Veterinary Immunology and Immunopathology 87 (2002) 207213

Immunity in neonates
Bror Moreina, Izzeldin Abusugraa, Gunilla Blomqvistb,*
Section of Virology, Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Biomedical Centre, Box 585, S-751 23 Uppsala, Sweden bVirology Department, National Veterinary Institute, Biomedical Centre, Box 585, S-751 23 Uppsala, Sweden
a

Abstract Passively derived maternal immunity hampers active immunization of newborns. Further, an immature immune system contributes to a weak and Th2 polarized immunity. This state of immunity in early life sustains endemic infections in man and continuous reinfections in animal herds. The endemic infections of the young occur preferentially when the immune system is still functionally immature and when the low levels of maternal antibodies are no longer protective but yet blocks protective immune responses. Vaccines overcoming these problems would have strong positive effects on the herd health and environmental benets. The Th2 bias of the newborn is mediated by high levels of progesterone and Th2 cytokines produced in the maternalfetal interface. The activity of the innate system is enhanced in the mother during the prepartus period, certainly having effects on the offspring. Newborn, 2-days-old, mice can be primed with Sendai virus envelope proteins as model antigens to induce Th1 or Th2 responses, dependent on the supplementation of the virus antigen formulation with Th1 or Th2 adjuvants. This priming has a strong life-long effect when complemented with subsequent boosts. However and importantly this priming effect can be modulated by adjuvants focusing for Th1 and Th2 when applied to the mice at 6 weeks of age, i.e. when they are immunologically adult. It has been shown in various species, besides mice, i.e. dog, sheep, horse and seal, that a strong Th1 driving adjuvant can induce immune response and protection in newborns when conventional vaccines fail. In conclusion, the Th2 bias prevailing around partus can be overcome by appropriate immunological treatments, permitting effective vaccination Keywords: Veterinary immunology; Neonate; Maternal immunity; Vaccine; Adjuvants; T helper cells; Th1/Th2 and protective immunity in the newborn. # 2002 Elsevier Science B.V. All rights reserved.

1. Introduction

animal herds. Infections of the young occur preferentially when the immune system is still functionally Active immunization of newborns by natural infec- immature and when the maternal antibodies have vanished to non-protective levels, but still block protion or vaccination is hampered by an immature tective immune responses. Vaccines overcoming the immune system and by passively derived maternal immunity. This early life situation prevails in most if problem with the immature immune status and blocknot all mammalian species and contributes to endemicing effects of passively transferred antibodies would infections of human and animal populations, sustain- have strong positive effects on the herd health, diminish the use of antibiotics and result in economical and ing a continuous prevalence of viral infections in environmental benets. It is becoming more and more evident that newborns are immune competent although with a biased * Th2 prole. In early days it was conceived that Corresponding author. Tel.: 46-18-674335;
fax: 46-18-4714517. E-mail address: gunilla.blomqvist@sva.se (G. Blomqvist).

0165-2427/02/$ see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 2 ) 0 0 0 7 8 - 8

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neonatal tolerance, a concept rst described by Medawer (Billingham et al., 1953), was a major component. This view was underlined by immunization experiments using alloantigens and peptide antigens (Schurmans et al., 1990; Forsthuber et al., 1996; Singh et al., 1996; Adkins, 1999).

