1. INTRODUCCIÓN
2.10. EQUIPOS DEL SISTEMA DE CONTROL
The overall pattern for compound mitogenicity at a successful cell yield appeared to be that B-lymphocyte stimulation increased along the series alanyl-glycine LAA monomer < dimer < trimer < Pam^Cys-Ser-Ser-Asn-Ala. Pam^Cys lipopeptides are derived from the N-terminal region o f bacterial lipoprotein, which constitutes the biologically active portion o f lipoprotein, and Pam^Cys-SSNA in particular, is identical to the N-terminus o f the native molecule with respect to its amino acid composition, and also exhibits a stimulatory activity comparable to native lipoprotein (Bessler et al
1985). The trimer 80k was most like Pam^Cys lipopeptides in composition, having
the alanine-glycine cross-linker o f bacterial cell walls, and a similar but distinct lipidic moiety, so would be expected to exhibit similar stimulant properties. However, at a more physiological dosage, the overall pattern shows approximately equal activity for
the three Boc-compounds, with greater activity for compound SOh, the free amine.
This cannot be explained by the free amine presenting a more biologically natural species, and therefore better able to penetrate splenocyte membrane, since the Boc- compounds displayed better mitogenic activity in the previous assays. Pam^Cys-SSNA was again superior in mitogenic activity at this dosage.
Unfortunately, Pam^Cys-Ala-Gly was not tested with the LAA compounds, particularly since it had been shown to be the most active stimulant o f the Pam^Cys
compounds (Reitermann et al 1989; Hoffmann et al 1989), and would therefore be a
useful control. It would also be interesting to compare the activity o f an alanyl-glycine
trimer with free amine under similar conditions, since compound 80h gave reasonable
results.
The three fatty acids o f Pam^Cys lipopeptides insert into biological membranes, including lymphocytes, and thus act as an anchor for the generally more polar
substituents attached (Metzger et al 1991), therefore it seems reasonable to assume that
LA A ’s acted similarly to display relatively good mitogenic properties. Thus, LA A ’s acted as a delivery system to penetrate splenocyte membrane, anchoring the alanyl- glycine moiety, however, further work is required (for example, with trimeric LA A ’s possessing free amine groups) to assess their use as a tool for elucidating the early events o f the immune system, since Pam^Cys lipopeptides have been established
as a successful group o f compounds constituting potent B-lymphocyte activators, and potent immunoadjuvants when covalently bound to antigens.
3.3 Immunological evaluation o f a lipidic amino acid based carrier-vaccine for
Chlamydia trachomatis.
The ocular infection trachoma is caused by three serovars (A, B/Ba, C) o f C.
trachomatis. Two o f the four variable domains (VD I and VD IV) o f the major Omp are immunodominant and surface exposed on the trachoma causing serovars (Zhong and Brunham 1991). In an attempt to develop an effective trachoma vaccine, LAA
(P I) and TCP (P2, P4) systems were evaluated as potential carrier-vaccines, using
monomer P3 for comparison with P4.
3.3.1 Production o f antibodies.
Antisera were raised to serovar B and C elementary bodies (EB) by injection o f C trachomatis EB. Anti-peptide antisera were raised in groups o f 4-6 mice from different strains (C57BL/10 (H-2"), BIO (H-2^), BIO.A (H-2"), B I0.D 2 (H-2‘*), BIO.BR (H-2''), SJL (H-2"), Balb/c (H-2"^) and CBA (H-2'')) injected with various peptide
preparations (P1-P4) emulsified in Freund’s complete adjuvant (FCA). Sera from each
group were collected and pooled 14 days after the final injection. BIO mice were also
injected with P4 and the adjuvant neuramidase/galactose oxidase (NAGO).
3.3.2 Immunogenicitv o f PI and P2 peptides.
A previous study (Toye et al 1990) determined that peptide sequence PI
(“^^SEATIFDVTTLNPTIAG^°^) contained both B cell epitopes and T helper sites. In this preliminary experiment, the peptide sequence PI was one o f five partially overlapping peptides o f a 61-mer sequence (from position 273 to 333) covering the
entire VD IV region o f serovar B Omp. The 61 amino acid peptide was found to recall an antigen-specific proliferation response o f T cells primed in vivo with chlamydial EB in BALB/c (H-2"^) mice. Fragment PI stimulated significant proliferation responses in both CBA (H-2") and C57BL/10 (H-2^) but not BALB/c (H-2‘*) mice.
Since the PI sequence contained both B cell epitopes and T helper sites, the
effects o f polymerization on immunogenicity o f the synthetic peptides PI and P2 were
evaluated. Previously, success was found in inducing high antibody response to polymerized synthetic peptides by incorporation into a polylysine core (Tam 1988; Tam et al 1990; Wang et al 1991). A refinement o f this was to conjugate three lipidic
amino acids to the polylysine core to give the TCP system P2. The immunogenicity
o f this peptide was compared with the same sequence coupled to two lipidic amino
acids to give the LAA system PI.
Peptides PI and P2 were used to immunize four congenic strains o f mice. The
antisera were assayed against serovar B EB whole organisms in an enzyme-linked
immunosorbent assay (ELISA) (Graph 3). PI elicited anti-EB antibodies with
relatively low titres, whereas P2 was able to raise antibodies with high titres in B 10.A
(H-2“) and BIO.BR (H-2'"), and intermediate titres in BIO (H-2^). B10.D2 (H-2"^) was a nonresponder to both PI and P2, which was consistent with the 11-2^* restriction o f T cell immune response with the PI sequence observed in the preliminary experiment.
In a pepscan assay (Graph 4), antisera (1:500 dilution) raised with P2 in the two high responder strains H-2^ and H -2 \ displayed similar epitope specificities as antisera (1:1200 dilution) raised with whole serovar B EB, except that more epitopes
were mapped with the P2 antisera.
These results suggest that the PI peptide sequence is a potential candidate sequence for providing a T helper cell site and a B cell sites covering B serogroup
serovars, and that the LCP preparation P2 significantly enhanced the immunogenicity