Correlation of Higher Antibody Levels to Pneumococcal Proteins with Protection from Pneumococcal Acute Otitis Media but not Protection from Nasopharyngeal Colonization in Young Children

Abstract

Objectives

We previously found that nasopharyngeal (NP) colonization by Streptococcus pneumoniae (Spn) elicits mucosal antibody responses to three protein vaccine candidates: pneumococcal histidine triad protein D (PhtD), pneumococcal choline binding protein A (PcpA), and detoxified pneumolysin (PlyD1). Here we sought to determine if mucosal antibody levels to the proteins correlated with protection from acute otitis media (AOM) and NP colonization.

Methods

228 NP samples were prospectively collected from 100 healthy infants at 6–24 months of age. Whenever children were diagnosed with AOM, middle ear fluids were collected to confirm the diagnosis by microbiologic culture. NP mucosal IgG and IgA were quantified by ELISA.

Results

Higher NP mucosal antibody levels to Spn proteins correlated with significantly decreased likelihood of developing AOM caused by Spn during 3 to 12 months of subsequent prospective monitoring. Specifically, children who did not experience AOM (n=111 samples) caused by Spn had 2-5-fold higher mucosal IgG levels to PcpA (all p values <0.01), 6-8-fold higher IgA to PhtD (all p values <0.05); 2-3-folder higher IgA to PcpA (all p values <0.05), and 2-3-fold higher IgA to PlyD1 (p=0.08, 0.03 and 0.08) compared with children who did experience AOM (n=18 samples). No association between mucosal antibody levels to the three proteins and NP colonization with Spn was found.

Conclusion

Higher NP mucosal IgG levels to PcpA, and IgA to PhtD, PcpA and PlyD1 correlate with reduced risk of development of Spn AOM infection but not with reduced risk of NP colonization in young children.

INTRODUCTION

Current licensed pneumococcal vaccines, including the 23-valent pneumococcal polysaccharide vaccine, and 7-, 10- and 13-valent pneumococcal conjugate vaccines (PCV-7, -10, -13) are serotype (polysaccharide)-based vaccines that protect against Streptococcus pneumoniae (Spn) infections caused by strains expressing the included serotypes. To date, there are 97 distinct serotypes according to capsular polysaccharide composition [29]. Within a few years of introduction of each of the PCVs, emergence of non-vaccine replacement serotypes has been noted in numerous studies [29, 10, 21]. Therefore, we and others have been evaluating next-generation purified pneumococcal protein vaccines that will be composed of highly conserved proteins expressed by virtually all Spn [10, 21]. We have studied three pneumococcal proteins: histidine triad protein D (PhtD), pneumococcal choline binding protein A (PcpA), and pneumolysin (Ply) and have shown that natural exposure to Spn following nasopharyngeal (NP) colonization elicits both serum and mucosal antibody responses in young children [22, 31]. We have also shown that vaccination with monovalent and trivalent vaccines containing PhtD, PcpA or detoxified Ply (PlyD1) and a combination of these three proteins confers protection against pneumonia and sepsis in a mouse model [33]. Our work and that of others has provided sufficient promise for the potential of a trivalent PhtD, PcpA, and PlyD1 vaccine that has entered human clinical trials [13, 6].

Pneumococcal protein vaccines have the potential to prevent Spn infections by all strains irrespective of the capsular serotype expressed. However, if pneumococcal protein vaccines, like PCVs, completely eliminate Spn NP colonization then a concern arises regarding the potential for providing a vacant niche that might be filled by other invasive bacteria such as Staphylococcus aureus [18]. We recently reported that higher mucosal antibody levels to PhtD, PcpA and PlyD1 in the NP was associated with reduced AOM caused by Spn in young children at onset of AOM [31]. Here we sought to determine if mucosal antibody levels to these three proteins correlated with protection from future risk of episodes of Spn AOM and/or Spn NP colonization over defined time spans.

