The Role of the 19-kDa Region of Merozoite Surface Protein 1 and Whole-Parasite-Specific Maternal Antibodies in Directing Neonatal Pups’ Responses to Rodent Malaria Infection¹

Young children and pregnant women in malaria endemic regions are the populations most at risk from the severe manifestations associated with malaria infection (1, 2). Infants up to 6 mo of age appear to be relatively resistant to the severe form of the disease (3, 4, 5). Several hypotheses have been proposed to explain this resistance, including the presence of fetal hemoglobin (which slows the growth of the parasite (6)), the lack of para-aminobenzoic acid in a complete milk diet (7, 8), the lack of exposure to mosquito bites (9), and the presence of maternally derived anti-parasite Abs in the infant (10, 11).

Although earlier studies suggested an association between decreasing levels of maternally derived malaria parasite-specific Ab and increased risk of clinical malaria, more recent studies have failed to duplicate this finding (12, 13). Studies performed in Nigeria and Tanzania were unable to find an association between the levels of cord blood Abs specific for various malaria Ags and the onset of clinical malaria or the time to patent parasitemia (12, 13). Similarly, another study in Liberia found no association between total malaria-specific Ab levels at birth and the risk of clinical disease (14). Interestingly, the study in Liberia and another in Kenya did demonstrate an association between the level of Abs at birth to the 19-kDa C-terminal fragment of the merozoite surface protein 1 (MSP1₁₉)⁴ and resistance to clinical malaria in infants (14, 15). A recent study in Ghana also failed to establish an association between Ab levels at birth and resistance to malaria infection, suggesting that the high Ab levels at birth were in fact a marker for risk of infection as opposed to protective immunity (16). Interestingly, the five infants with a high-density infection in this study were seronegative for MSP1₁₉-specific Abs at the time of infection. The potential role of maternally derived MSP1₁₉-specific Abs in infants is particularly interesting in light of data suggesting that natural Ab responses to MSP1₁₉ in malaria endemic regions increase with age and are correlated with protection in older children (17, 18).

Numerous studies have attempted to model the human situation by using rodent models of malaria (19, 20, 21, 22, 23, 24, 25, 26, 27). Initial studies in rats using the parasite Plasmodium berghei established that varying levels of resistance to malaria infection could be transferred from a mother to her offspring during lactation (20, 27). Two further studies in mice with P. vinckei and in rats with P. berghei also determined that this resistance was due to malaria parasite-specific Abs present in the milk (19, 26).

Although the actual role of maternally derived Abs remains to be established, their presence in the infant and neonate is not disputed. The route of transfer of Abs from mother to offspring differs between humans and mice. Humans acquire their Abs placentally (28), whereas rodent pups acquire theirs both placentally and in the milk (29), with the greatest acquisition occurring during the suckling period after birth (30). Although the route of transfer may differ, the presence of Abs at birth and for a limited duration after birth is similar.

It is yet to be determined whether the maternally derived Abs present in the neonate in the period immediately after birth affect the neonate’s ability to mount an immune response. With a number of malaria vaccine candidates entering clinical trials, it is essential to determine whether the maternal Abs raised against the vaccine and transferred to the offspring will have any effect on the neonate’s ability to produce an immune response against the parasite. To answer this question, we examined Abs against a malaria subunit vaccine candidate, MSP1₁₉, and against P. yoelii in the context of the mouse mother-offspring model. It was determined that the maternally derived Abs played a protective role in the pup and altered the specificity of the pups’ Ab response following parasite challenge. This suggests that the transfer of Abs specific for a malaria vaccine candidate from an immunized mother to her child should not prevent the infant from responding to malaria infection. However, the maternally derived Abs are likely to alter the response to critical epitopes on the parasite.

Female and male BALB/c (H-2^d) and C57BL/6 (H-2^b) mice were obtained from the Animal Resources Center (Willeton, Australia). Mice were housed in the Queensland Institute of Medical Research animal facility (Brisbane, Australia) under specific pathogen-free conditions. All experiments were performed in compliance with the Bancroft Centre Animal Ethics Committee requirements.

