Protection against an intranasal challenge by vaccines formulated with native and recombinant preparations of the Chlamydia trachomatis major outer membrane protein

To compare the ability of a native and a recombinant preparation of the major outer membrane protein of Chlamydia trachomatis mouse pneumonitis (MoPn; Ct-nMOMP and Ct-rMOMP) to protect against an intranasal (i.n.) challenge, BALB/c mice were vaccinated by the intramuscular (i.m.) and subcutaneous (s.c.) routes using CpG-1826 and Montanide ISA 720 as adjuvants. Animals inoculated i.n. with live elementary bodies (EB) of Chlamydia served as a positive control. Negative control groups were immunized with either Neisseria gonorrhoeae recombinant porin B (Ng-rPorB) or with minimal essential medium (MEM-0). Mice immunized with Ct-rMOMP, Ct-nMOMP and EB developed a strong immune response as shown by high levels of Chlamydia specific antibodies in serum and a strong T-cell lymphoproliferative response. Following the i.n. challenge with 10⁴ inclusion forming units (IFU) of C. trachomatis, mice immunized with Ct-nMOMP or Ct-rMOMP lost significantly less weight than the negative control animals immunized with Ng-rPorB or MEM-0 (P < 0.05). However, mice vaccinated with the Ct-nMOMP lost less weight than those immunized with the Ct-rMOMP (P < 0.05). Mice were euthanized at 10 days following the challenge, their lungs weighed and the number of IFU of Chlamydia determined. Based on the lung weight and number of IFU recovered, significant protection was observed in the groups of mice immunized with both Ct-nMOMP and the Ct-rMOMP (P < 0.05). Nevertheless, significantly better protection was achieved with the Ct-nMOMP in comparison with the Ct-rMOMP (P < 0.05). In conclusion, vaccination with a preparation of the nMOMP elicited a more robust protection than immunization with rMOMP, suggesting that the conformational structure of MOMP is critical for inducing strong protection.     

Induction of protective immunity by vaccination against Chlamydia trachomatis using the major outer membrane protein adjuvanted with CpG oligodeoxynucleotide coupled to the nontoxic B subunit of cholera toxin

The present study was undertaken to test the efficacy of immunization with the native major outer membrane protein (nMOMP) of Chlamydia trachomatis mouse pneumonitis (MoPn) serovar in combination with a novel immunostimulatory adjuvant consisting of CpG oligodeoxynucleotide (ODN) linked to the nontoxic B subunit of cholera toxin (CTB-CpG) to elicit a protective immune response to C. trachomatis. High levels of Chlamydia-specific IgG antibodies were detected in the sera from BALB/c mice immunized intramuscularly and subcutaneously (i.m. + s.c.) with the nMOMP/CTB-CpG vaccine or with nMOMP adjuvanted with a mixture of CT and CpG ODN (CT + CpG). Further, these immunization schemes gave rise to significant T-cell-mediated Chlamydia-specific immune responses. No Chlamydia-specific humoral or cell-mediated immune responses were detected in the control mice vaccinated with ovalbumin together with either CTB-CpG or CT + CpG. Following an intranasal challenge with C. trachomatis the groups of mice immunized with nMOMP plus CTB-CpG, CT + CpG or live C. trachomatis were found to be protected based on their change in body weight and lung weight as well as number of inclusion forming unit recovered from the lungs, as compared with control groups immunized with ovalbumin plus either adjuvants. Interestingly, IFN-γ-producing CD4⁺, but not CD8⁺, T-cells showed a significant correlation with the outcomes of the challenge. In conclusion, nMOMP in combination with the novel adjuvant CTB-CpG elicited a significant antigen-specific antibody and cell-mediated immune responses as well as protection against a pulmonary challenge with C. trachomatis.     

