Induction of protective immunity against Chlamydia muridarum intravaginal infection with the chlamydial immunodominant antigen macrophage infectivity potentiator

We previously reported that 5 Chlamydia muridarum antigens reacted with antisera from >90% mice urogenitally infected with C. muridarum and they are TC0660 (ABC transporter or ArtJ), TC0727 (outer membrane complex protein B or OmcB), TC0828 (macrophage infectivity potentiator or MIP), TC0726 (inclusion membrane protein or Inc) & TC0268 (hypothetical protein or HP). The orthologs of these antigens in Chlamydia trachomatis were also highly reactive with antisera from women urogenitally infected with C. trachomatis. In the current study, we evaluated these C. muridarum antigens for their ability to induce protection against a C. muridarum intravaginal challenge infection in mice. We found that only MIP induced the most pronounced protection against C. muridarum infection. The protection correlated well with robust C. muridarum MIP-specific antibody and Th1-dominant T cell responses. The MIP-immunized mice displayed significantly reduced live organism shedding from the lower genital tract and highly attenuated inflammatory pathologies in the upper genital tissues. These results demonstrate that MIP, an immunodominant antigen identified by both human and mouse antisera, may be considered a component of a multi-subunit chlamydial vaccine for inducing protective immunity.     

沙眼衣原体分泌性蛋白Pgp3滴鼻免疫增强小鼠对衣原体生殖道感染的保护作用

【目的】探讨不同免疫途径沙眼衣原体(Chlamydia trachomatis,Ct)分泌性蛋白Pgp3的免疫保护效果,分析其可能的保护机制,以确定Pgp3蛋白疫苗在Ct疫苗研制中的应用价值。【方法】分泌性蛋白Pgp3经滴鼻或肌注途径免疫雌性Balb/c小鼠,免疫60 d后,阴道接种鼠沙眼衣原体(Chlamydia muridarum,Cm)建立生殖道感染动物模型,在该模型中评价Pgp3蛋白疫苗抗Cm感染的保护效果,并探讨其机制。【结果】滴鼻或肌注免疫后,小鼠血清及生殖道中检测到了特异性抗体;小鼠脾淋巴细胞产生IFN-γ、IL-17及IL-5水平均明显高于对照组,且滴鼻免疫组IFN-γ水平升高较肌注组更显著(P<0.05);Pgp3蛋白滴鼻免疫组小鼠经Cm生殖道感染后,阴道带菌时间明显缩短,输卵管病理改变轻而肌注免疫组其保护作用不明显。【结论】Pgp3蛋白经滴鼻免疫可有效诱导小鼠产生明显的抗Cm保护效应。其可能的免疫保护机制与诱导Th1型为主的细胞免疫应答及高效价的特异性抗体有关,提示Pgp3蛋白疫苗具有潜在的疫苗研究与开发价值。     

Antigen-specific CD4+ T cells produce sufficient IFN-gamma to mediate robust protective immunity against genital Chlamydia muridarum infection

Chlamydia has been shown to evade host-specific IFN-gamma-mediated bacterial killing; however, IFN-gamma-deficient mice exhibit suboptimal late phase vaginal Chlamydia muridarum clearance, greater dissemination, and oviduct pathology. These findings introduce constraints in understanding results from murine chlamydial vaccination studies in context of potential implications to humans. In this study, we used mice deficient in either IFN-gamma or the IFN-gamma receptor for intranasal vaccination with a defined secreted chlamydial Ag, chlamydial protease-like activity factor (CPAF), plus CpG and examined the role of IFN-gamma derived from adoptively transferred Ag-specific CD4+ T cells in protective immunity against genital C. muridarum infection. We found that early Ag-specific IFN-gamma induction and CD4+ T cell infiltration correlates with the onset of genital chlamydial clearance. Adoptively transferred IFN-gamma competent CPAF-specific CD4+ T cells failed to enhance the resolution of genital chlamydial infection within recipient IFN-gamma receptor-deficient mice. Conversely, IFN-gamma production from adoptively transferred CPAF-specific CD4+ T cells was sufficient in IFN-gamma-deficient mice to induce early resolution of infection and reduction of subsequent pathology. These results provide the first direct evidence that enhanced anti-C. muridarum protective immunity induced by Ag-specific CD4+ T cells is dependent upon IFN-gamma signaling and that such cells produce sufficient IFN-gamma to mediate the protective effects. Additionally, MHC class II pathway was sufficient for induction of robust protective anti-C. muridarum immunity. Thus, targeting soluble candidate Ags via MHC class II to CD4+ T cells may be a viable vaccine strategy to induce optimal IFN-gamma production for effective protective immunity against human genital chlamydial infection.     

