Detergent extraction of herpes simplex virus type 1 glycoprotein D by zwitterionic and non-ionic detergents and purification by ion-exchange high-performance liquid chromatography.

Detergents (surfactants) are the key reagents in the extraction and purification of integral membrane proteins. Zwitterionic and non-ionic detergents were used for the extraction of recombinant glycoprotein D (gD-1) of herpes simplex virus type 1 (HSV-1) from insect cells infected with recombinant baculovirus. The highest yield was obtained with the two alkyl carboxybetaine detergents (N-dodecyl-N,N-dimethylammonio)undecanoate [DDMAU, critical micelle concentration (CMC) = 0.13 mM] and (N-dodecyl-N,N-dimethylammonio)butyrate (DDMAB, CMC = 4.3 mM). Therefore these zwitterionic detergents were used as additives to the elution buffers in ion-exchange high-performance liquid chromatography (HPIEC) to purify gD-1 of HSV-1 from the extracts. The non-ionic detergent pentaethyleneglycol monodecyl ether (C10E5) that was used in earlier studies [R.A. Damhof, M. Feijlbrief, S. Welling-Wester, G.W. Welling, J. Chromatogr. A, 676 (1994) 43] was used for comparison. Two columns were used, Mono Q and Resource Q, at 1 and 5 ml/min flow-rates, respectively. The results show that the detergents DDMAU and C10E5 are superior to DDMAB, when the detergents were used as additives to the elution buffers at 0.2% (w/v). With 0.2% DDMAB in the eluent, purification of HSV gD-1 was not possible. Detergents with a high CMC may be less suitable as additives in elution buffers. HPIEC at flow-rates of 1 and at 5 ml/min showed satisfactory results. At 5 ml/min HSV gD-1 was mainly concentrated in two eluent fractions. The highest recovery of gD-1 was obtained either by chromatography of a C10E5 extract using a Mono Q column at a flow-rate of 1 ml/min or by chromatography of a DDMAU extract using a Resource Q column at a flow-rate of 5 ml/min.

Kinetic analysis of synthetic analogues of linear-epitope peptides of glycoprotein D of herpes simplex virus type 1 by surface plasmon resonance.

The interaction between mAb A16 and glycoprotein D (gD) of herpes simplex virus type 1 was analyzed by studying the kinetics of binding with a surface-plasmon-resonance biosensor. mAb A16 belongs to group VII antibodies, which recognize residues 11-19 of gD. In a previous study, three critical residues, Asp13, Arg16 and Phe17, of this epitope were identified by screening a phage display library that contained a random 15-amino-acid insert with the antibody. The contribution to binding of these residues in the motif DXXRF was further analyzed by an amino-acid-replacement study of the epitope gD-(9-19)-peptide and of a gD-(9-19)-peptide mimotope, previously obtained by screening the phage display library. Amino acid residues of the motif were replaced by a neutral amino acid residue, an amino acid residue with opposite charge and a corresponding D-amino acid residue. Kinetic parameters of peptide analogues were determined with a surface plasmon-resonance biosensor. The kinetic parameters of the peptide analogues were compared with the kinetic parameters of the interaction between mAb A16 and the epitope gD-(9-19)-peptide. The minimal size of the gD epitope for mAb A16 was also determined in this study. The kinetic constants of the resulting gD-(11-17)-peptide were found to be similar to those of entire gD. The kinetic analysis precisely defined the epitope on gD for mAb A16 to residues 11-17, identified Arg16 as an essential residue and suggested that Asp13 and Phe17 are mainly involved in stabilization of the secondary structure of the peptide.

The use of dedicated peptide libraries permits the discovery of high affinity binding peptides.

