Immunological responses following administration of a genotype 1a/1b/2/3a quadrivalent HCV VLP vaccine.

The significant public health problem of Hepatitis C virus (HCV) has been partially addressed with the advent of directly acting antiviral agents (DAAs). However, the development of an effective preventative vaccine would have a significant impact on HCV incidence and would represent a major advance towards controlling and possibly eradicating HCV globally. We previously reported a genotype 1a HCV viral-like particle (VLP) vaccine that produced neutralizing antibodies (NAb) and T cell responses to HCV. To advance this approach, we produced a quadrivalent genotype 1a/1b/2a/3a HCV VLP vaccine to produce broader immune responses. We show that this quadrivalent vaccine produces antibody and NAb responses together with strong T and B cell responses in vaccinated mice. Moreover, selective neutralizing human monoclonal antibodies (HuMAbs) targeting conserved antigenic domain B and D epitopes of the E2 protein bound strongly to the HCV VLPs, suggesting that these critical epitopes are expressed on the surface of the particles. Our findings demonstrate that a quadrivalent HCV VLP based vaccine induces broad humoral and cellular immune responses that will be necessary for protection against HCV. Such a vaccine could provide a substantial addition to highly active antiviral drugs in eliminating HCV.

Mucosal-associated invariant T-cell activation and accumulation after in vivo infection depends on microbial riboflavin synthesis and co-stimulatory signals.

Despite recent breakthroughs in identifying mucosal-associated invariant T (MAIT) cell antigens (Ags), the precise requirements for in vivo MAIT cell responses to infection remain unclear. Using major histocompatibility complex-related protein 1 (MR1) tetramers, the MAIT cell response was investigated in a model of bacterial lung infection employing riboflavin gene-competent and -deficient bacteria. MAIT cells were rapidly enriched in the lungs of C57BL/6 mice infected with Salmonella Typhimurium, comprising up to 50% of αß-T cells after 1 week. MAIT cell accumulation was MR1-dependent, required Ag derived from the microbial riboflavin synthesis pathway, and did not occur in response to synthetic Ag, unless accompanied by a Toll-like receptor agonist or by co-infection with riboflavin pathway-deficient S. Typhimurium. The MAIT cell response was associated with their long-term accumulation in the lungs, draining lymph nodes and spleen. Lung MAIT cells from infected mice displayed an activated/memory phenotype, and most expressed the transcription factor retinoic acid-related orphan receptor γt. T-bet expression increased following infection. The majority produced interleukin-17 while smaller subsets produced interferon-γ or tumor necrosis factor, detected directly ex vivo. Thus the activation and expansion of MAIT cells coupled with their pro-inflammatory cytokine production occurred in response to Ags derived from microbial riboflavin synthesis and was augmented by co-stimulatory signals.

Characterization of a hepatitis C virus-like particle vaccine produced in a human hepatocyte-derived cell line.

An effective immune response against hepatitis C virus (HCV) requires the early development of multi-specific class 1 CD8+ and class II CD4+ T-cells together with broad neutralizing antibody responses. We have produced mammalian-cell-derived HCV virus-like particles (VLPs) incorporating core, E1 and E2 of HCV genotype 1a to produce such immune responses. Here we describe the biochemical and morphological characterization of the HCV VLPs and study HCV core-specific T-cell responses to the particles. The E1 and E2 glycoproteins in HCV VLPs formed non-covalent heterodimers and together with core protein assembled into VLPs with a buoyant density of 1.22 to 1.28 g cm-3. The HCV VLPs could be immunoprecipited with anti-ApoE and anti-ApoC. On electron microscopy, the VLPs had a heterogeneous morphology and ranged in size from 40 to 80 nm. The HCV VLPs demonstrated dose-dependent binding to murine-derived dendritic cells and the entry of HCV VLPs into Huh7 cells was blocked by anti-CD81 antibody. Vaccination of BALB/c mice with HCV VLPs purified from iodixanol gradients resulted in the production of neutralizing antibody responses while vaccination of humanized MHC class I transgenic mice resulted in the prodution of HCV core-specific CD8+ T-cell responses. Furthermore, IgG purified from the sera of patients chronically infected with HCV genotypes 1a and 3a blocked the binding and entry of the HCV VLPs into Huh7 cells. These results show that our mammalian-cell-derived HCV VLPs induce humoral and HCV-specific CD8+ T-cell responses and will have important implications for the development of a preventative vaccine for HCV.

