Exploring Changes in the Host Gut Microbiota During a Controlled Human Infection Model for Campylobacter jejuni.

Campylobacter jejuni infection is a leading cause of foodborne disease, common to children, adult travelers, and military populations in low- to middle-income countries. In the absence of a licensed vaccine, efforts to evaluate prophylactic agents are underway. The prophylactic efficacy of a twice-daily, 550 mg dose of the antibiotic rifaximin demonstrated no efficacy against campylobacteriosis in a controlled human infection model (CHIM); however, samples from the CHIM study were utilized to assess how the human gut microbiome responds to C. jejuni infection, and if a 'protective' microbiota exists in study participants not developing campylobacteriosis. Statistically significant, but minor, differences in study participant beta diversity were identified during the challenge period (p = 0.002, R2 = 0.042), but no significant differences were otherwise observed. Pre-challenge alpha diversity was elevated in study participants who did not develop campylobacteriosis compared to those who did (p < 0.001), but alpha diversity declined in all study participants from the pre-challenge period to post-discharge. Our work provides insight into gut microbiome shifts observed during a C. jejuni CHIM and following antibiotic treatment. This study utilized a high dose of 1.7 x 105 colony-forming units of C. jejuni; future work could include CHIM studies performed with inocula more closely mimicking natural exposure as well as field studies involving naturally-occurring enteric infections.

Distribution of Capsular Types of Campylobacter jejuni Isolates from Symptomatic and Asymptomatic Children in Peru.

Campylobacter jejuni is the leading bacterial cause of diarrhea worldwide. A capsular polysaccharide (CPS) conjugate vaccine is under development and requires determination of the valency. However, distribution of CPS types circulating globally is presently poorly described. We aimed to determine whether CPS type distribution in Peru differs from that in other endemic regions. We used a multiplex polymerase chain reaction (PCR) assay for the detection of CPS encoding genes capable of distinguishing all 35 CPS types on Campylobacter isolates in two prospective communities based studies conducted in cohorts of children less than 59 months of age in Peru. Results showed that CPS type HS4 complex was the most prevalent, followed by HS3 complex and HS15. Differences in CPS type for symptomatology were not statistically significant. Most subjects demonstrated repeated infections over time with different CPS types, suggesting that CPS types may confer of a level of homologous protective immunity. In this dataset, some differences in CPS type distribution were observed in comparison to other low-middle income countries. Further studies need to be conducted in endemic areas to increase our knowledge of CPS type distribution and guide vaccine development.

Anti-CTLA-4 synergizes with dendritic cell-targeted vaccine to promote IL-3-dependent CD4+ effector T cell infiltration into murine pancreatic tumors.

One successful class of cancer immunotherapies, immune checkpoint inhibitory antibodies, disrupts key pathways that regulate immune checkpoints, such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4). These agents unleash the potency of antigen-experienced T cells that have already been induced as a consequence of the existing tumor. But only 20% of cancers naturally induce T cells. For most cancers, vaccines are require to induce and mobilize T effector cells (Teffs ) to traffick into tumors. We evaluated the effects of anti-CTLA-4 given in combination with an antigen-specific dendritic cell vaccine on intratumoral Teffs in a murine pancreatic cancer model. The dendritic cell-targeted tumor antigen plus anti-CTLA-4 significantly increased the number of vaccine-induced CD4+ Teffs within the tumor. This increase was accompanied by a reduction in the size of the peripheral CD4+ Teff pool. We also found that IL-3 production by activated CD4+ T cells was significantly increased with this combination. Importantly, the CD4+ Teff response was attenuated in Il3-/- mice, suggesting mediation of the effect by IL-3. Finally, the induced T cell infiltration was associated with activation of the tumor endothelium by T cell-derived IL-3. Our findings collectively provide a new insight into the mechanism driving Teff infiltration and vascular activation in a murine pancreatic cancer model, specifically identifying a new role for IL-3 in the anticancer immune response.

Campylobacter jejuni capsule types in a Peruvian birth cohort and associations with diarrhoeal disease severity.

