Hybrid Immunity Improves the Immune Response after the Fourth COVID-19 Vaccine Dose in Individuals with Medical Conditions Predisposing to Severe COVID-19.

Data on immune responses following COVID-19 booster vaccinations and subsequent infections in the immunocompromised are limited. We studied antibody responses after the fourth dose and subsequent infections to define patient groups benefiting most from boosters. Fourth vaccine (booster) doses were, in Finland, first recommended for severely immunocompromised individuals, whom we invited to participate in our study in 2022. We assessed spike protein-specific IgG and neutralizing antibodies (NAb) against the ancestral and Omicron BA.1 strains one month after the fourth dose from 488 adult participants and compared them to the levels of 35 healthy controls after three doses. We used Bayesian generalized linear modeling to assess factors explaining antibody levels and assessed vaccine-induced and hybrid immunity six months after the last vaccine dose. Chronic kidney disease (CKD) and immunosuppressive therapy (IT) were identified as factors explaining sub-optimal antibody responses. The proportion of participants with a normal antibody response and NAbs was significantly lower regarding CKD patients compared to the controls. By the 6-month sampling point, one-third of the participants became infected (documented by serology and/or molecular tests), which notably enhanced antibody levels in most immunocompromised participants. Impaired antibody responses, especially NAbs against the Omicron lineage, suggest limited protection in individuals with CKD and highlight the need for alternative pharmaceutical preventive strategies. Vaccination strategies should take into account the development of robust hybrid immunity responses also among the immunocompromised.

Changes in SARS-CoV-2 seroprevalence and population immunity in Finland, 2020-2022.

Studying the prevalence of SARS-CoV-2 specific antibodies (seroprevalence) allows for assessing the impact of epidemic containment measures and vaccinations and estimating the number of infections regardless of viral testing. We assessed antibody-mediated immunity to SARS-CoV-2 induced by infections and vaccinations from April 2020 to December 2022 in Finland by measuring serum IgG to SARS-CoV-2 nucleoprotein (N-IgG) and spike glycoprotein from randomly selected 18-85-year-old subjects (n = 9794). N-IgG seroprevalence remained at <7% until the last quartile (Q) of 2021. After the emergence of the Omicron variant, N-IgG seroprevalence increased rapidly and was 31% in Q1/2022 and 54% in Q4/2022. Seroprevalence was highest in the youngest age groups from Q2/2022 onwards. We did not observe regional differences in seroprevalence in 2022. We estimated that 51% of the Finnish 18-85-year-old population had antibody-mediated hybrid immunity induced by a combination of vaccinations and infections by the end of 2022. In conclusion, major shifts in the COVID-19 pandemic and resulting population immunity could be observed by serological testing.

Strong Neutralizing Antibody Responses to SARS-CoV-2 Variants Following a Single Vaccine Dose in Subjects With Previous SARS-CoV-2 Infection.

Background: Previous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection primes the immune system; thus individuals who have recovered from infection have enhanced immune responses to subsequent vaccination (hybrid immunity). However, it remains unclear how well hybrid immunity induced by severe or mild infection can cross-neutralize emerging variants. We aimed to compare the strength and breadth of antibody responses in vaccinated recovered and uninfected subjects. Methods: We measured spike-specific immunoglobulin (Ig)G and neutralizing antibodies (NAbs) from vaccinated subjects including 320 with hybrid immunity and 20 without previous infection. From 29 subjects with a previous severe or mild infection, we also measured NAb responses against Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529/BA.1) variants following vaccination. Results: A single vaccine dose induced 2-fold higher anti-spike IgG concentrations and up to 4-fold higher neutralizing potency of antibodies in subjects with a previous infection compared with vaccinated subjects without a previous infection. Hybrid immunity was more enhanced after a severe than a mild infection, with sequentially decreasing NAb titers against Alpha, Beta, Delta, and Omicron variants. We found similar IgG concentrations in subjects with a previous infection after 1 or 2 vaccine doses. Conclusions: Hybrid immunity induced strong IgG responses, particularly after severe infection. However, the NAb titers were low against heterologous variants, especially against Omicron.

