Fine specificity and antigen receptor expression among influenza virus-specific cytolytic T lymphocyte clones

Influenza virus stimulates a vigorous cytolytic T lymphocyte (CTL) response in the mouse that is directed to several virion polypeptides. This report examines the fine specificity of a panel of murine influenza-specific CTL clones restricted by MHC class I products of the H-2d haplotype. Ten of 22 A/JAPAN/305/57-specific CTL clones analyzed were directed to the A/JAPAN/305/57 hemagglutinin protein as detected by using target cells infected with a recombinant vaccinia virus containing hemagglutinin gene. Based on their fine specificity of hemagglutinin recognition, these clones defined four functional epitopes on the hemagglutinin. The remaining 12 cytolytic clones exhibited cross-reactivity for type A influenza viruses of the major human subtypes, and approximately 60% of these clones were directed to the nucleocapsid protein. KJ16-133 monoclonal antibody analysis of the utilization of the T cell receptor V beta 8 gene segment subfamily revealed that members of this V beta gene subfamily are expressed by both hemagglutinin- and nucleocapsid-specific MHC class I-restricted CTL (and by influenza-specific MHC class II-restricted T lymphocytes as well). These results suggest that CTL detect several distinct antigenic sites on the hemagglutinin. In addition, these results reveal no direct correlation between viral antigenic specificity and V beta gene expression by these virus-specific CLT clones.     

Cytolytic T lymphocyte and antibody responses to synthetic peptides of influenza virus hemagglutinin

Two peptides corresponding to HA1(181-204) and HA2(103-123) of the A/Japan/305/57 influenza virus hemagglutinin (HA) were chemically synthesized by solid-phase methods and were tested for their ability to generate murine secondary anti-influenza cytolytic T lymphocytes (CTL) in vitro and to bind monoclonal anti-HA antibodies. Peptide HA1(181-204) could only generate CTL in the presence of helper factors contained in supernatant fluids from either Concanavalin A-stimulated mouse spleen cultures or WEHI-3 cells grown in vitro. Peptide HA2(103-123) stimulated the induction of anti-influenza CTL independent of helper factors, but the stimulation was also greatly increased if helper factors were added. A 10-fold molar excess of peptide HA2(103-123) was required to obtain optimal CTL activation over the quantities required in the HA1(181-204) system. This molar ratio remained unchanged, even in the presence of helper factors. Induction of influenza-specific CTL was antigen-dependent in both systems, even though some killing of noninfected target cells was also occasionally observed. Our results suggest that synthetic peptides can be recognized as antigenic determinants in the generation of H-2-restricted anti-viral CTL capable of killing appropriately infected target cells. The inability of peptide HA1(181-204) to generate sufficient help for CTL development suggests that certain regions of the HA can be recognized by CTL precursors, but not by all of the required helper cells. Peptide HA1(181-204) also reacted with three monoclonal anti-HA antibodies as well as mouse anti-influenza (A/Japan/305/57) immune sera. This antibody reactivity suggests the possibility of a shared antigenic epitope or region between T and B cells, and therefore provides new insight in our understanding of viral antigenicity.     

Class II-restricted T-cell clones to a synthetic peptide of influenza virus hemagglutinin differ in their fine specificities and in the ability to respond to virus.

Fifteen T-cell clones were derived from BALB/c or DBA/2 mice immunized with a synthetic peptide corresponding to the C-terminal 24 residues (residues 305 to 328) of the HA1 chain of H3 subtype influenza virus hemagglutinin. All of the clones proliferated when the peptide was presented in association with I-Ed. By using shorter homologs, it was shown that the T-cell response was focused predominantly on the region at the N-terminal end of the peptide encompassed by residues 306 to 319. Individual clones recognizing this region differed in their absolute requirements for residues at the extremities of the site and also in their patterns of efficiency of recognition of shorter homologs. One particular clone defined another site of T-cell recognition within residues 314 to 328. The response of the clones to peptide analogs identified certain residues within the sites that were critical for recognition, with the substitution Gln-311----Ser having a differential effect on clones responding to the N-terminal site. Only one of the clones responded well to influenza virus itself. This clone also required relatively low concentrations of the parent peptide for optimum stimulation and was suppressed by higher concentrations. The data demonstrate striking heterogeneity in the T-cell response even to a short synthetic peptide, with different T-cell clones recognizing slightly different but overlapping areas of the molecule.     

