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The Journal of Immunology, 2003, 171: 4287-4293.
Copyright © 2003 by The American Association of Immunologists

Mucosally Delivered E1-Deleted Adenoviral Vaccine Carriers Induce Transgene Product-Specific Antibody Responses in Neonatal Mice 1

Zhiquan Xiang*, Yan Li*, Guangping Gao{dagger}, James M. Wilson{dagger} and Hildegund C. J. Ertl2,*

* Wistar Institute and {dagger} University of Pennsylvania, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
E1-deleted adenoviral vectors of the human serotype 5 (AdHu5) and the chimpanzee serotype 68 (AdC68) expressing the rabies virus glycoprotein (rab.gp) were tested for induction of transgene product-specific Abs upon intranasal or oral immunization of newborn mice. Both vectors induced Abs to rabies virus that could be detected in serum and from mucosal secretions. Serum rabies virus neutralizing Ab titers sufficed to protect neonatally vaccinated mice against a subsequent challenge with rabies virus. The efficacy of the AdHu5rab.gp vector given orally to newborn mice born to AdHu5 virus-immune dams was not impaired by maternally transferred Abs to the vaccine carrier.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The development of vaccines for neonates is met by two target population-specific challenges. First, efficacious immunization of neonates is hampered by the relative immaturity of their immune system, especially their mucosal immune system that provides the first barrier to most pathogens (1, 2, 3, 4). Neonates mount an immune response to some vaccines or infections, but they have a propensity to generate Th2 rather than Th1-type immunity (5, 6) commonly less suited to ward off viral infections. Second, the induction of immune responses in neonates and young infants to common human pathogens is impaired by maternally transferred Abs that upon neutralization of the vaccine result in reduced stimulation of the infant’s B cells (1, 7).

Young infants are currently vaccinated routinely to a number of viral pathogens including polio virus, hepatitis B virus, and measles virus. Most of these vaccines require repeated administration to overcome the obstacles of raising potent immune responses in newborns, which raises practical and socioeconomic issues. Furthermore, infant vaccines with the exception of the Sabin polio vaccine are delivered by injection rather than by oral or intranasal routes. Mucosal immunization is not only more convenient but also safer compared with systemic vaccine delivery in underdeveloped countries where inadvertent transmission of other pathogens due to inappropriate sterilization of injection devises can negate the benefit of vaccines.

Novel vaccine carriers are being generated for pathogens for which traditional vaccine approaches based on inactivated or attenuated pathogens have failed or were deemed unsafe. DNA vaccines composed of simple plasmid vectors have yielded promising results in neonatal experimental animals where they induced transgene product-specific T and B cell-mediated immune responses that in some systems provided protection to subsequent challenge (8, 9, 10). We showed previously that mice immunized s.c. at birth with an E1-deleted adenovirus (Ad) 3 vector of the human serotype 5 (AdHu5) expressing the rabies virus glycoprotein developed protective Ab titers to rabies virus, which were only marginally reduced in pups born to rabies virus-immune compared with naive dams. Nevertheless, the pups’ B cell responses to the rabies virus glycoprotein were severely inhibited if their dams had been vaccinated to the AdHu5 vaccine carrier (11).

Because AdHu5 virus is a ubiquitous human pathogen and ~45% of the adult human population in the United States carries virus-neutralizing Abs to AdHu5 virus (12), we developed an alternative E1-deleted adenoviral vaccine carrier derived from an adenovirus that had been isolated from a chimpanzee (13). This virus, termed AdC68, does not circulate in humans nor do Abs to common human serotypes of adenovirus neutralize it (12). E1-deleted AdC68 recombinants similar to vectors based on AdHu5 virus induce potent transgene product-specific adaptive immune responses upon systemic immunization of adult mice (14, 15). In this study, we tested, using the rabies virus glycoprotein as our model Ag, both the AdHu5 and AdC68 vectors for induction of Abs to rabies upon mucosal, i.e., intranasal or oral, application to newborn mice born to naive or AdHu5 virus-immune dams.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Adult female and male ICR mice as well as time-pregnant ICR mice were purchased from Charles River Breeding Laboratories (Boston, MA) and kept at the Animal Facility of the Wistar Institute (Philadelphia, PA).

