B. pertussis gentamicin resistant strains used for challenge in the murine protection model included the reference Tohama phase I strain (CIP 8132, ptxP1-ptxA2-prn1), which expresses the adhesin pertactin (PRN), as well as clinical isolates (ptxP3-ptxA1-prn2) from the United States and Argentina that do not express the vaccine antigen pertactin (51–53). Bacteria were grown in Bordet–Gengou (BG) agar supplemented with 10% (v/v) defibrinated sheep blood (BGAS) and 50 μg/mL of gentamicin (BGAS-Gn)for 72 h at 36.5°C. Isolated colonies were replated in the same medium for 24 h and then resuspended in phosphate-buffered saline (PBS). The optical density at 650 nm was measured and serial 10-fold dilutions plated onto BG-blood agar to determine the number of bacteria in the challenge inoculum. To obtain OMVs, B. pertussis Tohama phase I strain CIP 8132, 24 h colonies grown in BGAS medium were used to seed liquid medium Stainer–Scholte (SS) (54).

OMVs used for vaccine formulation were isolated and characterized as previously described (37, 55). Briefly, culture samples from the decelerating growth phase in SS medium were centrifuged and the bacterial pellet obtained was resuspended in 20 mM Tris–HCl, 2 mM EDTA pH 8.5. The suspension was sonicated in cool water for 20 min. After two centrifugations at 10,000×g for 20 min at 4°C, the supernatant was pelleted at 100,000×g for 2 h at 4°C. This pellet was re-suspended in Tris buffer (20 mM pH 7.6). The samples obtained were negatively stained for electron microscope examination. Protein content was estimated by the Bradford method using bovine serum albumin as standard (56). The presence of the main immunogenic proteins in the OMVs was detected by immunoblot assays using specific antibodies as we previously described (not shown) (37, 40).

The vaccine formulation tested here combined 1/10 of the human dose of commercial ADACEL^® vaccine (aP, Sanofi Pasteur Limited) and 3µg of formalin detoxified OMVs derived from B. pertussis (hereafter named as combined). The pertussis component of commercial aP vaccine per human dose consists of 2.5 µg detoxified pertussis toxin (PTx), 5 µg filamentous hemagglutinin (FHA), 3 µg pertactin (PRN), 5 µg fimbriae types 2 and 3 (FIM).

Animal experiments were performed using BALB/c mice (4–6 weeks old), provided by the Faculty of Veterinary Sciences, La Plata, Argentina. Animals were kept in ventilated cages and housed under standardized conditions with regulated daylight, humidity, and temperature. They received food and water ad libitum. The animal protocols were authorized by the Ethical Committee for Animal Experiments of the Faculty of Science at La Plata National University (approval number 004 - 06-15, 003 - 06–15 extended its validity until August 10, 2027). A two-dose intramuscular immunization schedule, with doses administered 14 days apart, was used to immunize groups of 4–6 BALB/c mice with either the combined OMV+aP vaccine or 1/10 of the human dose of the commercial aP vaccine ADACEL^® ( Figure 1A ). Non-immunized mice served as the control group.

The MWG-test was carried out using groups of 6–8 BALB/c mice (14–16 g) which were vaccinated with the here tested vaccine formulation. Animals were observed for 7 days and body weight was recorded after 16 h, 3 and 7 days. Vaccines were considered non-toxic when passing the WHO and EP requirements (57).

In order to have an additional marker of pro-inflammatory capacity of the different formulations, serum was collected 4 h after each immunization by submandibular bleeding and serum separation. Serum IL - 6 was measured by ELISA using ELISA Flex: Mouse IL - 6 (HRP) Mabtech following manufacturer’s instructions.

The whole blood IL - 6 cytokine release assay was performed as described by Stoddard et al. (58). Briefly, a 245 μl of blood sample was dispensed into each well of a 96-well tissue culture plate. Serial dilutions of each vaccine were prepared in RPMI 1640 cell culture medium (GIBCO) in a range of 5–300 ng/ml, incubated with the whole blood and then briefly centrifuged. A sample of 55 μl of plasma from each well was removed and frozen pending quantification of the cytokines. IL - 6 levels were measured by ELISA using BD OptiEIA (BD Biosciences) following manufacturer instructions.