2. The polarization of immune response towards Th2 in newborns

The polarization towards Th2, with a relative deciency of Th1 and cytotoxic T lymphocyte (CTL) response is partly explained by reduced number and function of the accessory cells (Martinez et al., 1997; Adkins et al., 1994; Sarzotti et al., 1996; Astori et al., 1998; Delespesse et al., 1998; Marshall-Clarke et al., 2000). Above all, however, the immune status during the pregnancy is likely to be highly inuential. The 3. The blocking effect of passive immunity normal pregnancy is characterised by a down regula- on active immunization of newborns tion of the cell mediated Th1 response, an increased capacity of the innate immune system and an It is well recognized that passive immunity is an enhanced humoral response. This shift in Th1/Th2 obstacle for induction of active immunization. Most balance is in the most studied system, i.e. in the mouse vaccination regimens, e.g. in dogs, cats, large animals model, localised to the feto-placental tissues with and humans, have to consider the duration of maternal secretion of Th2 cytokines such as IL-4, IL-5 and antibodies before the vaccinations of the infants comIL-10 exerting Th1 tolerizing and/or suppressive mence. Possibly live vaccines are less effective than effects (Wegmann et al., 1993; Raghupathy, 1997). Also, placental factors such as PGE2 and progesterone non-replicating ones supplemented with strong adjuvants when passively transferred antibodies are precontribute to the prevailing Th2 environment in the decidual environment, i.e. progesterone by its down sent, as demonstrated in macaques with a subunit measles vaccine by van Binnendijk et al. (1997). regulation of IL-12 and upregulation of IL-10 (Sacks However, there are certainly exceptions from that rule. et al., 1999; Rukavina and Podack, 2000). The fetoplacental environment producing cytokines is likely to Because of this situation the breeding units are sensitive to the endemic or herd infections circulating in the inuence the fetus and subsequently the newborn, resulting in a regulation toward Th2 type of response environment. As stated in Section 1, a vaccination in early life associated with an increased susceptibility regimen would have a strong economical positive to infections by intracellular microorganisms such as effect if it could close the window allowing the viruses (Delespesse et al., 1998; Kovarik and Siegrist, endemic to sustain (Fig. 1). 1998). It is suggested that the immune system of the newborn is subjected to an educational process to obtain a Th1Th2 balance. This process is driven by the young individuals exposure to infections resulting in a long-lasting systemic and non-specic switch 4. The prohibitive effects of the neonate innate to include Th1 immune activity (Rook and Stanford, immature system on active immunization 1998). Most vaccines for children are; however, Th2 driving (e.g. diphtheria, tetanus and pertussis In the 50th Medawer and coworkers (Billingham et al., 1953) conceived that the immature immune system of the newborn is an obstacle for immune induction. This and other early works (Nossal, 1983) describe tolerance being induced by an active immunization of newborns. Recently the view of a low

vaccines) and the only licensed adjuvant for man is the Th2 driving mineral adjuvants (e.g. Al(OH)3) and MF52 an oil adjuvant. Although these vaccines provide protection against the infections for which they are used, they do not contribute to the educational process to a Th1 pathway. It is also considered that the dominant Th2 type of responses and lack of Th1 education are associated with the pathogenesis of allergic diseases (Rook and Stanford, 1998; De Carli et al., 1994; Mosmann and Sad, 1996; Grewe et al., 1998). Thus, the view is that the immune system of the newborn has a strong tendency to adopt a Th2 prole, and an early life immunization is conceived to conserve this polarization.

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Fig. 1. The window of susceptibility to infectious disease circulating in the herd. During the time period, marked by the arrows, neither the levels of maternal antibodies nor the endogenous produced antibodies are high enough to prevent infection.

competence of newborns to respond to active immunization has gradually changed to realize that the competence might be low, but it is there, and can be used if the conditions of the newborn is taken into consideration.

perarteriolar lymphoid sheath (PALS), germinal centers (GC) and B cell follicles develop late. Further MHC class II DCs and macrophages are sparse or undetectable and the antigen processing and presenting capacities are acquired late in ontogeny. Interestingly, in vitro, the antigen presenting capacity, by adding adult APCs (DC), having appropriate expression of CD40, can tip the immune response to adult 4.1. Defects of the neonate innate immune system levels. Simultaneous there is an upregulation of the corresponding ligand (CD40L) on the T cells. The initiation of the adapted immune response The neonatal B cells have also a number of defecoccurs in the innate system. There are a number of tives hampering their immunological capacity. B cells prerequisites required from this system for induction are also APCs. The B cell ligation is defective resultof a specic immune response, which are not fullled ing in incomplete signaling to acquire immune speby the newborn. The antigen presenting cells (APCs) cic immune response. Incomplete signaling is also most importantly the dendritic cells (DCs) of the due to low expression of MHC class II, lack of CD86 newborn have reduced capacity to express CD86 and its ligand on T cells lead to lack of costimulation and CD40 (Marshall-Clarke et al., 2000) and the and hampers BT cell cross talk. It should be born in corresponding ligands on the lymphocytes are mind that a B and T cell interaction is, in most cases, reduced. These surface molecules are required on required for specic B cell (antibody) response. The the APCs to give signal to the T cells via correspondlack of upregulation of CD40 and CD40L hampers the ing ligands (costimulation) to expand antigen specic B cell response and the B cell switch to different B cell clones. The upregulation of the MHC class II antigens classes and subclasses. These defects are likely to on the surface of the APCs is also reduced. The MHC cause anergy and possibly Th2 bias (Marshall-Clarke class II antigens are required to present the vaccine et al., 2000). antigens, after that the vaccine antigens have been ezymatically processed in the APC, to the T lymphocytes to obtain the specic T cell response. The neonate spleen is not developed. It lacks a structure (architecture) of the adult spleen. To achieve the mature structure the proinammatory cytokines lymphotoxin (LT) and tumor necrosis factor (TNF) are required. Structures missing in the neonate spleen are 5. The Th2 bias of the immature immune system There is conicting information about the Th2 bias and the tolerance of the immune system of the

