of November 23, 2014.This information is current as

Challengesfor Use in Neonates: Opportunities and Vaccines against Respiratory Viral Pathogens

Martha A. Alexander-Miller

http://www.jimmunol.org/content/193/11/5363doi: 10.4049/jimmunol.1401410

2014; 193:5363-5369; ;J Immunol 

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Vaccines against Respiratory Viral Pathogens for Use inNeonates: Opportunities and ChallengesMartha A. Alexander-Miller

The first six months of life reflect a time of high suscep-tibility to severe disease following respiratory virus in-fection. Although this could be improved significantlyby immunization, current vaccines are not approvedfor use in these very young individuals. This is the resultof the combined effects of poor immune responsivenessand safety concerns regarding the use of live attenuatedvaccines or potent adjuvants in this population. Vac-cines to effectively combat respiratory viral infectionideally would result in robust CD4+ and CD8+ T cellresponses, as well as high-affinity Ab. Inclusion of TLRagonists or single-cycle viruses is an attractive approachfor provision of signals that can act as potent stimula-tors of dendritic cell maturation, as well as direct acti-vators of T and/or B cells. In this article, I discuss thechallenges associated with generation of a robust im-mune response in neonates and the potential for adju-vants to overcome these obstacles. The Journal ofImmunology, 2014, 193: 5363–5369.

Respiratory infections are one of the leading causesof morbidity and mortality throughout the world.Among the most prevalent are infections with re-

spiratory syncytial virus (RSV), rhinovirus, and influenza virus(1). These infections are particularly problematic for infants,resulting in increased morbidity and mortality compared witholder children and adults. There are an estimated 11.9 millionepisodes of severe acute lower respiratory tract infection(ALRI) in young children each year (2). Children under 1 y ofage account for 6.4 million instances of severe ALRI andnearly 3 million cases that are grave enough to be consideredvery severe (2). Further, children ,12 mo of age exhibit a 3-fold increase in the rate of fatality following infection com-pared with children 12–59 mo of age (2). Not surprisingly,the likelihood of severe disease decreases as age increases. Forexample, in the case of RSV infection, approximately half ofchildren requiring hospitalization are #3 mo of age (3), andinfants younger than 27 d have the highest incidence ofALRI-associated disease (2). Together, these findings dem-onstrate the extreme susceptibility of the newborn to diseasecaused by respiratory pathogens.

The increased disease severity associated with respiratoryinfection in infants is the result of the naive status of theseindividuals, as well as the reduced ability of the immune systemto respond to infection. Defects in infant immunity span bothinnate and adaptive components, both of which are criticalcontributors to immune-mediated clearance of infection (4–6).Reported defects in the innate response include reduced mi-gration, phagocytosis, and bactericidal activity (6, 7). Adap-tive immune defects include decreased cytokine productionand costimulatory molecule expression by APCs, reducedT cell sensitivity following ligand engagement, decreasedT cell repertoire diversity, decreased T cell effector function,a bias toward Th2 development, and impaired B cell dif-ferentiation and survival (4–7) (Fig. 1).Effective control of respiratory virus infection begins with

a robust innate antiviral response that is dominated by theproduction of type I IFN. The production of this critical innateantiviral mediator is diminished in neonates as a result ofdecreased production on a per-cell basis, as well as a reductionin the number of plasmacytoid dendritic cells (DCs) (3, 8, 9),the cell type specialized for high-level type I IFN production.Beyond type I IFN, the innate response to virus infection thatresults in the production of cytokines and chemokines thatpromote inflammation and immune cell recruitment is de-creased in infants (10).Innate immune responses to virus infection are dependent on

activation through TLRs, as well as cytoplasmic innate sensors(e.g., RIG-I and MDA-5). Both TLR- and RIG-I–mediatedresponses are impaired in neonates (3, 9, 11–13). The reducedactivity of these innate sensors has implications for the gener-ation of the adaptive immune response because they areimportant mediators of DC maturation that promotescompetence for naive T cell activation. Specifically, DCs fromneonates produce low amounts of IL-12 and are impaired intheir ability to upregulate costimulatory molecules (e.g., CD80and CD86) following exposure to virus-derived signals (9).These deficiencies are the first obstacle in the generation of anefficacious adaptive immune response in the neonate.In addition to the impaired function of DCs, T lymphocytes

from neonates exhibit inherent defects in their ability to un-dergo activation and differentiation (14–16). Reported defectsinclude reduced levels of the signaling molecules lck and

Department of Microbiology and Immunology, Wake Forest School of Medicine,Winston-Salem, NC 27101

Received for publication June 2, 2014. Accepted for publication September 2, 2014.

