IDO Vaccine Ablates Immune-Suppressive Myeloid Populations and Enhances Antitumor Effects Independent of Tumor Cell IDO Status.

The immunosuppressive tumor microenvironment (TME) does not allow generation and expansion of antitumor effector cells. One of the potent immunosuppressive factors present in the TME is the indoleamine-pyrrole 2,3-dioxygenase (IDO) enzyme, produced mainly by cancer cells and suppressive immune cells of myeloid origin. In fact, IDO+ myeloid-derived suppressor cells (MDSC) and dendritic cells (DC) tend to be more suppressive than their IDO- counterparts. Hence, therapeutic approaches that would target the IDO+ cells in the TME, while sparing the antigen-presenting functions of IDO- myeloid populations, are needed. Using an IDO-specific peptide vaccine (IDO vaccine), we explored the possibility of generating effector cells against IDO and non-IDO tumor-derived antigens. For this, IDO-secreting (B16F10 melanoma) and non-IDO-secreting (TC-1) mouse tumor models were employed. We showed that the IDO vaccine significantly reduced tumor growth and enhanced survival of mice in both the tumor models, which associated with a robust induction of IDO-specific effector cells in the TME. The IDO vaccine significantly enhanced the antitumor efficacy of non-IDO tumor antigen-specific vaccines, leading to an increase in the number of total and antigen-specific activated CD8+ T cells (IFNγ+ and granzyme B+). Treatment with the IDO vaccine significantly reduced the numbers of IDO+ MDSCs and DCs, and immunosuppressive regulatory T cells in both tumor models, resulting in enhanced therapeutic ratios. Together, we showed that vaccination against IDO is a promising therapeutic option for both IDO-producing and non-IDO-producing tumors. The IDO vaccine selectively ablates the IDO+ compartment in the TME, leading to a significant enhancement of the immune responses against other tumor antigen-specific vaccines.

MEK inhibition reprograms CD8+ T lymphocytes into memory stem cells with potent antitumor effects.

Regenerative stem cell-like memory (TSCM) CD8+ T cells persist longer and produce stronger effector functions. We found that MEK1/2 inhibition (MEKi) induces TSCM that have naive phenotype with self-renewability, enhanced multipotency and proliferative capacity. This is achieved by delaying cell division and enhancing mitochondrial biogenesis and fatty acid oxidation, without affecting T cell receptor-mediated activation. DNA methylation profiling revealed that MEKi-induced TSCM cells exhibited plasticity and loci-specific profiles similar to bona fide TSCM isolated from healthy donors, with intermediate characteristics compared to naive and central memory T cells. Ex vivo, antigenic rechallenge of MEKi-treated CD8+ T cells showed stronger recall responses. This strategy generated T cells with higher efficacy for adoptive cell therapy. Moreover, MEKi treatment of tumor-bearing mice also showed strong immune-mediated antitumor effects. In conclusion, we show that MEKi leads to CD8+ T cell reprogramming into TSCM that acts as a reservoir for effector T cells with potent therapeutic characteristics.

Author Correction: PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1+CD38hi cells and anti-PD-1 resistance.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Review of Newly Identified Functions Associated With the Heat-Labile Toxin of Enterotoxigenic Escherichia coli.

Heat-labile toxin (LT) is a well-characterized powerful enterotoxin produced by enterotoxigenic Escherichia coli (ETEC). This toxin is known to contribute to diarrhea in young children in developing countries, international travelers, as well as many different species of young animals. Interestingly, it has also been revealed that LT is involved in other activities in addition to its role in enterotoxicity. Recent studies have indicated that LT toxin enhances enteric pathogen adherence and subsequent intestinal colonization. LT has also been shown to act as a powerful adjuvant capable of upregulating vaccine antigenicity; it also serves as a protein or antigenic peptide display platform for new vaccine development, and can be used as a naturally derived cell targeting and protein delivery tool. This review summarizes the epidemiology, secretion, delivery, and mechanisms of action of LT, while also highlighting new functions revealed by recent studies.

PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1+CD38hi cells and anti-PD-1 resistance.