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to enhance Th1 response, Th2 adjuvanted micelles by adsorbtion to Al(OH)3 and non-adjuvanted micelles formulation. The ISCOM formulation (SV-ISC) induced a Th1 prole, i.e. a prominent IFN-g response after a neonatal priming. Further the ISCOM modulated a neonatally induced primary Th2 immune response towards Th1 when given as a subsequent immunization. The Al(OH)3 formulation (SV-alMIC) induced in newborns a primary Th2 response with production of IL-5 and a detectable IgG1 antibody response and it modulated a neonatally induced Th1 response towards Th2. The non-adjuvanted micelle formulation (SV-MIC), requiring a 20-fold higher dose than adjuvanted formulations, induced Th1 supFig. 2. The placenta of the maternalfetal interface is a strong pression, which was overcome by a subsequent SVproducer of Th2 cytokines including IL-4, IL-10 and TGF-b driven ISC immunization. It is notable that only the Th2 by progesterone and prostaglandin E2. The Th2 inuence is conceived to prevent immunological driven abortion. driving adjuvant could induce detectable antibody response in 2-days-old mice conned to the IgG1 subclass. Others have reported that 1-week-old mice can respond with a primary specic IgG2a. It should be noted that Al(OH)3 has a depot effect which might newborns. There are certainly differences between extend the antigen release for several days. A single species and most information is derived from work in mice and in man. From above it can be concluded immunization with the Al(OH)3 formulation induced that a partially defective innate system may contributean immune response lasting as long as the mice to a Th2 bias. Besides, the placenta during the preg- were kept alive (up to 68 months). Thus, our ndings nancy period is likely to have a strong effect both on show that neonatal immunization to a large extent paves the way for a geared postnatal immunity but also the mother as well as on the offspring (Fig. 2). Th1 that responses are suppressed by placental products such as later immunizations do have potential to modify a progesterone, prostaglandin E2 and cytokines such as neonatally polarized immune response. The immune responses induced lasted as long as the mice were kept IL-4 and IL-10. (Wegmann et al., 1993; Sacks et al., 1999; Rukavina and Podack, 2000). It is likely that the alive (up to 68 months). Furthermore, the Th1Th2 Th2 inclination exerted by the placenta on the mother and probably also on the fetus has evolved to evade immunological rejection of the fetus.

5.1. Potent adjuvants can overcome the Th2 bias

Experiments were carried out in Uppsala to explore the rigidity of the dogma that the newborn has a strong tendency to adopt a Th2 prole, and that an early life Priming immunization conserves this polarization. Th1 and Th2 responses were induced to Sendai virus (SV) in neonate, i.e. 2-days-old mice, by aid of adjuvants. The aim was to investigate the nature of the primary SV-ISC neonate immunity and the inuence of the primary response on a subsequent adult secondary immuniza- SV-ISC SV-ISC tion. Further, the immune ontogeny was analyzed to SV-MIC explore the immune responses during an extended SV-MIC SV-MIC period of life. Thus, Balb/c mice were immunized with envelope proteins of SV in three different antigen SV-alMIC SV-alMIC presentation systems including ISCOMs with capacity SV-alMIC