Immunogenicity of a vaccine formulated with the Chlamydia trachomatis serovar F, native major outer membrane protein in a nonhuman primate model

To determine the ability of a vaccine formulated with the genital Chlamydia trachomatis, serovar F, native major outer membrane protein (Ct-F-nMOMP), to induce systemic and mucosal immune responses, rhesus macaques (Macaca mulatta) were immunized three times by the intramuscular (i.m.) and subcutaneous (s.c.) routes using CpG-2395 and Montanide ISA 720 VG, as adjuvants. As controls, another group of M. mulatta was immunized with ovalbumin instead of Ct-F-nMOMP using the same formulation and routes. High levels of Chlamydia-specific IgG and IgA antibodies were detected in plasma, vaginal washes, tears, saliva, and stools from the Ct-F-nMOMP immunized animals. Also, high neutralizing antibody titers were detected in the plasma from these animals. Monkeys immunized with ovalbumin had no detectable Chlamydia-specific antibodies. Furthermore, as measured by a lymphoproliferative assay, significant Chlamydia-specific cell-mediated immune responses were detected in the peripheral blood mononuclear cells (PBMC) from the rhesus macaques vaccinated with Ct-F-nMOMP when compared with the animals immunized with ovalbumin. In addition, the levels of two Th1 cytokines, IFN-γ and TNF-α, were significantly higher in the animals immunized with Ct-F-nMOMP when compared with those from the monkeys immunized with ovalbumin. To our knowledge, this is the first time that mucosal and systemic immune responses have been investigated in a nonhuman primate model using a subunit vaccine from a human genital C. trachomatis serovar.     

Identification of immunodominant antigens of Chlamydia trachomatis using proteome microarrays

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen in the world. In order to control this infection there is an urgent need to formulate a vaccine. Identification of protective antigens is required to implement a subunit vaccine. To identify potential antigen vaccine candidates, three strains of mice, BALB/c, C3H/HeN and C57BL/6, were inoculated with live and inactivated C. trachomatis mouse pneumonitis (MoPn) by different routes of immunization. Using a protein microarray, serum samples collected after immunization were tested for the presence of antibodies against specific chlamydial antigens. A total of 225 open reading frames (ORF) of the C. trachomatis genome were cloned, expressed, and printed in the microarray. Using this protein microarray, a total of seven C. trachomatis dominant antigens were identified (TC0052, TC0189, TC0582, TC0660, TC0726, TC0816 and, TC0828) as recognized by IgG antibodies from all three strains of animals after immunization. In addition, the microarray was probed to determine if the antibody response exhibited a Th1 or Th2 bias. Animals immunized with live organisms mounted a predominant Th1 response against most of the chlamydial antigens while mice immunized with inactivated Chlamydia mounted a Th2-biased response. In conclusion, using a high throughput protein microarray we have identified a set of novel proteins that can be tested for their ability to protect against a chlamydial infection.     

Evaluation of a multisubunit recombinant polymorphic membrane protein and major outer membrane protein T cell vaccine against Chlamydia muridarum genital infection in three strains of mice

An efficacious vaccine is needed to control Chlamydia trachomatis infection. In the murine model of Chlamydia muridarum genital infection, multifunctional mucosal CD4 T cells are the foundation for protective immunity, with antibody playing a secondary role. We previously identified four Chlamydia outer membrane proteins (PmpE, PmpF, PmpG and PmpH) as CD4 T cell vaccine candidates using a dendritic cell-based immunoproteomic approach. We also demonstrated that these four polymorphic membrane proteins (Pmps) individually conferred protection as measured by accelerated clearance of Chlamydia infection in the C57BL/6 murine genital tract model. The major outer membrane protein, MOMP is also a well-studied protective vaccine antigen in this system. In the current study, we tested immunogenicity and protection of a multisubunit recombinant protein vaccine consisting of the four Pmps (PmpEFGH) with or without the major outer membrane protein (MOMP) formulated with a Th1 polarizing adjuvant in C57BL/6, Balb/c and C3H mice. We found that C57BL/6 mice vaccinated with PmpEFGH + MOMP elicited more robust cellular immune responses than mice immunized with individual protein antigens. Pmps elicited more variable cellular immune responses than MOMP among the three strains of mice. The combination vaccine accelerated clearance in the three strains of mice although at different rates. We conclude that the recombinant outer membrane protein combination constitutes a promising first generation Chlamydia vaccine construct that should provide broad immunogenicity in an outbred population.     

