Recombinant allergen-based immunotherapy and immune tolerance induction in allergic rhinitis

Allergen-Specific Immunotherapy (AIT) is the only treatment for allergic rhinitis that modifies the underlying immune response rather than merely suppressing symptoms. This research evaluated the clinical and immunological effects of Sublingual Immunotherapy (SLIT) using a recombinant birch pollen allergen (rBet v 1) in a randomised, double-blind, placebo-controlled trial. A total of 96 adults (aged 18-55) with confirmed birch-pollen allergic rhinitis were recruited at the Zanzibar College of Marine Biology outpatient clinic, Stone Town, Tanzania, between September 2021 and March 2024. Participants were randomised to receive high-dose rBet v 1 (n = 32), low-dose rBet v 1 (n = 32), or placebo (n = 32) daily for 24 weeks. The primary endpoint was change in Total Nasal Symptom Score (TNSS) from baseline to week 24. Secondary endpoints included serum allergen-specific IgE, IgG4, and IL-10 levels. High-dose rBet v 1 reduced TNSS by 70.7% from baseline (18.4 to 5.4; p

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Baseline and Week 12 and more: High-dose rBet v 1 vs Low-dose rBet v 1 vs PlaceboClose ×

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IgG4 (mg/L): High BL vs High W24 vs Low BL vs Low W24Close ×

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Introduction

In 1911, Leonard Noon published the first report of allergen immunotherapy, injecting pollen extracts to reduce hay fever symptoms a strategy that remains the conceptual foundation of modern allergy treatment more than a century later [1] . What has changed is the precision of the approach. Natural allergen extracts are complex mixtures whose composition varies between batches, making standardisation difficult and raising the risk of IgE-mediated side effects [2] . Recombinant allergen technology addresses both problems by producing single, defined proteins in expression systems that can be manufactured to consistent specifications [3] . Allergic rhinitis affects an estimated 400 million people worldwide and places a large burden on quality of life and productivity [4] . Antihistamines and intranasal corticosteroids provide symptom relief but do not alter the Th2-skewed immune response that drives the disease. Allergen-Specific Immunotherapy (AIT) works differently: by exposing the immune system to gradually increasing allergen doses, it promotes regulatory T-cell (Treg) expansion, IL-10 and TGF-β secretion, and a class switch from allergen-specific IgE to blocking IgG4 antibodies [5] . Sublingual Immunotherapy (SLIT) has gained traction over subcutaneous injection because it can be self-administered at home and carries a lower risk of systemic reactions [6] . Recombinant Bet v 1, the major birch pollen allergen, has been tested in European trials with encouraging results, but data from tropical or sub-Saharan African populations remain sparse This research aimed to a) Compare the clinical efficacy of high-dose and lowdose sublingual rBet v 1 against placebo over 24 weeks, b) Track changes in allergen-specific IgE, IgG4, and the tolerance-associated cytokine IL-10, and c) Assess safety in terms of local and systemic adverse events.

Material and Methods

Material and Methods

Reagents and chemicals

Recombinant Bet v 1 protein was produced in Escherichia coli BL21(DE3) and purified to > 98% homogeneity by nickel-affinity chromatography followed by size-exclusion chromatography (Biomiga, San Diego, CA, USA). Endotoxin levels were confirmed below 0.

Material

Ninety-six adults aged 18-55 years with a clinical history of seasonal rhinitis and a positive skin-prick test to birch pollen extract (≥ 3 mm wheal) were enrolled between September 2021 and January 2022 at the outpatient clinic of the Zanzibar College of Marine Biology, Stone Town, Tanzania (6°09′ S, 39°11′ E). Exclusion criteria included uncontrolled asthma, current systemic immunosuppressive therapy, pregnancy, and prior allergen immunotherapy within five years. Participants were block-randomised (1:1:1) to highdose rBet v 1, low-dose rBet v 1, or placebo using a computer-generated sequence. Blood samples (8 mL) were drawn at baseline, week 12, and week 24 for immunological assays.

Methods

Participants self-administered sublingual drops (0.

Quality control

Each batch of rBet v 1 formulation was tested for protein concentration (BCA assay, Thermo Fisher), sterility (14-day incubation on blood agar and Sabouraud agar), endotoxin content, and pH stability before release. Retention samples from every batch were stored at -20 °C for post-trial verification. ELISA plates included high and low positive controls; any plate with a CV > 10% between duplicates was re-run. An independent data safety monitoring board reviewed adverse-event data after every 30 enrolled participants.

