Immunization with an mRNA DTP vaccine protects against pertussis in rats.

Bordetella pertussis is a Gram-negative bacterium that is the causative agent of the respiratory disease known as pertussis. Since the switch to the acellular vaccines of DTaP and Tap, pertussis cases in the US have risen and cyclically fallen. We have observed that mRNA pertussis vaccines are immunogenic and protective in mice. Here, we further evaluated the pertussis toxoid mRNA antigen and refined the formulation based on optimal pertussis toxin neutralization in vivo. We next evaluated the mRNA pertussis vaccine in Sprague-Dawley rats using an aerosol B. pertussis challenge model paired with whole-body plethysmography to monitor coughing and respiratory function. Female Sprague-Dawley rats were primed and boosted with either commercially available vaccines (DTaP or wP-DTP), an mRNA-DTP vaccine, or mock-vaccinated. The mRNA-DTP vaccine was immunogenic in rats and induced antigen-specific IgG antibodies comparable to DTaP. Rats were then aerosol challenged with a streptomycin-resistant emerging clinical isolate D420Sm1. Bacterial burden was assessed at days 1 and 9 post-challenge, and the mRNA vaccine reduced burden equal to both DTaP and wP-DTP. Whole-body plethysmography revealed that mRNA-DTP vaccinated rats were well protected against coughing which was comparable to the non-challenged group. These data suggest that an mRNA-DTP vaccine is immunogenic in rats and provides protection against aerosolized B. pertussis challenge in Sprague-Dawley rats.

Virus-like particles displaying the mature C-terminal domain of filamentous hemagglutinin are immunogenic and protective against Bordetella pertussis respiratory infection in mice.

Bordetella pertussis, the bacterium responsible for whooping cough, remains a significant public health challenge despite the existing licensed pertussis vaccines. Current acellular pertussis vaccines, though having favorable reactogenicity and efficacy profiles, involve complex and costly production processes. In addition, acellular vaccines have functional challenges such as short-lasting duration of immunity and limited antigen coverage. Filamentous hemagglutinin (FHA) is an adhesin of B. pertussis that is included in all multivalent pertussis vaccine formulations. Antibodies to FHA have been shown to prevent bacterial attachment to respiratory epithelial cells, and T cell responses to FHA facilitate cell-mediated immunity. In this study, FHA's mature C-terminal domain (MCD) was evaluated as a novel vaccine antigen. MCD was conjugated to virus-like particles via SpyTag-SpyCatcher technology. Prime-boost vaccine studies were performed in mice to characterize immunogenicity and protection against the intranasal B. pertussis challenge. MCD-SpyVLP was more immunogenic than SpyTag-MCD antigen alone, and in Tohama I strain challenge studies, improved protection against challenge was observed in the lungs at day 3 and in the trachea and nasal wash at day 7 post-challenge. Furthermore, a B. pertussis strain encoding genetically inactivated pertussis toxin was used to evaluate MCD-SpyVLP vaccine immunity. Mice vaccinated with MCD-SpyVLP had significantly lower respiratory bacterial burden at both days 3 and 7 post-challenge compared to mock-vaccinated animals. Overall, these data support the use of SpyTag-SpyCatcher VLPs as a platform for use in vaccine development against B. pertussis and other pathogens.

Immunogenicity, reactogenicity, and IgE-mediated immune responses of a mixed whole-cell and acellular pertussis vaccine schedule in Australian infants: A randomised, double-blind, noninferiority trial.

