Synthesis and immunological studies of alpha-conotoxin chimera containing an immunodominant epitope from the 268-284 region of HSV gD protein

We have synthesized and characterized new chimeric peptides by inserting an epitope of the glycoprotein D (gD) of herpes simplex virus (HSV) serotype 1 as 'guest' sequence in the 'host' structure of alpha-conotoxin G1, a 13-r esidue peptide (ECCNPACGRHYSC) isolated from the venom of Conus geographus. The 276-284 region of HSV gD-1 selected for these studies is highly hydrop hilic and adopts a beta-turn. The alpha-conotoxin G1 also contains a beta-t urn in the 8-12 region, stabilized by two disulfide bridges at positions 2- 7 and 3-13. Thus, the tetramer sequence of a-conotoxin. (8)Arg-His-Tyr-Ser( 12) has been replaced by Asp-Pro-Val-Gly (DPVC), identified previously as t he epitope core. The syntheses were performed by Fmoc strategy on Rink resi n and DTNB or air oxidation were applied for the formation of the first 3-1 3 disulfide bond in the presence of guanidinium hydrochloride. For the form ation of the second disulfide Cys(2)-Cys(7) three different oxidation proce dures [iodine in 95% acetic acid, air oxidation in dimethyl sulfoxide/l M H Cl or Tl(tfa)(3) in trifluoroacetic acid (TFE)] were compared. The high-per formance liquid chromatography purified peptides were characterized by elec trospray mass spectrometry and amino acid analysis. The bicyclic HSV-alpha- [Tyr(1)]-conotoxin chimeric peptide and native alpha-conotoxin G1 showed si milar circular dichroism spectra in phosphate-buffered saline (PBS) and in a PBS-TFE 1:1 (v/v) mixture, which might suggest that these compounds also share similar secondary structures. In immunologic studies the characterist ics of the primary and of the memory immunoglobulin (Ig) M- and IgG-type an tibody responses showed that the bicyclic HSV-alpha-[Tyr(1)]-conotoxin chim era is capable to induce strong antibody responses in C57/B1/6 mice but was poorly immunogenic in CBA and BALB/c mice. Data obtained with the C57/B1/6 serum indicate that the polyclonal antibodies recognize the DPVG motif pre sented in the bicyclic HSV-alpha-[Tyr(1)]-conotoxin and some reactivity was also found with the monocyclic but not with the linear form of the chimera . Results with two IgM type monoclonal antibodies from a bicyclic HSV-alpha -[Tyr(1)]-conotoxin immunized C57/B1/6 mouse also point to the specific: in teraction with the DPVG sequence;Taken together these studies suggest, that the relative intensity of DPVG-specific responses was found to be dependen t on the mouse strain and on the-conformation of the chimeric molecules. We found that: the IgM monoclonal antibodies are able to recognize the linear DPVG sequence, while the majority of IgG antibodies is directed to the sam e motif in a conformation stabilized by double cyclization.