Role of secretory antibodies in the defence against infections

Adaptive immunity mediated by secretory antibodies is important in the defence against mucosal infections. Specific secretory immunoglobulin A (SIgA) can inhibit initial pathogen colonization by performing immune exclusion both on the mucosal surface and within virus-infected secretory epithelial cells without causing tissue damage. Resistance against toxin-producing bacteria such as Vibrio cholerae appears to be particularly dependent on SIgA antibodies. Like natural infections, live topical vaccines or adequate combinations of inactivated vaccines and mucosal adjuvants give rise not only to SIgA antibodies, but also to long-standing serum IgG and IgA responses. The intranasal route of vaccine application could be particularly attractive to achieve this result, but only if successful stimulation is obtained without the use of toxic adjuvants. The degree of protection after vaccination may range from complete inhibition of reinfection to reduction of symptoms. In this scenario it is generally difficult to determine unequivocally the relative importance of SIgA versus serum antibodies. However, infection models in knockout mice strongly support the notion that SIgA exerts a decisive role in protection and cross-protection against a variety of infectious agents.     

Vaccine-induced protection against Helicobacter pylori in mice lacking both antibodies and interleukin-4

To test the hypothesis that a Th2 response to Helicobacter pylori is necessary for protection and to address the possibility that humoral and Th2 cellular responses may compensate for each other, we generated mice deficient in both interleukin-4 (IL-4) and antibodies. The immunized double-knockout mice were protected from H. pylori challenge, as were the parental strains and wild-type C57BL/6 mice. Neutralization of IL-4 in B-cell-deficient mice did not prevent protection. Immunized IL-5-deficient mice were also protected. Thus, IL-4 and IL-5 are not essential for protection.     

Vaccine-induced protection against Leishmania amazonensis is obtained in the absence of IL-12/23p40

Protozoa of the genus Leishmania are intracellular parasites of macrophages and may cause diverse clinical forms of leishmaniasis, including cutaneous, diffuse cutaneous, mucocutaneous and visceral leishmaniasis. Infection with L. major in mice indicates that a protective immune response is achieved when Th1 cells are developed. Thus, adoptive or vaccine-induced protection against leishmaniasis is largely dependent on cell-mediated immunity and IFN-gamma production. Induction of a Th1 response is dependent on the presence of IL-12 whilst lymphocytes are activated. This study was aimed at evaluating the role of IL-12 during infection with L. amazonensis and after vaccination with Leishvacin (killed Leishmania amazonensis promastigotes), since the role of this cytokine in vaccine-induced immunity with this preparation in experimental models or in humans is not yet elucidated. Hence, C57BL/6 interleukin-12-deficient mice (IL-12p40(-/-)) and wild-type controls (wt) were infected with L. amazonensis and the course of infection, parasite burden and cytokine production were compared. IL-12p40(-/-) mice were more susceptible to L. amazonensis than wt: lesions and parasite burden were larger in IL-12p40(-/-) when compared to wt. Interestingly, IL-4 was not produced in the absence of IL-12 in response to infection with L. amazonensis. To evaluate the role of IL-12 in the vaccine-induced immunity against L. amazonensis infection, IL-12p40(-/-) wt mice were vaccinated in the base of the tail and subsequently challenged with L. amazonensis in the footpads. Surprisingly, vaccinated IL-12p40(-/-) mice developed smaller lesions and had fewer parasites in footpads than non-vaccinated controls. Lymph node and spleen cells from vaccinated IL-12p40(-/-) mice did not produce high levels of IFN-gamma in response do in vitro stimulus with antigen. Hence, partial protection against infection with L. amazonensis could be obtained in the absence of functional IL-12 and a typical Th1 response.     

Universal vaccine against influenza virus: linking TLR signaling to anti-viral protection

A vaccine protecting against all influenza strains is a long-sought goal, particularly for emerging pandemics. As previously shown, vaccines based on the highly conserved extracellular domain of M2 (M2e) may protect against all influenza A strains. Here, we demonstrate that M2e-specific monoclonal antibodies (mAbs) protect mice from a lethal influenza infection. To be protective, antibodies had to be able to bind to Fc receptors and fix complement. Furthermore, mAbs of IgG2c isotype were protective in mice, while antibodies of identical specificity, but of the IgG1 isotype, failed to prevent disease. These findings readily translated into vaccine design. A vaccine targeting M2 in the absence of a toll-like receptor (TLR) 7 ligand primarily induced IgG1, whilst the same vaccine linked to a TLR7 ligand yielded high levels of IgG2c antibodies. Although both vaccines protected mice from a lethal challenge, mice treated with the vaccine containing a TLR7 ligand showed significantly lower morbidity. In accordance with these findings, vaccination of TLR7(-/-) mice with a vaccine containing a TLR7 ligand did not result in protection from a lethal challenge. Hence, the innate immune system is required to direct isotype switching toward the more protective IgG2a/c antibodies.     