Understanding resistance to antibody to programmed cell death protein 1 (PD-1; anti-PD-1) is crucial for the development of reversal strategies. In anti-PD-1-resistant models, simultaneous anti-PD-1 and vaccine therapy reversed resistance, while PD-1 blockade before antigen priming abolished therapeutic outcomes. This was due to induction of dysfunctional PD-1+CD38hi CD8+ cells by PD-1 blockade in suboptimally primed CD8 cell conditions induced by tumors. This results in erroneous T cell receptor signaling and unresponsiveness to antigenic restimulation. On the other hand, PD-1 blockade of optimally primed CD8 cells prevented the induction of dysfunctional CD8 cells, reversing resistance. Depleting PD-1+CD38hi CD8+ cells enhanced therapeutic outcomes. Furthermore, non-responding patients showed more PD-1+CD38+CD8+ cells in tumor and blood than responders. In conclusion, the status of CD8+ T cell priming is a major contributor to anti-PD-1 therapeutic resistance. PD-1 blockade in unprimed or suboptimally primed CD8 cells induces resistance through the induction of PD-1+CD38hi CD8+ cells that is reversed by optimal priming. PD-1+CD38hi CD8+ cells serve as a predictive and therapeutic biomarker for anti-PD-1 treatment. Sequencing of anti-PD-1 and vaccine is crucial for successful therapy.

Antibodies induced by enterotoxigenic Escherichia coli (ETEC) adhesin major structural subunit and minor tip adhesin subunit equivalently inhibit bacteria adherence in vitro.

Antibodies that block the adherence of enterotoxigenic Escherichia coli (ETEC) to host intestinal epithelial cells are protective. Multiepitope-fusion-antigens (MEFAs) carrying epitopes of ETEC adhesin major subunits or tip minor subunits induced antibodies against ETEC adherence. Adherence inhibition effectiveness of antibodies induced by major subunit epitopes versus minor tip subunit epitopes, however, has not been comparatively characterized. In this study, we immunized mice with a major subunit MEFA or a tip MEFA, evaluated MEFA anti-adhesin immunogenicity, and examined induced-antibodies against bacteria in vitro adherence or in vivo colonization in mice. Mice subcutaneously immunized with major subunit MEFA CFA/I/II/IV or tip MEFA showed no adverse effects and developed strong antigen-specific antibody responses. Data showed that antibodies derived from two MEFAs were equally effective against adherence of the bacteria expressing CS1, CS2, CS3, CS4/CS6, CS5/CS6, or CS6 adhesin in vitro. Subsequently, we immunized mice with CFA/I fimbriae, major subunit CfaB, or minor tip adhesin subunit CfaE. We found that antibodies induced by CFA/I, CfaB and CfaE equally inhibited in vitro adherence of ETEC strain H10407. Furthermore, we immunized mice with CFA/I fimbriae, CfaB, or CfaE, and then challenged the mice with H10407. Data showed that although not significantly, fewer H10407 bacteria colonized the immunized mice. These results suggest that ETEC adhesin major subunit and minor tip subunit should be equally effective in inducing neutralizing anti-adhesin antibodies, and that major subunit CFA/I/II/IV MEFA or tip MEFA, perhaps combined with toxoid fusion 3xSTaN12S-mnLTR192G/L211A, can be used for development of broadly protective vaccines against ETEC diarrhea.

Immunogenicity characterization of genetically fused or chemically conjugated heat-stable toxin toxoids of enterotoxigenic Escherichia coli in mice and pigs.

Enterotoxigenic Escherichia coli (ETEC) producing type Ib heat-stable toxin (STa) are a main cause of children's diarrhea and travelers' diarrhea, thus STa needs to be targeted in ETEC vaccine development. However, because this 19-amino acid STa is poorly immunogenic, attempts to genetically fuse or chemically couple it to carrier proteins have been made to enhance STa immunogenicity. In this study, we selected one genetic fusion and one chemical conjugate to comparatively evaluate STa immunogenicity. The genetic fusion is 3xSTaN12S-mnLTR192G/L211A carrying three toxoid (STaN12S) genetically fused to a double mutant LT monomer (mnLTR192G/L211A); the chemical conjugate is BSA-STaA14T, which has toxoid STaA14T chemically coupled to bovine serum albumin (BSA). We immunized mice with the STa toxoid fusion and chemical conjugates, and examined antibody responses. Furthermore, we immunized pigs and evaluated derived antibodies for efficacy to passively provide protection against ETEC diarrhea using a piglet model. Data showed that mice subcutaneously immunized with BSA-STaA14T or 3xSTaN12S-mnLTR192G/L211A developed a strong anti-STa antibody, and the induced antibodies exhibited equivalent toxin-neutralizing activities. Pigs immunized with 3xSTaN12S-mnLTR192G/L211A or BSA-STaA14T developed similar levels of anti-STa antibodies; piglets with passively acquired antibodies induced by the genetic fusion appeared better protected against STa + ETEC. Results from the current study indicate that the fusion and conjugate approaches are viable options for facilitating STa immunogenicity and developing ETEC vaccines.

