The immune response of pregnant sow after vaccination with crude fimbriae (F4) extracts vaccine and immunoprotection of nursery pig against pathogenic E. coli (F4+ETEC).

BACKGROUND: Neonatal and post-weaning diarrhea is a concern disease caused by enterotoxigenic Escherichia coli fimbriae F4 (F4+ETEC) in pig farms. Diarrhea outbreaks are often severe and costly due to the high prevalence and spread of the disease within the same herd. Vaccine is one of strategic solution in protecting pig against F4+ETEC infection in particular pig farm. In present study, we conducted two trials of vaccination with crude F4 fimbriae extract vaccine in pregnant sow and nursery pigs. METHODS: In experiment 1 (20 sows; non-vaccinated control, n=10), we vaccinated pregnant sows (n=10) twice at 4 wk and 2 wk before farrowing and evaluated impact of vaccination on maternal immunity. The sow serum and colostrum were collected before vaccination, 2 and 4 weeks after vaccination, 6 hours after farrowing, respectively, and the piglet's serum from both groups (2 piglet/sow, 10 piglets from each group) were also collected on 3 days old to measure F4 specific IgG, F4 specific IgA using in house ELISA kit. In experiment 2, to optimize doses and dosage of candidate vaccine in piglets, 18 piglets (3 piglets/group) were allocated into five immunized groups and one control group (unimmunized group), we immunized piglets twice at 4 and 6 weeks old with difference doses (i.e., 0, 50, 100, 150, 200 µg), and for a dose 150 µg, we immunized with two dosages at 1 ml and 2 ml. Piglets were challenged with a 3 ml dose of 3 × 109 CFU/ml bacterial culture of enterotoxigenic Escherichia coli (F4+ETEC) in order to evaluate the efficacy of vaccine. After challenging, the clinical sign of the piglets was daily observed and the rectal swab was performed every day for investigation of the fecal shedding of Escherichia coli (F4+ETEC) by using PCR technique. Serum were collected before, 2 and 4 weeks after vaccination and 1 week after challenge to measure F4 specific IgG, F4 specific IgA using in house ELISA kit and cytokines levels (i.e., IL-1 beta, IL-6, IL-8 and TNF alpha) before and 1 week after challenge using commercial ELISA kit. RESULTS: The levels of antibody results showed that in experiment 1, the anti-F4 antibody levels both F4 specific IgG and F4 specific IgA in serum and colostrum of vaccinated sow increased significantly after vaccination. The piglets of immunized sows have antibody level both F4 specific IgG and F4 specific IgA in their serum higher than those piglets of unimmunized sows significantly (p < 0.01). In experiment 2, irrespective of different doses and dosage, there is no difference in term of F4 specific IgG and F4 specific IgA levels among immunized groups. However, all of vaccinated piglets showed F4 specific IgG and F4 specific IgA levels higher and the elimination of Escherichia coli (F4+ETEC) in feces post challenge faster (< 3 days) than unvaccinated group (> 5 days). For cytokines levels, a higher level of IL-1 beta, IL-6, IL-8 and TNF alpha at 1 week after challenge in vaccinated groups was found when compared with the levels in non-vaccinated group. CONCLUSIONS: Our results suggest that crude F4 fimbriae extract autogenous vaccine is a candidate vaccine for protecting piglets against diarrhea disease caused by enterotoxigenic Escherichia coli (F4+ETEC) and vaccination the pregnant sow twice before farrowing is one of strategies to provide maternal derived antibody to the newborn piglets for against enterotoxigenic Escherichia coli (F4+ETEC) during early life.

Antibody-Mediated Targeting of Antigens to Intestinal Aminopeptidase N Elicits Gut IgA Responses in Pigs.