Immunization with chlamydial plasmid protein pORF5 DNA vaccine induces protective immunity against genital chlamydial infection in mice

To validate the immune protective efficacy of pORF5 DNA vaccine and to analyze potential mechanisms related to this protection. In this study, pORF5 DNA vaccine was constructed and evaluated for its protective immunity in a mouse model of genital chlamydial infection. Groups of BALB/c mice were immunized intranasally with pORF5 DNA vaccine. Humoral and cell mediated immune responses were evaluated. The clearance ability of chlamydial challenge from the genital tract and the chlamy- dia-induced upper genital tract gross pathology and histopathological characterization were also de- tected. The results showed that the total and the IgG2a anti-pORF5 antibody levels in serum were sig- nificantly elevated after pcDNA3.1-pORF5 vaccination, as were the total antibody and IgA levels in vaginal fluids. pcDNA3.1-pORF5 induced a significantly high level of Th1 response as measured by robust gamma interferon (IFN-γ). Minimal IL-4 was produced by immune T cells in response to the re-stimulation with pORF5 protein or the inactive elementary body in vitro. pcDNA3.1-pORF5-vacci- nated mice displayed significantly reduced bacterial shedding upon a chlamydial challenge and an accelerated resolution of infection. 100% of pcDNA3.1-pORF5 vaccinated mice successfully resolved the infection by day 24. pcDNA3.1-pORF5-immunized mice also exhibited protection against patho- logical consequences of chlamydial infection. The stimulated index was significantly higher than that of mice immunized with pcDNA3.1 and PBS (P<0.05). Together, these results demonstrated that immu- nization with pORF5 DNA vaccine is a promising approach for eliciting a protective immunity against a genital chlamydial challenge.     

Immunization with chlamydial plasmid protein pORF5 DNA vaccine induces protective immunity against genital chlamydial infection in mice

To validate the immune protective efficacy of pORF5 DNA vaccine and to analyze potential mechanisms related to this protection. In this study, pORF5 DNA vaccine was constructed and evaluated for its protective immunity in a mouse model of genital chlamydial infection. Groups of BALB/c mice were immunized intranasally with pORF5 DNA vaccine. Humoral and cell mediated immune responses were evaluated. The clearance ability of chlamydial challenge from the genital tract and the chlamydia-induced upper genital tract gross pathology and histopathological characterization were also detected. The results showed that the total and the IgG2a anti-pORF5 antibody levels in serum were significantly elevated after pcDNA3.1-pORF5 vaccination, as were the total antibody and IgA levels in vaginal fluids. pcDNA3.1-pORF5 induced a significantly high level of Th1 response as measured by robust gamma interferon (IFN-γ). Minimal IL-4 was produced by immune T cells in response to the re-stimulation with pORF5 protein or the inactive elementary body in vitro. pcDNA3.1-pORF5-vaccinated mice displayed significantly reduced bacterial shedding upon a chlamydial challenge and an accelerated resolution of infection. 100% of pcDNA3.1-pORF5 vaccinated mice successfully resolved the infection by day 24. pcDNA3.1-pORF5-immunized mice also exhibited protection against pathological consequences of chlamydial infection. The stimulated index was significantly higher than that of mice immunized with pcDNA3.1 and PBS (P<0.05). Together, these results demonstrated that immunization with pORF5 DNA vaccine is a promising approach for eliciting a protective immunity against a genital chlamydial challenge.     

A limited role for antibody in protective immunity induced by rCPAF and CpG vaccination against primary genital Chlamydia muridarum challenge

Mice deficient in B cells (μmT mice) were used to evaluate the role of antibody in enhanced chlamydial clearance and reduction of pathology afforded by vaccination with recombinant chlamydial protease-like activity factor (rCPAF). Enhanced, but comparable, chlamydial clearance was observed in μmT and wild-type (WT) mice after rCPAF+CpG vaccination. Chlamydia -induced pathology was present in mock-immunized animals, but at significantly greater levels in μmT than WT mice, whereas vaccinated μmT and WT mice exhibited similar reductions in pathology. Thus, antibodies may play a role in protection against chlamydial pathology after primary infection, but were largely dispensable in rCPAF+CpG-induced chlamydial clearance and reduction in pathology.     