The motif for peptide binding to monoclonal antibody mAb A16, which is known to be directed against glycoprotein D of Herpes simplex virus type 1, was determined using two dedicated peptide libraries. As a starting point for this study we used an A-16 binding lead sequence, which had previously been obtained from a phage display peptide library (Schellekens et al., 1994). Binding studies with different length variants of this peptide identified a 12mer as a suitable lead compound for our library study. Two incomplete dedicated resin-bound synthetic peptide libraries were generated. Both consisted of 2 x 10(6) 12mers, in which positions were alternately fixed (amino acids identical to the lead sequence) and random. The libraries were screened with mAb A16 and beads with binding peptides were sequenced using Edman degradation. This resulted in a unique peptide binding motif, essentially comprising a 7mer core sequence. Comparison of the sequence of the natural epitope with the binding motif revealed that its sequence was identical to the motif except for one position. Substitution of a methionine in the natural epitope by a tyrosine or a phenylalanine at that position, as dictated by the motif, resulted in a peptide with an affinity for binding to mAb A16 about 50 times higher than that of the natural epitope. Thus, if a lead sequence is available, the use of incomplete, dedicated synthetic peptide libraries provides a fast and powerful tool for the detection of high affinity peptides.

Identification of the core residues of the epitope of a monoclonal antibody raised against glycoprotein D of herpes simplex virus type 1 by screening of a random peptide library.

Random peptide libraries (RPL) displayed on the surface of a filamentous bacteriophage can be used to identify peptide ligands that interact with target molecules. We have used a 15-amino acid residue RPL displayed on bacteriophage M13 to identify the core residues within the epitope of a monoclonal antibody (mAb) A16 which interacts with a continuous epitope restricted to amino acid residues 9 to 19 in the N-terminal region of glycoprotein D of herpes simplex virus type 1 (gD-1). The single peptide sequence obtained after three rounds of selection contained identical residues at three positions compared to the authentic gD-1 sequence. Synthetic peptides were prepared based on the sequence of the original epitope and the phage-derived epitope. The binding constants (Ka) with mAb A16 were determined using surface plasmon resonance (SPR) biosensor technology. The RPL-derived peptide and peptide 9-19 of gD-1 had approximately the same affinity for mAb A16. This suggests that those residues within the epitope that are essential for binding were identified. The synthesis of shorter versions of the RPL-derived peptide restricted the binding region to seven amino acid residues. These results show that minimal information retrieved from the screening of an RPL combined with peptide synthesis can characterize the epitope of an mAb with high resolution. Immunization of mice with the phage-derived peptide protected against a challenge with a lethal dose of herpes simplex virus type 1 equally well as the gD-1 derived peptide.

Purification of the integral membrane glycoproteins D of herpes simplex virus types 1 and 2, produced in the recombinant baculovirus expression system, by ion-exchange high-performance liquid chromatography.

Selective elution of Sendai virus integral membrane proteins by ion-exchange high-performance liquid chromatography (HPIEC) using different detergent concentrations was reported before [S. Welling-Wester, M. Freijlbrief, D.G.A.M. Koedijk, M.A. Braaksma, B.R.K. Douma and G.W. Welling, J. Chromatogr., 646 (1993) 37]. In the present study this novel approach was applied to the purification of the integral membrane glycoprotein D of Herpes simplex virus type 1 and 2. The glycoproteins D of types 1 (gD-1) and 2 (gD-2) were cloned into the baculovirus expression system and produced in protein-free cultured insect cells. Detergent extracts of recombinant baculovirus-infected insect cells containing gD-1 or gD-2 were prepared using pentaethyleneglycol monodecyl ether, for extraction (final concentration 2%, w/v). The same detergent was used as additive in the elution buffers for HPIEC on a Mono Q HR 5/5 column. At low (0.005%) detergent concentration, most of the proteins present in the extract including part of gD were eluted with the sodium chloride gradient whereas a subsequent blank run using the same gradient at higher detergent concentration (0.1%) resulted in selective elution of pure gD.

Chimeric parvovirus B19 capsids for the presentation of foreign epitopes.

Chimeric proteins consisting of the VP2 capsid protein of human parvovirus B19 and defined linear epitopes from human herpes simplex virus type 1 and mouse hepatitis virus A59 inserted at the N-terminus and at a predicted surface region were expressed by recombinant baculoviruses. The chimeric proteins expressed the inserted epitopes and assembled into empty capsids. Immunoelectron microscopy indicated that the epitopes inserted in the loop were exposed on the surface of the chimeric particles. The chimeric capsids were immunogenic in mice and antibodies specific for the inserted sequences were induced. In the case of MHV, antibodies were produced that recognized the epitope in the context of native virus. Mice immunized with the chimeric capsids were partially protected against a lethal challenge infection with either MHV or HSV.

