Similar transfer experiments were also performed with spleen cells removed surgically from Ipups born from the earlier deliveries and injected into naïve mice, with the spleen cells from nonimmunized mice being used as a negative control for protection. One day after the transfer, the recipient mice were challenged with *B. pertussis*, and 7 days after the challenge the CFUs in the lungs

Figure 2 | Duration of protective immunity conferred by maternal immunization beyond the first pregnancy. (A) Schematic representation of the maternal vaccination and pup-challenge protocol. Female mice were vaccinated twice with commercial acellular pertussis (aP) at a 2-week interval; mated with male mice; and when the vaginal plug was detected, a third aP dose was administered. Non-immunized females were mated with male mice at the same time as the immunized females. The first birth was 3–4 weeks after the last immunization. Successive births occurred at intervals of 5–8 weeks. Mice born to aP-immunized or non-immunized females (the negative controls) were challenged with *Bordetella pertussis* at 4 weeks after birth. Mothers and pups were bled 1 day before pup challenge as indicated by the dotted vertical arrows. (B) Protection conferred to the offspring born in later pregnancies by maternal immunity acquired during the first pregnancy. Mice born to aP-immunized (Ipups *n* = 5) or non-immunized (Cpups *n* = 5) females were challenged with *B. pertussis* Tohama I 4 weeks after birth. The protection conferred to the offspring through maternal immunization during the first pregnancy was estimated by determining the number of bacteria recovered from mouse lungs. The bacterial counts expressed as the log10 of CFU per lungs, is plotted on the *ordinate* as a function of the number of births after the initial delivery indicated on the *abscissa*. \**p* < 0.001 Ipups versus Cpups. The anti-pertussis toxin (a-PTxA) IgG titers in the mothers and the offspring determined at the time of challenge are listed below the *abscissa*. The titers are expressed as the geometric mean of the data from each group (*n* = 5). *p* < 0.001 a-PTxA IgG titers in Ipups from the first and second deliveries versus those detected in Ipups from the third through the fifth deliveries. The results from one representative experiment are presented.

Figure 3 | Effect of passive immunization on protection through sera collected from Ipups. Pooled sera from either early pregnancy Ipups with high anti-pertussis toxin (a-PTxA) titer or late-pregnancy Ipups with low a-PTxA titer were tested by transfer to naive (Cpup *n* = 5) female mice. Pooled sera from Cpups transferred to naive female mice were used as negative control of protection. Twenty-four hours after transfer, the mice were infected with *Bordetella pertussis* and sacrificed 7 days later to determine the CFUs in the lungs. In the figure, the number of bacteria recovered from mouse lungs, expressed as the log10 of CFUs per lungs, is plotted on the *ordinate* for each of the two serum-donor groups (early- and late-pregnancies) indicated above the bars. \**p* < 0.05 Ipup serum versus Cpup serum. The IgG a-PTxA titers are listed for each pool below the *abscissa* (nd, not detected).

Figure 4 | Effect of passive immunization through spleen cells collected from Ipups on protection from infection. Whole spleen cells (20–50 × 106 ) from Ipups or Cpups (controls) were tested as possible vehicles of immune protection by passive transfer to naïve mice. Twenty-four hours after transfer, the mice were infected with *Bordetella pertussis* and sacrificed 7 days later to determine the CFUs in the lungs. Ipup-recipients were used as a positive control. In the figure, the number of bacteria recovered from the mouse lungs, expressed as the log10(mean CFUs ± SD of per lungs), is plotted on the *ordinate* for each of the spleen-cell-recipient groups (*n* = 7) indicated on the *abscissa*. The dotted horizontal line marks the lower limit of detection. \**p* < 0.05 Ipups versus both spleen-cell recipient groups.

of the recipients were counted. In this instance, no significant difference was found between the control group and the group injected with spleen cells from the Ipups (**Figure 4**).

#### Passive Immunization through Lactation

We also evaluated passive immunization through lactation. To achieve this aim, pups born to immune mothers (Ipups) were fostered to non-immune mothers (Ipups–Cmother), and pups born from control mothers were fostered to immune mothers (Cpups–Imother). Ipups breast-fed by their immune mother (Ipups–Imother) and Cpups breast-fed by their non-immune mother (Cpups–Cmother) were used as the respective positive and negative controls. After all the pups were suckled for 21 days, the pups' sera were collected to analyze the titers of a-PTxA and a-Bp IgGs before *B*. *pertussis* challenge. Pups were then challenged with a sublethal dose of *B. pertussis* to analyze the protection against infection. Through this experimental protocol and the resulting assays, we detected that pups from the Ipups–Imother and Cpups– Imother groups exhibited high titers of a-PTxA and a-Bp IgGs (**Figure 5A**) and also possessed a high degree of protection against *B. pertussis*—i.e., a reduction in the CFUs recovered from the lungs by more than six orders of magnitude (**Figure 5B**). In contrast, pups from the group Ipups–Cmother contained low titers of a-PTxA and a-Bp IgGs and retained a low degree of protection. The lowest titers of a-PTxA and a-Bp IgGs and degrees of protection were detected in the pups from the Cpups–Cmother group (**Figures 5A,B**).

#### Effect of Infant Vaccination in Mice Born to aP-Vaccinated Mothers on Protection

To evaluate the possible interference of maternal immunization with subsequent infant immune boosting, offspring born to aP-vaccinated mothers, upon weaning from their mothers, were split into four groups at 4 weeks after birth. Three groups were treated with a single dose of either the aP vaccine, a commercial wP vaccine, or the OMV vaccine (**Figure 6A**), while the remaining group was left untreated. Cpups that received one dose of the aP vaccine at 4 weeks after birth were also used as control. Two weeks after vaccination of the infant mice, the antibody titers to PTxA were measured. We observed that the titers in aP-vaccinated Cpups (given a single dose) were slightly higher than those detected in the non-vaccinated Cpups. In the mice born to aP-vaccinated mothers (i.e*.*, the vaccinated Ipups), the titers detected after vaccination with any of the three vaccines tested were lower than those quantified in Ipups left untreated (blunting effect) (**Figure 6B**). Moreover, that the titers in the non-vaccinated Ipups were higher than those detected in the vaccinated Cpups was most notable (**Figure 6B**). All the mice in this experiment, including the Cpups used as the negative control for protection, were then challenged with 106 –107 CFUs of *B. pertussis* 2 weeks after the postnatal immunization of the Ipups. We observed that at 7 days after challenge, the vaccinated Cpups exhibited a reduced bacterial burden in the lungs by 1.5 logs (32-fold) compared with the non-vaccinated Cpups (**Figure 6C**). Furthermore, aP vaccination of the infant mice born to vaccinated mothers did not interfere with the maternally transmitted protective immunity, as evidenced by a comparable reduction in the CFUs detected in the lungs of approximately 5 logs compared with the burden of the non-vaccinated Cpups. In these experiments, we also observed that the immunization of Ipups with vaccines of different antigenic compositions from that used in

Figure 5 | Effect of passive immunization through lactation on pertussis protection. Pups born from immune mothers (Ipups) were fostered to non-immune mothers (Ipups–Cmother), and pups born from control mothers were fostered to immune mothers (Cpups–Imother). Ipups breast-fed by their immune mother (Ipups–Imother) and Cpups breast-fed by their non-immune mother (Cpups–Cmother) were used as the respective positive and negative controls. All the pups (*n* = 6 for each group) were suckled for 21 days. (A) Sera from mothers and pups were collected at different time points to analyze the titers of anti-pertussis toxin (a-PTxA) and anti-whole-cell-*B. pertussis* (a-Bp) IgGs. The titers are expressed as the geometric mean of the data for each group (*n* = 6). (B) Pups were challenged with a sublethal dose of *Bordetella pertussis* Tohama phase I to analyze protection. In the figure, the number of bacteria recovered from the mouse lungs, expressed as the log10(mean CFUs ± SD of per lungs), is plotted on the *ordinate* for each of the dam-suckling groups indicated on the *abscissa*. The dotted horizontal line marks the lower limit of detection. Two independent experiments were performed. \**p* < 0.05 for Ipups–Imother and Cpups–Imother versus the other groups and for Ipups–Cmother versus Cpups–Cmother.

was detected, a third aP dose was administered. Four weeks after birth, the offspring received either no vaccine (*n* = 8) or one dose of the aP, OMV, or wP vaccine (*n* = 8 in each group), as indicated below the *abscissas* of panels (B,C). Two weeks later, the pups were challenged with *B. pertussis* and the bacterial burden in the lungs measured 7 days after challenge. Pups born to vaccinated or non-vaccinated mothers and receiving no postnatal vaccine served as controls. The pups (*n* = 5 for each group) were bled and the anti-pertussis toxin (a-PTxA) IgG titers determined 1 day before the challenge as designated by the dotted vertical arrow. (B) The titers, plotted on the *ordinate*, are expressed as the geometric mean of the data from each group specified on the *abscissa*. \**p* < 0.05 pups born to immunized mother versus others groups, and the nd signifies not detected. (C) In the figure, the number of bacteria recovered from the mouse lungs, expressed as the log10(mean CFUs ± SD per lung), is plotted on the *ordinate* for each group indicated on the *abscissa*. \**p* < 0.05 for negative control versus other groups, and aP-immunized pups born to non-immunized mother versus others groups. The dotted horizontal line marks the lower limit of detection. Of the three independent experiments that were performed, the results from a single representative one is presented.

the maternal immunization reciprocally did not interfere with the maternally transmitted protective immunity since after postnatal vaccination with either a commercial cellular vaccine (wP) or our previously designed OMV vaccine the protection conferred was similar to that seen in infant mice born to aP-vaccinated mothers with or without postnatal aP vaccination (**Figure 6C**).

## DISCUSSION

The vaccination of women with aP during pregnancy is expected to provide infants with a certain degree of protection from pertussis until they are old enough to be vaccinated themselves. Because of this strategy and the data reported on its safety, the Advisory Committee on Immunization Practices (ACIP) recommended in 2011 that unvaccinated pregnant women receive a dose of tetanus toxoid, reduced diphtheria toxoid, and aP vaccine (26). In an effort to reduce the pertussis burden in infants, in 2012, the ACIP recommended the use of aP during every pregnancy (27). In their publication of August 2015, the WHO stated that they considered the vaccination of pregnant women to be most likely the greatest cost-effective additional strategy for preventing disease in infants too young to be vaccinated, with that approach appearing to be more effective and favorable than the so-called *cocooning* tactic, the vaccination of adults in close contact with infants (6). Indeed, WHO recommended that the national programs might consider the vaccination of pregnant women with one dose of aP administered during the second or third trimester or at least 15 days before delivery as a control strategy in addition to routine primary infant pertussis vaccination either in entire countries, or in other more limited settings having a high or increasing infant morbidity and/or mortality from the disease. Although the present time is still early to assess the definitive effect of implementing this strategy on the disease in infants, reports in support of that approach have already appeared in the literature (28, 29). In particular, several studies evidenced the placental transfer of anti-*B. pertussis* antibodies from aP-vaccinated mothers to their infants where the infants born to those immunized mothers had a high level of antibodies during their first months of life (28, 29). A further report in England related the key observation that vaccine effectiveness against laboratory-confirmed pertussis had been sustained during the 3 years following the vaccine's introduction in 2012 (18). Also highly significant was the finding that the disease incidence in infants less than 3 months of age had remained low despite a high persistence in those aged 1 year and older (18). In 2017, a retrospective cohort study appeared that was designed to evaluate whether or not pertussis-infected infants born from 2011 through 2015 whose mothers had received aP vaccine during pregnancy had less severe pertussis than infants born to unvaccinated mothers. The authors concluded that the infants with pertussis whose mothers had been aP-vaccinated during pregnancy had a significantly lower risk of hospitalization and admission to intensive-care units as well as shorter hospital stays (30). Another promising aspect found in the Amirthalingam et al. (18) study was that additional protection from maternal immunization was retained in infants who subsequently received their first dose in the primary Amirthalingam series (18).