Fine specificity of antigen recognition by T cell hybridoma clones specific for poly-18: a synthetic polypeptide antigen of defined sequence and conformation

Antigen-specific T cell blasts to poly-18, a polypeptide antigen of defined sequence and conformation, were generated from lymph nodes of antigen-primed BALB/cCr mice. These blasts were fused with the BW5147 thymoma to obtain anti-poly-18-reactive T cell hybridomas. All of the hybridomas were IAd-restricted and secreted IL2 in the presence of IAd/poly-18. On the basis of fine specificity analysis, these hybridomas were classified into two groups. Group A hybridomas recognized a minimal peptide sequence of Glu-Tyr-Lys-(Glu-Tyr-Ala)3-Glu-Tyr-Lys, whereas Group B needed the sequence Glu-Tyr-Ala-(Glu-Tyr-Ala)3-Glu-Tyr-Lys/Ala for activation. Three critical residues were identified in Group A hybridomas: the alanine residue at position 9, the carboxy terminal lysine, and the lysine at position 3. In Group B hybridomas, the alanine at position 3 was found to be the critical residue. We suggest that the amino acid residue at position 3 (lysine/alanine) is the T cell receptor contact residue on the poly-18 antigen in BALB/cCr mice.     

The recognition specificity of a murine helper T cell for hemagglutinin of influenza virus A/PR/8/34

Human large granular lymphocytes (LGL), which are known to be responsible for natural killer (NK) cell activity, also produced a variety of lymphokines including interleukin 2 (IL 2), colony stimulating factor (CSF), and interferon (IFN) in response to phytohemagglutinin (PHA) or concanavalin A (Con A). Human peripheral blood LGL, which were purified by removal of monocytes adhering to plastic flasks and nylon columns, followed by separation on a discontinuous Percoll gradient, and additional treatment with anti-OKT3 and Leu-M1 plus complement, were more potent producers of these lymphokines than unseparated mononuclear cells (MNC), nylon column-eluted cells, or purified T lymphocytes. Moreover, IL 2 production by LGL could be further distinguished in that it was not enhanced by the addition of macrophages or macrophage-derived factor, i.e., IL 1, whereas addition of macrophages did potentiate IL 2 production by T lymphocytes. Further analysis of cells in the LGL population using various monoclonal antibodies revealed that removal of cells with OKT11 or AF-10, a monoclonal antibody against human HLA-DR antigen, decreased IL 2 production, whereas removal of OKT8+, OKM1+, Leu-M1+, or Leu-7+ cells led to enhanced IL 2 production. The LGL population is therefore heterogeneous and includes at least three functionally and phenotypically distinct subsets. An atypical T cell subset (OKT3-, Leu-1-, OKT11+) rather than the myeloid subset of LGL (Leu-M1+ or OKMI+) was the source of LGL-derived IL 2, whereas the latter subset and/or another subset of OKT8+ cells appear to regulate this IL 2 production. In addition to performing NK activity, LGL on a per cell basis seem to be more effective than T lymphocytes in producing lymphokines, namely, IL2, CSF, and IFN.     

The fine specificity of antigen and Ia determinant recognition by T cell hybridoma clones specific for pigeon cytochrome c

The activation of proliferative T lymphocytes normally involves the simultaneous recognition of a particular foreign antigen and a particular Ia molecule on the surface of antigen-presenting cells, the phenomenon of major histocompatibility complex (MHC) restriction. An analysis of T cell clones specific for pigeon cytochrome c, from B10.A and B10.S(9R) strains of mice, revealed the unusual finding that several of the clones could respond to antigen in association with Ia molecules from either strain. Using these cross-reactive clones, we performed experiments which demonstrated that both the Ia molecule and the T cell receptor contribute to the specificity of antigen recognition; however, MHC-linked low responsiveness to tuna cytochrome c (an immune response gene defect) could not be attributed solely to the efficacy with which the Ia molecules associated with the antigen. These results imply that antigen and Ia molecules are not recognized independently, but must interact at least during the process of T cell activation.     