Human dendritic cells pulsed with specific lipopeptides stimulate autologous antigen-specific T cells without the addition of exogenous maturation factors.

SUMMARY: Serum-free culture conditions to generate immature human monocyte-derived DC (Mo-DC) were optimized, and the parameters that influence their maturation after exposure to lipopeptides containing CD4(+) and CD8(+) T-cell epitopes were examined. The lipopeptides contained a single CD4(+) helper T-cell epitopes, one of a number of human leucocyte antigen (HLA)-A2-restricted cytotoxic T-cell epitope and the lipid Pam2Cys. To ensure complete maturation of the Mo-DC, we examined (i) the optimal lipopeptide concentration, (ii) the optimal Mo-DC density and (iii) the appropriate period of exposure of the Mo-DC to the lipopeptides. The results showed that a high dose of lipopeptide (30 microm) was no more efficient at upregulating maturation markers on Mo-DC than a low dose (6 microm). There was an inverse relationship between Mo-DC concentration and the mean fluorescence intensity of maturation markers. In addition, at the higher cell concentrations, the chemotactic capacity of the Mo-DC towards a cognate ligand, CCL21, was reduced. Thus, high cell concentrations during lipopeptide exposure were detrimental to Mo-DC maturation and function. The duration of exposure of Mo-DC to the lipopeptides had little effect on phenotype, although Mo-DC exposed to lipopeptides for 48 rather than 4 h showed an increased ability to stimulate autologous peripheral blood mononuclear cells to release interferon-gamma in the absence of exogenous maturation factors. These findings reveal conditions for generating mature antigen-loaded DC suitable for targeted immunotherapy.

Molecular and genetic characterisation of the host-protective oncosphere antigens of taeniid cestode parasites.

Highly effective recombinant vaccines have been developed against Taenia ovis infection in sheep, Taenia saginata infection in cattle, Taenia solium infection in pigs, Echinococcus granulosus and Echinococcus multilocularis infections in a variety of intermediate host species. These vaccines have been based on the identification and expression in Escherichia coli of antigens derived from the oncosphere life cycle stage, contained within the parasites' eggs. Investigation of the molecular aspects of these proteins and the genes encoding them have revealed a number of common features, including the presence of a predicted secretory signal sequence, and one or two copies of a fibronectin type III domain, each encoded by separate exons within the associated gene. Evidence has been obtained to confirm glycosylation of some of these antigens. Ongoing investigations will shed light on the biological roles played by the proteins within the parasites and the mechanism by which they make the parasites vulnerable to vaccine-induced immune responses.

Synthetic peptide epitope-based polymers: controlling size and determining the efficiency of epitope incorporation.

The assembly of synthetic peptide-based vaccines that incorporate multiple epitopes is a major goal of vaccine development, because such vaccines will potentially allow the immunization of outbred populations against a number of different pathogens. We have shown that free radical-induced polymerization of individual peptide epitopes results in the incorporation of multiple copies of the same or different epitopes into high molecular weight immunogens (O'Brien-Simpson, N.M., Ede, N.J., Brown, L.E., Swan, J. & Jackson, D.C. (1997) Polymerization of unprotected synthetic peptides: a view toward synthetic peptide vaccines. J. Am. Chem. Soc.119, 1183-1188; Jackson, D.C., O'Brien-Simpson, N., Ede, N.J. & Brown, L.E. (1997) Free radical induced polymerization of synthetic peptides into polymeric immunogens. Vaccine 15, 1697-1705). The ability to control the size of these polymers, to determine the physical and chemical properties of the backbone material and also to know the extent to which individual peptide epitopes are incorporated are important manufacturing considerations and form the subject of this study. We show here that the polymerization process is highly efficient with at least 70% of peptides incorporated into the resulting polymer, that acrylamide and acryloylated amino acids can be used as comonomers with peptide epitopes in the polymerization reaction and that the choice of the comonomer can influence the properties of the resulting polymer. We also show that the size of chain growth polymers is restricted in the presence of chain transfer agents, that the resulting polymer size can be predicted and that there is little or no difference in the immunogenicity of polymers that range in apparent molecular size between 18 kDa and 335 kDa. The successful polymerization of peptide epitopes with an acryloyl-amino acid creates the potential for introducing different physical and chemical properties into artificial protein immunogens.

The central role played by peptides in the immune response and the design of peptide-based vaccines against infectious diseases and cancer.

