Evidence that invasion-inhibitory antibodies specific for the 19-kDa fragment of merozoite surface protein-1 (MSP-1 19) can play a protective role against blood-stage Plasmodium falciparum infection in individuals in a malaria endemic area of Africa

The C-terminal 19-kDa fragment of Plasmodium falciparum merozoite surface protein-1 (MSP-1(19)) is a target of protective Abs against blood-stage infection and a leading candidate for inclusion in a human malaria vaccine. However, the precise role, relative importance, and mechanism of action of Abs that target this protein remain unclear. To examine the potential protective role of Abs to MSP-1(19) in individuals naturally exposed to malaria, we conducted a treatment time to infection study over a 10-wk period in 76 residents of a highland area of western Kenya during a malaria epidemic. These semi-immune individuals were not all equally susceptible to reinfection with P. falciparum following drug cure. Using a new neutralization assay based on transgenic P. falciparum expressing the P. chabaudi MSP-1(19) orthologue, individuals with high-level MSP-1(19)-specific invasion-inhibitory Abs (>75th percentile) had a 66% reduction in the risk of blood-stage infection relative to others in the population (95% confidence interval, 3-88%). In contrast, high levels of MSP-1(19) IgG or IgG subclass Abs measured by enzyme immunoassay with six different recombinant MSP-1(19) Ags did not correlate with protection from infection. IgG Abs measured by serology and functional invasion-inhibitory activity did not correlate with each other. These findings implicate an important protective role for MSP-1(19)-specific invasion inhibitory Abs in immunity to blood-stage P. falciparum infection, and suggest that the measurement of MSP-1(19) specific inhibitory Abs may serve as an accurate correlate of protection in clinical trials of MSP-1-based vaccines.     

Chimeras of labile toxin one and cholera toxin retain mucosal adjuvanticity and direct Th cell subsets via their B subunit

Native cholera toxin (nCT) and the heat-labile toxin 1 (nLT) of enterotoxigenic Escherichia coli are AB5-type enterotoxins. Both nCT and nLT are effective adjuvants that promote mucosal and systemic immunity to protein Ags given by either oral or nasal routes. Previous studies have shown that nCT as mucosal adjuvant requires IL-4 and induces CD4-positive (CD4+) Th2-type responses, while nLT up-regulates Th1 cell production of IFN-gamma and IL-4-independent Th2-type responses. To address the relative importance of the A or B subunits in CD4+ Th cell subset responses, chimeras of CT-A/LT-B and LT-A/CT-B were constructed. Mice nasally immunized with CT-A/LT-B or LT-A/CT-B and the weak immunogen OVA developed OVA-specific, plasma IgG Abs titers similar to those induced by either nCT or nLT. Both CT-A/LT-B and LT-A/CT-B promoted secretory IgA anti-OVA Ab, which established their retention of mucosal adjuvant activity. The CT-A/LT-B chimera, like nLT, induced OVA-specific mucosal and peripheral CD4+ T cells secreting IFN-gamma and IL-4-independent Th2-type responses, with plasma IgG2a anti-OVA Abs. Further, LT-A/CT-B, like nCT, promoted plasma IgG1 more than IgG2a and IgE Abs with OVA-specific CD4+ Th2 cells secreting high levels of IL-4, but not IFN-gamma. The LT-A/CT-B chimera and nCT, but not the CT-A/LT-B chimera or nLT, suppressed IL-12R expression and IFN-gamma production by activated T cells. Our results show that the B subunits of enterotoxin adjuvants regulate IL-12R expression and subsequent Th cell subset responses.     

Acquired immune responses to Plasmodium falciparum merozoite surface protein-1 in the human fetus.