Campylobacter jejuni is a leading cause of bacterial diarrhoea worldwide. The objective of this study was to examine the association between C. jejuni capsule types and clinical signs and symptoms of diarrhoeal disease in a well-defined birth cohort in Peru. Children were enrolled in the study at birth and followed until 2 years of age as part of the Malnutrition and Enteric Infections birth cohort. Associations between capsule type and clinical outcomes were assessed using the Pearson's χ2 and the Kruskal-Wallis test statistics. A total of 318 C. jejuni samples (30% from symptomatic cases) were included in this analysis. There were 22 different C. jejuni capsule types identified with five accounting for 49.1% of all isolates. The most common capsule types among the total number of isolates were HS4 complex (n = 52, 14.8%), HS5/31 complex (n = 42, 11.9%), HS15 (n = 29, 8.2%), HS2 (n = 26, 7.4%) and HS10 (n = 24, 6.8%). These five capsule types accounted for the majority of C. jejuni infections; however, there was no significant difference in prevalence between symptomatic and asymptomatic infection (all p > 0.05). The majority of isolates (n = 291, 82.7%) were predicted to express a heptose-containing capsule. The predicted presence of methyl phosphoramidate, heptose or deoxyheptose on the capsule was common.

Campylobacter jejuni transcriptional and genetic adaptation during human infection.

Campylobacter jejuni infections are a leading cause of bacterial food-borne diarrhoeal illness worldwide, and Campylobacter infections in children are associated with stunted growth and therefore long-term deficits into adulthood. Despite this global impact on health and human capital, how zoonotic C. jejuni responds to the human host remains unclear. Unlike other intestinal pathogens, C. jejuni does not harbour pathogen-defining toxins that explicitly contribute to disease in humans. This makes understanding Campylobacter pathogenesis challenging and supports a broad examination of bacterial factors that contribute to C. jejuni infection. Here, we use a controlled human infection model to characterize C. jejuni transcriptional and genetic adaptations in vivo, along with a non-human primate infection model to validate our approach. We found that variation in 11 genes is associated with either acute or persistent human infections and includes products involved in host cell invasion, bile sensing and flagella modification, plus additional potential therapeutic targets. In particular, a functional version of the cell invasion protein A (cipA) gene product is strongly associated with persistently infecting bacteria and we identified its biochemical role in flagella modification. These data characterize the adaptive C. jejuni response to primate infections and suggest therapy design should consider the intrinsic differences between acute and persistently infecting bacteria. In addition, RNA sequencing revealed conserved responses during natural host commensalism and human infections. Thirty-nine genes were differentially regulated in vivo across hosts, lifestyles and C. jejuni strains. This conserved in vivo response highlights important C. jejuni survival mechanisms such as iron acquisition and evasion of the host mucosal immune response. These advances highlight pathogen adaptability across host species and demonstrate the utility of multidisciplinary collaborations in future clinical trials to study pathogens in vivo.

Epidemiology of Campylobacter Infections among Children in Egypt.

Campylobacter is a frequently isolated bacterial pathogen among children with diarrhea. Data are lacking on the distribution and spectrum of disease associated with Campylobacter species and Campylobacter jejuni capsular polysaccharide (CPS) types. This information is essential because current vaccine research seeks to target specific CPS types. An effective CPS-conjugate vaccine will need to cover CPS types that are both common and associated with severe disease. The US Naval Medical Research Unit-3 conducted several prospective cohort studies researching diarrheal disease in Egypt from 1995 to 2003. In total, 1,057 children were enrolled and followed to a maximum age of 36 months. We analyzed Campylobacter-positive stool samples that were collected while subjects were symptomatic, along with corresponding clinical data. Of 441 Campylobacter isolates, 322 represented primary infections (189 C. jejuni, 127 Campylobacter coli, six unspeciated). There were 19 C. jejuni CPS types identified; eight accounted for 63.5% of primary C. jejuni infections. We also screened for the presence of the type-6 secretion system (T6SS), a putative virulence determinant. The T6SS was found in 18.0% of C. coli isolates and 57.6% of C. jejuni isolates (P < 0.001), and was not uniformly distributed among CPS types (P < 0.001). Strains with the T6SS were not associated with more severe disease. Clinical presentations across species and CPS types appeared similar. This study adds to the growing epidemiological data and also provides some analysis of the clinical spectrum associated with infection by specific Campylobacter species, C. jejuni capsule types, and possible virulence determinants.