High secondary attack rate and persistence of SARS-CoV-2 antibodies in household transmission study participants, Finland 2020-2021.

Background: Household transmission studies offer the opportunity to assess both secondary attack rate (SAR) and persistence of SARS-CoV-2 antibodies over time. Methods: In Spring 2020, we invited confirmed COVID-19 cases and their household members to four visits, where we collected nasopharyngeal and serum samples over 28 days after index case onset. We calculated SAR based on the presence of SARS-CoV-2 neutralizing antibodies (NAb) and assessed the persistence of NAb and IgG antibodies (Ab) against SARS-CoV-2 spike glycoprotein and nucleoprotein. Results: SAR was 45% (39/87), including 35 symptomatic secondary cases. During the initial 28-day follow-up, 62% (80/129) of participants developed NAb. Of those that seroconverted, 90% (63/70), 85% (63/74), and 78% (45/58) still had NAb to early B-lineage SARS-CoV-2 3, 6, and 12 months after the onset of the index case. Anti-spike IgG Ab persisted in 100% (69/69), 97% (72/74), and 93% (55/59) of seroconverted participants after 3, 6, and 12 months, while anti-nucleoprotein IgG Ab levels waned faster, persisting in 99% (68/69), 78% (58/74), and 55% (39/71) of participants, respectively. Conclusion: Following detection of a COVID-19 case in a household, other members had a high risk of becoming infected. NAb to early B-lineage SARS-CoV-2 persisted for at least a year in most cases.

Neutralizing antibodies to SARS-CoV-2 Omicron variant after third mRNA vaccination in health care workers and elderly subjects.

The emergence of SARS-CoV-2 Omicron variant (B.1.1.529) with major spike protein mutations has raised concern over potential neutralization escape and breakthrough infections among vaccinated and previously SARS-CoV-2-infected subjects. We measured cross-protective antibodies against variants in health care workers (HCW, n = 20) and nursing home residents (n = 9) from samples collected at 1-2 months, following the booster (3rd) dose. We also assessed the antibody responses in subjects infected before the Omicron era (n = 38) with subsequent administration of a single mRNA vaccine dose. Following booster vaccination, HCWs had high IgG antibody concentrations to the spike protein and neutralizing antibodies (NAb) were detectable against all variants. IgG concentrations among the elderly remained lower, and some lacked NAbs against the Beta and Omicron variants. NAb titers were significantly reduced against Delta, Beta, and Omicron compared to WT virus regardless of age. Vaccination induced high IgG concentrations and variable titers of cross-reactive NAbs in previously infected subjects, whereas NAb titers against Omicron were barely detectable 1 month postinfection. High IgG concentrations with cross-protective neutralizing activity were detected after three Coronavirus Disease 2019 (COVID-19) vaccine doses in HCWs. However, lower NAb titers seen in the frail elderly suggest inadequate protection against Omicron breakthrough infections, yet protection against severe COVID-19 is expected.

A Highly Sensitive and Specific SARS-CoV-2 Spike- and Nucleoprotein-Based Fluorescent Multiplex Immunoassay (FMIA) to Measure IgG, IgA, and IgM Class Antibodies.

