Targeting of antihapten antibodies to activated T cells via an IL-2-hapten conjugate prolongs cardiac graft survival.

Antihapten antibodies binding to ligand-hapten conjugates are able to mediate complement mediated lysis in vitro. Based on this observation we propose a new in vivo immunotherapy using molecules that combine a low molecular weight hapten binding to antibodies preexisting in serum and a cell specific ligand. The ligand-hapten conjugates are potential cytotoxic drugs which may (1) be specific for a given target cell, (2) be nonimmunogenic, (3) be of low molecular weight, (4) form soluble complexes with preexisting antibodies resulting in prolonged half life of the drug, and (5) induce a potent antibody mediated rejection of target cells. These novel compounds could be useful for the elimination of certain cell subsets involved in allograft rejection, cancers, infectious diseases, etc., without some of the pitfalls of conventional immunotherapies. The feasibility of this approach was demonstrated in an animal model using a compound consisting of one interleukin 2 and one fluorescein molecule (IL-2-FITC). BALB/c mice (H2d) previously immunized and expressing anti-FITC antibodies were transplanted with a fully mismatched C57BL/6 (H2b) heterotopic heart allograft. Untreated controls rejected their graft by day 9 (MSD = 9 +/- 0.7). Mice with preexisting anti-FITC antibodies treated with IL-2-FITC maintained their grafts for 38.7 +/- 7.1 days (P < 0.02). No prolongation of graft survival was observed in immunized animals that were treated with IL-2 alone (MSD = 10 +/- 1.4). Nonimmunized animals treated with IL-2-FITC rejected their grafts on day 9.4 +/- 1.1. This demonstrates that IL-2-FITC therapy specifically prolonged graft survival in animals with circulating anti-FITC antibodies. The data suggest that a ligand/hapten pair can redirect preexisting antihapten antibodies toward target cells in vivo. Such compounds may be developed for human use as alternatives to polyclonal or monoclonal antibody therapy.

Towards vaccine optimisation.

New molecular technologies have accelerated the search for sub-unit candidate vaccines. However, once identified the use of a candidate antigen must be optimised to reap the maximum benefit from the eventual vaccine. This optimisation should take into account both the needs of the target population, and the various ways of potentiating the protective immune response induced. One must be sure that the final product will be used. Hence, vaccine optimisation should strive toward meeting the needs of a specific epidemiological problem within the economic constraints of a given situation. This may be possible using novel delivery systems designed to limit the number of doses needed, improve the stability or facilitate the delivery of a particular vaccine. In meeting the needs of a target population in a field situation, one must also keep in mind certain safety factors that go beyond the usual regulatory constraints. The immune response to vaccine candidates can be potentiated in many ways. The ability to preferentially induce specific protective effector mechanisms: i.e., antibody isotypes, T-cell subsets, and T-cell sub-subsets, is becoming a reality. Carrier molecules designed to avoid the problems of epitope suppression and competition, and perhaps an eventual "carrier jam," are being developed. Adjuvants and immunostimulants may also help, but the critical issue here remains their acceptability for use in man. Finally novel strategies for the induction of the immune response may also potentiate the immune response in the optimisation of vaccines.

Differential effects of superantigen-induced "anergy" on priming and effector stages of a T cell-dependent antibody response.

The in vitro T cell nonresponsiveness or anergy to restimulation with staphylococcal enterotoxin B (SEB) following the in vivo injection of the superantigen is well characterized. Here we use mice transgenic for a V beta 8.2+ T cell receptor (TcR) (reactive with SEB) to establish a large population of anergic T cells in vivo. As expected, peripheral T cells from the SEB injected transgenic mice failed to proliferate or produce interleukin (IL)-2 following restimulation with the superantigen in vitro. However, in this system superantigen reactivity could be restored by either addition of exogenous IL-2, or stimulation with immobilized anti-TcR antibody. To evaluate the effects of superantigen-induced anergy in vivo, SEB-injected or noninjected control transgenic mice were immunized and boosted with the T cell-dependent antigen tetanus toxin (TT). SEB injection of the V beta 8.2+ transgenic mice 5 days prior to the TT immunization inhibited the anti-TT antibody response as measured over a 100-day period, whereas injection of a superantigen which does not interact with the V beta 8.2% TcR (such as SEA) did not. Furthermore, SEB injection of control nontransgenic mice did not interfere with the induction of a high titer anti-TT antibody response. In contrast to the inhibition seen when SEB was given prior to TT immunization, injection of transgenics with SEB either after the priming TT immunization or after the recall booster injection did not significantly influence the titers of anti-TT antibodies produced. These results demonstrate that the establishment of peripheral T cell anergy to superantigens inhibits the specific antigenic priming of helper T cells in vivo, but does not prevent primed T cells from helping B cells to mount an effective antibody response.