Animal models had been used earlier to obtain information about that strategy. Although the data reported once again had been scarce, evidence was nevertheless garnered for protection by the aP vaccine when used during pregnancy against intracerebral infections of *B. pertussis* and those contagions transmitted by aerosols to infant mice (22). In those studies, mice of from 6 to 10 days of age born to aP-immunized mothers were protected against an aerosol challenge with *B. pertussis* strain 18323. The authors ascertained that the protection was transferred from the dams to their offspring first through the placenta and then through the milk (22). More recently, Feunou et al. (31) confirmed that maternal immunization protected the offspring against *B. pertussis* challenge; but in their experimental paradigm, the protection waned and was eventually lost after the postnatal vaccination of the infant mice with the selfsame vaccine (31).

In the experiments reported here, we used just such a mouse model to enhance our understanding of that specific form of maternal immunization. In agreement with previous reports, we detected that maternal immunization with an aP vaccine—in our paradigm, administered in three doses (one being during the pregnancy)—led to offspring protection against *B. pertussis* infection (**Figure 1**). Moreover, we confirmed that the antibody levels to *B. pertussis* and PTxA were accordingly higher in those neonates (the Ipups) than in mice born to non-immunized females (the Cpups). The antibody titers in the offspring declined at 16 weeks relative to the levels at 4 weeks after birth, although the titers were high enough to protect the neonates against *B. pertussis* infection. Furthermore, the transferred antibodies, but not the spleen cells, from Ipups to naïve mice were sufficient to confer protection (**Figure 3**).

What was interesting to us was the observation that the immunity transferred to the offspring had a protective capacity even for pups born to mothers whose last dose of vaccine was given some weeks before pregnancy. Moreover, we detected that the immunity acquired during the first pregnancy was even capable of conferring protection to the offspring born in later pregnancies. We noted that although the titers of antibodies against PTxA were low, protection against pertussis in the Ipups born in those later deliveries was still significantly elevated (**Figure 2B**). These results, though having been obtained in a murine model, would underscore the need to revise the frequency with which human maternal immunization should be conducted.

In agreement with Oda et al. (22), we detected that the main protection was transferred through colostrum and/or milk. Those authors found that challenged infant mice born to mothers immunized with either the aP or the wP vaccine twice before mating evidenced the lowest increase in the number of CFUs per lung. Moreover, out of eight mice, seven deaths were registered in the non-immunized group, whereas five deaths occurred in the transplacentally immunized group, but only two in the transcolostrally immunized mice (22). In our protocol involving a schedule that included the administration of a third aP dose during pregnancy, we detected high degree of protection against *B.* pertussis through lactation (**Figure 5B**)*.* In particular, we detected that the Ipups– Imother and Cpups–Imother experimental groups exhibited high a-PTxA and a-*B. pertussis* IgG titers (**Figure 5A**) in combination with high resulting protection against *B. pertussis* infection (more than a 6-log reduction in the CFUs recovered from the lungs: cf. **Figure 5B**). The results obtained by Quinello et al., in agreement with ours, demonstrated that the *pertussis*-absorbed serum and the colostrum pools protected only 30% of the immunized mice, whereas purified IgGs protected some 65% (23). Although IgA was not measured in this study or in our work, its presence could contribute at least in part to protection. In fact, it was reported that IgA induced by oral or nasally delivered pertussis antigens formulated with mucosal adjuvants confers protection although at levels not so high than the equivalent parenterally delivered vaccines [reviewed in Ref. (32)].

Although the data garnered from our murine model underscored the significance of breastfeeding in protecting infants against pertussis infection, we must point out that this protection could be less substantial in humans since in *Homo sapiens* the majority of the maternal IgG is transferred to the fetus *in utero* during pregnancy (33) and not *via* the milk.

We also observed that, when infant mice born to aP-immunized mothers (the Ipups) were vaccinated at 3 weeks of age with the same aP vaccine or a different one (i.e., a commercial wP or the OMV vaccine); the titer of IgG against a-PTx decreased (**Figure 6B**), but the maternally derived protection was not reduced upon that subsequent vaccination, regardless of the type of vaccine administered. These results seem to be contradictory in principle to those reported by Feunou et al., but we must bear in mind that those authors applied two vaccine boosters, one at 7 days and another at 3 weeks of life (31). The blunting effect on protection that the authors observed could have resulted from the application of those boosters—and particularly the ones performed during neonatal life—at a time when the antibody titers in the pups were still high. In contrast, our results are in agreement with those observed in humans in whom the protective capacity conferred by maternal immunization was retained in neonates receiving their first dose of the primary series (18).

Though the use of mouse models to research maternal vaccination is not expected to completely replicate human physiology, the results obtained with a model of this design will enable a test of the proposed hypotheses under controlled conditions, where the forthcoming results can then refine those hypotheses for further validation in subsequent human studies. Here, in the present mouse model, we have demonstrated that immunization with aP during pregnancy or up to 5 weeks prior effectively protects newborns against pertussis. Although the titer of maternal antibodies in the infant offspring diminishes with time, protection is not reduced for at least up until 4 months of age. Moreover, subsequent vaccination of the infant mice with the same vaccine or a different one from the type used during pregnancy did not affect the transferred maternal protection (**Figure 6**). The potential

#### REFERENCES

blunting of protection conferred by maternal immunization through infant vaccination could be developed depending on the antibody levels in the infants. Another important finding here described was that maternal-vaccination-acquired immunity from the first pregnancy still conferred protection to offspring up to the fourth pregnancy. The results presented here reinforce the need to continue studying that blunting effect in humans as well as to revise the frequency of vaccination in successive pregnancies according to the time between each one.

#### ETHICS STATEMENT

The animal experiments were authorized by the Ethical Committee for Animal Experiments of the Faculty of Science at La Plata National University (approval number 004-06-15 and 003-06-15).

## AUTHOR CONTRIBUTIONS

DH planned the study, made the laboratory analysis, interpreted data, and drafted manuscript. DB, MG, and MZ planned the study, interpreted data, and revised figures and the manuscript. DS-M, EB, FC, PA, CC, and MSB performed certain experiments and laboratory analyses. All authors approved the final manuscript.

#### ACKNOWLEDGMENTS

Juan Guzman Cook and Vet. Clara Versellini provided excellent technical assistance. Dr. Donald F. Haggerty, a retired academic career investigator and native English speaker, edited the final version of the manuscript.

#### FUNDING

This work was supported by ANCPyT (PICT 2014-3617, PICT 2012-2719), CONICET, and CICBA (Argentina) grants to DH. DH, MG, and DB are members of the Scientific Career of CONICET. MZ, FC, PA, and EB are fellows from CONICET. DM and MB are fellows from ANPCyT and CC is a full-time Professor at Universidad Nacional de La Plata.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Gaillard, Bottero, Zurita, Carriquiriborde, Martin Aispuro, Bartel, Sabater-Martínez, Bravo, Castuma and Hozbor. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Whole Blood Profiling of Bacillus calmette–guérin-induced Trained innate immunity in infants identifies epidermal growth Factor, il-6, Platelet-Derived growth Factor-aB/ BB, and natural Killer cell activation

#### *Steven G. Smith <sup>1</sup> \*, Johanneke Kleinnijenhuis <sup>2</sup> , Mihai G. Netea <sup>2</sup> and Hazel M. Dockrell <sup>1</sup>*

*<sup>1</sup> Faculty of Infectious and Tropical Diseases, Department of Immunology and Infection, London School of Hygiene and Tropical Medicine, London, United Kingdom, 2Department of Internal Medicine, Radboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, Netherlands*

#### *Edited by:*

*Elizabeth Whittaker, Imperial College London, United Kingdom*

#### *Reviewed by:*

*António Gil Castro, University of Minho, Portugal Dennis Klinman, National Institutes of Health, United States*

*\*Correspondence: Steven G. Smith<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology*

*Received: 31 March 2017 Accepted: 17 May 2017 Published: 06 June 2017*

#### *Citation:*

*Smith SG, Kleinnijenhuis J, Netea MG and Dockrell HM (2017) Whole Blood Profiling of Bacillus Calmette–Guérin-Induced Trained Innate Immunity in Infants Identifies Epidermal Growth Factor, IL-6, Platelet-Derived Growth Factor-AB/BB, and Natural Killer Cell Activation. Front. Immunol. 8:644. doi: 10.3389/fimmu.2017.00644*

Vaccination of infants with bacillus Calmette–Guérin (BCG) activates both the innate and adaptive arms of the immune response. The antimycobacterial effects of these responses most likely account for the ability of BCG to protect against childhood forms of tuberculosis (TB). There is also evidence for a heterologous protective effect of BCG vaccination against TB-unrelated mortality in low birth weight infants. A possible mechanism of action of this effect, the induction of trained innate immunity, has been demonstrated when cells from BCG-vaccinated adults are restimulated *in vitro* with non-related microbial stimuli. Our aim was to examine an extensive panel of secreted immune biomarkers to characterize the profile of trained innate immunity in infants. Stimulation of whole blood for 48 h was performed 4 months after BCG vaccination, or in control unvaccinated infants. Stimulants were lipopolysaccharide; Pam3Cys (P3C); heat-killed *Candida albicans*, *Staphylococcus aureus*, *Escherichia coli*, and a lysate of *Mycobacterium tuberculosis*. Culture supernatants were tested for secreted cytokines and chemokines by 42-plex bead array and monocytes and natural killer (NK) cells assessed for expression of activation markers by flow cytometry. BCG-vaccinated infants displayed increases in 11 cytokines and chemokines in response to different non-specific innate immunity stimuli: epidermal growth factor (EGF); eotaxin; IL-6; IL-7; IL-8; IL-10; IL-12p40; monocyte chemotactic protein-3; macrophage inflammatory protein-1α; soluble CD40 ligand and platelet-derived growth factor (PDGF)-AB/BB. Although each stimulant induced a distinct response profile, three analytes, EGF, IL-6, and PDGF-AB/BB, were commonly higher after stimulation with Pam3Cys, *C. albicans*, and *S. aureus*. Conversely, certain cytokines such as interferon gamma-inducible protein-10, IL-2, IL-13, IL-17, GM-CSF, and GRO were suppressed in BCG-vaccinated infants, while no increases in TNFα or IL-1β production were detected. We did not observe a concomitant, BCG-associated change in monocyte surface activation markers in response to non-specific stimuli, but we detected a significant increase in CD69 expression on NK cells in response to Pam3Cys. Pam3Cys-induced NK cell activation correlated with the magnitude of IL-12p40 and IL-10 responses to the same stimulant. This study reveals a novel cytokine/chemokine biomarker signature of BCG-induced trained innate immunity in infants and the involvement of NK cells in these responses.