Many pathogens enter the host via the gut, causing disease in animals and humans. A robust intestinal immune response is necessary to protect the host from these gut pathogens. Despite being best suited for eliciting intestinal immunity, oral vaccination remains a challenge due to the gastrointestinal environment, a poor uptake of vaccine antigens by the intestinal epithelium and the tolerogenic environment pervading the gut. To improve uptake, efforts have focused on targeting antigens towards the gut mucosa. An interesting target is aminopeptidase N (APN), a conserved membrane protein present on small intestinal epithelial cells shown to mediate epithelial transcytosis. Here, we aimed to further optimize this oral vaccination strategy in a large animal model. Porcine APN-specific monoclonal antibodies were generated and the most promising candidate in terms of epithelial transcytosis was selected to generate antibody fusion constructs, comprising a murine IgG1 or porcine IgA backbone and a low immunogenic antigen: the F18-fimbriated E. coli tip adhesin FedF. Upon oral delivery of these recombinant antibodies in piglets, both mucosal and systemic immune responses were elicited. The presence of the FedF antigen however appeared to reduce these immune responses. Further analysis showed that F18 fimbriae were able to disrupt the antigen presenting capacity of intestinal antigen presenting cells, implying potential tolerogenic effects of FedF. Altogether, these findings show that targeted delivery of molecules to epithelial aminopeptidase N results in their transcytosis and delivery to the gut immune systems. The results provide a solid foundation for the development of oral subunit vaccines to protect against gut pathogens.

Escherichia coli type-1 fimbriae are critical to overcome initial bottlenecks of infection upon low-dose inoculation in a porcine model of cystitis.

Most uropathogenic Escherichia coli (UPEC) express type-1 fimbriae (T1F), a key virulence factor for urinary tract infection (UTI) in mice. Evidence that conclusively associates this pilus with uropathogenesis in humans has, however, been difficult to obtain. We used an experimental porcine model of cystitis to assess the role of T1F in larger mammals more closely related to humans. Thirty-one pigs were infected with UPEC strain UTI89 or its T1F deficient mutant, UTI89ΔfimH, at inoculum titres of 102 to 108 colony forming units per millilitre. Urine and blood samples were collected and analysed 7 and 14 days post-inoculation, and whole bladders were removed at day 14 and analysed for uroepithelium-associated UPEC. All animals were consistently infected and reached high urine titres independent of inoculum titre. UTI89ΔfimH successfully colonized the bladders of 1/6 pigs compared to 6/6 for the wild-type strain. Intracellular UPEC were detectable in low numbers in whole bladder explants. In conclusion, low doses of UPEC are able to establish robust infections in pigs, similar to what is presumed in humans. T1F are critical for UPEC to surpass initial bottlenecks during infection but may be dispensable once infection is established. While supporting the conclusions from mice studies regarding a general importance of T1F in successfully infecting the host, the porcine UTI models' natural high, more human-like, susceptibility to infection, allowed us to demonstrate a pivotal role of T1F in initial establishment of infection upon a realistic low-inoculum introduction of UPEC in the bladder.

Evaluation of the immunogenicity and protective ability of a pili subunit, SBP2', of Streptococcus suis serotype 2.

Streptococcus suis is an important zoonotic pathogen that leads to huge economic losses in the swine industry. Because of the enormous genetic and phenotypic diversity within S. suis, it is necessary to develop effective vaccines to control this zoonotic pathogen. SBP2' is a major pili subunit in S. suis that belongs to an srtBCD pili cluster and has already been reported to be associated with the pathogenesis of this bacterium. In this study, we aimed to evaluate the immunogenicity and protective ability of SBP2'. The rSBP2' protein was expressed by an Escherichia coli expression system and emulsified with Montanide ISA 201 adjuvant to prepare the subunit vaccine. Through active immune assays, the results showed that rSBP2' exhibited good immunogenicity and could protect mice from a lethal dose challenge. Additionally, the qRT-PCR data showed that the transcription levels of cytokines associated with systemic symptoms caused by S. suis were decreased, indicating that immunization with rSBP2' could protect the host from cytokine storms caused by S. suis. Furthermore, the passive immune assay showed that the humoral immunity induced by rSBP2' played an important role against S. suis infection. Taken together, SBP2' could provide proper immune protection against S. suis challenge and could be a candidate for S. suis subunit vaccine. The results of this study could provide new ideas for the development of effective vaccines against S. suis.

Akkermansia muciniphila and Its Pili-Like Protein Amuc_1100 Modulate Macrophage Polarization in Experimental Periodontitis.

