Diagnostic accuracy of SARS-CoV-2 rapid antigen self-tests in asymptomatic individuals in the omicron period: a cross-sectional study.

OBJECTIVES: To assess the performances of three commonly used antigen rapid diagnostic tests used as self-tests in asymptomatic individuals in the Omicron period. METHODS: We performed a cross-sectional diagnostic test accuracy study in the Omicron period in three public health service COVID-19 test sites in the Netherlands, including 3600 asymptomatic individuals aged ≥ 16 years presenting for SARS-CoV-2 testing for any reason except confirmatory testing after a positive self-test. Participants were sampled for RT-PCR (reference test) and received one self-test (either Acon Flowflex [Flowflex], MP Biomedicals (MPBio), or Siemens-Healthineers CLINITEST [CLINITEST]) to perform unsupervised at home. Diagnostic accuracies of each self-test were calculated. RESULTS: Overall sensitivities were 27.5% (95% CI, 21.3-34.3%) for Flowflex, 20.9% (13.9-29.4%) for MPBio, and 25.6% (19.1-33.1%) for CLINITEST. After applying a viral load cut-off (≥5.2 log10 SARS-CoV-2 E-gene copies/mL), sensitivities increased to 48.3% (37.6-59.2%), 37.8% (22.5-55.2%), and 40.0% (29.5-51.2%), respectively. Specificities were >99% for all tests in most analyses. DISCUSSION: The sensitivities of three commonly used SARS-CoV-2 antigen rapid diagnostic tests when used as self-tests in asymptomatic individuals in the Omicron period were very low. Antigen rapid diagnostic test self-testing in asymptomatic individuals may only detect a minority of infections at that point in time. Repeated self-testing in case of a negative self-test is advocated to improve the diagnostic yield, and individuals should be advised to re-test when symptoms develop.

Head-to-head comparison of the accuracy of saliva and nasal rapid antigen SARS-CoV-2 self-testing: cross-sectional study.

BACKGROUND: The diagnostic accuracy of unsupervised self-testing with rapid antigen diagnostic tests (Ag-RDTs) is mostly unknown. We studied the diagnostic accuracy of a self-performed SARS-CoV-2 saliva and nasal Ag-RDT in the general population. METHODS: This large cross-sectional study consecutively included unselected individuals aged ≥ 16 years presenting for SARS-CoV-2 testing at three public health service test sites. Participants underwent molecular test sampling and received two self-tests (the Hangzhou AllTest Biotech saliva self-test and the SD Biosensor nasal self-test by Roche Diagnostics) to perform themselves at home. Diagnostic accuracy of both self-tests was assessed with molecular testing as reference. RESULTS: Out of 2819 participants, 6.5% had a positive molecular test. Overall sensitivities were 46.7% (39.3-54.2%) for the saliva Ag-RDT and 68.9% (61.6-75.6%) for the nasal Ag-RDT. With a viral load cut-off (≥ 5.2 log10 SARS-CoV-2 E-gene copies/mL) as a proxy of infectiousness, these sensitivities increased to 54.9% (46.4-63.3%) and 83.9% (76.9-89.5%), respectively. For the nasal Ag-RDT, sensitivities were 78.5% (71.1-84.8%) and 22.6% (9.6-41.1%) in those symptomatic and asymptomatic at the time of sampling, which increased to 90.4% (83.8-94.9%) and 38.9% (17.3-64.3%) after applying the viral load cut-off. In those with and without prior SARS-CoV-2 infection, sensitivities were 36.8% (16.3-61.6%) and 72.7% (65.1-79.4%). Specificities were > 99% and > 99%, positive predictive values > 70% and > 90%, and negative predictive values > 95% and > 95%, for the saliva and nasal Ag-RDT, respectively, in most analyses. Most participants considered the self-performing and result interpretation (very) easy for both self-tests. CONCLUSIONS: The Hangzhou AllTest Biotech saliva self Ag-RDT is not reliable for SARS-CoV-2 detection, overall, and in all studied subgroups. The SD Biosensor nasal self Ag-RDT had high sensitivity in individuals with symptoms and in those without prior SARS-CoV-2 infection but low sensitivity in asymptomatic individuals and those with a prior SARS-CoV-2 infection which warrants further investigation.

Diagnostic accuracy of covid-19 rapid antigen tests with unsupervised self-sampling in people with symptoms in the omicron period: cross sectional study.