Significant contribution of subtype G to HIV-1 genetic complexity in Nigeria identified by a newly developed subtyping assay specific for subtype G and CRF02_AG.

While abundant sequence information is available from human immunodeficiency virus type 1 (HIV-1) subtypes A, B, C and CRF01_AE for HIV-1 vaccine design, sequences from West Africa are less represented. We sought to augment our understanding of HIV-1 variants circulating in 6 Nigerian cities as a step to subsequent HIV-1 vaccine development.The G/CRF02_AG multi-region hybridization assay (MHA) was developed to differentiate subtype G, CRF02_AG and their recombinants from other subtypes based on 7 HIV-1 segments. Plasma from 224 HIV-1 infected volunteers enrolled in a cohort examining HIV-1 prevalence, risk factor, and subtype from Makurdi (30), Abuja (18), Enugu (11), Kaduna (12), Tafa (95), and Ojo/Lagos (58) was analyzed using MHA. HIV-1 genomes from 42 samples were sequenced to validate the MHA and fully explore the recombinant structure of G and CRF02_AG variants.The sensitivity and specificity of MHA varied between 73-100% and 90-100%, respectively. The subtype distribution as identified by MHA among 224 samples revealed 38% CRF02_AG, 28% G, and 26% G/CRF02_AG recombinants while 8% remained nontypeable strains. In envelope (env) gp120, 38.84% of the samples reacted to a G probe while 31.25% reacted to a CRF02 (subtype A) probe. Full genome characterization of 42 sequences revealed the complexity of Nigerian HIV-1 variants.CRF02_AG, subtype G, and their recombinants were the major circulating HIV-1 variants in 6 Nigerian cities. High proportions of samples reacted to a G probe in env gp120 confirms that subtype G infections are abundant and should be considered in strategies for global HIV-1 vaccine development.

Updated Campylobacter jejuni Capsule PCR Multiplex Typing System and Its Application to Clinical Isolates from South and Southeast Asia.

Campylobacter jejuni produces a polysaccharide capsule that is the major determinant of the Penner serotyping scheme. This passive slide agglutination typing system was developed in the early 1980's and was recognized for over two decades as the gold standard for C. jejuni typing. A preliminary multiplex PCR technique covering 17 serotypes was previously developed in order to replace this classic serotyping scheme. Here we report the completion of the multiplex PCR technology that is able to identify all the 47 Penner serotypes types known for C. jejuni. The number of capsule types represented within the 47 serotypes is 35. We have applied this method to a collection of 996 clinical isolates from Thailand, Cambodia and Nepal and were able to successfully determine capsule types of 98% of these.

Targeting of the non-mutated tumor antigen HER2/neu to mature dendritic cells induces an integrated immune response that protects against breast cancer in mice.

INTRODUCTION: Given their relative simplicity of manufacture and ability to be injected repeatedly, vaccines in a protein format are attractive for breast and other cancers. However, soluble human epidermal growth factor receptor (HER2)/neu protein as a vaccine has not been immunogenic. When protein is directly targeted to antigen uptake receptors, such as DEC205 (DEC), efficient processing and presentation of antigen take place. The aim of this study was to determine the immunogenicity of a HER2 protein vaccine that directly targets to DEC+ dendritic cells (DCs) in a mouse breast cancer model. METHODS: We genetically engineered the HER2 extracellular domain into a monoclonal antibody specific for DEC (DEC-HER2). Mice of various genetic backgrounds were immunized with DEC-HER2 in combination with DC maturation stimuli (poly IC ± CD40 Ab). Vaccine-induced T cell immunity was determined by analyzing the ability of CD4+/CD8+ T cell to produce interferon (IFN)-gamma and proliferate upon antigen rechallenge. Sera were assessed for the presence of antigen specific antibody (Ab). For vaccine efficacy, FVB/N mice were immunized with DEC-HER2 in combination with poly IC and protection against neu-expressing mammary tumors was assessed. Protection mechanisms and tumor-specific T cell responses were also evaluated. RESULTS: We demonstrate that DEC-HER2 fusion mAb, but not Ctrl Ig-HER2, elicits strong, broad and multifunctional CD4+ T cell immunity, CD8+ T cell responses, and humoral immunity specific for HER2 antigen. Cross-reactivity to rat neu protein was also observed. Importantly, mice xeno-primed with DEC-HER2 were protected from a neu-expressing mammary tumor challenge. Both CD4+ and CD8+ T cells mediated the tumor protection. Robust anti-tumor T cell immunity was detected in tumor protected mice. CONCLUSIONS: Immunization of mice with HER2 protein vaccine targeting DEC+ DCs in vivo induced high levels of T- and B-cell immunity. Non-targeted HER2 protein was poorly immunogenic for CD4+ and CD8+ T cells. This vaccination approach provided long-term survival benefit for mice challenged with neu-expressing tumor following as little as 2.7 µg of HER2 protein incorporated in the vaccine. Vaccine-induced CD4+ and CD8+ T cells were both essential for tumor protection. This immunization strategy demonstrates great potential towards the development of vaccines for breast cancer patients.

