Multivalent mRNA-DTP vaccines are immunogenic and provide protection from Bordetella pertussis challenge in mice

Abstract

Acellular multivalent vaccines for pertussis (DTaP and Tdap) prevent symptomatic disease and infant mortality, but immunity to Bordetella pertussis infection wanes significantly over time resulting in cyclic epidemics of pertussis. The messenger RNA (mRNA) vaccine platform provides an opportunity to address complex bacterial infections with an adaptable approach providing Th1-biased responses. In this study, immunogenicity and challenge models were used to evaluate the mRNA platform with multivalent vaccine formulations targeting both B. pertussis antigens and diphtheria and tetanus toxoids. Immunization with mRNA formulations were immunogenetic, induced antigen specific antibodies, as well as Th1 T cell responses. Upon challenge with either historical or contemporary B. pertussis strains, 6 and 10 valent mRNA DTP vaccine provided protection equal to that of 1/20th human doses of either DTaP or whole cell pertussis vaccines. mRNA DTP immunized mice were also protected from pertussis toxin challenge as measured by prevention of lymphocytosis and leukocytosis. Collectively these pre-clinical mouse studies illustrate the potential of the mRNA platform for multivalent bacterial pathogen vaccines.

Fig. 1. Bacterial mRNA constructs are translated into protein in mammalian cells in vitro and are immunogenic in mice.

a Schematic of mRNA design for each antigen, describing (1) the protein sequence, (2) the signal sequence and (3) toxin-inactivating mutations included in the construct. The expected molecular weight of the translated protein is included in parentheses. b Following transfection of Expi293F cells with individual mRNA constructs, supernatants were collected 48 h later and evaluated for detection of secreted pertussis, tetanus and diphtheria proteins using the JESS system and antigen specific antibodies. c–h Sera collected from 8-week-old C57BL/6 J mice one-month post-boost were evaluated for antigen specific antibodies using a Luminex assay which has beads coated with native antigens purified from (d–f) Bordetella pertussis (FHA, FIM 2/3, PRN) and (c, g, h) detoxified PT, DT, and TT toxoids. The mRNA-DTP-6 vaccine was able to induce Bp antigen specific antibodies comparable to DTaP vaccination. Bar graphs are mean with error bars shown as ±SD. A two-way ANOVA with Tukey’s post-hoc test was used to calculate significant difference. **p < 0.0021, ****p < 0.0001. LNP-ncRNA= Lipid Nanoparticle- noncoding RNA; mRNA-DTP-6= mRNA- Diphtheria Tetanus Pertussis 6 antigen.

Fig. 2. mRNA-DTP-6 vaccination induces more splenic CD4 and CD8 memory T cells than DTaP vaccination.

Flow cytometry was used to measure Splenic memory T cells. mRNA-DTP-6 elicited statistically higher levels of CD4⁺CXCR3⁺ TEM (a) and TCM (b) and CD8⁺CXCR3⁺ TEM (c) and TCM (d) than DTaP vaccinated mice. T cell cytokine responses were also measured with flow cytometry and mRNA-DTP-6 induced higher levels of CD4⁺IFNγ⁺ (e) which suggest a more Th1-skewed cell mediated response than DTaP vaccinated mice. There was no statistical difference between mRNA-DTP-6 and DTaP CD4⁺IL-13⁺ T cells (f). Error bars are mean ± SEM. A Mann–Whitney two-tailed with 95% confidence interval test was used to determine statistical difference from mRNA-DTP-6; **p < 0.0021, ***p < 0.0002, ****p < 0.0001. LNP-ncRNA= Lipid nanoparticle- noncoding mRNA; mRNA-DTP-6= mRNA- Diphtheria, Tetanus, and Pertussis 6 antigen vaccine.

Fig. 3. IgG antibody titers in the serum, bacterial burden in the respiratory tract, and pulmonary IL-6 production 3 days after B. pertussis isolate UT25Sm1 challenge using a C57BL/6 J model.

