Reduced Bordetella pertussis-specific CD4+ T-Cell Responses at Older Age.

Pertussis, a human-specific respiratory infectious disease caused by the Gram-negative bacterium Bordetella pertussis (Bp), remains endemic with epidemic years despite high vaccination coverage. Whereas pertussis vaccines and natural infection with Bp confer immune protection, the duration of protection varies and is not lifelong. Recent evidence indicates a considerable underestimation of the pertussis burden among older adults. Whereas the impact of increasing age on Bp-specific humoral immunity has been demonstrated, little is known on immunosenescence of CD4+ T-cell responses in the context of Bp. Here, we aimed to address whether increasing age impacts responsiveness of the Bp-specific CD4+ T-cells in the memory pool following a clinically symptomatic pertussis infection in whole cell vaccine-primed pediatric and adult cases. Cytokine and proliferative responses and phenotypical profiles of CD4+ T cells specific for Bp antigens at an early and late convalescent timepoint were compared. Responses of various Th cytokines, including IFNγ, were significantly lower in older adults at early and late timepoints post diagnosis. In addition, we found lower frequencies of Bp-specific proliferated CD4+ T cells in older adults, in the absence of differences in replication profile. Phenotyping of Bp-specific CD4+ T cells suggested reduced expression of activation markers rather than increased expression of co-inhibitory markers. Altogether, our findings show that the magnitude and functionality of the Bp-specific memory CD4+ T-cell pool decrease at older age. Declined CD4+ T-cell responsiveness to Bp is suggested to contribute to the burden of pertussis in older adults.

Detection of opsonizing antibodies directed against a recently circulating Bordetella pertussis strain in paired plasma samples from symptomatic and recovered pertussis patients.

Correlates of protection (CoPs) against the highly contagious respiratory disease whooping cough, caused by Bordetella pertussis, remain elusive. Characterizing the antibody response to this pathogen is essential towards identifying potential CoPs. Here, we evaluate levels, avidity and functionality of B. pertussis-specific-antibodies from paired plasma samples derived from symptomatic and recovered pertussis patients, as well as controls. Natural infection is expected to induce protective immunity. IgG levels and avidity to nine B. pertussis antigens were determined using a novel multiplex panel. Furthermore, opsonophagocytosis of a B. pertussis clinical isolate by neutrophils was measured. Findings indicate that following infection, B. pertussis-specific antibody levels of (ex-) pertussis patients waned, while the avidity of antibodies directed against the majority of studied antigens increased. Opsonophagocytosis indices decreased upon recovery, but remained higher than controls. Random forest analysis of all the data revealed that 28% of the opsonophagocytosis index variances could be explained by filamentous hemagglutinin- followed by pertussis toxin-specific antibodies. We propose to further explore which other B. pertussis-specific antibodies can better predict opsonophagocytosis. Moreover, other B. pertussis-specific antibody functions as well as the possible integration of these functions in combination with other immune cell properties should be evaluated towards the identification of CoPs against pertussis.

Impact of age and vaccination history on long-term serological responses after symptomatic B. pertussis infection, a high dimensional data analysis.

Capturing the complexity and waning patterns of co-occurring immunoglobulin (Ig) responses after clinical B. pertussis infection may help understand how the human host gradually loses protection against whooping cough. We applied bi-exponential modelling to characterise and compare B. pertussis specific serological dynamics in a comprehensive database of IgG, IgG subclass and IgA responses to Ptx, FHA, Prn, Fim2/3 and OMV antigens of (ex-) symptomatic pertussis cases across all age groups. The decay model revealed that antigen type and age group were major factors determining differences in levels and kinetics of Ig (sub) classes. IgG-Ptx waned fastest in all age groups, while IgA to Ptx, FHA, Prn and Fim2/3 decreased fast in the younger but remained high in older (ex-) cases, indicating an age-effect. While IgG1 was the main IgG subclass in response to most antigens, IgG2 and IgG3 dominated the anti-OMV response. Moreover, vaccination history plays an important role in post-infection Ig responses, demonstrated by low responsiveness to Fim2/3 in unvaccinated elderly and by elevated IgG4 responses to multiple antigens only in children primed with acellular pertussis vaccine (aP). This work highlights the complexity of the immune response to this re-emerging pathogen and factors determining its Ig quantity and quality.

The Use of Innovative Two-Component Cluster Analysis and Serodiagnostic Cut-Off Methods to Estimate Prevalence of Pertussis Reinfections.

Bordetella pertussis circulates even in highly vaccinated countries affecting all age groups. Insight into the scale of concealed reinfections is important as they may contribute to transmission. We therefore investigated whether current single-point serodiagnostic methods are suitable to estimate the prevalence of pertussis reinfection. Two methods based on IgG-Ptx plasma levels alone were used to evaluate the proportion of renewed seroconversions in the past year in a cohort of retrospective pertussis cases ≥ 24 months after a proven earlier symptomatic infection. A Dutch population database was used as a baseline. Applying a classical 62.5 IU/ml IgG-Ptx cut-off, we calculated a seroprevalence of 15% in retrospective cases, higher than the 10% observed in the population baseline. However, this method could not discriminate between renewed seroconversion and waning of previously infection-enhanced IgG-Ptx levels. Two-component cluster analysis of the IgG-Ptx datasets of both pertussis cases and the general population revealed a continuum of intermediate IgG-Ptx levels, preventing the establishment of a positive population and the comparison of prevalence by this alternative method. Next, we investigated the complementary serodiagnostic value of IgA-Ptx levels. When modelling datasets including both convalescent and retrospective cases we obtained new cut-offs for both IgG-Ptx and IgA-Ptx that were optimized to evaluate renewed seroconversions in the ex-cases target population. Combining these cut-offs two-dimensionally, we calculated 8.0% reinfections in retrospective cases, being below the baseline seroprevalence. Our study for the first time revealed the shortcomings of using only IgG-Ptx data in conventional serodiagnostic methods to determine pertussis reinfections. Improved results can be obtained with two-dimensional serodiagnostic profiling. The proportion of reinfections thus established suggests a relatively increased period of protection to renewed infection after clinical pertussis.