Protection of pigs against Chlamydia trachomatis challenge by administration of a MOMP-based DNA vaccine in the vaginal mucosa

Plasmid DNA (pWRG7079::MOMP) expressing the major outer membrane protein of a human Chlamydia trachomatis serovar E strain was tested for the ability to induce an immune response and protect against experimental genital infection with the same serovar. The vaccine was tested in pigs, as they are genetically and physiologically related to humans and suitable for studying C. trachomatis infection of the genital system. To increase the immune response, GM-CSF, LTA and B and CpG motives were used as adjuvants. GM-CSF was administered seven days before immunization, while the other adjuvants were administered together with the vaccine. Ten pigs were randomly divided into two groups. One group received an intravaginal primo-vaccination and a booster of 500 μg pWRG7079::MOMP, while the other group received the placebo vaccine pWRG7079. All animals were challenged with 10⁸ TCID₅₀ of C. trachomatis serovar E. Pigs immunized with the DNA vaccine showed significantly less macroscopic lesions, vaginal excretion and chlamydial replication in the genital tract, as compared to placebo-vaccinated controls. However, infection could not be completely cleared.     

Characterization of protective immune responses promoted by human antigen targets in a urogenital Chlamydia trachomatis mouse model

A vaccine against genital tract infections caused by Chlamydia trachomatis is urgently needed. We have previously identified a number of immunodominant human T- and/or B-cell antigen targets in patients with a C. trachomatis infection. Herein we use a urogenital C. trachomatis mouse model to investigate the protective efficacy of these antigens. C3H/HeN mice were immunized with recombinant antigens formulated in the adjuvant CAF01. Immunity post vaccination was analyzed and the protective efficacy against vaginal challenge with C. trachomatis was monitored by vaginal swabbing. All antigens elicited a significant immune response when administered in CAF01 but the balance between CMI and humoral responses differed markedly for the different antigens. Six (CT443, CT043, CT858, CT610, CT004 and CT681) antigens were found to be protective. We demonstrated by T-cell depletion studies that the protection promoted by the two antigens CT043 and CT004 was mediated by CD4⁺ T-cells. Both antigens are frequently recognized by T-cells during a natural Chlamydia infection in humans and if included in a future multi-component Chlamydia vaccine probably would operate mainly through the induction of a CMI response.     

Enhancement of the protective efficacy of a Chlamydia trachomatis recombinant vaccine by combining systemic and mucosal routes for immunization

Chlamydia trachomatis causes respiratory and sexually transmitted infections. Here, we tested a vaccine formulated with the recombinant major outer membrane protein from C. trachomatis mouse pneumonitis (CT-MoPn) for its ability to protect mice against an intranasal (i.n.) challenge. The adjuvants CpG and Montanide were used for systemic routes, intramuscular (i.m.) and subcutaneous (s.c.), and cholera toxin for mucosal routes, sublingual (s.l.) and colonic (c.l.). Mucosal immunizations were performed either alone or in combination with systemic routes. Mice inoculated i.n. with 10⁴ inclusion-forming units (IFU) of CT-MoPn served as a positive control and the Neisseria gonorrhoeae recombinant porin B (Ng-rPorB) as the negative antigen control. Immunized animals were challenged i.n. with 10⁴ IFU of CT-MoPn. Following immunization the combination groups showed high chlamydial serum IgG titers (s.l. + i.m. + s.c. 25,600; c.l + i.m. + s.c. 102,400) and the IgG2a/IgG1 ratios indicated a Th1 response. Following the i.n. challenge the s.l. + i.m. + s.c. group showed the best protection as demonstrated by an increase in body weight of 0.3% over the 10 day course of infection. A statistically significant difference was found when compared with the Ng-rPorB immunized animals that had lost 20% of their original body weight (P < 0.05). In addition, the repeated measures ANOVA test showed significant difference in body weight change for the combined immunized groups vs their mucosal counterparts and also the systemic immunized group. A statistically significant difference (P < 0.05) was also observed in the number of IFUs recovered from the lungs when the s.l. + i.m. + s.c. (2.8 × 10⁶) and c.l. + i.m. + s.c. (3.4 × 10⁶) groups were compared to their respective mucosal only groups (s.l.: 61.9 × 10⁶ and c.l: 136.2 × 10⁶) and the control Ng-rPorB immunized mice (198.2 × 10⁶) (P < 0.05). In conclusion, a combined systemic plus mucosal vaccination provides better protection against a respiratory challenge with C. trachomatis than either systemic or mucosal immunizations alone.     