The human cancer antigen mesothelin is more efficiently presented to the mouse immune system when targeted to the DEC-205/CD205 receptor on dendritic cells.

To develop a tumor vaccine directly targeting tumor antigen to dendritic cells in situ, we engineered human mesothelin (MSLN) into an antibody specific for mouse DEC-205, a receptor for antigen presentation. We then characterized both T cell and humoral responses to human MSLN and compared immunizing efficacy of DEC-205-targeted MSLN to nontargeted protein after a single-dose immunization. Targeting human MSLN to DEC-205 receptor induced stronger CD4(+) T-cell responses compared to high doses of mesothelin protein. Approximately 0.5% CD4(+) T cells were primed to produce IFN-gamma, tumor necrosis factor-alpha, and IL-2 via intracellular cytokine staining, and the T cells also could proliferate rapidly. The immune response exhibited breadth because the primed CD4(+) T cells responded to at least three epitopes in the H-2(b) background. Targeting MSLN protein to DEC-205 receptor also resulted in cross-presentation to CD8(+) T cells. Antibody responses against human MSLN were also detected in serum from primed mice by ELISA assays. In summary, targeting of MSLN to DEC-205 improves the induction of CD4(+) and CD8(+) T-cell immunity accompanied by an antibody response. DEC-205-targeting could be valuable for enhancing immunity to MSLN in cancers where this nonmutated protein is expressed.

The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells.

DNA vaccines promote an immune response by providing antigen-encoding DNA to the recipient, but the efficacy of such vaccines needs improving. Many approaches have considerable potential but currently induce relatively weak immune responses despite multiple high doses of DNA vaccine. Here, we asked whether targeting vaccine antigens to DCs would increase the immunity and protection that result from DNA vaccines. To determine this, we generated a DNA vaccine encoding a fusion protein comprised of the vaccine antigen and a single-chain Fv antibody (scFv) specific for the DC-restricted antigen-uptake receptor DEC205. Following vaccination of mice, the vaccine antigen was expressed selectively by DCs, which were required for the increased efficacy of MHC class I and MHC class II antigen presentation relative to a control scFv DNA vaccine. In addition, a DNA vaccine encoding an HIV gag p41-scFv DEC205 fusion protein induced 10-fold higher antibody levels and increased numbers of IFN-gamma-producing CD4+ and CD8+ T cells. After a single i.m. injection of the DNA vaccine encoding an HIV gag p41-scFv DEC205 fusion protein, mice were protected from an airway challenge with a recombinant vaccinia virus expressing the HIV gag p41, even with 1% of the dose of nontargeted DNA vaccine. The efficacy of DNA vaccines therefore may be enhanced by inclusion of sequences such as single-chain antibodies to target the antigen to DCs.

Heterologous expression of the Treponema pallidum laminin-binding adhesin Tp0751 in the culturable spirochete Treponema phagedenis.

