Nucleoside-modified mRNA vaccines yield robust blocking antibody responses against major house dust mite allergens.

To the Editor,

The capacity of nucleoside‐modified mRNA vaccines to generate potent neutralizing antibody responses against viruses, including SARS‐CoV‐2, could be harnessed for the design of new allergen‐specific immunotherapy (AIT) protocols to promote high levels of blocking antibodies. In the present study, two lipid nanoparticle (LNP)‐formulated nucleoside‐modified mRNAencoding secreted hypoallergenic forms of house dust mite (HDM) allergen Der p 1 (ProDer p 1, pDp1) and Der p 2 (Dp2K96A) were synthesized (Figures S1 and S2). BALB/c mice were intramuscularly (i.m.) immunized with 20 μg of each mRNA‐LNP at weeks 0, 3 and 6 and antibody responses were followed for 15 total weeks (Figure 1A).

FIGURE 1

Immunogenicity and blocking IgG antibody capacity of monovalent mRNA‐pDp1‐LNP and mRNA‐Dp2K96A‐LNP. (A) Immunization and bleeding schedule; (B) pDp1‐ and Dp2‐specific IgG1 and IgG2a antibody titers; the dotted horizontal lines represent the lowest serum dilution tested. * p < .05; (C) Blocking capacity of specific IgG triggered by mRNA‐pDp1‐LNP or mRNA‐Dp2K96A‐LNP at 1/40 or 1/200 dilution. * p < .05; (D) Inhibition of RBL‐SX38 cell degranulation by specific IgG induced by mRNA‐pDp1‐LNP or mRNA‐Dp2‐LNP immunization. RBL‐SX38 cells, primed with five Der p 1‐ or Der p 2‐positive sera, were activated with 0.01 μg/ml nDer p 1 or rDer p 2 preincubated or not with pooled mouse sera (preimmune or week 6) diluted 20 or 200 times. *p < .05. One representative of two similar experiments is shown. N = 6 animals per experimental group. p values were calculated using the Mann–Whitney t‐test or Two‐way anova.

Both mRNA‐LNP formulations triggered potent allergen‐specific IgG1/IgG2a titers (Figure 1B), with the highest binding magnitude reached after the first boosting dose (p < .05, week 6). A second booster vaccination did not increase the antibody levels (p > .05). At the time of animal necropsy (week 15), the decrease in specific antibody titers was not significant for Der p 1‐specific IgG1 and Der p 2‐specific IgG2a but was significant for Der p 1‐specific IgG2a and Der p 2‐specific IgG1 (p < .05). Remarkably, Der p 1‐ or Der p 2‐specific IgE were not detectable in any tested sera (data not shown).

Allergen‐specific IgGs were capable to inhibit the binding of human specific IgE (Table S1) to coated natural Der p 1 (nDer p 1) or rDer p 2 (Figure 1C). The magnitude of blocking capacity of IgG antibodies to Der p 1 or Der p 2 peaked at week 9 (three immunizations), with inhibition percentage of around 85%–90% and 60%–75% for 1/40 and 1/200 immune serum dilution, respectively. At the time of animal necropsy (week 15), the binding of IgE to coated allergen could still be reduced by around 70% and 20% at the same tested serum dilutions. The antibodies at week 9 were able to inhibit the degranulation of RBL‐SX38 cells triggered by nDer p 1 or rDer p 2, the highest magnitude of inhibition (around 80%) being observed with the highest dilution of mice sera (1/200 dilution) (Figure 1D).

As HDM allergics are predominantly co‐sensitized with Der p 1 and Der p 2, we next characterized the immunogenicity of a bivalent vaccine (a 1:1 mix of pDp1 ‐ Dp2K96A mRNA‐LNP) and investigated the difference in antibody responses between three different doses (10, 2 or 0.4 μg) (Figure 2A). Statistically significant difference in specific IgG1 and IgG2a titers was observed between the different dose groups after the first and the second immunization (Figure 2B). Strikingly, a third immunization largely attenuated the dose effect as comparable antibody titers were detected at all tested dose (with the exception of Der p 2‐specific IgG1 at week 6) up to week 13 (p > .05). Two immunizations with 10 or 2 μg but not with 0.4 μg mRNA mix induced Der p 1‐ and Der p 2‐specific IgG capable to inhibit IgE binding to natural Der p 1 by around 75% and 55% and to rDer p 2 by around 50% and 30%, respectively (Figure 2C). The blocking IgG responses peaked after the second boosting dose (around 85% inhibition in the 10 μg dose group, p < .05) and the levels of inhibition were maintained up to week 13. Der p 1‐/Der p 2 specific antibodies, after three immunizations, displayed dose‐dependent RBL degranulation inhibitory capacities (Figure 2D). The basophil activation was reduced by more than 90% and 80% with the Der p 1‐ and Der p 2‐specific IgG, respectively, in the 10 μg group (1/200 dilution, p < 0.05). Finally, splenocyte restimulations evidenced that mRNA vaccinations triggered strict Th1 response as judged by IFNγ production and the absence of detectable levels of IL‐5 (Figure 2E). The trend for a dose‐dependent IFNγ secretion was not stastistically significant.