For protection assays, mice were intranasally challenged with a sublethal dose of B. pertussis (as specified in the figure legends). Animals were euthanized one-week post-challenge, and bacterial loads in lungs and nasal tissue were quantified. Organs were aseptically excised, serially diluted, and plated for colony counting according to previously established protocols (31, 40, 48). Specifically, lungs and the nasal tissue was aseptically excised to minimize contamination from environmental bacteria.

Once extracted, the biological material was mechanically homogenized in a known volume of sterile phosphate-buffered saline (PBS). Subsequently, serial 10-fold dilutions of the homogenates were prepared and plated onto BGAS. The plates were incubated at 36.5°C for 72 hours. After incubation, colony-forming units (CFU) were counted to determine the bacterial load per lungs and nose. A minimum of three independent experiments were performed.

To study protection conferred by passive transfer, pooled serum (100 µl) or spleen cells (2.5 × 10⁷) derived from non-immunized or immunized mice were transferred intraocularly into 4-week-old naïve recipient mice. Twenty-four hours later, the mice were infected with B. pertussis and protection assessed on day 7 as described above.

Blood samples were collected two weeks after the final immunization and at sacrifice.

To detect antibody levels induced in mice by vaccination, Enzyme-linked immunosorbent assay (ELISA) was performed as we previously described (31, 40, 59). Plates (Nunc A/S, Roskilde, Denmark) were coated with sonicated B. pertussis Tohama phase I (whole cell lysates) or purified recombinant PTxA both at 3 µg/mL in 0.5 M carbonate buffer, pH 9.5 by overnight incubation at 4°C. Blocked plates with 3% milk in PBS (2 h 37°C) were incubated with serially diluted samples of mouse serum (1 h 37°C). Sera was obtained after leaving the blood samples to clot for 1 h at 37°C followed by centrifuging for 10 min at 6,000xg. Total IgG, IgG-isotypes and IgA from individual serum or pooled sera bound to the plates were detected after a 2-h incubation with goat anti–mouse-IgG–linked horseradish peroxidase (1:8,000 Invitrogen, USA) and goat anti–mouse-IgA–linked horseradish peroxidase (1:3,000) (Abcam). For measuring IgG isotypes, detection of bound antibody was determined using HRP labeled subclass-specific anti-mouse IgG1 (1:3,000), IgG2a (1:2,000) (Invitrogen USA). As substrate 1.0 mg/mL o-phenylendiamine (OPD, Bio Basic Canada Inc) in 0.1 M citrate-phosphate buffer, pH 5.0 containing 0.1% hydrogen peroxide was used. Optical densities (ODs) were measured with BioTek 800 TS microplate reader (BioTek, Agilent Technologies, US) at 490 nm. From the experimental protocol performed in triplicate, one representative experiment is presented in the Results.

Avidity was measured by an ELISA elution assay as the overall strength of binding between antibody and antigen, using plates incubated for 10 min with increasing concentration of ammonium thiocyanate (NH₄SCN) from 0 to 1 M. Antibody avidity was defined as the amount (percentage) of antibody retained for each increment of NH₄SCN concentration relative to that measured in absence of NH₄SCN.

After B. pertussis challenge, spleens from untreated and immunized mice were passed through a 40-mm cell strainer to obtain a single-cell suspension. Spleen cells were seeded in 48 well culture plates in a final volume of 500 µl/well RPMI 1640 with 10% fetal bovine serum, containing 100 IU/mL penicillin and 100 µg/mL streptomycin (44). All cell samples were stimulated with heat killed bacteria suspension 5x10⁶ UFC/well (HK_Bp), PTx subunit A (PTxA1 μg/mL) or medium only. After 72 h of incubation (37 °C and 5% CO₂), IFN-γ, IL - 17, IL - 22 and IL - 5 concentrations were quantified in supernatants by ELISA (Mabtech, USA) using conditions recommended by the manufacturer.

