Dependence of the murine antibody response to an anti-CDR2 VH peptide on immunogen formulation.

A peptide corresponding to the second complementarity determining region of the heavy chain (CDR2 VH) from a murine anti-CD4 monoclonal antibody, designated L202, was synthesized by solid phase methodology in a number of different antigenic forms, for the purpose of comparing the effectiveness of different adjuvant-carrier systems in the induction of a murine antibody response against the immunizing peptide and parent antibody molecule. Two of the synthetic constructs contained the palmitoyl and N-palmitoyl-cysteinyl-S-(2,3-palmitoyloxy)-propanediol (PAM3Cys) moieties, respectively, attached to the peptide amino terminus with the immunogen comprising liposomal formulations of each. A third immunogen consisted of the CDR2 VH peptide admixed with the PAM3Cys non-covalently and incorporated into liposomes (PAM3Cys + CDR2 VH). A fourth composition comprised the CDR2 VH peptide conjugated to KLH via the sulfhydryl of an added N terminal cysteine (KLH-CDR2 VH) and injected with Complete Freund's adjuvant (CFA). A fifth immunogen consisted of the CDR2 VH peptide synthesized on an octameric, branched polylysine core as a multiple antigenic peptide (MAP-CDR2 VH) injected in the presence of Freund's adjuvant. Groups of five mice were injected intramuscularly with each of these immunogens and bled at two week intervals. The highest anti-peptide gamma-immunoglobulin (IgG) responses (against uncoupled peptide by ELISA) after 56 days were obtained with mice receiving the PAM3Cys-CDR2 VH peptide. However, when screened against the CDR2 V(H) peptide present as the MAP derivative by ELISA, IgG raised against the cognate MAP-CDR2 peptide was much more reactive than IgG raised against the liposomal PAM3Cys-CDR2 VH immunogen. In either case, IgG raised against the KLH-CDR2VH conjugate was poorly reactive. These differences in reactivity to the two forms of the CDR2 VH peptide by ELISA did not correspond to major differences in reactivities to the intact L202 Ab by ELISA. Although the IgG against the MAP immunogen was slightly more reactive than the other antisera against the l202 Ab, all titers were less than 1:100. These data illustrate some limitations of using anti-peptide responses as indicators of potential reactivity against the native protein, but suggest that alternate formulations including lipoidal peptides are more effective than corresponding KLH-peptide conjugates in eliciting Ab responses against poorly immunogenic epitopes.

Fine specificity of the murine antibody response to HIV-1 gp160 determined by synthetic peptides which define selected epitopes.

In this report, we assess the humoral immune response in inbred strains of mice immunized with baculovirus-derived recombinant HIV-1 gp160 (rgp160). Six inbred strains of mice were each immunized with two different concns (5 and 50 micrograms) of rgp160, and the antibody response to rgp160 and synthetic peptides which define distinct gp160 epitopes was examined. Within a given inbred strain of mice, no significant difference in antibody titers to gp160 was observed in those groups receiving either 5 or 50 micrograms of rgp160 per injection. Following three immunizations with rgp160, differences in anti-gp160 titers were observed among the various inbred strains; however, these differences became less apparent after additional injections with rgp160. In addition, each mouse strain exhibited a unique reactivity pattern to seven gp160 epitopes defined by synthetic peptides. Multiple injections with rpg160 were required to induce responses to certain gp160 epitopes. The observed differences in the fine specificity of the humoral immune response to distinct gp160 epitopes among the six inbred strains suggest a genetic basis for regulating the antibody response to these epitopes. This apparent regulation can be overcome by multiple injections with rgp160.

A simplified method for determination of peptide-protein molar ratios using amino acid analysis.

In this report, we have described methods to improve the efficiency of coupling synthetic peptides to keyhole limpet hemocyanin (KLH) and for the analysis of the composition of the resulting peptide-protein conjugates. KLH was first dissolved in buffers containing 3 M guanidine hydrochloride to maintain solubility and derivatized with either of two water soluble, heterobifunctional crosslinkers, m-maleimido-benzoyl-N-hydroxy-sulfosuccinimide ester (SMBS), or sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SSMCC) (300:1 molar excess over KLH). Synthetic peptides containing an amino terminal cysteine were then crosslinked to the modified KLH via sulfhydryl reaction with the crosslinker maleimide groups. Following dialysis to remove free peptide, the amino acid composition of the conjugate was determined. The molar ratio of peptide to protein within the conjugate was obtained by comparing the conjugate composition with that of both the KLH and peptide analyzed separately, and by a multiple regression, least squares analysis of the data. This method is generally applicable to the analysis of the molar ratios of protein-protein conjugates of unknown sequence or composition, and requires only the prior determination of the experimental amino acid composition of each component of the conjugate separately.