The malaria parasite used was P. yoelii YM. The parasites were maintained by i.p. passaging of 10⁶ parasitized erythrocytes into recipient mice. The parasites, as infected RBCs, were cryopreserved in liquid nitrogen.

Recombinant MSP1₁₉ corresponding to P. yoelii YM protein was produced with a C-terminal His₆ tag in Saccharomyces cerevisiae (yMSP1₁₉) (31). It was provided as a generous gift by Dr. Anthony Stowers (Malaria Vaccine Development Unit, National Institutes of Health, Rockville, MD).

Female mice were immunized s.c. with 20 μg of MSP1₁₉ in CFA. Equal amounts (10 μg at each site) were delivered to the tail base and abdomen. The mice were boosted with 20 μg of MSP1₁₉ in IFA s.c. on day 21 at the base of the neck and then i.p. on days 42 and 56. They were given a final boost i.p. on day 63 with the same amount of MSP1₁₉ in PBS. Upon completion of the immunization schedule, blood was collected from the mice every 5 days for 30 days. The sera from the individual mice were pooled and the Ab titer was determined by ELISA. The pooled hyperimmune sera (HIS) were used in passive transfer studies.

Mice were infected i.p. with 1 × 10⁵ P. yoelii YM parasitized erythrocytes. When the parasitemia had reached ∼40%, the mice were treated on 3 consecutive days with 0.2 ml of 1 mg/ml pyrimethamine dissolved in PBS/Tween 80. This infection and drug cure regime was repeated an additional four times with 3 wk between the last day of drug treatment and the new cycle of infection and drug cure. Three weeks after the completion of the infection and drug cure regime, blood was collected from the mice every 5 days for a period of 30 days. The sera from the individual mice were pooled and the Ab titer was determined by ELISA. The pooled HIS were used in passive transfer studies.

Female mice were immunized according to the schedule outlined above with the following changes: the day 42 boost with MSP1₁₉ in IFA was given s.c. and the day 56 boost with MSP1₁₉ was given i.p. in PBS. This was followed by a boost with MSP1₁₉ in PBS at day 63. A separate group of female mice underwent infection and drug cure according to the schedule outlined above.

Female mice were hormone treated to facilitate timed breeding experiments. They were given 100 μl of a 20 IU/ml solution of Serum Gonadotrophin (Folligon; Intervet, Bendigs, Australia) followed 46–48 h later by 100 μl of a 20 IU/ml solution of Chorionic Gonadotrophin (Chorulon; Intervet). Hormone-treated female mice were housed with male mice. Nineteen to 21 days later, female mice were monitored for delivery of pups. Pups were weaned at 16–21 days after birth.

Pups were challenged i.p. with 1 × 10⁵ P. yoelii YM parasitized erythrocytes. Parasitemia was monitored by microscopic examination of stained blood films every second day after challenge infection. Five microliters of blood was collected from pups by tail snip, mixed 1/10 with 45 μl of PBS, and spun at 13,000 rpm for 10 min to obtain the diluted serum. This was collected for the duration of the experiment and stored frozen until it was assayed for Ab levels.

Serum Ab levels were assessed by ELISA. Ninety-six-well plates were coated with 50 μl of 0.5 μg/ml MSP1₁₉ or 10 μg/ml crude P. yoelii YM parasite Ag in bicarbonate coating buffer (pH 9.6) and incubated at 4°C overnight. The plates were then blocked with 100 μl of 1% BSA/PBS for 1 h at 37°C. After washing with 0.05% Tween 20/PBS, serum was added to the first well for a starting dilution of 1/500, serially diluted across the plate (the Ab titer cutoff was 1,024,000), and incubated for 1 h at 37°C. The plates were washed again, and 50 μl of a goat anti-mouse IgG HRP conjugate (The Binding Site, Birmingham, U.K.) was added at a dilution of 1/3,000 in 1% BSA/PBS and incubated for 1 h at 37°C. After further washing, 50 μl of substrate solution (ABTS; Sigma-Aldrich, St. Louis, MO) was added. After a 30-min incubation at room temperature, the OD was determined at 405 nm on a Tecan Spectra plate reader (Tecan, Salzburg, Austria).

