Natural infection with herpes simplex virus type 1 (HSV-1) induces humoral and T cell responses to the HSV-1 glycoprotein H:L complex

The glycoproteins of herpes simplex virus type 1 (HSV-1) are important targets for the immune system in the control of HSV-1 infections. The humoral and T cell responses to the glycoprotein (g)H(t(His)):gL complex of HSV-1 were st

Size-exclusion high-performance liquid chromatography of integral membrane proteins: Effect of detergents on immunological activity

A mixture of integral membrane proteins from Sendai virus was used as a model to study the effect of various detergents and organic solvent on the purification by size-exclusion high-performance liquid chromatography (HPLC) on two tandemly linked Superose-6 columns. The best separations were obtained with either 0.1% sodium dodecyl sulphate, 0.05% sarkosyl or 0.1% lauryldimethylamineoxide in the eluent. In addition, the effect of the eluent on the immunological activity as a measure of intact structure was studied. A considerable part of the protein structure remained intact after HPLC and was able to react with antibodies directed against the intact virus protein

The influence of different adjuvants on the immune response to a synthetic peptide comprising amino acid residues 9–21 of herpes simplex virus type 1 glycoprotein D

The immuno-modulating properties of different adjuvant systems on the murine humoral and cellular immune response to a synthetic peptide comprising amino acid residues 9–21 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were investigated. For immunization, the peptide was conjugated to ovalbumin or bovine serum albumin by glutaraldehyde and the adjuvants used in this study were Freund's complete adjuvant (FCA), aluminium hydroxide, the Ribi adjuvant system (RAS) and two non-ionic block polymer surfactants, viz. L101 and 31R1, in oil in water emulsions. High anti-peptide antibody titers were obtained after immunization with FCA, aluminium hydroxide, RAS and L101. All adjuvants, except RAS, stimulated the induction of delayed type hypersensitivity obtained after immunization with peptide 9–21 coupled to ovalbumin and elicited by injection of purified HSV-1 virions in the footpad. Challenge with a lethal dose of HSV-1 showed that mice immunized with peptide 9–21 coupled to ovalbumin in combination with FCA, RAS and L101, respectively, were significantly protected. Although immunization with peptide 9–21 coupled to ovalbumin combined with aluminium hydroxide stimulated induction of delayed type hypersensitivity, no significant protective immunity against the challenge was generate

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 (C(10)E(5)) 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 C(10)E(5) 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 C(10)E(5) 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. (C) 1998 Elsevier Science B.V. All rights reserved

Effect of different amounts of the non-ionic detergents C10E5 and C12E5 present in eluents for ion-exchange high-performance liquid chromatography of integral membrane proteins of Sendai virus

Non-ionic detergents (0.03-0.5%) are used as additives to the eluents when integral membrane proteins are subjected to ion-exchange high-performance liquid chromatography (HPIEC). It is not known whether this concentration should bear some relation to the critical micelle concentration (CMC) of a detergent (the concentration at which micelles begin to form) or that only the amount of detergent is of importance in order to maintain the membrane proteins in solution. This was investigated with a detergent extract of Sendai virus which contains two integral membrane proteins, the fusion protein and the haemagglutinin-neuraminidase protein. Two polyoxyethylene alkyl ethers (C10E5 and C12E5) were used both for extraction (2% final concentration) and as additives in the elution buffers for HPIEC on Mono Q with ''classical'' HPLC and the micro-HPLC Smart System (Pharmacia-LKB). The CMCs of the two non-ionic detergents C10E5 and C12E5 are 0.026 and 0.002%, respectively. Concentrations below and above the CMC were used in the eluent. The results showed that the concentration of the detergent should be 2-26 times the CMC in order to avoid aggregation. The integral membrane proteins of Sendai virus remain on the column when the detergent concentration is less than 0.026-0.05%, independent of the CMC of the detergent. This may be utilized in HPIEC strategies: at low detergent concentration, hydrophilic proteins are eluted with the salt gradient and a subsequent blank run with the same gradient at higher detergent concentrations results in elution of the integral membrane proteins