Table 1 Ratio of SV specic IgG2a and IgG1 serum antibodies of mice 8 weeks and 68 months after neonatal priming at 48 h of age and boost at 6 weeks of age Boosting n IgG2a/IgG1 ratio 6 Weeks 8 Weeks 68 months 0.92 0.89 0.74 0.84 0.71 0.51 0.85 0.67 0.70

SV-ISC SV-MIC SV-alMIC SV-ISC SV-MIC SV-alMIC SV-ISC SV-MIC SV-alMIC

3 4 4 5 5 5 5 4 4

0.88 0.81 0.50 0.56 0.36 0.14 0.76 0.52 0.57

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prole was kept, but with a more prominent increase neonatal immunization with SV envelope proteins in of the IgG2a antibodies (Table 1). Our results do not ISCOMs was analyzed. It was found that maternal support the proposal that re-exposure to antigen after immunity of mothers immunized with Al(OH)3 adjua neonatal priming generally results in a Th2 dominated vanted micelles of SV suppressed an endogenous SV immune response (Barrios et al., 1996; Adkins and specic antibody production regardless the offspring Du, 1998). was immunized at neonatal (2 days) of age or as adults (6 weeks of age). In contrast, a signicant suppression of the Th1 cell response, measured as IFN-g production was recorded after neonatal but not after adult immunization. The inhibition of the IL-5 production 6. Maternal immunity inuences the antibody was on the other hand insignicant. These suppressive and cell mediated immune responses effects by maternal immunity was only partial, since after immunization of the offspring the neonatal immunization primed for both IgG2a antibody and T cell (IFN-g) responses recorded as The inuence of the presence of maternal antibodies on the immune responses in offspring after

Fig. 3. The maternal immune status caused by vaccination inuences the T cell immune response of the offspring to the ISCOM vaccine measured by IFN-g/IL-5 ratio.

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Table 2 Experimental vaccines in neonates Viral antigen Herpes Equi 2 Parvovirus Canine distemper Rotavirus (oral) Sendai virus Subject 10-Days-old foals 3-Weeks-old dogs Day old seals 1-Week-old lambs 2-Days-old mice Outcome Protection High levels of HI antibody Protection Immune response/protection? T and B cell responses

Can lymphatic organ architecture be influenced and established earlier e.g. by proinflammatory cytokines? Potent adjuvants can overcome some of these problems.