Analogues of peptide 9-21 of glycoprotein D of herpes simplex virus and their binding to group VII monoclonal antibodies.

Several analogues of the amino acid sequence of peptide 9-21 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were synthesized and investigated for reactivity with different group VII monoclonal antibodies, Mabs LP14, ID3, 170, HD4, A16, EII-24 and Ev-10, in a competition enzyme-linked immunosorbent assay (ELISA). Replacement of Arg at position 16 by His resulted in a loss of binding with the group VII Mabs. Substitution of Pro at residue 14 by Leu gave a reduced binding for a number of Mabs and loss of binding for Mab A16. Substitution of Lys at position 10 by Glu gave reduced binding for three out of the seven Mabs. In addition substitutions of Met at position 11 by norleucine and oxidized Met were studied. The boundaries of the epitope cluster were mapped by studying synthetic variants of peptide 9-21 which were shorter either at the C-terminus or at the N-terminus, or both. Peptide 10-18 and peptide 9-17 were the shortest peptides, which were still reactive with the group VII Mabs. Mab HD4 requires the N-terminus of peptide 9-21 for effective binding, while for binding of other Mabs contribution of the residues in the C-terminal part of this peptide is more important.

Comparison of detergents for extraction and ion-exchange high-performance liquid chromatography of Sendai virus membrane proteins.

The integral membrane proteins of Sendai virus, haemagglutinin-neuraminidase (HN) and fusion protein (F) were extracted from purified virions with a non-ionic and two zwitterionic detergents, i.e., pentaethylene glycol monolauryl ether (C12E5), lauryldimethylamine oxide (LDAO) and dodecyldimethylammoniopropane-1-sulphonate (SB12), respectively. The extracts were subjected to ion-exchange high-performance liquid chromatography (HPIEC) using 0.1% of the detergent in the eluent on four different columns (MA7Q, Zorbax BioSeries SAX, Mono Q and PL-SAX) with a quaternary amine as interacting ligand and with different pore sizes: non-porous and 30, 80 nm and 400 nm, respectively. The relative recoveries of protein were similar for all the columns. The highest recovery of HN and F protein and the best separation were obtained with C12E5. Analysis of HPIEC fractions with monoclonal antibodies directed against conformational epitopes showed that C12E5 had less effect on the conformation than the other two detergents.

Reactivity of human sera with overlapping synthetic peptides of herpes simplex virus type 1 glycoprotein D.

Thirty eight human sera, seropositive for herpes simplex virus (HSV) and 56 human sera, seronegative for HSV by immunofluorescence and by ELISA, were investigated for reactivity with a series of overlapping synthetic peptides of HSV type 1 glycoprotein D (gD-1). Thirty four out of the 38 human sera positive for HSV reacted with peptides located between residues 300 and 369; the HSV-negative sera reacted with six of the gD-1 peptides, but with none of the peptides within residues 300 to 369.

Immunological properties of multiple repeats of a linear epitope of herpes simplex virus type 1 glycoprotein D.

Several peptides containing the amino acid sequence 9-21 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were synthesized and investigated for reactivity with monoclonal antibody LP14 in a competition enzyme-linked immunosorbent assay (ELISA). Peptides containing two or four repeats of sequence 9-21 reacted at least one order of magnitude better with LP14 than with the monomeric form of sequence 9-21. Dimers in which one of the repeats of one or more essential residues were absent did not show this increased reactivity. Antisera obtained from rabbits immunized with a peptide containing two repeats of sequence 9-21 coupled to bovine serum albumin showed high antipeptide antibody titers with this peptide and were able to neutralize virus infectivity in vitro. Sera obtained from rabbits immunized with the free dimer could not neutralize virus infectivity.

Antibodies against synthetic peptides of herpes simplex virus type 1 glycoprotein D and their capability to neutralize viral infectivity in vitro.