IL-4 depletion enhances host resistance and passive IgA protection against tuberculosis infection in BALB/c mice

The influence of Th2 cytokines in tuberculosis has been a matter of dispute. Here we report that IL-4 has a profound regulatory effect on the infection of BALB/c mice with Mycobacterium tuberculosis. Depletion of IL-4 with a neutralizing mAb caused only evanescent reduction of lung infection, but when combined with i.n. inoculations of IgA anti-mycobacterial alpha-crystallin mAb and mouse rIFN-gamma, we observed a 40-fold reduction of the bacterial counts in the lungs at 3 wks following i.n. infection (p<0.001). In genetically deficient IL-4-/- BALB/c mice, infection in both lung and spleen was substantially reduced for up to 8 wks without further treatment. Reconstitution of IL-4-/- mice with rIL-4 increased bacterial counts to wild-type levels and made the mice refractory to protection by IgA/IFN-gamma. Analysis of the lungs showed increased granulomatous infiltration and proinflammatory mediators in anti-IL-4/IgA/IFN-gamma-treated and infected mice. We conclude that the action of IL-4 in tuberculosis is targeted at macrophages and that it may include an antagonistic effect on their IgA/IFN-gamma-induced activation and nitric oxide production. The described novel immunotherapy, combining treatments with anti-IL-4, IgA antibody and IFN-gamma, has potential for translation toward the passive immunoprophylaxis of tuberculosis.     

宿主microRNA调节细菌感染免疫应答的研究进展

MicroRNAs (miRNAs)作为重要的基因表达调节因子广泛参与发育、凋亡、肿瘤、免疫和机体-微生物相互作用等生理病理过程.miRNAs可对宿主固有免疫和适应性免疫进行精细调控.目前几种常见致病菌(幽门螺旋杆菌、沙门氏菌、李斯特氏菌、分枝杆菌)感染中宿主miRNAs调节免疫应答的相关研究表明,miRNAs在调节宿主抗细菌感染免疫应答中发挥重要作用.明确机体抗感染免疫中miRNAs如何调节免疫应答将有助于开发作用于miRNAs或其靶标的新的治疗手段.     

Long-term treatment with anti-alpha 4 integrin antibodies aggravates colitis in G alpha i2-deficient mice

Targeted deletion of the heterotrimeric G protein, Galphai2, in mice induces lethal colitis closely resembling ulcerative colitis. In chronic colitis, migration of circulating leukocytes into the intestinal mucosa is partially dependent on alpha4 integrins. In previous studies, short-term administration of anti-alpha4 integrin antibodies has been shown to attenuate intestinal inflammation, and here we elucidate the effect of long-term administration of anti-alpha4 integrin antibodies on colitis in Galphai2(-/- )mice. Long-term blockade of alpha4 integrin significantly increased the severity of colitis in Galphai2(-/-) mice. The inflammation was confined to the colon, associated with increased cancer in situ, destruction of crypt architecture, and increased production of IL-1beta, TNF-alpha and IFN-gamma. Blockade of alpha4 integrin reduced the recruitment of activated T cells to the small intestine. In strong contrast, there were significantly higher numbers of activated T cells in the colonic lamina propria and epithelium, most probably due to in situ proliferation. Furthermore, treatment with alpha4 integrin antibodies induced decreased levels of total IgA and IgG in sera, whereas total IgM levels were unchanged. These new findings may have implications in the understanding of the progression of chronic intestinal inflammation.     

B and CD4+ T‐cell expression of TLR2 is critical for optimal induction of a T‐cell‐dependent humoral immune response to intact Streptococcus pneumoniae

TLR2^?/? mice immunized with Streptococcus pneumoniae (Pn) elicit normal IgM, but defective CD4⁺ T‐cell‐dependent type 1 IgG isotype production, associated with a largely intact innate immune response. We studied the T‐cell‐dependent phosphorylcholine (PC)‐specific IgG3 versus the T‐cell‐independent IgM response to Pn to determine whether TLR2 signals directly via the adaptive immune system. Pn‐activated TLR2^?/? BMDC have only a modest defect in cytokine secretion, undergo normal maturation, and when transferred into naïve WT mice elicit a normal IgM and IgG3 anti‐PC response, relative to WT BMDC. Pn synergizes with BCR and TCR signaling for DNA synthesis in purified WT B and CD4⁺T cells, respectively, but is defective in cells lacking TLR2. Pn primes TLR2^?/? mice for a normal CD4⁺ T‐cell IFN‐γ recall response. Notably, TLR2^?/? B cells transferred into RAG‐2^?/? mice with WT CD4⁺T cells, or TLR2^?/? CD4⁺T cells transferred into athymic nude mice, each elicit a defective IgG3, in contrast to normal IgM, anti‐PC response relative to WT cells. These data are the first to demonstrate a major role for B‐cell and CD4⁺ T‐cell expression of TLR2 for eliciting an anti‐bacterial humoral immune response.     