Co-administered Tag-Less Toxoid Fusion 3xSTaN12S-mnLTR192G/L211A and CFA/I/II/IV MEFA (Multiepitope Fusion Antigen) Induce Neutralizing Antibodies to 7 Adhesins (CFA/I, CS1-CS6) and Both Enterotoxins (LT, STa) of Enterotoxigenic Escherichia coli (ETEC).

Enterotoxigenic Escherichia coli (ETEC) bacteria remain a leading cause of children's diarrhea and travelers' diarrhea. Vaccines that induce antibodies to block ETEC bacterial adherence and to neutralize toxin enterotoxicity can be effective against ETEC-associated diarrhea. Recent studies showed that 6xHis-tagged CFA/I/II/IV multiepitope fusion antigen (MEFA) induced broad-spectrum antibodies to inhibit adherence of the seven most important ETEC adhesins (CFA/I, CS1 to CS6) (Ruan et al., 2014a) and 6xHis-tagged toxoid fusion antigen 3xSTaN12S-mnLTR192G/L211A (previously named as 3xSTaN12S-dmLT) elicited antibodies to neutralize both heat-labile toxin (LT) and heat-stable toxin (STa) produced by ETEC strains (Ruan et al., 2014b). In this study, we constructed two new genes to express tag-less toxoid fusion 3xSTaN12S-mnLTR192G/L211A and tag-less CFA/I/II/IV MEFA and then examined immunogenicity of each tag-less protein in mouse immunization. We further combined two tag-less proteins and investigated antigen co-administration in mice. Data showed that mice immunized with tag-less 3xSTaN12S-mnLTR192G/L211A or tag-less CFA/I/II/IV MEFA developed antigen-specific IgG antibody responses, and mice co-administered with two tag-less proteins induced neutralizing antibodies against seven adhesins and both toxins. These results indicated tag-less toxoid fusion 3xSTaN12S-mnLTR192G/L211A and tag-less CFA/I/II/IV MEFA administered individually or combined induced neutralizing antitoxin and/or anti-adhesin antibodies, and suggested the potential application of two tag-less proteins for ETEC vaccine development.

Enterotoxigenic Escherichia coli Adhesin-Toxoid Multiepitope Fusion Antigen CFA/I/II/IV-3xSTaN12S-mnLTG192G/L211A-Derived Antibodies Inhibit Adherence of Seven Adhesins, Neutralize Enterotoxicity of LT and STa Toxins, and Protect Piglets against Diarrhea.

Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children's diarrhea and travelers' diarrhea. Vaccines inducing antibodies to broadly inhibit bacterial adherence and to neutralize toxin enterotoxicity are expected to be effective against ETEC-associated diarrhea. 6×His-tagged adhesin-toxoid fusion proteins were shown to induce neutralizing antibodies to several adhesins and LT and STa toxins (X. Ruan, D. A. Sack, W. Zhang, PLoS One 10:e0121623, 2015, https://doi.org/10.1371/journal.pone.0121623). However, antibodies derived from His-tagged CFA/I/II/IV-2xSTaA14Q-dmLT or CFA/I/II/IV-2xSTaN12S-dmLT protein were less effective in neutralizing STa enterotoxicity and were not evaluated in vivo for efficacy against ETEC diarrhea. Additionally, His-tagged proteins are considered less desirable for human vaccines. In this study, we produced a tagless adhesin-toxoid MEFA (multiepitope fusion antigen) protein, enhanced anti-STa immunogenicity by including a third copy of STa toxoid STaN12S, and examined antigen immunogenicity in a murine model. Moreover, we immunized pregnant pigs with the tagless adhesin-toxoid MEFA protein and evaluated passive antibody protection against STa+ or LT+ ETEC infection in a pig challenge model. Results showed that tagless adhesin-toxoid MEFA CFA/I/II/IV-3xSTaN12S-mnLTR192G/L211A induced broad antiadhesin and antitoxin antibody responses in the intraperitoneally immunized mice and the intramuscularly immunized pigs. Mouse and pig serum antibodies significantly inhibited adherence of seven colonization factor antigen (CFA) adhesins (CFA/I and CS1 to CS6) and effectively neutralized both toxins. More importantly, suckling piglets born to the immunized mothers acquired antibodies and were protected against STa+ ETEC and LT+ ETEC diarrhea. These results indicated that tagless CFA/I/II/IV-3xSTaN12S-mnLTR192G/L211A induced broadly protective antiadhesin and antitoxin antibodies and demonstrate that this adhesin-toxoid MEFA is a potential antigen for developing broadly protective ETEC vaccines.

