Characteristics of anti-pertussis toxin antibodies after vaccination and infection

Pertussis or whooping cough is a human respiratory tract infection caused by Bordetella pertussis bacteria. Despite extensive vaccinations, pertussis incidence has increased during the last decades. The reasons behind this resurgence are not entirely understood. Multiple factors may influence the resurgence, such as improved diagnostic methods, the adaptation of circulating B. pertussis strains, the waning of vaccination-induced immunity, and asymptomatic pathogen transmission. The shift from whole-cell to acellular pertussis vaccines (aPV) has been speculated as one of the reasons for the quickly waning vaccination-induced immunity. The reduced reactogenicity of the aPVs is achieved by chemically treating the vaccine components. The treatments alter the structure of the vaccine antigens and may affect the functional properties of antibodies generated after vaccination. Currently, no internationally established immunological correlates of protection exist for evaluating pertussis vaccine-induced protection. This study compared the antibody responses to pertussis toxin (PT) after aPV and infection. PT is an exotoxin that can elicit many biological activities and is included in all current aPVs. Binding strength, binding location, and the neutralizing activity of these antibodies were evaluated with newly developed immunoassays for patient and vaccination samples in Finnish, Danish, and Dutch populations. Infection and aPV-derived antibodies recognized different epitopes of PT. The binding strength of antibodies was higher after aPVs compared to infection. Elevated concentrations of PT-neutralizing antibodies remained one year after aPV. These characteristics were influenced by the detoxification method of PT, amount of PT included in aPV, number of vaccination doses received, existing immunological memory of PT, and recency of the latest vaccination. The studied antibody characteristics may contribute as potential correlates of protection and thus aid the evaluation of the next generation of vaccines, vaccination programs, and diagnostics.Hinkuyskätoksiinivasta-aineiden ominaisuudet rokotuksen ja infektion jälkeen Hinkuyskä on hengitystieinfektio, jonka aiheuttaa Bordetella pertussis -bakteeri. Jo pitkään jatkuneista rokotusohjelmista huolimatta hinkuyskän ilmaantuvuus on kasvanut viime vuosikymmeninä. Syitä tähän ei täysin ymmärretä, mutta siihen voivat vaikuttaa parempi diagnostiikka, kiertävien B. pertussis -kantojen muuntuminen, rokotuksen aiheuttaman immuniteetin nopea heikkeneminen sekä taudinaiheuttajan oireeton leviäminen väestössä. Vaihtoa kokosolurokotteista soluttomiin hinkuyskärokotteisiin arvellaan keskeiseksi tekijäksi nopeasti heikkenevään immuniteettiin. Soluttomien rokotteiden matala reaktogeenisyys saavutetaan käsittelemällä rokotekomponentteja kemiallisesti, mikä johtaa niiden proteiinirakenteiden muuttumiseen. Tämä voi edelleen vaikuttaa rokotuksen jälkeen muodostuvien vasta-aineiden erilaisiin ominaisuuksiin.Tällä hetkellä hinkuyskärokotteista saatavan suojan arvioimiseksi ei ole olemassa kansainvälisesti vakiintuneita immunologisia vastemuuttujia. Tässä tutkimuksessa verrattiin vasta-ainevasteita hinkuyskätoksiinin (PT, engl. pertussis toxin) soluttoman hinkuyskärokotteen ja infektion jälkeen. Vasta-aineiden sitomisvoimaa, sitoutumispaikkaa ja neutralisointiaktiivisuutta arvioitiin varta vasten kehitetyillä immunomäärityksillä rokote- ja potilasnäytteistä Suomen, Tanskan ja Hollannin väestössä. Rokotteista ja infektiosta muodostuneet vasta-aineet tunnistivat erilaisia epitooppeja. Vasta-aineiden sitoutumisvoimakkuus oli korkeampi pian rokotuksen jälkeen infektioon verrattuna. PT:tä neutraloivien vasta-aineiden pitoisuudet olivat merkittävästi koholla vielä vuoden kuluttua rokotuksen jälkeen. Näihin tutkittuihin ominaisuuksiin vaikuttivat PT:n eri kemialliset käsittelymenetelmät, rokotuksessa käytetyn PT:n määrä, rokoteannosten määrä, immunologinen muisti PT:lle ja viimeisimmän rokotuksen ajankohta. Tämän tutkimuksen perusteella vasta-aineiden eri ominaisuuksia voitaisiin käyttää seuraavan sukupolven rokotteiden, rokotusohjelmien ja diagnostiikan kehittämisessä

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.</p

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 studie</p

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 eval-uated 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

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

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

Pertussis 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.</p

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. </div

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.</p

A novel whole blood assay to quantify the release of T cell associated cytokines in response to Bordetella pertussis antigens.