Viruses

The AdHu5rab.gp recombinant, an E1-deleted Ad recombinant of the human serotype 5 expressing the glycoprotein of the Evelyn Rokitniki Abelseth (ERA) strain of rabies virus, has been described previously (16). Generation of the more recently developed E1-deleted AdC68rab.gp vaccine expressing the same transgene product in a simian Ad virus vector has been reported (14). Viral recombinants, including AdHu5 and AdC68 vectors expressing green fluorescent protein (GFP) as well as wild-type AdHu5 virus, were propagated in 293 cells. The viral recombinants were harvested by freeze thawing of infected 293 cells followed by pelleting of the cell debris. In addition, the AdHu5 virus was purified by CsCl centrifugation and virus particles per milliliter were determined by spectrophotometry at 260 nm as described elsewhere (12). AdHu5 virus and adenoviral recombinants were titrated on 293 cells to determine PFU. The ERA strain of rabies virus was grown on BHK-21 cells, purified by gradient centrifugation, and inactivated by treatment with {beta}-propiolactone (BPL). The protein content of the inactivated rabies virus (ERA-BPL) was determined and adjusted to 1 mg/ml. The CVS-11 strain of rabies virus was propagated and titrated on BHK-21 cells. Mice were challenged with the challenge virus strain CVS-N2C of rabies (17), a variant of the CVS-24 strain which is closely related to the ERA strain but is highly virulent in mice. The virus was derived from the brains of neonatally infected ICR mice and titrated by intranasal challenge of young adult ICR mice.

Immunization of mice

Adult female ICR mice were immunized once with 109 PFU of AdHu5 virus given intranasally and 2 days later cohoused with males. Males were separated from pregnant females before birth of the pups. Pups were immunized within 24–48 h after birth intranasally or orally with 107 PFU of AdHu5rab.gp, AdC68rab.gp, or an AdC68 control vector expressing GFP (AdC68GFP). Alternatively, pups were left unvaccinated to establish titers of maternally transferred Abs.

Preparation of samples

Blood was harvested by retro-orbital puncture. Sera were prepared and heat inactivated at 56°C for 30 min. Sera were tested for virus neutralization against rabies virus or adenovirus starting at a 1/5 dilution. Vaginal lavage was harvested by rinsing the vaginal cavity three times with 50 µl of PBS. Sample were centrifuged at 10,000 rpm for 10 min to remove debris and then tested by ELISA starting with a 1/2 dilution. Sera were analyzed by ELISA starting with a 1/200 dilution. Ab isotypes were tested with a 1/400 dilution of sera or a 1/2 dilution of vaginal lavage (18).

ELISA

Sera were tested on virus-coated plates as described previously (17). Briefly, round-bottom microtiter plate wells were coated overnight with 0.2 µg of ERA-BPL virus or 5 x 109 virus particles of purified AdHu5 virus diluted in 100 µl of coating buffer (15 mM Na2CO3, 35 mM NaHCO3, and 3 mM Na2N, pH 9.6). The next day plates were treated for 24 h with PBS containing 3% BSA. The following day plates were washed twice with 150 µl of PBS, dried, and kept at -20°C. Sera were serially diluted in PBS containing 3% BSA. The different dilutions of sera were incubated in duplicates at 100 µl/well on the ERA-BPL-coated plates for 1 h at 4°C. Plates were washed and incubated for 20 min with the substrate (10 mg d-nitrophenyl phosphate disodium dissolved in 10 ml of 1 mM MgCl2, 3 mM NaN3, and 0.9 M diethanolamine, pH 9.8). Plates were then read in an automated ELISA reader at 405 nm. Isotypes of Abs to rabies virus were tested on ERA-BPL-coated plates with the Calbiochem isotyping kit (Calbiochem, La Jolla, CA), which has comparable sensitivity for different Ab isotypes (19). Isotype ELISAs were read at 450 nm.

