John E. Powell

A method for preparing β-hCG COOH peptide-carrier conjugates of predictable composition

Abstract A method for coupling peptides related to the beta subunit of human chorionic gonadotropin (hCG) to macromolecular carriers that permits covalent conjugation in a predictable fashion was developed. Peptides representing hCG beta subunit residues 109–145 and 111–145 were coupled to tetanus toxoid, flagellin, synthetic polypeptides and Ficoll. Carrier compounds containing amino groups but devoid of sulfhydryl groups were reacted with 6-maleimido caproic acyi N -hydroxy succinimide ester (MCS) under conditions that result in the bifunctional reagent attached to carrier amino groups with the stable maleimido group free. Subsequently, peptides containing sulfhydryl groups were reacted with the reagent modified carrier whereby the peptides coupled to the carrier via the reaction between sulfhydryl groups on peptide and the maleimido group on carriers. Peptides devoid of sulfhydryl groups were thiolated using homocysteine thiolactone. The efficiency of coupling was confirmed by amino acid analysis and SDS polyacrylamide electrophoresis. Results indicated that the coupling of peptides to carriers could be regulated by the number of moles of MCS reacted with carrier to yield free maleimido groups. Sulfhydryl group reaction with maleimido groups was stoichiometric. Conjugates prepared by this method were used to immunize rabbits and significant levels of antibodies to the hCG peptides have been attained.

Preparation and Formulation of a Human Chorionic Gonadotropin Antifertility Vaccine: Selection of Adjuvant and Vehicle*

ABSTRACT: Peptides representing sequences of the beta subunit of HCG (Cys‐(Pro)6‐111–145 and 109–145) were coupled to tetanus toxoid (TT) and the resultant conjugates used to test the ability of eight experimental adjuvants and six different vehicles for enhancing antibody responses to the peptides in inbred strains of mice (C3H/He, C57BL/6, DBA/1, and SJL) and outbred rabbits. Antibody levels reactive to both 125I‐peptides and 125I‐HCG were used as criteria of efficacy of these adjuvants and vehicles. In most experiments, groups of animals were immunized with conjugates in complete Freunds adjuvant (CFA) for comparison. Two hydrophilic adjuvants‐NAc‐nor‐Mur‐L.ala‐D.isoGln, CGP 11,637 and NGlycol‐nor‐Mur‐L.α‐abu‐D.isoGln, DT‐1‐were consistently the most effective in stimulating antibody production to both peptides and HCG. An emulsion containing squalene‐water stabilized with arlacel A proved to be superior to other vehicles tested for administering conjugates and adjuvants to animals. Antibody levels produced by animals with different genetic backgrounds were shown to be polymorphic with respect to experimental adjuvants, but responses among groups using the same adjuvant and different vehicles were more consistent. Data were obtained suggesting that the coupling of a peptide antigen and a peptide adjuvant to the same carrier holds promise as a model for vaccine development.

The identification of peptide sequences of human chorionic gonadotropin containing a conformational epitope

A series of overlapping peptides were synthesized representing the entire amino acid sequence of the beta-subunit of human chorionic gonadotropin (hCG) and these were reacted with a monoclonal antibody shown to be specific for hCG. One linear peptide (residues 40-52 of the sequence) reacted significantly with the monoclonal antibody but a conjugate of this peptide to diphtheria toxoid (DT) failed to elicit significant levels of antibodies reactive to hCG in rabbits. The subsequent preparation of an extended peptide (residues 38-57) in which the two cysteines were oxidized to form a loop peptide yielded a highly immunogenic antigen when conjugated to DT. Antibody levels reactive with hCG from loop peptide immunizations of rabbits exceeded those found after immunization with a 37 residue peptide representing the carboxyl terminus of the beta-hCG subunit. The antisera did not react with pituitary glycoprotein hormones with similar sequences.