Waning and aging of cellular immunity to Bordetella pertussis.

While it is clear that the maintenance of Bordetella pertussis-specific immunity evoked both after vaccination and infection is insufficient, it is unknown at which pace waning occurs and which threshold levels of sustained functional memory B and T cells are required to provide long-term protection. Longevity of human cellular immunity to B. pertussis has been studied less extensively than serology, but is suggested to be key for the observed differences between the duration of protection induced by acellular vaccination and whole cell vaccination or infection. The induction and maintenance of levels of protective memory B and T cells may alter with age, associated with changes of the immune system throughout life and with accumulating exposures to circulating B. pertussis or vaccine doses. This is relevant since pertussis affects all age groups. This review summarizes current knowledge on the waning patterns of human cellular immune responses to B. pertussis as addressed in diverse vaccination and infection settings and in various age groups. Knowledge on the effectiveness and flaws in human B. pertussis-specific cellular immunity ultimately will advance the improvement of pertussis vaccination strategies.

Identification of pertussis-specific effector memory T cells in preschool children.

Whooping cough remains a problem despite vaccination, and worldwide resurgence of pertussis is evident. Since cellular immunity plays a role in long-term protection against pertussis, we studied pertussis-specific T-cell responses. Around the time of the preschool acellular pertussis (aP) booster dose at 4 years of age, T-cell memory responses were compared in children who were primed during infancy with either a whole-cell pertussis (wP) or an aP vaccine. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with pertussis vaccine antigens for 5 days. T cells were characterized by flow-based analysis of carboxyfluorescein succinimidyl ester (CFSE) dilution and CD4, CD3, CD45RA, CCR7, gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α) expression. Before the aP preschool booster vaccination, both the proliferated pertussis toxin (PT)-specific CD4(+) and CD8(+) T-cell fractions (CFSE(dim)) were higher in aP- than in wP-primed children. Post-booster vaccination, more pertussis-specific CD4(+) effector memory cells (CD45RA(-) CCR7(-)) were induced in aP-primed children than in those primed with wP. The booster vaccination did not appear to significantly affect the T-cell memory subsets and functionality in aP-primed or wP-primed children. Although the percentages of Th1 cytokine-producing cells were alike in aP- and wP-primed children pre-booster vaccination, aP-primed children produced more Th1 cytokines due to higher numbers of proliferated pertussis-specific effector memory cells. At present, infant vaccinations with four aP vaccines in the first year of life result in pertussis-specific CD4(+) and CD8(+) effector memory T-cell responses that persist in children until 4 years of age and are higher than those in wP-primed children. The booster at 4 years of age is therefore questionable; this may be postponed to 6 years of age.

Age related differences in dynamics of specific memory B cell populations after clinical pertussis infection.

For a better understanding of the maintenance of immune mechanisms to Bordetella pertussis (Bp) in relation to age, we investigated the dynamic range of specific B cell responses in various age-groups at different time points after a laboratory confirmed pertussis infection. Blood samples were obtained in a Dutch cross sectional observational study from symptomatic pertussis cases. Lymphocyte subpopulations were phenotyped by flowcytometry before and after culture. Memory B (Bmem) cells were differentiated into IgG antibody secreting cells (ASC) by polyclonal stimulation and detected by an ELISPOT assay specific for pertussis antigens pertussis toxin (Ptx), filamentous haemagglutinin (FHA) and pertactin (Prn). Bp antigen specific IgG concentrations in plasma were determined using multiplex technology. The majority of subjects having experienced a clinical pertussis episode demonstrated high levels of both Bp specific IgG and Bmem cell levels within the first 6 weeks after diagnosis. Significantly lower levels were observed thereafter. Waning of cellular and humoral immunity to maintenance levels occurred within 9 months after antigen encounter. Age was found to determine the maximum but not base-line frequencies of Bmem cell populations; higher levels of Bmem cells specific for Ptx and FHA were reached in adults and (pre-) elderly compared to under-fours and schoolchildren in the first 6 weeks after Bp exposure, whereas not in later phases. This age effect was less obvious for specific IgG levels. Nonetheless, subjects' levels of specific Bmem cells and specific IgG were weakly correlated. This is the first study to show that both age and closeness to last Bp encounter impacts the size of Bp specific Bmem cell and plasma IgG levels.

Loss of multi-epitope specificity in memory CD4(+) T cell responses to B. pertussis with age.

Pertussis is still occurring in highly vaccinated populations, affecting individuals of all ages. Long-lived Th1 CD4(+) T cells are essential for protective immunity against pertussis. For better understanding of the limited immunological memory to Bordetella pertussis, we used a panel of Pertactin and Pertussis toxin specific peptides to interrogate CD4(+) T cell responses at the epitope level in a unique cohort of symptomatic pertussis patients of different ages, at various time intervals after infection. Our study showed that pertussis epitope-specific T cell responses contained Th1 and Th2 components irrespective of the epitope studied, time after infection, or age. In contrast, the breadth of the pertussis-directed CD4(+) T cell response seemed dependent on age and closeness to infection. Multi-epitope specificity long-term after infection was lost in older age groups. Detailed knowledge on pertussis specific immune mechanisms and their insufficiencies is important for understanding resurgence of pertussis in highly vaccinated populations.