Effect of immunization during pregnancy and pre-existing immunity on diphtheria-tetanus-acellular pertussis vaccine responses in infants.

Immunization during pregnancy (IP) against pertussis is recommended in many countries to protect infants. Although maternal antibodies can influence the infants' antibody responses to primary vaccinations, their effect on the development of functional antibodies and B cells remain poorly studied. We investigated the maternal immune response to IP and the effect of IP and pre-existing antibodies on infants' primary vaccine responses in an open-label, non-randomized trial. Forty-seven mothers received tetanus-diphtheria-acellular pertussis (Tdap) vaccine during pregnancy, and 22 mothers were included as controls. Sixty-nine infants received primary doses of DTaP at three and five months of age. Geometric mean concentrations of antibodies to pertussis toxin, filamentous haemagglutinin, pertactin, diphtheria, and tetanus toxins, pertussis toxin neutralizing antibodies (PTNAs), and plasma and memory B-cell frequencies were studied at delivery, and at three, five and six months. Levels of antibodies, PTNAs, and frequencies of memory B-cells were significantly increased at delivery and up to six months after in mothers with IP compared to those without IP (all p < 0.05, except for PT-specific memory B-cells). In vaccinated pregnant women, high pre-existing antibody levels were positively correlated with higher antibody responses after IP. IP blunted the infants' antibody and plasma B-cell responses to all vaccine antigens, except for tetanus toxin. This blunting effect was the strongest in infants with high concentrations of maternal antibodies. In conclusion, IP resulted in significantly higher concentrations of antibodies in infants up to three months of age (all p < 0.05); but was associated with blunting of various infants' vaccine responses.

Oral fluid-based lateral flow point-of-care assays for pertussis serology.

Introduction. Current serological diagnosis of pertussis is usually performed by ELISA, which is typically performed in larger diagnostic or reference laboratories, requires trained staff, and due to sample batching may have longer turnaround times.Hypothesis and Aim. A rapid point-of-care (POC) assay for pertussis serology would aid in both the diagnosis and surveillance of the disease.Methodology. A quantitative lateral flow (LF)-based immunoassay with fluorescent Eu-nanoparticle reporters was developed for the detection of anti-pertussis toxin (PT) and adenylate cyclase toxin (ACT) antibodies from oral fluid samples (N=100), from suspected pertussis cases with respiratory symptoms.Results. LF assay results were compared to those obtained with anti-PT IgG oral fluid ELISA. For an ELISA cut-off value of 50 arbitrary units, the overall agreement between the assays was 91/100 (91 %), the sensitivity was 63/70 (90 %) and the specificity was 28/30 (93 %). No ACT-specific antibodies were detected from oral fluid samples; however, the signal readout positively correlated to those patients with high anti-PT IgG antibodies.Conclusion. The developed LF assay was a specific, sensitive and rapid test for serological diagnosis of pertussis with anti-PT antibodies and is a suitable POC test using oral fluid samples.

Antibody avidity to pertussis toxin after acellular pertussis vaccination and infection.

Pertussis toxin (PT) is a unique virulence factor of Bordetella pertussis, and therefore a key component of acellular pertussis vaccines. Although immunity after infection seems to persist longer than after vaccination, the exact mechanisms are not fully known. In this study the overall binding strength (avidity) of anti-PT IgG antibodies was compared after acellular booster vaccination and infection, as a parameter to evaluate long-lasting protection.Danish and Finnish serum samples from a total of 134 serologically confirmed patients and 112 children who received acellular booster vaccines were included in this study. The concentration of anti-PT IgG was first determined by ELISA, followed by two separate ELISAs to evaluate antibody avidity: either with a dilution series of urea as a bond-breaking agent of antibody and antigen binding and a constant anti-PT IgG concentration between the samples or with a constant dilution ratio of sera and detergent. In addition to urea, the use of diethylamine and ammonium thiocyanate as disruptive agents were first compared between each other.A strong Spearman correlation (R > 0.801) was noted between avidity and concentration of anti-PT IgG antibodies if a constant serum dilution method was used, and avidity was noted to be higher in patients in comparison to vaccinees in Denmark, but not in Finland. However, no correlation between antibody concentration and avidity was found if a constant anti-PT IgG concentration was used (R = -0.157). With this method, avidity after vaccination was significantly higher in comparison to that after infection in both Danish and Finnish subjects (p < 0.01). A shorter time since the latest booster vaccination was found to affect avidity positively on the next PT-antigen exposure with either vaccination or infection.

