Published clinical studies of peptide immunotherapy in allergic disease are restricted to peptides from two model allergens, Fel d1 from cats and Api m1 (PLA2) from bee venom. The reported clinical and mechanistic outcomes of peptide immunotherapy are shown in Fig. 2. Studies are summarized in Table 1.
A number of PIT studies have been performed in subjects allergic to cats. In four studies the safety and efficacy of a mixture of two polypeptides from Fel d 1 was evaluated. In the first of these to be reported, peptides were administered subcutaneously to 95 cat allergic subjects at 3- to 4-day intervals. Three peptide doses were employed (7.5, 75 and 750 μg per injection) (45). Improvements in lung and nasal symptom scores were observed at higher doses. Treatment was associated with a large number of adverse events occurring minutes to hours after peptide administration. Related in vitro studies showed reduced IL-4 production in peptide-specific T-cell lines in vitro following therapy (46).
In further studies, reduction in allergen PD20 (provocative dose of inhaled allergen resulting in a 20% reduction in forced expiratory volume in 1 s; FEV1) was observed in response to high-dose (individual doses of 750 μg up to a total dose of between 1500 and 4500 μg) and medium-dose (individual doses of 75 μg up to a total dose of between 150 and 450 μg) regimens when compared with baseline, but not when compared with placebo (47). Treatment was also associated with reduced allergen-induced IL-4 production from peripheral blood mononuclear cells (PBMC).
In a double-blind, placebo-controlled, parallel-group study, peptides or placebo was administered weekly by subcutaneous injection (four doses of 250 μg) to 42 subjects with cat-allergic rhinitis and/or asthma (48). Treatment was associated with adverse events, primarily late-onset symptoms of rhinitis, asthma and pruritis. PBMC cytokine secretion profiles did not differ between peptide-treated and placebo-treated subjects. No changes in early and late-phase skin responses to allergen challenge were observed.
In the largest of the four studies, 133 cat allergic subjects received eight subcutaneous injections of 750 μg of the peptide mixture. The only significant clinical outcome was an improvement in pulmonary function observed in individuals with reduced baseline FEV1 (49). This improvement was only evident at a single time point (3 weeks) after therapy. Several adverse events were reported in common with other studies.
More recently, a series of clinical studies have been performed using mixtures of shorter peptides from Fel d 1 (50–56). Peptides were administered intradermally to cat allergic asthmatic subjects of mild to moderate disease severity. Peptide administration significantly reduced the magnitude of the cutaneous late-phase reaction to intradermal allergen challenge. Evaluation of PBMC responses to allergen in vitro demonstrated reductions in both Th2 cytokines and γ-interferon (IFN-γ) (50).
Subsequently, in a double-blind, placebo-controlled study, 24 cat allergic asthmatic subjects underwent cutaneous allergen challenge, inhaled methacholine PC20 and inhaled allergen PD20 (52). Subjects received a total of 90 μg of each of 12 peptides, covering most of the Fel d 1 molecule, in divided incremental doses starting at 5 μg and administered at 3- to 4-day intervals. There was a significant reduction in both early and late-phase cutaneous reactions to intradermal challenge with allergen when compared with placebo. Reduced proliferative responses and Th1 and Th2 cytokine production were observed in the active treatment group. Reductions in pro-inflammatory cytokines were associated with a concomitant increase in IL-10. Subjects treated with peptides felt significantly better able to tolerate exposure to cats after therapy. No significant improvements were observed in PD20 or PC20, but the study may not have been sufficiently powered to detect such differences which were secondary, rather than primary, outcome measures.
However, a significant improvement in PC20 was observed in a small open-label study using a similar peptide preparation delivered at 2-week intervals rather than 3- to 4-day intervals (53). Peptides were administered by intradermal injection but at 2-week intervals and a lower dosing regimen was applied (total dose: 41.1 μg). A significant reduction in the magnitude of the cutaneous late-phase reaction was also observed following allergen challenge in the skin. There was a significant increase in the number of CD25+ cells in allergen challenge skin sites compared with placebo challenge after peptide immunotherapy. The number of CD4+/IFN-γ+ cells also increased, suggesting that recruitment of Th1 cells to the skin may be an important mechanism. No increases in IL-10+ cells were observed in the skin but expression of TGF-β mRNA was increased.