This work was supported by National Institutes of Health Grant R01AI098339.

Address correspondence and reprint requests to Dr. Martha A. Alexander-Miller, De-partment of Microbiology and Immunology, Wake Forest School of Medicine, Room

2E-018, Wake Forest BioTech Place, 575 North Patterson Avenue, Winston-Salem, NC27101. E-mail address: marthaam@wakehealth.edu

Abbreviations used in this article: ALRI, acute lower respiratory tract infection; BCG,bacillus Calmette–Guerin; DC, dendritic cell; RSV, respiratory syncytial virus.

Copyright� 2014 by TheAmerican Association of Immunologists, Inc. 0022-1767/14/$16.00

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ZAP-70 (17), as well as a decrease in AP-1–mediated tran-scription (18). The combined deficiencies in DC maturationand T cell responsiveness are likely contributors to impairedT cell responses observed in vivo following infection or vac-cination (19, 20).Ab responses are also significantly decreased in neonates (4,

21). Ab responses in young infants are largely IgM, with IgGproduction generally weak for the first year of life (22). Al-though increased relative to IgG, IgM responses are also im-paired, as exemplified by RSV infection of human infants inwhom both IgM and IgG responses are poor (23). Similarfindings were reported in murine models (24). Isotype anal-ysis showed a skewing toward IgG1, indicating a Th2-biasedresponse (24). Important contributors to the poor Ab re-sponse in infants are impaired accessory cells [i.e., T follicularhelper cells (25) and follicular DCs (26)], as well as inherentdefects in B cell survival and differentiation (27). A potentialcontributor to the latter is the reduced expression of BCMAand BAFF-R on neonate B cells (28). Both of these receptorsbind to BAFF, with BCMA having an additional ligandAPRIL (29). Engagement of BAFFR or BCMA on B cellspromotes survival through upregulation of antiapoptoticbcl-2 family members, together with downregulation of theproapoptotic factors bim and bad (30). The survival of plas-mablasts and differentiation into long-lived Ab-secreting cellsin the neonate are likely hampered by decreased levels ofAPRIL and BCMA (31). In the context of influenza virusinfection, the absence of BAFF and APRIL was shown toresult in an overall reduction in antiviral IgG (32). Whetherthere are defects in the initial activation of neonatal B cells isa matter of debate, although there are reports from studies ofcord blood cells that suggest competence in this arena (33).

Infant immune response to vaccination against respiratory viruses

Although data from the analysis of respiratory virus vaccineresponses in very young infants are limited, what are availablesupport the inadequacy of current vaccine approaches for usein this population. These studies predominantly analyzed theAb response, undoubtedly for practical reasons associated withsampling in this population. Delivery of the trivalent inacti-vated influenza vaccine in infants between 3 and 5 mo of ageresults in poor generation of Ab (34, 35). An initial dose ofvaccine was not capable of inducing seroconversion for most

strains (as defined by a fold-fold increase in Ab) (34). Thislow responsiveness was not the result of maternal Ab, becauseall individuals had prevaccination titers ,1:8. A second doseresulted in a seroconversion rate of 27–32% for H1N1 strainsand 17–93% across H3N2 strains. Not surprisingly, a corre-lation was observed between age and the rate of conversion,with older infants converting at a higher rate than youngerinfants (34). In a second study, conversion was assessed fol-lowing completion of two doses of vaccine, with a reportedconversion rate of 42–43% for H1N1 and 39–67% forH3N2 strains (35). For comparison, published studies assess-ing responses in older children (11–16 y) reported that .90%of individuals experienced a 4-fold increase in titer aftera single vaccination (36).Vaccine responsiveness in young infants also has been