Effects of steroid hormones in fetal bovine serum on plating and cloning of human cells in vitro

SummaryFetal bovine sera from each of three different commercial sources were tested for their ability to support cloning of human fibroblastoid cells in vitro. Cloning efficiencies varied according to serum source. Serum (10 samples) from company A did not support growth, while sera (10 samples) from companies B and C provided adequate to excellent conditions for cloning and growth. Cells from neonatal foreskin or embryonic lung responded to each serum similarly. Bovine serum albumin type H7 from company C supported cell growth in media without serum.Sera containing 1.0 ng per ml or more of progesterone inhibited growth, whereas sera containing less than 1.0 ng per ml supported cloning and growth. In the low progesterone sera, the concentration of 17-β-estradiol exceeded 100 pg per ml. Growth supporting sera could be made non-supportive by adding 0.1 μg per ml of progesterone. The addition to non-supportive sera of 0.1 μg per ml of 17-β-estradiol or hydrocortisone made these sera supportive of cell growth.Addition of estrogen or hydrocortisone to a culture medium that inhibits growth, with subsequent reversal of the inhibitory effect, implies that these hormones competitively regulate growth of responsive cells in vitro.

Preparation and Formulation of a Human Chorionic Gonadotropin Antifertility Vaccine: Selection of a Peptide Immunogen*

ABSTRACT: Peptides representing the amino acid sequence of the carboxy‐terminal of the human chorionic gonadotropin (HCG) beta subunit were used in studies to determine whether an immunogen could be prepared that would be suitable for an HCG antifertility vaccine. Peptides of varying length were conjugated to several macro‐molecular carriers, in varying peptide‐carrier ratios, via an N‐ or C‐terminal group, with and without amino acid spacers. Antibody levels to peptides and intact HCG were used as the criteria for conjugate utility. Immunizations with a peptide of 37 amino acid residues elicited the highest antibody levels to HCG. No differences were found between N‐ or C‐terminal conjugates, but peptides with amino acid spacers elicited higher responses than peptides without spacer. Conjugates with a peptide:carrier ratio greater than 20 peptides/105 daltons carrier were more immunogenic than those with a lower ratio. Diphtheria and tetanus toxoids were consistently the most effective carriers for enhancing antibody responses to peptides. It was concluded that conjugates of β‐HCG peptide 109–145 coupled to a toxoid carrier via its N‐terminus in a ratio of 20–30 peptides per 105 daltons carrier was a suitable immunogen for further studies for the development of an anti‐fertility vaccine.

Characteristics of antibodies raised to carboxy-terminal peptides of hCG beta subunit

Abstract Natural and synthetic peptides representing COOH terminal sequences of the beta subunit of human chorionic gonadotropin (hCG) were coupled to tetanus toxoid and rabbits immunized with the conjugates. Sera were evaluated for antibody levels, antibody affinity and antibody class. Conditions for appropriate estimation of these parameters were also studied. Findings revealed that iodination of hCG or peptides did not alter their immunological properties and that reaction of labelled antigens with antisera require differing periods of time for the establishment of equilibrium. Peak titers to the hCG antigens were reached at 70 days of immunization. Antibodies to natural peptide 109–145 of β-hCG and synthetic peptides 109–145, 111–145, and 115–145 reacted to hCG approximately 90% as well as they reacted to the peptide used for immunizations whereas antibodies to synthetic peptide 125–145 reacted only 60–70% as well. Mean antibody affinities to peptides were not significantly higher than affinities of the same sera to hCG. The affinity of antisera to synthetic peptide 125–145 to both antigens and the mean ratio of hCG affinity : peptide affinity was somewhat lower than those to longer peptides. After 70 days of immunization, antibodies were predominantly of the IgG class. The findings indicate that antibodies raised to natural or synthetic peptides of the COOH region of hCG beta subunit are highly cross-reactive to native hCG but have affinities somewhat lower than those generated to the intact hormone or its beta subunit.

Serum levels of a placental protein during gestation in the baboon

A placental specific protein (Sp-1) isolated from human placentas was used to establish a radioimmunoassay for measurement of a baboon protein in the sera of pregnant baboons. No reaction was observed with any known pituitary or placental hormone in this assay system. Levels of Sp-1 were detected at 17 to 18 days of gestation and reached high levels by the end of the first trimester of pregnancy. A slow increase in serum levels continued thereafter until parturition. Disappearance of Sp-1 post partum was very slow and required 26 to 34 days to become undetectable in the serum. No function or role of Sp-1 in pregnancy has been suggested, but possible use of its measurement for detecting abnormal placental function deserves investigation.