MEFA (multiepitope fusion antigen)-Novel Technology for Structural Vaccinology, Proof from Computational and Empirical Immunogenicity Characterization of an Enterotoxigenic Escherichia coli (ETEC) Adhesin MEFA.

Vaccine development often encounters the challenge of virulence heterogeneity. Enterotoxigenic Escherichia coli (ETEC) bacteria producing immunologically heterogeneous virulence factors are a leading cause of children's diarrhea and travelers' diarrhea. Currently, we do not have licensed vaccines against ETEC bacteria. While conventional methods continue to make progress but encounter challenge, new computational and structure-based approaches are explored to accelerate ETEC vaccine development. In this study, we applied a structural vaccinology concept to construct a structure-based multiepitope fusion antigen (MEFA) to carry representing epitopes of the seven most important ETEC adhesins [CFA/I, CFA/II (CS1-CS3), CFA/IV (CS4-CS6)], simulated antigenic structure of the CFA/I/II/IV MEFA with computational atomistic modeling and simulation, characterized immunogenicity in mouse immunization, and examined the potential of structure-informed vaccine design for ETEC vaccine development. A tag-less recombinant MEFA protein (CFA/I/II/IV MEFA) was effectively expressed and extracted. Molecular dynamics simulations indicated that this MEFA immunogen maintained a stable secondary structure and presented epitopes on the protein surface. Empirical data showed that mice immunized with the tagless CFA/I/II/IV MEFA developed strong antigen-specific antibody responses, and mouse serum antibodies significantly inhibited in vitro adherence of bacteria expressing these seven adhesins. These results revealed congruence of antigen immunogenicity between computational simulation and empirical mouse immunization and indicated this tag-less CFA/I/II/IV MEFA potentially an antigen for a broadly protective ETEC vaccine, suggesting a potential application of MEFA-based structural vaccinology for vaccine design against ETEC and likely other pathogens.

Antibodies derived from a toxoid MEFA (multiepitope fusion antigen) show neutralizing activities against heat-labile toxin (LT), heat-stable toxins (STa, STb), and Shiga toxin 2e (Stx2e) of porcine enterotoxigenic Escherichia coli (ETEC).

Enterotoxigenic Escherichia coli (ETEC) strains are the main cause of diarrhea in pigs. Pig diarrhea especially post-weaning diarrhea remains one of the most important swine diseases. ETEC bacterial fimbriae including K88, F18, 987P, K99 and F41 promote bacterial attachment to intestinal epithelial cells and facilitate ETEC colonization in pig small intestine. ETEC enterotoxins including heat-labile toxin (LT) and heat-stable toxins type Ia (porcine-type STa) and type II (STb) stimulate fluid hyper-secretion, leading to watery diarrhea. Blocking bacteria colonization and/or neutralizing enterotoxicity of ETEC toxins are considered effective prevention against ETEC diarrhea. In this study, we applied the MEFA (multiepitope fusion antigen) strategy to create toxoid MEFAs that carried antigenic elements of ETEC toxins, and examined for broad antitoxin immunogenicity in a murine model. By embedding STa toxoid STaP12F (NTFYCCELCCNFACAGCY), a STb epitope (KKDLCEHY), and an epitope of Stx2e A subunit (QSYVSSLN) into the A1 peptide of a monomeric LT toxoid (LTR192G), two toxoid MEFAs, 'LTR192G-STb-Stx2e-STaP12F' and 'LTR192G-STb-Stx2e-3xSTaP12F' which carried three copies of STaP12F, were constructed. Mice intraperitoneally immunized with each toxoid MEFA developed IgG antibodies to all four toxins. Induced antibodies showed in vitro neutralizing activities against LT, STa, STb and Stx2e toxins. Moreover, suckling piglets born by a gilt immunized with 'LTR192G-STb-Stx2e-3xSTaP12F' were protected when challenged with ETEC strains, whereas piglets born by a control gilt developed diarrhea. Results from this study showed that the toxoid MEFA induced broadly antitoxin antibodies, and suggested potential application of the toxoid MEFA for developing a broad-spectrum vaccine against ETEC diarrhea in pigs.