Infants born in areas of stable malaria transmission are relatively protected against severe morbidity and high density Plasmodium falciparum blood-stage infection. This protection may involve prenatal sensitization and immunologic reactivity to malaria surface ligands that participate in invasion of red cells. We examined cord blood T and B cell immunity to P. falciparum merozoite surface protein-1 (MSP-1) in infants born in an area of stable malaria transmission in Kenya. T cell cytokine responses to the C-terminal 19-kDa fragment of MSP-1 (MSP-1(19)) were detected in 24 of 92 (26%) newborns (4-192 IFN-gamma and 3-88 IL-4-secreting cells per 10(6)/cord blood lymphocytes). Peptide epitopes in the N-terminal block 3 region of MSP-1 also drove IFN-gamma and/or IL-13 production. There was no evidence of prenatal T cell sensitization to liver-stage Ag-1. A total of 5 of 86 (6%) newborns had cord blood anti-MSP-1(19) IgM Abs, an Ig isotype that does not cross the placenta and is therefore of fetal origin. The frequency of neonatal B cell sensitization was higher than that indicated by serology alone, as 5 of 27 (18%) cord blood samples contained B cells that produced IgG when stimulated with MSP-1(19) in vitro. Neonatal B cell IgG responses were restricted to the Q-KNG allele of MSP-1(19), the major variant in this endemic area, whereas T cells responded to all four MSP-1(19) alleles evaluated. In utero sensitization to MSP-1 correlated with the presence of malaria parasites in cord blood (chi(2) = 20, p < 0.0001). These data indicate that prenatal sensitization to blood-stage Ags occurs in infants born in malaria endemic areas.     

Induction of genital immunity by DNA priming and intranasal booster immunization with a replication-defective adenoviral recombinant.

Mice immunized through different routes such as i.m., intradermally, or intratracheally with a DNA vaccine to rabies virus developed high titers of serum Ab but only borderline levels of mucosal Abs determined from vaginal secretions. DNA vaccines given by either route enhanced vaginal IgA and IgG2a secretion upon a subsequent intranasal booster immunization with an E1-deleted adenoviral recombinant expressing the same Ag of rabies virus. DNA vaccine priming reduced the Ab response to the adenoviral Ags and counterbalanced the impaired B cell response to the rabies virus Ag expressed by the adenoviral recombinant in mice preimmune to adenovirus. The vaginal B cell response could further be enhanced by using the Th2-type cytokines IL-4 or IL-5 as genetic adjuvants concomitantly with the DNA vaccine before intranasal booster immunization with the recombinant vaccine.     

Genetic polymorphism and natural selection in the malaria parasite Plasmodium falciparum.

We have studied the genetic polymorphism at 10 Plasmodium falciparum loci that are considered potential targets for specific antimalarial vaccines. The polymorphism is unevenly distributed among the loci; loci encoding proteins expressed on the surface of the sporozoite or the merozoite (AMA-1, CSP, LSA-1, MSP-1, MSP-2, and MSP-3) are more polymorphic than those expressed during the sexual stages or inside the parasite (EBA-175, Pfs25, PF48/45, and RAP-1). Comparison of synonymous and nonsynonymous substitutions indicates that natural selection may account for the polymorphism observed at seven of the 10 loci studied. This inference depends on the assumption that synonymous substitutions are neutral, which we test by analyzing codon bias and G+C content in a set of 92 gene loci. We find evidence for an overall trend towards increasing A+T richness, but no evidence for mutation bias. Although the neutrality of synonymous substitutions is not definitely established, this trend towards an A+T rich genome cannot explain the accumulation of substitutions at least in the case of four genes (AMA-1, CSP, LSA-1, and PF48/45) because the Gleft and right arrow C transversions are more frequent than expected. Moreover, the Tajima test manifests positive natural selection for the MSP-1 and, less strongly, MSP-3 polymorphisms; the McDonald-Kreitman test manifests natural selection at LSA-1 and PF48/45. We conclude that there is definite evidence for positive natural selection in the genes encoding AMA-1, CSP, LSA-1, MSP-1, and Pfs48/45. For four other loci, EBA-175, MSP-2, MSP-3, and RAP-1, the evidence is limited. No evidence for natural selection is found for Pfs25.     