For ELISAs using allotype-specific reagents, the above method was used with the following changes. The serum (previously diluted 1/10) was added to plate to give a dilution of 1/500. The biotin-conjugated goat anti-mouse IgG2a^a or IgG2a^b Abs (BD PharMingen, San Diego, CA) were added at a concentration of 0.2 μg/ml after washing serum from the plate and then incubated at 37°C for 45 min. After washing, the streptavidin-peroxidase HRP conjugate (BioSource International, Camarillo, CA) was added at a concentration of 0.1 μg/ml and incubated at room temperature for 45 min. After washing, the substrate was added and the plate was read according to the method outlined above.

For competition ELISAs, 96-well plates previously coated with MSP1₁₉ and blocked with 1% BSA/PBS were incubated overnight with 200 μg/ml mAb302. After washing of the plates, serum taken from pups on the day of P. yoelii YM challenge and on day 23 postchallenge were added to the plate at a dilution of 1/500 and incubated for 1 h at 37°C. The ELISA was then performed according to the method outlined for the ELISA using the allotypic specific reagents. (All serum samples were assayed with and without the addition of mAb302.) The serum samples on the day of parasite challenge were assayed for their IgG2a^a component as an indicator of the maternally derived Ab specificity. The serum samples at the end of the experiment were assayed for their IgG2a^b component, which reflected the development of the pups’ Ab response. Serum samples from pups born to naive mothers and subsequently infected with P. yoelii YM and drug cured were used to determine the specificity of the pup’s Ab response to parasite infection in the absence of maternally derived Abs.

Multiscreen Assay plates (Millipore, Bedford, MA) were coated with 100 μl of 0.5 μg/ml MSP1₁₉ in bicarbonate coating buffer (pH 9.6) and incubated overnight at 4°C. After emptying the wells, they were blocked for 2 h at 37°C with 200 μl of 5% FCS. After three washes in 0.05% Tween 20/PBS, the wells were filled with 200 μl of medium and incubated at 37°C for 30 min. This washing step was repeated. After a wash in PBS, 200 μl of spleen or lymph node cell suspensions were added at a concentration of 2 × 10⁶ cells/ml. The plates were incubated for 5 h at 37°C, 5% CO₂, in a humid atmosphere. The cell suspensions were removed from the plates and the wells were washed three times with PBS. One hundred microliters of a 1/500 dilution of biotinylated goat anti-mouse IgG2a^b Ab (BD PharMingen) in 5% FCS/PBS was added to the wells and incubated overnight at 4°C. After three washes in PBS, 100 μl of a 1/500 dilution of streptavidin alkaline-phosphatase (BD PharMingen) in 5% FCS/PBS was added to the wells and incubated at 37°C for 2 h. After washing in PBS, 100 μl of substrate solution (5-bromo-4-chloro-3-indoyl phosphate/nitro blue tetrazolium alkaline phosphatase; Sigma-Aldrich) was added and the plates were incubated in the dark at room temperature for 15 min. The reaction was stopped with water. Plates were read using a stereomicroscope.

Data from the experiments in this article were analyzed using the following statistical tests: Fisher’s exact, Mann-Whitney U, χ², Kruskal-Wallis, and Spearman rank correlation.

It has been shown that specific Abs administered directly to immunocompetent adult mice can protect them from lethal malaria infection (32). As a prelude to examining the effect of maternally derived Abs on the immune response to parasite infection in the pup, we wished to determine whether whole-parasite- or MSP1₁₉-specific Abs directly administered to mouse pups would result in protection similar to that observed in adult mice.