evaluated for measles and mumps. This vaccine is routinelygiven at 12 mo of age. Administration at an earlier time(i.e., 6 or 9 mo of age) resulted in a significantly reduced Abresponse (37). A parallel impairment in the T cell responsealso was observed, with virus-specific cells exhibiting a re-duction in the amount of IFN-g produced in response tostimulation (37). Importantly, as with influenza virus, thereduced responsiveness in these individuals could not beaccounted for by the presence of maternal Ab. The re-duction in responsiveness in 6- and 9-mo-old infants sug-gests that the immune system continues to be impaired tosome extent throughout the first 9 mo of life. It is difficultto state definitively when the immune system of the infantreaches full maturity. In this regard, four doses of the oralpolio vaccine given to infants resulted in reduced IFN-g–producing T cell responses compared with adults receivinga single dose (38), whereas infants administered bacillusCalmette–Guerin (BCG) vaccine had responses similar toadults (39). These findings suggest that the time at whichresponses in children can approximate adults varies withthe nature/strength of the challenge (27). Although there isoften significant impairment in immunity in young infants,the ability to obtain some degree of responsiveness fol-lowing vaccination and instances in which responsiveness isrelatively robust offer hope that the provision of additionalstimulatory agents in the context of vaccination may be ableto boost the response to levels that are protective in theseindividuals.

FIGURE 1. Neonates exhibit multiple adaptive

immune defects that contribute to poor responses

following infection or vaccination. Potent adaptive

immune responses are dependent on the capacity

of DCs to undergo maturation, together with the

robust activation, differentiation, and survival of

T and B cells. Defects encompassing a broad range

of these attributes have been reported in cells

from neonates. As a result, responses following in-

fection and vaccination are qualitatively and quan-

titatively reduced in these individuals compared with

adults.

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Desirable attributes of immune responses elicited by respiratory virus–specific vaccines and lung-specific challenges

The goal of vaccination is the generation of long-lived pro-tective immunity. In the case of respiratory virus infection, thisideally includes the generation of high-affinity neutralizing Ab,central memory T cells, and lung-resident memory cells. Thisis a tall order in the context of adult vaccination and is evenmore challenging in the neonate.We have an increasing appreciation for the importance of

lung-resident memory T cells in the control of virus infection(40–42). The presence of these cells is the result of thecombined effects of production in the local lymph node andin BALT. Although the benefit of vaccination strategies thatcould induce local immune responses in BALT is clear,approaches that could achieve this goal are less so. Lung-targeted delivery of a vaccine is certainly a difficult under-taking in a neonate. A further point of caution is that,although induction of BALT to facilitate immune responsesis potentially advantageous, it is unclear how/whether thepresence at very early ages of the strong inflammatory signalsnecessary for the induction of BALT (43) would impact es-tablishment of regulatory processes in the lung. For example,an overly robust inflammatory challenge was shown to inducelong-term changes in airway macrophages (i.e., these cellsexhibit decreased responsiveness to TLR agonists, reducedphagocytosis, and increased production of IL-10) (44). Thesechanges can be conferred to newly recruited macrophages,thereby maintaining altered function for extended times (44).This is clearly an area in which additional studies are neededto assess the potential for direct targeting of the lung forvaccination in this population.In standard vaccine-delivery approaches, resulting memory

T cells must be recruited to the lung airway following reac-tivation in the draining lymph node, an event that is a criticalcomponent of effective clearance and protection (45). In thisregard, analysis of influenza infection of mouse neonatesshowed that T cells were impaired in their ability to migratefrom the interstitium to the airways (46). Thus, efficienttrafficking of effector T cells may pose an added obstacle toefficacious responses following infection.An additional challenge in the lung is the apparent ability of

the lung environment to negatively regulate effector cell func-tion. The loss of function in effector cells was reported to occurin the context of a number of infectious processes, includingRSV (47, 48), parainfluenza virus 5 (49), and murine pneu-movirus (50). Our work in this area suggests that this is anintrinsic property of the lung (51, 52). Functional inactivationis observed even in the face of the inflammatory environmentpresent following infection. That said, negative regulation isless apparent at early times postinfection when viral load andinflammation are high, suggesting that the presence of an in-flammatory environment may dampen the inhibitory effect(47, 49). The extent to which this effect occurs in the lungs ofneonates has not been explored. However, it seems likely it willbe in play or even enhanced given the propensity for negativeregulation of the immune response in this population.