Virus neutralization assay

Sera were tested on BHK-21 cells for neutralization of CVS-11 virus, which is antigenically closely related to the ERA virus (17). Sera were tested on 293 cells for neutralization of adenovirus using Ad recombinants expressing GFP in a plaque reduction assay (20).

Statistical analyses

Experiments were conducted at least twice using at least five mice per group. Sera were tested by ELISA or neutralization assay in duplicates or triplicates. Results show the means ± SDs. Significance was calculated using Student’s t test for two to three dilutions. Data with p values below 0.05 were considered to reflect a statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Neonatal mice develop transgene product-specific Abs upon mucosal immunization with E1-deleted adenoviral recombinants of human or simian serotypes

Groups of 8–10 pups from naive ICR dams were immunized within 24–48 h after birth with 107 PFU of AdHu5rab.gp or AdC68rab.gp virus given intranasally or orally. In some experiments, a control virus expressing GFP was included as a negative control. Pups were bled 3 wk later and serum Ab titers (Fig. 1A) and isotype profiles (Fig. 1B) were determined by ELISAs. Pups immunized with either of the recombinant Ad viruses expressing the rabies virus glycoprotein given through the mucosal routes developed serum Abs to rabies virus. Such a response was not elicited by the control vector (data not shown). Both recombinants gave comparable responses upon intranasal administration to neonates while upon oral immunization the AdHu5rab.gp vector elicited a higher Ab response compared with the AdC68rab.gp vector. The isotypes of Abs to rabies virus were mixed in pups immunized orally or intranasally with the AdHu5rab.gp vector and composed of approximately equal levels of IgG1 and IgG2a. Upon oral or intranasal immunization, the AdC68rab.gp vector induced by 3 wk of age a pronounced IgG1 response with no detectable IgG2a to the rabies virus Ag (Fig. 1B).



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  		FIGURE 1. ICR pups (n = 5–8) were vaccinated within 24–48 h after birth with 107 PFU of AdHu5rab.gp ({blacksquare} in A and bars in B) or AdC68rab.gp virus ({square} in A and bars in B) given orally or intranasally. Pups were bled 3 wk later. A, Ab titers to rabies virus were determined by an ELISA (A) in comparison to a normal mouse serum (x) on plates coated with inactivated rabies virus. B, Isotypes of Abs to rabies virus were tested by an ELISA. Both graphs shows mean adsorption ± SD for pooled sera.

 
As mucosal delivery of vaccines to neonatal mice is technically challenging, we tested a number of individual sera harvested from 3-wk-old female pups immunized at birth with the AdC68rab.gp vaccine given either intranasally or orally. Although titers varied in individual pups, which may in part reflect inaccurate delivery of the vaccine dose to neonatal mice as well as genetic differences of the cohort of outbred pups, all of the pups had readily detectable titers to rabies virus (Fig. 2A). In orally immunized pups, these titers were remarkably stable and comparable when tested again from the same cohort of animals 6 mo later. Titers had increased by then in intranasally vaccinated pups (Fig. 2B), suggesting a delay in responsiveness or a longer persistence of the vaccine-encoded Ag upon airway delivery.



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  		FIGURE 2. Individual sera of 4- and 27-wk-old ICR mice immunized at birth with 107 PFU of AdC68rab.gp virus given orally (n = 5–7) or intranasally (n = 5) were tested for Abs to rabies virus by an ELISA on plates coated with inactivated rabies virus. x, Pooled normal mouse serum. The graph shows mean adsorption ± SD.

 
Virus-neutralizing Ab (VNA) titers tested 3 wk after immunization of pups were low but detectable in all groups (Fig. 3). VNA titers did not correlate with ELISA titers and were comparable in all of the groups except for the group immunized by the AdHu5rab.gp vector given intranasally. VNA titers to rabies virus correlate with protection and titers above 0.5 IU are known to prevent disease following a peripheral challenge with an otherwise lethal dose of rabies virus. VNA titers of pooled sera from all of the vaccinated groups exceeded the 0.5 IU benchmark.



