL activity was observed in mice immunized with gp96 from uninfected animals. Masses corresponding to one N-f-met peptide were found by matrix-assisted laser desorption/ionization-mass spectrometry on gp96 isolated from C57BL/6 mice infected with L. monocytogenes, but not on gp96 from noninfected mice. Therefore, bacterial N-f-met peptides from intracellular bacteria can bind to gp96 in the infected host, and gp96 loaded with these peptides can generate N-f-met-peptide-specific CTL. We assume a unique role of gp96 in Ag processing through the H2-M3 pathway.