Vaccines are one of the most cost effective methods of improving public health thereby increasing the quality of life. Prophylactic and therapeutic treatment by vaccines can prevent infectious diseases and some cancers and could also be used in the treatment of autoimmune disorders. An appreciation of this potential has resulted in a burgeoning literature which not only describes the scientific efforts being made into designing new and improved vaccines but also drives the efforts being made by public health organizations world-wide in delivering vaccines to the community. At the forefront of technologies being applied to the design of vaccines is the use of synthetic peptides; the chemical technologies used to assemble peptides have made great strides over the last decade and assembly of hi-fidelity peptides which can be of high molecular weight, multimeric or even branched is now almost routine. Together with the advances in peptide technology our understanding of the molecular events that are necessary to induce immune responses has also made great strides. The central role that peptides play in immune recognition is now recognised and rules are emerging that are being applied to the construction of peptide-based vaccines that, in the right context, can induce humoral (antibody) and cellular (cytotoxic and helper T cell) immune responses. Synthetic peptides are exquisitely placed to answer questions about immune recognition and along the way to provide us with new and improved vaccines.

Identification of canine helper T-cell epitopes from the fusion protein of canine distemper virus.

The fusion protein of canine distemper virus (CDV-F), a 662 amino-acid envelope protein, was used as the target molecule for identification of canine T helper (Th) epitopes. A library of 94 peptides, each 17 residues in length overlapping by 10 residues and covering the entire sequence of CDV-F, was screened using a lymphocyte proliferation assay with peripheral blood mononuclear cells (PBMC) obtained from dogs inoculated with canine distemper virus (CDV) vaccine. Initially we observed low and inconsistent proliferation of PBMC in response to these peptides, even when using cells obtained from dogs that had received multiple doses of CDV. Subsequently, the use of expanded cell populations derived by in vitro stimulation of canine PBMC with pools of peptides allowed the identification of a number of putative canine Th-epitopes within the protein sequence of CDV-F. There were two major clusters of Th-epitopes identified close to the cleavage site of the F0 fusion protein, while some others were scattered in both the F1 and F2 fragments of the protein. Some of these peptides, in particular peptide 35 (p35), were stimulatory in dogs of different breeds and ages. The identification of such promiscuous canine Th-epitopes encouraged us to assemble p35 in tandem with luteinising hormone releasing hormone (LHRH) a 10 amino-acid residue synthetic peptide representing a B-cell epitope which alone induces no antibody in dogs. The totally synthetic immunogen was able to induce the production of very high titres of antibodies against LHRH in all dogs tested. These results indicate that p35 could be an ideal candidate for use as a Th-epitope for use in outbred dogs.

Assembly of synthetic peptide vaccines by chemoselective ligation of epitopes: influence of different chemical linkages and epitope orientations on biological activity.

In this paper, we describe the assembly of synthetic peptide vaccines composed of a T helper cell epitope and a B cell epitope that were synthesized separately and then attached using three different chemoselective ligation methods: oxime bond formation, thioether bond formation and disulfide bond formation. The resulting vaccines were tested in animals to investigate their efficacy. We found that thioether bond formation gave the highest yield of material and that the chemistry involved did not adversely affect immunogenicity and biological activity of the peptide vaccine. Ligation of epitopes by oxime bond formation did not diminish biological activity either, but the yields of peptide vaccine were lower than when thioether bond formation was used. The vaccines in which a disulfide bond was used to attach the two epitopes resulted in the lowest yield and produced vaccines that also generated a weaker immune response with sub-optimal biological activity. Connecting the T helper epitope via its N-terminus or its C-terminus to the N-terminus of the B cell epitope had little influence on resulting immunogenicity and biological activity.

Differential effect of CD8(+) and CD8(-) dendritic cells in the stimulation of secondary CD4(+) T cells.