Skewed T cell receptor V alpha repertoire among superantigen reactive murine T cells.

Reactivity of murine T cells with viral or bacterial superantigens is clearly correlated with the expression of TCR V beta domains. Thus, T cells responding to the minor lymphocyte stimulatory locus (Mls-1a) or staphylococcal enterotoxin B (SEB) express predominantly TCR V beta 6 or V beta 8.2 respectively. We have investigated the involvement of the other major variable element of the TCR, the V alpha domain, in these superantigen responses. Using a panel of anti-TCR V alpha mAbs, it is demonstrated that the TCR V alpha repertoire among superantigen stimulated V beta 6+ or V beta 8.2+ blasts (responding to Mls-1a or SEB respectively in vitro) is altered in comparison with anti-CD3 stimulated cells expressing the same V beta domains. Furthermore, the TCR V alpha repertoire is strongly skewed in TCR V beta 8.2 transgenic mice that have undergone extensive peripheral clonal deletion after SEB injection. These data imply that the V alpha domain influences superantigen recognition by the TCR.

Redirecting circulating antibodies via ligand-hapten conjugates eliminates target cells in vivo.

The elimination of cell populations in vivo often relies on reagents that are self-limiting, are difficult to design and produce or contain highly toxic components. Here we describe a novel immunotherapy using molecules that combine a cell-specific ligand and a hapten binding to preexisting antibodies in serum. The F(ab')2 fragment of a polyclonal anti-thymocyte globulin (ATG) preparation was used as a T-cell-specific ligand, and fluorescein isothiocyanate (FITC), as the hapten. Clearance of ligand-hapten conjugates from the circulation through formation of immune complexes was prevented through controlled synthesis of conjugates so that they contained one F(ab')2 fragment and one FITC molecule. Administration of a single dose of F(ab')2 or F(ab')2ATG-FITC into naive mice had no effect on the number of circulating T cells. In contrast, injection of F(ab')2ATG-FITC into mice with circulating anti-FITC antibodies resulted in the elimination of peripheral T cells. The reduction in cell numbers was equivalent to that obtained with a corresponding dose of intact ATG. Experiments in thymectomized mice demonstrated that the reduction of circulating T cells was due to target-cell elimination and not to immunomodulation or cellular sequestration. The adaptability of the model to other sources of effector antibodies and more useful ligands is discussed.

Peripheral clonal deletion of superantigen-reactive T cells is enhanced by cortisone.

The T cell receptor (TcR) V beta-specific expansion, deletion and induction of nonresponsiveness among murine T cells responding to superantigens in the periphery has been well characterized. Here we demonstrate that clonal deletion of staphylococcal enterotoxin (SE) B-reactive V beta 8.2+ cells can be significantly increased when mice are injected with hydrocortisone (HC) following superantigen stimulation in vivo. The induced sensitivity to HC persists for at least 30 days after SEB injection, making it unlikely that proliferating cells were uniquely responsible for the enhanced deletion. Superantigen-induced HC sensitivity was a general phenomenon and could also be observed among V beta 11+ cells after the injection of SEA. Experiments conducted on thymectomized mice indicated that HC-sensitive, SEB-responsive cells could not be accounted for by rapidly produced, immature lymphocytes recently exported from the thymus. Further, V beta 8.1+ peripheral lymphocytes from TcR transgenic mice expressing the Mls-1a superantigen were sensitive to HC. These results imply that the majority of cells remaining after superantigen-induced clonal expansion and deletion in vivo have indeed reacted with the superantigen. Implications for differential superantigen recognition by T cells expressing the same TcR V beta domain, perhaps due to a significant V alpha contribution to the interaction in vivo, are discussed.