Human cord blood CD4+CD25hi regulatory T cells suppress prenatally acquired T cell responses to Plasmodium falciparum antigens

In malaria endemic regions, a fetus is often exposed in utero to Plasmodium falciparum blood-stage Ags. In some newborns, this can result in the induction of immune suppression. We have previously shown these modulated immune responses to persist postnatally, with a subsequent increase in a child's susceptibility to infection. To test the hypothesis that this immune suppression is partially mediated by malaria-specific regulatory T cells (T(regs)) in utero, cord blood mononuclear cells (CBMC) were obtained from 44 Kenyan newborns of women with and without malaria at delivery. CD4(+)CD25(lo) T cells and CD4(+)CD25(hi) FOXP3(+) cells (T(regs)) were enriched from CBMC. T(reg) frequency and HLA-DR expression on T(regs) were significantly greater for Kenyan as compared with North American CBMC (p < 0.01). CBMC/CD4(+) T cells cultured with P. falciparum blood-stage Ags induced production of IFN-γ, IL-13, IL-10, and/or IL-5 in 50% of samples. Partial depletion of CD25(hi) cells augmented the Ag-driven IFN-γ production in 69% of subjects with malaria-specific responses and revealed additional Ag-reactive lymphocytes in previously unresponsive individuals (n = 3). Addition of T(regs) to CD4(+)CD25(lo) cells suppressed spontaneous and malaria Ag-driven production of IFN-γ in a dose-dependent fashion, until production was completely inhibited in most subjects. In contrast, T(regs) only partially suppressed malaria-induced Th2 cytokines. IL-10 or TGF-β did not mediate this suppression. Thus, prenatal exposure to malaria blood-stage Ags induces T(regs) that primarily suppress Th1-type recall responses to P. falciparum blood-stage Ags. Persistence of these T(regs) postnatally could modify a child's susceptibility to malaria infection and disease.     

Prenatal malaria immune experience affects acquisition of Plasmodium falciparum merozoite surface protein-1 invasion inhibitory antibodies during infancy

African infants are often born of mothers infected with malaria during pregnancy. This can result in fetal exposure to malaria-infected erythrocytes or their soluble products with subsequent fetal immune priming or tolerance in utero. We performed a cohort study of 30 newborns from a malaria holoendemic area of Kenya to determine whether T cell sensitization to Plasmodium falciparum merozoite surface protein-1 (MSP-1) at birth correlates with infant development of anti-MSP-1 Abs acquired as a consequence of natural malaria infection. Abs to the 42- and 19-kDa C-terminal processed fragments of MSP-1 were determined by serology and by a functional assay that quantifies invasion inhibition Abs against the MSP-1(19) merozoite ligand (MSP-1(19) IIA). Infants had detectable IgG and IgM Abs to MSP-1(42) and MSP-1(19) at 6 mo of age with no significant change by age 24-30 mo. In contrast, MSP-1(19) IIA levels increased from 6 to 24-30 mo of age (16-29%, p < 0.01). Infants with evidence of prenatal exposure to malaria (defined by P. falciparum detection in maternal, placental, and/or cord blood compartments) and T cell sensitization at birth (defined by cord blood lymphocyte cytokine responses to MSP-1) showed the greatest age-related increase in MSP-1(19) IIA compared with infants with prenatal exposure to malaria but who lacked detectable T cell MSP-1 sensitization. These data suggest that fetal sensitization or tolerance to MSP-1, associated with maternal malaria infection during pregnancy, affects the development of functional Ab responses to MSP-1 during infancy.     