Keywords: bacillus Calmette–Guérin, vaccination, heterologous effects, trained immunity, infants, cytokines, chemokines, natural killer

#### INTRODUCTION

*Mycobacterium bovis* bacillus Calmette–Guérin (BCG) is currently the only licensed vaccine for tuberculosis (TB). Although its protective efficacy against adult pulmonary TB is variable (1–3), BCG affords more reliable protection against childhood forms of the disease when administered to infants (4). The immune mechanisms responsible for this protection are not fully understood, although many studies have described BCGinduced, antigen-specific immune responses that may play a part (5–12).

Evidence also exists of a beneficial effect for the BCG vaccine against several different diseases and outcomes other than TB, including as a therapeutic agent against bladder cancer when instilled directly into the bladder (13) and protection against all-cause mortality in low birth weight infants (14). These effects are heterologous (non-specific) as they do not rely on mycobacteria-specific adaptive immune responses. Exactly how BCG mediates these effects is not clear, although different pathways are probably involved. Non-specific protective effects against mortality in low birth weight infants are a result of resistance to pathogenic microorganisms that are responsible for death due to neonatal sepsis and respiratory infections (15, 16). The immune responses mediating this effect could be either heterologous T-cells (i.e., T-cells induced by an epitope from one organism but with cross-reactivity to others) or the ability of BCG to potentiate the responsiveness of the innate immune system to later infections: a biological process termed trained innate immunity which represents a *de facto* innate immune memory (17–19). Such a phenomenon is thought to have existed for millions of years as acquired systemic resistance in plants and also in invertebrates, neither of which have adaptive immune systems (17). Several studies have revealed enhancements of the neonatal innate immune response to Gram-positive and Gramnegative infections following previous encounters, mediated by different toll-like receptor pathways (20) or initiated by *in utero* inflammatory conditions such as histologic chorioamnionitis (21). Studies in adults show that trained innate cells, including monocytes and natural killer (NK) cells, appear to be epigenetically and metabolically reprogrammed to produce increased amounts of pro-inflammatory cytokines and display higher levels of surface activation markers in response to restimulation with toll-like receptor ligands or different whole microorganisms (22–25).

If trained immunity is to provide a mechanism of action for the non-specific protective effects of BCG, evidence of trained immunity in infants or infant innate cells is needed. The immune system of the newborn infant differs from that of the adult in its constitution as well as in its propensity to respond to different stimuli; differences that reflect the unique physiological challenge of transitioning from the intrauterine environment to the outside world where some microorganisms are beneficial commensals and some are life-threatening pathogens (26, 27). Newborn innate immune responses are characterized by a reduced capacity for pro-inflammatory cytokines and dendritic cell differentiation and activation, but a greater propensity to produce regulatory cytokines (28, 29). More data are needed on how these differences impact upon the generation of trained innate immunity by BCG.

Our aim in this study was to probe the infant immune response to mycobacteria-unrelated stimuli following BCG vaccination for potential mediators of trained immunity. We used a multiplex bead array approach for the detection of secreted cytokines and chemokines from diluted whole blood following stimulation with a panel of innate stimuli. The rationale for this was to maximize the variety of immune cells available for stimulation as well as the potential to detect a broad array of soluble mediators. Our data provide a description of previously unreported profiles of trained immunity in infants and a role for activated NK cells.

#### MATERIALS AND METHODS

#### Study Participants and Sample Collection

Healthy, UK-born infants were recruited following ethical approval from the National Research Ethics Service Committee London-East (11/LO/0363) and from the Ethics Committee of the London School of Hygiene and Tropical Medicine (ref. 4068). Written consent was obtained from parents prior to recruitment. Infants were recruited from two regions of South East England: Redbridge, where a single dose of intradermal BCG (BCG Vaccine Danish Strain 1331, Staten Serum Institute, Copenhagen, Denmark) was administered to infants at approximately 6 weeks of age in local vaccination clinics and West Essex where infants do not routinely receive BCG. Heparinized venous blood was obtained 4 months post-vaccination or from unvaccinated infants at an age-matched time point. This exploratory study was part of a larger study (11) and 4-month post-vaccination samples available were *n* = 11, vaccinated infants; and *n* = 10, unvaccinated infants for Luminex analysis and *n* = 10 and *n* = 8, respectively, for flow cytometry analysis.

#### Diluted Whole Blood Assays for Cytokine Responses to Innate Stimuli

Venous blood was diluted 1/5 in RPMI 1640 (Invitrogen) supplemented with 2 mM l-glutamine (Invitrogen) and cultured at 37°C for 48 h in 96-well U-bottomed plates in a final volume of 200 µl. Duplicate wells were incubated alone (medium only negative control) or with the following stimuli: lipopolysaccharide (LPS; 10 ng/ml); (S)-(2,3-bis(palmitoyloxy)-(2RS) -propyl)- *N*-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys4-OH, trihydrochloride [Pam3Cys; 10 µg/ml]; heat-killed (HK) *Candida albicans* (*C. albicans*; 106 microorganisms/ml); HK *Staphylococcus aureus* (*S. aureus*; 106 microorganisms/ml); HK *Escherichia coli* (*E. coli*; 106 microorganisms/ml); and sonicated *Mycobacterium tuberculosis* H37Rv [*Mtb* lysate; 1 µg/ml end concentration]. Concentrations used for each stimulus were optimized in previous experiments.

## Tissue Culture Supernatant Harvest and Multiplex Bead Array Assay

After 48 h, plates were centrifuged at 400 *g* for 5 min. Supernatants were removed from duplicate wells, pooled, and stored in aliquots at −80°C prior to analysis. Thawed supernatants were subjected to multiplex bead array analysis using the human cytokine/chemokine Milliplex™ MAP 42-plex premixed kit (Merck Millipore) and following the manufacturer's instructions. The pre-mixed bead set included the following panel: IL-1α, IL-1β, IL-1Ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17, epidermal growth factor (EGF), eotaxin, Flt-3L, FGF-2, fractalkine, G-CSF, GM-CSF, GRO, IFNα2, IFNγ, IP-10, MCP-1, MCP-3, MDC, MIP-1α, MIP-1β, platelet-derived growth factor (PDGF)-AA, PDGF-AB/BB, RANTES, sCD40L, sIL-2Ra, TGFα, TNFα, TNFβ, and VEGF. Data were acquired using the Biorad Luminex® 100 system and Bioplex Manager Software version 6.1 (Biorad).

## Peripheral Blood Leukocyte (PBL) Harvest and Flow Cytometry

Following the removal of supernatants from diluted whole blood assay plates, remaining PBLs were harvested and cryopreserved for later analysis. EDTA in PBS (2 mM) was added to assay wells, which were incubated at room temperature for 15 min to detach adherent cells. Wells were mixed and cells from duplicate wells were pooled and then incubated with 10× volume of 1× FACS Lysing Buffer (BD Biosciences) at room temperature for 10 min. Following red cell lysis, PBLs were pelleted, resuspended in fetal bovine serum with 10% dimethylsulfoxide, and cryopreserved in liquid nitrogen. For flow cytometric analysis, thawed cells were washed in PBS with 0.1% bovine serum albumin and 0.01% sodium azide (both Sigma Aldrich) and stained for 30 min at 4°C with the following antibodies: CD3-BV510 (clone UCHT1); CD25-PerCP-Cy™5.5 (clone M-A251); CD56-PE-Cy™7 (clone B159); CD206-PE-CF594 (clone 19.2); HLA-DR-BV605 (clone G46-6) (all from BD Biosciences); CD11b-FITC (clone ICRF44); CD69-PE (clone FN50); CD163-APC (clone eBioGHI/61) (all from Affymetrix/eBiosciences); CD14-BV650 (clone M5E2; Biolegend). Following a further wash, cells were resuspended in PBS with 1% paraformaldehyde for acquisition. Cells were acquired using a BD LSR II flow cytometer (BD Biosciences) equipped with blue (488 nm), red (633 nm), and violet (405 nm) lasers and FACSDiva 6.1.3 software. Compensation was carried out using BD™ CompBead Plus compensation particles stained separately with each antibody conjugate and fluorescence minus one control stains were used to determine the position of phenotype and activation marker analysis gates.

## Data Analysis, Management, and Statistical Analysis

Multiplex bead array data for each stimulation condition were background subtracted using values measured in unstimulated controls. Background levels of each cytokine/chemokine are given in Table S1 in Supplementary Material. Final concentrations below the lower limit of quantitation were adjusted to the value of the lowest standard (3.2 pg/ml) and data above the upper limit of quantitation were adjusted to the highest standard (10,000 pg/ml). The compensation matrix was generated and flow cytometric data were analyzed using FlowJo™ 10.2 software (FlowJo LLC). Monocytes were gated based on CD14 and HLA-DR expression as well as on size and granularity. NK cells were gated based on CD56 expression and lack of CD3 expression (see Figure S1 in Supplementary Material). Multiplex bead array data were analyzed using IBM® SPSS® Statistics version 23 software (IBM Corp.) and Prism 7 for Windows (GraphPad Software Inc.). Flow cytometric data were further analyzed using a combination of Spice 5.35 (30) and Prism. Statistical comparisons between vaccinated and unvaccinated infant groups for both multiplex bead array and flow cytometry data were by Mann–Whitney *U* test and correlations were by Spearman's rank correlation coefficient.

## RESULTS

## Infant BCG Vaccination Is Associated with Enhanced Cytokine and Chemokine Responses to Non-Specific Innate Stimuli

Diluted whole blood from BCG-vaccinated and unvaccinated infants was stimulated with a panel of non-specific innate stimuli and with a preparation of *Mtb* lysate. Secreted cytokines and chemokines were measured in tissue culture supernatants by multiplex bead array after 48 h.

Previous reports described TNFα, IL-6, and IL-1β as the characteristic cytokines of BCG-induced trained innate immunity in adults (24, 31). However, when we investigated this signature in infants, only IL-6 was significantly increased in response to non-specific stimuli. Re-stimulation with the mycobacterial stimulant *Mtb* lystae did produce a significant increase in TNFα (**Figure 1**). As expected, *Mtb* lystae induced the most extensive upregulation of responses in BCG-vaccinated infants including 27 cytokines and chemokines that characterize both innate and adaptive T-cell responses (**Figures 1** and **2**; **Table 1**). We detected 11 cytokines and chemokines that were significantly increased and 6 that were significantly decreased in BCG-vaccinated infants in response to non-specific stimulants (**Figure 2**; **Table 1**). Each non-specific stimulant induced a distinct response profile. For example, increases in 10 cytokines and chemokines were detected following Pam3Cys stimulation

whereas *C. albicans* stimulation revealed BCG-associated increases in four analytes: EGF, IL-6, PDGF-AB/BB, MCP-3, but decreases in four further analytes: IL-2, IL-13, IL-17, IP-10. Despite distinct response profiles for each non-specific stimulant, we observed a common signature of increases in EGF, IL-6, and PDGF-AB/BB in response to three different stimulants: Pam3Cys, *C. albicans*, and *S. aureus*.