a Vaccination and challenge model utilized to evaluate mRNA vaccine immunogenicity and protection. b Schematic on novel mRNA constructs included in mRNA vaccination. c A chart listing greater combination of mRNA and antigens included. Serum IgG antibody titers to B. pertussis (Bp) (d) and PT (e) were determined by ELISA assay. B. pertussis was quantified by counting serially diluted CFUs following challenge with isolate UT25Sm1. CFU counts were determined from lung and trachea (L &T) homogenate (f). g Pro-inflammatory cytokine, IL-6, was quantified in the lungs at 3 days post-challenge using a V-Plex cytokine kit. Kruskal-Wallis with Dunn’s post-hoc test (d and e) or a one-way ANOVA with a Tukey’s post-hoc test (f, g) were used to calculate differences to MVC; *p < 0.05, **p < 0.005, ***p < 0.005. Another Kruskal-Wallis with Dunn’s post-hoc test (d and e) or a one-way ANOVA with a Tukey’s post-hoc test (f and g) were used to calculate differences between multivalent vaccine formulation; ^#p < 0.05, ^##p < 0.005. Box and whisker plots display minimum to maximum values with all data points. MVNC = mock-vaccinated and non-challenged; MVC = mock-vaccinated and challenged; SEM = standard error of the mean; SD = standard deviation; ns= non-significant, p > 0.05. n = 5 per treatment group. Dotted lines indicate lowest limit of detection. Consult Table 1 for vaccine antigen description and Table 2 for vaccine name.

Fig. 4. Immunological response quantified post-prime and post-boost.

a Immunization and serum collection timeline. b Serum IgG antibody titers to B. pertussis (Bp) strain UT25 were analyzed by ELISA assay two weeks after the prime and boost immunization. Post-boost Th1/Th2 IgG antibodies in the sera quantified via ELSA assay. Post-boost anti-B. pertussis (Bp) IgG1 (c), IgG2a (d), IgG2b (e) response after boost. f Heat map depicting serological IgG titers recognizing pertussis, diphtheriae, and tetanus antigens 1-day post-challenge in BALB/c mice. IgG antibodies to B. pertussis strains UT25 and D420, PT, FHA, PRN, RTX, DT, and TT depicted in log scale. MVMC= mock-vaccinated, mock-challenged; MVC=mock-vaccinated and challenged; n = 10 per treatment group, except for MVMC n = 5. Kruskal–Wallis with Dunn’s post-hoc test was used to calculate differences to MVC; *p < 0.05, ***p < 0.005, ****p < 0.001 indicate differences from MV and #p < 0.05 indicates differences from DTaP. A Student’s t test was performed to compare differences in IgG titers between prime and boost for each vaccine. Box and whisker plots display minimum to maximum values with all data points. MV=mock-vaccinated; SD= standard deviation. n = 5–6 per treatment group. Dotted lines indicate lowest limit of detection.

Fig. 5. Bacterial burden and white blood cell count after challenge with two different B. pertussis strains.

Bacterial burden and white blood cell count (WBC) were enumerated after challenge with two strains of B. pertussis in BALB/c mice at 3 time points. a Timeline for vaccination, bacterial challenge, and end-point assays. b, d CFU’s were calculated at days 1, 3, and 7 post-challenge from the lung and trachea homogenates of mice challenged with Bp UT25 (b) and D420 (d). c and e WBC counts were quantified in blood at days 1, 3 and 7 post-UT25 (c) and D420 challenge (e) using an IDEXX Procyte. b, d Dotted line indicates lowest limit of CFU detection, while dotted line in (c) and (e) indicates the average WBC count of non-challenged mice. A one-way ANOVA with Tukey’s post-hoc test calculated differences to MVC. *indicates a significant difference from MVC at day 1, ^ indicates difference from MVC at day 3, and # indicates a difference from MVC at day 7. MVC = mock-vaccinated and challenged; SEM=standard error of the mean. n = 5 per treatment group. Consult Table 1 for vaccine antigen description and Table 2 for vaccine composition.

Fig. 6. Complete blood cell counts and anti-PT IgG titers in mice after PT challenge.

a Schematic of vaccination and PT (2 µg) challenge using BALB/c mice. b White blood cell (WBC), lymphocyte (c) and neutrophil (d) counts were quantified in blood 3 days post-PT challenge using an IDEXX Procyte. e Post-boost sera were analyzed for IgG antibody titers to holotoxin PT and PTx-S1 antibodies by ELISA. Dotted line in panels (b-d) depicts average WBC, neutrophil and lymphocyte counts in the blood for mock-vaccinated and non-challenged mice and in panel (e) dotted line depicts lower limit of detection. Box and whisker plots display minimum to maximum values with all data points. A one-way ANOVA with Tukey’s post-hoc test calculated differences in (b–d) (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001). Kruskal-Wallis with Dunn’s post-hoc test was used to calculate differences to MVC in panel (e); **p < 0.01, ***p < 0.005, ****p < 0.001 indicate differences from MV MVPTC = mock-vaccinated and PT challenged. Consult Table 1 for vaccine antigen description and Table 2 for vaccine composition.