Endogenous IFN-gamma production is induced and required for protective immunity against pulmonary chlamydial infection in neonatal mice

Chlamydia trachomatis infection in neonates, not adults, has been associated with the development of chronic respiratory sequelae. Adult chlamydial infections induce Th1-type responses that subsequently clear the infection, whereas the neonatal immune milieu in general has been reported to be biased toward Th2-type responses. We examined the protective immune responses against intranasal Chlamydia muridarum challenge in 1-day-old C57BL/6 and BALB/c mice. Infected C57BL/6 pups displayed earlier chlamydial clearance (day 14) compared with BALB/c pups (day 21). However, challenged C57BL/6 pups exhibited prolonged deficits in body weight gain (days 12-30) compared with BALB/c pups (days 9-12), which correlated with continual pulmonary cellular infiltration. Both strains exhibited a robust Th1-type response, including elevated titers of serum antichlamydial IgG2a and IgG2b, not IgG1, and elevated levels of splenic C. muridarum-specific IFN-gamma, not IL-4, production. Additionally, elevated IFN-gamma, not IL-4 expression, was observed locally in the infected lungs of both mouse strains. The immune responses in C57BL/6 pups were significantly greater compared with BALB/c pups after chlamydial challenge. Importantly, infected mice deficient in IFN-gamma or IFN-gamma receptor demonstrated enhanced chlamydial dissemination, and 100% of animals died by 2 wk postchallenge. Collectively, these results indicate that neonatal pulmonary chlamydial infection induces a robust Th1-type response, with elevated pulmonary IFN-gamma production, and that endogenous IFN-gamma is important in protection against this infection. The enhanced IFN-gamma induction in the immature neonatal lung also may be relevant to the development of respiratory sequelae in adult life.     

Chlamydia muridarum infection subverts dendritic cell function to promote Th2 immunity and airways hyperreactivity

There is strong epidemiological evidence that Chlamydia infection can lead to exacerbation of asthma. However, the mechanism(s) whereby chlamydial infection, which normally elicits a strong Th type 1 (Th1) immune response, can exacerbate asthma, a disease characterized by dominant Th type 2 (Th2) immune responses, remains unclear. In the present study, we show that Chlamydia muridarum infection of murine bone marrow-derived dendritic cells (BMDC) modulates the phenotype, cytokine secretion profile, and Ag-presenting capability of these BMDC. Chlamydia-infected BMDC express lower levels of CD80 and increased CD86 compared with noninfected BMDC. When infected with Chlamydia, BMDC secrete increased TNF-alpha, IL-6, IL-10, IL-12, and IL-13. OVA peptide-pulsed infected BMDC induced significant proliferation of transgenic CD4(+) DO11.10 (D10) T cells, strongly inhibited IFN-gamma secretion by D10 cells, and promoted a Th2 phenotype. Intratracheal transfer of infected, but not control noninfected, OVA peptide-pulsed BMDC to naive BALB/c mice, which had been i.v. infused with naive D10 T cells, resulted in increased levels of IL-10 and IL-13 in bronchoalveolar lavage fluid. Recipients of these infected BMDC showed significant increases in airways resistance and decreased airways compliance compared with mice that had received noninfected BMDC, indicative of the development of airways hyperreactivity. Collectively, these data suggest that Chlamydia infection of DCs allows the pathogen to deviate the induced immune response from a protective Th1 to a nonprotective Th2 response that could permit ongoing chronic infection. In the setting of allergic airways inflammation, this infection may then contribute to exacerbation of the asthmatic phenotype.     