These data led us to conclude that BCG vaccination of infants, in this setting, induces a complex reprogramming of the innate immune system to respond mainly in an enhanced fashion when exposed *in vitro* to a panel of heterogeneous, non-specific stimuli. For a few cytokines, downregulation of production was observed after vaccination. Overall, this effect is consistent with trained immunity which is known to be mediated by cells of the innate immune system.

## NK Cell CD69 Expression Is Increased in BCG-Vaccinated Infants following Pam3Cys Re-Stimulation *In Vitro*

To determine which innate immune cells were associated with altered cytokine and chemokine responses to non-specific stimuli in BCG-vaccinated infants, we stained PBLs recovered from 48 h diluted whole blood assays for markers of monocyte and NK cell phenotype and activation.

Consistent with BCG-associated increases in a broad array of cytokine and chemokine responses following *Mtb* lystae restimulation, we also detected concomitant increases in expression of CD11b and CD206 on monocytes (**Figure 3A**). There were no significant, BCG vaccination-associated changes to monocyte activation markers in response to non-specific stimuli. In addition

\**p* < 0.05.

to its effect on monocytes, *Mtb* lystae stimulation also induced increased surface expression of the activation marker CD69 on NK cells (**Figure 3B**). Although there were no significant changes to monocyte activation markers in response to non-specific stimuli, BCG-vaccinated infants displayed a significant increase in NK cell CD69 expression in response to Pam3Cys (**Figure 3B**).

Based on these data, we conclude that, accompanying the cytokine/chemokine profile of BCG-induced trained immunity in infants, there is a role for activated NK cells but that their involvement depends upon the nature of the restimulating nonspecific ligand.

## Pam3Cys-Induced NK Cell Activation Correlates with the Secretion of IL-12p40 and IL-10

Pam3Cys is a ligand for TLR2 which is found on the surface of NK cells. We speculated that enhanced Pam3Cys-induced NK cell activation in BCG-vaccinated infants could be a result of changes to the cytokine milieu that this ligand induces (**Figure 2**) or due to intrinsic changes to NK cells following BCG vaccination that alter their responsiveness to TLR2-mediated Pam3Cys stimulation or a combination of the two. Although the scope of this study did

Figure 2 | Distinct patterns of cytokine and chemokine secretion characterize bacillus Calmette–Guérin (BCG)-induced trained immunity in response to different, non-specific stimuli. The concentrations of 42 cytokines and chemokines were measured in 48 h, diluted whole blood assay supernatants using multiplex bead array. (A) Heat maps represent the vaccination effect [the log2 median fold change in analyte concentration in the BCG-vaccinated infants (*n* = 11) compared to unvaccinated infants (*n* = 10)] on the indicated cytokine/chemokine responses to different stimuli: ML, *Mtb* lysate; LPS, lipopolysaccharide; EC, *E. coli*; P3C, Pam3Cys; CA, *C. albicans*; SA, *S. aureus*. (B) Volcano plots represent the vaccination effect on cytokine/chemokine responses to different indicated stimuli which are plotted on the *x*-axis against the −log10 *p* value on the *y*-axis (calculated by Mann–Whitney *U* test comparisons of responses in BCG-vaccinated and unvaccinated groups). Data points represent individual cytokines and chemokines. Significant responses of interest are labeled where clarity permits. Horizontal dotted lines represent a *p* value of 0.05.

Table 1 | BCG vaccination-associated cytokine and chemokine responses induced by specific and non-specific stimuli in 48 h diluted whole blood assays.

not allow us to address the intrinsic changes hypothesis, we were able to look at associations between the magnitude of Pam3Cysmediated NK cell activation and the changes in cytokine and chemokine release in response to the same stimulant. Of the 10 analytes that were significantly upregulated in BCG-vaccinated infants in response to Pam3Cys, IL-12p40 and IL-10 secretion

Figure 3 | Bacillus Calmette–Guérin (BCG) vaccination of infants is associated with enhanced natural killer (NK) cell activation in response to Pam3Cys. Following 48 h, diluted whole blood assay stimulation of samples from BCG-vaccinated (□, *n* = 10) and unvaccinated (○, *n* = 8) infants, cell pellets were cryopreserved and later analyzed by flow cytometry. (A) Monocytes were identified based on high CD14 and HLA-DR expression as well as on size and granularity (see Figure S1A in Supplementary Material) and stained for the indicated markers of monocyte activation and differentiation. Data points represent the fold change in MFI for samples incubated with the indicated stimulants compared to unstimulated samples. (B) CD56+ CD3− NK cells were gated (see Figure S1B in Supplementary Material) and the expression of the activation markers CD69 and CD25 analyzed. Data points represent the fold change in percent of activation marker expressing NK cells for samples incubated with the indicated stimulants compared to unstimulated samples. Responses in vaccinated and unvaccinated groups were compared using the Mann–Whitney *U* test: \**p* < 0.05; \*\**p* < 0.01.

demonstrated significant correlation with the extent of NK cell CD69 expression (**Figure 4**).

From these data, we conclude that cytokine secretion, most notably IL-12, may account at least in part for the TLR2-mediated activation of NK cells in BCG-vaccinated infants.

#### DISCUSSION

Trained innate immunity has, to date, been demonstrated and characterized largely in adults. Isolated PBMC released more TNFα, IL-6, and IL-1β when exposed to non-specific innate stimuli 3 months after BCG vaccination in adults, and monocytes displayed higher levels of activation markers (24). Mechanisms of trained immunity such as underlying epigenetic regulation and shifts in the metabolic pathways used have similarly been revealed in vaccinated adults and in adult cellular models *in vitro* (22, 32). If trained innate immunity is to provide a causal link between BCG vaccination of infants and non-specific protection against mortality due to infectious diseases other than those with a mycobacterial etiology (15, 16), then a detailed description of the phenomenon in infants and infant cells is needed.

In this paper, we have profiled the infant whole blood cytokine and chemokine secretome of trained immunity following BCG vaccination using a panel of non-specific innate stimuli and reveal a differentially expressed signature. A total of 11 analytes were overexpressed and 6 under-expressed in vaccinated infants, with different innate stimuli inducing distinct combinations of response elements: for example, the Pam3Cys-induced response included the overexpression of 10 analytes, while *S. aureus* only induced three analytes. Four analytes were overexpressed and four under-expressed in response to *C. albicans*; LPS and *E. coli* each induced one overexpressed and two under-expressed analytes. These distinctions most likely reflect the different characteristics of the stimuli involved. Two of them, LPS and Pam3Cys, are defined biochemical compounds that interact with specific pattern recognition receptors: TLR4 and TLR2/1 heterodimer, respectively. *C. albicans*, *S. aureus*, and *E. coli* are all HK preparations of microorganisms containing complex mixtures of pathogen-associated molecular patterns that will interact with an array of different pathogen recognition receptors or other innate antigen receptors. It has been suggested that different microorganisms act on multiple, distinct TLRs simultaneously in a manner likened to the playing of a unique "chord" on a "molecular piano" (17) and the different response profiles generated by different stimuli described here may be an illustration of that effect.

The pathways by which BCG mediates its training effect *in vivo* are yet to be fully elucidated, although a role for the mycobacterial cell wall component muramyl dipeptide, acting *via* the NOD2 receptor, has been described (24). β-glucan, another activator of trained immunity mediates its effects *via* a dectin-1/Raf1 dependant pathway (33), yet the role of dectin-1 in recognizing mycobacteria is probably minor at most. The emerging picture is one in which the recall of a trained immune response may be restimulated *via* different receptors to those that prime that same

response and that the cytokine/chemokine signature of that recall is determined by the receptors involved. Although little is known at this time about the mechanisms responsible, this should be a focus of future studies.

Despite the differences described above, we described here a biosignature of BCG-induced trained innate immunity in infants comprising increases in EGF, IL-6, and PDGF-AB/BB, all of which were apparent in responses to three different innate stimuli. This is different to the characteristic signature of trained innate immunity in adults, which includes TNFα and IL-1β as well as IL-6. It must be however underlined that in adults not all the cytokines reported here were measured, and some of them may be upregulated as well in older individuals. The absence of canonical innate cytokines such as TNFα in this infant biosignature may reflect the maturation of the infant innate immune response, whereby the ability to make certain cytokines develop over the first 9 months of life (34). Despite the most extensive response profile, *Mtb* lystae stimulation did not upregulate EGF or PDGF-AB/BB. This may reflect the involvement of a broader array of pattern recognition receptors in response to *Mtb* lystae with downstream effects that manifest themselves as a distinct pattern of upregulated and downregulated markers. These observations illustrate the importance of an unbiased, multiplex approach to analyzing these responses. A more directed study focused on TNFα, IL-6, and IL-1β would have underestimated the effect of trained immunity in infants and would have missed the previously unreported markers EGF and PDGF-AB/BB, as well as the finding that *C. albicans*-induced trained immunity recall in BCG-vaccinated infants involves reduced IL-2, IL-13, IL-17, and IP-10 responses. It is interesting that IL-17 responses to *C. albicans* are reduced in vaccinated infants. Th-17 cells are known to be important in immune responses to *C. albicans* (35). BCG vaccination has been shown to enhance non-specific (innate-mediated) protection against candidiasis in SCID mice (24) and adult BCG vaccination activates heterologous Th-17 responses specific for non-mycobacterial ligands including *C. albicans* (31). Our data suggest that BCG is exerting a different influence on the infant immune response, steering it away from a bias toward Th-17 development that is known to exist in early life (36). Detailed analyses of epigenetic and transcriptional programs of training or tolerance induction have revealed upregulation and downregulation of genes that characterize these responses hence it is unsurprising that the profiles we report here involve both upregulation and downregulation of different cytokines and chemokines associated with both T-cell and inflammatory monocyte responses to stimuli such as *C. albicans* as well as to *E. coli* and LPS which have both been associated with the induction of tolerance (37, 38).

In contrast to our findings here, another report of infant BCGassociated, non-specific cytokine responses described increases in TNFα and IL-1β as well as in IL-6 in response to Pam3Cys (39). Differences between study designs that might explain this difference are that Jensen et al. carried out their study in a lowincome country and in low birth weight infants. In addition, the genetic backgrounds of the populations studied were different. Their sampling time point of 4 weeks was earlier than that used here and there were some methodological differences in their whole blood assay. An interesting possible explanation is that, unlike Jensen et al., infants in our study had received DTP vaccination through the national vaccination program by the time of sampling which is thought to negatively affect the positive nonspecific effects of BCG (40). Finally, compared to Jensen et al., our study was relatively small and exploratory and was possibly underpowered to detect small differences in cytokine responses. It should be noted that in agreement with our findings and those of others (7), Jensen et al. found no BCG-associated increase in TNFα in response to non-specific LPS stimulation.