METHODS

Study cohort and sample collection

This study derives from a cohort of children prospectively enrolled during a 10-year time span (2006–2015) to evaluate immunity to Spn and non-typeable Haemophilus influenzae (NTHi) NP colonization and AOM in young children. The subject enrollments, samples collections, and AOM diagnosis criteria have been described previously [22, 31]. Briefly, healthy infants without previous episodes of AOM were enrolled at 6 months of age in a private pediatric practice in Rochester, NY. NP swabs and nasal wash (NW) samples were collected at 7 prospective visits of children at 6, 9, 12, 15, 18, 24 and 30–36 months of age. Whenever the children were diagnosed with AOM, tympanocentesis was performed and middle ear fluid (MEF) samples collected to confirm the diagnosis with microbiologic culture for otopathogens. Identification of the major bacterial AOM pathogens was determined by standard culture methodology. All of the children received routine vaccinations according to the U.S. schedule including PCV-7- or -13 (Prevnar, Wyeth Pharmaceuticals, Collegeville, PA) at the appropriate age. The study was approved by the Institutional Review Board (IRB) of Rochester General Hospital, and written informed consent was obtained from parents or guardians of all children.

From 589 eligible children we randomly selected 100 children to assess the correlation of NP mucosal IgA and IgG antibody titers to PhtD, PcpA and PlyD1 with AOM incidence; the sample size was based on statistical power calculations based on our prior work [31]. The average age of the 100 children was 14.5 ± 5.9 months and 37, 32, 42, 36, 40, 39 samples were tested when children were age at 6, 9, 12, 15,18 and 24 months of age, respectively. The characteristics of the children are summarized in Table 1.

Table 1.

Characteristics of study cohorts

	All children enrolled N (%)	Children for ELISA N (%)
Total	589 (100%)	100(100%)
Female	282 (48%)	47(47%)
Male	307 (52%)	53(53%)
White	401 (68%)	68 (68%)
Non-white	188 (32%)	32(32%)
Breast feeding > 6M	241 (41%)	40(40%)
Formula or breast feeding < 6M	348 (59%)	60(60%)
Daycare	164 (28%)	29(29%)
Non-daycare	425 (72%)	71(71%)
Otitis-prone (OP) children*	47 (8%)	7(7%)
Non-OP children	542 (92%)	93(93%)

^*Children experiencing 3 episodes within 6 months or 4 episodes in 12 months

NW samples from the prospective visits were analyzed for IgG and IgA antibody to Spn proteins as described below. Then we determined if an episode of AOM or NP colonization caused by Spn occurred during the subsequent 3, 6, or 12 months of follow up. In this way a measurement of NP mucosal antibody could be correlated with future risk of AOM or NP colonization by Spn over defined time spans.

ELISA

Pneumococcal protein-specific IgG and IgA antibody, and total IgG and IgA were determined in the NW by quantitative ELISA, and results expressed as geometric means (GM) with 95% confidence intervals (CI) of ratios of specific IgG to total IgG or specific IgA to total IgA (ng/µg) as previously described [31]. Samples with a total IgG or IgA < 0.05 µg/ml were excluded because in preliminary studies we determined that such samples were from children with a difficult or failed sampling process, and had undetectable antigen-specific antibodies.

Statistics

Statistical analysis was performed with R Project version 2.13.2 and graphs were made with GraphPad Prism 6.0. For the purpose of statistical analysis, samples with undetectable specific antibody were arbitrarily assigned a value equivalent to half the lower limit of detection of corresponding specific antibodies. Antibody levels between groups were compared using two-tailed Mann-Whitney Test. Because antibodies were found to correlate significantly with age, antibody levels were adjusted for age using a GEE model, assuming a within-subject auto-regressive correlation. Association of NP antibody levels with future Spn colonization and Spn AOM was confirmed using the Regression Estimating Wald Test. P < 0.05 was considered statistically significant.

RESULTS

Comparison of NP mucosal antibody levels to pneumococcal proteins between children did and did not develop AOM infection caused by Spn

Among 228 samples, 116 were collected from children who had Spn colonization detected and 112 were collected from children who did not have Spn colonization detected. Overall, the children with detected colonization had less AOM infections in the following 12 months of prospective monitoring (21% (24/116) than children who had not had detected colonization (35% (39/112), p=0.02), indicating that prior colonization led to less AOM infections as expected since colonization is an immunizing event [22].