Validation and standardization of accurate serological assays are crucial for the surveillance of the coronavirus disease 2019 (COVID-19) pandemic and population immunity. We describe the analytical and clinical performance of an in-house fluorescent multiplex immunoassay (FMIA) for simultaneous quantification of antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein and spike glycoprotein. Furthermore, we calibrated IgG-FMIA against World Health Organization (WHO) International Standard and compared FMIA results to an in-house enzyme immunoassay (EIA) and a microneutralization test (MNT). We also compared the MNT results of two laboratories. IgG-FMIA displayed 100% specificity and sensitivity for samples collected 13 to 150 days post-onset of symptoms (DPO). For IgA- and IgM-FMIA, 100% specificity and sensitivity were obtained for a shorter time window (13 to 36 and 13 to 28 DPO for IgA- and IgM-FMIA, respectively). FMIA and EIA results displayed moderate to strong correlation, but FMIA was overall more specific and sensitive. IgG-FMIA identified 100% of samples with neutralizing antibodies (NAbs). Anti-spike IgG concentrations correlated strongly (ρ = 0.77 to 0.84, P < 2.2 × 10-16) with NAb titers, and the two laboratories' NAb titers displayed a very strong correlation (ρ = 0.95, P < 2.2 × 10-16). Our results indicate good correlation and concordance of antibody concentrations measured with different types of in-house SARS-CoV-2 antibody assays. Calibration against the WHO international standard did not, however, improve the comparability of FMIA and EIA results. IMPORTANCE SARS-CoV-2 serological assays with excellent clinical performance are essential for reliable estimation of the persistence of immunity after infection or vaccination. In this paper we present a thoroughly validated SARS-CoV-2 serological assay with excellent clinical performance and good comparability to neutralizing antibody titers. Neutralization tests are still considered the gold standard for SARS-CoV-2 serological assays, but our assay can identify samples with neutralizing antibodies with 100% sensitivity and 96% specificity without the need for laborious and slow biosafety level 3 (BSL-3) facility-requiring analyses.

Persistence of neutralizing antibodies a year after SARS-CoV-2 infection in humans.

Most subjects develop antibodies to SARS-CoV-2 following infection. In order to estimate the duration of immunity induced by SARS-CoV-2 it is important to understand for how long antibodies persist after infection in humans. Here, we assessed the persistence of serum antibodies following WT SARS-CoV-2 infection at 8 and 13 months after diagnosis in 367 individuals. The SARS-CoV-2 spike IgG (S-IgG) and nucleoprotein IgG (N-IgG) concentrations and the proportion of subjects with neutralizing antibodies (NAb) were assessed. Moreover, the NAb titers among a smaller subset of participants (n = 78) against a WT virus (B) and variants of concern (VOCs): Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) were determined. We found that NAb against the WT virus persisted in 89% and S-IgG in 97% of subjects for at least 13 months after infection. Only 36% had N-IgG by 13 months. The mean S-IgG concentrations declined from 8 to 13 months by less than one third; N-IgG concentrations declined by two-thirds. Subjects with severe infection had markedly higher IgG and NAb levels and are expected to remain seropositive for longer. Significantly lower NAb titers against the variants compared to the WT virus, especially after a mild disease, suggests reduced protection against VOCs.

Multilaboratory Comparison of Pneumococcal Multiplex Immunoassays Used in Immunosurveillance of Streptococcus pneumoniae across Europe.

Surveillance studies are required to estimate the impact of pneumococcal vaccination in both children and the elderly across Europe. The World Health Organization (WHO) recommends use of enzyme immunoassays (EIAs) as standard methods for immune surveillance of pneumococcal antibodies. However, as levels of antibodies to multiple serotypes are monitored in thousands of samples, a need for a less laborious and more flexible method has evolved. Fluorescent-bead-based multiplex immunoassays (MIAs) are suitable for this purpose. An increasing number of public health and diagnostic laboratories use MIAs, although the method is not standardized and no international quality assessment scheme exists. The EU Pneumo Multiplex Assay Consortium was initiated in 2013 to advance harmonization of MIAs and to create an international quality assessment scheme. In a multilaboratory comparison organized by the consortium, agreement among nine laboratories that used their own optimized MIA was assessed on a panel of 15 reference sera for 13 pneumococcal serotypes with the new WHO standard 007sp. Agreement was assessed in terms of assay accuracy, reproducibility, repeatability, precision, and bias. The results indicate that the evaluated MIAs are robust and reproducible for measurement of vaccine-induced antibody responses. However, some serotype-specific variability in the results was observed in comparisons of polysaccharides from different sources and of different conjugation methods, especially for serotype 4. On the basis of the results, the consortium has contributed to the harmonization of MIA protocols to improve reliability of immune surveillance of Streptococcus pneumoniaeIMPORTANCE Serology of Streptococcus pneumoniae is challenging due to existence of multiple clinically relevant serotypes and the introduction of multivalent vaccines in national immunization programs. Multiplex immunoassays (MIAs) are applied as high-throughput cost-effective methods for serosurveillance, and yet laboratories use their own protocols. The aims of this study were to assess the agreement of results generated by MIAs in different laboratories within the EU Pneumo Multiplex Assay Consortium, to analyze factors contributing to differences in outcome, and to create a harmonized protocol. The study demonstrated good agreement of results of MIAs performed by laboratories using controlled assays for determination of levels of vaccine-induced pneumococcal antibodies. The EU Pneumo Multiplex Assay Consortium is open to everyone working in public health services, and it aims to facilitate efforts by participants to run and maintain a cost-effective, reproducible, high-quality MIA platform.