Dendritic cells (DC), in their role in initiation of the adaptive immune response, have been extensively studied for their capacity to interact and stimulate naive T cells. Subsets of mature murine DC isolated directly from the spleen have been shown to differ in their ability to induce proliferative responses in both primary CD4(+) and primary CD8(+) T cells; the myeloid-related CD8alpha(-) DC induce a more intense or prolonged proliferation of naive T cells than do the lymphoid-related DC bearing CD8alpha despite similar expression of MHC and co-stimulatory molecules. Here we examine the interaction of these DC subpopulations with T cells already in the activated or memory state which are known to have greater sensitivity to antigen stimulation and bear receptors with increased capacity for signal transduction. We show that influenza virus-specific CD4(+) T cell clones and splenic T cells from peptide-primed animals proliferated in response to antigen presented by separated splenic CD8(-) DC. In contrast, these T cells showed only weak, if any, proliferation in response to CD8(+) DC despite observable cluster formation in the cultures. The differential between the two DC types in inducing proliferation was even more pronounced than previously seen with primary T cells and did not reflect differential longevity of the DC in culture, altered response kinetics or deviation from IL-2 to IL-4 induction with CD8(+) DC, but was related to the levels of IL-2 induced. The deficiency in the CD8(+) DC was not overcome by using infectious virus rather than synthetic peptide as the antigen source. These results show that lymphoid-related CD8(+) splenic DC, despite their mature phenotype, fail to provide appropriate signals to secondary CD4(+) T cells to sustain their proliferation.

Trichostrongylus colubriformis extract upregulates TNF-alpha receptor expression and enhances TNF-alpha sensitivity of L929 cells.

Many pathogens have developed strategies to avoid the host's immune system and hence improve their long-term survival. These strategies include antigenic variation, mimicry of host regulatory proteins and production of immunoregulatory molecules. The ruminant gastrointestinal nematode Trichostrongylus colubriformis produces several factors with homology to human immunoregulatory proteins. However, direct immunomodulation by T. colubriformis proteins has not yet been unequivocally demonstrated. Results in the present paper demonstrate that soluble T. colubriformis factors promote proliferation of the TNF-susceptible mouse fibrosarcoma cell line L929, while inhibiting proliferation of all other cell types tested. In addition, T. colubriformis homogenate enhanced the susceptibility of L929 cells to the cytotoxic action of ovine TNF-alpha. Within 1 h of exposure, T. colubriformis factors bind L929 cells in a stable fashion, yet it takes up to 24 h for the cells to become sensitised to TNF-alpha. Interestingly, the increase of both TNF-alpha sensitivity and proliferation of treated L929 cells correlated with an upregulation in expression of TNF-alpha p55 and p75 receptors.

Multi-epitope schistosome vaccine candidates tested for protective immunogenicity in mice.

The major challenge in the development of anti-schistosome vaccines is to use defined antigens to stimulate an appropriate immune response that leads to resistance. Several promising candidate vaccine antigens including the glycolytic enzyme triose-phosphate isomerase (SmTPI), a 28 kDa glutathione-S-transferase (Sm28), the myofibrilar protein paramyosin (Sm97), an integral membrane protein (Sm23) and calpain (Smcalpain) have been characterised and their primary sequences derived for Schistosoma mansoni. Furthermore, sequences are available for synthetic peptides mimicking epitopes on these molecules capable of inducing schistosome-specific T- and B-cell responses. These schistosome vaccine candidates have generally been tested with varying degrees of success as single components, with only one report of the use of a multivalent antigen or multi-epitope approach. We describe the assembly of multiple defined and different epitopes of S. mansoni into a variety of single covalent structures; these included a DNA vaccine encoding different epitopes in tandem, the polyprotein itself that is encoded by this DNA and branched synthetic peptide epitope-based polymers in which the individual epitopes are pendant from an inert backbone. Each of the vaccine constructs examined, with the exception of the DNA vaccine, generated antibodies that were capable of binding to a tandem sequence of the epitopes. Although these results were encouraging, none of the constructs protected animals from subsequent challenge infection, indicating that the immune responses elicited were inadequate or inappropriate for parasite killing in vivo.

The geometry of synthetic peptide-based immunogens affects the efficiency of T cell stimulation by professional antigen-presenting cells.

In the pathway leading to antibody production there are two points at which CD4(+) T(h) cells need to be recruited. The first of these is priming of T cells by their interaction with dendritic cells (DC) bearing antigen presented on MHC class II molecules and the second is the collaborative interaction of these primed T cells with B cells presenting the same antigen. We have previously shown that the configuration of T and B cell determinants within synthetic peptide immunogens can greatly influence the amount of immunogen required to produce an antibody response. Here we investigate whether the difference in potency of different immunogens is related to their ability to be presented by either DC or B cells. We show that determinants in a branched configuration, which are the most efficient at eliciting antibody in vivo, are presented to T cell clones by splenic CD8(-) DC 10-fold more efficiently than the corresponding determinants in a tandem linear arrangement. B cells also showed preferential presentation of branched immunogens to one T cell clone but in contrast to DC, not to a second T cell clone, indicating differences between the two antigen-presenting cell types. We also show that branched immunogens have a greater stability in serum compared to linear peptides, which may further enhance the differences in their in vivo potency.