Clonal anergy to staphylococcal enterotoxin B in vivo: selective effects on T cell subsets and lymphokines.

Injection of bacterial superantigens such as staphylococcal enterotoxin B (SEB) in adult mice results in initial proliferation of SEB-responsive V beta 8+ T cells followed by induction of a state of non-responsiveness frequently referred to as clonal anergy. We show here that SEB-induced anergy involves selective changes in lymphokine production and that it affects CD4+ V beta 8+ and CD8+ V beta 8+ T cells in different fashions. Whereas both CD4+ V beta 8+ and CD8+ V beta 8+ cells from anergic mice exhibit strongly reduced proliferative capacity and interleukin(IL)-2 production upon restimulation with SEB either in vivo or in vitro the CD8+ subset from SEB-injected mice produces other lymphokines (such as interferon(IFN)-gamma) at normal or slightly increased levels in response to SEB. Changes in the levels of production of IL-2 and IFN-gamma protein correlated well with mRNA accumulation both in vivo and in vitro. Collectively these data suggest that superantigen-induced anergy involves selective changes in signal transduction and/or gene regulation in T lymphocytes.

Use of a tuberculin purified protein derivative--Asn-Ala-Asn-Pro conjugate in bacillus Calmette-Guérin primed mice overcomes H-2 restriction of the antibody response and avoids the need for adjuvants.

Because of its immunodominancy, and because it is conserved in different geographical isolates of Plasmodium falciparum, the repetitive sequence of the circumsporozoite protein, (Asn-Ala-Asn-Pro)n [(NANP)n], has been envisaged for the development of an anti-falciparum malaria subunit vaccine. However, the murine immune response to (NANP)n peptides, either carrier-free or coupled to carrier proteins, was shown to be inducible only by using strong (e.g., Freund's) adjuvants. Furthermore, response to the carrier-free peptide, administered in adjuvant, is genetically restricted to I-Ab mice. In the present paper, we report that high titers of antibodies against the NANP repetitive epitope were obtained in responder C57BL/6 (H-2b) mice when they were primed with live BCG (bacillus Calmette-Guérin Mycobacterium tuberculosis var. bovis) and immunized once with the synthetic peptide (NANP)40 coupled to tuberculin purified protein derivative (PPD) without the use of any adjuvant. This approach also led to the production of high titers of anti-NANP antibodies in ASW (H-2s), B10.RIII (H-2r), BALB/c (H-2d), C3H/He (H-2k), and DBA/1 (H-2q) nonresponder mice after two injections of the conjugate. In both cases, BCG priming was obligatory for the induction of antibodies reacting with the synthetic peptide. The levels of anti-NANP antibodies in nonresponder BALB/c mice were demonstrated to be comparable to the levels induced after PPD-(NANP)40 immunization in Freund's complete or incomplete adjuvant. The antibodies induced were also capable of recognizing P. falciparum sporozoites in immunofluorescence assays and, furthermore, these antibodies inhibited the penetration of live sporozoites into human hepatocytes in vitro. This system functioned independently of the subjects' resistance or susceptibility to BCG infection. Given the widespread natural exposure to mycobacterial antigens and the extensive use of BCG and PPD in the human population, this approach might be envisaged for vaccination with malaria peptides.

Mycobacterial heat-shock proteins as carrier molecules. II: The use of the 70-kDa mycobacterial heat-shock protein as carrier for conjugated vaccines can circumvent the need for adjuvants and Bacillus Calmette Guérin priming.