Since we have previously shown that Spn colonization elicits significant mucosal antibody responses to pneumococcal proteins [31], we used the samples that had Spn colonization detected to further analyze the correlation of mucosal antibody levels and incidence of AOM. We compared NW IgG and IgA levels to PhtD, PcpA and PlyD1 of children that did and did not have at least one episode of AOM in the following 3 to 12 months of prospective monitoring. We found that children with no AOM infections during prospective monitoring had significantly higher geometric mean (GM) mucosal levels of IgG to PcpA (Fig. 1C) and IgA levels to PhtD and PcpA at all three time points, and IgA levels to PPlyD1 at 6 months of prospective monitoring (Fig 1B, D, F) compared to children who developed AOM infections caused by Spn. GM IgG levels in the children without Spn AOM infections at time points of 3, 6 and 12 months of prospective monitoring had 4.4-fold (0.48 ng/µg, p = 0.002), 4.8-fold (0.43 ng/µg, p = 0.0002), and 3.9-fold (0.53 ng/µg, p = 0.0005) higher levels of GM antibody levels to PcpA than the children who developed Spn AOM infections (2.09 ng/µg) (Fig 1C). No significant difference was found in GM of IgG levels to PhtD and PlyD1 between children without Spn AOM and those with Spn AOM infection (all p values >0.05) (Fig.1A, 1E). The GM IgA levels to PhtD for children without Spn AOM infection at time points of 3, 6 and 12 months of prospective monitoring were 6.0-fold (0.96 ng/µg, p = 0.013), 7.8-fold (0.74 ng/µg, p = 0.0003) and 7.2-fold (0.80 ng/µg, p<0.0001) higher, respectively, than GM levels of children who developed an Spn AOM infection (5.78 ng/µg) (Fig 1B). The GM IgA levels to PcpA for children without Spn AOM infection at time points of 3, 6 and 12 months were 2.6-fold (1.01 ng/µg, p = 0.04), 3.4-fold (0.77 ng/µg, p = 0.006) and 2.8-fold (0.92 ng/µg, p = 0.004) higher, respectively, than children who developed an Spn AOM infection (2.59 ng/µg) (Fig 1D). GM IgA levels to PlyD1 among children without Spn AOM infection at time points of 3, 6 and 12 months of prospective monitoring were 2.5-fold (0.15 ng/µg, p = 0.08), 2.8-fold (0.13 ng/µg, p = 0.03), and 2.2-fold (0.17 ng/µg, p = 0.08) higher than children who developed an Spn AOM infection (0.37 ng/µg) (Fig 1F).

Figure 1. Mucosal antibody levels were negatively associated with possibility of future episode of Spn AOM.

NP wash samples were collected from healthy children at 6–24 months of age. Antibody levels were determined by quantitative ELISA and expressed as the ratios of pneumococcal specific to total IgG and total IgA and compared between children who had at least one future episode of AOM in 3, 6 and 12 months later and children who did not have any AOM in entire study period. A, anti-PhtD IgG; B, anti-PhtD IgA; C, anti-PcpA IgG; D, anti-PcpA IgA; E, anti-PlyD1 IgG; F, anti-PlyD1 IgA.

Comparison of NP mucosal antibody levels to pneumococcal proteins between children did and did not have Spn colonization

We also compared GM of NP mucosal IgG and IgA antibody levels to PhtD, PcpA and PlyD1 in children who did and did not have Spn colonization during prospective monitoring (Fig 2). We did not observe a correlation of antibody levels with Spn colonization events. There were no differences in GM of either IgG or IgA to any of the three proteins for children who did and did not have Spn colonization events within 3 (Fig. 2A-B) and 6 (Fig.2C-D) months of prospective monitoring (all p values >0.05) except IgG to PhtD (Fig.2A).

Figure 2. Mucosal antibody levels were not associated with prevention of future Spn colonization.