New multi-determinant strategy for a group A streptococcal vaccine designed for the Australian Aboriginal population.

Infection with group A streptococci can result in acute and post-infectious pathology, including rheumatic fever and rheumatic heart disease. These diseases are associated with poverty and are increasing in incidence, particularly in developing countries and amongst indigenous populations, such as Australia's Aboriginal population, who suffer the highest incidence worldwide. Immunity to group A streptococci is mediated by antibodies against the M protein, a coiled-coil alpha helical surface protein of the bacterium. Vaccine development faces two substantial obstacles. Although opsonic antibodies directed against the N terminus of the protein are mostly responsible for serotypic immunity, more than 100 serotypes exist. Furthermore, whereas the pathogenesis of rheumatic fever is not well understood, increasing evidence indicates an autoimmune process. To develop a suitable vaccine candidate, we first identified a minimum, helical, non-host-cross-reactive peptide from the conserved C-terminal half of the protein and displayed this within a non-M-protein peptide sequence designed to maintain helical folding and antigenicity, J14 (refs. 8,9). As this region of the M protein is identical in only 70% of group A streptococci isolates, the optimal candidate might consist of the conserved determinant with common N-terminal sequences found in communities with endemic group A streptococci. We linked seven serotypic peptides with J14 using a new chemistry technique that enables the immunogen to display all the individual peptides pendant from an alkane backbone. This construct demonstrated excellent immunogenicity and protection in mice.

Totally synthetic lipid-containing polyoxime peptide constructs are potent immunogens.

A synthetic peptide corresponding to a sequence from influenza hemagglutinin was used as a model antigen to study the immunogenicity of polyoxime constructs. In the absence of any adjuvant, tetrameric forms of different polyoxime constructs did not elicit an antibody response. High and long-lasting levels of antibody were induced, however, by polyoxime constructs to which Pam3Cys (tripalmitoyl-S-glyceryl cysteine) was attached. Comparable serum antibody levels were achieved with Tetraoxime-Pam3Cys administered by the intraperitoneal or intranasal routes to those obtained when the monomeric peptide was administered by the intraperitoneal route in complete Freund's adjuvant (CFA). Mice receiving Tetraoxime-Pam3Cys and Pam3Cys-peptide intranasally developed peptide-specific antibody secreting cells (ASCs) in their lungs and mediastinal lymph nodes. At low dose, the Tetraoxime-Pam3Cys induced higher levels of antibody compared to those elicited by the monomeric Pam3Cys-peptide delivered by either route. These results show that lipo-tetraoxime constructs assembled by polyoxime chemistry can be potent inducers of systemic and mucosal immunity.

Mice lacking the transcription factor subunit Rel can clear an influenza infection and have functional anti-viral cytotoxic T cells but do not develop an optimal antibody response.

Rel, a haemopoietic cell-restricted member of the NF-kappaB/Rel family of transcription factors, has recently been shown to be important in the function of B and T lymphocytes. In an attempt to understand the role of this protein in the immune response, we examined the ability of Rel(-/-) mice to counter an influenza virus infection. Normal levels of virus-specific cytotoxic T cells induced in Rel(-/-) mice were able to clear virus from the lungs, albeit with somewhat delayed kinetics compared to normal mice. Rel(-/-) mice did, however, display a markedly reduced T cell proliferative response to the virus, and exhibited impaired local and systemic influenza virus-specific antibody responses. This defect was sufficient to result in an inability of vaccinated mice, but not of previously infected mice, to acquire antibody-dependent protective immunity to reinfection with the same virus. These findings establish that during the response to influenza virus, Rel function allows optimal development of humoral immunity, a role that apparently cannot be fulfilled by other NF-kappaB/Rel proteins.

Antigenic and immunogenic properties of totally synthetic peptide-based anti-fertility vaccines.

In this study we describe the results of experiments in which a variety of totally synthetic luteinizing hormone releasing hormone (LHRH) vaccines were assembled and examined for their abilities to elicit antibody responses and induce sterility in mice. It is shown that totally synthetic vaccines consisting of a 15 residue-defined T cell epitope and the 10 residue LHRH epitope not only induced high titers of antibody but also induced sterility. This effect did not appear to correlate with antibody titer, antibody isotype or comparative antibody affinity, but may be related to the length of time for which antibodies are present to exert their influence.