Passive antibodies derived from intramuscularly immunized toxoid fusion 3xSTaN12S-dmLT protect against STa+ enterotoxigenic Escherichia coli (ETEC) diarrhea in a pig model.

Enterotoxigenic Escherichia coli (ETEC) strains are among the most common causes of children's diarrhea and travelers' diarrhea. Developing effective vaccines against ETEC associated diarrhea becomes a top priority. ETEC heat-labile toxin (LT) and heat-stable toxin (STa) toxoid fusion 3xSTaN12S-dmLT was demonstrated recently to induce neutralizing antitoxin antibodies in intraperitoneally or subcutaneously immunized mice. However, whether antibodies derived from this toxoid fusion are protective against ETEC diarrhea has not been examined. In this study, we intramuscularly immunized pregnant gilts with toxoid fusion 3xSTaN12S-dmLT, challenged suckling piglets with a STa-positive ETEC strain, and assessed protective efficacy of passive acquire antitoxin antibodies against ETEC diarrhea. Data showed all three immunized gilts developed anti-STa IgG and IgA antibodies, and piglets born to the immunized dams acquired anti-STa and anti-LT antibodies. When challenged with a STa+ ETEC strain, none of the piglets born to the immunized dams developed watery diarrhea, with 20 piglets remained normal and the other 8 piglets developed mild diarrhea indicated with stained butt. In contrast, the control dams and born piglets had no anti-STa or anti-LT antibodies detected, and 26 out 32 piglets developed watery diarrhea after challenge of the STa+ ETEC strain. These results indicated that passive acquired anti-STa antibodies are protective against ETEC diarrhea, and suggested potential application of toxoid fusion 3xSTaN12S-dmLT in ETEC vaccine development.

Enterotoxigenic Escherichia coli heat-stable toxin and heat-labile toxin toxoid fusion 3xSTaN12S-dmLT induces neutralizing anti-STa antibodies in subcutaneously immunized mice.

Enterotoxigenic Escherichia coli (ETEC) bacteria producing heat-stable toxin (STa) and/or heat-labile toxin (LT) are among top causes of children's diarrhea and travelers' diarrhea. Currently no vaccines are available for ETEC associated diarrhea. A major challenge in developing ETEC vaccines is the inability to stimulate protective antibodies against the key STa toxin that is potently toxic and also poorly immunogenic. A recent study suggested toxoid fusion 3xSTaN12S-dmLT, which consists of a monomer LT toxoid (LTR192G/L211A) and three copies of STa toxoid STaN12S, may represent an optimal immunogen inducing neutralizing antibodies against STa toxin [IAI 2014, 82(5):1823-32]. In this study, we immunized mice with this fusion protein following a different parenteral route and using different adjuvants to further characterize immunogenicity of this toxoid fusion. Data from this study showed that 3xSTaN12S-dmLT toxoid fusion induced neutralizing anti-STa antibodies in the mice following subcutaneous immunization, as effectively as in the mice under intraperitoneal route. Data also indicated that double mutant LT (dmLT) can be an effective adjuvant for this toxoid fusion in mice subcutaneous immunization. Results from this study affirmed that toxoid fusion 3xSTaN12S-dmLT induces neutralizing antibodies against STa toxin, suggesting this toxoid fusion is potentially a promising immunogen for ETEC vaccine development.