Periodontitis is a chronic inflammatory disease triggered by dysbiosis of the oral microbiome. Porphyromonas gingivalis is strongly implicated in periodontal inflammation, gingival tissue destruction, and alveolar bone loss through sustained exacerbation of the host response. Recently, the use of other bacterial species, such as Akkermansia muciniphila, has been suggested to counteract inflammation elicited by P. gingivalis In this study, the effects of A. muciniphila and its pili-like protein Amuc_1100 on macrophage polarization during P. gingivalis infection were evaluated in a murine model of experimental periodontitis. Mice were gavaged with P. gingivalis alone or in combination with A. muciniphila or Amuc_1100 for 6 weeks. Morphometric analysis demonstrated that the addition of A. muciniphila or Amuc_1100 significantly reduced P. gingivalis-induced alveolar bone loss. This decreased bone loss was associated with a proresolutive phenotype (M2) of macrophages isolated from submandibular lymph nodes as observed by flow cytometry. Furthermore, the expression of interleukin 10 (IL-10) at the RNA and protein levels was significantly increased in the gingival tissues of the mice and in macrophages exposed to A. muciniphila or Amuc_1100, confirming their anti-inflammatory properties. This study demonstrates the putative therapeutic interest of the administration of A. muciniphila or Amuc_1100 in the management of periodontitis through their anti-inflammatory properties.

T4 Pili Promote Colonization and Immune Evasion Phenotypes of Nonencapsulated M4 Streptococcus pyogenes.

Streptococcus pyogenes (group A Streptococcus [GAS]) is an important human pathogen causing a broad spectrum of diseases and associated with significant global morbidity and mortality. Almost all GAS isolates express a surface hyaluronic acid capsule, a virulence determinant that facilitates host colonization and impedes phagocyte killing. However, recent epidemiologic surveillance has reported a sustained increase in both mucosal and invasive infections caused by nonencapsulated GAS, which questions the indispensable role of hyaluronic acid capsule in GAS pathogenesis. In this study, we found that pilus of M4 GAS not only significantly promotes biofilm formation, adherence, and cytotoxicity to human upper respiratory tract epithelial cells and keratinocytes, but also promotes survival in human whole blood and increased virulence in murine models of invasive infection. T4 antigen, the pilus backbone protein of M4 GAS, binds haptoglobin, an abundant human acute-phase protein upregulated upon infection and inflammation, on the bacterial surface. Haptoglobin sequestration reduces the susceptibility of nonencapsulated M4 GAS to antimicrobial peptides released from activated neutrophils and platelets. Our results reveal a previously unappreciated virulence-promoting role of M4 GAS pili, in part mediated by co-opting the biology of haptoglobin to mitigate host antimicrobial defenses.IMPORTANCE Group A Streptococcus (GAS) is a strict human pathogen causing more than 700 million infections globally each year. The majority of the disease-causing GAS are encapsulated, which greatly guarantees survival and dissemination in the host. Emergence of the capsule-negative GAS, such as M4 GAS, in recent epidemiologic surveillance alarms the necessity to elucidate the virulence determinants of these pathogens. Here, we found that M4 pili play an important role in promoting M4 GAS adherence and cytotoxicity to human pharyngeal epithelial cells and keratinocytes. The same molecule also significantly enhanced M4 GAS survival and replication in human whole blood and experimental murine infection. T4 antigen, which composes the backbone of M4 pili, was able to sequester the very abundant serum protein haptoglobin to further confer M4 GAS resistance to antibacterial substances released by neutrophils and platelets.

A multivalent vaccine candidate targeting enterotoxigenic Escherichia coli fimbriae for broadly protecting against porcine post-weaning diarrhea.