Treponema pallidum subsp. pallidum, the causative agent of syphilis, is an unculturable, genetically intractable bacterium. Here we report the use of the shuttle vector pKMR4PEMCS for the expression of a previously identified T. pallidum laminin-binding adhesin, Tp0751, in the nonadherent, culturable spirochete Treponema phagedenis. Heterologous expression of Tp0751 in T. phagedenis was confirmed via reverse transcriptase PCR analysis with tp0751 gene-specific primers and immunofluorescence analysis with Tp0751-specific antibodies; the latter assay verified the expression of the laminin-binding adhesin on the treponemal surface. Expression of Tp0751 within T. phagedenis was functionally confirmed via laminin attachment assays, in which heterologous Tp0751 expression conferred upon T. phagedenis the capacity to attach to laminin. Further, specific inhibition of the attachment of T. phagedenis heterologously expressing Tp0751 to laminin was achieved by using purified antibodies raised against recombinant T. pallidum Tp0751. This is the first report of heterologous expression of a gene from an unculturable treponeme in T. phagedenis. This novel methodology will significantly advance the field of syphilis research by allowing targeted investigations of T. pallidum proteins purported to play a role in pathogenesis, and specifically host cell attachment, in the nonadherent spirochete T. phagedenis.

Defining the interaction of the Treponema pallidum adhesin Tp0751 with laminin.

Various invasive pathogens attach to host tissues via the extracellular matrix component laminin, the major glycoprotein found within basement membranes. Previous investigations identified the laminin-binding adhesin Tp0751 within the spirochete bacterium Treponema pallidum. In the current study, Tp0751 was shown to attach to a variety of laminin isoforms that are widely distributed throughout the host, including laminins 1, 2, 4, 8, and 10. Such universal attachment is conducive for an adhesin present within a highly invasive pathogen that encounters a variety of tissue sites during the course of infection. Additional studies systematically identified the amino acid residues within Tp0751 that contribute to laminin binding using synthetic peptides designed from the mature protein sequence. The minimum laminin-binding region of the adhesin was localized to 10 amino acids; peptides containing these residues inhibited attachment of Tp0751 and T. pallidum to laminin. Further, Tp0751-specific antibodies inhibited attachment of T. pallidum to laminin. This study furthers our knowledge of the interaction of T. pallidum with laminin, an association that is proposed to facilitate bacterial traversal of basement membranes and subsequent entry into the circulation and tissue invasion. As such, these investigations will reveal new targets for possible prevention of bacterial dissemination and establishment of chronic infection.

Treponema pallidum fibronectin-binding proteins.

Putative adhesins were predicted by computer analysis of the Treponema pallidum genome. Two treponemal proteins, Tp0155 and Tp0483, demonstrated specific attachment to fibronectin, blocked bacterial adherence to fibronectin-coated slides, and supported attachment of fibronectin-producing mammalian cells. These results suggest Tp0155 and Tp0483 are fibronectin-binding proteins mediating T. pallidum-host interactions.

Ultrastructure of a novel eurytele nematocyst of Carybdea alata Reynaud (Cubozoa, Cnidaria).

The ultrastructural characteristics of nematocysts from the cubozoan Carybdea alata Reynaud, 1830 (Hawaiian box jellyfish) were examined using light, scanning and transmission electron microscopy. We reclassified the predominant nematocyst in C. alata tentacles as a heterotrichous microbasic eurytele, based on spine, tubule and capsule measurements. These nematocysts exhibited a prominent and singular stylet, herein referred to as the lancet. Discharged nematocysts from fixed tentacle preparations displayed the following structures: a smooth shaft base, lamellae, a hemicircumferential fissure demarking the proximal end of a stratified lancet, and a gradually tapering tubule densely covered with large triangularly shaped spines. The lancet remained partially adjoined to the shaft base in a hinge-like fashion in rapidly fixed, whole-tentacle preparations. In contrast, this structure was not observed in discharged nematocyst preparations which involved multiple transfer steps prior to fixation. Various approaches were designed to detect this structure in the absence of fixative. Detached lancets were located in proximity to discharged tubules in undisturbed coverslip preparations of fresh tentacles. In addition, examination of embedded nematocysts from fresh tentacles laid on polyacrylamide gels revealed still-attached lancets. To examine the function of this structure in prey capture, Artemia sp. laden tentacles were prepared for scanning electron microscopy. While carapace exteriors exhibited structures proximal to the lancet, i.e., the nematocyst capsule and shaft base, neither tubule nor lancet structures were visible. Taken together, the morphological data suggested a series of events involved in the discharge of a novel eurytele from C. alata.