Naive BALB/c mice were bred and their pups were treated as follows: MSP1₁₉ HIS, P. yoelii HIS, or normal mouse serum (NMS) was administered directly to the pup, and 0.250-ml injections of serum were given on days −1, 0, and +1 relative to the day of challenge. Pups were challenged at 2 wk of age with 1 × 10⁵ P. yoelii YM parasitized erythrocytes and the development of parasitemia was monitored. The pups that were given NMS succumbed to challenge, whereas all of the pups that received MSP1₁₉ HIS and P. yoelii HIS were protected from lethal malaria infection (Fig. 1). Pups given P. yoelii HIS exhibited the greatest protection. The pups given MSP1₁₉ HIS exhibited the longest prepatent period in contrast with the pups receiving NMS, which had the shortest.

Once we had established that Abs directly administered to the pups could protect them from malaria infection, we next examined whether Abs transferred from a P. yoelii or MSP1₁₉ immune mother could also protect the pups after parasite challenge. BALB/c female mice immune to challenge with P. yoelii (by virtue of having undergone multiple cycles of parasite infection and drug cure) or immunized with MSP1₁₉ and naive mothers were bred with naive BALB/c males. At birth, one-half of the litters were transferred between the immune and nonimmune mothers so that each litter consisted of natural and cross-fostered pups. This postbirth transfer of pups allowed the route of transfer of maternal Abs (placental and milk) to the pup to be confirmed. Litters were challenged at 2, 3, 4, 5, or 8 wk of age with 1 × 10⁵ P. yoelii YM parasitized erythrocytes to determine the effect of maternally derived whole-parasite-specific Abs on parasite growth in the pup.

Pups suckled on the P. yoelii immune mother, but not pups suckled on a nonimmune mother, were protected when challenged at 2 and 3 wk of age (Fig. 2, A and B). Pups suckled on an immune mother and challenged at 4, 5, and 8 wk of age exhibited parasitemia curves similar to those of the age-matched pups suckled on nonimmune mothers (Fig. 2, C-E).

The serum level of whole-parasite-specific Abs acquired from suckling on the immune mother was greatest in the pups at 2 and 3 wk of age (Fig. 3, A and B, left column), after which it decreased. The level of total Ab present in the pups was very similar between natural (filled symbols) and cross-fostered pups (open symbols), confirming that there is minimal acquisition of Abs via the placenta. There was a significant difference when comparing the Ab titers at the time of challenge in surviving pups and nonsurviving pups (p < 0.001, Mann-Whitney U test). These results suggest that maternally derived milk Abs are important in protecting the pups.

Pups suckled on MSP1₁₉-immune mothers (immunized with a protocol known to induce complete protection of the mothers (33)) and challenged at 2, 3, or 4 wk of age experienced a significant delay in the onset of parasitemia compared with their age-matched counterparts suckled on a nonimmune mother (Fig. 4, A–C). These pups also had the highest levels of Abs present at the time of challenge (Fig. 5, A and B, left column). The levels of Abs in the natural (filled symbols) and cross-fostered pups (open symbols) that were suckled on immune mothers were similar. There was a significant correlation between the length of the prepatent period and the Ab titer at the time of challenge (r = 0.840, p < 0.001, Spearman rank correlation). This supports a role for maternally derived MSP1₁₉-specific Abs in the protection of the rodent pups.

Because the results presented thus far indicated a protective role for maternally derived Abs in the pup, it was of interest to determine the effect of these P. yoelii- and MSP1₁₉-specific Abs on the development of the pups’ Ab response to the parasite. To examine this, we exploited differences in murine strain allotype genetics that allowed differentiation between maternally derived Abs and pup Abs (34). BALB/c mice (Igh-1a) produce IgG2a^a, C57BL/6 mice (Igh-1b) produce IgG2a^b, and pups derived from a BALB/c female × C57BL/6 male mating produce both IgG2a^a and IgG2a^b. Any IgG2a^a P. yoelii- or MSP1₁₉-specific Abs present in these F₁ pups before challenge would be of maternal origin. P. yoelii immune or MSP1₁₉-immunized BALB/c female mice were mated with naive C57BL/6 males. Litters born to MSP1₁₉ immune mothers were designated Groups A and B, whereas litters born to P. yoelii immune mothers were designated Groups C, D, and E. These litters were challenged at 2 wk of age with P. yoelii YM.