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  		FIGURE 3. The same pooled sera shown in Fig. 1 were tested at serial dilutions (starting with a 1/5 dilution) for neutralization of rabies virus of the CVS strain against a standardized WHO reference serum. Data are expressed as IU calculated by extrapolation against the WHO serum. A batch of pooled normal mouse serum tested in parallel as a control had a titer below 0.2 IU.

 
To determine whether pups developed mucosally secreted Abs upon neonatal immunization, vaginal lavage was harvested from 2-mo-old female pups and tested for Ab isotypes to rabies virus. As shown in Fig. 4, either vaccine given orally or intranasally resulted in vaginal Abs to rabies virus. Isotypes were mixed with the AdHu5rab.gp vector, again showing a higher propensity to induce Th1-linked Abs of the IgG2a isotype than the AdC68rab.gp vector. Results were especially promising in intranasally AdC68rab.gp-vaccinated pups that had substantial levels of rabies virus-specific IgA Abs in their vaginal lavage.



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  		FIGURE 4. Vaginal lavage of female pups immunized at birth orally or intranasally with AdHu5rab.gp ({blacksquare}, n = 5) or AdC68rab.gp vector ({square}, n = 5) was harvested from 2-mo-old female pups by rinsing of the vaginal cavity with a total of 150 µl of saline. The cleared vaginal lavage fluids were pooled and tested for isotypes of Abs to rabies virus by an ELISA starting with a 1/2 dilution. Vaginal lavage fluid harvested and pooled from age-matched naive ICR mice (third column) were tested in parallel.

 
Effect of pre-exposure of dams to AdHu5 virus on the B cell response of neonatal mice to mucosal immunization with the E1-deleted adenoviral recombinants

We developed the simian E1-deleted adenoviral vector initially to circumvent interference by VNAs against vaccine carriers based on adenoviruses derived from common human serotypes such as the AdHu5 vector. These Abs are present in a large percentage of adult humans (12) and one would expect that women transfer such Abs to their offspring, which in turn would impair the infants’ ability to mount an immune response to the transgene product of AdHu5 vector-based vaccines. We had indeed shown previously that pups born to dams that had been immunized with an AdHu5 vector responded poorly to the AdHu5rab.gp vaccine delivered by s.c. injection (11).

To test whether induction of a rabies virus-specific Ab response upon oral delivery of the AdHu5rab.gp vector was affected by maternal transfer of Abs to the vaccine carrier, female ICR dams were immunized intranasally with 109 PFU of AdHu5 virus and bred to ICR males. Pups from naive and AdHu5-immune dams were vaccinated within 24–48 h after birth with 107 PFU of AdHu5rab.gp virus given orally. Some pups from the immune dams were left unvaccinated and used to assess titers of maternally transferred Abs to AdHu5 virus once pups were 3 wk old. As shown in Fig. 5B, unvaccinated pups had serum Ab titers to the AdHu5 virus that could be detected by ELISA, but were below those detectable by a neutralization assay (i.e., <1/20) Titers were substantially lower than those found in their vaccinated dams that were bled and tested in parallel. The pooled sera from the dams had a neutralizing titer to AdHu5 virus of 1/160. Maternal Abs to adenovirus were in part secreted at mucosal surfaces; as such, Abs were detected in the vaginal lavage of female pups born to AdHu5-immune dams (Fig. 5B).



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  		FIGURE 5. A, Female ICR mice were immunized intranasally with ~109 PFU of AdHu5 virus and mated 2 days later. Three weeks after delivery of the pups, the dams ({square}, n = 4) and some of their pups that had not been vaccinated ({blacksquare}, n = 10) were bled and their serum Ab titers to AdHu5 virus were determined by an ELISA on plates coated with purified AdHu5 virus against a normal ICR mouse serum. B, Vaginal lavage was harvested from 3-wk-old pups of AdHu5-immune dams ({blacksquare}, n = 4) and from age-matched pups born to naive dams ({square}, n = 4). Neither group of pups had been vaccinated at birth. Ab titers to AdHu5 virus were determined by an ELISA on plates coated with purified adenovirus. Both graphs show mean adsorption ± SD.