BACKGROUND: In many countries, infant vaccination with acellular pertussis (aP) vaccines has replaced use of more reactogenic whole-cell pertussis (wP) vaccines. Based on immunological and epidemiological evidence, we hypothesised that substituting the first aP dose in the routine vaccination schedule with wP vaccine might protect against IgE-mediated food allergy. We aimed to compare reactogenicity, immunogenicity, and IgE-mediated responses of a mixed wP/aP primary schedule versus the standard aP-only schedule. METHODS AND FINDINGS: OPTIMUM is a Bayesian, 2-stage, double-blind, randomised trial. In stage one, infants were assigned (1:1) to either a first dose of a pentavalent wP combination vaccine (DTwP-Hib-HepB, Pentabio PT Bio Farma, Indonesia) or a hexavalent aP vaccine (DTaP-Hib-HepB-IPV, Infanrix hexa, GlaxoSmithKline, Australia) at approximately 6 weeks old. Subsequently, all infants received the hexavalent aP vaccine at 4 and 6 months old as well as an aP vaccine at 18 months old (DTaP-IPV, Infanrix-IPV, GlaxoSmithKline, Australia). Stage two is ongoing and follows the above randomisation strategy and vaccination schedule. Ahead of ascertainment of the primary clinical outcome of allergist-confirmed IgE-mediated food allergy by 12 months old, here we present the results of secondary immunogenicity, reactogenicity, tetanus toxoid IgE-mediated immune responses, and parental acceptability endpoints. Serum IgG responses to diphtheria, tetanus, and pertussis antigens were measured using a multiplex fluorescent bead-based immunoassay; total and specific IgE were measured in plasma by means of the ImmunoCAP assay (Thermo Fisher Scientific). The immunogenicity of the mixed schedule was defined as being noninferior to that of the aP-only schedule using a noninferiority margin of 2/3 on the ratio of the geometric mean concentrations (GMR) of pertussis toxin (PT)-IgG 1 month after the 6-month aP. Solicited adverse reactions were summarised by study arm and included all children who received the first dose of either wP or aP. Parental acceptance was assessed using a 5-point Likert scale. The primary analyses were based on intention-to-treat (ITT); secondary per-protocol (PP) analyses were also performed. The trial is registered with ANZCTR (ACTRN12617000065392p). Between March 7, 2018 and January 13, 2020, 150 infants were randomised (75 per arm). PT-IgG responses of the mixed schedule were noninferior to the aP-only schedule at approximately 1 month after the 6-month aP dose [GMR = 0·98, 95% credible interval (0·77 to 1·26); probability (GMR > 2/3) > 0·99; ITT analysis]. At 7 months old, the posterior median probability of quantitation for tetanus toxoid IgE was 0·22 (95% credible interval 0·12 to 0·34) in both the mixed schedule group and in the aP-only group. Despite exclusions, the results were consistent in the PP analysis. At 6 weeks old, irritability was the most common systemic solicited reaction reported in wP (65 [88%] of 74) versus aP (59 [82%] of 72) vaccinees. At the same age, severe systemic reactions were reported among 14 (19%) of 74 infants after wP and 8 (11%) of 72 infants after aP. There were 7 SAEs among 5 participants within the first 6 months of follow-up; on blinded assessment, none were deemed to be related to the study vaccines. Parental acceptance of mixed and aP-only schedules was high (71 [97%] of 73 versus 69 [96%] of 72 would agree to have the same schedule again). CONCLUSIONS: Compared to the aP-only schedule, the mixed schedule evoked noninferior PT-IgG responses, was associated with more severe reactions, but was well accepted by parents. Tetanus toxoid IgE responses did not differ across the study groups. TRIAL REGISTRATION: Trial registered at the Australian and New Zealand Clinical 207 Trial Registry (ACTRN12617000065392p).

Association of serum pertussis antibodies with acute asthma attacks in children.

Objective: The aim of this study was to examine the serum antibody levels against pertussis toxin (PT) in children experiencing an acute asthma attack and to explore the potential association between these levels and asthma. Methods: A prospective investigation was conducted, which involved 107 children with acute asthma attacks and 77 children diagnosed with bronchitis. The serum immunoglobulin G (IgG) antibody levels specific to PT were measured by using an in-house enzyme-linked immunosorbent assay. Based on the serum PT-IgG antibody levels, the children with asthma were categorized into three groups: non-pertussis infected, suspected pertussis infected, and recent pertussis infected. The clinical manifestations and pulmonary function of pediatric patients diagnosed with asthma were assessed and compared across various groups. Results: Of the total asthma group, 25 patients tested positive for PT-IgG, whereas only six patients in the bronchitis group were PT-IgG positive. The prevalence of recent pertussis infection was observed to be higher in the asthma group compared with the bronchitis group. Within the asthma group, those with recent pertussis infection exhibited a higher likelihood of experiencing wheezing and impaired lung function in comparison with the non-pertussis infection group. Conclusion: Pertussis infection is relatively common in children with asthma and correlates with the severity of asthma.

