Protective efficacy of antibodies to the Staphylococcus aureus type 5 capsular polysaccharide in a modified model of endocarditis in rats.

The protective efficacy of antibodies to the Staphylococcus aureus type 5 capsular polysaccharide (CP5) was examined in a modified model of catheter-induced endocarditis. Rats were catheterized by surgically passing a polyethylene catheter through the right carotid artery and aortic valve into the left ventricle. The following day, the rats were injected by the intraperitoneal (i.p.) route with immunoglobulin G (IgG) purified from nonimmunized rabbits or from rabbits immunized with a conjugate vaccine composed of CP5 and CP8 linked covalently to recombinant Pseudomonas aeruginosa exotoxoid A. One day after passive immunization, the animals were challenged i.p. with one of three serotype 5 S. aureus isolates (strain Reynolds, Lowenstein, or VP) or nontypeable strain 521. Protection was evaluated by comparing quantitative cultures of blood, endocardial vegetations, and kidneys from control and immune animals. For experiments performed with S. aureus Reynolds and Lowenstein, rats given capsular antibodies (645 microg of CP5-specific IgG) showed a significantly (P < 0.05) lower prevalence of endocarditis than rats injected with nonimmune IgG. Similarly, quantitative cultures of the blood, kidneys, and aortic valve vegetations revealed that fewer S. aureus cells were recovered from rats given capsule-specific IgG than from rats administered nonimmune IgG. Rats challenged with strain VP were protected with 1.145 mg of CP5-specific IgG. Capsular antibodies did not protect against infection elicited by a nontypeable strain. These results demonstrate that capsular antibodies elicited by immunization with a polysaccharide-protein conjugate vaccine protect experimental animals against serotype 5 S. aureus infection in a modified model of endocarditis.

Evidence that the antigen receptors of cytotoxic T lymphocytes interact with a common recognition pattern on the H-2Kb molecule.

Recognition of class I MHC antigens involves interaction between TCRs of cytotoxic T lymphocytes (CTL) and the two alpha helices of MHC molecules. Using a combined panel of H-2Kb mutants selected by either a CTL clone or MAbs, we have shown evidence that the TCRs of 59 Kb-specific CTL clones shared a common binding pattern on the H-2Kb molecule. Mutations of amino acid residues at the C-terminal regions, but not the N-terminal regions, of the alpha helices abrogated the recognition by the majority of the clones. The data suggests that TCRs predominantly recognize the class I MHC molecule with an orientation that is parallel to the beta-pleated strands and diagonal to the alpha helices.

Effect of conjugation methodology, carrier protein, and adjuvants on the immune response to Staphylococcus aureus capsular polysaccharides.

Conjugate vaccines were prepared with S. aureus type 8 capsular polysaccharide (CP) using three carrier proteins: Pseudomonas aeruginosa exotoxin A (ETA), a non-toxic recombinant ETA (rEPA), and diphtheria toxoid (DTd). Adipic acid dihydrazide (ADH) or N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) was used as a spacer to link the CP to carrier protein. All conjugates gave a high immune response with a boost after the second immunization. Conjugates prepared with ADH gave higher antibody titers than conjugates prepared with SPDP. IgG1 was the primary subclass elicited by all conjugates regardless of the carrier protein or the conjugation method used to prepare the vaccines. The non-immunogenic CP and the conjugates were formulated with either monophosphoryl lipid A (MPL), QS21, or in Novasomes and evaluated in mice. While the adjuvants failed to improve the immunogenicity of the nonconjugated CP, a more than fivefold increase in the antibody levels was observed when these adjuvants were used with the conjugates. Significant rises in IgG2b and IgG3 were observed with all formulations. The enhancement of the immunogenicity and the IgG subclass shift, as seen with some adjuvants, may prove to be important in immunocompromised patients.

Presentation of a horse cytochrome c peptide by multiple H-2b class I major histocompatibility complex (MHC) molecules to C57BL/6- and bm1-derived cytotoxic T lymphocytes: presence of a single MHC anchor residue may confer efficient peptide-specific CTL recognition.