Genetic control of anti-idiotypic vaccination against coronavirus infection

The idiotypic network can be experimentally altered to induce protective immune responses against microbial pathogens. Both internal image and noninternal image anti-idiotypic (anti-Id) antibodies have been shown to trigger antigen (Ag)-specific immune responses. Therefore, mechanisms of anti-Id vaccination appear to go beyond structural mimicry of Ag, but remain undefined. Using the neurotropic murine coronavirus animal model, we have previously shown that a polyclonal noninternal image anti-Id (Ab2) could vaccinate BALB/c mice. To characterize its mode of action, we have examined the immune modulating capability of this Ab2 in vivo in strains of mice with different H-2 haplotypes. Even though only internal image anti-Id are expected to induce non-genetically restricted immunity, this noninternal image Ab2 induced protective immunity in four of eight genetically different strains of mice susceptible to coronavirus infection. These were BALB/c (H-2^d), DBA/1 (H-2^d), DBA/1 (H-2^q), and SWR (H-2^q) mice. Protection was generally correlated with the induction of specific antiviral Ab (Ab3) that showed biological properties, such as virus neutralization in vitro, similar to the initial Ab1. To evaluate the genetic implication of the H-2 haplotypes in protection, congenic mice were also tested. Vaccination profiles suggest that cooperation between background gene(s) of the BALB/c mouse with H-2^d and H-2^q loci is necessary for an optimal protective immune response, although the main genetic element(s) regulating the antiviral response to Ab2 inoculation appeared to be located outside the major histocompatibility complex. These results are consistent with the ability of Ab2 to induce protective antiviral antibodies in genetically different animals by biological mimicry.     

Mucosal adjuvant activity of flagellin in aged mice

We evaluated the ability of flagellin, a highly effective mucosal adjuvant in mice and non-human primates, to promote mucosal innate and adaptive immunity in aged mice. We found that intratracheal instillation of flagellin induced a stronger respiratory innate response in aged mice than in young mice, and that intranasal instillation of flagellin was equally effective at triggering recruitment of T and B lymphocytes to the draining lymph nodes of young and aged mice. Intranasal immunization of aged mice with flagellin and the Yersinia pestis protein F1 promoted specific IgG and IgA production, but at lower levels and lower avidities than in young mice. Although intranasal instillation of flagellin and F1 antigen increased germinal center formation and size in young mice, it did not do so in aged mice. Our findings are consistent with the conclusion that flagellin can promote adaptive immune responses in aged mice, but at a less robust level than in young mice.     

Interleukin-15 mediates protection against experimental tuberculosis: a role for NKG2D-dependent effector mechanisms of CD8+ T cells

CD8+ T cells are involved in protection against Mycobacterium tuberculosis infection and represent a promising target for new vaccine strategies. Because IL-15 is important for the homeostasis of CD8+ T cells, we studied the immune response in IL-15-deficient mice during tuberculosis. In the absence of IL-15, CD8+ T cells failed to efficiently accumulate in draining lymph nodes and at the site of infection. The expression of antigen-specific effector functions, such as the production of interferon-gamma and cytotoxicity, were impaired in CD8+ T cells, but not CD4+ T cells, from IL-15-deficient mice. This defect was associated with an increased mortality of IL-15-deficient mice during the chronic phase of infection. The lectin-like stimulatory receptor natural killer group 2D (NKG2D) was up-regulated on CD8+ T cells only from wild-type mice, but not from IL-15-deficient mice. Mechanistically, blocking NKG2D function with an mAb inhibited M. tuberculosis-directed CD8+ T cell responses in vitro. We conclude that in addition to regulating the expansion of CD8+ T cells, IL-15 is also necessary for inducing effector mechanisms in CD8+ T cells that depend on NKG2D expression. Hence, our results implicate IL-15 and NKG2D as promising targets for modulating CD8+ T cell-mediated protection against tuberculosis.     