Assessment of an Antibody-in-Lymphocyte Supernatant Assay for the Etiological Diagnosis of Pneumococcal Pneumonia in Children.

New diagnostic tests for the etiology of childhood pneumonia are needed. We evaluated the antibody-in-lymphocyte supernatant (ALS) assay to detect immunoglobulin (Ig) G secretion from ex vivo peripheral blood mononuclear cell (PBMC) culture, as a potential diagnostic test for pneumococcal pneumonia. We enrolled 348 children with pneumonia admitted to Patan Hospital, Kathmandu, Nepal between December 2015 and September 2016. PBMCs sampled from participants were incubated for 48 h before harvesting of cell culture supernatant (ALS). We used a fluorescence-based multiplexed immunoassay to measure the concentration of IgG in ALS against five conserved pneumococcal protein antigens. Of children with pneumonia, 68 had a confirmed etiological diagnosis: 12 children had pneumococcal pneumonia (defined as blood or pleural fluid culture-confirmed; or plasma CRP concentration ≥60 mg/l and nasopharyngeal carriage of serotype 1 pneumococci), and 56 children had non-pneumococcal pneumonia. Children with non-pneumococcal pneumonia had either a bacterial pathogen isolated from blood (six children); or C-reactive protein <60 mg/l, absence of radiographic consolidation and detection of a pathogenic virus by multiplex PCR (respiratory syncytial virus, influenza viruses, or parainfluenza viruses; 23 children). Concentrations of ALS IgG to all five pneumococcal proteins were significantly higher in children with pneumococcal pneumonia than in children with non-pneumococcal pneumonia. The concentration of IgG in ALS to the best-performing antigen discriminated between children with pneumococcal and non-pneumococcal pneumonia with a sensitivity of 1.0 (95% CI 0.73-1.0), specificity of 0.66 (95% CI 0.52-0.78) and area under the receiver-operating characteristic curve (AUROCC) 0.85 (95% CI 0.75-0.94). Children with pneumococcal pneumonia were older than children with non-pneumococcal pneumonia (median 5.6 and 2.0 years, respectively, p < 0.001). When the analysis was limited to children ≥2 years of age, assay of IgG ALS to pneumococcal proteins was unable to discriminate between children with pneumococcal pneumonia and non-pneumococcal pneumonia (AUROCC 0.67, 95% CI 0.47-0.88). This method detected spontaneous secretion of IgG to pneumococcal protein antigens from cultured PBMCs. However, when stratified by age group, assay of IgG in ALS to pneumococcal proteins showed limited utility as a test to discriminate between pneumococcal and non-pneumococcal pneumonia in children.

Comparison of serological assays using pneumococcal proteins or polysaccharides for detection of Streptococcus pneumoniae infection in children with community-acquired pneumonia.