References

Adkins, B., 1999. T-cell function in newborn mice and humans. Immunol. Today 20 (7), 330335. Adkins, B., Du, R.Q., 1998. Newborn mice develop balanced Th1/ Th2 primary effector responses in vivo but are biased to Th2 clear cut boost effects by the subsequent adult immu- secondary responses. J. Immunol. 160, 42174224. Adkins, B., Ghanei, A., Hamilton, K., 1994. Up-regulation of nization. murine neonatal T helper cell responses by accessory cell When the quotas of spleen cell production of IFN-g factors. J. Immunol. 153 (8), 33783385. vs IL-5 of neonatally immunized mice born to mothers Astori, M., Finke, D., Karapetian, O., Acha-Orbea, H., 1998. immunized with the three different delivery systems Development of TB cell collaboration in neonatal mice. Int. Immunol. 11, 445451. described above of SV-antigen were compared, a Barrios, C., Brandt, C., Berney, M., Lambert, P.H., Siegrist, C.A., signicant correlation (r 0:997 at a p < 0:05) 1996. Partial correction of the TH2/TH1 imbalance in neonatal between the IgG2a/IgG1 quotas of the mothers and murine responses to vaccine antigens through selective the IFN-g/IL-5 levels of the offspring was recorded adjuvant effects. Eur. J. Immunol. 26, 26662670. (Fig. 3). These results suggest a maternal inuence on Billingham, R.E., Brent, L., Medawar, P.B., 1953. Actively acquired tolerance of foreign cells. Nature 172, 603606. the development of Th1/Th2 immunity in neonates. De Carli, M., Elios, M.M.D., Zancuoghi, G., Romagnani, S., Del Prete, G., 1994. Human Th1 and Th2 cells: functional properties regulation of development and role in autoimmunity. Autoimmunity 18, 301308. Delespesse, G., Yang, L.P., Ohshima, Y., Demeure, C., Shu, U., Byun, D.G., Sarfati, M., 1998. Maturation of human neonatal CD4 and CD8 T lymphocytes into Th1/Th2 effectors. 7. Experimental vaccines have induced potent Vaccine 16, 14151419. immune responses in neonates Forsthuber, T., Yip, H.C., Lehmann, P.V., 1996. Induction of TH1 and TH2 immunity in neonatal mice. Science 271, 17281730. In Table 2 a number of experimental vaccines are Grewe, M., Bruijnzeel-Koomen, C.A.F.M., listed which have induced potent immune responses inSchopf, E., Thepen, T., neonates. It should be noted that six different antigens Langeveld-Wildschut, A.G., Ruzicka, T., Krutmann, J., 1998. A and Th2 the immunopathogenesis of atopic and ve different species are included which indicates role for Th1Immunol. cells in 19 (8), 359361. dermatitis. Today a generality i.e. that potent adjuvant can be used to Kovarik, J., Siegrist, C.A., 1998. Immunity in early life. Immunol. overcome at least some conceived difculties to ef- Today 19 (4), 150152. Marshall-Clarke, S., Reen, D., Tasker, L., Hassan, J., 2000. ciently induce immune protection in neonates. Neonatal immunity: how well has it grown up? Immunol. Today 21 (1), 3541. Martinez, X., Brandt, C., Saddallah, F., Tougne, C., Barrios, C., Wild, F., Dougan, G., Lambert, P.H., Siegrist, C.A., 1997. DNA immunization circumvents decient induction of T helper type 1 and cytotoxic T lymphocyte responses in neonates and during 8. Conclusions and question marks early life. Proc. Natl. Acad. Sci. USA 94 (16), 87268731. Mosmann, T.R., Sad, S., 1996. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol. Today 17 (3), 138146. Maternal immunity is an obstacle for vaccination of Nossal, G.J., 1983. Cellular mechanisms of immunologic tolerance. newborns. Ann. Rev. Immunol. 1, 3362.

To what an extent is the immature immune system of newborns a consequence of immunological unexperience? MHC class II expression and costimulation (e.g. CD86) can be upregulated in T cell responses in newborns. Can Ig switch be established earlier?

B. Morein et al. / Veterinary Immunology and Immunopathology 87 (2002) 207213 Raghupathy, R., 1997. Th1-type immunity is incompatible with successful pregnancy. Immunol. Today 18 (10), 478482. Rook, G.A.W., Stanford, J.L., 1998. Give us this day our daily germs. Immunol. Today 19 (3), 113116. Rukavina, D., Podack, E.R., 2000. Abundant perforin expression at the maternalfetal interface: guarding the semiallogeneic transplant? Immunol. Today 21 (4), 160163. Sacks, G., Sargent, I., Redman, C., 1999. An innate view of human pregnancy. Immunol. Today 20 (3), 114118. Sarzotti, M., Robbins, D.S., Hoffman, P.M., 1996. Induction of protective CTL responses in newborn mice by a murine retrovirus. Science 271, 17261728. Schurmans, S., Heusse, C.H., Qin, H., Merino, J., Brighouse, G., Lambert, P.H., 1990. In vivo effects of anti-IL-4 monoclonal antibody on neonatal induction of tolerance and on an

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associated autoimmune syndrome. J. Immunol. 160, 24652473. Singh, R.R., Hahn, B.H., Sercarz, E.E., 1996. Neonatal peptide exposure can prime T cells and, upon subsequent immunization induce their immune deviation: implications for antibody vs. T cell-mediated autoimmunity. J. Exp. Med. 183 (4), 16131621. van Binnendijk, R.S., Peolen, M.C.M., van Amerongen, G., de Vries, P., Osterhaus, A.D.M.E., 1997. Protective immunity in macaques vaccinated with live attenuated, recombinant and subunit measles vaccine in the presence of passively acquired antibodies. J. Infect. Dis. 175, 534542. Wegmann, T.G., Lin, H., Guilbert, L., Mosmann, T.R., 1993. Bidirectional cytokine interactions in the maternalfetal relationship is successful pregnancy a TH2 phenomenon? Immunol. Today 14 (7), 353356.