Autoantibodies with a protective function: polyreactive antibodies against alkaline phosphatase in bacterial infections

In patients with acute bacterial infections antibodies directed against a particular bacterial antigen were detected. The molecular mass of this bacterial antigen was 50 kDa as determined by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis. By comparison of the NH₂-terminal amino acid sequence, the 50-kDa antigen was identified as alkaline phosphatase (AP). Affinity-purified antibodies from patients' sera directed against the bacterial AP (anti-alpha) were also shown to react with human and animal AP, which have different structures. Anti-alpha are IgG subtype 3 immunoglobulins, and their light chains are of the kappa type. Upon isoelectric focussing, the anti-alpha formed a scalariform pattern with five to seven bands in the pH range 7–9. The anti-alpha have an opsonic activity and cause a five- to eightfold increase of phagocytosis of gram-positive and gram-negative bacteria. According to their polyreactivity, their sudden rise early in infection, their oligoclonality, as well as their opsonizing properties, they are assumed to be permanently available natural antibodies that take part in early defence mechanisms. Received: 29 May 1997     

Value of urinary neopterin in the differential diagnosis of bacterial and viral infections

Summary Neopterin is released by stimulated macrophages. In this study we analyzed the diagnostic potential of urinary neopterin concentrations in patients with bacterial and viral infection. All but one of 17 patients with viral infection had increased urinary neopterin concentrations. Patients with bacterial urinary tract infection also showed increased neopterin concentrations, whereas patients with bacterial pneumonia had significantly lower neopterin levels. In addition, patients with acute bacterial pneumonia had lower neopterin levels than patients with protracted infection. A significant inverse correlation between urinary neopterin and hemoglobin concentrations was found. Neopterin concentrations could serve as a helpful additional marker of infectious diseases. Combined with other clinical and laboratory parameters it is a useful parameter for distinguishing between viral and bacterial origins of infection, as was shown by multivariate stepwise linear discriminant analysis.Abbreviations ESR erythrocyte sedimentation rate     

An evolutionarily-conserved role for murine Ly-1 B cells in protection against bacterial infections.

The murine Ly-1 B cell lineage, although comprising only a minority of peripheral IgM+ B cells, secretes a major proportion of the IgM antibodies occurring naturally in serum. Ly-1 B cells also seed a large number of IgA+ plasma cells to the gut walls, thereby contributing significantly to production of natural IgA antibodies in response to chronic stimulation by the normal gut flora. Apart from these naturally-produced antibodies, Ly-1 B cells also produce specific antibodies following deliberate immunisation with the bacterial cell wall antigens, phosphorylcholine and dextran. The inability of the X-linked immunodeficient CBA/N mice to produce antibody responses to these two antigens is overcome by reconstitution with normal Ly-1 B cells from the parental CBA strain. Ly-1 B cells therefore appear to play a dominant role in natural immunity and protection against bacterial infections. The compartmentalisation of development and function within murine B cells is suggestive of an evolutionary structuring of the murine immune system, with Ly-1 B cells representing a conserved, primitive B cell lineage and retaining key, associated functions.     

Competition between colitogenic Th1 and Th17 cells contributes to the amelioration of colitis

Th17 cells and Th1 cells coordinate to play a critical role in the formation of inflammatory bowel diseases. To examine how Th17 and Th1 cells are regulated at inflammatory sites, we used Th1‐dominant CD4⁺CD45RB^high T cell‐transferred RAG‐2^?/? and Th1/Th17‐mixed IL‐10^?/? mice. Interestingly, not only did colitic RAG‐2^?/? mice that were parabiosed with WT mice show significant amelioration of colitis, but amelioration of disease was also observed in those parabiosed with colitic IL‐10^?/? mice. To assess the interference between Th1 and Th17 colitogenic T cells, we co‐transferred colitogenic CD4⁺ T cells from the lamina propria (LP) of CD4⁺CD45RB^high T cell‐transferred RAG‐2^?/? mice and IL‐10^?/? mice into RAG‐2^?/? mice. Surprisingly, the co‐transferred RAG‐2^?/? mice showed a vast cellular infiltration of LP CD4⁺ T cells similar to that seen in RAG‐2^?/? mice re‐transferred with the cells from colitic RAG‐2^?/? mice alone, but the co‐transferred RAG‐2^?/? mice did not have the wasting symptoms, which are also absent in RAG‐2^?/? mice transferred with cells from colitic IL‐10^?/? mice alone. Furthermore, the percentages of Th1 and Th17 cells originating from IL‐10^?/? mice and those of Th1 cells originating from colitic RAG‐2^?/? mice were all significantly decreased in the co‐transferred mice as compared with the singly‐transferred paired RAG‐2^?/? mice, suggesting that Th1 and Th17 cells are in competition, and that their orchestration results in a merged clinical phenotype of the two types of murine colitis.     