Structure-activity relationships of cysteine-lacking pentapeptide derivatives that inhibit ras farnesyltransferase.

Mutational activation of ras has been found in many types of human cancers, including a greater than 50% incidence in colon and about 90% in pancreatic carcinomas. The activity of both native and oncogenic ras proteins requires a series of post-translational processing steps. The first event in this process is the farnesylation of a cysteine residue located in the fourth position from the carboxyl terminus of the ras protein, catalyzed by the enzyme farnesyltransferase (FTase). Inhibitors of FTase are potential candidates for development as antitumor agents. Through a high-volume screening program, the pentapeptide derivative PD083176 (1), Cbz-His-Tyr(OBn)-Ser(OBn)-Trp-DAla-NH2, was identified as an inhibitor of rat brain FTase, with an IC50 of 20 nM. Structure-activity relationships were carried out to determine the importance of the side chain and chirality of each residue. This investigation led to a series of potent FTase inhibitors which lack a cysteine residue as found in the ras peptide substrate. The parent compound (1) inhibited the insulin-induced maturation of Xenopus oocytes (concentration: 5 pmol/oocyte), a process which is dependent on the activation of the ras pathway.

Inhibition of lymphokine-activated killer activity during HIV infection: role of HIV-1 gp41 synthetic peptides.

Lymphokine-activated killer (LAK) activity was analyzed in 31 human immune deficiency virus 1 (HIV-1)-infected patients. It was found to be reduced in all groups of patients, being more pronounced in those with acquired immune deficiency syndrome (AIDS) and AIDS-related complex compared to HIV-1-seropositive, asymptomatic individuals. Only high doses of interleukin-2 were able to restore LAK activity comparable to that of normal controls. In addition, HIV-1 gp41 synthetic peptide sequences 735-752 and 846-860 were able to significantly inhibit normal LAK activity at all the effector:target ratios tested. HIV-1-positive serum and the supernatant fluids from cultured peripheral-blood mononuclear cells from HIV-1-infected patients had the same inhibitory effect on normal LAK activity. These data provide evidence that (1) LAK activity appears to be impaired during the course of HIV-1 infection and (2) HIV-1-positive serum and HIV-1 components could exert a profound inhibition of this functional activity.

Synthetic peptides define the fine specificity of the human immunodeficiency virus (HIV) gp160 humoral immune response in HIV type 1-infected chimpanzees.

The fine specificities of antibodies produced against human immunodeficiency virus type 1 (HIV-1) gp160 were examined in sera from 23 HIV-1-infected chimpanzees. These animals had been infected with one of six isolates of HIV-1. Sera were screened by enzyme-linked immunosorbent assay for reactivity against seven synthetic peptides corresponding to regions of gp160. Chimpanzees appear to remain healthy after infection with HIV-1, suggesting that these animals may prevent extensive spread of the virus in vivo through immunologic mechanisms. Antibody specificity to gp160 epitopes may play a key role in the defense against HIV-1-related disease. Approximately one-half of all chimpanzee sera contained antibodies reactive with peptide 846-860, which corresponds to the carboxyl terminus of gp41. Less than 10% of sera from HIV-1-infected humans that were examined contained antibodies reactive with peptide 846-860, suggesting that this region is not highly immunogenic in humans. Of the human sera containing antibodies reactive with this peptide, all were from individuals classified as Walter Reed stages 1 to 3. No sera from humans with advanced stages of the disease contained antibodies reactive with peptide 846-860. Peptide 600-611, which reportedly reacts with nearly all sera from HIV-infected humans, was reactive with less than one-half of sera from HIV-1-infected chimpanzees. The observed differences in antibody reactivity to gp160 peptides in sera from HIV-1-infected chimpanzees and humans suggest that each may generate antibodies against differing sets of HIV-1 epitopes. These differences may contribute to the lack of disease progression in chimpanzees after infection with HIV-1.