Acid-induced conformational modification of the hemagglutinin molecule alters interaction of influenza virus with antigen-presenting cells

Brief exposure of influenza virus to pH 5 was found to have extensive effects upon presentation of viral Th cell antigenic determinants. This acidity, comparable to that encountered in host cell endosomes, was known to effect conformational changes in the viral hemagglutinin (HA) which alter the molecule's fusion activity, antigenicity, and susceptibility to enzymes. Three major effects of low pH upon presentation of viral T cell determinants were observed: first, acid pretreatment permitted presentation by pre-fixed APC of two of three linear T cell sites of the HA molecule, bypassing the APC activity required to present untreated virus; second, the two determinants presented in this manner disappeared rapidly from APC surfaces; third, acid-pretreated virus was not efficiently utilized by active APC in the normal pathway of viral antigen presentation. These observations suggest that the pH-induced conformational transition of HA may constitute sufficient processing for certain linear determinants of the molecule and additionally influences the processes involved in the general formation and presentation of viral T cell sites.     

The role of IL 1 in the antigen-specific activation of murine class II-restricted T lymphocyte clones

The role of IL 1 in the antigen-specific activation of class II-restricted T lymphocytes was examined by using a model system consisting of cloned WEHI 5 B lymphoma accessory cells and class II-restricted, soluble antigen- or alloantigen-reactive T cell clones. The addition of exogenous recombinant IL 1 to the T cell cultures resulted in a significant enhancement of the antigen-specific T cell proliferation response, but at best, only small increases in IL 2 release. Goat IgG anti-IL 1 antibodies were added to the T cell cultures to assess their effect on T cell activation. The IL 1 enhancement of the T cell proliferation response was inhibited by the anti-IL 1 antibodies in a dose-dependent manner. In contrast, only modest levels (10 to 25%) of proliferation inhibition were observed in T cell cultures containing either WEHI 5 or splenocyte accessory cells but no exogenous IL 1. When the anti-IL 1 antibodies were added to primary mixed lymphocyte cultures stimulated by WEHI 5 cells in the absence of exogenous IL 1, no significant inhibition of proliferation was observed. A small but statistically significant proliferation inhibition was observed when anti-IL 1 antibodies were added to mixed lymphocyte reaction cultures stimulated by splenocytes. Two-color cytofluorometric analysis of the effects of IL 1 on antigen-activated T cell clones demonstrated that under suboptimal stimulation conditions, IL 1 stimulated a small but significant increase in the number of T cells bearing IL 2 receptors. In the presence of optimal numbers of WEHI 5 accessory cells, IL 1 enhanced T cell proliferation in the absence of a detectable increase in the number of T cells bearing IL 2 receptors, the number of IL 2 receptors per T cell, or the levels of IL 2 released. Finally, exogenous IL 1 can be added as late as 18 to 24 hr after culture initiation without significantly reducing its ability to enhance the T cell proliferation response. These data indicate that IL 1 has pleiotropic effects on murine T lymphocytes and can function to enhance T cell activation at multiple points during the activation sequence.     

Theoretical studies of the interaction between influenza virus hemagglutinin and its small molecule ligands

Hemagglutinin (HA) is a membrane protein present on the influenza viral envelope. It is responsible for molecular recognition between the viral particle and the host cell, as well as fusion of the viral envelope to the endosome bilayer. Because it is essential for influenza viral infection and replication, it has become a target for the design of anti-influenza drugs. Previous studies have identified two small molecule HA ligands (CL-385319 and 1L) that inhibit infection with pseudovirus H5N1 with different potency. In order to compare their different inhibitory activities and shed light on drug design targeting the HA protein, we conducted a variety of theoretical calculations, including docking, molecular dynamics simulations, free energy calculations, as well as quantum calculations to investigate interactions between these two ligands and the HA protein. We found that molecule 1L has stronger π–π interactions with the side chains of residues F110₂ and M24₁ compared with molecule CL-385319. We propose that these stronger π–π interactions are responsible for the higher inhibitory activity of molecule 1L. Our calculations will aid drug design studies targeting the HA protein.

Induction and activity of class II-restricted, Lyt-2+ cytolytic T lymphocytes specific for the influenza H5 hemagglutinin