Protective Activity and Safety of Experimental Acellular Pertussis Vaccines Based on Antigenic Complexes Isolated from Biofilm and Planktonic Cultures of Bordetella pertussis.

Continued circulation of the whooping cough pathogen, even in countries with high vaccine coverage, can be related to persistence of Bordetella pertussis biofilms in the respiratory tract. The films differ from planktonic cells by increased resistance to the host immune system and antibacterial drugs. The available acellular pertussis vaccines (aPV) containing antigens isolated from planktonic cultures of B. pertussis protect from severe forms of whooping cough, but do not effectively influence circulation of virulent strains in the subclinical forms of the disease and asymptomatic carriage. It is promising to create new generation aPV based on antigens isolated from biofilm cultures of B. pertussis capable of more effectively controlling the entire infectious cycle of whooping cough, including colonization, persistence, and transmission of the pathogen. From antigenic complexes isolated from the culture medium of biofilm and planktonic cultures of the strain B. pertussis No. 317 (serotype 1.2.3), experimental aPV were made: aPV-B and aPV-P, respectively. In intracerebral infection of mice with a virulent strain of B. pertussis, aPV-B demonstrated 2.5-fold higher protective activity than aPV-P and also more effectively reduced colonization of the lungs by B. pertussis cells in mice after intranasal infection with a virulent strain. Both vaccine preparations were safe and did not cause death in mice after administration of histamine.

Structural basis for antibody binding to adenylate cyclase toxin reveals RTX linkers as neutralization-sensitive epitopes.

RTX leukotoxins are a diverse family of prokaryotic virulence factors that are secreted by the type 1 secretion system (T1SS) and target leukocytes to subvert host defenses. T1SS substrates all contain a C-terminal RTX domain that mediates recruitment to the T1SS and drives secretion via a Brownian ratchet mechanism. Neutralizing antibodies against the Bordetella pertussis adenylate cyclase toxin, an RTX leukotoxin essential for B. pertussis colonization, have been shown to target the RTX domain and prevent binding to the αMß2 integrin receptor. Knowledge of the mechanisms by which antibodies bind and neutralize RTX leukotoxins is required to inform structure-based design of bacterial vaccines, however, no structural data are available for antibody binding to any T1SS substrate. Here, we determine the crystal structure of an engineered RTX domain fragment containing the αMß2-binding site bound to two neutralizing antibodies. Notably, the receptor-blocking antibodies bind to the linker regions of RTX blocks I-III, suggesting they are key neutralization-sensitive sites within the RTX domain and are likely involved in binding the αMß2 receptor. As the engineered RTX fragment contained these key epitopes, we assessed its immunogenicity in mice and showed that it elicits similar neutralizing antibody titers to the full RTX domain. The results from these studies will support the development of bacterial vaccines targeting RTX leukotoxins, as well as next-generation B. pertussis vaccines.

Leveraging serology to titrate immunisation programme functionality for diphtheria in Madagascar.

Diphtheria is a potentially devastating disease whose epidemiology remains poorly described in many settings, including Madagascar. Diphtheria vaccination is delivered in combination with pertussis and tetanus antigens and coverage of this vaccine is often used as a core measure of health system functioning. However, coverage is challenging to estimate due to the difficulty in translating numbers of doses delivered into numbers of children effectively immunised. Serology provides an alternative lens onto immunisation, but is complicated by challenges in discriminating between natural and vaccine-derived seropositivity. Here, we leverage known features of the serological profile of diphtheria to bound expectations for vaccine coverage for diphtheria, and further refine these using serology for pertussis. We measured diphtheria antibody titres in 185 children aged 6-11 months and 362 children aged 8-15 years and analysed them with pertussis antibody titres previously measured for each individual. Levels of diphtheria seronegativity varied among age groups (18.9% of children aged 6-11 months old and 11.3% of children aged 8-15 years old were seronegative) and also among the districts. We also find surprisingly elevated levels of individuals seropositive to diphtheria but not pertussis in the 6-11 month old age group suggesting that vaccination coverage or efficacy of the pertussis component of the DTP vaccine remains low or that natural infection of diphtheria may be playing a significant role in seropositivity in Madagascar.