The aim of this study was to compare the results of serological assays using pneumococcal proteins or polysaccharides for the detection of pneumococcal infection in childhood pneumonia. Serological assays measured IgG against eight pneumococcal proteins (Ply,CbpA,PspA1,PspA2,PcpA,PhtD,StkP-C,PcsB-N), C-polysaccharide [in the whole study population, n = 183], or 19 pneumococcal capsular polysaccharides (1,2,4,5,6B,7F,8,9 V,10A,11A,12F,14,15B,17F,18C,19F,20,23F,33F) [only in a subgroup of patients, n = 53] in paired serum samples of children aged <5 years-old hospitalized with clinical and radiological diagnosis of community-acquired pneumonia. We also performed an inhibition of binding test with the anti-capsular polysaccharide assay in order to confirm the specificity of the antibody responses detected. Invasive pneumococcal pneumonia was investigated by blood culture and PCR (ply-primer). Among 183 children, the anti-protein assay detected antibody response in 77/183(42.1%) patients and the anti-C-polysaccharide assay in 28/183(15.3%) patients. In a subgroup of 53 children, the anti-protein assay detected response in 32/53(60.4%) patients, the anti-C-polysaccharide assay in 11/53(20.8%) patients, and the anti-capsular polysaccharide in 25/53(47.2%) patients. Simultaneous antibody responses against ≥2 different capsular polysaccharides were detected in 11/53(20.8%) patients and this finding could not be explained by cross-reactivity between different serotypes. Among 13 patients with invasive pneumococcal pneumonia, the sensitivity of the anti-protein assay was 92.3%(12/13), of the anti-C-polysaccharide assay 30.8%(4/13), and of the anti-capsular polysaccharide assay 46.2%(6/13). The serological assay using pneumococcal proteins is more sensitive for the detection of pneumococcal infection in children with pneumonia than the assay using pneumococcal polysaccharides. Future studies on childhood pneumonia aetiology should consider applying serological assays using pneumococcal proteins.

A fluorescent multiplexed bead-based immunoassay (FMIA) for quantitation of IgG against Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis protein antigens.

Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are pathogens commonly associated with infectious diseases in childhood. This study aimed to develop a fluorescent multiplexed bead-based immunoassay (FMIA) using recombinant proteins for the quantitation of serum IgG antibodies against these bacteria. Eight pneumococcal proteins (Ply, CbpA, PspA1, PspA2, PcpA, PhtD, SP1732-3 and SP2216-1), 3 proteins of H. influenzae (NTHi Protein D, NTHi0371-1, NTHi0830), and 5 proteins of M. catarrhalis (MC Omp CD, MC_RH4_2506, MC_RH4_1701, MC_RH4_3729-1, MC_RH4_4730) were used to develop the FMIA. Optimal coupling concentrations for each protein, comparison of singleplex and multiplex assays, specificity, reproducibility, and correlation to ELISA for six pneumococcal antigens were determined for validation. FMIA was then used to analyze acute and convalescent paired serum samples of 50 children with non-severe pneumonia. The coupling concentrations varied for different antigens, ranging from 1.6 to 32µg of protein/million beads. Correlation between singleplexed and multiplexed assays was excellent, with R≥0.987. The FMIA was specific, reaching >92% homologous inhibition for all specificities; heterologous inhibition ≥20% was found only in six cases. The assay was repeatable, with averages of intra-assay variation ≤10.5%, day-to-day variation ≤9.7% and variation between technicians ≤9.1%. Comparison with ELISA for pneumococcal antigens demonstrated good correlation with R ranging from 0.854 (PspA2) to 0.976 (PcpA). The samples from children showed a wide range of antibody concentrations and increases in convalescent samples. In conclusion, the FMIA was sensitive, specific, and repeatable, using small amounts of recombinant proteins and sera to detect antibodies against S. pneumoniae, H. influenzae and M. catarrhalis. The methodology would be suitable for studies investigating etiological diagnosis and in experimental vaccine studies.

Concentration and high avidity of pneumococcal antibodies persist at least 4 years after immunization with pneumococcal conjugate vaccine in infancy.