In influenza A virus infections, CTL are a significant component of the host immune response which limits viral replication and promotes recovery. To examine the CTL response to the influenza virus A/Ty/Ont/7732/66[H5N9], particularly the H5 hemagglutinin, a long term CTL line was generated from spleen cells of A/Ty/Ont-immune Balb/c [H-2d] mice secondarily stimulated in vitro with A/Ty/Cal/Hurst-2/71[H5N2]. This CTL line was highly specific for influenza viruses of the H5 subtype. From this line, clones were isolated by limiting dilution and shown to be H5 hemagglutinin-specific based on recognition of an H5 vaccinia virus recombinant (H5 Vac). The clones exhibited the classical CTL surface phenotype Lyt-1-2+L3T4-; however, unlike the typically class I-restricted Lyt-2+ CTL, they were restricted in antigen recognition by class II (I-E) MHC molecules based on target cell recognition and antibody blocking of cytotoxicity. The clones recognized both infectious and non-infectious A/Ty/Ont presented by class II+ target cells. In adoptive transfer studies to assess the biologic role of the clones in vivo, these class II-restricted clones did not appear to alter mortality. However, these cells significantly reduced both morbidity and virus titers in the lungs of infected animals at 5 days post-infection. Thus, in the immune response to this virus, class II-restricted Lyt-2+ CTL specific for the H5 hemagglutinin were readily generated and their biologic role in vivo involved viral clearance.     

Genetic control and fine specificity of the immune response to a synthetic peptide of influenza virus hemagglutinin.

The immune response to a synthetic peptide, H3 HA1(305-328), representing the C'-terminal 24 amino acid residues of the HA1 chain of the hemagglutinin of the H3 subtype of influenza virus is controlled by genes in the I region of the major histocompatibility complex. Mice of the H-2d haplotype are high responders and produce antibody for several months after a single injection of peptide without carrier. Mice of the H-2b, H-2k, and H-2q haplotypes are low antibody responders. Investigation of recombinant and congenic mouse strains revealed that high responsiveness requires the genes that encode the I-Ed molecule. Immunoassays, involving direct binding to analogs of this peptide and inhibition by both these analogs and synthetic epitopes, were used to analyze the specificity of the polyclonal response. In BALB/c mice, the primary antibody response is directed principally against the antigenic site 314-LKLAT-318, whereas the secondary response after a boost is predominantly directed to a distinct site, 320-MRNVPEKQT-328. The T-cell response to the peptide H3 HA1(305-328), as measured by antigen-induced proliferation of primed T cells in vitro, is also I-Ed restricted in high-responder H-2d mice and is directed against an antigenic site that does not require the four C-terminal residues unique to the H3 influenza subtype. A different epitope appears to be recognized by T cells from CBA (H-2k) mice, which proliferate to a moderate extent on exposure to the peptide but, nevertheless, do not provide help for an antibody response.     

Immunogenicity of loop-structured short synthetic peptides mimicking the antigenic site A of influenza virus hemagglutinin

In order to assess the relevance of conformation for the antigenic site A of the hemagglutinin of influenza virus we synthesized two peptides, comprising two variant sequences of the central part of site A (amino acids 140 - 146 of subunit HA1) inserted into an artificial peptide skeleton, which imposes a loop-like structure on the respective sequence stretch. Assuming that the loop structure in the synthetic peptides would roughly approximate to the structure of the cognate protein sequence we tried to raise protein-reactive anti-peptide antibodies. The antibodies obtained indeed showed reactivity against influenza virus, although the discriminating specificity with regard to a mutation at position 144 was lost for virus binding in contrast to the highly specific peptide binding. Considering the failures in raising anti-hemagglutinin antibodies against the site A by immunization with short flexible peptide our results support the hypothesis that conformation makes a major contribution to the immunogenic and antigenic characteristics of site A in influenza hemagglutinin.     

T cell clones for antigen selection and lymphokine production in murine Schistosomiasis mansoni.

The role of T cells in immunity to murine schistosomiasis was examined through the use of T cell clones that recognize the live schistosomulum stage of Schistosoma mansoni. T cell clones of three different phenotypes were isolated and expanded into long term culture from lymph nodes of C57B1/6J mice vaccinated with irradiated S. mansoni larvae. They were characterized by surface markers, lymphokine production, and functional assays. The m.w. range of the Ag recognized by one clone was identified through nitrocellulose blotting and confirmed with a preparation of the putative protein made by immunoaffinity purification. All but one of the clones were CD4+, CD5+, Th cells. One clone, 35, produced Il-2 and IFN-gamma and was designated a TH1 clone. The others were designated TH2 clones because of Il-4 production. One clone was CD8+ and failed to show help. Clone 35 recognized live schistosomula and produced Il-2 when presented a 27-kDa protein from nitrocellulose. It proliferated in response to purified Ag. Clone 35 participated along with macrophages to induce up to 98% killing of live schistosomula in vitro. IFN-gamma and TNF-alpha were essential to the killing mechanism whereas Il-1, Il-2, and Il-4 were not required. This study has approached Ag identification for vaccine development from the point of view of T cells and showed that TH1 cells are essential to in vitro macrophage killing of schistosomula in murine schistosomiasis.     