Overcoming Waning Immunity in Pertussis Vaccines: Workshop of the National Institute of Allergy and Infectious Diseases.

Despite high vaccine coverage in many parts of the world, pertussis is resurging in a number of areas in which acellular vaccines are the primary vaccine administered to infants and young children. This is attributed in part to the suboptimal and short-lived immunity elicited by acellular pertussis vaccines and to their inability to prevent nasal colonization and transmission of the etiologic agent Bordetella pertussis In response to this escalating public health concern, the National Institute of Allergy and Infectious Diseases held the workshop "Overcoming Waning Immunity in Pertussis Vaccines" in September 2019 to identify issues and possible solutions for the defects in immunity stimulated by acellular pertussis vaccines. Discussions covered aspects of the current problem, gaps in knowledge and possible paths forward. This review summarizes presentations and discussions of some of the key points that were raised by the workshop.

Mucosal Immunization with DTaP Confers Protection against Bordetella pertussis Infection and Cough in Sprague-Dawley Rats.

Pertussis is a respiratory disease caused by the Gram-negative pathogen, Bordetella pertussis. The transition from a whole-cell pertussis vaccine (wP and DTP) to an acellular pertussis vaccine (aP, DTaP, and Tdap) correlates with an increase in pertussis cases, despite widespread vaccine implementation and coverage, and it is now appreciated that the protection provided by aP rapidly wanes. To recapitulate the localized immunity observed from natural infection, mucosal vaccination with aP was explored using the coughing rat model of pertussis. Overall, our goal was to evaluate the route of vaccination in the coughing rat model of pertussis. Immunity induced by both oral gavage and intranasal vaccination of aP in B. pertussis challenged rats over a 9-day infection was compared to intramuscular wP (IM-wP)- and IM-aP-immunized rats that were used as positive controls. Our data demonstrate that mucosal immunization of aP resulted in the production of anti-B. pertussis IgG antibody titers similar to IM-wP- and IM-aP-vaccinated controls postchallenge. IN-aP also induced anti-B. pertussis IgA antibodies in the nasal cavity. Immunization with IM-wP, IM-aP, IN-aP, and OG-aP immunization protected against B. pertussis-induced cough, whereas OG-aP immunization did not protect against respiratory distress. Mucosal immunization by both intranasal and oral gavage administration protected against acute inflammation and decreased bacterial burden in the lung compared to mock-vaccinated challenge rats. The data presented in this study suggest that mucosal vaccination with aP can induce a mucosal immune response and provide protection against B. pertussis challenge. This study highlights the potential benefits and uses of the coughing rat model of pertussis; however, further questions regarding waning immunity still require additional investigation.

Intranasal Immunization with Acellular Pertussis Vaccines Results in Long-Term Immunity to Bordetella pertussis in Mice.

Bordetella pertussis colonizes the respiratory mucosa of humans, inducing an immune response seeded in the respiratory tract. An individual, once convalescent, exhibits long-term immunity to the pathogen. Current acellular pertussis (aP) vaccines do not induce the long-term immune response observed after natural infection in humans. In this study, we evaluated the durability of protection from intranasal (i.n.) pertussis vaccines in mice. Mice that convalesced from B. pertussis infection served as a control group. Mice were immunized with a mock vaccine (phosphate-buffered saline [PBS]), aP only, or an aP base vaccine combined with one of the following adjuvants: alum, curdlan, or purified whole glucan particles (IRI-1501). We utilized two study designs: short term (challenged 35 days after priming vaccination) and long term (challenged 6 months after boost). The short-term study demonstrated that immunization with i.n. vaccine candidates decreased the bacterial burden in the respiratory tract, reduced markers of inflammation, and induced significant serum and lung antibody titers. In the long-term study, protection from bacterial challenge mirrored the results observed in the short-term challenge study. Immunization with pertussis antigens alone was surprisingly protective in both models; however, the alum and IRI-1501 adjuvants induced significant B. pertussis-specific IgG antibodies in both the serum and lung and increased numbers of anti-B. pertussis IgG-secreting plasma cells in the bone marrow. Our data indicate that humoral responses induced by the i.n. vaccines correlated with protection, suggesting that long-term antibody responses can be protective.