Antibodies derived from an enterotoxigenic Escherichia coli (ETEC) adhesin tip MEFA (multiepitope fusion antigen) against adherence of nine ETEC adhesins: CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS21 and EtpA.

Diarrhea continues to be a leading cause of death in children younger than 5 years in developing countries. Enterotoxigenic Escherichia coli (ETEC) is a leading bacterial cause of children's diarrhea and travelers' diarrhea. ETEC bacteria initiate diarrheal disease by attaching to host receptors at epithelial cells and colonizing in small intestine. Therefore, preventing ETEC attachment has been considered the first line of defense against ETEC diarrhea. However, developing vaccines effectively against ETEC bacterial attachment encounters challenge because ETEC strains produce over 23 immunologically heterogeneous adhesins. In this study, we applied MEFA (multiepitope fusion antigen) approach to integrate epitopes from adhesin tips or adhesive subunits of CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS21 and EtpA adhesins and to construct an adhesin tip MEFA peptide. We then examined immunogenicity of this tip MEFA in mouse immunization, and assessed potential application of this tip MEFA for ETEC vaccine development. Data showed that mice intraperitoneally immunized with this adhesin tip MEFA developed IgG antibody responses to all nine ETEC adhesins. Moreover, ETEC and E. coli bacteria expressing these nine adhesins, after incubation with serum of the immunized mice, exhibited significant reduction in attachment to Caco-2 cells. These results indicated that anti-adhesin antibodies induced by this adhesin tip MEFA blocked adherence of the most important ETEC adhesins, suggesting this multivalent tip MEFA may be useful for developing a broadly protective anti-adhesin vaccine against ETEC diarrhea.

Inhibition of Salmonella-induced apoptosis as a marker of the protective efficacy of virulence gene-deleted live attenuated vaccine.

Vaccination is one of the best protection strategies against Salmonella infection in humans and chickens. Salmonella bacteria must induce apoptosis prior to initiating infection, pathogenesis and evasion of host immune responses. In this study, we evaluated the efficacy of vaccinating chickens against Salmonella Enteritidis (SE) using a vaccine candidate strain (JOL919), constructed by deleting the lon and cpxR genes from a wild-type SE using an allelic exchange method. In present study day old chickens were inoculated with 1×10(7)cfu (colony forming unit) of JOL919 per os. We measured cell-mediated immunity, protective efficacy and extent of apoptosis induction in splenocytes. Seven days post-immunization, the number of CD3+CD4+ and CD3+ CD8+ T cells was significantly higher in the immunized group compared to the control group, indicating a significant augmentation of systemic immune response. The internal organs of chickens immunized with JOL919 had a significantly lower challenge-strain recovery, indicating effective protection and clearance of the challenge strain. Post-challenge, the number of apoptotic cells in the immunized group was significantly lower than in the control group. Additionally, AV/PI (Annexin V/propidium iodide) staining was performed to differentiate between apoptotic cells and necrotic cells, which corroborated TUNEL-assay (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling) results. The proportions of AV+/PI- and AV+/PI+ cells, which represent the proportions of early apoptotic and late apoptotic/early necrotic cells present, respectively, were significantly lower in the immunized group. Our findings suggest that the apoptotic splenocytes in immunized chickens significantly decreased in number, which occurred concomitantly with a significant rise in systemic immune response and bacterial clearance. This suggests that inhibition of apoptosis may be a marker of protection efficacy in immunized chickens.

Comparison of a live attenuated Salmonella Enteritidis vaccine candidate secreting Escherichia coli heat-labile enterotoxin B subunit with a commercial vaccine for efficacy of protection against internal egg contamination by Salmonella in hens.