Separate litters of pups born to MSP1₁₉-immunized mothers (Groups A and B) had medium levels of total MSP1₁₉-specific IgG in their blood at the time of challenge, low levels of IgG2a^a, and no detectable IgG2a^b (Fig. 6, A and B, right column). In response to challenge, the total MSP1₁₉-specific IgG levels in these pups decreased. However, the P. yoelii- and MSP1₁₉-specific IgG2a^b increased, indicating that the pups were able to respond to malaria infection in the presence of maternally derived MSP1₁₉-specific Abs (Fig. 6, A and B, left and right columns). Interestingly, there was no production of P. yoelii- or MSP1₁₉-specific IgG2a^a in the pups after challenge with the malaria parasite.

Separate litters of pups born to P. yoelii immune mothers had high levels of MSP1₁₉-specific IgG and IgG2a^a in their blood, but no detectable IgG2a^b at the time of challenge (Fig. 6, C-E, right column). They also had medium levels of P. yoelii-specific IgG and IgG2a^a, but no detectable IgG2a^b in their serum (Fig. 6, C–E, left column). In response to challenge, the total MSP1₁₉- and P. yoelii-specific IgG and IgG2a^a levels decreased, whereas specific IgG2a^b levels increased, indicating that the pups were able to respond to malaria infection in the presence of maternally derived P. yoelii-specific Abs. However, there was no production of P. yoelii- or MSP1₁₉-specific IgG2a^a in the pups. These data indicated that in the presence of maternally derived P. yoelii- and MSP1₁₉-specific Abs, pups could develop an Ab response following parasite challenge.

The F₁ pups suckled on either MSP1₁₉ or P. yoelii immune mothers were protected from malaria infection. With the exception of one pup each from Groups A and B succumbing to challenge, all other mice survived, even though some developed quite high parasitemia (data not shown).

Although F₁ pups derived from a mating between naive BALB/c female × C57BL/6 male mice produce both an IgG2a^a and an IgG2a^b response after infection with P. yoelii (data not shown), pups born to either MSP1₁₉ or P. yoelii immune mothers produced only IgG2a^b in response to parasite infection and failed to generate a specific IgG2a^a response. We asked whether the difference in the pups’ allotype production in the presence of maternally derived Abs might also reflect a difference in Ab specificity. Serum samples from pups born to MSP1₁₉-immunized mothers, P. yoelii immune mothers, and naive mothers that were subsequently infected with P. yoelii YM and drug cured were studied.

Initially, we approached this question of specificity by first establishing whether mAb302 (IgG3) could interfere with the binding of the pups’ sera to MSP1₁₉. Monoclonal Ab302 binds to a region in the first epidermal growth factor (EGF)-like domain of MSP1₁₉ and confers protection when passively transferred to mice (35). We have previously demonstrated that Abs in the sera of mice immunized with MSP1₁₉ could interfere with the binding of mAb302 to MSP1₁₉. Therefore, if mAb302 was shown to interfere with the binding of the Abs in the pups’ sera to MSP1₁₉, this would indicate a common specificity for a protective epitope.

Serum from pups born to naive mothers and subsequently infected with P. yoelii YM and drug cured was added to MSP1₁₉-coated plates that had been previously incubated with or without mAb302, and both the MSP1₁₉-specific IgG2a^a and IgG2a^b levels were assayed. We found that the addition of mAb302 could significantly interfere with the binding of the MSP1₁₉-specific IgG2a^a and IgG2a^b Abs in the serum (p = 0.009 and 0.009, respectively, Mann-Whitney test) (Fig. 7 A). Thus, in the absence of maternally derived Abs, the pups produced Abs of both allotypes that recognized the same epitope as mAb302 in response to parasite infection.