Approaches to overcome the reduced responsiveness to vaccination inneonates

Virus infection often results in long-lasting, protective im-munity. Arguably, the closest that we have come to achieving

this goal in the context of vaccination against viral pathogens isthrough the use of live attenuated constructs. This approachhas resulted in the eradication of smallpox and large reductionsin infections with viral pathogens previously associated withchildhood disease (e.g., measles, mumps, rubella, and chickenpox). The success of live attenuated vaccines is likely a con-sequence of elicitation of both robust Ab and CD8+ T cellresponses (53–55), a goal that has not yet been realized withinactivated/subunit vaccines. However, although the ability toachieve both humoral and cell-mediated immune responses ishighly desirable, the use of live attenuated constructs inneonates is undesirable because of safety concerns, includingthe potential for undiagnosed immune deficiencies. Conse-quently, alternative approaches that can elicit both arms of theimmune response, combined with a superior safety profile, aresorely needed.Generation of potent cell-mediated and humoral immune

responses requires the participation of multiple cell types: ata minimum, DCs, CD4+ T cells, CD8+ T cells, and B cells.Optimal activation of these populations can be facilitated bymediators that act directly on individual cells (direct), as wellas those that modulate function in accessory cells that sub-sequently provide activating signals to T cells in the form ofcell surface molecules or cytokines (indirect). For example,vaccines that target DC maturation will promote T cell ac-tivation. However, T and B cells also can receive direct signalsvia stimulatory cell surface receptors. Targeting T and B cellsby the combination of direct and indirect activation signals inthe context of vaccination may aid in overcoming defectsassociated with neonatal immune responsiveness.Vaccine responsiveness can be significantly improved by

inclusion of adjuvants. Approved adjuvants in the UnitedStates and/or Europe include aluminum salts, oil-in-wateremulsions (MF59, AS03, an AF03), virosomes, and AS04(monophosphoryl lipid A preparation with aluminum salt)(56). Excellent reviews on the actions of adjuvants were re-cently published (56, 57). An area of intense focus in adjuvantdevelopment is the use of TLR agonists. TLRs are sensors ofpathogen associated molecular patterns that survey the envi-ronment through residence at both the cell membrane and theendosome. Ten TLRs have been characterized in humans(58). Cell surface TLRs, including TLR1/2 and TLR2/6heterodimers, together with TLR4, TLR5, and TLR10 homo-dimers, recognize a variety of pathogen associated molecularpatterns associated with bacterial or viral infection. Endo-somal TLRs (TLR3, 7, 8, 9) sense pathogen-derived nucleicacids and are key players in the context of virus infection (59).Respiratory viruses contain ligands for multiple TLRs, a por-tion of which are shared across many viruses and, thus, areattractive for vaccine development. For example, influenzavirus and RSV, pathogens of major clinical importance inneonates, activate TLR3 and TLR7 (60, 61). Ligands havebeen identified for each TLR, with the exception of TLR10.Not surprisingly, work is underway to exploit these ligands inthe context of vaccination.Individual TLRs can be widely distributed on immune cells.

For example, TLR7 is expressed by human T cells (62), B cells(63), and plasmacytoid DCs (64), and TLR8 is expressed onmonocytes/macrophages and myeloid DCs (65, 66). As such,a TLR7/8 agonist would provide both direct and indirectactivation signals for the elicitation of T and B cells following