To discriminate blood-borne circulating cells from tissue-localized cells we administered intravenously anti-mouse PE-CD45 Ab (eBioscience) 10 min before they were euthanized as previously described (52). After the enzymatic disruption of tissue for 1 h at 37 °C with Collagenase D (1 mg/mL; Sigma-Aldrich) and DNAse I (20 U/mL; Sigma-Aldrich) and the lysis of red blood cells, cells from lungs and noses were incubated with CD16/CD32 FcgRIII (1:100) to block IgG Fc receptors. Cells were incubated with LIVE/DEAD Aqua (Invitrogen), followed by specific surface TRM-CD4+ T cells staining with CD45.2-BV650 (BD), CD3-PeCy7 (BD), CD4-FITC (Invitrogen), CD44-PE (BD), CD62L-PE-CF594 (BD), CD103-APC-EF780 (Invitrogen), CD69-APC (Invitrogen) ( Supplementary Figure S1 ).

Fluorescence minus one or non-specific isotype Abs were used as controls. Flow cytometric analysis was performed on an LSR Fortessa, and data were acquired using Diva software (BD Biosciences). The results were analyzed using FlowJo software (TreeStar) (60).

The data were evaluated statistically by t- Student test, two-way or one-way analysis of variance (ANOVA) followed by Bonferroni for multiple comparisons (via the GraphPad Prism^® software). Differences were considered significant at a p <0.05.

Although it may seem straightforward to assume that vaccine combinations offer clear benefits for both individuals and public health systems, formulations that include multiple antigens must overcome unforeseen challenges, even when combining vaccines with well-established efficacy. Immunological, physical, and/or chemical interactions among the combined components may alter the host immune response (61). Therefore, a key challenge in the development of combination vaccines is the potential reduction in efficacy or safety compared to the individual components. Accordingly, our first experiment aimed to evaluate the safety of a combination of the commercial acellular vaccine and outer membrane vesicles derived from B. pertussis (hereafter referred to as combined) using a murine intranasal challenge model. Since the aim of this study is to address the limitations of the commercial acellular vaccine (aP) through a combined formulation including both aP and OMVs, all experiments presented herein were conducted in direct comparison with the aP vaccine. As a negative control, we included a group of animals that received no immunogens.

For the combined formulation, we used doses previously shown to be safe and immunogenic when administered individually in the same animal model. As in subsequent experiments, groups of BALB/c mice were immunized intramuscularly (i.m.) following a two-dose schedule with a 14-day interval between the first and second doses ( Figure 1A ).

Vaccine safety in the murine model was assessed by both monitoring body weight gain and measuring serum levels of the pro-inflammatory cytokine IL - 6 at 4 hours post-administration of the first dose ( Figures 1B, C ). For the weight gain test, animals were weighed at 24 h, 3 days, and 7 days post-immunization. As expected, all PBS-treated mice steadily gained weight at all time points analyzed.

While the acellular vaccine did not cause any reduction in weight at any of the assessed time points, the combined vaccine induced a slight, non-significant decrease in body weight at 24 and 72 hours. By day 7, mice in both vaccinated groups had recovered and showed more than 60% of the weight gain observed in the non-immunized control group.

Taken together, these results indicate that both the combined and acellular vaccine formulations meet the safety criteria based on the weight gain curve and can be considered non-toxic in this model.

Furthermore, serum IL - 6 levels, used as a marker of pro-inflammatory response, reached 257.0 ± 47.5 pg/mL at 4 hours post-immunization with the combined vaccine. A similar level was observed with the commercial aP vaccine used at 1/10 of the human dose (405.7 ± 47.5 pg/mL). Importantly, both values were not significantly different from those measured in the untreated control group. Additionally, in in vitro human whole-blood assays, we observed that IL - 6 levels induced by OMV doses ranging from 0.25 to 3 µg (combined with 1/10 of the human aP dose) ranged from 2 ng/mL to 12 ng/mL for the lowest and highest OMV quantities tested. These levels were lower than those induced by an approved commercial whole-cell vaccine (~60 ng/mL). No cytokine stimulating activity was detected for the commercial aP vaccine. Moreover, no visible signs of local reactogenicity at the injection site, such as erythema, swelling, or induration, were observed in any of the vaccinated animals. This visual assessment, performed by trained personnel, further supports the safety profile of both the combined and aP formulations in this model.