T cell receptor junctional regions and the MHC molecule affect the recognition of antigenic peptides by T cell clones.

Nine independent pigeon cytochrome c-specific T cell clones were analyzed by using a panel of antigenic peptide analogs presented in association with three allelic IE-encoded MHC glycoproteins. Eight of the T cell clones expressed a TCR composed of a unique alpha- and beta-chain amino acid sequence, and concordantly, each of these T cell clones exhibited a unique Ag specificity. This was true for several clones which differed only in TCR V-J junctional regions. Interestingly, for a given clone, the response to some of the peptide analogs depended to a large extent on the allelic form of the presenting MHC molecule. A simple interpretation of these data would suggest that certain positions of the peptide Ag are most important for Ag-MHC molecule interactions, and that these specific interactions can influence the antigenic epitope recognized by the TCR. We suggest that an antigenic peptide binds to an MHC glycoprotein in a distinct way, but may retain a measure of flexibility.     

The interaction of nominal antigen with T cell antigen receptors. I. Specific binding of multivalent nominal antigen to cytolytic T cell clones

In this report, we describe an experimental strategy for analyzing the interaction of nominal antigen with antigen-specific T cell clones. Our approach was based on the notion that low affinity interactions between nominal antigen and T cell antigen receptors might be detected by using a highly multivalent form of the antigen in which a large number of identical, appropriately spaced epitopes are attached to a polymer backbone. Antigens of this kind should be capable of multivalent binding to receptors on the T cell, resulting in a marked enhancement of the overall avidity of the interaction. To examine this possibility, we established a series of murine cytolytic T cell (Tc) clones specific for the readily detectable hapten fluorescein isothiocyanate (FL). These clones lysed FL-conjugated target cells in an antigen-specific fashion and also showed specificity for target cell MHC gene products. The interaction of these clones with the nominal antigen FL was assessed by flow cytometry, using a series of water-soluble FL-conjugated polymers varying in polymer backbone and FL isomer. High m.w. (600 to 2000 Kd) polymers of acrylamide, dextran, or Ficoll conjugated with 300 to 800 FL groups/molecule bound specifically to anti-FL Tc clones. There was little binding to syngeneic spleen cells, thymocytes, noncytolytic T cell clones, or T cell clones specific for other haptens such as NIP. Polymer concentrations in the 1 to 10 micrograms/ml range produced readily detectable binding within minutes at 20 degrees C, and the binding approached plateau levels at polymer concentrations of between 100 and 300 micrograms/ml. Studies with closely related FL isomers showed that the same antigen fine specificity was operative in both lysis of FL-conjugated target cells and in binding of FL-conjugated polymers. The functional significance of the observed binding was assessed by measuring the effect of FL-conjugated polymers on lymphokine secretion by the clones. High m.w. FL-conjugated polymers caused a dose-dependent increase in the production of macrophage activation factor (MAF) by anti-FL Tc clones, but did not increase MAF production by an NIP-specific clone. In contrast, concanavalin A induced MAF production by both FL-specific and NIP-specific clones. Thus, the observed binding is both specific and functionally significant. These results suggest that soluble nominal antigen, in an appropriately multivalent form, can bind specifically to antigen receptors on Tc clones.     

Heterogeneity among human T cell clones recognizing an HLA-DR4,Dw4-restricted epitope from the 18-kDa antigen of Mycobacterium leprae defined by synthetic peptides.

Synthetic peptides have been used to exactly define a T cell epitope region from the immunogenic 18-kDa protein of Mycobacterium leprae. Four M. leprae reactive CD4+ T cell clones, isolated from two healthy individuals vaccinated with killed M. leprae, recognized a determinant initially defined by the peptide (38-50). However, fine mapping of the minimal sequence required for T cell recognition revealed heterogeneity among the T cell clones with regard to the N- and carboxyl-terminal boundaries of the epitopes recognized. MHC restriction analysis showed that the immunogenic peptides were presented to the T cells in an HLA-DR4,Dw4-restricted manner in all cases. The results suggest that a polyclonal T cell response representing different fine specificities is directed toward a possible immunodominant epitope from the M. leprae 18-kDa Ag in individuals carrying this MHC haplotype.     

