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Cytosolic dsRNA improves neonatal innate immune responses to adjuvants in use in pediatric vaccines

Abstract Pattern recognition receptors (PRRs) of the innate immune system represent the critical front‐line defense against pathogens, and new vaccine formulations target these PRR pathways to boost vaccine responses, through activation of cellular/Th1 immunity. The majority of pediatric vaccines contain aluminum (ALUM) or monophosphoryl lipid A (MPLA) as adjuvants to encourage immune activation. Evidence suggests that elements of the innate immune system, currently being targeted for vaccine adjuvanticity do not fully develop until puberty and it is likely that effective adjuvants for the neonatal and pediatric populations are being overlooked due to modeling of responses in adult systems. We recently reported that the activity of the cytosolic nucleic acid (CNA) sensing family of PRRs is strong in cord blood and peripheral blood of young children. This study investigates the function of CNA sensors in subsets of neonatal innate immune cells and shows that myeloid cells from cord blood can be activated to express T cell costimulatory markers, and also to produce Th1 promoting cytokines. CD80 and CD86 were consistently up‐regulated in response to cytosolic Poly(I:C) stimulation in all cell types examined and CNA activation also induced robust Type I IFN and low levels of TNFα in monocytes, monocyte‐derived macrophages, and monocyte‐derived dendritic cells. We have compared CNA activation to adjuvants currently in use (MPLA or ALUM), either alone or in combination and found that cytosolic Poly(I:C) in combination with MPLA or ALUM can improve expression of activation marker levels above those observed with either adjuvant alone. This may prove particularly promising in the context of improving the efficacy of existing ALUM‐ or MPLA‐containing vaccines, through activation of T cell‐mediated immunity.


INTRODUCTION
Along with clean water, the development of vaccines has widely been lauded as one of the world's greatest health interventions. According to the WHO, immunization currently prevents 2-3 million deaths every year from diseases like diphtheria, tetanus, pertussis, influenza, and measles. However, it is also estimated that approximately 20 million people, mostly infants, have insufficient access to vaccines. 1 In areas of the developing world, including areas of conflict, a drop-off rate of up to 22% is observed between dose 1 and dose 3 of diphtheria, tetanus, and pertussis vaccine and a rate of 46% between dose 1 and 2 of measlescontaining vaccines. 2 One way of improving access to vaccines, and thereby decreasing deaths associated with some of these infectious diseases, would be to cut down on the need for boosters by making vaccines more efficient, and also tailoring vaccines and adjuvants for deployment in very early life when contact with a healthcare worker is more accessible.
Vaccines in their simplest form are comprised of an antigen alone.
Historically, inactivated or live-attenuated whole cell vaccines did not need additional adjuvants to boost the immune response, as bacterial or viral signatures within the whole cell vaccine activated the immune system very effectively. More recently, a move toward highly purified subunit vaccines has improved the safety of vaccines. One downside to these highly purified vaccines is a lack of immunostimulatory properties, leading to a need for addition of adjuvants, which can encourage immune memory and also allow for lower doses of vaccines to be given. 3 Empirically, and quite by accident, aluminum (ALUM) was found to improve vaccine responses and encourage immunologic memory 4 and has been the adjuvant of choice for over 90 years. ALUM is still the most common adjuvant in current vaccine formulations. The mechanism of action of ALUM has been widely investigated and debated. Several mechanisms have been proposed to explain the adjuvant activity of ALUM, including the "depot effect," enhanced antigen uptake by antigen presenting cells, NLPR3 inflammasome activation, and others. 5 ALUM adjuvants effectively drive humoral immunity through various mechanisms including IL-33 production, 6 but induce low levels of cellular immune responses, possibly due to the ability of ALUM to inhibit IL-12 and drive IL-10 in some instances. 7,8 In recent years, there has been a move toward developing adjuvants that are better inducers of proinflammatory cytokines and Type I IFN, in the hope of generating a stronger Th1 response. 9 This increased Th1 response would in turn lead to increased cellular immunity, which is particularly beneficial in combating intracellular infections. TLR ligands and other pathogen-associated molecular patterns became an obvious target due to their ability to induce Type I IFN and proinflammatory cytokine production. 10 Of note, monophosphoryl lipid A (MPLA), a TLR4 agonist, formulated with ALUM triggers an enhanced Th1 response and is approved for use in Fendrix (Hepatitis B) and Cervarix (Human Papilloma Virus) in Europe. Previous literature has shown that many TLR responses, including TLR3, are attenuated or absent in neonatal and pediatric populations. [11][12][13][14][15][16] One notable exception to this are TLR7/8 responses, which tend to remain intact and are being investigated for their adjuvant properties. 17,18 Interestingly, combinations of TLR agonists with other innate immune activators such as C-type lectin receptor agonists have shown promise in activating neonatal immune cells. 19 At present, most neonatal or pediatric vaccines contain either ALUM or MPLA as an adjuvant, and these may not be optimal for use in neonatal or pediatric populations. Development of pediatric-relevant, effective adjuvants, or combinations of adjuvants could increase efficacy and decrease the need for boosters or delayed vaccination.
Poly(I:C) is a synthetic dsRNA sequence that is associated with viral infections. It can activate TLR3 on the surface of immune cells 20 or, when transfected into cells, can activate cytosolic nucleic acid (CNA) sensors such as RIG-I. 21 In both cases, it induces a robust Type I IFN response in adults. Previous literature has shown that TLR3 responses to Poly(I:C) are attenuated in neonates; however, we have previously shown that intracellular Poly(I:C) responses are intact in mixed populations of neonatal immune cells. 25 Slavica et al. 26 have previously shown that TLR3 mRNA levels are notably decreased in cord blood mononuclear cells (CBMCs) compared with adult blood mononuclear cells, whereas RIG-I mRNA levels are similar. TLR3 is also absent at the protein level in isolated cord blood NK cells 26 ; however, protein expression levels have not been investigated on cord blood monocytes, monocyte-derived macrophages (MDMs), or monocyte-derived dendritic cells (moDCs).
Targeting different mechanisms that activate the immune response, through combining adjuvants, may result in a more effective or longer lasting immune response. 27  with MPLA or ALUM can induce CD4+ T cell proliferation. This implies that Poly(I:C) can activate T cells both directly and through myeloid cells. Although further work is necessary, these results indicate that Poly(I:C)/dsRNA is a very favorable candidate adjuvant for inclusion in pediatric vaccine formulation.