Characterization of solubilized microvillous membrane proteins and glycoproteins from human placental syncytiotrophoblast

Microvillous membrane fractions from human term placentae were prepared by differential centrifugation. Extration of membranes with PBS-EDTA or KCI removed soluble cytoplasmic components and serum proteins excepting trace amounts of albumin and transferrin. PAGE-SDS revealed 11 components in the Triton solubilized crude fraction after PBS-EDTA extraction. Membrane components solubilized with Triton were not fractionated by gel filtration on Bio-Gel A-50 m but DEAE-cellulose chromatography partially resolved these components. Three fractions were obtained by stepwise elution of absorbed materials using increasing concentrations of NaCl in the equilibrating buffer. These fractions were characterized using SDS-PAGE. The material unabsorbed to the DEAE contained two components of small molecular weight and one of them showed a positive PAS stain. The first eluted protein peak showed nine components, seven of which stained with PAS. The bulk of glycoproteins with molecular weights greater than 130 000 daltons were found in this fraction. The second eluted peak from DEAE was rich in components with molecular weights less than 42 000 daltons. Four components in this fraction were not identified in the other two ion-exchange fractions. Bands representing mobilities of albumin, transferrin and alkaline phosphatase were observed in DEAE-cellulose fractions; however, 12 components of unknown structure were revealed.

THE ROLE OF CARBOXY-TERMINAL PORTION OF BETA SUBUNIT OF HUMAN CHORIONIC GONADOTROPIN IN HUMAN IMMUNODEFICIENCY VIRUS INFECTION

Human chorionic gonadotropin (hCG) and the beta subunit of this dimer glycoprotein hormone (beta hCG) have been reported by us to inhibit HIV replication. In order to identify the active site responsible for the antiviral activity, twelve overlapping peptides spanning across beta hCG were examined for their effect against HIV-caused cell death. Although the NH2-terminus of beta hCG appeared to contribute to activity, the core region was biologically inert. The most potent activity was observed with the fragment representing the carboxy-terminus of beta hCG. The dose response curve to serial dilutions of the peptide, containing amino acid residues 106-145, had a bell-shaped appearance - characteristic of hCG and beta hCG. The peak of activity corresponded to 100 ng/ml - the dose at which two thirds of virus-exposed MT-4 T lymphocytes survived. None of the tested peptides were toxic to MT-4. While the mechanism of action remains unclear, the results suggest that the COOH-terminal portion, unique to beta hCG, confers anti-HIV activity.

Folding and immunogenicity of zinc-finger peptide constructs corresponding to loop regions of the protein antigens LDH-C4 and β-hCG

This paper describes our continuing studies on stabilization of peptide structures in supersecondary conformations that are designed to mimic conformational antigenic epitopes. In this work we have used the consensus Cys2His2 zinc-finger peptide motif as a template to engineer and synthesize antigenic loop peptide segments from two protein antigens, lactate dehydrogenase C4 isozyme (LDH-C4) and human chorionic gonadotropin β subunit (β-hCG). Confirmation that the engineered peptide constructs assumed a zinc-finger conformation was obtained by absorption spectroscopy of the Co2+ complexes. The circular dichroism (CD) spectra of the free peptides show random coil conformations, while the Zn2+-complexed peptides acquired the zinc-finger motif upon titration with Zn2+, as evidenced by the appearance of absorbances indicating α-helix and some β-conformation. No peptide aggregation was observed, as these peptides were monomeric under all conditions tested. In order to examine the immunogenicity of the zinc-finger constructs, one sequence from LDH-C4 (ZFLMVF) and two sequences from β-hCG (ZF2TT3 and ZF4TT3) were selected and chimeras were synthesized to incorporate promiscuous T-cell epitopes from either tetanus toxoid or measles virus. The ZFLMVF construct was highly immunogenic in rabbits, and the ZF2TT3 and ZF4TT3 peptides were highly immunogenic in both mice and rabbits, eliciting high-titer antipeptide antibodies specific for their immunogenic sequences. However, the antibodies raised to the zinc-finger constructs showed minimal reactivity against their respective native protein antigens as determined by ELISA. This is surprising in the case of β-hCG, since the ZF2 zinc-finger peptide was an effective inhibitor of binding of anti-β-hCG-loop(38–57) antibodies to whole hCG, as assessed by a competitive inhibition radioimmunoassay. This implies that, although the cyclized 40–52 sequence from βhCG and the zinc-finger peptide ZF2 exhibit similar conformations in solution, the zinc-finger engineered loop is apparently not in a sufficiently correct conformation for antibody recognition of native hCG. Our results with the LDH-C4 zinc finger loop imply that antibody recognition of antigen involves specific side-chain interactions that must be maintained by a precise conformation.