Triggering Receptor Expressed on Myeloid Cells-1 (TREM-1) Contributes to Bordetella pertussis Inflammatory Pathology.

Whooping cough (pertussis) is a severe pulmonary infectious disease caused by the bacteria Bordetella pertussis. Pertussis infects an estimated 24 million people annually, resulting in >150,000 deaths. The NIH placed pertussis on the list of emerging pathogens in 2015. Antibiotics are ineffective unless administered before the onset of the disease characteristic cough. Therefore, there is an urgent need for novel pertussis therapeutics. We have shown that sphingosine-1-phosphate receptor (S1PR) agonists reduce pertussis inflammation without increasing bacterial burden. Transcriptomic studies were performed to identify this mechanism and allow for the development of pertussis therapeutics that specifically target problematic inflammation without sacrificing bacterial control. These data suggested a role for triggering receptor expressed on myeloid cells-1 (TREM-1). TREM-1 cell surface receptor functions as an amplifier of inflammatory responses. Expression of TREM-1 is increased in response to bacterial infection of mucosal surfaces. In mice, B. pertussis infection results in Toll-like receptor 9 (TLR9)-dependent increased expression of TREM-1 and its associated cytokines. Interestingly, S1PR agonists dampen pulmonary inflammation and TREM-1 expression. Mice challenged intranasally with B. pertussis and treated with ligand-dependent (LP17) and ligand-independent (GF9) TREM-1 inhibitors showed no differences in bacterial burden and significantly reduced tumor necrosis factor-α (TNF-α) and C-C motif chemokine ligand 2 (CCL-2) expression compared to controls. Mice receiving TREM-1 inhibitors showed reduced pulmonary inflammation compared to controls, indicating that TREM-1 promotes inflammatory pathology, but not bacterial control, during pertussis infection. This implicates TREM-1 as a potential therapeutic target for the treatment of pertussis.

Immunogenicity of a new enhanced tetanus-reduced dose diphtheria-acellular pertussis (Tdap) vaccine against Bordetella pertussis in a murine model.

BACKGROUND: The necessity of the tetanus-reduced dose diphtheria-acellular pertussis (Tdap) vaccine in adolescence and adults has been emphasized since the resurgence of small-scale pertussis in Korea and worldwide due to the waning effect of the vaccine and variant pathogenic stains in the late 1990s. GreenCross Pharma (GC Pharma), a Korean company, developed the Tdap vaccine GC3111 in 2010. Recently, they enhanced the vaccine, GC3111, produced previously in 2010 to reinforce the antibody response against filamentous hemagglutinin (FHA). In this study, immunogenicity and efficacy of the enhanced Tdap vaccine compared and evaluated with two Tdap vaccines, GC3111 vaccine produced in 2010 previously and commercially available Tdap vaccine in a murine model. METHODS: Two tests groups and positive control group of Balb/c mice were primed with two doses of the diphtheria-tetanus-acellular pertussis (DTaP) vaccine followed by a single booster Tdap vaccine at 9 week using the commercially available Tdap vaccine or 2 Tdap vaccines from GC Pharma (GC3111, enhanced GC3111). Humoral response was assessed 1 week before and 2 and 4 weeks after Tdap booster vaccination. The enhanced GC3111 generated similar humoral response compare to the commercial vaccine for filamentous hemagglutinin (FHA). The interferon gamma (IFN-γ) (Th1), interleukin 5 (IL-5) (Th2) and interleukin 17 (IL-17) (Th17) cytokines were assessed 4 weeks after booster vaccination by stimulation with three simulators: heat inactivated Bordetella pertussis (hBp), vaccine antigens, and hBp mixed with antigens (hBp + antigen). A bacterial challenge test was performed 4 weeks after booster vaccination. RESULTS: Regarding cell-mediated immunity, cytokine secretion differed among the three simulators. However, no difference was found between two test groups and positive control group. All the vaccinated groups indicated a Th1 or Th1/Th2 response. On Day 5 post-bacterial challenge, B. pertussis colonies were absent in the lungs in two test groups and positive control group. CONCLUSIONS: Our results confirmed the immunogenicity of GC Pharma's Tdap vaccine; enhanced GC3111 was equivalent to the presently used commercial vaccine in terms of humoral response as well as cell-mediated cytokine expression.