OBJECTIVE: To assess the performance of rapid antigen tests with unsupervised nasal and combined oropharyngeal and nasal self-sampling during the omicron period. DESIGN: Prospective cross sectional diagnostic test accuracy study. SETTING: Three public health service covid-19 test sites in the Netherlands, 21 December 2021 to 10 February 2022. PARTICIPANTS: 6497 people with covid-19 symptoms aged ≥16 years presenting for testing. INTERVENTIONS: Participants had a swab sample taken for reverse transcription polymerase chain reaction (RT-PCR, reference test) and received one rapid antigen test to perform unsupervised using either nasal self-sampling (during the emergence of omicron, and when omicron accounted for >90% of infections, phase 1) or with combined oropharyngeal and nasal self-sampling in a subsequent (phase 2; when omicron accounted for >99% of infections). The evaluated tests were Flowflex (Acon Laboratories; phase 1 only), MPBio (MP Biomedicals), and Clinitest (Siemens-Healthineers). MAIN OUTCOME MEASURES: The main outcomes were sensitivity, specificity, and positive and negative predictive values of each self-test, with RT-PCR testing as the reference standard. RESULTS: During phase 1, 45.0% (n=279) of participants in the Flowflex group, 29.1% (n=239) in the MPBio group, and 35.4% ((n=257) in the Clinitest group were confirmatory testers (previously tested positive by a self-test at own initiative). Overall sensitivities with nasal self-sampling were 79.0% (95% confidence interval 74.7% to 82.8%) for Flowflex, 69.9% (65.1% to 74.4%) for MPBio, and 70.2% (65.6% to 74.5%) for Clinitest. Sensitivities were substantially higher in confirmatory testers (93.6%, 83.6%, and 85.7%, respectively) than in those who tested for other reasons (52.4%, 51.5%, and 49.5%, respectively). Sensitivities decreased from 87.0% to 80.9% (P=0.16 by χ2 test), 80.0% to 73.0% (P=0.60), and 83.1% to 70.3% (P=0.03), respectively, when transitioning from omicron accounting for 29% of infections to >95% of infections. During phase 2, 53.0% (n=288) of participants in the MPBio group and 44.4% (n=290) in the Clinitest group were confirmatory testers. Overall sensitivities with combined oropharyngeal and nasal self-sampling were 83.0% (78.8% to 86.7%) for MPBio and 77.3% (72.9% to 81.2%) for Clinitest. When combined oropharyngeal and nasal self-sampling was compared with nasal self-sampling, sensitivities were found to be slightly higher in confirmatory testers (87.4% and 86.1%, respectively) and substantially higher in those testing for other reasons (69.3% and 59.9%, respectively). CONCLUSIONS: Sensitivities of three rapid antigen tests with nasal self-sampling decreased during the emergence of omicron but was only statistically significant for Clinitest. Sensitivities appeared to be substantially influenced by the proportion of confirmatory testers. Sensitivities of MPBio and Clinitest improved after the addition of oropharyngeal to nasal self-sampling. A positive self-test result justifies prompt self-isolation without the need for confirmatory testing. Individuals with a negative self-test result should adhere to general preventive measures because a false negative result cannot be ruled out. Manufacturers of MPBio and Clinitest may consider extending their instructions for use to include combined oropharyngeal and nasal self-sampling, and other manufacturers of rapid antigen tests should consider evaluating this as well.

SARS-CoV-2 RNA and antibody dynamics in a Dutch household study with dense sampling frame.

This study investigated the dynamics of SARS-CoV-2 infection and diagnostics in 242 household members of different ages and with different symptom severity after SARS-CoV-2 exposure early in the pandemic (March-April 2020). Households with a SARS-CoV-2 confirmed positive case and at least one child in the Netherlands were followed for 6 weeks. Naso (NP)- and oropharyngeal (OP) swabs, oral fluid and feces specimens were analyzed for SARS-CoV-2 RNA and serum for SARS-CoV-2-specific antibodies. The dynamics of the presence of viral RNA and the serological response was modeled to determine the sampling time-frame and sample type with the highest sensitivity to confirm or reject a SARS-CoV-2 diagnosis. In children higher viral loads compared to adults were detected at symptom onset. Early in infection, higher viral loads were detected in NP and OP specimens, while RNA in especially feces were longer detectable. SARS-CoV-2-specific antibodies have 90% probability of detection from 7 days (total Ig) and 18 days (IgG) since symptom onset. For highest probability of detection in SARS-CoV-2 diagnostics early in infection, RT-PCR on NP and OP specimens are more sensitive than on oral fluid and feces. For SARS-CoV-2 diagnostics late after infection, RT-PCR on feces specimens and serology are more valuable.