Given the known properties of OMVs to induce Th1, Th2, and Th17 immune profiles (31, 43, 44, 46, 62), as well as their adjuvant and immunomodulatory capabilities (49), we evaluated the humoral immune response specific to pertussis toxin (in particular PTxA) and a heat-killed B. pertussis lysate (HK_Bp). Specifically, we comparatively assessed the levels of IgG and the IgG1 and IgG2a isotypes specific to these antigens in response to the combined vaccine formulation and the commercial aP vaccine.

Using the previously described two-dose immunization schedule ( Figure 1A ), triplicate experiments demonstrated that the combined formulations elicited a more robust antibody response ( Figure 2 ). Notably, mice immunized with the combined vaccine exhibited significantly higher serum IgG antibody titers against both PTxA (p < 0.05) and HK_Bp (p < 0.0001) compared to those receiving the aP treatment alone. As expected, IgG levels in unimmunized mice were markedly lower (data not shown). The NH₄SCN avidity assay using the chaotropic agent showed that the antibodies induced by the combined vaccine (anti-PTxA IgG) were of slightly higher quality than those elicited by the aP vaccine, although the difference was not statistically significant (not shown).

Regarding IgG isotype profiles, mice immunized with the aP vaccine exhibited marginally higher (though statistically insignificant) levels of PTxA-specific IgG1 compared to those receiving the combined formulation, while PTxA-specific IgG2a levels were significantly lower (p < 0.05). This pattern aligns with the established Th2-skewed response characteristic of aP vaccines, whereas the combined vaccine preferentially induced a Th1-biased response. For HK_Bp-specific antibodies, both IgG1 and IgG2a titers were significantly reduced in aP-vaccinated mice relative to the combined vaccine group (p< 0.001). Notably, the combined formulation elicited slightly higher IgG2a than IgG1 levels against HK_Bp, further supporting its Th1-polarizing capacity ( Figures 2E, F ).

These results are of particular interest as they demonstrate that the addition of OMVs to the aP vaccine enhances the humoral immune response and modulates the immune profile toward Th1 polarization.

To further investigate the ability of OMVs to modulate the immune response of combined vaccine formulations toward a Th1 profile, we performed spleen cell stimulation assays using splenocytes from mice immunized with different vaccine formulations, as well as from non-immunized controls ( Figure 3 ). The stimuli used were the A subunit of pertussis toxin (PTxA) and the HK_Bp. After the incubation period, we evaluated IFN-γ levels as a marker of Th1-type response ( Figure 3A ). Additionally, we assessed IL - 17 [Th17 profile marker, Figure 3B )] and IL - 5 (Th2 profile marker, Figure 3C ) levels to further characterize the immune response induced by the tested combined formulations.

The results were obtained from determinations performed after bacterial intranasal challenge, in alignment with the 3Rs principle, as the same challenged animals were used to evaluate protection against colonization.

Figure 3A shows that, for both stimuli, the presence of OMVs in the combined vaccines induced significantly higher IFN-γ levels compared to the aP formulation (68,415 ± 18,244 pg/mL vs. 8,217 ± 1,129 pg/mL, p<0.001 for PTxA stimulation; 157,291 ± 40,285 pg/mL vs. 29,610 ± 2,581 pg/mL for HK_Bp stimulation, p<0.05 between combined and aP, respectively). Similarly, IL - 17 levels ( Figure 3B ) were significantly higher in samples from mice immunized with the combined vaccine compared to those from mice vaccinated with aP (2,260 ± 568.8 pg/mL vs. 132.8 ± 30.67 pg/mL, p < 0.001 for PTxA stimulation; 49,772 ± 15,278 pg/mL vs. 4,625 ± 475.2 pg/mL for HK_Bp stimulation, p < 0.05).

In contrast, IL - 5 levels ( Figure 3C ) were significantly elevated in the aP group relative to the combined vaccine group (p < 0.001 for PTxA stimulation and p < 0.05 for HK_Bp stimulation).