This study compared a new live attenuated Salmonella Enteritidis vaccine candidate secreting Escherichia coli heat-labile enterotoxin B subunit (SE-LTB) with a commercial Salmonella Enteritidis (SE) vaccine for efficacy of protection against SE infection in laying hens. Chickens were divided into 3 groups of 20 each. Group A chickens were inoculated orally with phosphate-buffered saline and served as controls, group B chickens were inoculated orally with the vaccine candidate, and group C chickens were inoculated intramuscularly with a commercial vaccine, the primary inoculation in groups B and C being at 10 wk of age and the booster at 16 wk. Groups B and C showed significantly higher titers of plasma immunoglobulin G, intestinal secretory immunoglobulin A, and egg yolk immunoglobulin Y antibodies compared with the control group, and both vaccinated groups showed a significantly elevated cellular immune response. After virulent challenge, group B had significantly lower production of thin-shelled and/or malformed eggs and a significantly lower rate of SE contamination of eggs compared with the control group. Furthermore, the challenge strain was detected significantly less in all of the examined organs of group B compared with the control group. Group C had lower gross lesion scores only in the spleen and had lower bacterial counts only in the spleen, ceca, and ovary. These findings indicate that vaccination with the SE-LTB vaccine candidate can efficiently reduce internal egg and internal organ contamination by Salmonella and has advantages over the commercial vaccine.

La présente étude visait à comparer un nouveau vaccin candidat vivant atténué de Salmonella Enteritidis secrétant la sous-unité B de l'entérotoxine thermolabile d'Escherichia coli (SE-LTB) avec un vaccin commercial contre Salmonella Enteritidis (SE) pour leur efficacité de protection contre une infection par SE chez des poules pondeuses. Les poulets ont été séparés en trois groupes de 20 animaux chacun. Les poulets du groupe A furent inoculés oralement avec de la saline tamponnée et ont servi de témoins, les poulets du groupe B furent inoculés oralement avec le vaccin candidat, et les poules du groupe C furent inoculés par voie intramusculaire avec le vaccin commercial, une première immunisation dans les groupes B et C étant faite à l'âge de 10 semaines et un rappel à 16 semaines. Comparativement aux oiseaux du groupe témoin, ceux des groupes B et C avaient des titres significativement plus élevés d'immunoglobulines G plasmatiques, d'immunoglobulines A sécrétoires intestinales et d'immunoglobulines Y du jaune d'oeuf, et les animaux des deux groupes immunisés présentaient également une réponse immunitaire cellulaire supérieure. Suite à un challenge avec une souche virulente, les oiseaux du groupe B avaient une production significativement plus faible d'oeufs à coquille mince ou d'oeufs malformés, et un taux significativement plus faible d'oeufs contaminés par SE comparativement au groupe témoin. De plus, la souche servant au challenge était détectée de manière significativement moindre dans tous les organes examinés des animaux du groupe B comparativement au groupe témoin. Les animaux du groupe C avaient des pointages de lésions macroscopiques inférieurs uniquement pour la rate et avaient des dénombrements bactériens plus bas seulement dans la rate, les caeca, et les ovaires. Ces données indiquent que la vaccination avec le vaccin candidat SE-LTB peut réduire de manière efficace la contamination des organes internes et de l'intérieur des oeufs par Salmonella et possède certains avantages par rapport au vaccin commercial.(Traduit par Docteur Serge Messier).

Cross-protection against Salmonella Typhimurium infection conferred by a live attenuated Salmonella Enteritidis vaccine.

In this study, a genetically engineered live attenuated Salmonella Enteritidis (SE) vaccine was evaluated for its ability to protect against Salmonella Typhimurium (ST) infection in chickens. The birds were orally primed with the vaccine on the 1st day of life and given an oral booster at 5 wk of age. Control birds were orally inoculated with phosphate-buffered saline. Both groups of birds were orally challenged with a virulent ST strain at 9 wk of age. Compared with the control chickens, the vaccinated chickens had significantly higher levels of systemic IgG and mucosal IgA against specific ST antigens and a significantly greater lymphoproliferative response to ST antigens. The excretion of ST into the feces was significantly lower in the vaccinated group than in the control group on days 9 and 13 d after challenge. In addition, the vaccinated group had significantly fewer pronounced gross lesions in the liver and spleen and lower bacterial counts in the internal organs than the control group after challenge. These data indicate that genetically engineered live attenuated SE may induce humoral and cellular immune responses against ST antigens and may confer protection against virulent ST challenge.