Detection of SARS-CoV-2 infection in the general population by three prevailing rapid antigen tests: cross-sectional diagnostic accuracy study.

BACKGROUND: Rapid antigen diagnostic tests (Ag-RDTs) are the most widely used point-of-care tests for detecting SARS-CoV-2 infection. Since the accuracy may have altered by changes in SARS-CoV-2 epidemiology, indications for testing, sampling and testing procedures, and roll-out of COVID-19 vaccination, we evaluated the performance of three prevailing SARS-CoV-2 Ag-RDTs. METHODS: In this cross-sectional study, we consecutively enrolled individuals aged >16 years presenting for SARS-CoV-2 testing at three Dutch public health service COVID-19 test sites. In the first phase, participants underwent either BD-Veritor System (Becton Dickinson), PanBio (Abbott), or SD-Biosensor (Roche Diagnostics) testing with routine sampling procedures. In a subsequent phase, participants underwent SD-Biosensor testing with a less invasive sampling method (combined oropharyngeal-nasal [OP-N] swab). Diagnostic accuracies were assessed against molecular testing. RESULTS: Six thousand nine hundred fifty-five of 7005 participants (99%) with results from both an Ag-RDT and a molecular reference test were analysed. SARS-CoV-2 prevalence and overall sensitivities were 13% (188/1441) and 69% (129/188, 95% CI 62-75) for BD-Veritor, 8% (173/2056) and 69% (119/173, 61-76) for PanBio, and 12% (215/1769) and 74% (160/215, 68-80) for SD-Biosensor with routine sampling and 10% (164/1689) and 75% (123/164, 68-81) for SD-Biosensor with OP-N sampling. In those symptomatic or asymptomatic at sampling, sensitivities were 72-83% and 54-56%, respectively. Above a viral load cut-off (≥5.2 log10 SARS-CoV-2 E-gene copies/mL), sensitivities were 86% (125/146, 79-91) for BD-Veritor, 89% (108/121, 82-94) for PanBio, and 88% (160/182, 82-92) for SD-Biosensor with routine sampling and 84% (118/141, 77-89) with OP-N sampling. Specificities were >99% for all tests in most analyses. Sixty-one per cent of false-negative Ag-RDT participants returned for testing within 14 days (median: 3 days, interquartile range 3) of whom 90% tested positive. CONCLUSIONS: Overall sensitivities of three SARS-CoV-2 Ag-RDTs were 69-75%, increasing to ≥86% above a viral load cut-off. The decreased sensitivity among asymptomatic participants and high positivity rate during follow-up in false-negative Ag-RDT participants emphasise the need for education of the public about the importance of re-testing after an initial negative Ag-RDT should symptoms develop. For SD-Biosensor, the diagnostic accuracy with OP-N and deep nasopharyngeal sampling was similar; adopting the more convenient sampling method might reduce the threshold for professional testing.

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.

The Human CD4+ T Cell Response against Mumps Virus Targets a Broadly Recognized Nucleoprotein Epitope.