Whole blood based point-of-care assay for the detection of anti-pertussis toxin IgG antibodies.

Current serological diagnosis of pertussis is usually done by ELISA to determine serum specific anti-pertussis toxin (PT) IgG antibodies. However, the ELISAs are often central-laboratory based, require trained staff, and have long turnaround times. A rapid point-of-care (POC) assay for pertussis serology would aid in both diagnosis and surveillance of the disease. In this study, a quantitative lateral flow assay (LFA) with fluorescent Eu-nanoparticle reporters was used for the detection of anti-PT antibodies from whole blood. The assay was evaluated by testing overall 141 samples including 25 before and 116 one month after acellular pertussis booster vaccination. LFA results were compared to those obtained with standardized anti-PT IgG ELISAs with paired serum samples. Correlation between the assays was high (Pearson R = 0.832), and the achieved analytical sensitivity of the LFA was 29 IU/mL, which would be sufficient for clinically relevant cutoffs for determining recent infections. The paired samples, collected pre- and post-booster, demonstrated a significant increase in anti-PT IgG antibodies similar to that detected by ELISA. The developed LFA opens up several alternatives for a suitable POC test also in middle- and low-income countries.

Memory B Cell Activation Induced by Pertussis Booster Vaccination in Four Age Groups of Three Countries.

Background: Immunogenicity of acellular pertussis (aP) vaccines is conventionally assessed by measuring antibody responses but antibody concentrations wane quickly after vaccination. Memory B cells, however, are critical in sustaining long-term protection and therefore may be an important factor when assessing pertussis immunity after vaccination. Aim: We studied pertussis specific memory B cell (re)activation induced by an aP booster vaccination in four different age groups within three countries. Materials and methods: From a phase IV longitudinal interventional study, 268 participants across Finland, the Netherlands and the United Kingdom were included and received a 3-component pertussis booster vaccine: children (7-10y, n=53), adolescents (11-15y, n=66), young adults (20-34y, n=74), and older adults (60-70y, n=75). Memory B cells at baseline, day 28, and 1 year post-vaccination were measured by a pertussis toxin (Ptx), filamentous haemagglutinin (FHA), and pertactin (Prn) specific ELISpot assay. Antibody results measured previously were available for comparison. Furthermore, study participants were distributed into groups based on their baseline memory B cell frequencies, vaccine responses were monitored between these groups. Results: Geometric mean (GM) memory B cell frequencies for pertussis antigens at baseline were low. At 28 days post-vaccination, these frequencies increased within each age group and were still elevated one year post-booster compared to baseline. Highest frequencies at day 28 were found within adolescents (GM: 5, 21, and 13, for Ptx, FHA and Prn, respectively) and lowest within older adults (GM: 2, 9, and 3, respectively). Moderate to strong correlations between memory B cell frequencies at day 28 and antibody concentrations at day 28 and 1 year were observed for Prn. Memory B cell frequencies > 1 per 100,000 PBMCs at baseline were associated with significantly higher memory responses after 28 days and 1 year. Conclusions: An aP booster vaccine (re)activated memory B cells in all age groups. Still elevated memory B cell frequencies after one year indicates enhanced immunological memory. However, antigen specific memory B cell activation seems weaker in older adults, which might reflect immunosenescence. Furthermore, the presence of circulating memory B cells at baseline positively affects memory B cell responses. This study was registered at www.clinicaltrialsregister.eu: No. 2016-003678-42.