The involvement of monocytes in adult trained immunity has been demonstrated using stimulating ligands that interact with receptors known to be found on monocytes and more directly by the demonstration that markers of monocyte activation and differentiation are upregulated following training (24). It is also possible to isolate adult blood monocytes and train them directly with BCG and β-glucan *in vitro* (32). Although the assay described in this paper was designed primarily to look at secreted cytokines and chemokines, it was possible to harvest PBLs and to examine monocytes and NK cells for evidence of BCG vaccination-associated trained innate activation. We examined four markers of monocyte activation, but found no significant differences in their expression following non-specific stimulation of samples from vaccinated and unvaccinated infants. Stimulation with *Mtb* lystae, which also induced increases in 27 cytokines and chemokines in vaccinated infants, did increase levels of CD11b and CD207 on the surface of monocytes. Unlike responses induced by non-specific ligands in these experiments, it is likely that antigen-specific T-cell help is involved in these changes in monocyte activation. It is interesting that monocytes from BCG-vaccinated infants show increased expression of CD207 (mannose receptor), as this is usually found only on certain populations of differentiated macrophages and dendritic cells (41).

Natural killer cells from BCG-vaccinated infants displayed increased levels of the activation marker CD69 in response to Pam3Cys, as well as to *Mtb* lystae. Previous work has described long-lived mycobacteria-specific NK cells following infant BCG as well as a role for NK cells in BCG-induced trained immunity in adults and in BCG-vaccinated SCID mice which show increased resistance to *C. albicans* infection (25, 42). However, this is the first time their involvement in trained immunity to heterologous stimuli in infants has been described. NK cells express the TLR2 receptor *via* which they are reported to interact with mycobacteria (43, 44). NK cells are also activated in response to type 1 cytokines (45). Although we cannot determine the pathway of NK cell activation from the current data, we did observe a correlation between CD69 expression, IL-12p40 secretion, and IL-10 secretion in response to Pam3Cys. IL-12 is known to be a soluble activator of NK cells, while a role for IL-10 is less obvious. A limitation of this study was the small volume of blood available from infants which meant it was not possible to identify the cell type producing the cytokines and chemokines described. Clearly it would be interesting to determine whether the NK responses described above or other populations were a source, either by depletion studies or intracellular cytokine staining and future studies should address this. Additionally, as well as stimulation of the TLR2/1 heterodimer using Pam3Cys, it would be useful in future to determine more precisely the role of TLR2 either in homodimeric form or as a heterodimer with TLR6 also using specific ligands for these receptors.

This exploratory study was not designed to investigate the heterologous effects of infant BCG vaccination on clinical outcomes; however, if the non-specific immune responses we describe are to provide a mechanism for these effects, a causal relationship will need to be demonstrated in a larger study. The setting of such a study will need to be carefully considered as to date, the results of clinical trials have been mixed. Heterologous, BCG-induced protection against clinical outcomes related to infectious disease has been observed in a low-income setting in West Africa (15, 46) but was not recapitulated in another trial in a high income, European setting (47, 48).

In conclusion, we have described a novel whole blood signature of BCG-induced, trained innate immunity in infants that includes secretion of EGF, IL-6, and PDGF-AB/BB and involves activated NK cells. We also show distinct patterns of cytokine and chemokine release in response to different innate ligands. The data show different patterns to previously published descriptions of BCG-induced trained innate immunity in adults and low birth weight infants and suggest that more, larger scale studies of this effect in different populations are required for a more complete understanding.

## ETHICS STATEMENT

This study was carried out in accordance with the recommendations of the National Research Ethics Service Committee London-East and the ethics committee of the London School of Hygiene and Tropical Medicine with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the National Research Ethics Service Committee London-East and the ethics committee of the London School of Hygiene and Tropical Medicine.

## AUTHOR CONTRIBUTIONS

SS performed the experiments, was responsible for the analysis and interpretation of data, and wrote the manuscript. JK and MN produced the panel of stimuli used in experiments. SS, JK, MN, and HD all contributed to the conception and design of the work, critically read and contributed to revisions of the manuscript, approved the final version of the manuscript, and agreed to be accountable for all aspects of the work including its accuracy and integrity.

## ACKNOWLEDGMENTS

The authors would like to thank all the infants and parents who participated in this study, Mrs. Rose Blitz who was responsible for participant recruitment, and sample collection and the project phlebotomist Mrs. Shakuntala Patel.

## FUNDING

This work was supported by the European Commission within the seventh framework program (FP7) NEWTBVAC (grant number HEALTH-F3-2009-241745), by the European Tuberculosis Vaccine Initiative (grant number TBVI-12 NORAD), and by the European Commission within Horizon2020 TBVAC2020 (grant number H2020 PHC-643381). MN was supported by an ERC Consolidator Grant (310372) and a Spinoza Grant of the Netherlands Organization for Scientific Research.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/fimmu.2017.00644/ full#supplementary-material.

## REFERENCES

low-birth weight infants in Guinea-Bissau: a randomized-controlled trial. *J Infect Dis* (2014) 211(6):956–67. doi:10.1093/infdis/jiu508

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Smith, Kleinnijenhuis, Netea and Dockrell. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

#### *Alessandro Borghesi1,2,3\*, Mauro Stronati3 and Jacques Fellay1,2*

*1School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland, 2Swiss Institute of Bioinformatics, Lausanne, Switzerland, 3Neonatal Intensive Care Unit, San Matteo Hospital, Pavia, Italy*

Only a small proportion of newborn infants exposed to a pathogenic microorganism develop overt infection. Susceptibility to infection in preterm infants and infants with known comorbidities has a likely multifactorial origin and can be often attributed to the concurrence of iatrogenic factors, environmental determinants, underlying pathogenic processes, and probably genetic predisposition. Conversely, infection occurring in otherwise healthy full-term newborn infants is unexplained in most cases. Microbial virulence factors and the unique characteristics of the neonatal immune system only partially account for the interindividual variability in the neonatal immune responses to pathogens. We here suggest that neonatal infection occurring in otherwise healthy infants is caused by a failure of the specific protective immunity to the microorganism. To explain infection in term and preterm infants, we propose an extension of the previously proposed model of the genetic architecture of infectious diseases in humans. We then focus on group B streptococcus (GBS) disease, the best characterized neonatal infection, and outline the potential molecular mechanisms underlying the selective failure of the immune responses against GBS. In light of the recent discoveries of pathogen-specific primary immunodeficiencies and of the role of anticytokine autoantibodies in increasing susceptibility to specific infections, we hypothesize that GBS disease occurring in otherwise healthy infants could reflect an immunodeficiency caused either by rare genetic defects in the infant or by transmitted maternal neutralizing antibodies. These hypotheses are consistent with available epidemiological data, with clinical and epidemiological observations, and with the state of the art of neonatal physiology and disease. Studies should now be designed to comprehensively search for genetic or immunological factors involved in susceptibility to severe neonatal infections.

Keywords: newborn infant, life-threatening, primary immunodeficiency, genetic predisposition to disease, Mendelian diseases, monogenic, infection, group B streptococcus

## INTRODUCTION

Neonates are commonly thought to be vulnerable to pathogens because of neonatal immaturity, immune tolerance, or immune deviation, a developmentally regulated transitional state (1–4). These concepts, while useful to describe the highest incidence of infection during the neonatal age at the population level, do not take into account interindividual variability. Even if the highest incidence of

#### *Edited by:*

*Kirsty Le Doare, Imperial College London, UK*

#### *Reviewed by:*

*Manish Sadarangani, University of British Columbia, Canada Pia Pannaraj, Children's Hospital Los Angeles, USA*

> *\*Correspondence: Alessandro Borghesi<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology*

*Received: 29 November 2016 Accepted: 15 February 2017 Published: 07 March 2017*

#### *Citation:*

*Borghesi A, Stronati M and Fellay J (2017) Neonatal Group B Streptococcal Disease in Otherwise Healthy Infants: Failure of Specific Neonatal Immune Responses. Front. Immunol. 8:215. doi: 10.3389/fimmu.2017.00215*

**166**

infection is observed during the first 28 days of life, the majority of newborn infants are resistant to common pathogens, and only a small proportion of infants exposed to a given microorganism develop overt disease.

It is very clear from epidemiological studies that multiple risk factors contribute to the individual risk of developing neonatal infections. Based on them, neonates can be classified into highand low-risk groups; individual risks can be estimated; and preventive protocols can be designed for infants who are at high risk of suffering from severe infections (5–7).

Despite their proven clinical utility, most preventive protocols are only partially effective. This can be explained in part by incomplete adherence by healthcare practitioners and missed opportunities (8–10). However, another critical limitation is the inability of current protocols to accurately predict susceptibility to severe infection at the individual level. Furthermore, infections that occur in the absence of any recognizable factors are currently unpredictable.

Many research groups are focusing on the mechanisms of host susceptibility and resistance to pediatric and adult infections (11, 12). Conversely, neonatal infections have been much less studied from a host susceptibility perspective. Several layers of complexity have indeed prevented researchers from fully understanding the neonatal-specific protective immunity, beyond maternal protection of the neonate through transplancentally transmitted antibodies (Abs). Considering the global burden of neonatal infectious diseases, this looks like a missed opportunity to address a critical public health problem.

The study of neonatal infections raises several practical and ethical issues and is challenging from a scientific perspective. First, the neonatal immune system is a rapidly evolving entity, as is every other organ and system soon after birth (13). Second, and possibly more importantly, there is a complex immune interplay between the mother and the child. The maternal environment (the maternal immune system and microbiome) is intertwined with physiological and pathological processes occurring in the fetal and neonatal tissues (e.g., the maturation of fetal and neonatal immune responses, the composition of the neonatal microbiome) (13–17).

To date, little is known about the mechanisms leading to individual vulnerability and resistance to specific pathogens in the neonatal age. We here propose novel, testable hypotheses that could explain the interindividual differences in pathogen susceptibility and help dissect the molecular and cellular bases of severe neonatal infections.

#### EPIDEMIOLOGY OF NEONATAL INFECTIONS

The *Global Burden of Disease Study 2015* reports that "sepsis and other neonatal infections" account for 336,300 neonatal deaths each year worldwide (18).

The distribution of infecting microorganisms varies between term and preterm infants and is different in the neonatal period compared to other age groups. Early-onset and late-onset infections are defined as infection occurring during and after the first 6 days of life, respectively. According to other definitions, 48–96 h of life could be used as cutoff (19).

Group B streptococcus (GBS), or *Streptococcus agalactiae*, is one of the leading pathogens in neonatal infections occurring in full-term newborn infants during the first week of life (9). It is also the most frequent cause of sepsis and meningitis in young infants after the first week of life (20, 21). Recent reports show an increase in the proportion of *Escherichia coli* infection, mostly associated with urinary tract infection, in previously healthy, full-term infants aged 1 week to 3 months (22, 23). Other pathogens responsible for invasive infection in full-term infants include Gram-positive (*Staphylococcus aureus*, *Streptococcus* spp., *Enterococcus* spp., and, less frequently, *Listeria monocytogenes*) and Gram-negative microorganisms (*Klebsiella* spp., *Citrobacter* spp., *Serratia marcescens*, *Salmonella* spp., *Haemophilus influenzae*) (22, 23). Deep organ infections by *Candida* spp. and other fungal microorganisms are exceedingly rare in full-term infants.