Studies evaluating upper and lower airway outcomes following a higher dose PIT protocol have also been performed (55). Cat allergic asthmatic subjects were screened by inhaled incremental allergen challenge to establish whether they developed a single early asthmatic response or a dual early and late asthmatic response. Those with dual responses were recruited into a small double-blind, placebo-controlled (n = 16; 8 active, 8 placebo) study whilst those displaying single early responses (n = 12) were recruited into an open study. Both groups underwent the same treatment protocol receiving approximately 300 μg of each of 12 peptides delivered as a mixture intradermally in saline. Outcomes were evaluated 4–8 weeks after the completion of therapy and again at 3–4 months. Treatment significantly reduced cutaneous late-phase reactions to intradermal allergen challenge as in previous studies. The effect of PIT on late asthmatic responses was evaluated by comparison of bolus inhaled allergen challenge before and after treatment. No effect was observed on the early asthmatic reaction but a significantly reduced late asthmatic reaction was observed within the treatment group (FEV1; area under curve 2–8 h postchallenge). In the open study allergen PD20 was evaluated by incremental inhaled allergen challenge. Nasal outcomes were measured after inhaled allergen challenge. No change was observed in allergen PD20 nor histamine PC20. However, nasal allergen challenge revealed a significant reduction in outcome scores (sneezing, weight of nasal secretions and nasal blockage). Additionally, significant improvements were observed in three of seven rhinitis quality of life questionnaire fields.
The effect of peptide immunotherapy on peripheral blood CD4+ responses and CD4+CD25+ regulatory T-cell suppression of allergen-stimulated cultures has also been evaluated (54). Proliferative responses and IL-13 production from PBMC cultured with allergen in vitro were significantly reduced following PIT as in previous studies. Regulatory T-cell activity was assessed by mixing with autologous CD4+CD25− cells. No change in the suppressive activity of CD4+CD25+ cells was observed. Thus, CD4+CD25+ regulatory T cells may not play a significant role in the mechanism of action of peptide immunotherapy.
The role of PIT on antigen-specific inducible regulatory T cells was addressed by mixing CD4+ T cells with CD4negative cells (56). The two populations were labelled with different fluorescent dyes (CD4+ were labelled red and CD4negative were labelled green). CD4+ cells from before and after PIT were mixed with CD4negative cells from before and after therapy, in all possible combinations. The results demonstrated that CD4+ cells isolated after PIT could suppress the proliferative response of baseline CD4negative cells. These data provide evidence that peptide immunotherapy induces a population of CD4+ T cells with regulatory activity.
Insect venom allergy
The first study of PIT in insect venom allergic individuals employed peptides from Api m 1. Five bee venom allergic subjects received divided incremental doses of a mixture of three peptides at weekly intervals (57). Ten subjects treated with conventional bee venom immunotherapy served as controls. A cumulative peptide dose of 397.1 μg was given. Subcutaneous challenge with 10 μg of whole Api m 1 was tolerated without systemic allergic symptoms by all subjects. Three of five tolerated a wild bee sting challenge without reaction, the remaining two subjects developed mild systemic allergic reactions. No changes were observed in levels of allergen-specific serum IgE or IgG4 during PIT but following subcutaneous challenge with whole allergen 1 week after the last peptide injection, serum concentrations of both antibody isotypes increased sharply.
Texier et al. determined the major T-cell epitopes of Api m 1 using direct binding of peptides to purified major histocompatibility complex class II molecules (58). Four peptides were identified, which were administered to bee venom allergic individuals with mild disease in a controlled, open-label, single-blind study (59). A total dose of 431.1 μg of each of the four peptides was administered to 12 individuals. Treatment was well tolerated and no adverse events were observed. Allergen-specific T-cell proliferative responses to whole bee venom and purified allergen were reduced following treatment. IL-13 and IFN-γ responses of PBMC cultured with allergen were reduced after treatment whereas IL-10 was increased. Late-phase cutaneous reactions to both whole bee venom and Api m 1 were significantly reduced. There was a small, significant but transient increase in allergen-specific IgG and IgG4 during peptide immunotherapy. No subjective outcome measures were analysed.
In another study, bee venom allergic subjects were treated according to a RUSH protocol with three synthetic polypeptides (long synthetic peptides) covering the whole Api m 1 molecule and ranging in length from 45 to 60 amino acids in length (60). Patients received approximately 250 μg in incremental divided doses at 30 min intervals starting with 0.1 μg. Up to five maintenance injections of 100 μg, or in some cases 300 μg, were subsequently given. No significant change in skin sensitivity to intradermal allergen challenge was observed. A transient increase in T-cell proliferation to the peptides was observed during therapy in the active treatment group. IFN-γ and IL-10 levels but not Th2 cytokines increased. Allergen-specific IgG4 levels increased throughout the study period. Peptide-specific IgE was induced in some patients during the study. Some local and disseminated erythema with occasional hand palm pruritis was observed in two subjects.
In conclusion, improved understanding of the mechanisms of immunological tolerance and the mechanisms underlying the efficacy of SIT have allowed substantial progress to be made in peptide immunotherapy. There are numerous studies in both allergic and autoimmune diseases that support the potential of this approach. More studies are required to define the optimal dose range, dose interval and route of administration. Initial concerns over the incidence of adverse events may be allayed through use of short peptides and low doses.