Fimbriae-mediated initial adherence is the initial and critical step required for enterotoxigenic Escherichia coli (ETEC) infection. Therefore, vaccine candidates have been developed that target these fimbriae and induce specific anti-fimbriae antibodies to block initial ETEC attachment. While this vaccine effectively protects against ETEC-associated post-weaning diarrhea (PWD), developing a broadly effective vaccine against initial ETEC attachment remains a challenging problem, owing to the immunological heterogeneity among these antigens. Here, we applied multi-epitope fusion antigen (MEFA) technology to construct a FaeG-FedF-FanC-FasA-Fim41a MEFA using the adhesive subunits of predominant fimbriae K88 and F18 as the backbone, which also integrated epitopes from adhesive subunits of the rare fimbriae K99, 987P, and F41; we then generated a MEFA computational model and tested the immunogenicity of this MEFA protein in immunized mice. We next evaluated the potential of the fimbriae-targeted MEFA as a vaccine candidate to effectively prevent PWD using in vitro assessment of its anti-fimbriae, antibody-directed inhibition of bacterial adherence. Computational modeling showed that all relevant epitopes were exposed on the MEFA surface and mice subcutaneously immunized with the MEFA protein developed IgG antibodies to all five fimbriae. Moreover, anti-fimbriae antibodies induced by the MEFA protein significantly inhibited the adhesion of K88+, F18+, K99+, 987P+, and F41+ ETEC strains to piglet small intestinal IPEC-1 and IPEC-J2 cell lines. Taken together, these results indicate that FaeG-FedF-FanC-FasA-Fim41a MEFA protein induced specific anti-fimbriae neutralizing antibodies against the five targeted fimbriae. Critically, these results show the potential of fimbriae-targeted MEFA and indicate their promise as a broad, effective vaccine against PWD.

Generating mucosal and systemic immune response following vaccination of vibrio cholerae adhesion molecule against shigella flexneri infection.

Introduction: Previous studies have shown 37.8 kDa pili subunit protein of Vibrio cholerae and 49.8 kDa pili subunit protein of Shigella flexneri can act as an adhesion molecule to initiate infection. These molecules also have the ability to agglutinate blood. The present study assessed mucosal and systemic immunity following vaccination using 37.8 kDa V. cholerae and protection against S. flexneri. Subjects and Methods: Haemagglutination test was performed after purification of V. cholerae protein, followed by an anti-haemagglutination test. The intestinal weight and colony count were used to validate the protective effect on balb/c mice which were divided into the naive group, Shigella-positive control group, Vibrio-positive control group, V. cholerae infected-Vibrio-vaccinated group and S. flexneri-infected-Vibrio-vaccinated group. Th17, Treg, interleukin (IL) IL-17A, ß-defensin and secretory-immunoglobulin A (s-IgA) were also measured to determine the systemic and mucosal immunity after vaccination. Results: The haemagglutination and anti-haemagglutination tests showed that the 37.8 kDa protein could inhibit 49.8 kDa of the S. flexneri pili subunit. Decreased intestinal weight and colony count of vaccinated group compared to naive group also support cross reaction findings. Vaccination also generates higher level of Th17, Treg, IL-17A, ß-defensin and s-IgA significantly. Conclusions: 37.8 kDa subunit pili can act as a homologous vaccine candidate to prevent V. cholerae and S. flexneri infection.

Aggregative Adherence Fimbriae II of Enteroaggregative Escherichia coli Are Required for Adherence and Barrier Disruption during Infection of Human Colonoids.

Symptomatic and asymptomatic infection with the diarrheal pathogen enteroaggregative Escherichia coli (EAEC) is associated with growth faltering in children in developing settings. The mechanism of this association is unknown, emphasizing a need for better understanding of the interactions between EAEC and the human gastrointestinal mucosa. In this study, we investigated the role of the aggregative adherence fimbriae II (AAF/II) in EAEC adherence and pathogenesis using human colonoids and duodenal enteroids. We found that a null mutant in aafA, the major subunit of AAF/II, adhered significantly less than wild-type (WT) EAEC strain 042, and adherence was restored in a complemented strain. Immunofluorescence confocal microscopy of differentiated colonoids, which produce an intact mucus layer comprised of the secreted mucin MUC2, revealed bacteria at the epithelial surface and within the MUC2 layer. The WT strain adhered to the epithelial surface, whereas the aafA deletion strain remained within the MUC2 layer, suggesting that the presence or absence of AAF/II determines both the abundance and location of EAEC adherence. In order to determine the consequences of EAEC adherence on epithelial barrier integrity, colonoid monolayers were exposed to EAEC constructs expressing or lacking aafA Colonoids infected with WT EAEC had significantly decreased epithelial resistance, an effect that required AAF/II, suggesting that binding of EAEC to the epithelium is necessary to impair barrier function. In summary, we show that production of AAF/II is critical for adherence and barrier disruption in human colonoids, suggesting a role for this virulence factor in EAEC colonization of the gastrointestinal mucosa.