In very low-birth-weight (VLBW; <1,500 g at birth) infants, Gram-negative pathogens are the most frequently isolated microorganism in early-onset infections, while Gram-positive bacteria are the most frequently isolated pathogens in late-onset infections in the neonatal intensive care units (NICUs), followed by Gramnegative bacteria and fungal organisms (24–29).

#### SUSCEPTIBILITY TO NEONATAL INFECTIONS

#### Maturation of the Neonatal Immune System

The neonatal immune responses differ in many aspects from immune responses in other age groups. A fine-tuning is required to balance the need for tolerance to beneficial antigens (microbial flora and nutrients) and the need for defense against harmful microorganisms.

The cord blood is enriched in CD4+CD25+ T regulatory cells with potent suppressor activity (30, 31) and other immunosuppressive cell populations including some B cell populations and CD71+ erythroid cells (32, 33).

Despite this strong immunosuppressive component, the neonatal immune system has been demonstrated to be able to mount pro-inflammatory responses that are appropriate for the protection against common pathogens in most infants (34). The two main components of the adaptive immune system, the T and B cell compartments, undergo maturation during human fetal life, with progressive and regulated acquisition of B and T cell repertoire diversity and complexity (35). In addition, the human cord blood possesses several pro-inflammatory cell populations, including newborn-specific interleukin (IL)8-producing T cells (36) and a population of CD4+ T cells with a memory-like phenotype and a variety of effector functions (37).

Cells of the neonatal adaptive immune system are capable of mounting a wide range of responses, from poor or "deviant" T helper 2 (Th2)-skewed antiinflammatory responses to balanced Th1/Th2 responses, and even strong adult-like pro-inflammatory responses (2–4, 38).  This phenomenon is linked to extensive epigenetic modifications at the Th2 locus (*IL5*, *IL13*, and *IL4* genes) and in particular to hypomethylation of the conserved non-coding sequence 1 locus, an enhancer and coordinate regulator of Th2 cytokine production (38, 41). Despite this Th2 bias, neonatal adaptive immune responses can shift toward a dominant Th1 and pro-inflammatory response depending on the type of innate responses and the conditions of antigen exposure (38, 42).

Adaptive immune responses require, however, several days to take place. Neonates cannot rely on preexistent immunological memory because exposure to foreign antigens and pathogens is limited during intrauterine life (34, 42). Furthermore, humoral immunity largely depends on maternally transmitted antimicrobial IgG Abs during the first days of life. The rapid decline of maternal IgG in the neonatal plasma after birth (with a half-life of 21–30 days) is accompanied by a relatively slow maturation of both T-dependent and T-independent B-cell responses throughout the first months of life (13, 43).

Infections occurring in the neonatal period are, by definition, primary infections, and neonates mostly rely on the innate immune responses that provide a first line of defense against invading pathogenic microorganisms (34, 44, 45).

A number of studies demonstrated that the neonatal innate immune responses are characterized by dampened Th1-polarizing and pro-inflammatory responses [low amounts of tumor necrosis factor (TNF) upon toll-like receptor (TLR) stimulation] and by increased production of Th2-polarizing and antiinflammatory cytokines (higher IL6/TNF ratio compared to adult responses) (45–48). Furthermore, decreased phosphorylation of signal transducer and activation of transcription 1 in response to interferon gamma (IFN-γ) (49) and developmental maturation of specific dendritic cell subsets (50) contribute to the neonatalspecific Th2-polarizing innate immunity.

Interestingly, full-term healthy newborn infants do not appear specifically vulnerable to deep infection by microorganisms typically causing disease in immunodeficient patients, most notably *Nocardia* and fungi-like *Aspergillus*, *Candida*, *Cryptococcus*, *Pneumocystis*, and other opportunistic pathogens, suggesting a substantial maturation of the specific antifungal protective responses in most full-term newborn infants.

Altogether, the characterization of the neonatal immune responses over the past two decades has shown profound differences with adult immunity that in part explain the overall increased susceptibility to life-threatening infection of newborn infants. However, little is known so far about the interindividual differences in the immune protection against pathogens in the neonatal age.

#### Heritability of Neonatal Sepsis

There is controversy over the heritability of susceptibility to neonatal sepsis. In one study, comparing the concordance of lateonset sepsis in same-sex vs. unlike-sex twin pairs, no evidence was found of a genetic component of susceptibility to late-onset sepsis among VLBW infants (51). Conversely, another study compared sepsis concordance rates between monozygotic and dizygotic twins; the authors found that 49% of the variance in liability to late-onset sepsis could be explained by genetic factors alone and 51% by residual environmental factors (52). Both studies focused on cohorts of very preterm/VLBW infants. No study so far addressed the question of heritability of neonatal sepsis in late-preterm and full-term newborn infants.

The question of the role of the genetic background on neonatal host susceptibility to infection has been addressed by several studies. All published studies, included in a recent metaanalysis, used a candidate gene approach on cohorts of preterm infants (53). One genome-wide association study (GWAS) is ongoing (54). Such studies are useful to investigate the contribution of host genetics in the setting of a likely multifactorial pathogenesis, as it is probably the case for most infections occurring in preterm infants. Different approaches are needed to find the genetic determinants of susceptibility to life-threatening infections occurring in full-term infants with no underlying medical conditions in which susceptibility to infection is largely unexplained.

#### Lessons from Inborn Errors of Immunity in Pediatric Infections

Inborn errors of immunity or primary immunodeficiencies (PIDs) are a group of genetic disorders characterized by increased susceptibility to infection. Historically, the so-called conventional PIDs have been the first PIDs described and dissected from a molecular perspective (55). They are typically Mendelian diseases, caused by highly penetrant single-gene defects. They often occur in families or in the presence of consanguinity and are characterized by a profound defect in one or more arms of the immune system leading to susceptibility to recurrent infections by a broad range of microorganisms (56).

Over the past two decades, it has become clear that infectious diseases previously thought to be due to the sole virulence of the pathogen may be the expression of a monogenic disorder underlying a PID. Inborn errors of immunity resulting from single-gene defects have been shown to underlie multiple bacterial infections [myeloid differentiation primary response 88 (MYD88) and interleukin 1 receptor-associated kinase 4 (IRAK4) deficiency], monogenic susceptibility to mycobacterial disease (deficiency of genes in the IL12/IFN-γ loop), herpes simplex encephalitis (defect in TLR3-dependent immune responses), and severe primary *Influenza* virus infection (interferon regulatory factor 7 deficiency) (56–60).

These "non-conventional" PIDs are distinguished from conventional PIDs as they often occur in sporadic cases without any family history of severe infection. Individuals affected by non-conventional PIDs are often otherwise healthy. The immunological phenotype is not detectable with first-line immunological studies, and the disease might manifest as a single episode of severe and potentially lethal infection caused by a common or opportunistic pathogen, mostly during primary infection (56, 58, 59).

The discovery of non-conventional PIDs suggested that monogenic conditions might underlie infectious diseases of infancy and childhood more frequently than previously thought (11). The model of the genetic architecture of human infectious diseases that has been proposed based on these observations suggests that infections occurring early in life are more likely to be caused by single-gene disorders (61).

#### PIDs in Neonatal Infections

The proportion of neonatal infections that can be explained by known PIDs is unknown. However, there is evidence from case reports or small case studies that life-threatening infections occurring early in life may represent the first phenotypic manifestation of an inborn error of immunity.

The role of conventional PIDs in conferring susceptibility to infection in the neonatal age has been recently reviewed by Walkovich and Connelly (62). It is important here to remember that a high index of suspicion is required, given that the infectious and potential extraimmunological phenotypes may be only partially expressed during the neonatal period.

Non-conventional PIDs have also been shown to underlie life-threatening neonatal infections. Pyogenic infections occurring during the first few weeks of life have been described as the first phenotypic manifestation of IRAK4 and MYD88 deficiencies (63–65). *Klebsiella pneumoniae* infection often striking in neonatal units as a fulminant and fatal disease, has been linked in some pediatric patients to IL12 receptor subunit beta 1 deficiency (66).

Loss-of-function mutations in interferon induced with helicase c domain 1 (*IFIH1*), a cytosolic sensor of the viral RNA, have been implicated as causative factors in lower respiratory tract infections (pneumonitis, bronchiolitis) caused by RNA viruses (67). Interestingly, the phenotype of IFIH1 deficiency is narrow (restricted to few related RNA viruses), transient (recurrence was found in one of eight patients), and organ specific (only affects the lungs).

Variants in single Ig And TIR domain containing (*SIGIRR*) have been implicated as a possible causative or facilitating factor of necrotizing enterocolitis (NEC) (68), but fulminant and infection-associated NEC (69) in infants with no other identifiable facilitating iatrogenic factor or medical condition has not been linked yet to a genetic condition.

#### Spectrum of Neonatal Infections

From a clinical perspective, newborn infants suffering from lifethreatening infections may be divided in two major groups:

(1) Newborn infants with a known medical condition. This group includes all infants admitted to a NICU (therefore exposed to nosocomial pathogens) and specifically very preterm (<32 weeks gestational age) and extremely preterm (<28 weeks gestational age) infants, infants undergoing surgery, infants with organ disease (e.g., urinary tract malformations, neurological conditions), and infants receiving medical procedures or treatments that are *per se* sufficient to explain an increased vulnerability to colonizing microorganisms. Infections in this group are multifactorial or linked to one specific known factor of vulnerability, and only a small proportion of the risk is probably explained by individual genetic variation.

(2) Otherwise healthy, full-term, or late-preterm newborn infants with no identifiable medical conditions. Severe infections in these infants occur without any apparent risk or facilitating factor and, from a host perspective, can be considered idiopathic diseases.

Most of these infections occur as isolated events (the spectrum of susceptibility is extremely narrow, in most cases restricted to a single microorganism) and rarely recur.

Some infections are almost never observed in healthy children after the first year of life or in adults. These include neonatal GBS disease, viral bronchiolitis, and rare cases of infection-related NEC in late preterm and full-term infants. Conversely, other infections are not age specific, but may occur with particular frequency and severity in the neonatal period and infancy. These include infections by *E. coli*, *Klebsiella* spp., *Listeria monocytogenes*, and other Gram-negative and Gram-positive pathogens.

The biological underpinnings of the interindividual differences in resistance and vulnerability to specific pathogens in otherwise healthy infants are currently unknown.

## Hypothetical General Model for Neonatal Infections

A general model to explain susceptibility to neonatal infections in full-term and preterm infants is lacking.

Single factors with high effect size explain some of the most severe diseases occurring in infants without known comorbidities. A prime example in neonatal medicine is the rare occurrence of rapidly progressive neonatal jaundice and kernicterus in otherwise healthy, full-term babies, which is due to neonatal hemolysis resulting from either monogenic defects (e.g., spherocytosis, G6PD deficiency) or alloimmune maternal Abs (anti-Rh, anti-ABO) (70). Conversely, hemolysis leading to kernicterus in extremely preterm infants is more likely to be multifactorial, depending on the combined contribution of common genetic polymorphisms, underlying medical conditions, iatrogenic factors, and other environmental determinants (71).

As a general observation, single-gene or single-factor disorders are more likely to underlie severe neonatal disease phenotypes in otherwise healthy full-term infants, while a multifactorial pathogenesis is more likely to explain mild-to-severe neonatal disease in the presence of comorbidities or iatrogenic factors, with severity depending on the underlying pathogenic process (**Table 1**).