In this study the immunogenic tryptic fragment from a horse cytochrome c (cyt c) digest recognized by cytotoxic T lymphocytes (CTL) induced by in vitro peptide stimulation from C57BL/6 (B6) and mutant B6.C-H-2bm1 (bm1) mice is identified. An identical sequence, p40-53, is recognized by CTL from both B6 and bm1 mice. In addition, both B6 bm1 cloned CTL lines display unusual major histocompatibility complex (MHC) class I-restricted recognition of this peptide in that they respond to it in the context of H-2Kb, H-2Db, and H-2Kbm1 class I molecules, although the sequence lacks the usual structural Kb and Db peptide-binding motifs. Truncated analogues which resemble the lengths of naturally processed MHC class I-presented peptides, confer reactivity for B6 and bm1 CTL against EL4 (H-2b) targets as well as the L cell transfectants, L+Kb, L+Db, and L+Kbm1. The antigenic peptide with the greatest potency is p41-49, which appears to be generated by angiotensin converting enzyme cleavage of the full-length p40-53 tryptic peptide. The minimum antigenic peptide recognized by both B6 and bm1 CTL, and which targets lysis on each of the transfectants, is the hexamer p43-48 peptide from horse cyt c. Residues Pro44 and Thr47, which occupy polymorphic positions with respect to other species-variant cyt c molecules, influence recognition of these peptides differently for the B6 and bm1 CTL. The ability of H-2Kb, H-2Db, and mutant H-2Kbm1 class I molecules to present the same peptide to a single cloned CTL is discussed in the context of current knowledge of peptide anchor residues and side chain-specific binding pockets in the MHC class I peptide-binding site.

Induction of the cell cycle in baby rat kidney cells by adenovirus type 5 E1A in the absence of E1B and a possible influence of p53.

From previous studies on the induction of DNA synthesis in quiescent primary baby rat kidney cells by adenovirus type 5 (Ad5) E1A deletion mutants, we concluded that induction is prevented only when cellular proteins p300 and pRb are both uncomplexed with E1A (J.A. Howe, J.S. Mymryk, C. Egan, P.E. Branton, and S.T. Bayley, Proc. Natl. Acad. Sci. USA 87:5883-5887, 1990). We have now examined induction by these same mutants in virus lacking the E1B region, so that cellular p53 was no longer complexed to the E1B 55-kDa protein. E1A mutants that fail to bind pRb induced DNA synthesis at a significantly lower level in Ad5 lacking E1B than in Ad5 containing E1B. Apparently, therefore, uncomplexed p53 can partially replace p300 in cooperating with pRb to suppress DNA synthesis in baby rat kidney cells.

Selective reactivity of CD8-independent T lymphocytes to a cytotoxic T lymphocyte-selected H-2Kb mutant altered at position 222 in the alpha 3 domain.

To study the structural basis for specificity and affinity of cytotoxic T lymphocytes for major histocompatibility complex/peptide complexes, we have employed a cytotoxic T lymphocyte (CTL)-mediated immunoselection approach to obtain H-2Kb structural mutants which are resistant to lysis by a Kb-specific alloreactive CTL clone. In this study we describe the Kb structural mutant, designated R8.60.14, recovered following immunoselection using the CD8-dependent CTL clone 60 as a selective agent. Although serologically unaltered with respect to Kb expression, R8.60.14 is not recognized by CD8-dependent, Kb-specific CTL. DNA sequence analysis revealed a single Glu----Lys amino acid substitution at position 222 in the Kb alpha 3-domain of this variant. To determine if a direct correlation exists between CD8 dependence of a Kb specific CTL and its failure to respond to R8.60.14, we examined the lytic response against R8.60.14 by CD8-independent, Kb-specific CTL obtained from long-term culture in the presence of anti-CD8 monoclonal antibody, 3.155. CD8-independent CTL exhibit no difference in their response against the R8 parent and R8.60.14 variant. This study demonstrates unequivocally that Kb-specific recognition of R8.60.14 by CD8-independent CTL is unaltered, while the response by CD8-dependent CTL is completely abrogated. Thus, the sole basis for emergence of this variant in the CTL-mediated immunoselection approach used in this study resides in the alteration of a single CD8-binding site residue at position 222 in the Kb alpha 3 domain. The functional importance of this Glu222 residue for the interaction between the CD8 molecule on CD8-dependent CTL and the Kb alpha 3 domain is further reinforced by virtue of the recovery of the R8.60.14 variant on the basis of its resistance to lysis by a CD8-dependent CTL clone in this CTL-mediated immunoselection approach.

Identification of an autologous insulin B chain peptide as a target antigen for H-2Kb-restricted cytotoxic T lymphocytes.

We have examined the CD8+ peripheral T cell repertoire of C57BL/6 (H-2b) mice for cytotoxic T lymphocyte (CTL) reactivities to insulin, using in vitro immunization with a chymotryptic digest of reduced bovine insulin. The results presented in this study demonstrate that potentially autoreactive H-2Kb-restricted cytotoxic T cells specific for an autologous insulin B chain peptide are present in the preimmune splenic T cell repertoire. The immunogenic peptide comprises residues 7-15 from the insulin B chain and has features in common with naturally processed Kb-restricted peptides identified by others. The minimal peptide sequence recognized by these cytotoxic T cells is 10-15, which is highly conserved in mammalian species and constitutes a self-peptide in mice. The presence of class I major histocompatibility complex-restricted CTLs with potentially autoreactive specificities in preimmune animals raises the possibility of a role for such cells in autoimmune disease states. Possible mechanisms for the in vivo expansion of insulin peptide-specific CTLs are discussed.