IgG responses to Anopheles gambiae salivary antigen gSG6 detect variation in exposure to malaria vectors and disease risk

Assessment of exposure to malaria vectors is important to our understanding of spatial and temporal variations in disease transmission and facilitates the targeting and evaluation of control efforts. Recently, an immunogenic Anopheles gambiae salivary protein (gSG6) was identified and proposed as the basis of an immuno-assay determining exposure to Afrotropical malaria vectors. In the present study, IgG responses to gSG6 and 6 malaria antigens (CSP, AMA-1, MSP-1, MSP-3, GLURP R1, and GLURP R2) were compared to Anopheles exposure and malaria incidence in a cohort of children from Korogwe district, Tanzania, an area of moderate and heterogeneous malaria transmission. Anti-gSG6 responses above the threshold for seropositivity were detected in 15% (96/636) of the children, and were positively associated with geographical variations in Anopheles exposure (OR 1.25, CI 1.01-1.54, p?=?0.04). Additionally, IgG responses to gSG6 in individual children showed a strong positive association with household level mosquito exposure. IgG levels for all antigens except AMA-1 were associated with the frequency of malaria episodes following sampling. gSG6 seropositivity was strongly positively associated with subsequent malaria incidence (test for trend p?=?0.004), comparable to malaria antigens MSP-1 and GLURP R2. Our results show that the gSG6 assay is sensitive to micro-epidemiological variations in exposure to Anopheles mosquitoes, and provides a correlate of malaria risk that is unrelated to immune protection. While the technique requires further evaluation in a range of malaria endemic settings, our findings suggest that the gSG6 assay may have a role in the evaluation and planning of targeted and preventative anti-malaria interventions.     

High Antibody Responses against Plasmodium falciparum in Immigrants after Extended Periods of Interrupted Exposure to Malaria

Background

Malaria immunity is commonly believed to wane in the absence of Plasmodium falciparum exposure, based on limited epidemiological data and short-lived antibody responses in some longitudinal studies in endemic areas.

Methods

A cross-sectional study was conducted among sub-Saharan African adults residing in Spain for 1 up to 38 years (immigrants) with clinical malaria (n=55) or without malaria (n=37), naïve adults (travelers) with a first clinical malaria episode (n=20) and life-long malaria exposed adults from Mozambique (semi-immune adults) without malaria (n=27) or with clinical malaria (n=50). Blood samples were collected and IgG levels against the erythrocytic antigens AMA-1 and MSP-1₄₂ (3D7 and FVO strains), EBA-175 and DBL-α were determined by Luminex. IgG levels against antigens on the surface of infected erythrocytes (IEs) were measured by flow cytometry.

Results

Immigrants without malaria had lower IgG levels than healthy semi-immune adults regardless of the antigen tested (P≤0.026), but no correlation was found between IgG levels and time since migration. Upon reinfection, immigrants with malaria had higher levels of IgG against all antigens than immigrants without malaria. However, the magnitude of the response compared to semi-immune adults with malaria depended on the antigen tested. Thus, immigrants had higher IgG levels against AMA-1 and MSP-1₄₂ (P≤0.015), similar levels against EBA-175 and DBL-α, and lower levels against IEs (P≤0.016). Immigrants had higher IgG levels against all antigens tested compared to travelers (P≤0.001), both with malaria.

Conclusions

Upon cessation of malaria exposure, IgG responses to malaria-specific antigens were maintained to a large extent, although the conservation and the magnitude of the recall response depended on the nature of the antigen. Studies on immigrant populations can shed light on the factors that determine the duration of malaria specific antibody responses and its effect on protection, with important implications for future vaccine design and public health control measures.     

Amelioration of mercury-induced autoimmunity by 4-1BB

In certain strains of mice, subtoxic doses of HgCl2 (mercuric chloride; mercury) induce a complex autoimmune condition characterized by the production of antinucleolar IgG Abs, lymphoproliferation, increased serum levels of IgG1/IgE Abs, and deposition of renal immune complexes. 4-1BB is an important T cell costimulatory molecule that has been implicated in T cell proliferation and cytokine production, especially production of IFN-gamma. To elucidate T cell control mediated by the 4-1BB signaling pathway in this syndrome, we assessed the effect of administering agonistic anti-4-1BB mAb on mercury-induced autoimmunity. Groups of A.SW mice (H-2s) received mercury/control Ig or mercury/anti-4-1BB or PBS alone. Anti-4-1BB mAb treatment resulted in a dramatic reduction of mercury-induced antinucleolar Ab titers, serum IgG1/IgE induction, and renal Ig deposition. These effects may be related to the present finding that anti-4-1BB mAb decreases B cell numbers and function. The anti-4-1BB mAb-treated mercury group also showed a marked reduction in Th2-type cytokines but an increase in Th1-type cytokines and chemokines. Increased IFN-gamma production due to anti-4-1BB mAb treatment appears to be responsible for the observed B cell defects because neutralization of IFN-gamma in vivo substantially restored B cell numbers and partly restored IgG1/IgE. Collectively, our results indicate that 4-1BB mAb can down-regulate mercury-induced autoimmunity by affecting B cell function in an IFN-gamma-dependent manner and thus, preventing the development of autoantibody production and tissue Ig deposition.     