We also measured IL - 22 levels ( Figure 3D ), given its recognized role as a key mediator of early mucosal defense and protection against epithelial lung damage (49, 50). Consistently, IL - 22 levels in samples from mice immunized with the combined vaccine were significantly higher than those from the aP group following HK_Bp stimulation (25,982 ± 7,104 pg/mL vs. 8,391 ± 3,153 pg/mL, p < 0.05). Notably, IL - 22 levels in aP-immunized mice were not significantly different from those in non-immunized controls. When the spleen cells were stimulated with PTxA, no differences were detected among the studied treatment.

Flow cytometry analysis revealed a significantly higher number of lung- ( Figure 3E ) and nasal-resident ( Figure 3F ) memory CD4⁺ T cells in OMV+aP-treated mice compared to those that received the aP vaccine alone. These findings indicate that the inclusion of OMVs in the aP formulation enhances the recruitment and establishment of memory T cells in the respiratory mucosa, a response not achieved with the aP vaccine alone.

Altogether, these results demonstrate that the addition of OMVs to combined vaccine formulation is advantageous over the standalone aP formulation, not only in terms of enhancing the humoral response but also by promoting a Th1/Th17/Th22 immune profile, increasing epitope diversity due to their complex composition and resident memory CD4⁺ T cells.

To evaluate the protective capacity of the different vaccine formulations tested in this study, groups of mice immunized with two-dose schedule were intranasally challenged with a sublethal dose of B. pertussis (1×10⁷–5×10⁷ CFU) 14 days after receiving the final dose ( Figure 4A ). Seven days post-challenge, all animals, including those in the non-immunized control group, were euthanized by cervical dislocation to assess bacterial colonization in the upper and lower respiratory tracts.

The challenge was performed using B. pertussis strains with distinct genotypic and phenotypic characteristics. These included the reference Tohama I phase I strain (ptxP1-ptxA2-prn1), which expresses the adhesin pertactin (PRN), and two ptxP3-ptxA1-prn2 strains that lack PRN expression. One of the PRN-deficient strains (PRN^-) corresponds to a locally circulating isolate, while the other derives from the 2012 U.S. outbreak, kindly provided by Dr. Lucia Tondella (CDC).

As expected, the non-immunized control group exhibited the highest bacterial burden in the lungs (3.02×10⁵ CFU/lungs, Figure 4B ). Immunized groups showed significant reductions in lung colonization by the Tohama strain compared to controls (p < 0.0001). The highest reduction was observed in animals immunized with the combined OMV+aP formulation, which exhibited a 4.45-log decrease (complete bacterial clearance, Figure 4B ). In contrast, mice immunized with the aP vaccine alone showed a 2.86-log₁₀ reduction ( Figure 4B ).

When challenged with the U.S. PRN⁻ strain, mice receiving the combined vaccine showed complete bacterial clearance from the lungs (4.39-log decrease, p< 0.0001 Figure 4C ). In contrast, aP-immunized mice exhibited bacterial loads close to those of the non-immunized group (1.43-log decrease, Figure 4C ). A similar trend was observed with the locally circulating PRN⁻ strain ( Figure 4D ), though all groups showed higher residual bacterial loads compared to those seen with the U.S. PRN⁻ strain, suggesting intrinsic differences in virulence or immune evasion capacity between the PRN-deficient isolates.

To dissect the respective contributions of humoral and cellular responses induced by the combined OMV+aP vaccine, passive transfer experiments were conducted using sera or splenocytes from immunized donor mice. Naïve recipient mice were subsequently challenged intranasally with B. pertussis Tohama phase I strain, and bacterial burden, antibody titers, and immune cell populations were assessed.

Transfer of sera ( Figure 5A ) from OMV+aP-immunized mice conferred significantly higher protection in the lungs compared to sera from aP-immunized or PBS-treated donors ( Figure 5B , p<0.0001). Lung bacterial counts were undetectable in recipients of sera from OMV+aP-immunized mice, indicating complete protection. In contrast, mice receiving sera from aP-vaccinated donors showed residual bacterial loads in the lungs (4.1 10²CFU/lungs), while PBS controls exhibited the highest bacterial burden (3.1 10⁴CFU/lungs).