Participants
Umbilical cord blood samples were obtained from term births following normal pregnancy, labor, and delivery at National Maternity Hospital (NMH), Holles St, Dublin 2. All infants had an uncomplicated postnatal course and Apgar scores of 9 at 5 min. Ethics approval was obtained from the Ethics Committees of NMH and informed consent was obtained from each participant.

Cell viability
A lactate dehydrogenase (LDH) cytotoxicity assay kit (Pierce) was used to measure cell death in response to stimulation as per manufacturer's instructions. The absorbance was read at 490 nm on a 96 well plate spectrophotometer and background (absorbance at 680 nm) was subtracted.

Flow cytometry
Monocytes, MDMs, moDCs, or T cells were assessed for purity or were

RIG-I is abundantly expressed and functional on cord blood monocytes, MDMs, and moDCs, whereas TLR3 is absent
Our previous work showed that CBMCs had intact responses to CNA sensor activation through Poly(I:C). 25  are predominantly due to CNA sensor activation rather than TLR3 activation.

Cytosolic Poly(I:C) is more efficient at inducing Type I IFN production in human neonatal innate immune cells than adjuvants currently used in pediatric vaccines
Cytosolic Poly(I:C) activation of CNA receptors is a potent activator of Type I IFN responses in neonatal cord blood cells. 25 We were interested in comparing the activation of a variety of neona-  Given the low responsiveness of the neonatal cells to ALUM, we assayed IL-33, which is known to be induced in the peritoneal lavage of mice injected with ALUM. 6 Though not significant, there was a clear induction of IL-33 in neonatal monocytes in response to ALUM, 5-fold over unstimulated, leading us to the conclusion that ALUM was functioning in our neonatal cell systems but simply not to anticipated levels ( Figures S3(A)-S3(C)). To further demonstrate that ALUM was active in our experimental setting, we carried out inflammasome activation assays in adult PBMCs and observed significant IL-1β production in response to ALUM, in LPS-primed PBMCs ( Figure S3(D)).