Prevention of nosocomial Acinetobacter baumannii infections with a conserved immunogenic fimbrial protein.

Acinetobacter baumannii, one of the most life-threatening nosocomial drug-resistant pathogens, imposes high morbidity and mortality rates, thus highlighting immunization-based treatments or prevention measures. The selection of appropriate antigens can elicit protective immunity. The gene encoding a fimbrial protein introduced via reverse vaccinology was cloned, expressed and evaluated for immunogenicity in a murine model. Mice immunized with the recombinant protein were challenged with A. baumannii ATCC 19606. Adherence to A549 cell line of specific anti-sera treated A. baumannii was also assessed. Passive immunity was evaluated in a murine pneumonia model. Indirect ELISA showed a high specific antibody titre. Adherence of A. baumannii to A549 cell line decreased by 40% after incubation with 1:250 dilution of specific anti-sera. All the actively immunized mice survived. Bacterial load in the spleen and liver of the immunized mice was 3-fold lower than those of the control. The number of bacteria in the lungs of passively immunized mice was about 6-fold lower than the control mice. The fimbrial protein could be considered as a promising protective immunogen against A. baumannii.

Coimmunization with Two Enterotoxigenic Escherichia coli (ETEC) Fimbrial Multiepitope Fusion Antigens Induces the Production of Neutralizing Antibodies against Five ETEC Fimbriae (F4, F5, F6, F18, and F41).

Fimbriae mediate the initial adherence of enterotoxigenic Escherichia coli (ETEC) to the piglet small intestine and play an important role in development of ETEC-driven postweaning diarrhea (PWD). PWD inflicts huge economic losses on the swine industry each year, making development of alternative treatment and prevention measures for PWD essential. Vaccine candidates that induce antifimbria antibodies that block the initial attachment and colonization of ETEC pathogens with fimbriae are one approach that could help prevent PWD. In this study, we constructed two multiepitope fusion antigens (MEFAs) that carried, expressed, and displayed representative epitopes of F4, F5, F6, F18, and F41 ETEC fimbriae. These MEFAs used either the F4 major subunit FaeG or the F18 adhesive subunit FedF as a backbone. To assess the potential of these MEFAs as antifimbria vaccine candidates that could help prevent PWD, we generated computational models of the MEFAs, constructed them, and then tested their immunogenicity by using them to immunize mice. Computational modeling showed that all relevant epitopes were exposed on the MEFA surface. We found that coadministration of our MEFAs in mice successfully induced five fimbria-specific antibodies in accordance with the epitopes included in the MEFA constructs. Furthermore, the induced antibodies can significantly inhibit the ability of ETEC strains that express F4, F5, F6, F18, and F41 fimbriae to adhere to piglet small intestinal IPEC-1 and IPEC-J2 cells. Our findings indicate that the antifimbria antibodies induced by our FaeG-Fim41a-FanC-FasA and FedF-FasA-Fim41a-FanC fimbria MEFAs blocked adherence of five ETEC fimbriae, suggesting these multivalent fimbria MEFAs may be useful for developing broadly protective antifimbria vaccines against PWD caused by ETEC infections.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC)-associated postweaning diarrhea (PWD) is still a leading disease in recently weaned piglets. Vaccination is considered to be the most ideal and efficacious strategy for preventing PWD. Recently, a commercialized live monovalent F4 oral vaccine and a bivalent F4/F18 oral vaccine have been demonstrated to effectively protect piglets in the F4-positive (F4+) and F18+ ETEC challenge models. However, they will not provide cross-protection against F5+, F6+, or F41+ ETEC-associated PWD cases, as they lack all five fimbria antigens. Thus, a multivalent vaccine containing all five ETEC fimbriae would be more effective in preventing ETEC-driven PWD. In this study, we designed two fimbria-targeted MEFAs using the MEFA technology, and further study demonstrated that these coadministered MEFAs in mice can induce protective antibodies against the five fimbriae expressed by ETEC. These MEFAs could be used as an efficient PWD vaccine candidate; furthermore, MEFA-based structural technology provides an alternative and promising strategy for the development of vaccines against pathogens with heterogeneous virulence factors.