乙型脑炎病毒单克隆抗体制备及其特性鉴定

目的制备乙脑病毒单克隆抗体并对其各项生物学性质进行鉴定。方法通过免疫、融合、克隆和筛选等方法制备乙脑单抗,使用ELISA、IFA、中和试验和Westernblot等方法鉴定单抗的敏感性、特异性、种内反应广谱性以及中和活性。结果最终获得3株稳定分泌的乙脑单克隆抗体细胞株,其滴度均超过106。3株单抗只与乙脑病毒反应,与其他9种虫媒病毒皆不反应。ELISA相加试验证明3株单抗的作用位点非常相近,选择其中的F12.37与10个代表性乙脑病毒株反应,F12.37能够敏感地检测到所有10个在细胞内复制的病毒株。F12.37还能够中和乙脑P3株(基因Ⅲ型)和SH03-103株(基因Ⅰ型),保护50%细胞不产生病变的稀释度分别为1∶3.2×105和1∶105,Westernblot结果显示F12.37与乙脑病毒E蛋白相互作用。结论本研究获得了3个高滴度、高特异性的乙脑单克隆细胞株,其中F12.37具有较高的敏感性和较好的种内反应广谱性,能够中和两个基因型乙脑病毒,其作用位点位于乙脑病毒E蛋白。     

Microbial colonization induces oligoclonal expansions of intraepithelial CD8 T cells in the gut

Two populations of CD8(+) IEL generally express restricted, but apparently random and non-overlapping TCR repertoires. Previous studies in mice suggested that this could be explained by a dual origin of CD8(+) IEL, i.e. that CD8alphabeta(+) IEL derive from a few peripheral CD8(+) T cell lymphoblasts stimulated by microbial antigens in gut-associated lymphoid tissue, whereas CD8alphaalpha(+) IEL descend from an inefficient intestinal maturation pathway. We show here that the gut mucosa, instead, becomes seeded with surprisingly broad and generally non-overlapping CD8 IEL repertoires and that oligoclonality is induced locally after microbial colonization. In germ-free (GF) rats, both CD8alphabeta(+) and CD8alphaalpha(+) IEL displayed surprisingly diverse TCR Vbeta repertoires, although beta-chain diversity tended to be somewhat restricted in the CD8alphaalpha(+) subset. CDR3 length displays in individual Vbeta-Cbeta and Vbeta-Jbeta combinations generally revealed polyclonal distributions over 6-11 different lengths, similar to CD8(+) lymph node T cells, and CDR3beta sequencing provided further documentation of repertoire diversity. By contrast, in ex-GF rats colonized with normal commensal microflora, both CD8alphabeta(+) and CD8alphaalpha(+) IEL displayed oligoclonal CDR3 length distributions for most of the Vbeta genes analyzed. Our data suggest that microbial colonization induces apparently random clonal expansions of CD8alphabeta(+) and CD8alphaalpha(+) IEL locally in the gut.     

IL‐7 is essential for lymphopenia‐driven turnover of colitogenic CD4+ memory T cells in chronic colitis

We previously demonstrated that IL‐7 is essential for the persistence of T‐cell‐mediated colitis, by showing that adoptive transfer of CD4⁺CD45RB^high T cells into IL‐7^?/?×RAG‐1^?/? mice did not induce colitis; and that intestinal IL‐7 is not essential for this colitis model, by showing that IL‐7^?/?×RAG‐1^?/? mice parabiosed with colitic CD4⁺CD45RB^high T‐cell‐transferred RAG‐1^?/? mice developed colitis. Here, we investigated the role of IL‐7 in the maintenance of colitogenic CD4⁺ T cells by surgically separating these parabionts. Surprisingly, the separated IL‐7^?/?×RAG‐1^?/? mice were consistently diseased after separation, although no IL‐7 mRNA was detected in the tissues of separated IL‐7^?/?×RAG‐1^?/? partners. CD4⁺ T cells isolated from the separated RAG‐1^?/? or IL‐7^?/?×RAG‐1^?/? mice were then transferred into new RAG‐1^?/? or IL‐7^?/?×RAG‐1^?/? mice. Regardless of the source of donor cells, RAG‐1^?/? recipients developed colitis, whereas IL‐7^?/?×RAG‐1^?/? recipients did not. Collectively, these results demonstrate that IL‐7 is essential for lymphopenia‐driven turnover of colitogenic CD4⁺ T cells rather than the maintenance of those cells in established colitic mice. They also provide a basis for the timing of IL‐7/IL‐7R blockade for the treatment of inflammatory bowel diseases.