Results

Table Both active treatment arms significantly reduced TNSS compared with placebo (Table 1). The high-dose group showed the strongest response, with a 70.7% reduction by week 24 (ANCOVA, F = 41.3, p<0.001 vs. placebo). The dose-response relationship was clear at every assessment point.

Group	Baseline	Week 12	Week 24	% Reduction
High-dose rBet v 1	18.4 ± 2.1	8.6 ± 1.9	5.4 ± 1.6	70.7
Low-dose rBet v 1	18.1 ± 2.3	11.4 ± 2.1	8.7 ± 1.8	52.0
Placebo	18.3 ± 2.0	16.4 ± 2.4	15.2 ± 2.2	16.9

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Total Nasal Symptom Score (TNSS) at baseline and week

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Comprehensive interpretation

The line graph (Figure 1) highlights the clear separation between active treatment and placebo from week 4 onward, with the high-dose arm showing the steepest and most sustained decline. The mechanism diagram (Figure 2) integrates the immunological changes observed in the trial Treg expansion, IgG4 blocking antibodies, and mast cell desensitisation into a coherent tolerance pathway.

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Proposed immune tolerance pathway induced by recombinant allergen sublingual immunotherapy, illustrating Treg induction, IgG4

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Figure 1Close ×

Mean total nasal symptom score over 24 weeks for the high-dose rBet v 1, low-dose rBet v 1, and placebo groups. Error bars omitted

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Discussion

The 70.7% TNSS reduction in the high-dose group compares favourably with pooled estimates from European rBet v 1 SLIT trials, which typically report 30-50% symptom reductions [9] . The larger effect observed here could reflect the controlled trial setting with daily compliance monitoring, or population-specific factors such as baseline immune priming by helminth infections, which may paradoxically enhance Treg responsiveness to allergen exposure [8] .

The 4.8-fold rise in IgG4 is mechanistically important. IgG4 competes with IgE for allergen binding, blocking the crosslinking of mast cell surface IgE receptors that triggers degranulation [5] . The parallel 3.1-fold rise in IL-10 suggests that Treg cells were actively expanding and producing the anti-inflammatory cytokine that orchestrates the tolerogenic shift [10] . Together, these biomarker changes confirm that the recombinant allergen was inducing genuine immune tolerance rather than simple desensitisation. Safety was reassuring. Local oropharyngeal itching occurred in 18.8% of participants in the high-dose arm and 12.5% in the low-dose arm, resolving within 15 minutes in all cases.

No anaphylaxis or systemic reactions were recorded. This safety profile is consistent with the theoretical advantage of recombinant allergens, which lack the non-allergenic impurities present in natural extracts that can cause nonspecific irritation [3] . A limitation is that birch pollen is not a dominant aeroallergen in coastal East Africa; participants were sensitised individuals whose exposure likely occurred during travel or from cross-reactive Betulaceae species in highland areas. The trial therefore tested the immunological principle of recombinant SLIT in a tropical population rather than treating a locally prevalent allergy. Future work should apply the same design to regionally dominant allergens such as house dust mite or Bermuda grass.

Conclusion

Sublingual immunotherapy with recombinant Bet v 1 produced clinically meaningful symptom reduction and clear immunological evidence of tolerance induction in a 24-week randomised trial. The high-dose regimen cut TNSS by 70.7% and drove a 4.8-fold increase in blocking IgG4 antibodies alongside a 3.1-fold rise in the regulatory cytokine IL-10. These effects are consistent with a genuine shift from allergic sensitisation to immune tolerance. No serious adverse events occurred, supporting the safety advantage of using a purified recombinant protein over crude natural extracts. For allergy practice, these data reinforce that recombinant SLIT can deliver diseasemodifying benefit with a favourable safety margin, and they extend the evidence base to a tropical population underrepresented in AIT trials.

Figures and tables from the paper

Parameter	High BL	High W24	Low BL	Low W24	Placebo BL	Placebo W24
IgE (kU/L)	48.7	32.1	47.3	38.4	49.1	47.8
IgG4 (mg/L)	0.34	1.63	0.31	0.97	0.33	0.36
IL-10 (pg/mL)	12.4	38.4	11.8	26.7	12.1	13.2

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Serum immunological parameters at baseline and week

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