Partial IgA-deficiency with increased Th2-type cytokines in TGF-beta 1 knockout mice.

Though it has been shown that TGF-beta 1 directs B cells to switch to IgA in vitro, no studies have assessed TGF-beta 1 effects on mucosal vs systemic immunity in vivo. When the B cell functions of TGF-beta 1 gene-disrupted (TGF-beta 1-/-) mice were analyzed, significantly decreased IgA levels and increased IgG and IgM levels in serum and external secretions were observed. Further, analysis of Ab forming cells (AFC) isolated from both mucosal and systemic lymphoid tissue showed elevated IgM, IgG, and IgE, with decreased IgA AFC. A lack of IgA-committed B cells was seen in TGF-beta 1-/- mice, especially in the gastrointestinal (GI) tract. Splenic T cells triggered via the TCR expressed elevated Th2-type cytokines and, consistent with this observation, a 31-fold increase in serum IgE was seen in TGF-beta 1-/- mice. Thus, uncontrolled B cell responses, which include elevated IgE levels, a lack of antiinflammatory IgA, and an excess of complement-binding IgG and IgM Abs, will promote inflammation at mucosal surfaces in TGF-beta 1-/- mice and likely contribute to pulmonary and GI tract lesions, ultimately leading to the early death of these mice.     

Antibody repertoire development in fetal and neonatal piglets. VIII. Colonization is required for newborn piglets to make serum antibodies to T-dependent and type 2 T-independent antigens

Cesarean-derived piglets were reared for 5 wk under germfree conditions or monoassociated with a benign Escherichia coli (G58-1) or a enterohemorrhagic strain (933D) derived from O157:H7, and immunized i.p. with the T-dependent (TD) Ags fluorescein-labeled (FL) keyhole limpet hemocyanin or trinitrophenylated (TNP) keyhole limpet hemocyanin and the type 2 T-independent Ags TNP-Ficoll or FL-Ficoll. Only colonized piglets showed an increase in serum IgG, IgA, and IgM and had serum Abs to FL, TNP, and colonizing bacteria. While serum Abs to FL or TNP appeared following colonization alone, secondary responses were restricted to piglets immunized using TD carriers. While animals colonized with 933D had significantly higher total serum IgG and IgM levels and specific IgG Abs than those colonized with G58-1, no differences were seen in serum IgA levels, B cell diversification in the ileal Peyer's patches, and specific activity (ELISA activity per micrograms of Ig) of pre-boost serum IgG and IgM anti-TNP and anti-FL Abs. Serum IgA Abs to TNP, FL, or bacteria were not detected. Ag-driven responses, as measured by an increase in specific Ab activity, were only observed in secondary responses to TD Ags and to colonizing, pathogenic E. coli. We propose that germline-encoded, isotype-switched B cells in newborn piglets differentiate to Ab-secreting cells 1) after stimulation by bacteria-activated APCs or 2) through direct stimulation by bacterial products. We further propose that Ag-driven systemic responses require both bacterial colonization and TD Ags translocated to the peritoneum.     