Induction of pluripotent protective immunity following immunisation with a chimeric vaccine against human cytomegalovirus

Based on the life-time cost to the health care system, the Institute of Medicine has assigned the highest priority for a vaccine to control human cytomegalovirus (HCMV) disease in transplant patients and new born babies. In spite of numerous attempts successful licensure of a HCMV vaccine formulation remains elusive. Here we have developed a novel chimeric vaccine strategy based on a replication-deficient adenovirus which encodes the extracellular domain of gB protein and multiple HLA class I & II-restricted CTL epitopes from HCMV as a contiguous polypeptide. Immunisation with this chimeric vaccine consistently generated strong HCMV-specific CD8(+) and CD4(+) T-cells which co-expressed IFN-gamma and TNF-alpha, while the humoral response induced by this vaccine showed strong virus neutralizing capacity. More importantly, immunization with adenoviral chimeric vaccine also afforded protection against challenge with recombinant vaccinia virus encoding HCMV antigens and this protection was associated with the induction of a pluripotent antigen-specific cellular and antibody response. Furthermore, in vitro stimulation with this adenoviral chimeric vaccine rapidly expanded multiple antigen-specific human CD8(+) and CD4(+) T-cells from healthy virus carriers. These studies demonstrate that the adenovirus chimeric HCMV vaccine provides an excellent platform for reconstituting protective immunity to prevent HCMV diseases in different clinical settings.     

Plasmid-Encoded Pgp5 Is a Significant Contributor to Chlamydia muridarum Induction of Hydrosalpinx

We have previously shown that the plasmid-encoded Pgp3 is a major virulence factor for C. muridarum induction of hydrosalpinx. We now report that Pgp5 also plays a significant role in the development of hydrosalpinx following C. muridarum induction. Pgp5 deficiency was introduced via either in-frame deletion (CM-Δpgp5) or premature stop codon installation (CM-pgp5S). Mice infected with either CM-Δpgp5 or CM-pgp5S developed hydrosalpinges at significantly reduced levels with an incidence rate of <40% and a mean severity score of 2 or less. In contrast, 80% or more mice developed hydrosalpinx with a severity score of >3 when mice were infected with Pgp5-sufficient C. muridarum (plasmid-competent wild type or plasmid-free C. muridarum transformed with a full plasmid or depleted of pgp7 gene). The attenuated pathogenicity of the Pgp5-deficient C. muridarum correlated with a significantly reduced level of ascending infection in the oviduct tissue despite the similar overall shedding courses between mice infected with Pgp5-deficeint versus sufficient C. muridarum. Furthermore, in the oviducts of mice infected with Pgp5-deficient C. muridarum, significantly lower levels of inflammatory cell infiltration and cytokine production were detected. Thus, Pgp5 is a significant plasmid-encoded virulence factor for C. muridarum pathogenicity in the upper genital tract.     

Induction of protective immunity to experimental hepatic amoebic infection in hamsters

Immunisation of hamsters with the Sephadex G-200 chromatographed Fraction-I of the crude amoebic extract afforded protection in 58% of the animals against an intrahepatic challenge with a virulent subline of axenic Entamoeba histolytica NIH-200 (V). The protected animals had variable levels of anti-FI antiamoebic antibodies, while none of the infected controls had such antibodies. Findings suggest that chromatographed FI of crude amoebic extract might function as a potent immunogen affording protection in amoebic infection.     

CpG oligodeoxynucleotides stimulate protective innate immunity against pulmonary Klebsiella infection

Bacterial pneumonia is a leading cause of mortality in the United States. Innate immune responses, including type-1 cytokine production, are critical to the effective clearance of bacterial pathogens from the lung. Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotide motifs (CpG ODN), which mimic the effects of bacterial DNA, have been shown to enhance type-1 cytokine responses during infection due to intracellular pathogens, resulting in enhanced microbial clearance. The role of CpG ODN in modulating protective innate immunity against extracellular pathogens is unknown. Using a murine model of Gram-negative pneumonia, we found that CpG ODN administration stimulated protective immunity against Klebsiella pneumoniae. Specifically, intratracheal (i.t.) administration of CpG ODN (30 microg) 48 h before i.t. K. pneumoniae challenge resulted in increased survival, compared with animals pretreated with control ODN or saline. Pretreatment with CpG ODN resulted in enhanced bacterial clearance in lung and blood, and higher numbers of pulmonary neutrophils, NKT cells, gammadelta-T cells, and activated NK1.1+ cells and gammadelta-T lymphocytes during infection. Furthermore, pretreatment with CpG ODN enhanced the production of TNF-alpha, and type-1 cytokines, including IL-12, IFN-gamma, and the IFN-gamma-dependent ELR- CXC chemokines IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma in response to Klebsiella challenge, compared with control mice. These findings indicate that i.t. administration of CpG ODN can stimulate multiple components of innate immunity in the lung, and may form the basis for novel therapies directed at enhancing protective immune responses to severe bacterial infections of the lung.     