There was a low level of MSP1₁₉-specific IgG2a^a Ab present in the serum of the pups born to MSP1₁₉ immune mothers (before challenge of the pups), which was further decreased when mAb302 was added to the wells (p = 0.009) (Fig. 7,B). In contrast, the level of MSP1₁₉-specific IgG2a^b Ab present in the serum of these pups at 23 days postchallenge was not significantly affected by the addition of the mAb (p = 0.144) (Fig. 7 B), suggesting that maternally derived Abs recognized epitopes similar to mAb302, whereas in the presence of maternally derived Abs, the pups generated Abs that recognized alternate epitopes.

The detection of MSP1₁₉-specific IgG2a^a Abs present in the serum of pups born to P. yoelii immune mothers was also significantly decreased when mAb302 was added to the wells (p = 0.009) (Fig. 7,C). These findings indicated that the maternally derived IgG2a^a MSP1₁₉-specific Ab in the pup at day 0 shared specificities for the Ag similar to those of mAb302. In contrast, the detection of MSP1₁₉-specific IgG2a^b Abs present in the serum of the pups at day 23 was not significantly affected in the presence of mAb302 (p = 0.121) (Fig. 7 C), suggesting that the majority of MSP1₁₉-specific IgG2a^b Abs present in the pups 3 wk after challenge had a different specificity than either mAb302 or maternally derived IgG2a^a Abs present in the pups at the time of challenge.

Previous studies in mice have shown that maternal cells may be transferred placentally to the fetus and by lactation to the neonate (36, 37, 38). The results from studies on the frequency of cell trafficking have not been consistent, possibly due to the different methods and technologies used (37, 38). Studies in malaria parasite-infected rodents have shown that prenatal sensitization to blood-stage malaria parasites or Ags occurs and that lymphocytes from newborn animals can transfer malaria-specific immunity to naive recipients (21, 39).

We asked whether the development of the pups’ Ab response to malaria infection was either due to maternally derived MSP1₁₉-specific B cells or the result of prebirth exposure of neonatal B cells to Ag (or both). ELISPOT assays were performed using goat anti-mouse IgG1 and IgG2a^b to detect maternally derived MSP1₁₉-specific Ab-secreting B cells (IgG1) or pup MSP1₁₉-specific Ab-secreting B cells (IgG1 and IgG2a^b) in naive pups born to MSP1₁₉ immune or nonimmune mothers. Preliminary experiments to detect IgG1- and IgG2a^a-secreting B cells in the spleens and lymph nodes of adult and infant mice after MSP1₁₉ immunization showed that there was a greater number of MSP1₁₉-specific IgG1-secreting B cells compared with IgG2a^a-secreting B cells (data not shown). For this reason, IgG1 detection was performed in preference to IgG2a^a detection.

MSP1₁₉-immunized and naive BALB/c female mice were bred with C57BL/6 male mice, offspring were sacrificed at 2, 3, and 4 wk of age, and their inguinal and popliteal lymph nodes and spleens were removed. The pups had not been infected with P. yoelii YM or immunized with MSP1₁₉. Four pups were sampled from each mother at every time point. Adult BALB/c and F₁ BALB/c × C57BL/6 pups previously immunized with MSP1₁₉ were used as positive controls for detection of IgG1 and IgG2a^b, respectively.

B cells secreting MSP1₁₉-specific IgG1 and IgG2a^b were identified from the spleens of the previously immunized adult BALB/c and F₁ BALB/c × C57BL/6 mice, respectively (Fig. 8). No IgG1 or IgG2a^b MSP1₁₉-specific Ab-secreting B cells were isolated from the naive pups or pups born to and suckled on MSP1₁₉-immunized mothers. A similar trend was observed with the cells derived from the lymph nodes (data not shown). This indicates that the MSP1₁₉-specific Abs generated in the pups after infection could not have been of maternal B cell origin or a result of pre-exposure of neonatal B cells to malaria Ag that might have been transferred from mother to fetus.