Schistosoma mansoni and alpha-galactosylceramide: prophylactic effect of Th1 Immune suppression in a mouse model of Graves' hyperthyroidism

Graves' hyperthyroidism, an organ-specific autoimmune disease mediated by stimulatory thyrotropin receptor (TSHR) autoantibodies, has been considered a Th2-dominant disease. However, recent data with mouse Graves' models are conflicting. For example, we recently demonstrated that injection of BALB/c mice with adenovirus coding the TSHR induced Graves' hyperthyroidism characterized by mixed Th1 and Th2 immune responses against the TSHR, and that transient coexpression of the Th2 cytokine IL-4 by adenovirus skewed Ag-specific immune response toward Th2 and suppressed disease induction. To gain further insight into the relationship between immune polarization and Graves' disease, we evaluated the effect of Th2 immune polarization by helminth Schistosoma mansoni infection and alpha-galactosylceramide (alpha-GalCer), both known to bias the systemic immune response to Th2, on Graves' disease. S. mansoni infection first induced mixed Th1 and Th2 immune responses to soluble worm Ags, followed by a Th2 response to soluble egg Ags. Prior infection with S. mansoni suppressed the Th1-type anti-TSHR immune response, as demonstrated by impaired Ag-specific IFN-gamma secretion of splenocytes and decreased titers of IgG2a subclass anti-TSHR Abs, and also prevented disease development. Similarly, alpha-GalCer suppressed Ag-specific splenocyte secretion of IFN-gamma and prevented disease induction. However, once the anti-TSHR immune response was fully induced, S. mansoni or alpha-GalCer was ineffective in curing disease. These data support the Th1 theory in Graves' disease and indicate that suppression of the Th1-type immune response at the time of Ag priming may be crucial for inhibiting the pathogenic anti-TSHR immune response.     

Correlation of T-cell response and lymphokine profile with RESA peptides of Plasmodium falciparum containing a universal T-cell epitope and an immunopotentiator, polytuftsin.

Two RESA repeat sequences, (EENVEHDA)2 and (DDEHVEEPTVA)2, were chemically linked to a universal T-cell epitope, CS.T3 and polytuftsin, and a natural immunopotentiator, was physically mixed with these conjugates. The immunogens were studied for in vitro antigen-induced T-cell proliferation, and cytokine levels were measured in the culture supernatants. The RESA peptide(s)-CS.T3 conjugate containing polytuftsin showed the highest stimulation index (SI) as compared to the RESA peptide-CS.T3 conjugates or RESA peptides alone. Spleen cells from mice primed with either RESA peptide(s)-CS.T3 conjugate or RESA peptide-CS.T3 conjugate containing polytuftsin, when pulsed in vitro with the respective RESA peptide, showed a higher proliferation index as compared to spleen cells primed and pulsed in vitro with the respective RESA peptides. This observation has an important relevance during natural reinfection for boosting the immune response. The culture supernatants from the cells primed and pulsed in vitro with RESA peptide-CS.T3 conjugate and RESA peptide-CS.T3 conjugate containing polytuftsin showed higher IL-2 and IFN-gamma levels as compared to the RESA peptides alone. Very low IL-4 levels were detected with the above formulations. The cytokine profile is suggestive of a CD4+ TH1 type of immune response, which is ideal for the killing of intracellular pathogens like the malarial parasite.     

Differential requirement for the CD40-CD154 costimulatory pathway during Th cell priming by CD8 alpha+ and CD8 alpha- murine dendritic cell subsets

Dendritic cells (DCs) regulate the development of distinct Th populations and thereby provoke appropriate immune responses to various kinds of Ags. In the present work, we investigated the role CD40-CD154 interactions play during the process of Th cell priming by CD8 alpha(+) and CD8 alpha(-) murine DC subsets, which have been reported to differently regulate the Th response. Adoptive transfer of Ag-pulsed CD8 alpha(+) DCs induced a Th1 response and the production of IgG2a Abs, whereas transfer of CD8 alpha(-) DCs induced Th2 cells and IgE Abs in vivo. Induction of distinct Th populations by each DC subset was also confirmed in vitro. Although interruption of CD80/CD86-CD28 interactions inhibited Th cell priming by both DC subsets, disruption of CD40-CD154 interactions only inhibited the induction of the Th1 response by CD8 alpha(+) DCs in vivo. CD40-CD154 interactions were not required for the proliferation of Ag-specific naive Th cells stimulated by either DC subset, but were indispensable in the production of IL-12 from CD8 alpha(+) DCs and their induction of Th1 cells in vitro. Taken together, in our immunization model of Ag-pulsed DC transfer, CD40-CD154 interactions play an important role in the development of CD8 alpha(+) DC-driven Th1 responses but not CD8 alpha(-) DC-driven Th2 responses to protein Ags.     