Different effects of simple anoxic lactic acidosis and simulated in vivo anoxic acidosis on turtle heart.

We compared responses of turtle heart at 20 degrees C to an anoxic lactic acidosis solution (LA) containing 35 mM lactic acid in an otherwise normal turtle Ringers equilibrated with 3% CO2/97% N2 at pH 7.0) to a solution simulating in vivo anoxic acidosis (VA), with elevated concentrations of lactate, Ca2+, Mg2+, and K+, and decreased Cl-, equilibrated with 10.8% CO2/89.2% N2 at pH 7.0. We examined mechanical properties on cardiac muscle strips and determined intracellular pH (pHi) and high energy phosphates on perfused hearts using 31P-NMR. Maximum active force (Fmax) and the maximum rate of force development (dF/dtmax) of muscle strips were significantly higher during VA than during LA superfusion. An elevation of Ca2+ alone (to 6 mM) in LA significantly increased both Fmax and dF/dtmax but the effects diminished toward the end of the exposure; however, hypercapnic anoxic lactic acidosis (addition of 20 mM HCO3- to LA, equilibrated with 10.8% CO2/89.2% N2, pH 7.0) did not significantly affect Fmax or dF/dtmax. During VA perfusion, pHi (6.73 +/- 0.01) was significantly higher than that during LA perfusion (pHi 6.69 +/- 0.013), but the difference is probably too small to have physiological significance. ATP, creatine phosphate, and inorganic phosphate were not significantly different in the two anoxic solutions. We conclude that the reduction of cardiac mechanical function in vivo is minimized by the integrated effects of changes of ionic concentrations, but the observed changes in Ca2+ and pHi cannot fully explain the effect.

Avoidance of self-reactivity results in skewed CTL responses to rare components of synthetic immunogens.

In studying the CTL recognition of peptide determinants derived from the nuclear Ag La (SS-B), we observed significant skewing of the response toward rare components present within the immunogen. Thus, priming of naive mouse lymphocytes in vitro with a synthetic H-2Kb-binding peptide comprising human La (hLa) residues 51-58 resulted in class I-restricted cytotoxic T cells that failed to recognize naturally presented hLa 51-58 peptide. Instead, the majority of T hybrids recognized a low abundance (< or = 1%) contaminant present at picomolar concentrations in the original synthesis and identified as a peptide adduct containing N,4-t-butyl asparagine at position 6 of the hLa 51-58 sequence. The preferred T cell recognition of the butyl adduct was not due to increased affinity of this peptide for the H-2Kb molecule or to the antagonism of CTL recognizing the unmodified determinant. Rather, the bias in the immune response appeared to be the result of partial self-tolerance to the homologous mouse La 51-58 determinant, which differs from its human counterpart by only a single amino acid at position 1 (T-->I). Accordingly, the CTL response appeared to be focused on "non-self" ligands present within the synthesis, even though they were present at very low concentrations. These observations have significant implications for the use of synthetic peptide vaccines, especially those designed to manipulate responses to self peptides such as tumor Ags in which self-tolerance may result in unexpected reactivity.

CTL recognition of an altered peptide associated with asparagine bond rearrangement. Implications for immunity and vaccine design.

The extent to which peptides containing chemically and post-translationally modified amino acid side chains are recognized by primed CTL has not been clearly defined. We report on the CTL recognition of a MHC class I-restricted peptide containing a cyclized asparagine (succinimide) residue. This modification of the asparagine side chain is a common intermediate structure during deamidation, isomerization, and bond rearrangements of amide-containing amino acids and also occurs as a side reaction in peptide synthesis. The CTL specifically recognized the succinimide-containing peptide showing only weak cross-reactivity at high concentrations of the parent peptide containing unmodified asparagine. Similarly, CTL raised against the parent peptide did not recognize the succinimide derivative of this peptide. Naturally processed forms of these structures are likely to occur given the importance and frequency of deamidation both in vitro and in vivo. Moreover, since succinimide intermediates of deamidated peptides can occasionally be very stable, these peptides have the potential to act as altered self-Ags with significant implications for autoimmunity. In addition, unwanted and potentially hazardous specificities may be elicited when using synthetic peptides in subunit vaccines in which succinimide residues may form spontaneously during storage or chemical synthesis.