Peptides corresponding to residues 1-13, 9-21, 18-30, 82-93, 137-150, 181-197, 232-243, 235-243, 267-281, 271-281 and 302-315 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were chemically synthesized. These peptides were coupled to carrier proteins, and the resulting conjugates were used to immunize rabbits. An enzyme-linked immunosorbent assay was used to determine antipeptide antibody titers in serum collected after immunization. All peptides appeared to be immunogenic in rabbits. Western immunoblot analysis with detergent extracts of HSV-1-infected Vero cells showed that antibodies against each of the peptides were able to react with the parent glycoprotein under denaturing conditions. Antisera against peptides 1-13, 9-21, and 18-30 neutralized HSV-1 infectivity in vitro, peptide 9-21 being the most successful in this respect. Immunization with a mixture of peptides 9-21 and 267-281 yielded antisera which reacted strongly with glycoprotein gD in Western blot analysis and showed a more solid virus-neutralizing activity in vitro.

Growth-stimulating effects of somatomedin-/insulin-like peptides in Snell dwarf mice.

The effect of the somatomedin-/insulin-like growth factors IGF-I, IGF-II and N2, as well as of semi-purified SM fractions separated by isoelectric focusing derived from human Cohn IV on different growth parameters, have been studied in the Snell dwarf mouse. HPLC-pure IGF-II, N2 and IGF-I stimulate to a similar extent the sulphate incorporation into costal cartilage, the osteochondral junction and epiphyseal cartilage. After 4 weeks of treatment, increase in body length and weight as well as the weights of several organs is obtained with SM fractions, focusing at acid and neutral pH, and containing mainly IGF-II- and less than 5% IGF-I-like peptides. Fractions containing mainly IGF-I-like peptides and focusing at basic pH at the dosage used seem to be less stimulatory on most of these parameters. The rump/tail ratio and weight/length ratio is comparable to that obtained after treatment with human growth hormone (hGH). hGH induced a significant stimulation of the weight of the liver, kidneys, heart, thymus and spleen. The acid and neutral SM fractions induced growth of the liver, kidneys and spleen. The basic fractions only produced a significant weight gain in kidneys and spleen. The skinfold thickness is stimulated by the SM preparations and only slightly by hGH.

Structural properties and reactivity of N-terminal synthetic peptides of herpes simplex virus type 1 glycoprotein D by using antipeptide antibodies and group VII monoclonal antibodies.

To investigate the contribution of individual amino acids to the antigenicity of the N-terminal region of herpes simplex virus type 1 glycoprotein D, a series of 14 overlapping synthetic peptides within residues 1 to 30 were examined for their reactivity with monoclonal antibody LP14 (a group VII monoclonal antibody; in herpes simplex virus mutants resistant to LP14, arginine 16 is substituted by histidine) and two antipeptide antisera (antipeptide 9-21 and antipeptide 1-23). Maximal binding was achieved with peptides 9-21, 10-30, 9-30, and 8-30 and the chymotryptic fragment 9-17 of peptide 9-21, suggesting that a major antigenic site is located within residues 10 through 17. Lysine 10 was shown to be essential for high reactivity, either by binding directly to the antibody molecule or by stabilizing an ordered structure of the peptide. The importance of ordered structure was demonstrated by a decrease in reactivity after sodium dodecyl sulfate treatment of peptides 9-21 and 8-30.

Growth and differentiation of pluripotent embryonal carcinoma cells in the Snell dwarf mouse.

To investigate the influence of hormones on the process of cellular differentiation the growth and differentiation of a transplantable tumour, induced by inoculation of pluripotent mouse embryonal carcinoma (EC) cells have been studied in athymic nude mice and, normal and hypopituitary Snell dwarf mice. All athymic nude mice developed tumours independent of the numbers of cells inoculated. In contrast, the tumour percentage in normal Snell mice was lower, showing a dose-dependent increase of takes. In dwarfs tumour percentage was comparable with that observed in normal Snell mice. The morphological differentiation of teratocarcinomas grown in athymic nude mice, normal and dwarfed Snell mice shows derivatives of all three germ layers next to undifferentiated embryonal carcinoma cells. This suggests that the pituitary hormonal deficiencies of the dwarfs (growth hormone, thyroid stimulating hormone and prolactin) did not influence the tumour induction nor the development of the different tissues present in this type of tumour.