The diacylated lipopeptide FSL-1 induces TLR2-mediated Th2 responses

The diacylated lipopeptide FSL-1 enhanced the generation of IgG antibodies in TLR2(+/+) mice, but not in TLR2(-/-) mice, when administered together with hen egg lysozyme as an antigen. Escherichia coli lipopolysaccharide enhanced the generation of antigen-specific antibodies in both TLR2(-/-) and TLR2(+/+) mice. In TLR2(+/+) mice, the level of enhancement due to FSL-1 was similar to that caused by lipopolysaccharide. Analysis of the IgG antibodies subclass demonstrated that the level of Th2-type IgG1 antibodies was higher than that of Th1-type IgG2a antibodies. Both FSL-1 and lipopolysaccharide induced production of IL-10 and IL-6 by splenocytes from TLR2(+/+) mice. Lipopolysaccharide also induced production of these cytokines by splenocytes from TLR2(-/-) mice, but FSL-1 did not. Neither FSL-1 nor lipopolysaccharide induced IL-12p70 production by splenocytes from either type of mice. FSL-1 upregulated B7.2 expression in B220(+) cells from TLR2(+/+) mice but not those from TLR2(-/-) mice, whereas lipopolysaccharide upregulated B7.2 expression in B220(+) cells from both types of mice. FSL-1 and, to a lesser extent, lipopolysaccharide activated mitogen-activated protein kinases in splenocytes. FSL-1 and, to a lesser extent, lipopolysaccharide induced the expression of c-Fos, which is known to be involved in Th2-type responses, in splenocytes. Thus, this study demonstrated that FSL-1 possessed TLR2-mediated Th2-type responses in vivo.     

Influence of Mycobacterium bovis bacillus Calmette-Guérin on antibody and cytokine responses to human neonatal vaccination.

The immaturity of the immune system increases the susceptibility of young infants to infectious diseases and prevents the induction of protective immune responses by vaccines. We previously reported that Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccination induces a potent Th1 response to mycobacterial Ags in newborns. In this study, we evaluated the influence of BCG on the response to unrelated vaccines given in early life. Newborns were randomly allocated to one of three study groups receiving BCG at birth, when infants received their first dose of hepatitis B and oral polio vaccines; at 2 mo of age, when infants received their first dose of diphtheria and tetanus vaccines; or at 4.5 mo of age, when immune responses to vaccines were measured. Administration of BCG at the time of priming markedly increased the cellular and Ab responses to the hepatitis B vaccine, but had only a limited influence on the cytokine response to tetanus toxoid and no effect on the Ab responses to tetanus and diphtheria toxoids. Although BCG induced a potent Th1-type response to mycobacterial Ags, it promoted the production of both Th1- and Th2-type cytokines in response to unrelated vaccines. The effect of BCG was apparent at the systemic level, as it increased the Ab response to oral polio vaccine. These results demonstrate that BCG influences the immune response to unrelated Ags in early life, likely through its influence on the maturation of dendritic cells.     