Cytosolic Poly(I:C) increases costimulatory marker expression on human neonatal monocytes and MDMs in combination with MPLA or ALUM
One downstream consequence of Type I IFN production is the upregulation of costimulatory molecules, such as CD80 and CD86, on myeloid cells. 28 CD80 and CD86 are found on the surface of many immune cells and work together to bind to CD28 on T cells to provide a costimulatory signal to these T cells. 29 Having examined cytokine production from neonatal myeloid cells in response to Poly(I:C), MPLA, and ALUM, we were interested in looking at the expression of these important costimulatory markers on neonatal cells in response to activation with these adjuvants. CD80 expression on moDCs was increased following Poly(I:C) transfection compared with unstimulated cells, though not significantly (p = 0.07). When moDCs were stimulated with combinations of MPLA or ALUM with Poly(I:C), a modest but not significant increase in CD80 expression was seen (Figure 6(B)).
CD83 is a marker of DC maturation, expressed on functionally mature DCs, and is involved in promoting prolonged activation of CD8+ T cells. moDCs treated with MPLA showed an increase in expression of CD83 and this increase was enhanced when MPLA was combined with Poly(I:C), though Poly(I:C) induced very little CD83 expression alone. ALUM did not appear to have any effect on CD83 levels alone, but a significant increase was observed when ALUM was combined with Poly(I:C) (Figure 6(C)).
In the case of HLA-DR, a molecule essential for antigen presentation of peptide to the TCR, Poly(I:C) and MPLA, but not ALUM, induced a significant increase in expression on moDCs. The combination of Poly(I:C) with MPLA also induced a significant increase in HLA-DR expression (Figure 6(D)). CD40 expression on moDCs was also analyzed and whereas some increase in expression was seen with Poly(I:C), MPLA, or combinations of Poly(I:C) with MPLA or ALUM, there was no significant increase with any stimulation (Figure S4(B)).  (Figures 7(A) and 7(B)).

The interaction between moDCs and T cells plays an instructive
These data confirm that Poly(I:C), in combination with established adjuvants, can act on innate immune cells to enhance their capacity to initiate CD4+ T cell responses. Interestingly, no differences in cytokine levels produced by CD4+ T cells were detected in this assay (data not shown) therefore to determine whether tested adjuvants could also influence qualitative responses we used direct stimulation assays.
When treating T cells directly, we unexpectedly found that Poly(I:C), or MPLA alone, was capable of significantly increasing the amount of IFNγ produced from TCR-activated isolated CD4+ T cells (Figure 7(C)).
Surprisingly, no such effects were observed when both stimuli were used in combination. Over recent years, Th17 responses have been implicated in many infectious diseases, along with many autoimmune disorders and recognition of the significance of this proinflammatory cytokine in vaccine immunology has steadily grown. 30 Interestingly, These data demonstrate that potential vaccine adjuvants under investigation have the capacity to directly stimulate activated neonatal CD4+ T cell responses.

DISCUSSION
PRR stimulation is now an accepted mechanism for immune activation by potential vaccine adjuvants. 9 An example of the successful targeting of PRR pathways for adjuvanticity is activation of TLR4 through MPLA, which is now a component of several licensed vaccines including Cervarix. 31 The issue arising when targeting many PRR pathways as potential adjuvants is their compromised efficacy at activating the immune system in newborns and young children . 14 shown. 47  Our previous work has shown that mixed populations of cord blood cells produced IFNγ in response to Poly(I:C). In this study, we examined myeloid cells for Poly(I:C)-induced IFNγ production and found that IFNγ production was below the level of detection (∼2 pg/ml, data not shown). Interestingly however, Poly(I:C) can induce IFNγ from naïve CD4+ T cells directly. Poly(I:C) alone was also able to induce some IL-17A production from naïve CD4+ T cells. When Poly(I:C) and MPLA were combined to treat CD4+ T cells, IFNγ was no longer induced. The decreased IFNγ response in the presence of Poly(I:C) and MPLA could be due to the inhibitory effect of IL-10 produced in response to MPLA.
While IFNγ was no longer induced, IL-17A production was significantly increased with the combination of adjuvants. Historically, IL-17A has been deemed very important in the mucosa but IL-17A also plays a critical role in vaccine-induced immunity against bacterial infections. 30 The role of IL-17A in the context of vaccines against bacterial infections seems to be in the induction of chemokines that recruit Th1 cells, neutrophils, macrophages, and results in enhanced phagocytosis. 48,49 Although it has previously been shown that IL-10 can suppress Th17 cytokines secreted by T cells, 50