NP wash samples were collected from healthy children at 6–24 months of age. Antibody levels were determined by quantitative ELISA, expressed as the ratios of pneumococcal specific to total IgG and total IgA and compared between children who had future Spn colonization and children who did not have future Spn colonization in 3 and 6 months later. Lines represent GM with 95% CI. A, 3 month, IgG; B, 3 month, IgA; C, 6 month, IgG; D, 6 month, IgA.

DISCUSSION

Mucosal immunity plays a crucial role in prevention of respiratory tract infections. NP colonization with Spn as an immunizing event elicits systemic and mucosal antibody responses to Spn proteins as we reported previously [22, 31] as have others [27, 23]. Here for the first time we show that significantly higher mucosal IgG levels to Spn protein vaccine candidates induced by NP colonization correlate with protection against Spn AOM infections but that these same antibody levels do not protect against NP colonization.

It is widely accepted that colonization in the NP is a necessary pre-requisite for development of Spn infections. The concept of a disassociation between antibody levels that protect against infection but do not prevent NP colonization is new [21]. Up to now, it was presumed that protection from Spn infections by pneumococcal protein vaccines would be the same as PCVs, demonstrating a strong correlation with induction of high serum and mucosal antibodies that provide protection from acquisition of NP colonization and infection. Elimination from the NP of strains of Spn expressing the capsular types included in the vaccines by PCVs provides herd protection thereby enhancing the cost effectiveness of vaccination. How can the novel results of this work showing a positive effect of higher mucosal anti- Spn protein antibody levels on reduced development of infection but no effect on detection of Spn colonization be understood?

We have new findings that point to an explanation. We have recently conducted studies in a mouse model of Spn/Influenzae virus co-infection in the upper-airways and found that vaccination with PhtD does not eliminate Spn colonization in the upper-airways but does prevent the bacterial load of Spn from exceeding a pathogenic threshold to cause invasive Spn infection [15]. Thus, anti-Spn protein antibody may be able to prevent escalated growth of Spn in the NP during viral URI and subsequent dissemination to the middle ear to cause AOM, but not completely eliminate colonization of the organism in the NP. Our work in mice showed that a reduction in Spn load in the NP of about 1 log was sufficient to result in protection from infection

Bacterial loads and viral infection are very important for pathogenesis of Spn infections [19, 7]. The pathogenesis of AOM begins in the upper-airways and is triggered by a viral URI co-infection that facilitates the transition of asymptomatic bacterial carriage to an infection [20]. Spn is most often a commensal in the respiratory tract [5, 16]. During health, Spn colonization is asymptomatic and tolerogenic [4]. It elicits a mild NP epithelial inflammation that allows controlled immune surveillance in the NP, leading to the clearance of colonization without developing immunopathology [34]. Viral infection triggers pathogenesis [19, 7]. Prior to Spn AOM infections, virtually all children and adults have a viral respiratory co-infection [19]. When a viral infection occurs, an inflammatory response induced by the viral co-infection promotes growth and transmission of Spn if the organism is alreadypresent in the NP prior to viral infection [24, 25], or facilitates colonization and escalation of growth of new colonized Spn post viral infection [26]. Higher Spn and viral loads in the NP are positively associated with development of AOM [3]. When the loads reach a threshold that the host physiological immune response cannot contain, Spn disseminates to middle ear, lung and other sterile sites to cause Spn infection.

We found that antibody-dependent immunity is an important mechanism to prevent pneumococcal infections. Our previous studies in a mouse model showed that antibody responses to pneumococcal cell surface proteins enhance phagocytosis by neutrophils during early pathogenesis after Spn infection, resulting in an increase of bacterial clearance [33]. We previously reported in children that natural colonization by Spn as an immunization event elicits serum and mucosal antibody response to pneumococcal proteins [32, 22, 31]. Both naturally-occurring and vaccine-induced anti-pneumococcal protein antibodies confer passive protection against Spn infection [9]. Antibody-mediated inhibition of Spn adherence to epithelial cells is one mode of protection [14]. We did not study a role of cellular immune responses in preventing Spn infection. Wilson, et al. have shown that both humoral and cellular immune responses are required to protect against Spn infection [30], Antibody-independent immunity such as CD4-mediated and IL-17-mediated T-cell response and TLR-mediated innate responses may play critical roles in protection against Spn infection [28, 2, 12, 30, 11, 8, 15].