To provide more extensive evidence of long-term effects of vaccination on immunity against Streptococcus pneumoniae, a follow-up study of the Finnish Otitis Media (FinOM) Vaccine Trial was conducted. One of the objectives was to assess the persistence and avidity of pneumococcal antibodies 4 years after pneumococcal vaccination given in infancy. Children with complete follow-up in the FinOM trial up to 24 months of age were invited to a single visit in their fifth year of life. A blood sample was taken from all children for determination of anticapsular antibody concentrations to vaccine serotypes and avidity of antibodies to three serotypes. Children had been vaccinated at 2, 4, 6, and 12 months of age with 7-valent pneumococcal capsular polysaccharide, CRM197 conjugate vaccine (PCV7), or a control vaccine. Serum IgG antibody concentrations to vaccine serotypes remained significantly higher in children who had received PCV7 than in control children for 4 years after the fourth PCV7 dose. Concentrations of antibodies to frequently carried serotypes (6B and 19F) declined less than those of antibodies to a rarely carried serotype (4), suggesting that natural boosting contributed to antibody persistence. Furthermore, antibody avidity was significantly higher in PCV7 than control vaccine recipients. Four doses of PCV7 given in infancy elicit long-lasting antibody responses with high avidity. (This study has been registered at ClinicalTrials.gov under registration no. NCT00378417.).

Serum IgM antibodies contribute to high levels of opsonophagocytic activities in toddlers immunized with a single dose of the 9-valent pneumococcal conjugate vaccine.

In immunogenicity trials of pneumococcal conjugate vaccines (PCVs), only IgG antibody concentrations to pneumococcal capsular polysaccharides (PPSs) are usually determined, along with the opsonophagocytic activity (OPA) of antipneumococcal antibodies. We aimed to determine the role of both IgG and IgM in OPA in toddlers receiving one dose of 9-valent PCV (PCV9). The IgG and IgM antibody concentrations to PPSs of serotypes 6A, 9V, 14, 19F, and 23F were measured by enzyme immunoassay in sera from toddlers (ages 18 to 35 months) 1 month after a single PCV9 dose. The OPA for the same serotypes was measured by multiplexed opsonophagocytosis assay (MOPA). Further, IgG and IgM concentrations and MOPA were measured to PPS of serotypes 6A, 14, and 19F in sera collected 12 months after vaccination. The detected MOPA titers were high in comparison to the IgG concentrations 1 month after immunization. The IgM concentrations were higher than IgG concentrations for serotypes 6A and 14 (P < 0.001) and as high as IgG for serotypes 9V, 19F, and 23F. Correlation of the IgM antibody concentrations with MOPA (r = 0.35 to 0.65) was stronger compared to that of the IgG antibodies (r = 0.07 to 0.41). The depletion of IgG antibodies in three sets of pooled sera only slightly decreased the OPA activity against serotype 14. At 12 months after immunization, 50 to 100% of serum samples still showed detectable MOPA activity against serotypes 6A, 14, and 19F. Our results suggest that IgM contributes to OPA 1 month after a single PCV9 vaccination in toddlers and that functionally active IgM and IgG antibodies persist for at least a year.

Altered phenotype of peripheral blood dendritic cells in pediatric type 1 diabetes.

OBJECTIVE: Dendritic cells (DCs) are largely responsible for the activation and fine-tuning of T-cell responses. Altered numbers of blood DCs have been reported in type 1 diabetes (T1D). We aimed at characterizing the less well-known phenotypic properties of DCs in T1D. RESEARCH DESIGN AND METHODS: In a case-control setting, samples from a total of 90 children were studied by flow cytometry or by quantitative real-time PCR (qPCR). RESULTS: We found decreased numbers of myeloid DCs (mDCs) (8.97 vs. 13.4 cells/µL, P = 0.009, n = 31) and plasmacytoid DCs (pDCs) (9.47 vs. 14.6 cells/µL, P = 0.018, n = 30) in recent-onset T1D. Using a panel of antibodies against functionally important DC markers, we detected a decreased expression of CC chemokine receptor 2 (CCR2) on mDCs (percentage above negative control, P = 0.002, n = 29) and pDCs (median intensity, P = 0.003, n = 30) from T1D patients. In an independent series of children, the reduced expression of CCR2 was confirmed by qPCR in isolated mDCs (P = 0.043, n = 20). Serum concentrations of CCR2 ligands monocyte chemotactic protein-1 and -3 did not differ between the groups. A trend for an enhanced responsiveness of the nuclear factor-κB pathway (P = 0.063, n = 39) was seen in mDCs from children with ß-cell autoantibodies, which is possibly related to the reduced CCR2 expression, since CCR2 on mDCs was downregulated by nuclear factor-κB-activating agents. CONCLUSIONS: Given the role of CCR2 in DC chemotaxis and in DC-elicited Th1 differentiation, our results may indicate a functionally important DC abnormality in T1D affecting the initiation and quality of immune responses.