In humans, the passive transfer of humoral-mediated immunity from mother to child occurs during pregnancy. This transfer of Abs stops at birth, with levels in the newborn decreasing at a constant rate (40). It is believed that these maternally derived Abs act to protect the neonate during the time when it is immunologically vulnerable. It has been suggested that immunizing pregnant women with a malaria vaccine may result in an increase in the level of Abs transferred to the neonate as well as an increased development of the newborn’s memory T cells via maternally transferred cytokines (15). It has been shown that after malaria infection the level of maternally derived anti-MSP1₁₉ IgG in the infant is correlated with a decrease in placental malaria and a delay in the infant’s first detectable malaria infection (41). What is not clear, however, is the effect that maternally derived Abs generated as a result of vaccination may have on the infant’s ability to respond to malaria infection.

Using a rodent model of malaria, the present study addresses a number of issues regarding passively transferred Abs, either directly or maternally derived, and the development of the neonatal immune response to malaria infection. Initially it was shown that the direct transfer of P. yoelii-specific and MSP1₁₉-specific Abs to the pup was sufficient to protect against lethal challenge. This established that the protection by the direct transfer of specific Ab demonstrated in previous studies using adult mice (32, 34) could also be achieved in infant mice (Fig. 1).

Additional experiments examined whether this protection could be duplicated if the passive transfer of Abs was via the mother to her offspring either placentally or by suckling. It was shown that whole-parasite-specific Abs could protect pups from parasite challenge at 2 and 3 wk of age when suckled on a P. yoelii immune mother (Fig. 2). The cross-fostering of pups at birth between immune and nonimmune mothers confirmed earlier studies in rats and mice that demonstrated that the majority of Abs were transferred via suckling and that Abs acquired via the placenta alone could not protect against lethal malaria infection. Interestingly, the levels of Abs present at the time of challenge in 2-, 3-, and 4-wk-old pups were very similar, but survival from challenge was seen only in the 2- and 3-wk-old pups. The slightly lower Ab titer seen in 4-wk-old pups may not have slowed the parasite growth sufficiently to allow for the pups to generate their own immune response against the parasite.

For BALB/c pups suckling on MSP1₁₉-immunized mothers, the specific Abs present in the pups at 2, 3, and 4 wk of age were insufficient to protect the mice from parasite challenge (Fig. 4). However, they were able to suppress the growth of the parasite when compared with pups suckled on nonimmune mothers. This extended prepatent period is a feature of MSP1₁₉-specific Ab mediated immunity (32). MSP1₁₉-specific Abs act by suppressing parasite growth, allowing the immune system time to generate a response against the parasite. If sufficient MSP1₁₉ Abs are present at the time of challenge, mice are protected. These pups had lower MSP1₁₉-specific Ab titers at the time of challenge than did the pups that received the Ab directly in the form of HIS and survived parasite challenge (data not shown). A lower Ab titer at the time of challenge may not have suppressed the parasite growth sufficiently to allow the pup to develop its own protective immune response against the parasite.

This study did show, by using allotypic reagents, that 2-wk-old pups produced Abs in response to malaria infection in the presence of both maternally derived MSP1₁₉- and P. yoelii-specific Abs (Fig. 6). However, the presence of maternal Abs affected the fine specificity of the immune response following parasite challenge (Fig. 7). The Abs generated by the pups in response to infection recognized an epitope different from that of mAb302 and the maternally derived Abs. Previous work assessing the specificity and protective ability of mAbs specific for MSP1₁₉ determined that there were at least two distinct epitopes that could be defined (42). All four of the MSP1₁₉-specific mAbs were able to modify the parasitemia, with the most protective sharing the same subclass (IgG3) and specificity (binding to an epitope in the first EGF-like domain) as mAb302. The other two mAbs were of different subclasses (IgG1 and IgG2b), bound to epitopes that required both C-terminal EGF-like domains for their formation, and were only partially effective at suppressing parasite growth. Although these studies demonstrated that Abs specific for at least two distinct epitopes are capable of modifying parasite growth, further work is required to address the protective ability of the Abs generated by the pup in the absence of the protective maternally derived Abs.