Lack of evidence supporting a role of IFN-ß and TGF-ß in differential polarization of Bordetella pertussis specific-T cell responses.

Bordetella Pertussis (BP) vaccine-induced immunity is waning worldwide despite excellent vaccine coverage. Replacement of the whole-cell inactivated vaccine (wP) by an acellular subunit vaccine (aP) is thought to play a major role and to be associated with the recurrence of whooping cough. Previously, we detected that the polarization towards a Th2 and Th1/Th17 response in aP and wP vaccinees, respectively, persists upon aP boosting in adolescents and adults. Additionally, IL-9 and TGF-ß were found to be up-regulated in aP-primed donors and network analysis further identified IFN-ß as a potential upstream regulator of IL-17 and IL-9. Based on these findings, we hypothesized that IFN-ß produced following aP vaccination may lead to increased IL-9 and decreased IL-17 production. Also, due to the well characterized role of TGF-ß in both Th17 and Th9 differentiation, we put forth that TGF-ß addition to BP-stimulated CD4 + T cells might modulate IL-17 and IL-9 production. To test this hypothesis, we stimulated in vitro cultures of PBMC or isolated naive CD4 + T cells from aP vs wP donors with a pool of BP epitopes and assessed the effect of IFN-ß or TGF-ß in proliferative responses as well as in the cytokine secretion of IL-4, IL-9, IL-17, and IFN-γ. IFN-ß reduced BP-specific proliferation in PBMC as well as cytokine production but increased IL-9, IL-4, and IFN-γ cytokines in naïve CD4 + T cells. These effects were independent of the childhood vaccination received by the donors. Similarly, TGF-ß reduced BP-specific proliferation in PBMC but induced proliferation in naïve CD4 + T cells. However, stimulation was associated with a generalized inhibition of cytokine production regardless of the original aP or wP vaccination received by the donors. Our study suggests that key T cell functions such as cytokine secretion are under the control of antigen stimulation and environmental cues but molecular pathways different than the ones investigated here might underlie the long-lasting differential cytokine production associated with aP- vs wP-priming in childhood vaccination.

Impact of pertussis-specific IgA, IgM, and IgG antibodies in mother's own breast milk and donor breast milk during preterm infant digestion.

BACKGROUND: The survival of antibody isotypes specific to pertussis toxin (PT) and filamentous hemagglutinin (FHA) from mother's own milk (MBM) and donor breast milk (DBM) during preterm infant digestion was investigated. METHODS: Feed, gastric, and stool samples were collected from 20 preterm mother-infant pairs at 8-9 days and 21-22 days postpartum. Samples were analyzed via ELISA for anti-FHA or anti-PT immunoglobulin A (IgA), IgM, and IgG. RESULTS: Anti-PT IgA, anti-FHA IgG, and anti-PT IgG were lower in MBM than DBM at 8-9 days postpartum, whereas anti-FHA IgM was higher in MBM than DBM. Anti-PT IgA, anti-PT IgG, and anti-FHA IgG in DBM decreased in gastric contents at both postpartum times but those antibodies in MBM were stable or increased during gastric digestion. Anti-FHA-specific IgA and IgM were higher in gastric contents from infants fed MBM than from infants fed DBM at 8-9 days. All pertussis antibodies were detected in infant stools at both postpartum times. CONCLUSIONS: Pertussis-specific antibodies from MBM were stable during infant digestion, whereas anti-pertussis IgA and IgG from DBM decreased in gastric contents. The constant region and variable region of antibodies and maternal immunization appear to be the critical factors for their stability to proteolytic digestion and pasteurization. IMPACT: Pertussis-specific antibodies from mother's breast milk were stable during infant digestion, whereas anti-pertussis IgA and IgG from donor breast milk decreased in gastric contents. The constant region and variable region of pertussis-specific antibodies and the maternal immunization (previous infections and vaccinations) appear to be the critical factors for their stability to proteolytic digestion and pasteurization. Pertussis-specific antibodies from either mother's breast milk or donor breast milk survived during preterm infant digestion and both types of milk will compensate for the lower IgG transplacental transfer in preterm infants compared with term infants.