Transfer of splenocytes ( Figure 5A ) from OMV+aP-vaccinated donors resulted in enhanced protection in the lungs colonization (3.1 -log decrease in comparison with control group, p< 0.0001) compared to recipients of aP- (1.3-log decrease in comparison with control group, p< 0.01) or PBS- splenocytes ( Figure 5B ). Mice receiving aP splenocytes exhibited only partial protection.

Consistently, sera from mice that received OMV+aP-derived splenocytes showed significantly elevated anti-B. pertussis IgG titers 7 days post-challenge, as determined by ELISA (p < 0.01), indicating the ability of transferred lymphocytes to support antibody production upon infection (Figure 5C). We also observed that post-challenge, recipients of immune cells from donor animals vaccinated with the combined vaccine showed significantly higher IFN-γ levels compared to those receiving cells from the aP-vaccinated group (359,802 ± 133,612 pg/mL vs. 69,517± 7,314 pg/mL; p<0.05 between combined and aP groups, respectively) ( Figure 5D ). Similarly, IL - 17 and IL - 22 levels were significantly elevated in mice that received splenocytes from combined-vaccinated animals versus those receiving splenocytes from aP-vaccinated animals (p<0.05 Figure 5D ). No significant differences were observed for IL - 5.

Collectively, these data highlight the immunological superiority of the OMV+aP vaccine, which elicits robust humoral and cellular responses and confers enhanced protection not only against PRN⁺ strains but also against PRN⁻ isolates. Passive transfer studies support a mechanistic role for both antibodies and antigen-experienced lymphocytes in mediating protection, reinforcing the rationale for combined vaccine strategies.

Reducing the transmission of B. pertussis has long been a central goal of vaccination strategies, particularly given the extremely high contagiousness of the pathogen. The basic reproduction number (R₀) for B. pertussis has been estimated at approximately 17, making it one of the most transmissible bacterial infections.

Pioneering studies by Warfel et al. (21) using the non-human primate baboon model demonstrated that acellular pertussis vaccines, while effective at protecting against disease, fail to reduce bacterial colonization in the upper respiratory tract (URT), thereby allowing continued transmission despite immunization. This finding underscored the need to develop vaccines capable of inducing mucosal immunity and limiting nasopharyngeal colonization. Here we investigated whether the addition of OMVs to the aP vaccine formulation could enhance its ability to reduce B. pertussis colonization in the nasal cavity. Mice were immunized with a two-dose schedule, as previously described, and then challenged intranasally with the B. pertussis Tohama phase I strain using two different inoculum doses ( Figure 6A ):

The high-dose challenge was included to assess the extent to which the treatments could reduce nasal colonization.

In non-immunized control animals, both doses resulted in comparable levels of colonization in the nasal cavity (~2.24 × 10⁴ CFU/nose). This finding suggests a local niche limitation that constrains bacterial growth in the URT regardless of the inoculum size. Mice immunized with the aP vaccine alone showed practically no reduction in nasal colonization under either challenge condition. In contrast, the combined OMV+aP vaccine demonstrated significant protective efficacy in the URT, with bacterial reductions of 1.66 logs (p < 0.001) and 0.9 log (p < 0.001) following challenge with 3 × 10³ CFU and 5 × 10⁷ CFU, respectively. These results indicate that the inclusion of OMVs may overcome the limitations of aP vaccines in controlling colonization and, by extension, transmission.

Given the established role of mucosal IgA in preventing colonization by B. pertussis, particularly in the nasal epithelium (63), we evaluated serum IgA levels post-vaccination. Elevated levels of antigen-specific IgA were observed in mice immunized with the combined formulation ( Figure 6B , p< 0.001), both in sera and in nasal tissue homogenates, consistent with the improved control of bacterial colonization.

Together, these findings support the hypothesis that adding OMVs to aP vaccines enhances mucosal immunity, promoting IgA production and reducing colonization in the URT, a critical step toward curbing transmission of pertussis.