New putative vaccine candidates against Acinetobacter baumannii using the reverse vaccinology method.

Infections caused by multi-drug resistance Acinetobacter baumannii are increasing worldwide. Discovery of the vaccine against this bacterium as a cost-effective and preventive strategy seems necessary. This study has introduced 11 new putative vaccine candidates against A. baumannii using the reverse vaccinology method. We considered 33 genomes of A. baumannii strains and selected the outer membrane and secreted proteins as putative vaccine candidates using Vaxign web tool. Finally, 11 proteins were confirmed as promising vaccine candidates. These targets belonged to proteins involved in cell division (NlpD), fimbria or pili assembly (FimA, PapC, and PapC associated with usher system), iron acquisition (FhuA, BfnH, FatA-like protein, and IutA), DcaP-like protein and two novel hypothetical proteins (HP-1 and HP-2). The analysis of linear and conformational B-cell epitopes showed that the outer membrane proteins including DcaP-like protein and HP-2 had high conserved surface-exposed epitopes that they can consider as excellent putative vaccine targets in the upcoming immunological assays.

Application of a Novel Epitope- and Structure-Based Vaccinology-Assisted Fimbria-Toxin Multiepitope Fusion Antigen of Enterotoxigenic Escherichia coli for Development of Multivalent Vaccines against Porcine Postweaning Diarrhea.

Enterotoxigenic Escherichia coli (ETEC) strains producing K88 (F4) or F18 fimbriae and enterotoxins are the predominant cause of pig postweaning diarrhea (PWD). We recently identified neutralizing epitopes of fimbriae K88 and F18, heat-labile toxin (LT), heat-stable toxins type I (STa) and type II (STb), and Shiga toxin 2e (Stx2e). In this study, we explored a novel epitope- and structure-based vaccinology platform, multiepitope fusion antigen (MEFA), for PWD vaccine development. By using an epitope substitution LT toxoid, which lacks enterotoxicity but retains immunogenicity, as the backbone to present neutralizing epitopes of two ETEC fimbriae and four toxins, we generated PWD fimbria-toxin MEFA to mimic epitope native antigenicity. We then examined MEFA protein immunogenicity and evaluated MEFA application in PWD vaccine development. Mice subcutaneously immunized with PWD MEFA protein developed strong IgG responses to K88, F18, LT, and STb and moderate responses to the toxins Stx2e and STa. Importantly, MEFA-induced antibodies inhibited adherence of K88 or F18 fimbrial bacteria to pig intestinal cells and also neutralized LT, STa, STb, and Stx2e toxicity. These results indicated that PWD fimbria-toxin MEFA induced neutralizing antibodies against an unprecedent two fimbriae and four toxins and strongly suggested a potential application of this MEFA protein in developing a broadly protective PWD vaccine.IMPORTANCE ETEC-associated postweaning diarrhea (PWD) causes significant economic losses to swine producers worldwide. Currently, there is no effective prevention against PWD. A vaccine that blocks ETEC fimbriae (K88 and F18) from attaching to host receptors and prevents enterotoxins from stimulating water hypersecretion in pig small intestinal epithelial cells can effectively protect against PWD and significantly improves pig health and well-being. The fimbria-toxin MEFA generated from this study induced neutralizing antibodies against both ETEC fimbriae and all four ETEC toxins, suggesting a great potential of this fimbria-toxin MEFA in PWD vaccine development and further supporting the general application of this novel MEFA vaccinology platform for multivalent vaccine development.

Expression of the Nontypeable Haemophilus influenzae Type IV Pilus Is Stimulated by Coculture with Host Respiratory Tract Epithelial Cells.