There is a considerable amount of literature addressing the effect of perinatal exposure to Ids/anti-idiotypic Abs on the host’s subsequent immune response (43, 44). Traditionally, these experiments have studied the effect of direct administration of monoclonal anti-idiotypic Abs on the fine specificity of the recipient’s subsequent immune response (45, 46, 47), although the effect on a pup’s immune response after maternal transfer of anti-idiotypic Abs has also been examined (48). In the instance of the current study, it is conceivable that the transfer of MSP1₁₉ maternally derived Abs of a particular fine specificity (as defined by mAb302) resulted in the production of anti-idiotypic Abs by the pup. Consequently, the pup would not make Abs expressing this Id. This could explain the altered fine specificity of the pup’s Ab response to MSP1₁₉ after parasite challenge in the presence of maternally derived Abs. Preliminary data from an experiment examining the effect of maternally derived MSP1₁₉-specific Abs on Ab responses to MSP1₁₉ vaccination in the pup have indicated that, in the presence of high-titer maternally derived Abs, the pup’s response to the epitope bound by the maternally derived Abs and mAb302 is also completely blocked.

Another important aspect of this study is the effect of maternally derived Abs on the resulting allotypic response in the pup after malaria infection. In the presence of maternally derived Abs, the pups recognize an alternate epitope following malaria infection, but only IgG2a^b of this altered fine specificity is produced. It is possible that allotypic exclusion/suppression may be occurring in these pups, preventing them from producing Abs expressing the maternal allotype. Additional experiments have been designed to examine the exact mechanism responsible for this altered Ab response.

It would be of great interest to examine whether the perinatal exposure to maternally derived Abs affects protective immunity or the specificity of the Ab responses to allelic variants of P. yoelii MSP1₁₉, because this has very important implications for the development of a human malaria vaccine. Previous studies in mice have indicated that administration of mAb302 enables them to survive a normally lethal homologous challenge (35) and offers protection against infection with three of the five P. yoelii strains (49). In this instance, one might predict that if the epitope recognized by mAb302 and MSP1₁₉-specific maternally derived Abs is indeed the same (as is indicated by our study), the presence of these maternally derived Abs would not necessarily preclude the pup from responding to the epitope in the other two P. yoelii strains. The protection offered by the maternally derived Abs to the other two strains is unknown. In humans it has been reported that serum Abs recognize epitopes that are conserved or cross-reactive between two commonly occurring allelic forms of P. falciparum MSP1₁₉ (50). Therefore, it is possible that Abs induced by vaccination with one or another of the allelic forms of the protein could recognize MSP1₁₉ from multiple strains of P. falciparum. However, this observed cross-reactivity in conjunction with our own data suggests that interference by vaccine-induced maternally derived Abs in the neonate may also occur after infection with multiple P. falciparum strains.

The effect of maternally derived Abs directed against a vaccine Ag on the neonatal pup’s immune response to the same Ag in the context of a live infection is a unique observation. Further studies need to be conducted focusing on other mouse models of infectious disease in concert with the mother-offspring model to determine whether this phenomenon is universal. If it is shown to be widespread, this will have very important implications for the current approach to protecting neonates via maternal immunization.

Although the exact role of maternal Abs in human infants is still undetermined, studies have shown a correlation between the presence of MSP1₁₉-specific Abs and resistance to clinical malaria. If this is the case, immunization of mothers with MSP1₁₉ may be one way to protect infants from the serious consequences of malaria infection while they develop their own protective immune response. However, the results presented in this study suggest that maternally derived Abs generated as a result of malaria infection or immunization with MSP1₁₉ interfere with the infant pups’ response to malaria infection in that they affect the fine specificity of the immune response after challenge with the malaria parasite. In a heterogeneous population, such as humans, in which the fine specificity may differ between individuals and may be critical for protection, the immune status of the mother may qualitatively alter the newborn’s response after parasite infection.

We thank Dr. Carole Long for providing mAb302 and Dr. Michelle Gatton for advice on statistical analysis.