BACKGROUND: Bordetella pertussis continues to cause whooping cough globally even in countries with high immunisation coverage. Booster vaccinations with acellular pertussis vaccines are thus used in children, adolescents, and adults. T cell immunity is crucial for orchestrating the immune response after vaccination. However, T cell assays can be expensive and difficult to implement in large clinical trials. In this study, a whole blood (WB) stimulation assay was developed to identify secreted T cell associated cytokines in different age groups after acellular pertussis booster vaccination. MATERIAL AND METHODS: Longitudinal WB samples were collected from a small set of subjects (n = 38) aged 7-70 years participating in a larger ongoing clinical trial. For assay development, samples were diluted and incubated with purified inactivated pertussis toxin (PT), filamentous haemagglutinin (FHA), inactivated B. pertussis lysate, and complete medium (M) as stimulating conditions, with anti-CD28 and anti-CD49d as co-stimulants. Different timepoints around the vaccination (D0, D7, D14, D28), WB dilution factor (1:2, 1:4) and incubation time (24 h, 48 h, 72 h) were compared. Responses to 15 cytokines were tested with Luminex/multiplex immunoassay. RESULTS: The optimized assay consisted of WB incubation with M, PT, and FHA (including the two co-stimulants). After 48 h incubation, supernatants were collected for measurement of seven selected T cell associated cytokines (IL-2, IL-5, IL-10, IL-13, IL-17 A, IL-17F, and IFN-y) from samples before and 28 days after vaccination. PT stimulation showed a trend for upregulation of IL-2, IL-13, and IL-17 A/F for adult subjects, whereas the responses of all cytokines were downregulated for the paediatric subjects. Furthermore, PT and FHA-stimulated WB showed diverse cytokine producing profiles. CONCLUSIONS: The developed WB-based cytokine assay was shown to be less costly, easy to perform, and functional in differently aged individuals. Further, it requires only a small amount of fresh blood, which is beneficial especially for studies including infants. Our results support the use of this assay for other immunological studies in the future

Serologisten lateraalivirtausmääritysten kehittäminen hinkuyskädiagnostiikkaan

Hinkuyskä on maailmanlaajuisesti merkittävä infektiotauti. Kattavasta rokote-järjestelmästä huolimatta maailmanlaajuisesti 30 miljoonaa ihmistä sairastuu ja 195 000 lasta kuolee hinkuyskään vuosittain. Useimmat hinkuyskäkuolemat tapahtuvat kehitysmaissa, joissa diagnoosi perustuu pelkkään oireiden tulkintaan. Tämän lisäksi nykyiset laboratoriomenetelmät ovat hitaita, kalliita tai vaativat koulutetun henkilökunnan. Täten hinkuyskälle on tarvetta kehittää uudenlaisia ja yksinkertaisia vieritestausmenetelmiä. Vieritestit auttaisivat myös hinkuyskän maailmanlaajuisen epidemiologian ja rokotusstrategioiden seuraamisessa. Tässä työssä kehitettiin nopea ja yksinkertainen lateraalivirtausmääritys hinkuyskän IgG-vasteen serologiseen testaamiseen. Lateraalivirtausmäärityksiä kehitettiin kaksi kappaletta, ja ne perustuivat joko sekundäärivasta-aineen tai kaksoisantigeeniperiaatteen käyttämiseen vasta-aineiden havaitsemiseksi. Lateraalivirtausalustalle valmistettiin hinkuyskätoksiinista koostuva testiviiva, joka sitoo potilasnäytteestä hinkuyskää vastaan muodostuneet spesifiset vasta-aineet. Seuraavaksi vasta-aineet havaittiin fluoresoiviin europium-nanopartikkeleihin konjugoiduilla IgG-sekundäärivasta-aineilla tai hinkuyskätoksiinilla. ELISA:lla varmennettujen kliinisten seeruminäytteiden (n = 109) toimivuus testattiin molemmilla määritystavoilla. Molempien määritysten tulokset korreloivat ELISA:n tulosten kanssa. Täten pystyttiin osoittamaan, että hinkuyskäspesifisiä vasta-aineita voidaan mitata kliinisistä näytteistä lateraalivirtausperiaatteella. Kaksoisantigeenimäärityksestä saatiin kehitettyä yksinkertainen ja nopea (40 minuuttia), mutta määrityksen herkkyys ei ole vielä riittävällä tasolla kenttädiagnostiikkaan. Määrityksen herkkyys oli 75,0 % ja spesifisyys 82,9 %. Sekundäärivasta-ainemääritys sen sijaan on monivaiheisempi ja pesuja vaativa testi, mutta se saavutti hyvän korrelaation ELISA:n tulosten kanssa (0,84). Määrityksen herkkyys oli 91,7 % ja spesifisyys 95,3 %. Tätä testiä voitaisiin käyttää kvantitatiivisiin tai vähintäänkin semi-kvantitatiivisiin käyttötarkoituksiin. Testejä voitaisiin edelleen kehittää monialyyttimääritykseksi tai pyrkiä yksinkertaistamaan kenttädiagnostiikkaan sopivammiksi näytematriisia ja leimamolekyyliä muuttamalla.Siirretty Doriast