 
AdHu5rab.gp-vaccinated pups were also bled at 3 wk of age and vaginal lavage was harvested at 6 wk of age from female pups. Titers (Fig. 6A) and isotypes (Fig. 6B) of Abs to rabies virus were tested by an ELISA and a neutralization assay from sera and by an ELISA from vaginal lavage (Fig. 6C) using pooled samples. Immunization of dams with AdHu5 virus did not impair the transgene product-specific Ab response detected in sera or vaginal lavage of pups vaccinated orally with the AdHu5rab.gp vector but rather caused an increase in serum Ab titers to rabies virus as was shown by ELISA (Fig. 6, A and B) and by neutralization assay using pooled sera (Fig. 6C). This was assessed further by testing titers of individual pups by ELISA. The results confirmed that maternal Abs had not reduced the Ab response of the pups to rabies virus (Fig. 7). Preimmunization did not have a major effect on the isotype profile of the early B cell response to the rabies virus glycoprotein expressed by the AdHu5rab.gp vaccine although pups born to immune dams developed a slightly higher ratio of IgG2a:IgG1 ratio in sera and higher levels of IgA in vaginal lavage compared with those born to naive dams (Fig. 6B). Another interesting observation that was reproducible and for which we currently do not have an explanation was that female pups born to naive dams responded better to oral vaccination compared with male pups; this difference was statistically significant (p = 0.01 for the 1/400 and p = 0.006 for the 1/800 dilution of serum) and not observed in pups born to AdHu5 virus-immune dams (p = 0.156 for the 1/400 dilution of serum and p = 0.123 for the 1/800 dilution of serum; Fig. 7). Male pups born to AdHu5-immune dams developed higher Ab titers than those born to naive dams, although this difference did not reach statistical significance (p = 0.057 at the 1/400 serum dilution and p = 0.72 at the 1/800 serum dilution).



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  		FIGURE 6. Pups born to naive or AdHu5-immune dams were vaccinated within 24–48 h after birth with 107 PFU of AdHu5 virus. Their sera were harvested 3 wk later and tested for Abs to rabies virus. A, Titers of sera (left graph) and vaginal lavage (right graph; female pups) from AdHu5-immune ({blacksquare}, n = 10) and naive ({square}, n = 10) dams were tested for Ab titers to rabies virus by an ELISA on plates coated with inactivated rabies virus. B, The same set of pooled sera was tested for Ab isotypes by an ELISA using a 1/400 dilution of serum. {square}, Pups born to naive dams; {blacksquare}, pups born to AdHu5-immune dams; , pups born to naive dams. C, The same set of pooled sera was tested for neutralization of rabies virus against a WHO standard serum. Data are expressed as IU.

 


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  		FIGURE 7. Individual sera of male ({blacksquare}, n = 10) or female ({square}, n = 10) ICR pups born to naive (left) or AdHu5-immune dams (right) and vaccinated at birth with 107 PFU of AdHu5rab.gp vector orally were tested at 3 wk of age for Ab titers to rabies virus by an ELISA in comparison to a pooled normal mouse serum (x).

 
Pups born to naive or AdHu5-immune dams that had been vaccinated orally at birth with the AdHu5rab.gp vector were fully protected against challenge with 10 mean lethal doses of rabies virus given intranasally 2 mo later (Fig. 8).



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  		FIGURE 8. Ten pups each from naive ({blacksquare}) or AdHu5-immune ({square}) dams vaccinated at birth with AdHu5 virus were challenged at 8 wk of age with 10 LD50 of CVS virus given intranasally. Nine naive age-matched pups were also challenged (x). The one naive pup that survived became ill on day 13. The graph shows disease-free survival.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The immune system of neonatal mammals is immature at birth and thus prone to the development of tolerance as is best exemplified by the unresponsiveness of newborn mice to allogenic lymphocytes. This can be circumvented by presenting the alloantigens by dendritic cells, indicating that the induction of neonatal tolerance in this system is a reflection of the relative lack of stimulator cells rather than an immaturity of the responding T cell populations (21). Other Ags if given at high doses have been shown to induce apparent tolerance of CD8+ T cells by favoring induction of Th2 responses (22, 23). Immunization of neonates with a DNA vaccine expressing an Ag of malaria resulted in Ag-specific tolerance (24) while the same vaccine induced a potent immune response in adult mice (25). Applying the DNA vaccine with GM-CSF in the form of a genetic adjuvant evoked an immune response in neonates again suggestive of a primary defect of APCs as the underlying mechanism for tolerance induction (26).