Aging reduces the functionality of anti-pneumococcal antibodies and the killing of Streptococcus pneumoniae by neutrophil phagocytosis.

We evaluated the effect of aging on the functional activity of naturally acquired anti-pneumococcal antibodies, the function of neutrophils in phagocytic killing of opsonized pneumococci, and the complement activity. Opsonic activities of antibodies to all tested pneumococcal serotypes were significantly lower and phagocytic killing of pneumococci by neutrophils was significantly impaired among the elderly, whereas the complement activity was slightly higher in the elderly than in the young adults. The reduced functional activity of serotype-specific antibodies and the compromised function of neutrophils in the opsonophagocytosis of pneumococci are likely to contribute to the increased susceptibility of the elderly to pneumococcal diseases.

Multilaboratory comparison of Streptococcus pneumoniae opsonophagocytic killing assays and their level of agreement for the determination of functional antibody activity in human reference sera.

Antibody-mediated killing of Streptococcus pneumoniae (pneumococcus) by phagocytes is an important mechanism of protection of the human host against pneumococcal infections. Measurement of opsonophagocytic antibodies by use of a standardized opsonophagocytic assay (OPA) is important for the evaluation of candidate vaccines and required for the licensure of new pneumococcal conjugate vaccine formulations. We assessed agreement among six laboratories that used their own optimized OPAs on a panel of 16 human reference sera for 13 pneumococcal serotypes. Consensus titers, estimated using an analysis-of-variance (ANOVA) mixed-effects model, provided a common reference for assessing agreement among these laboratories. Agreement was evaluated in terms of assay accuracy, reproducibility, repeatability, precision, and bias. We also reviewed four acceptance criterion intervals for assessing the comparability of protocols when assaying the same reference sera. The precision, accuracy, and concordance results among laboratories and the consensus titers revealed acceptable agreement. The results of this study indicate that the bioassays evaluated in this study are robust, and the resultant OPA values are reproducible for the determination of functional antibody titers specific to 13 pneumococcal serotypes when performed by laboratories using highly standardized but not identical assays. The statistical methodologies employed in this study may serve as a template for evaluating future multilaboratory studies.

Functional antibodies elicited by two heptavalent pneumococcal conjugate vaccines in the Finnish Otitis Media Vaccine Trial.

In the Finnish Otitis Media Vaccine Trial, the now-licensed pneumococcal conjugate vaccine containing polysaccharides conjugated to protein CRM(197) (PncCRM) and the experimental pneumococcal polysaccharide-meningococcal outer membrane protein complex conjugate vaccine (PncOMPC), showed similar efficacy profiles against acute otitis media despite different antibody concentrations in sera. We now report the opsonophagocytic activities (OPA) in these sera. OPA, antibody concentration, and avidity for serotypes 6B, 19F, and 23F were determined in sera of infants who received either pneumococcal conjugate (PCV) or control vaccine at 2, 4, and 6 months of age and either the homologous or pneumococcal polysaccharide vaccine at 12 months of age. OPA varied by vaccine and serotype. The majority of PCV recipients had positive OPA after the fourth dose, while OPA was undetectable in the control group. Coinciding with the efficacy data, the concentration of antibodies required for 50% killing was low for 6B and high for 19F for both PCVs. Contradictory to the efficacy data, PncOMPC induced lower functional capacity to 23F than PncCRM. OPA correlated with antibody concentration, while avidity and functional capacity of antibodies showed no correlation. The OPA data provide valuable additional information for serotype-specific differences in protection and when evaluating serotype-specific immunogenicity and should thus be considered when defining serological correlates of protection.