Transcriptional profiling of human macrophages during infection with Bordetella pertussis.

Bordetella pertussis, a strictly human re-emerging pathogen and the causative agent of whooping cough, exploits a broad variety of virulence factors to establish efficient infection. Here, we used RNA sequencing to analyse the changes in gene expression profiles of human THP-1 macrophages resulting from B. pertussis infection. In parallel, we attempted to determine the changes in intracellular B. pertussis-specific transcriptomic profiles resulting from interaction with macrophages. Our analysis revealed that global gene expression profiles in THP-1 macrophages are extensively rewired 6 h post-infection. Among the highly expressed genes, we identified those encoding cytokines, chemokines, and transcription regulators involved in the induction of the M1 and M2 macrophage polarization programmes. Notably, several host genes involved in the control of apoptosis and inflammation which are known to be hijacked by intracellular bacterial pathogens were overexpressed upon infection. Furthermore, in silico analyses identified large temporal changes in expression of specific gene subsets involved in signalling and metabolic pathways. Despite limited numbers of the bacterial reads, we observed reduced expression of majority of virulence factors and upregulation of several transcriptional regulators during infection suggesting that intracellular B. pertussis cells switch from virulent to avirulent phase and actively adapt to intracellular environment, respectively.

Development and validation of magnetic bead pentaplex immunoassay for simultaneous quantification of murine serum IgG antibodies to acellular pertussis, diphtheria and tetanus antigens used in combination vaccines.

We describe here a magnetic bead-based multiplex (pentaplex) immunoassay (MIA) platform developed as an alternative to enzyme-linked immunosorbent assays (ELISA) used in immunogenicity testing of DTaP/TdaP vaccine in animals. MIA simultaneously measures the concentration of serum (IgG) antibodies against B. Pertussis antigens; pertussis toxin, filamentous hemagglutinin (FHA), pertactin (PRN) and tetanus (T) and diphtheria (D) toxoid in the Tdap vaccine immunized animals. Assay validation experiments were done using a panel of serum samples. The results are expressed in IU/ml using WHO reference mice serum. The standard curve was linear with 4PL logistic fit over an eight 2-fold dilution range with LOQ of 0.003, 0.022, 0.005 IU/ml for PT, FHA and PRN and 0.016 U/ml for T and D antigens indicating sensitivity. No interference was observed in monoplex versus multiplex measurements. Specificity was demonstrated by ≥90% homologous and ≤15% heterologous inhibition for all the antigens. The assay was reproducible, with a mean coefficient of variation (CV) of ≤10% for intra-assay duplicates and ≤25% for interassays using different lots of beads and analyst. Accuracy was demonstrated wherein the ratio of observed vs. assigned unitages were within 80-120%. The study suggests that the Pentaplex (MIA) platform meets all the criteria for the serological assay combination vaccines with additional advantages of high throughput, reduced sample volumes, faster analysis with reduced manpower in contrast to conventional monoplex ELISA.

Immunogenicity and safety of a DTaP-IPV/Hib pentavalent vaccine given as primary and booster vaccinations in healthy infants and toddlers in Japan.

BACKGROUND: Globally, the use of single DTaP-IPV/Hib vaccines that combine DTaP-IPV and Hib is widespread, but in Japan vaccination is usually concomitant at separate sites. The immunogenicity and safety of a primary vaccination series and booster of a combined pentavalent DTaP-IPV/Hib vaccine were evaluated and compared to separate administration of DTaP-IPV and Hib in Japanese infants. METHODS: Healthy Japanese infants were administered DTaP-IPV/Hib (Group A: N = 207) or DTaP-IPV + Hib (Group B: N = 207) by the subcutaneous (SC) or DTaP-IPV/Hib by the intramuscular (IM) route (Group C: N = 10). All subjects received a 3-dose primary vaccination series and a booster. Non-inferiority (Group A versus Group B) was tested post-primary series and subsequent post hoc analyses were performed for anti-Hib. Safety was assessed by parental reports. RESULTS: Non-inferiority for SC administration of Group A versus Group B for the primary series was demonstrated for antibody responses to all antigens except Hib using the threshold of 1.0 µg/mL. Post hoc analyses for anti-Hib demonstrated non-inferiority for the primary series response using 0.15 µg/mL, and for pre-booster antibody persistence and the booster response using 0.15 µg/mL and 1.0 µg/mL. The immune response was similar for each antigen following SC or IM administration. There were no safety concerns in any group, and a lower incidence of injection sites for the IM route was observed as expected. CONCLUSIONS: These data show the good immunogenicity and safety profile of the DTaP-IPV/Hib vaccine as a 3-dose infant primary series followed by a booster in the second year of life in Japan.