Neonates despite the immaturity of their immune system can respond to Ags. Neonatal lymphocytes secrete cytokines upon activation (27, 28), although there is a relative deficiency in the production of IFN-{gamma} (29), which favors induction of Th2-type immune responses. Ig- pre-B cells that are more susceptible to tolerization are frequent in neonates (30); nevertheless, neonates also have mature B cells that secrete specific Abs upon activation.

Functional maturation of the mucosal immune system lags behind that of the systemic immune system (2, 3, 4), potentially to allow a window for induction of tolerance to harmless Ags such as normal gut flora, food, or air pollutants. As a consequence, gastrointestinal infections with pathogenic bacteria are the leading cause of infant death worldwide. Notwithstanding, 10-day-old mice can produce IgA Abs to enteric infections (31, 32).

Using the AdHu5rab.gp vector, we had previously shown that mice vaccinated systemically at birth developed protective Ab titers to rabies virus unless they had maternally transferred Abs to the vaccine carrier (8). Adenoviral vectors can thus in principle overcome the relative immaturity of the neonatal immune system, which may relate to the vectors’ ability to infect dendritic cells, driving their maturation into APCs (33, 34). Maturation of dendritic cells upon adenoviral infection depends on NF-{kappa}B activation (35), suggesting that adenovirus may drive differentiation of dendritic cells through a pathway similar to that triggered upon engagement of pathogen recognition receptors such as Toll-like receptors (36). Adenoviral vectors also induce a pronounced inflammatory reaction and rapid release of cytokines such as IL-6, TNF-{alpha}, and IL-12 (36, 37, 38), which may compensate for some of the defects of the immature immune system of neonates.

In this study, we tested the AdHu5rab.gp vector as well as a recently developed E1-deleted AdC68 recombinant derived from a chimpanzee for induction of Abs to rabies virus upon intranasal or oral administration to newborn outbred mice. Pups developed rabies virus-specific Ab titers to both vaccines given through either of the mucosal routes. Interestingly, unlike adult mice that generate markedly higher titers upon intranasal than oral vaccination with the Ad vectors (44), pups developed equivalent or higher titers upon oral application of the vaccines. This may be a reflection of the increased intestinal permeability of neonates (39, 40), which facilitates uptake of macromolecules such as Abs, cytokines, and antibacterial factors present in breast milk. We reported formerly that the AdC68rab.gp vector given i.m. has a higher propensity to induce Th1-associated Ab isotypes in sera compared with the AdHu5rab.gp vector (14), which was attributed to the more pronounced inflammatory reaction and higher levels of IFN-1 induced by the simian compared with the human adenoviral vector. In contrast, upon mucosal immunization, especially through the oral route, the AdC68rab.gp vector failed to induce Th1-associated IgG2a Abs to rabies virus, confirming that the type of immune response is markedly influenced by the route of vaccine administration (14, 18). Both vectors induced mucosal Abs in neonates within 3 wk, including IgA in vaginal lavage. Highest levels of IgA were achieved upon intranasal vaccination with the AdC68rab.gp vector, while oral immunization favored induction of vaginal IgG Abs. This is in agreement with previous findings in adult mice that showed that intranasal immunization favors stimulation of Abs secreted at the mucosa of the genital tract (18).