Functional activity of antibodies after immunization of Finnish and Israeli infants with an 11-valent pneumococcal conjugate vaccine.

Finnish and Israeli infants received an 11-valent mixed carrier pneumococcal conjugate vaccine (11PCV) with or without aluminum adjuvant at the age of 2, 4, 6, and 12 months. We measured opsonophagocytic activity (OPA) of antibodies to pneumococcal strains of serotypes 4, 6B, 14, 19F, and 23F. At 7 months, OPA was clearly detected for all the serotypes. At 13 months, OPAs increased further and the proportion of individuals with a positive OPA ranged between 81 and 100%. The adjuvant improved functional activity of antibodies to serotype 6B pneumococci. In conclusion, immunization of infants with the 11PCV induced functionally active antibodies.

Kinetics and avidity of antibodies evoked by heptavalent pneumococcal conjugate vaccines PncCRM and PncOMPC in the Finnish Otitis Media Vaccine Trial.

The licensure of new pneumococcal conjugate vaccines (PCVs) relies on immunogenicity data. When defining correlates of protection, vaccine efficacy data must be included. In the FinOM Vaccine Efficacy Trial, the PncOMPC vaccine showed an efficacy profile similar to that of the licensed PncCRM vaccine despite different antibody responses after primary and booster vaccinations. We determined antibody kinetics and avidities in a subgroup of infants participating in the FinOM trial. A total of 166 infants in three vaccine groups were immunized at 2, 4, 6, and 12 months of age with 7-valent PCV, PncCRM or PncOMPC, or hepatitis B vaccine. Concentrations of serum immunoglobulin G (IgG) against pneumococcal capsular polysaccharides were determined at 2, 4, 6, 7, 12, 13, and 24 months of age, and the avidity index (AI) to serotypes 6B, 19F, and 23F were determined at 7, 12, 13, and 24 months of age by enzyme immunoassay. Both PCVs were highly immunogenic, but they demonstrated different kinetics of antibody response; the concentration of IgG against serotypes 6B, 19F, and 23F declined faster after the third and fourth doses of vaccine in the PncCRM group than in the PncOMPC group. For both PCVs, the mean AI of anti-6B and -23F, but not of anti-19F, increased during the follow-up, which is in line with serotype-specific protection in the FinOM trial. Our data suggest that the kinetics and avidities of antibodies should be considered, in addition to antibody responses, when defining correlates of protection.

Long-term persistence of immunity after immunisation with Haemophilus influenzae type b conjugate vaccine.

Although Haemophilus influenzae type b (Hib) conjugate vaccines, after licensure in 1987, are now recommended for world-wide use, the duration of protective immunity afforded by them is not known. We therefore assessed the immunogenity at 9-10 years of age in 37 children who had received the first Hib conjugate, PRP-D, in infancy (the Hib-conjugate group) and were now given a dose of Hib polysaccharide (PS) as a test vaccine. The anti-Hib PS antibodies (Hib-ab) were measured before and after this test vaccination, and the values compared to those in 37 control children who had not previously received any Hib vaccine and in 13 children who had received Hib PS vaccine in infancy (the Hib-PS group). Prior to the test vaccination, the Hib-ab concentrations in the Hib-conjugate group were 3.6-fold higher than in the control group. After the test vaccination, the Hib-conjugate group had higher total Hib-ab concentrations, higher proportion of IgG and higher avidity of Hib-ab than the control or the Hib-PS group, suggesting persisting immunological memory in a Hib-c group. A mathematical model, including memory, predicted accurately the Hib-ab concentrations, which are maintained through anamnestic responses to intervening stimuli (Hib or cross-reacting bacteria).