Mumps outbreaks among vaccinated young adults stress the need for a better understanding of mumps virus (MuV)-induced immunity. Antibody responses to MuV are well characterized, but studies on T cell responses are limited. We recently isolated a MuV-specific CD4+ T cell clone by stimulating peripheral blood mononuclear cells (PBMCs) from a mumps case with the viral nucleoprotein (MuV-N). In this study, we further explored the identity and relevance of the epitope recognized by the CD4+ T cell clone and ex vivo by T cells in a cohort of mumps cases. Using a two-dimensional matrix peptide pool of 15-mer peptides covering the complete MuV-N, we identified the epitope recognized by the T cell clone as MuV-N110-124 GTYRLIPNARANLTA, present in a well-conserved region of the viral protein. Upon peptide-specific stimulation, the T cell clone expressed the activation marker CD137 and produced gamma interferon, tumor necrosis factor, and interleukin-10 in a HLA-DR4-restricted manner. Moreover, the CD4+ T cells exerted a cytotoxic phenotype and specifically killed cells presenting MuV-N110-124 Furthermore, the identified peptide is widely applicable to the general population since it is predicted to bind various common HLA-DR molecules, and epitope-specific CD4+ T cells displaying cytotoxic/Th1-type properties were found in all tested mumps cases expressing different HLA-DR alleles. This first broadly recognized human MuV-specific CD4+ T cell epitope could provide a useful tool to detect and evaluate virus-specific T cell responses upon MuV infection or following vaccination.IMPORTANCE Recent outbreaks of mumps among vaccinated young adults have been reported worldwide. Humoral responses against mumps virus (MuV) are well characterized, although no correlate of protection has been elucidated, stressing the need to better understand cellular MuV-specific immunity. In this study, we identified the first MuV T cell epitope, which is derived from the viral nucleoprotein (MuV-N) and was recognized by a cytotoxic/Th1 CD4+ T cell clone that was isolated from a mumps case. Moreover, the epitope was predicted to bind a broad variety of common HLA-DRB1 alleles, which was confirmed by the epitope-specific cytotoxic/Th1 CD4+ T cell responses observed in multiple mumps cases with various HLA-DRB1 genotypes. The identified epitope is completely conserved among various mumps strains. These findings qualify this promiscuous MuV T cell epitope as a useful tool for further in-depth exploration of MuV-specific T cell immunity after natural mumps virus infection or induced by vaccination.

Bordetella pertussis outer membrane vesicle vaccine confers equal efficacy in mice with milder inflammatory responses compared to a whole-cell vaccine.

The demand for improved pertussis vaccines is urgent due to the resurgence of whooping cough. A deeper understanding of the mode of action of pertussis vaccines is required to achieve this improvement. The vaccine-induced effects of a candidate outer membrane vesicle vaccine (omvPV) and a classical protective but reactogenic whole cell vaccine (wPV) were comprehensively compared in mice. The comparison revealed essential qualitative and quantitative differences with respect to immunogenicity and adverse effects for these vaccines. Both vaccines stimulated a mixed systemic Th1/Th2/Th17 response. Remarkably, omvPV evoked higher IgG levels, lower systemic pro-inflammatory cytokine responses and enhanced splenic gene expression than wPV. The omvPV-induced transcriptome revealed gene signatures of the IFN-signaling pathway, anti-inflammatory signatures that attenuate LPS responses, anti-inflammatory metabolic signatures, and IgG responses. Upon intranasal challenge, both immunized groups were equally efficient in clearing Bordetella pertussis from the lungs. This study importantly shows that immunization with omvPV provides a milder inflammatory responses but with equal protection to bacterial colonization and induction of protective antibody and Th1/Th17 type immune responses compared to wPV. These results emphasize the potential of omvPV as a safe and effective next-generation pertussis vaccine.

Immunological Signatures after Bordetella pertussis Infection Demonstrate Importance of Pulmonary Innate Immune Cells.

Effective immunity against Bordetella pertussis is currently under discussion following the stacking evidence of pertussis resurgence in the vaccinated population. Natural immunity is more effective than vaccine-induced immunity indicating that knowledge on infection-induced responses may contribute to improve vaccination strategies. We applied a systems biology approach comprising microarray, flow cytometry and multiplex immunoassays to unravel the molecular and cellular signatures in unprotected mice and protected mice with infection-induced immunity, around a B. pertussis challenge. Pre-existing systemic memory Th1/Th17 cells, memory B-cells, and mucosal IgA specific for Ptx, Vag8, Fim2/3 were detected in the protected mice 56 days after an experimental infection. In addition, pre-existing high activity and reactivation of pulmonary innate cells such as alveolar macrophages, M-cells and goblet cells was detected. The pro-inflammatory responses in the lungs and serum, and neutrophil recruitment in the spleen upon an infectious challenge of unprotected mice were absent in protected mice. Instead, fast pulmonary immune responses in protected mice led to efficient bacterial clearance and harbored potential new gene markers that contribute to immunity against B. pertussis. These responses comprised of innate makers, such as Clca3, Retlna, Glycam1, Gp2, and Umod, next to adaptive markers, such as CCR6+ B-cells, CCR6+ Th17 cells and CXCR6+ T-cells as demonstrated by transcriptome analysis. In conclusion, besides effective Th1/Th17 and mucosal IgA responses, the primary infection-induced immunity benefits from activation of pulmonary resident innate immune cells, achieved by local pathogen-recognition. These molecular signatures of primary infection-induced immunity provided potential markers to improve vaccine-induced immunity against B. pertussis.