[Studying the possibility of using immuno-enzy test system for evaluating anti-pertussis immunity.]

A research objective - to study the possibility of using the ELISA Anti-K enzyme immunoassay system to evaluate anti-pertussis immunity. А comparative assessment of the content of co-crank antibodies in the blood serum of adults, pregnant women and children 6 years old in the agglutination test, in the test system "Anti-K ELISA" and test systems of foreign production was carried out. The "Anti-K" IFA test system makes it possible to detect the level of specific antibodies to both the whole cell and cell-free pertussis component of the vaccine at any stage of the vaccination cycle. This diagnostic test can be used to determine the tactics of immunization, and to assess population immunity.

Peptidoglycan Recognition Protein 4 Suppresses Early Inflammatory Responses to Bordetella pertussis and Contributes to Sphingosine-1-Phosphate Receptor Agonist-Mediated Disease Attenuation.

Incidence of whooping cough (pertussis), a bacterial infection of the respiratory tract caused by the bacterium Bordetella pertussis, has reached levels not seen since the 1950s. Antibiotics fail to improve the course of disease unless administered early in infection. Therefore, there is an urgent need for the development of antipertussis therapeutics. Sphingosine-1-phosphate receptor (S1PR) agonists have been shown to reduce pulmonary inflammation during Bordetella pertussis infection in mouse models. However, the mechanisms by which S1PR agonists attenuate pertussis disease are unknown. We report the results of a transcriptome sequencing study examining pulmonary transcriptional responses in B. pertussis-infected mice treated with S1PR agonist AAL-R or vehicle control. This study identified peptidoglycan recognition protein 4 (PGLYRP4) as one of the most highly upregulated genes in the lungs of infected mice following S1PR agonism. PGLYRP4, a secreted, innate mediator of host defenses, was found to limit early inflammatory pathology in knockout mouse studies. Further, S1PR agonist AAL-R failed to attenuate pertussis disease in PGLYRP4 knockout (KO) mice. B. pertussis virulence factor tracheal cytotoxin (TCT), a secreted peptidoglycan breakdown product, induces host tissue damage. TCT-oversecreting strains were found to drive an early inflammatory response similar to that observed in PGLYRP4 KO mice. Further, TCT-oversecreting strains induced significantly greater pathology in PGLYRP4-deficient animals than their wild-type counterparts. Together, these data indicate that S1PR agonist-mediated protection against pertussis disease is PGLYRP4 dependent. Our data suggest PGLYRP4 functions, in part, by preventing TCT-induced airway damage.

Commensal Microbes Affect Host Humoral Immunity to Bordetella pertussis Infection.

As important players in the host defense system, commensal microbes and the microbiota influence multiple aspects of host physiology. Bordetella pertussis infection is highly contagious among humans. However, the roles of the microbiota in B. pertussis pathogenesis are poorly understood. Here, we show that antibiotic-mediated depletion of the microbiota results in increased susceptibility to B. pertussis infection during the early stage. The increased susceptibility was associated with a marked impairment of the systemic IgG, IgG2a, and IgG1 antibody responses to B. pertussis infection after antibiotic treatment. Furthermore, the microbiota impacted the short-lived plasma cell responses as well as the recall responses of memory B cells to B. pertussis infection. Finally, we found that the dysbiosis caused by antibiotic treatment affects CD4+ T cell generation and PD-1 expression on CD4+ T cells and thereby perturbs plasma cell differentiation. Our results have revealed the importance of commensal microbes in modulating host immune responses to B. pertussis infection and support the possibility of controlling the severity of B. pertussis infection in humans by manipulating the microbiota.