Along the same lines, we here suggest that single factors with high effect size may underlie life-threatening infections in otherwise healthy, full-term, or late-preterm babies, while a polygenic/ multifactorial model may better explain the occurrence and severity of infections in very and extremely preterm infants.

Accordingly, we propose an extension of the model of the genetic architecture of infectious diseases proposed by Alcais et al. (61) to include full-term and preterm infants (**Figure 1**).

Additional host factors (maternal antimicrobial protective Abs, vaginal and breast microbiome, epigenetic, and maturational changes in the immune system) and determinants of microbial virulence may also modulate disease severity (17).

#### Table 1 | Mechanisms of disease in term and preterm infants.

*This table reports examples of hematological and non-hematological neonatal disease phenotypes that can be explained by either monofactorial or multifactorial conditions. The list is non-comprehensive, and other conditions explained by the same mechanisms include neonatal hyperthyroidism, arrhythmias and neuromuscular disorders. Monofactorial conditions, that include monogenic disorders and pathogenic maternal Abs are, in general, severe, often explain disease in full-term infants but can also underlie disease in preterm infants. Disease phenotypes linked to multifactorial conditions can be mild to severe and are generally found in infants with co-morbidities.*

*Abs, antibodies; GSPDH, glucose-6-phosphate dehydrogenase; TPO, thyroperoxidase; TSHr, thyroid-stimulating hormone receptor; TG, thyreoglobulin.*

threatening infectious diseases is mostly observed during childhood, while complex interactions between environmental influences and polygenic susceptibility (blue lines) play a more important role for infections occurring later in life. We propose a specular trend for the contribution of human genetic variants to infection susceptibility with decreasing gestational age. In newborn infants at extremely low gestational ages, exogenous factors play a major role, while host genetic defects are more likely to explain life-threatening infection in full-term, otherwise healthy babies.

#### Current Evidence Supporting the Model

In preterm infants, multiple factors are well known to contribute to both the occurrence and the severity of infections. Colonization of deep mucosal tissues by hospital-acquired microorganisms and translocation to the bloodstream is facilitated by several factors: biomedical devices (endotracheal or nasogastric tubes), invasive procedures, thin skin and mucosal layers, central catheters, total parenteral nutrition, drugs (histamine type 2 receptor-antagonists, steroids, antibiotics), delayed initiation of enteral nutrition with formula milk, associated diseases, male gender, an incomplete maturation of the preterm immune system (6, 35, 72–74), and, possibly, a weak polygenic predisposition (52).

In full-term infants, supporting evidence for a role of single host factors in determining susceptibility to infection is provided by the example of urinary tract malformation as one single, high effect-size factor, in determining susceptibility to urosepsis (75) independently of other protective or risk factors. In infants with urinary tract malformation, the effect of the alterations in urinary flow on the facilitation of urosepsis exceeds by far the effect of other potentially modulating factors.

Where no apparent determinant of higher susceptibility to infection is identified, a failure of the individual specific protective innate immune responses can be hypothesized. The failure of specific arms of the immune system that are non-redundant in the neonatal defense against a given microorganism would exceed in effect size the modulating potential of other protective factors.

The view of single-gene defects contributing to the burden of neonatal infections in otherwise healthy infants is supported by the growing body of evidence in the literature describing neonatal infections as the first phenotypic manifestation of a known conventional or non-conventional PID (62–65, 67, 76). However, the great majority of neonatal infections still need to be characterized from a host molecular perspective.

## NEONATAL GBS DISEASE

In the past years, given its predominant role among neonatal infections, neonatal GBS disease has been extensively characterized from an epidemiological standpoint. The elucidation of the mechanisms underlying neonatal vulnerability to GBS may serve as a model to understand the pathogenesis of other neonatal infectious diseases. In the following paragraphs, we discuss the unique susceptibility to GBS infection of some young infants and propose that it could be due to genetic or immune factors.

#### Epidemiology and Clinical Characteristics

Group B streptococcus is a Gram-positive, β-hemolytic bacterium frequently colonizing the human gastrointestinal and genitourinary tracts. Invasive GBS disease is extremely rare in healthy adults, with a reported incidence of 10/100,000 non-pregnant individuals (20, 21). Young infants, pregnant and post-partum women, and older adults with underlying medical conditions display higher rates of invasive disease (77).

The global incidence of neonatal GBS disease is estimated to be as high as 0.53/1,000 live births (78). The incidence is highest in infants during the first 3 months of life and dramatically declines afterward (7). Early-onset GBS disease (EOD, onset during the first 6 days of life) occurs after vertical transmission of the bacterium through ascending infection or during delivery through a GBS-colonized birth canal. Risk of EOD can be reduced by administration of antibiotics to the mother during labor. Lateonset GBS disease (LOD) (onset between 7 and 89 days of life) is thought to result from horizontal transmission in most cases. The source of GBS can been identified in some cases. Potential routes of transmission include persistent mucous membrane and skin colonization from acquisition of GBS at birth or after birth from mothers with vaginal colonization; gut colonization through ingestion of infected breast milk from mothers with or without mastitis; or the community or hospital environment (15, 79–81). No preventive strategy exists for LOD. After the introduction of *intrapartum* antibiotic prophylaxis in clinical practice, the incidence of EOD has dropped in the United States from 1.7/1,000 live births in 1993 to ~0.3/1,000 live births, but the incidence of LOD remained stable (7, 82). Clinically, neonatal GBS disease has the features of a severe, life-threatening bacterial infection with systemic disease (sepsis), often associated with organ involvement (meningitis, osteoarthritis, NEC), requiring admission to a NICU. Untreated, it is almost always fatal with multiorgan failure due to septic shock and disseminated intravascular coagulopathy. Case-fatality ratio was as high as 50% in the 1970s (7) and has now dropped to <10% (78, 82), thanks to improvements in neonatal intensive care techniques and the prompt detection of clinical signs of infection and immediate initiation of antibiotic treatment.

#### Established Risk Factors for Human Neonatal GBS Disease and Gaps in Knowledge

Approximately 50% of infants born to GBS-colonized mothers (10–30% of all pregnancies) are in turn colonized. Of these, only 1–2% develops overt EOD (7). Data on the proportion of GBSexposed infants developing LOD are lacking, but it is probably low, given a likely increase in the cumulative exposure/colonization rate with age and a concurrent decline in the incidence of GBS disease.

During the past decades, epidemiological studies led to the identification of several risk factors for EOD, including maternal colonization with GBS and bacteriuria, prematurity, chorioamnionitis, and/or *intrapartum* fever, prolonged (>18 h) premature rupture of membranes (PROM), low maternal anticapsular polysaccharide GBS Abs (29, 83–88), and GBS disease in an older sibling (89). Established risk factors for LOD include prematurity and gut colonization by the pathogen (80, 90).

In many cases, invasive GBS infection develops in otherwise healthy, full-term newborn infants, with as many as 42% of early-onset cases (91) and most late-onset cases occurring in the absence of any established risk factor. Known risk factors are therefore unable to reliably predict the occurrence of GBS disease at the individual level. Rather, they identify groups of infants enriched for determinants of susceptibility, but the nature of such determinants has remained elusive.

#### GBS Microbial Load and Virulence Factors

Fetal and neonatal exposure to the microorganism is the *sine qua non-for* neonatal colonization and subsequent infection. Heavy maternal vaginal colonization has since long been recognized as a risk factor for EOD, possibly due to greater bacterial inoculum to the lungs (7). High bacterial load in maternal milk has been linked to neonatal gut colonization and subsequent invasive LOD (80). The determinants of maternal carriage and the maternal bacterial overgrowth are poorly understood. Mild maternal disease may accompany heavy maternal colonization: maternal GBS urinary tract infection in pregnancy is considered a sign of heavy colonization (7), and maternal mastitis may be responsible for high bacterial load in maternal milk (80).

Additional microbial factors, beyond bacterial load, contribute to the development of invasive disease. Ten different GBS serotypes have been described (Ia, Ib, II–IX), based on the capsular polysaccharide antigen. Serotypes Ia, Ib, II, III, and V are most frequently found in EOD; serotype III is the most frequently isolated serotype in LOD and meningitis, but all serotypes can cause neonatal infection (19, 82, 92, 93). The capsular polysaccharide is thought to contribute to the virulence of the microorganism by aiding to escape the host immune responses. Deeper investigation on GBS isolates through multilocus sequence typing and grouping of genetically related sequence types (STs) into clonal complexes (CCs) has shown that most human isolates belong to few CCs (CC1, CC10, CC17, CC19, CC23, and CC26) (94–98) (http://pubmlst.org/sagalactiae/). The hypervirulent CC17 strains (including the hypervirulent ST-17 strain) are newborn specific. They possess the adhesin HvgA and other surface proteins conferring the ability to invade the neonatal central nervous system and are responsible for most LOD with meningitis, but are usually not responsible for adult disease (99). Strains belonging to all the six CCs have been reported in EOD (82, 98, 99).

The neonatal-specific hypervirulence of some bacterial strains and the bacterial load may explain in part the occurrence of neonatal disease. Nonetheless, individual susceptibility is not fully explained by bacterial virulence, especially in cases in which infection is caused by non-hypervirulent strains.

#### Protective Immunity to GBS

One fundamental and yet-unanswered question in the field is which are the non-redundant pathways of the innate immune system conferring neonatal protection to GBS.

Several different methodologies in *in vitro* and animal models have been used to attempt to answer this question.

Both knockout mouse and *in vitro* models of GBS infection identified a critical role for TLR and IL1 receptor signaling and/or signaling through MYD88 in bacterial clearance, TNF-mediated inflammation, septic shock, and microglia activation and neurodegeneration (100–109). Specifically, TLR2 and IL1R signaling have been shown to be both beneficial and harmful, depending on the experimental conditions (101, 110–112). A role for IL6, IL10, IL12, and IL18 has been demonstrated in mouse models of GBS infection through administration of anticytokine specific Abs (113–116).

The relevance of the studied pathways in the experimental settings may largely depend on the experimental conditions. Conversely, the non-redundant role of the studied signaling pathways in the human model in natural (as opposed to experimental) conditions still needs to be elucidated (117).

One human study suggested that a null polymorphism in sialic acid-binding immunoglobulin-like lectin 14 (*SIGLEC14*) influences human inflammatory responses to GBS in neutrophils and amniotic membranes and is possibly correlated with GBS-related preterm birth (118), but no data are available on the possible role of SIGLEC proteins in the pathogenesis of GBS infection.

#### HYPOTHESIS

Despite advances in the understanding of both the host and the microbial sides of neonatal GBS infection, currently available data are not able to fully explain neonatal susceptibility to infection at the individual level.

We hypothesize that susceptibility to neonatal GBS disease in otherwise healthy infants is due to a failure of the specific neonatal protective innate immune responses to GBS. This neonatal immunodeficiency could be either intrinsic (genetic defect in the infant) or extrinsic/environmental (interference of maternal Abs). In the next paragraphs, we present the genetic and the "maternal antibody" hypotheses of GBS disease and explain how these fit with current evidence.