The type IV pilus (Tfp) of nontypeable Haemophilus influenzae (NTHI) mediates adherence, colonization, motility, and biofilm formation, and the major protein subunit, PilA, is a promising vaccine candidate. Thus, it is crucial to understand how Tfp expression is regulated within the microenvironments of the human nasopharynx, which NTHI colonizes asymptomatically, and the more distal regions of the respiratory tract where NTHI-induced diseases occur. Here, we examined the effects of coculture of NTHI with human airway epithelial cells and heme availability on Tfp expression at temperatures typical of the human nasopharynx (34°C) or warmer anatomical sites during infection (37°C). Tfp expression was estimated by pilA promoter activity, pilA gene expression, and relative abundances of PilA and pilin protein. The results revealed that at both temperatures, NTHI cocultured with airway epithelial cells demonstrated significantly greater expression of pilA, PilA/pilin protein, and likely, fully assembled Tfp than NTHI cultured on an abiotic surface. Because NTHI is a heme auxotroph, we hypothesized that availability of heme from host cells might be a signal for Tfp expression. Thereby, we cultured NTHI in iron-limited medium, and we observed that supplementation with heme significantly increased pilA promoter activity. Collectively, our data suggested that NTHI Tfp expression was stimulated by soluble factor(s) released by epithelial cells, which are present in all microenvironments of the respiratory tract. The expression of this target antigen under conditions that mimic the human airway strongly supports the rationale for the use of PilA as a vaccine immunogen to prevent NTHI-induced diseases of the respiratory tract.

Characterization of two putative Dichelobacter nodosus footrot vaccine antigens identifies the first lysozyme inhibitor in the genus.

The Gram-negative anaerobic bacterium Dichelobacter nodosus (Dn) causes footrot in ruminants, a debilitating and highly contagious disease that results in necrotic hooves and significant economic losses in agriculture. Vaccination with crude whole-cell vaccine mixed with multiple recombinant fimbrial proteins can provide protection during species-specific outbreaks, but subunit vaccines containing broadly cross-protective antigens are desirable. We have investigated two D. nodosus candidate vaccine antigens. Macrophage Infectivity Potentiator Dn-MIP (DNO_0012, DNO_RS00050) and Adhesin Complex Protein Dn-ACP (DNO_0725, DNO_RS06795) are highly conserved amongst ~170 D. nodosus isolates in the https://pubmlst.org/dnodosus/ database. We describe the presence of two homologous ACP domains in Dn-ACP with potent C-type lysozyme inhibitor function, and homology of Dn-MIP to other putative cell-surface and membrane-anchored MIP virulence factors. Immunization of mice with recombinant proteins with a variety of adjuvants induced antibodies that recognised both proteins in D. nodosus. Notably, immunization with fimbrial-whole-cell Footvax vaccine induced anti-Dn-ACP and anti-Dn-MIP antibodies. Although all adjuvants induced high titre antibody responses, only antisera to rDn-ACP-QuilA and rDn-ACP-Al(OH)3 significantly prevented rDn-ACP protein from inhibiting lysozyme activity in vitro. Therefore, a vaccine incorporating rDn-ACP in particular could contribute to protection by enabling normal innate immune lysozyme function to aid bacterial clearance.

Antibody responses to flagellin C and Streptococcus gallolyticus pilus proteins in colorectal cancer.

Antibodies to Streptococcus gallolyticus subspecies gallolyticus (SGG) have been associated with colorectal cancer (CRC). Because SGG may correlate with impaired gut epithelia, we assessed the association of antibodies to bacterial flagellin C (FliC), a measure potentially related to this impairment, with CRC and the CRC-specific interaction with antibodies to SGG proteins. Antibodies to FliC and SGG pilus proteins Gallo2178 and Gallo2179 were measured in two independent studies, a combined study from Nijmegen and Detroit (93 CRC cases, 74 controls) and a replication data set including 576 cases and 576 controls from the Spanish multicenter multicase-control study (MCC-Spain). Logistic regression was applied to assess whether antibodies to FliC were associated with CRC and modified the association of antibodies to SGG proteins with CRC. Antibodies to FliC were associated with those to SGG Gallo2178 among CRC cases, resulting in an interaction in the association of antibodies to Gallo2178 with CRC (p = 0.007). This association was only present among individuals with high antibody responses to FliC (OR: 2.42, 95% CI: 1.45-4.06). In conclusion, our findings suggest that colorectal tumorigenesis could be accompanied by an impaired integrity of the epithelium that could result in associated increased antibody responses to bacterial proteins.