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vaccination. In contrast, in humans, TLR5 is expressed onDCs and T cells but not B cells (67). As a result, agonists forthis receptor would deliver activating signals to a more limitednumber of cells. Thus, the choice of TLR agonist will de-termine the cells targeted during vaccination.The importance of TLR engagement in the generation of

efficacious vaccine responses is suggested by studies of Polackand colleagues (68). The enhanced respiratory disease thatoccurred following vaccination of children with formalin-inactivated RSV was a major setback in vaccine develop-ment. The low-avidity Ab generated was found to be theresult of deficient TLR stimulation (68). These data stronglysupport the critical role for TLR in the generation of pro-tective immune responses in the context of vaccination.There are a number of ongoing trials to assess the efficacy

of TLR agonists in the context of adult vaccination withpromising results. The question then arises whether this isa valuable avenue of investigation in the context of neonates.Our understanding of the neonate response to TLR engage-ment is far from complete. Available data suggest that theneonate expresses TLRs at levels that are relatively similar tothose of adults (69, 70). Despite this, many studies using cellsfrom cord blood or neonatal mice reported hyporesponsive-ness to TLR engagement. Analysis of DCs from neonatesrevealed impaired maturation, as measured by cytokine pro-duction and upregulation of the costimulatory moleculesCD80 and CD86 in response to TLR agonists (9, 11, 12).The decreased responsiveness is associated with reduced ex-pression of MyD88, suggesting that impaired signaling maycontribute to the failure to undergo appropriate maturation(69). Although admittedly decreased, the ability to promotesome degree of maturation following TLR engagement allowsfor the possibility that increasing the strength of signalingthrough these receptors may be able to overcome the observeddeficits. In support of this, there are instances in which in-creasing the level of TLR agonist results in cytokine pro-duction that is similar to levels observed in adults (12, 71).Flagellin is a potent TLR5 agonist that has shown great

promise as an adjuvant in the context of adult vaccination (67);it is currently in clinical trails as a component of vaccinesagainst plague and influenza. In human adult-derived cells,TLR5 was found to both promote efficient maturation ofDCs (72) and increase activation of T lymphocytes (62). Inthe context of the neonate, when purified cord blood T cellswere treated with flagellin together with TCR ligation, in-creased proliferation and higher levels of IFN-g and lyticcomponents were observed (73). Flagellin exposure also wasreported to induce CD45RO and CCR4 expression in cordblood–derived T cells, demonstrating that TLR stimulationcan trigger maturation and may alter trafficking in T cells(74). Surprisingly, adult T cells did not show these samechanges, suggesting inherent differences in neonate and adultresponses. The efficacy of flagellin as an adjuvant in the set-ting of infant vaccination has been explored in young (4–6-mo-old) monkeys (75). Inclusion of flagellin in a vaccineagainst Pseudomonas aeruginosa resulted in increased Abresponses and significantly reduced pathogen loads followingchallenge (75). Thus, flagellin holds promise, and furtherstudies to evaluate its usefulness in neonates are merited.There is evidence that TLR2 agonists also may be beneficial

in neonates. Inclusion of the TLR2 agonist Pam3Cys increased

activation of T cells in this population (73). Further, selectTLR ligands can induce maturation of APCs, approaching thelevel observed in adults (12, 76). For example, the TLR8agonist 3M-002 induces potent upregulation of CD40,CD80, CD83, and CD86, as well as production of the Th1-polarizing cytokine IL-12p70, in cells from neonates (12). Acontributor to the effectiveness of TLR8 agonists in the contextof neonate-derived cells appears to be the resistance of thispathway to inhibition by adenosine (12), a known inhibitoryimmune modulator in the blood of newborns (77).An alternative strategy for increasing the efficacy of these

immune modulators is the delivery of multiple TLR agonists.Simultaneous engagement of several TLRs has been shown tochange DC maturation qualitatively and quantitatively (78, 79).T cells derived from human cord blood stimulated concurrentlywith TLR2 and TLR5 agonists underwent greater proliferationand cytokine production compared with cells stimulated witheither agonist alone (73). Although unknown, studies of tu-berculosis vaccination may suggest that this approach is useful inthe context of newborn vaccination. The tuberculosis vaccine(BCG), which is routinely delivered within 48 h of birth, is oneof a limited number of vaccines that has shown success in veryyoung infants. BCG contains ligands for five distinct TLRs(TLR1, TLR2, TLR4, TLR6, and TLR9) (80). It is tempting tospeculate that the engagement of multiple TLRs may bea contributing factor to the ability of this vaccine to induceimmune responses in these very young infants.Although TLR agonists hold great promise as adjuvants,