Antigen-specific human T lymphocyte clones: induction, antigen specificity, and MHC restriction of influenza virus-immune clones

Human peripheral blood lymphocytes from an HLA-Dw1,3 individual were primed in vitro with influenza A virus (A/Texas/1-77/x-49) and subsequently cloned by limiting dilution in TCGF. Of the 96 TLCs originally obtained, nine were characterized in detail. TLCs were antigen specific, responding to influenza A virus, not to influenza B, TGAL, GAT, tetanus toxoid, or KLH, and only when antigen was presented by cells unable to form rosettes with AET-treated SRBC. Presentation of antigen by unseparated PBL often resulted in significant "back stimulation," probably via production of growth factors. The MHC requirements for the induction of TLC proliferation were analyzed. Of four representative clones analyzed, three required Dw1;DR1 compatibility for successful presentation of viral antigens by a panel of antigen-presenting cells. In contrast, one TLC showed an unusual pattern of response that could not be correlated to a particular HLA haplotype. Monoclonal anti-T cell antibody analysis of the surface phenotype of two TLCs maintained in continuous culture for 5 mo indicated that they were OKT3+, 4+, and 8-, consistent with an inducer/helper phenotype. To confirm the clonal nature of TLCs, data on the functional properties of TLC subclones are also presented.     

Influenza virus-specific human cytotoxic T cell clones: Heterogeneity in antigenic specificity and restriction by class II MHC products

Human cytotoxic T lymphocytes specific for A/JAP/57 (H2N2) influenza virus were cloned from in vitro stimulations of peripheral blood lymphocytes. Analysis of the viral specificity in cytotoxic function revealed one clone that killed all type A influenza-infected targets, another clone that was specific for the hemagglutinin subtype of the immunizing influenza virus, and the third clone that demonstrated cytotoxicity restricted to the hemagglutinin of A/JAP/57 and A/JAP/62 (H2N2) and not other type A influenza strains with the H2N2 subtypes. The phenotype of these three clones was Leu 2^?, Leu 3⁺, Leu 4⁺; MHC restriction of their cytotoxic function was mapped to HLA-DR by a panel of target cells as well as by inhibition of cytotoxicity with monoclonal antibodies. Proliferation of these clones, examined in a tritiated thymidine incorporation assay, was found to be driven by antigen in the absence of exogenous lymphokines. For all three clones antigen-dependent production and secretion of lymphokines with IL-2 activity was demonstrated. The antigen specificity of proliferation and factor production was shown to be identical to the pattern that each clone revealed in its cytotoxic function.     

Membrane fusion activity of the influenza virus hemagglutinin: interaction of HA2 N-terminal peptides with phospholipid vesicles

We have investigated the interaction of a number of synthetic 20-residue peptides, corresponding to the HA2 N-terminus of the influenza virus hemagglutinin (X31 strain), with phospholipid vesicles and monolayers. Besides the wild-type sequence, two peptides were studied with mutations corresponding to those previously studied in entire HA's expressed in transfected cells [Gething et al., (1986) J. Cell. Biol. 102, 11-23]. These mutations comprised a single Glu replacement for Gly at the N-terminus ("El" mutant) or at position 4 ("E4") of the HA2 subunit and were shown to produce striking alterations in virus-induced hemolysis and syncytia formation, especially for E1. The X31 "wild-type" (wt) peptide and its E4 variant are shown here to have the capacity to insert into phosphatidylcholine (POPC) large unilamellar vesicle (LUV) membranes in a strictly pH-dependent manner, penetration being marginal at pH 7.4 and significant at pH 5.0. Bilayer insertion was evident from a shift in the intrinsic Trp fluorescence of the wt and E4 peptides and from the induction of calcein leakage from POPC LUV and correlated well with the peptides' ability at pH 5.0 to penetrate into POPC monolayers at initial surface pressures higher than 30 mN/m. By contrast, the E1 peptide was found, at pH 5.0, to bind less tightly to vesicles (assessed by a physical separation method) and to cause much less leakage of POPC LUV than the wt, even under conditions where the peptides were bound to approximately the same extent. Consistent with the correlation between leakage and penetration observed for the wt peptide at pH 5 versus 7, the E1 peptide, even at low pH, showed much less lipid-vesicle-induced shift of its Trp fluorescence than wt, caused a much slower rate of leakage of vesicle contents, and did not insert into POPC monolayers at surface pressures beyond 28.5 mN/m. Circular dichroism spectroscopy measurements of peptides in POPC SUV showed that the conformations of all three peptides are sensitive to pH, but only the wt and E4 peptides became predominantly alpha-helical at acid pH.