Effective mucosal immunity to anthrax: neutralizing antibodies and Th cell responses following nasal immunization with protective antigen

Mucosal, but not parenteral, immunization induces immune responses in both systemic and secretory immune compartments. Thus, despite the reports that Abs to the protective Ag of anthrax (PA) have both anti-toxin and anti-spore activities, a vaccine administered parenterally, such as the aluminum-adsorbed anthrax vaccine, will most likely not induce the needed mucosal immunity to efficiently protect the initial site of infection with inhaled anthrax spores. We therefore took a nasal anthrax vaccine approach to attempt to induce protective immunity both at mucosal surfaces and in the peripheral immune compartment. Mice nasally immunized with recombinant PA (rPA) and cholera toxin (CT) as mucosal adjuvant developed high plasma PA-specific IgG Ab responses. Plasma IgA Abs as well as secretory IgA anti-PA Abs in saliva, nasal washes, and fecal extracts were also induced when a higher dose of rPA was used. The anti-PA IgG subclass responses to nasal rPA plus CT consisted of IgG1 and IgG2b Abs. A more balanced profile of IgG subclasses with IgG1, IgG2a, and IgG2b Abs was seen when rPA was given with a CpG oligodeoxynucleotide as adjuvant, suggesting a role for the adjuvants in the nasal rPA-induced immunity. The PA-specific CD4(+) T cells from mice nasally immunized with rPA and CT as adjuvant secreted low levels of CD4(+) Th1-type cytokines in vitro, but exhibited elevated IL-4, IL-5, IL-6, and IL-10 responses. The functional significance of the anti-PA Ab responses was established in an in vitro macrophage toxicity assay in which both plasma and mucosal secretions neutralized the lethal effects of Bacillus anthracis toxin.     

Critical role of preconceptional immunization for protective and nonpathological specific immunity in murine neonates

Expression of Th2 immunity against environmental Ags is the hallmark of the allergic phenotype and contrasts with the Th1-like pattern, which is stably expressed in healthy adults throughout life. Epidemiological studies indicate that the prenatal environment plays an important and decisive role in the development of allergy later in life. Since the underlying mechanisms were unclear, an animal model was developed to study the impact of maternal allergy on the development of an allergic immune response in early life. An allergic Th2 response was induced in pregnant mice by sensitization and aerosol allergen exposure. Both, IgG1 and IgG2a, but not IgE, Abs cross the placental barrier. Free allergen also crosses the placental area and was detected in serum and amniotic fluids of neonatal F(1) mice. These F(1) mice demonstrated a suppressed Th1 response, as reflected by lowered frequencies and reduced levels of IFN-gamma production. Development of an IgE response against the same allergen was completely prevented early in life. This effect was mediated by diaplacental transfer of allergen-specific IgG1 Abs. In contrast, allergic sensitization against a different allergen early in life was accelerated in these mice. This effect was mediated by maternal CD4 and OVA-specific Th2 cells induced by allergic sensitization during pregnancy. These data indicate a critical role for maternal T and B cell response in shaping pre- and postnatal maturation of specific immunity to allergens.     

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide stimulate the induction of Th2 responses by up-regulating B7.2 expression.

Vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), two structurally related neuropeptides produced within the lymphoid microenvironment, modulate several immunologic functions. We have recently demonstrated that VIP and PACAP enhance the macrophage costimulatory activity for naive CD4+ T cells exposed to allogeneic or anti-CD3 stimuli through the differential regulation of the B7 costimulatory molecules. In this study, we report on the role of VIP and PACAP on macrophage B7 expression and costimulatory function for Ag-primed CD4+ T cells, and on the macrophage-induced regulation of Th1/Th2 differentiation in vitro and in vivo. VIP and PACAP up-regulate the costimulatory activity of macrophages for Ag-primed CD4+ T cells. VIP-/PACAP-treated macrophages gain the ability to induce Th2-type cytokines such as IL-4 and IL-5 and reduce Th1-type cytokines such as IFN-gamma and IL-2. In vivo administration of VIP or PACAP in Ag-immunized mice reduce the numbers of IFN-gamma-secreting cells and enhance the numbers of IL-4-secreting cells. One of the consequences of the VIP-/PACAP-induced shift in cytokine profile is a change in the Ag-specific Ig isotype, increasing IgG1 and decreasing IgG2a levels. Finally, the preferential differentiation into Th2 effector cells after Ag stimulation induced by VIP-/PACAP-treated macrophages is mediated through the up-regulation of B7.2 expression.