Vaccine-Mediated Activation of Human TLR4 Is Affected by Modulation of Culture Conditions during Whole-Cell Pertussis Vaccine Preparation.

The potency of whole-cell pertussis (wP) vaccines is still determined by an intracerebral mouse protection test. To allow development of suitable in vitro alternatives to this test, insight into relevant parameters to monitor the consistency of vaccine quality is essential. To this end, a panel of experimental wP vaccines of varying quality was prepared by sulfate-mediated suppression of the BvgASR master virulence regulatory system of Bordetella pertussis during cultivation. This system regulates the transcription of a range of virulence proteins, many of which are considered important for the induction of effective host immunity. The protein compositions and in vivo potencies of the vaccines were BvgASR dependent, with the vaccine containing the highest amount of virulence proteins having the highest in vivo potency. Here, the capacities of these vaccines to stimulate human Toll-like receptors (hTLR) 2 and 4 and the role these receptors play in wP vaccine-mediated activation of antigen-presenting cells in vitro were studied. Prolonged BvgASR suppression was associated with a decreased capacity of vaccines to activate hTLR4. In contrast, no significant differences in hTLR2 activation were observed. Similarly, vaccine-induced activation of MonoMac-6 and monocyte-derived dendritic cells was strongest with the highest potency vaccine. Blocking of TLR2 and TLR4 showed that differences in antigen-presenting cell activation could be largely attributed to vaccine-dependent variation in hTLR4 signalling. Interestingly, this BvgASR-dependent decrease in hTLR4 activation coincided with a reduction in GlcN-modified lipopolysaccharides in these vaccines. Accordingly, expression of the lgmA-C genes, required for this glucosamine modification, was significantly reduced in bacteria exposed to sulfate. Together, these findings demonstrate that the BvgASR status of bacteria during wP vaccine preparation is critical for their hTLR4 activation capacity and suggest that including such parameters to assess consistency of newly produced vaccines could bring in vitro testing of vaccine quality a step closer.

Transcriptome signature for dampened Th2 dominance in acellular pertussis vaccine-induced CD4(+) T cell responses through TLR4 ligation.

Current acellular pertussis (aP) vaccines promote a T helper 2 (Th2)-dominated response, while Th1/Th17 cells are protective. As our previous study showed, after adding a non-toxic TLR4 ligand, LpxL1, to the aP vaccine in mice, the Bordetella pertussis-specific Th2 response is decreased and Th1/Th17 responses are increased as measured at the cytokine protein level. However, how this shift in Th response by LpxL1 addition is regulated at the gene expression level remains unclear. Transcriptomics analysis was performed on purified CD4(+) T cells of control and vaccinated mice after in vitro restimulation with aP vaccine antigens. Multiple key factors in Th differentiation, including transcription factors, cytokines, and receptors, were identified within the differentially expressed genes. Upregulation of Th2- and downregulation of follicular helper T cell-associated genes were found in the CD4(+) T cells of both aP- and aP+LpxL1-vaccinated mice. Genes exclusively upregulated in CD4(+) T cells of aP+LpxL1-vaccinated mice included Th1 and Th17 signature cytokine genes Ifng and Il17a respectively. Overall, our study indicates that after addition of LpxL1 to the aP vaccine the Th2 component is not downregulated at the gene expression level. Rather an increase in expression of Th1- and Th17-associated genes caused the shift in Th subset outcome.

Association of Vitamin D Receptor Polymorphism with Susceptibility to Symptomatic Pertussis.

Pertussis, caused by infection with the gram negative B. pertussis bacterium, is a serious respiratory illness that can last for months. While B. pertussis infection rates are estimated between 1-10% in the general population, notifications of symptomatic pertussis only comprise 0.01-0.1% indicating that most individuals clear B. pertussis infections without developing (severe) clinical symptoms. In this study we investigated whether genetic risk factors are involved in the development of symptomatic pertussis upon B. pertussis infection. Single-nucleotide polymorphisms (SNPs) in candidate genes, MBL2, IL17A, TNFα, VDR, and IL10 were genotyped in a unique Dutch cohort of symptomatic clinically confirmed (ex-)pertussis patients and in a Dutch population cohort. Of the seven investigated SNPs in five genes, a polymorphism in the Vitamin D receptor (VDR) gene (rs10735810) was associated with pertussis. The VDR major allele and its homozygous genotype were more present in the symptomatic pertussis patient cohort compared to the control population cohort. Interestingly, the VDR major allele correlated also with the duration of reported pertussis symptoms. Vitamin D3 (VD3) and VDR are important regulators of immune activation. Altogether, these findings suggest that polymorphisms in the VDR gene may affect immune activation and the clinical outcome of B. pertussis infection.