#### The Genetic Hypothesis of GBS Disease

Several reports, recently reviewed (76), demonstrate that adult and neonatal GBS infection may be a phenotypic expression of both conventional (Kostmann disease, transient hypogammaglobulinemia of infancy, chronic granulomatous disease, activated phosphatidylinositol 3-kinase δ syndrome—like immunodeficiency, C2 and IgG4 subclass deficiency, and isolated congenital asplenia) and non-conventional (IRAK4 and MYD88 deficiency) PIDs. Even when occurring in the context of a nonconventional PID, neonatal GBS infection may be one of the several manifestations of a broader phenotype that, for MYD88 and IRAK4 deficiency, includes susceptibility to multiple pyogenic bacteria. Conversely, most cases of neonatal GBS disease occur as an isolated infection, indicating that the susceptibility to GBS is pathogen specific and not linked to a more general state of immunosuppression.

We hypothesize that inborn errors of the primary innate immune responses to GBS, i.e., monogenic susceptibility to GBS disease, underlie some cases of isolated neonatal GBS infection occurring in otherwise healthy neonates. The clinical and immunological phenotypes of isolated neonatal GBS disease may indeed be consistent with those of non-conventional PIDs (57): (i) GBS disease is a potentially lethal infection striking early in life; (ii) the infecting strain/serotype and its virulence factors, while accounting for some variability in the occurrence and severity of infection (92, 119, 120), are not sufficient to explain susceptibility and resistance at the individual level; (iii) the spectrum of susceptibility is extremely narrow, restricted to GBS; and (iv) in most cases, there are no immunological defects at first-line immunological studies that would be consistent with conventional PIDs. In addition, GBS infection usually strikes once in life and only rarely recurs (~1% of cases) (121). This observation is consistent with a low recurrence rate in the subset of non-conventional PIDs characterized by immunodeficiency of the protective immunity to primary infections (57).

The highest incidence of GBS disease during the first 3 months of life would be explained by the high likelihood of being exposed to GBS in the perinatal period and/or by the full penetrance of the genetic defects in this age group.

Recurrence of GBS infection concerns only a small percentage of cases, both singletons and twins, and has been linked to re-exposure to GBS through maternal milk or other sources, to inappropriate treatment, or to persistence of GBS on skin and mucosal surfaces after the first infectious episode (122–125). A genetic explanation for recurrence is also plausible. In some PIDs of protective immunity to primary infection, the genotype has been shown to influence the recurrence rate (126). Therefore, recurrence of neonatal GBS disease may indicate a more severe phenotype or represent the phenotypic manifestation of a specific genetic defect.

The occurrence of GBS disease in siblings (89), as well as the recurrence described in a consanguineous family (76), suggests that the genetic hypothesis may be a plausible explanation for some cases. Infection by poorly virulent strains, the presence of other cases with overlapping phenotypes in the family, consanguinity in the parents, recurrence and severity of the clinical signs, and slow or absent response to antimicrobials despite appropriate treatment strengthen (although their absence does not exclude) the hypothesis of a PID underlying GBS infection.

#### The "Maternal Antibody" Hypothesis of GBS Disease

The highest incidence of GBS disease is registered during the first 3 months of life, with most cases (77–78%) occurring during the first week of life (20, 21). This observation, together with a known role of GBS in prenatal disease, both prematurely and at full term of pregnancy (GBS-related stillbirth, term or preterm PROM, chorioaminionitis), suggest that a maternal factor might be particularly important for perinatal infection.

Recently, neutralizing anticytokine auto-Abs have been found in adult and pediatric patients suffering from life-threatening infections, revealing novel mechanisms of unusual susceptibility to specific pathogens (127–130). Auto-Abs against IL17 and/or IL22 have been associated with chronic mucocutaneous candidiasis; anti-IFN-γ auto-Abs with adult-onset immunodeficiency; anti-IL6 auto-Abs with recurrent skin infection; and auto-Abs against GM-CSF with pulmonary alveolar proteinosis (131).

The clinical phenotypes resulting from anticytokine auto-Abs partially (anti-IFN-γ, anti-IL6) or completely (anti-IL17, anti-GM-CSF) overlap with known monogenic conditions affecting the same pathways, demonstrating that Ab-mediated diseases may be immunophenocopies of monogenic immune disorders.

In neonates, autoimmunity is an exceedingly rare condition, but Ab-mediated disease due to transplacental crossing of maternal auto- or allo-Abs is a well-recognized and relatively frequent mechanism of organ dysfunction. This has been shown in the thyroid (congenital hypothyroidism), the blood (fetal and neonatal hemolytic disease and fetal and neonatal auto- and alloimmune thrombocytopenia), the neuromuscular junction (transient neonatal myasthenia gravis), the heart (congenital heart block due to SSA/Ro Abs), and other organs and tissues (132–137). We therefore hypothesize that neonatal GBS disease may be caused by yet-undiscovered neutralizing maternal auto-Abs or allo-Abs against components of the fetal and neonatal immune system that are non-redundant in conferring neonatal protection against GBS. The progressive decay of circulating maternal Abs in the infant plasma might then explain the decreasing incidence of infection over the first 3 months of life. Furthermore, the presence of pathogenic circulating Abs in the maternal blood would be consistent with the occurrence of mild disease in the mother (GBS-related urinary tract infection or mastitis) that is often associated with neonatal GBS disease, as well as with the well-documented higher risk of GBS-EOD in infants with a previous sibling with GBS disease (7). Finally the removal, with exchange transfusion, of pathogenic Abs from neonatal plasma could be an additional explanation to the efficacy of the procedure in infants with septic shock (138).

The proposed mechanism could in part explain neonatal GBS disease in full-term infants. Despite transplacental transfer of Abs is reduced at low gestational ages, allo- or autoimmune pathogenic maternal Abs have been demonstrated to be able to cause disease in the preterm infant or during gestation (71, 134). Therefore, the "maternal antibody" hypothesis could also explain some cases of neonatal GBS disease occurring in preterm infants.

#### Testing the Hypotheses—Possible Study Methodologies

Previous studies that addressed the role of genetics in the susceptibility to neonatal infection focused on the associations between selected common single-nucleotide polymorphisms and infectious outcomes (139). A more integrated approach including genomics, transcriptomics, proteomics, and functional studies is required to uncover the precise molecular determinants of susceptibility to specific pathogens causing neonatal infections.

*Ad hoc* studies should be designed depending on the phenotype under investigation.

Multicenter GWASs may provide some insight into the pathogenesis of suspected multifactorial infections as, for instance, those occurring in preterm infants. GWAS are currently ongoing on neonatal cohorts (54).

Exome or genome sequencing studies have potential to uncover the cause of suspected monogenic disorders. Cases should be prioritized based on the clinical profile most suggestive of a monogenic etiology, including extreme severity, consanguinity, recurrence of infection, and familial presentation. Depending on the design of the study, analysis of the trio (proband and parents) and of the family or cohort studies should be carried out to uncover the individual, rare (<1% in the general population), and functionally deleterious genetic variants that best fit the most likely genetic model (*de novo*, autosomal dominant with complete or incomplete penetrance, autosomal recessive with mono- or biallelic mutations). This approach could shed light on the pathways that are non-redundant in neonatal protection against GBS.

Functional follow-up will be required to validate candidate variants and confirm their causative role. These studies should be designed to assess the integrity of the molecular pathways affected by the mutations and determine how they are relevant to the neonatal immune responses in primary cells and/or immortalized cell lines.

Laboratory experiments will be also needed to investigate the possible interfering effect of maternal plasma on the neonatal immune responses. The laboratory tests could include cytokine production assays, detailed analyses of RNA (transcriptome analysis) and protein expression in *ex vivo* samples (blood collected during sepsis), and *in vitro* experiments (stimulation of patient and control cells with different ligands, cell differentiation

#### Table 2 | Comparison of neonatal hemolytic disease and neonatal group B streptococcus (GBS) disease.

*EOD, early-onset GBS disease; LOD, late-onset GBS disease. Incidence is expressed as number per 1,000 (or 100,000) live births.*

assays) in the presence of maternal or control plasma. Specific assays should be used for the detection of specific Abs in the maternal and in the perinatal plasma.

Ultimately, these experiments should aim at demonstrating a causative link between the molecular findings, the observed cellular phenotypes, and the patient's clinical phenotype.

## CONCLUSION

Transient susceptibility to a narrow range of infections during the neonatal age may be explained by inborn errors of immunity, in the context of a relatively immature, non-redundant immune system. The early recognition of a PID as an essential contributing factor to a severe neonatal infection is clinically very relevant, as it may change the management and allow the referral of the patient to the clinical immunologist for specific follow-up and family counseling.

In parallel fields, the discovery of concurrent genetic and auto-/alloimmune mechanisms for several neonatal diseases has dramatically changed practice, as exemplified by the development of highly effective screening and diagnostic procedures for neonatal hemolysis, which reduced the incidence of fetal erythroblastosis and neonatal bilirubin encephalopathy by two orders of magnitude, from ~1/1,000 to ~1/100,000 live births (**Table 2**) (140). Similar observations can be made for congenital hypothyroidism and other common and rare neonatal diseases (**Table 1**).

Current prevention efforts, although invaluable for neonatal health, only had a limited impact on the global incidence of neonatal infections (9, 141) (**Table 2**). A more complete understanding of the mechanisms underlying the interindividual variability in the neonatal innate immune responses to pathogens is required to develop highly effective, pathogen-specific and individualtailored preventive protocols.

## AUTHOR CONTRIBUTIONS

AB conceived the manuscript; conducted the literature search; and drafted, edited, and approved the final version of the paper. MS participated in the discussion of ideas, helped with the writing, revised critically, and approved the final version of the manuscript. JF participated in the discussion of ideas, edited, revised critically, and approved the final version of the manuscript.

## ACKNOWLEDGMENTS

The authors wish to thank the colleagues who collaborate with them on ongoing projects on GBS disease and supported them with suggestions and critical discussions. From San Matteo Hospital, Pavia (Italy), Neonatal Intensive Care Unit: Stefania Longo and Iolanda Mazzucchelli. From Ecole Polytechnique Fédérale de Lausanne (Switzerland), Fellay lab: Samira Asgari, Nimisha Chaturvedi, Yann Dubois, Christian Hammer, Thomas Junier, Olivier Naret, Petar Scepanovic, and Christian Thorball. From the Swiss Pediatric Sepsis Study (SPSS): Luregn Schlapbach (Mater Children's Hospital, Brisbane, Australia) and Eric Giannoni (Centre hospitalier universitaire vaudois, Lausanne, Switzerland). From the Rockefeller University, New York (USA): Janet Markle. The authors are obliged to Jean-Laurent Casanova (Laboratory of Human Genetics of Infectious Diseases, Rockefeller University, New York and Institut Imagine, Paris, France) for his precious advices and hints on human genetics of infectious diseases. The authors are grateful to all the colleagues from neonatal and pediatric units who struggle with them in the attempt to understand the molecular bases of neonatal infections. The authors' most heart-felt thanks are due to the little patients and their families, who are their most important collaborators.

## FUNDING

The authors did not receive any specific financial support for the writing of the submitted work. JF is the recipient of an SNF Professorship from the Swiss National Science Foundation (PP00P3\_133703).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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