other approaches are under active investigation. Given theremarkable success of live attenuated viruses as vaccines, mucheffort has focused on the production of viral constructs that canachieve similar levels of immune stimulation while obviatingthe safety concerns associated with replicating virus. Onepromising area is the generation of single-cycle virus con-structs. These constructs enter cells and produce significantamounts of viral RNA, as well as protein (81). This results ineffective Ag presentation, as well as DC maturation and in-flammatory cytokine production (82). Although not yettested, it seems likely that a contributor to the efficacy ofsingle-cycle vaccines is the induction of innate immune responsessimilar to those resulting from virus infection (i.e., activation ofendosomal TLRs [TLR3, TLR7, and TLR8] and RLR [e.g.,RIG-I or MDA-5]). However, because these constructs areincapable of making infectious virus and, as such, do notresult in spread of virus beyond the initially infected cell, theyhave significantly reduced safety concerns.Vaccines generated using single-cycle virus constructs showed

usefulness in mice and nonhuman primates, eliciting both cell-mediated and Ab responses (82–86). In addition, they showpromise in the context of the neonate. Infant mice vaccinatedwith a single-cycle HSV-1 variant within 24 h of birth haddramatically improved CD4+ and CD8+ T cell responses andwere protected from virus challenge (87). In addition, infantrhesus macaques vaccinated with chimeric Venezuelan equineencephalitis/Sindbis virus replicon particles generated neu-tralizing Ab and were protected from disease following virusexposure, even at 1 y postvaccination (88). Protection fromvirus challenge at this time is consistent with the generation oflong-lasting memory responses in infants, a goal that hasproven challenging in the setting of the neonate. Despite theirsuccess in experimental models, from a practical standpoint

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there may be hurdles to the use of these adjuvants given publicwariness. This presents a challenge for the real-world appli-cation of these approaches.

Experimental challenges to forward movement in the development ofvaccines that are effective in neonates

One of the significant challenges associated with a morecomplete understanding of the defects in the neonatal immuneresponse and the development of vaccines is the limitationsassociated with current experimental models. Neonatal mice,although extremely tractable, have a highly abbreviated periodof infancy, making assessment of responses following prime-boost strategies difficult. A limitation to their use in the de-velopment of TLR agonists as adjuvants is the differentialdistribution and function of these molecules in mice versushumans. For example, in contrast to humans, murine T cellsdo not respond to TLR5 agonists (89). Further, mice do nothave a functional TLR10 (90), and they express TLRs forwhich functional human equivalents appear to be lacking.The distribution of TLRs among DC subsets and B cells alsodiffers between mice and humans (91, 92).With regard to T cell differentiation, there is evidence that

mice may overestimate the Th2 bias of the neonate. Specifically,the strong Th2 bias apparent in neonatal mice following vac-cination with acellular pertussis is not replicated in human infantswhere a more balanced Th1/Th2 response is observed (93).Human studies are also challenging as a result of the under-

standable difficulty in obtaining cells from neonates and re-striction to in vitro analyses. Cells derived from cord blood havebeen used as a surrogate, but responses in these cells may notreflect the newborn past the first few days of birth, because theirfunction is likely modulated by transient changes associated withdelivery (e.g., the increase in adenosine) (10). New models (e.g.,nonhuman primates) could markedly benefit this area of researchbecause innate sensor distribution and function more closelyresemble those of humans. In addition, the more extended pe-riod of infancy allows assessment of prime-boost regimens.

ConclusionsAlthough there is still much to learn with regard to the infantimmune system, there is evidence that, under the right cir-cumstances, the neonate can make a strong and effective re-sponse to challenge. At its core, the goal of a vaccine is toreproduce the quantity and quality of the immune response thatis generated following pathogen clearance. As our understand-ing of the receptors and pathways involved in pathogen recogni-tion and immune activation continues to expand, we willundoubtedly gain an appreciation for new targets and strategiesthat can be exploited to generate vaccines capable of providingprotection in the highly vulnerable neonate population.

AcknowledgmentsI thank Dr. Karen Haas for helpful comments regarding the manuscript.

DisclosuresThe author has no financial conflict of interest.

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