We have shown previously that pups born to AdHu5-immune animals responded poorly to the AdHu5rab.gp vaccine given systemically (8). Maternally transferred Abs to rabies virus had no such effect, which presumably reflects that the rabies virus Ag is not displayed by the adenoviral vaccine but requires infection of cells and transcription and translation of the adenoviral genome. Therefore, we tested here for the effect of maternally transferred Abs to the vaccine carrier, that is the AdHu5 virus for which humans carry neutralizing Abs, rather than to the vaccine Ag. Interestingly, unlike in our previous study, maternally transferred Abs to the AdHu5 vector failed to reduce the Ab response to rabies virus presented by the AdHu5 vaccine given orally. A similar observation was reported previously upon mucosal immunization of poxvirus-immune mice with a vaccinia virus recombinant (41). In our system, pooled sera from pups born to AdHu5-immune dams showed higher serum Ab titers to rabies virus, which mainly reflected an increase in IgG2a Abs. Titration of sera from individual pups born to naive dams or adenovirus-exposed dams revealed a gender difference in the response to the vaccine, with male pups born to AdHu5-immune dams responding better to the vaccine compared with male pups born to naive dams. The tendency of male pups especially to develop better Ab responses in the presence of maternally transferred immunity remains to be investigated further and we can currently only speculate on the underlying mechanism. M cells within the intestinal epithelium transport Ag from the apical surface, i.e., the gut lumen, to the underlying gut-associated lymphoid tissue. A recently identified receptor that awaits further characterization was shown to bind secretory IgA but not IgG or IgM (42), suggesting that this receptor may mediate the transport of IgA or IgA-Ag complexes from the gut lumen to the underlying lymphoid tissues. In our system, maternal secretory IgA specific for adenoviral Ags ingested by suckling pups may have facilitated uptake of the adenoviral vectors, which could have increased production of the rabies virus glycoprotein and thus the antigenic load. This in turn could result in an improved immune response to the vaccine Ag. Neonatal vaccines based on a carrier that does not circulate in the human population such as the AdC68 vector, are not recognized by maternally transferred Abs unless such vaccines become available for widespread use in adult humans. We did not formally test whether the efficacy of AdC68 vectors given to neonates with maternally transferred Abs to the AdC68 virus was affected differentially depending on the route of immunization, but, considering the similarities between the two vaccine carriers, assume a similar pattern.

Targeting the mucosal immune system of infants through intranasal or oral application of vaccines has logistic advantages and the benefit of sponsoring induction of Abs at mucosal surfaces, the most common port of entry for infectious agents. Most traditional vaccines based on attenuated or inactivated pathogens are unsuited for oral delivery. Novel vaccine carriers, such as E1-deleted adenoviral vectors or recombinant bacteria (43), have shown efficacy upon oral application in experimental animals. In addition, as shown here, oral immunization of neonatal mice is remarkably efficient at inducing systemic and mucosal transgene product-specific Abs and can circumvent interference by maternal Abs. The efficacy of oral vaccination on the induction of transgene product-specific CD8+ T cells which do not play a role in vaccine-induced protection against rabies virus, but are thought to be crucial to ward off other infectious agents and the effect of maternally transferred Abs on stimulation of this arm of the immune system, remains to be investigated. Maternally transferred Ab titers that suffice to impair the efficacy of vaccination are below those needed to provide reliable protection to the infants against the corresponding pathogens, leaving infants with declining maternal Ab titers highly susceptible to infections. Oral vaccines, such as those based on E1-deleted adenoviral recombinants, may circumvent the calamity of maternal Abs. The superb immunogenicity of E1-deleted adenoviral vectors combined with the good safety profile demonstrated for AdHu5 vectors in adult humans adds to their attractiveness for further development as vaccines for infants.


   Acknowledgments
 
We thank the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health, for their support to the Wistar Institute.


   Footnotes
 
1 This work was funded by grants from National Institutes of Health/National Institute of Allergy and Infectious Diseases. Back

2 Address correspondence and reprint requests to Dr. Hildegund C. J. Ertl, Tumor Immunology Program, Wistar Institute, University of Pennsylvania, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address: ertl{at}wistar.upenn.edu Back

3 Abbreviations used in this paper: Ad, adenovirus; ERA, Evelyn Rokitniki Abelseth; GFP, green fluorescent protein; BPL, {beta}-propiolactone; VNA, virus-neutralizing Ab. Back

Received for publication June 19, 2003. Accepted for publication August 12, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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