Pertussis toxin neutralizing antibody response after an acellular booster vaccination in Dutch and Finnish participants of different age groups.

ABSTRACTPertussis incidence has increased in many countries and the disease occurs among all age groups, suggesting the need for booster immunizations through life. In addition to determining the concentration of anti-pertussis toxin (PT) antibodies, the ability of PT neutralizing antibodies (PTNAs) could be used to assess vaccine responses.Altogether 258 participants [7-10-year-old (N = 73), 11-15-year-old (N = 85), 20-35-year-old (N = 50) and 60-70-year-old (N = 50)] were included. Sera were collected before, one month, and one year after a single dose of a three pertussis component containing acellular pertussis vaccine. The adolescents were primed in childhood either by acellular or whole-cell vaccination. PTNA titres were determined by a Chinese hamster ovary cell assay and anti-PT IgG/IgA antibody concentrations by multiplex immunoassay.In all age groups, a significant increase in levels of PTNAs and anti-PT IgG was observed one month after vaccination and remained at least two-fold higher one year post-booster, in comparison to pre-booster. Young adults had the lowest response. The strongest increase in PTNAs was observed in participants who had ≥10 IU/mL concentration of anti-PT IgG antibodies pre-booster. At pre-booster, whole-cell-primed adolescents had higher PTNAs than acellular-primed peers (p = 0.047). One year post-booster, the Finnish whole-cell-primed adolescents had a higher level of PTNAs than acellular-primed adolescents (p = 0.049), however, this was not observed in Dutch adolescents. In conclusion, PTNAs increased after vaccination in all age groups, and the strongest increase was related to the presence of high pre-booster antibodies.

Evaluation of Anti-PT Antibody Response after Pertussis Vaccination and Infection: The Importance of Both Quantity and Quality.

Pertussis toxin (PT) is considered the main virulence factor causing whooping cough or pertussis. The protein is widely studied and its composition was revealed and sequenced already during the 1980s. The human immune system creates a good response against PT when measured in quantity. However, the serum anti-PT antibodies wane rapidly, and only a small amount of these antibodies are found a few years after vaccination/infection. Therefore, multiple approaches to study the functionality (quality) of these antibodies, e.g., avidity, neutralizing capacity, and epitope specificity, have been investigated. In addition, the long-term B cell memory (Bmem) to PT is crucial for good protection throughout life. In this review, we summarize the findings from functional PT antibody and Bmem studies. These results are discussed in line with the quantity of serum anti-PT antibodies. PT neutralizing antibodies and anti-PT antibodies with proper avidity are crucial for good protection against the disease, and certain epitopes have been identified to have multiple functions in the protection. Although PT-specific Bmem responses are detectable at least five years after vaccination, long-term surveillance is lacking. Variation of the natural boosting of circulating Bordetella pertussis in communities is an important confounding factor in these memory studies.

Simultaneous Determination of Antibodies to Pertussis Toxin and Adenylate Cyclase Toxin Improves Serological Diagnosis of Pertussis.

Serological diagnosis of pertussis is mainly based on anti-pertussis toxin (PT) IgG antibodies. Since PT is included in all acellular vaccines (ACV), serological assays do not differentiate antibodies induced by ACVs and infection. Adenylate cyclase toxin (ACT) is not included in the ACVs, which makes it a promising candidate for pertussis serology with the specific aim of separating infection- and ACV-induced antibodies. A multiplex lateral flow test with PT and ACT antigens was developed to measure serum antibodies from pertussis-seropositive patients (n = 46), healthy controls (n = 102), and subjects who received a booster dose of ACV containing PT, filamentous hemagglutinin, and pertactin (n = 67) with paired sera collected before and one month after the vaccination. If the diagnosis was solely based on anti-PT antibodies, 98.5-44.8% specificity (before and after vaccination, respectively) and 78.2% sensitivity were achieved, whereas if ACT was used in combination with PT, the sensitivity of the assay increased to 91.3% without compromising specificity. No increase in the level of anti-ACT antibodies was found after vaccination. This exploratory study indicates that the use of ACT for serology would be beneficial in combination with a lower quantitative cutoff for anti-PT antibodies, and particularly in children and adolescents who frequently receive booster vaccinations.