Dans la présente étude on évalua la capacité d'un vaccin vivant atténué génétiquement modifié de Salmonella Enteritidis (SE) à protéger contre une infection par Salmonella Typhimurium (ST) chez le poulet. Les poulets furent inoculés oralement avec le vaccin à leur premier jour de vie et reçurent un rappel à 5 semaines d'âge. Les oiseaux témoins reçurent par voie orale de la saline tamponnée. Les deux groupes furent challengés par voie orale avec une souche virulente de ST à 9 sem d'âge. Comparativement aux oiseaux témoins, les poulets vaccinés avaient des taux significativement plus élevés d'IgG systémiques et d'IgA locaux contre des antigènes spécifiques de ST et une réponse lympho-proliférative significativement plus importante aux antigènes de ST. L'excrétion de ST dans les fèces était significativement moindre dans le groupe vacciné comparativement au groupe témoin aux jours 9 et 13 après le challenge. De plus, après le challenge le groupe vacciné avait significativement moins de lésions macroscopiques marquées dans le foie et la rate et des dénombrements bactériens moindres dans les organes internes que le groupe témoin. Ces résultats indiquent que le vaccin SE vivant génétiquement modifié peut induire une réponse immunitaire humorale et cellulaire contre des antigènes de ST et peut conférer une protection contre un challenge avec une souche de ST virulente.(Traduit par Docteur Serge Messier).

Comparative evaluation of safety and efficacy of a live Salmonella gallinarum vaccine candidate secreting an adjuvant protein with SG9R in chickens.

We previously reported JOL916, a live attenuated Salmonella gallinarum (SG), as a vaccine candidate for protection from fowl typhoid (FT). In the present study, we evaluated JOL1355, an SG that secretes heat-labile enterotoxin B subunit protein, for safety, immunogenicity, and protective efficacy against FT. In a single intramuscular inoculation, live attenuated SG (JOL916) and commercial live attenuated SG9R immunized groups showed gross lesions and bacterial persistence up to 21 days post-immunization. However, the JOL1355 immunized group showed gross lesions and bacterial persistence up to only 3 and 7 days post-immunization, respectively. In addition, several birds in the JOL916 and SG9R immunized group shown clinical signs after immunization, while JOL1355 immunized birds did not show any adverse effects. In a subsequent study, birds were primed and boosted at 4 and 8 weeks of age, respectively, and compared with control birds inoculated with sterile phosphate-buffered saline. The immunized groups B (JOL916), C (SG9R), and D (JOL1355) exhibited significantly higher humoral and cellular immune responses compared to those in the unimmunized control group A. In addition, the birds of each group were challenged with virulent SG at 11 weeks of age, and significantly increased survival rates were observed in all immunized groups compared with the control group. These results indicated that JOL1355 was able to efficiently induce an acquired immune response to protect birds after challenge, and may be safer than JOL916 and the commercial vaccine SG9R in chickens.

Construction of a recombinant-attenuated Salmonella Enteritidis strain secreting Escherichia coli heat-labile enterotoxin B subunit protein and its immunogenicity and protection efficacy against salmonellosis in chickens.

A live attenuated Salmonella Enteritidis (SE) strain secreting Escherichia coli heat-labile enterotoxin B subunit (LTB) protein was constructed as a new vaccine candidate. The comparative effect of this vaccine candidate was evaluated with a previously reported SE vaccine, JOL919. An asd+, p15A ori plasmid containing eltB-encoding LTB was introduced into a ΔlonΔcpxRΔasd SE strain, and designated as JOL1364. In a single immunization experiment, group A chickens were orally inoculated with phosphate-buffered saline as a control, group B chickens were orally immunized with JOL919, and group C chickens were orally immunized with JOL1364. The immunized groups B and C showed significantly higher systemic, mucosal and cellular immune responses as compared to those of the control group. In addition, the immunized group C showed significantly higher mucosal and cellular immune responses as compared to those of the immunized group B at the 1st week post-immunization. In the examination of protection efficacy, the immunized groups B and C showed lower gross lesion scores in the liver and spleen, and lower bacterial counts of SE challenge strain in the liver, spleen, and caeca as compared to those of the control group. The number of SE-positive birds was significantly lower in the immunized group C as compared to that of the control group at the 14th day post-challenge. In addition, the number of birds carrying the challenge strain in the caeca was significantly lower in the immunized group C than those in the immunized group B and control group at the 7th and 14th day post-challenge. These results indicate that immunization with the JOL1364 vaccine candidate can induce higher mucosal and cellular immune responses than those of the JOL919 for efficient protection against salmonellosis.