Identification of dendritic cell subsets responding to genital infection by Chlamydia muridarum

Dendritic cells (DCs) are central for the induction of T-cell responses needed for chlamydial eradication. Here, we report the activation of two DC subsets: a classical CD11b+ (cDC) and plasmacytoid (pDC) during genital infection with Chlamydia muridarum . Genital infection induced an influx of cDC and pDC into the genital tract and its draining lymph node (iliac lymph nodes, ILN) as well as colocalization with T cells in the ILN. Genital infection with C. muridarum also stimulated high levels of costimulatory molecules on cDC central for the activation of naïve T cells in vivo . In contrast, pDC expressed low levels of most costimulatory molecules in vivo and did not secrete cytokines associated with the production of T helper (Th)1 cells in vitro . However, pDC upregulated inducible costimulatory ligand expression and produced IL-6 and IL-10 in response to chlamydial exposure in vitro . Our findings show that these two DC subsets likely have different functions in vivo . cDCs are prepared for induction of antichlamydial T-cell responses, whereas pDCs have characteristics associated with the differentiation of non-Th1 cell subsets.     

Stimulation of the cytosolic receptor for peptidoglycan, Nod1, by infection with Chlamydia trachomatis or Chlamydia muridarum

Infection of epithelial cells by the intracellular pathogen, Chlamydia trachomatis, leads to activation of NF-kappaB and secretion of pro-inflammatory cytokines. We find that overexpression of a dominant-negative Nod1 or depletion of Nod1 by RNA interference inhibits partially the activation of NF-kappaB during chlamydial infection in vitro, suggesting that Nod1 can detect the presence of Chlamydia. In parallel, there is a larger increase in the expression of pro-inflammatory genes following Chlamydia infection when primary fibroblasts are isolated from wild-type mice than from Nod1-deficient mice. The Chlamydia genome encodes all the putative enzymes required for proteoglycan synthesis, but proteoglycan from Chlamydia has never been detected biochemically. Since Nod1 is a ubiquitous cytosolic receptor for peptidoglycan from Gram-negative bacteria, our results suggest that C. trachomatis and C. muridarum do in fact produce at least the rudimentary proteoglycan motif recognized by Nod1. Nonetheless, Nod1 deficiency has no effect on the efficiency of infection, the intensity of cytokine secretion, or pathology in vaginally infected mice, compared with wild-type controls. Similarly, Rip2, a downstream mediator of Nod1, Toll-like receptor (TLR)-2, and TLR4, increases only slightly the intensity of chlamydial infection in vivo and has a very mild effect on the immune response and pathology. Thus, Chlamydia may not produce sufficient peptidoglycan to stimulate Nod1-dependent pathways efficiently in infected animals, or other receptors of the innate immune system may compensate for the absence of Nod1 during Chlamydia infection in vivo.     

LIGHT信号通路在鼠衣原体生殖道感染中的作用

【目的】初步探讨与单纯疱疹病毒糖蛋白D竞争结合疱疹病毒侵入介体的淋巴毒素类似物(lymphotoxin-like inducible protein that competes with glycoprotein D for herpesvirus entry on T cells,LIGHT)在抗衣原体感染免疫及介导衣原体生殖道病理损伤过程的作用。【方法】用1×104IFUs的Mo Pn经生殖道感染野生型(wild type,wt)、LIGHT KO小鼠,每组一半小鼠于感染后49d,再次感染相同剂量的Mo Pn。每隔3-4 d取生殖道分泌物,测定其中衣原体包涵体的数量。初次感染后80d,处死小鼠,眼眶取血,分离血清,用间接免疫荧光法测定其中抗体类型及效价;同时分离生殖道,肉眼观察其输卵管、子宫角水肿程度,然后甲醛固定、切片,H&E染色后,显微镜下观察各组织炎性浸润程度和管腔水肿程度。分离小鼠脾细胞,体外用衣原体EB刺激,测定上清中IL-4、IL-5、IL-17和IFN-γ等细胞因子水平。【结果】LIGHT KO小鼠阴道带菌时间与wt组相当,大部分小鼠均在原发感染后28d左右完全清除感染,且均产生对再次感染的免疫力。LIGHT KO和wt小鼠子宫角和输卵管均出现一定程度的病变,但差异无统计学意义。两组小鼠在原发和继发感染Mo Pn后,均产生高效价的特异性抗Mo Pn Ig G抗体,总抗体及各Ig G抗体亚类效价差异均无统计学意义(P>0.05),且Ig G2a/Ig G1比值均大于1。和wt小鼠一样,LIGHT KO小鼠脾淋巴细胞经衣原体再次刺激后均可产生较高水平的IFN-γ和IL-17,且未能检测到IL-4和IL-5。【结论】小鼠抗Mo Pn生殖道感染及Mo Pn引起的生殖道病理损伤不依赖于LIGHT信号通路。     