FIGURE 2

Immunogenicity, blocking IgG antibody capacity and reactogenicity of bivalent mRNA‐pDp1‐LNP/mRNA‐Dp2K96A‐LNP. (A) Immunization and bleeding schedule; (B) pDp1‐ and Dp2‐specific IgG1 and IgG2a antibody titers; the dotted horizontal lines represent the lowest serum dilution tested. * p < .05; (C) Blocking capacity of specific IgG triggered by bivalent mRNA‐LNP at week 6 and at 1/40 or 1/200 dilution. * p < .05; (D) Inhibition of RBL‐SX38 cell degranulation by specific IgG induced by bivalent mRNA‐LNP. RBL‐SX38 cells, primed with five Der p 1‐ or Der p 2‐positive sera, were activated with 0.01 μg/ml nDer p 1 or rDer p 2 preincubated or not with pooled mouse sera (preimmune or week 6) diluted 20 or 200 times. * p < .05; (E) IFNγ and IL‐5 secretion by splenocytes from immunized mice restimulated with rpDp1 or rDp2. (F) Sensitization and challenge schedule to test the reactogenicity of mRNA‐LNP; (G) Body temperature change (Mean and SEM) monitored by infrared thermometer for 60 min following the challenge. * p < .05; (H) Anaphylactic symptom score observed 20 min following the challenge. * p < .05; (I) Serum levels of mMCPT‐1 (Mean and SD) measured before and 60 min following challenge. * p < .05. One representative of two similar experiments is shown. N = 6 animals per experimental group. p values were calculated using the Mann–Whitney t‐test or Two‐way anova.

Finally, we evaluated the reactogenicity of mRNA‐LNP in BALB/c mice intraperitoneally (i.p.) co‐sensitized with nDer p 1/rDer p 2 adsorbed to alum. Two weeks post‐sensitization, animals were intramuscularly injected with a mix of 10 μg pDp1‐Dp2K96A mRNA‐LNP or Poly(C) control mRNA‐LNP. As a positive control, sensitized mice were challenged intraperitoneally with unadjuvanted nDer p 1/rDer p 2 (Figure 2F). The i.p. nDer p 1/rDer p 2 challenge performed in sensitized mice induced, within 20 min, an anaphylactic response characterized by a drop in body temperature, symptom development and high MCPT‐1 serum levels (Figure 2G–I). These cardinal features of anaphylaxis were not observed in mRNA‐LNP‐challenged animals. Mouse monitoring performed 5 h post‐mRNA‐LNPs injection, a time point where the mRNA translation to antigen production commonly peaks, did not evidence any change in mouse behavior and body temperature. Mice remained as well in good health conditions at time points 24 and 48 h following mRNA‐LNP injection.

In conclusion, our results show the great potential of the synthetic nucleoside‐modified mRNA‐LNP platform for the development of potent allergen‐specific blocking IgG responses. Moreover, our first preclinical safety data, combined with the short‐lived in‐vivo antigen expression commonly observed in mRNA‐LNP‐immunized mice, suggest that AIT based on mRNA‐LNP administration could be safe. Future studies will aim to measure the efficacy of AIT protocols based on mRNA‐encoding Der p 1 and Der p 2 in mouse models of HDM‐induced allergic airway inflammation.

CONFLICT OF INTEREST

N.P. and D.W. are named as co‐inventors on a patent describing the use of nucleoside‐modified mRNA in lipid nanoparticles as a vaccine platform (WO 2016/176330 A1). P.J.C.L is employee at Acuitas Therapeutics. The remaining authors declare that they have no conflicts of interest.

Appendix S1

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