It should be noted that high levels of IgA to the studied proteins were also observed in children with AOM (Figure 1). Also, in the case of anti-PhtD IgA, there seems to be a bi-modal distribution of IgA levels in all three AOM groups. The results suggest that lack of previous exposure to Spn (resulting in low antibody levels) as well as lack of existing Spn colonization (high antibody levels in absence of Spn carriage) may both be associated with children progressing to AOM. The latter explanation cannot be excluded taking into consideration results of the study published by Auranen [1].

We observed that mucosal IgG levels (p=0.004) were higher in children who had future Spn colonization within 3 months compared to those who did not (p=0.004 in anti-PhtD IgG, p=0.09 in anti-PlyD1 IgG and p=0.22 in anti-PcpA IgG). This phenomenon can be explained by the fact that NP colonization often persisted despite measurable mucosal antibody. We analyzed the association of current and future colonization events of Spn in this cohort, and found that 58% of children who had an identified initial Spn colonization event were still colonized by Spn three months later. Among children who were still colonized with Spn, 60% were colonized with the same serotype (data not shown).

In our previous study antibody levels were compared at onset of AOM versus healthy children and both IgG and IgA to the vaccine proteins correlated with protection from AOM. In this study we compared antibody levels before AOM versus healthy children and only IgA levels were consistently correlated with protection from AOM. We attribute this difference to events during pneumococcal pathogenesis. Specifically at onset to AOM there is a concurrent viral upper respiratory infection and the nasopharynx has a pro-inflammatory environment resulting in marked transudation of antibody from serum to the NP whereas between AOM events the NP is in homeostasis and transudation of IgG is minimal whereas locally-produced IgA predominates.

In the current study we used traditional qualitative cultures of Spn in the NP. This method would not detect bacterial load differences. Similar to our work, Prevaes, et al. used traditional qualitative cultures of Spn in the NP and reported that Spn colonization elicits serum antibody response to 14 pneumococcal surface proteins, but none of those antibodies were associated with a reduction in risk of subsequent carriage [23]. Lebon, et al. have found NP colonization induced serum antibody responses against 17 pneumococcal protein vaccine candidates, and higher antibody levels were associated with decreased number of respiratory tract infection, but not decreased asymptomatic colonization [17]. Both studies identified bacterial pathogens by traditional culture. A quantitative approach like qPCR for bacterial and viral loads would be needed to evaluate protection of anti Spn protein antibodies against NP colonization.

Our study has limitations. The NP colonization events in our study were defined by bacterial cultures at a moment in time when samples were collected at 6–24 months of age. Almost certainly there were children who had Spn NP colonization events before we commenced samplings at age 6 months and who had NP colonization events between the timing of scheduled samplings. We did not determine NP bacterial or viral loads which have proven to be critical variants influencing host immune responses [18, 3]. In addition, antibody levels in this study were ELISA titers. We did not measure functional antibody titers which are may be closely related with protection.

In summary, NP mucosal antibody levels to Spn proteins PhtD, PcpA and PlyD1 correlated with protection against AOM, but not with protection against NP colonization. We are pursuing additional new sample collections to study the correlation of mucosal antibody levels with Spn bacterial load and viral load under the hypothesis that protection against Spn infection occurs when mucosal antibody levels are sufficient to prevent disease pathogenesis during a viral URI but not sufficient to eliminate all Spn from the NP. Based on the results of this work, we propose that anti-Spn protein antibody may prevent escalated growth of Spn in the NP and subsequent dissemination to the lung or middle ear to cause pneumonia or AOM, even though such antibodies do not completely eliminate the organism from the NP. This may be a favorable result because complete elimination of Spn from the NP may leave behind an ecological niche that would be filled by other potentially pathogenic bacteria. Further study is needed to understand differences in antibody-mediated protection from Spn colonization induced by protein-based vaccines compared to glyconjugate vaccines since gylcoconjugate vaccines completely eliminate strains expressing the targeted capsular types from the NP.