Recent Advances in Pathogenic Streptococcus Vaccine Development.

Streptococcus pneumoniae (Spn) and Streptococcus pyogenes (Spy) cause many invasive and noninvasive diseases responsible for high morbidity and mortality worldwide. Safe, efficacious and affordable vaccines could have a significant, positive impact on the global infectious disease burden. Since the implementation of pneumococcal vaccine in the 1980s, the incidence of Spn infection has decreased significantly. Still so, these currently used multivalent polysaccharides and conjugated pneumococcal vaccines have some limitations. For Spy, there are even no vaccines available yet. There is an urgent need of new vaccines against Spn and Spy. Encouragingly, with the hard work of many investigators worldwide, a number of new vaccines candidates are developed with promising results. Of them, many have already entered the clinical trial stage. This review will describe the current status of Spn and Spy vaccine development, with particular focus on protein-based strategy.

Biology of the Gonococcus: Disease and Pathogenesis.

Neisseria gonorrhoeae infection is a major public health problem worldwide. The increasing incidence of gonorrhea coupled with global spread of multidrug-resistant isolates of gonococci has ushered in an era of potentially untreatable infection. Gonococcal disease elicits limited immunity, and individuals are susceptible to repeated infections. In this chapter, we describe gonococcal disease and epidemiology and the structure and function of major surface components involved in pathogenesis. We also discuss the mechanisms that gonococci use to evade host immune responses and the immune responses following immunization with selected bacterial components that may overcome evasion. Understanding the biology of the gonococcus may aid in preventing the spread of gonorrhea and also facilitate the development of gonococcal vaccines and treatments.

Vaccine acquired pertussis immunity was weakened at 4 years of age and asymptomatic pertussis infection was suspected based on serological surveillance.

Serological surveillance of pertussis antibodies was performed in 118 children aged 1-12 years. The positivity of pertussis toxin (PT) antibodies was low at 4-6 years and significantly higher at 8-9 years, compared with those at 6 years. Fimbriae 2 (Fim2) antibody showed similar response to the PT antibody. Higher antibody titers against Fim3 were observed among subjects ≥5 years and highest at 8 years. Data demonstrated that the vaccine-induced antibodies decayed by 4-5 years and subclinical pertussis infection was suspected thereafter, suggesting the need for additional dose at around 4-5 years.

A Double-Strand Break Does Not Promote Neisseria gonorrhoeae Pilin Antigenic Variation.

The major subunit of the type IV pilus (T4p) of Neisseria gonorrhoeae undergoes antigenic variation (AV) dependent on a guanine quadruplex (G4) DNA structure located upstream of the pilin gene. Since the presence of G4 DNA induces genome instability in both eukaryotic and prokaryotic chromosomes, we tested whether a double-strand break (DSB) at the site of the pilE G4 sequence could substitute for G4-directed pilin AV. The G4 motif was replaced by an I-SceI cut site, and the cut site was also introduced to locations near the origin of replication and the terminus. Expression of the I-SceI endonuclease from an irrelevant chromosomal site confirmed that the endonuclease functions to induce double-strand breaks at all three locations. No antigenic variants were detected when the G4 was replaced with the I-SceI cut site, but there was a growth defect from having a DSB in the chromosome, and suppressor mutations that were mainly deletions of the cut site and/or the entire pilE gene accumulated. Thus, the pilE G4 does not act to promote pilin AV by generating a DSB but requires either a different type of break, a nick, or more complex interactions with other factors to stimulate this programmed recombination system.IMPORTANCENeisseria gonorrhoeae, the causative agent of gonorrhea, possesses a DNA recombination system to change one of its surface-exposed antigens. This recombination system, known as antigenic variation, uses an alternate DNA structure to initiate variation. The guanine quadruplex DNA structure is known to cause nicks or breaks in DNA; however, much remains unknown about how this structure functions in cells. We show that inducing a break by different means does not allow antigenic variation, indicating that the DNA structure may have a more complicated role.