In a recent work, we have shown that mycobacterial heat-shock proteins (hsp) of 65-kDa (GroEL-type) and 70-kDa (DnaK-type) acted as carrier molecules in mice, previously primed with Mycobacterium tuberculosis var. bovis (bacillus Calmette-Guérin, BCG), for the induction of high and long-lasting titers of IgG against the repetitive malaria synthetic peptide (NANP)40. Anti-peptide antibodies were induced when the malaria peptide, conjugated to the mycobacterial hsp, was given in the absence of any adjuvants (Lussow et al., Eur. J. Immunol. 1991. 87:2960). In this report, we show that mice immunized with peptides or oligosaccharides conjugated to the 70-kDa hsp produced high titers of IgG antibodies in the absence of any previous priming with BCG. The anti-peptide antibody response persisted for at least 1 year. This adjuvant-free carrier effect of the 70-kDa hsp was T cell dependent, since no anti-peptide nor anti-70-kDa IgG antibodies were induced in athymic nu/nu mice. Previous immunization of mice with the 65-kDa or 70-kDa hsp did not have any negative effect on the induction of anti-peptide IgG antibodies after immunization with hsp-peptide conjugates in the absence of adjuvants. Furthermore, preimmunization with the 65-kDa hsp could substitute for BCG in providing an effective priming for the induction of anti-(NANP) antibodies. Finally, both the 65-kDa and 70-kDa hsp acted as carrier molecules for the induction of IgG antibodies to group C meningococcal oligosaccharides, in the absence of adjuvants. These findings strongly suggest that the use of hsp as carriers in conjugated constructs for the induction of anti-peptide and anti-oligosaccharide antibodies could be of value in the design of new vaccines for eventual use in humans.

Successful primate immunization with peptides conjugated to purified protein derivative or mycobacterial heat shock proteins in the absence of adjuvants.

We have previously shown in mice that antibodies can be induced to synthetic malaria peptides conjugated to mycobacterial antigens, such as purified protein derivative (PPD) or heat shock proteins (hsp), and given in the absence of adjuvants after a previous priming with bacille Calmette-Guérin (BCG). In the present study we investigated this model of immunization in the non-human primates, Saimiri sciureus monkeys. Monkeys primed with BCG subcutaneously and then immunized subcutaneously with the Plasmodium falciparum sporozoite (NANP)40 synthetic peptide conjugated to PPD or mycobacterial hsp of 65 or 70 kD, in the absence of adjuvants, produced antipeptide and anti-sporozoite IgG antibodies. Interestingly, the carrier effect of the hsp of 70 kD for the induction of anti-(NANP)40 antibodies was also observed in the absence of a previous priming with BCG. These data suggest that such a vaccination strategy may be applied to humans.

Mycobacterial heat-shock proteins as carrier molecules.

We have previously shown that the priming of mice with live Mycobacterium tuberculosis var. bovis (Bacillus Calmette-Guérin, BCG) and immunization with the repetitive malaria synthetic peptide (NANP)40 conjugated to purified protein derivative (PPD), led to the induction of high and long-lasting titers of anti-peptide IgG antibodies, overcoming the requirement of adjuvants and the genetic restriction of the antibody response to the peptide (Lussow et al., Proc. Natl. Acad. Sci. USA 1990. 87:2960). This initial work led us to the following observations. BCG had to be live for priming to lead to the induction of anti-peptide antibodies. Surprisingly, priming with other living microorganisms which chronically infect the macrophage (e.g. Salmonella typhimurium and Leishmania major) also induced anti-peptide antibodies in mice immunized with PPD-(NANP)40 conjugate. It was, thus, hypothesized that molecules expressed during active infection and also known to be highly conserved between species, namely the heat-shock proteins (hsp), could mediate the T cell sensitization required for the production of anti-peptide antibodies. In fact, when the PPD protion of the conjugate was replaced by a highly purified recombinant protein corresponding to the 65-kDa (GroEL-type) hsp of M. bovis, this resulted in the production of anti-(NANP) IgG antibodies in BCG-primed mice, irrespective of the major histocompatibility complex-controlled responsiveness to the (NANP) sequence itself. Further, similar induction of anti-peptide antibody response was also obtained with a recombinant 70-kDa (DnaK-type) hsp of M. tuberculosis, but not with a small molecular mass (18 kDa) of M. leprae. Finally, an adjuvant-free carrier effect for anti-peptide IgG antibody production in BCG-primed mice, was also exerted by the GroEL hsp of Escherichia coli. This finding that hsp can act as carrier molecules without requiring conventional adjuvants is of potential importance in the development of vaccine strategies.