Development of an IFNγ ELISPOT for the analysis of the human T cell response against mumps virus.

In the last decade, mumps virus (MuV) causes outbreaks in highly vaccinated populations. Sub-optimal T cell immunity may play a role in the susceptibility to mumps in vaccinated individuals. T cell responses to mumps virus have been demonstrated, yet the quality of the MuV-specific T cell response has not been analyzed using single cell immunological techniques. Here we developed an IFNγ ELISPOT assay to assess MuV-specific T cell responses in peripheral blood mononuclear cells (PBMC) of healthy (vaccinated) donors and mumps patients. Various in vitro MuV-specific stimulation methods of PBMC were compared, using either live or inactivated MuV alone or MuV-infected autologous antigen presenting cells, i.e. Epstein Barr Virus-transformed B lymphoblastoid cell lines (EBV-BLCL) or (mitogen pre-activated) PBMC, for their ability to recall IFNγ-producing responder cells measured by ELISPOT. For the detection of MuV-specific T cell responses, direct exposure (24h) to live MuV was the preferred stimulation method when assay sensitivity and practical reasons were considered. Notably, flowcytometric confirmation of data revealed that primarily T cells and NK cells produce IFNγ upon live MuV stimulation. Depleting PBMC from CD56(+) NK cells prior to stimulation with live MuV led to the enumeration of MuV-specific T cell responses by ELISPOT. Our assay constitutes a tool to evaluate memory MuV-specific T cell responses in MuV vaccinated or infected persons. Furthermore, this study provides evidence that live MuV not only induces IFNγ production by T cells, but also by NK cells.

Roads to the development of improved pertussis vaccines paved by immunology.

Current acellular pertussis vaccines have various shortcomings, which may contribute to their suboptimal efficacy and waning immunity in vaccinated populations. This calls for the development of new pertussis vaccines capable of inducing long-lived protective immunity. Immunization with whole cell pertussis vaccines and natural infection with Bordetella pertussis induce distinct and more protective immune responses when compared with immunization with acellular pertussis vaccines. Therefore, the immune responses induced with whole cell vaccine or after infection can be used as a benchmark for the development of third-generation vaccines against pertussis. Here, we review the literature on the immunology of B. pertussis infection and vaccination and discuss the lessons learned that will help in the design of improved pertussis vaccines.

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.

Ex vivo peptide-MHC II tetramer analysis reveals distinct end-differentiation patterns of human pertussis-specific CD4(+) T cells following clinical infection.

Pertussis is occurring in highly vaccinated populations, suggesting insufficient protective memory CD4(+) T cells to Bordetella (B.) pertussis. P.69 Pertactin (P.69 Prn) is an important virulence factor of B. pertussis, and P.69 Prn7-24 is an immunodominant CD4(+) T cell epitope in mice and broadly recognized in humans. P.69 Prn7-24 peptide-MHC II tetramers (DRB4*0101/IVKT) were designed to ex vivo interrogate the presence and differentiation state of P.69 Prn7-24 specific CD4(+) T cells in six symptomatic pertussis cases. Cases with relatively more CD45RA(-)CCR7(+) central memory CD4(+)DRB4*0101/IVKT(+) T cells secreted Th1 cytokines, while cases with more CD45RA(-)CCR7(-) effector memory CD4(+)DRB4*0101/IVKT(+) T cells secreted both Th1 and Th2 cytokines upon peptide stimulation. CD45RA(+)CCR7(-) terminal differentiation pattern was associated with low or non-functionality based on cytokine secretion. This study provides proof of principle for further peptide-MHC II tetramer guided approaches in the elucidation of limited immunological memory to B. pertussis and the resurgence of pertussis.

Modulation of the CD4(+) T cell response after acellular pertussis vaccination in the presence of TLR4 ligation.