First demonstration of protective immunity against foetopathy in cattle with latent Neospora caninum infection

The parasite Neospora caninum is an important cause of abortion in cattle world-wide. Chronically infected dams transmit the parasite transplacentally and infected foetuses may be aborted or born chronically infected but clinically normal. Chronically infected cows repeatedly transmit the parasite to foetuses in several pregnancies and some may abort more than once suggesting that the immune response in these cattle is compromised during pregnancy. To investigate the nature of the immune response in chronically infected cattle, five naturally, chronically infected cows were challenged with N. caninum tachyzoites at 10 weeks of gestation. No foetopathy occurred and all five delivered live calves at full-term. In four naive pregnant cows challenged at the same time, all four foetuses died within 3-5 weeks of challenge. Of the five live calves born to the chronically infected challenged cows, three were transplacentally infected with N. caninum. The kinetics of the maternal anti-N. caninum antibody responses during gestation suggested that these transplacental infections were not the result of the superimposed challenge, but the result of the recrudescence of the maternal chronic infection-which occurred concurrently in non-challenged, chronically infected pregnant controls. These data provide the first experimental evidence that protective immunity occurs in neosporosis. They also suggest that whilst immunity to a pre-existing infection will protect against an exogenous challenge, this protective immunity will not prevent transplacental infection. This implies that a subtle form of concomitant immunity exists in chronically infected cattle and has important implications for vaccine development.     

Polyclonal and specific antibodies mediate protective immunity against enteric helminth infection

Anti-helminth immunity involves CD4+ T cells, yet the precise effector mechanisms responsible for parasite killing or expulsion remain elusive. We now report an essential role for antibodies in mediating immunity against the enteric helminth Heligmosomoides polygyrus (Hp), a natural murine parasite that establishes chronic infection. Polyclonal IgG antibodies, present in naive mice and produced following Hp infection, functioned to limit egg production by adult parasites. Comparatively, affinity-matured parasite-specific IgG and IgA antibodies that developed only after multiple infections were required to prevent adult worm development. These data reveal complementary roles for polyclonal and affinity-matured parasite-specific antibodies in preventing enteric helminth infection by limiting parasite fecundity and providing immune protection against reinfection, respectively. We propose that parasite-induced polyclonal antibodies play a dual role, whereby the parasite is allowed to establish chronicity, while parasite load and spread are limited, likely reflecting the long coevolution of helminth parasites with their hosts.     

Induction of protective immunity to Brugia pahangi in jirds by drug-abbreviated infection.

Protective immunity of homologous challenge infection was examined in jirds after drug-abbreviated infection with Brugia pahangi. Mebendazole (MBZ) treatment at the early prepatent (5-7 weeks of post infection) or the late prepatent (7-9 weeks of post infection) period was highly effective in causing almost complete eradication of the primary infection. After challenge infection, the worm burden was significantly reduced 19% (31.1 in average) and 77% (9.5) to that of the controls (38.8 and 41.7), respectively. The magnitude of eosinophil response paralleled the degree of protection. No or only a few microfilariae were seen after challenge infection in jirds treated during the prepatent periods. They were also resistant to intravenous challenge with the microfilariae of B. pahangi. MBZ treatment at the patent period was, on the contrary, incomplete against primarily infected adult worms, and was not able to induce either significant protection (30.1 vs 33.1 in control) or eosinophil response to the challenge infection.