Differences in epitope-specific antibodies to pertussis toxin after infection and acellular vaccinations.

OBJECTIVES: Pertussis toxin (PT) is a component of all acellular pertussis vaccines. PT must be detoxified to be included in acellular vaccines, which results in conformational changes in the functional epitopes of PTs. Therefore, induced epitope-specific antibodies to PT may vary after vaccinations or natural infections, and this information could reveal biomarkers implicated for protection and successful immunisation. METHODS: Pertussis toxin epitope-specific antibodies in sera from 152 vaccinated children and 72 serologically confirmed patients were tested with a blocking ELISA, based on monoclonal antibodies that target protective PT epitopes. RESULTS: All study groups induced considerable antibody titres to subunit 1 (S1). Of interest, S3 7E10-specific antibodies were present in patients, but not after vaccinations (P < 0.001). The impact of glutaraldehyde treatment of PT was visible on epitope 1D7 (S1), whereas epitopes 1B7 (S1) and 10D (S1) were more preserved. Antibodies to these epitopes were higher after three primary vaccine doses than after a single booster dose. CONCLUSION: The high amount of 7E10-specific antibodies in patients suggests this epitope might be functionally relevant in protection. The overall characteristics of epitope-specific antibodies are influenced by infection or vaccination background, by the used detoxification method of PT and by the amount of the toxin used in immunisation.

Multiplex Point-of-Care Tests for the Determination of Antibodies after Acellular Pertussis Vaccination.

Most of the current serological diagnosis of pertussis is based on pertussis toxin (PT) IgG antibodies and does not differentiate between vaccination and infection-induced antibodies. PT is included in all of acellular pertussis vaccines available in the world. Multiplex testing of non-vaccine antigen-related antibodies has the potential to improve the diagnostic outcome of these assays. In this study, we developed a quantitatively spatial multiplex lateral flow immunoassay (LFIA) for the detection of IgG antibodies directed against PT, pertactin (PRN), and filamentous hemagglutinin (FHA). The assay was evaluated with serum samples with varying anti-PT, anti-PRN, and anti-FHA IgG levels and the result was compared to those obtained with standardized ELISA. The developed assay showed good specificity with PT and PRN antibodies and semiquantification throughout the antigen combinations. This exploratory study indicates that the multiplex LFIA is specific and sensitive, and a similar test platform with alternative antigens could be suitable for new type of pertussis serology.

A rapid lateral flow immunoassay for serological diagnosis of pertussis.

Current serological diagnosis of pertussis is usually done by ELISA. However, the ELISAs are often central-laboratory based, require trained staff and have long turnaround times. A rapid point-of-care (POC) assay for pertussis serology would aid in both diagnosis and surveillance of the disease. While lateral flow immunoassays (LFIA) are simple to use and ideal for point-of-care diagnostics, they were limited to qualitative assays until recently. In this study, we developed a quantitative LFIA with fluorescent Eu-nanoparticle reporters for the detection of anti-pertussis toxin (PT) IgG. The assay was evaluated by testing 198 serum samples with varying anti-PT IgG levels and the result was compared to those obtained with standardized anti-PT IgG ELISA. At the diagnostic cutoff of 100 IU/mL in ELISA, the LFIA had a concordance of 92% with the ELISA, with a specificity of 96% [95% confidence interval (CI): 89-99%] and a sensitivity of 88% [CI: 77-94%]. The developed LFIA has a turnaround time of one hour and requires only a simple manipulation by the user and an instrument for the quantitative detection of the signal. We conclude that the LFIA is specific and sensitive for serological diagnosis of pertussis and is suitable for a POC test.