Whole cell pertussis (wP) vaccines are gradually being replaced by aluminum salt-adjuvanted acellular pertussis (aP) vaccines. These promote CD4(+) T cell responses with a non-protective Th2 component, while protective immune mechanisms to B. pertussis may rather involve long-lived Th1/Th17 type CD4(+) T cells. Here we asked whether addition of a non-toxic meningococcal LPS derivative, LpxL1, as adjuvant can favorably modulate the aP-induced pertussis-specific CD4(+) T cell response in mice. To assess the effect of TLR4 ligation, Th type, quantity, and memory potential of pertussis-specific CD4(+) T cells were determined at the single-cell level after aP and aP+LpxL1 vaccination using intracellular cytokine staining and MHC class II tetramers. Adding LpxL1 to the aP vaccine weakened the Th2 component and strengthened the Th1/Th17 component of the specific CD4(+) T cell response. Notably, LpxL1 addition also induced higher frequencies of tetramer positive CD4(+) T cells in draining lymph nodes or blood, depending on the phase after vaccination. Moreover, there was a net profit in the number of CD4(+) T cells with a central memory phenotype, preferred for long-term immunity. Thus, adding a TLR4 ligand as adjuvant to a current aP vaccine was associated with a more favorable pertussis-specific CD4(+) T cell response.

Bordetella pertussis naturally occurring isolates with altered lipooligosaccharide structure fail to fully mature human dendritic cells.

Bordetella pertussis is a Gram-negative bacterium and the causative agent of whooping cough. Despite high vaccination coverage, outbreaks are being increasingly reported worldwide. Possible explanations include adaptation of this pathogen, which may interfere with recognition by the innate immune system. Here, we describe innate immune recognition and responses to different B. pertussis clinical isolates. By using HEK-Blue cells transfected with different pattern recognition receptors, we found that 3 out of 19 clinical isolates failed to activate Toll-like receptor 4 (TLR4). These findings were confirmed by using the monocytic MM6 cell line. Although incubation with high concentrations of these 3 strains resulted in significant activation of the MM6 cells, it was found to occur mainly through interaction with TLR2 and not through TLR4. When using live bacteria, these 3 strains also failed to activate TLR4 on HEK-Blue cells, and activation of MM6 cells or human monocyte-derived dendritic cells was significantly lower than activation induced by the other 16 strains. Mass spectrum analysis of the lipid A moieties from these 3 strains indicated an altered structure of this molecule. Gene sequence analysis revealed mutations in genes involved in lipid A synthesis. Findings from this study indicate that B. pertussis isolates that do not activate TLR4 occur naturally and that this phenotype may give this bacterium an advantage in tempering the innate immune response and establishing infection. Knowledge on the strategies used by this pathogen in evading the host immune response is essential for the improvement of current vaccines or for the development of new ones.

Molecular signatures of the evolving immune response in mice following a Bordetella pertussis infection.

Worldwide resurgence of pertussis necessitates the need for improvement of pertussis vaccines and vaccination strategies. Since natural infections induce a longer-lasting immunity than vaccinations, detailed knowledge of the immune responses following natural infection can provide important clues for such improvement. The purpose was to elucidate the kinetics of the protective immune response evolving after experimental Bordetella pertussis (B. pertussis) infection in mice. Data were collected from (i) individual analyses, i.e. microarray, flow cytometry, multiplex immunoassays, and bacterial clearance; (ii) twelve time points during the infection; and (iii) different tissues involved in the immune responses, i.e. lungs, spleen and blood. Combined data revealed detailed insight in molecular and cellular sequence of events connecting different phases (innate, bridging and adaptive) of the immune response following the infection. We detected a prolonged acute phase response, broad pathogen recognition, and early gene signatures of subsequent T-cell recruitment in the lungs. Activation of particular transcription factors and specific cell markers provided insight into the time course of the transition from innate towards adaptive immune responses, which resulted in a broad spectrum of systemic antibody subclasses and splenic Th1/Th17 memory cells against B. pertussis. In addition, signatures preceding the local generation of Th1 and Th17 cells as well as IgA in the lungs, considered key elements in protection against B. pertussis, were established. In conclusion, molecular and cellular immunological processes in response to live B. pertussis infection were unraveled, which may provide guidance in selecting new vaccine candidates that should evoke local and prolonged protective immune responses.