Intranasal immunization with a mixture of PspA and a Toll-like receptor agonist induces specific antibodies and enhances bacterial clearance in the airways of mice

To develop an effective nasal vaccine for Streptococcus pneumoniae, the effects of a panel of Toll-like receptor (TLR) agonists in combination with pneumococcal surface protein A (PspA) on induction of PspA-specific antibodies and bacterial clearance were compared in mice. Mice were nasally immunized with 10 μg of TLR agonist (TLR 2–4 and 9) and 2.5 μg of PspA once per week for 3 weeks. Significantly increased levels of PspA-specific immunoglobulin G (IgG) and IgA in the airways and PspA-specific IgG in plasma were found in mice administered PspA plus each TLR agonist, compared with mice administered PspA alone. In a sub-lethal pneumonia model using a serotype 3 pneumococcal strain, bacterial density in the lungs of mice was significantly reduced in mice administered PspA plus each TLR agonist, compared with mice administered either PspA alone or phosphate-buffered saline alone 3 h after bacterial challenge. Similarly, enhanced bacterial clearance was found in the nasopharynx of mice administered PspA plus each TLR agonist 1 day after infection with a serotype 19F strain. Our data suggest that PspA-specific antibody induced by nasal immunization with PspA plus TLR agonist is capable of reducing the bacterial load in both the nasopharynx and lungs after challenge with pneumococci with different serotypes. Despite the skewed Th1/Th2 immune responses, the effects of nasal immunization with PspA plus each TLR agonist on bacterial clearances from the lungs 3 h after infection and from nasopharynx 1 day after infection in mice were equivalent.     

Induction of protection against vaginal shedding and infertility by a recombinant Chlamydia vaccine

A vaccine formulated with the Chlamydia muridarum recombinant major outer membrane protein, plus the adjuvants CpG and Montanide, was tested for its ability to protect BALB/c mice against a vaginal challenge. Mice were immunized by mucosal [intravaginal (i.vag.) plus colonic (col.), or intranasal (i.n.) plus sublingual (s.l.)], or systemic [intramuscular (i.m.) plus subcutaneous (s.c.)] routes, and a combination of mucosal priming and systemic boosting routes. A negative control group was vaccinated with the Neisseria gonorrhoeae porin B (Ng-rPorB) and a positive control group was inoculated in the nares with live Chlamydia. The strongest Chlamydia-specific humoral and cell-mediated immune responses were observed in the groups immunized by a combination of mucosal and systemic routes. Following the vaginal challenge, groups immunized using mucosal priming followed by systemic immunization had a significant decrease in the number of mice with positive vaginal cultures. For example, of the mice immunized i.n./s.l. + i.m./s.c., 24% had positive cultures during the six weeks of the experiment versus 69% for the negative control group immunized with Ng-rPorB (P < 0.05). Similarly, the groups of mice primed by the mucosal routes and boosted by the systemic routes had significantly less IFU in the vaginal cultures when compared to the Ng-rPorB animals (P < 0.05). These combination groups were also protected against infertility. The two groups had fertility rates of 100% (i.n./s.l. + i.m./s.c.) and 81% (i.vag./col. + i.m./s.c.) equivalent to the positive-control group immunized with live Chlamydia (100% fertility; P > 0.05). These results show the importance of the schedule and routes of vaccination and represent the first study to show protection against infertility by a Chlamydia recombinant subunit vaccine.     

Comparison of the nine polymorphic membrane proteins of Chlamydia trachomatis for their ability to induce protective immune responses in mice against a C. muridarum challenge

ObjectivesTo test vaccines, formulated with novel antigens, to protect mice against Chlamydia infections.MethodsTo determine the ability of polymorphic membrane proteins (Pmps) to induce cross-species protective immune responses, recombinant fragments from all nine C. trachomatis serovar E Pmps were used to vaccinate BALB/c mice utilizing CpG-1826 and Montanide ISA 720 as adjuvants. C. muridarum recombinant MOMP and PBS, formulated with the same adjuvants, were used as positive and negative controls, respectively. Mice were challenged intranasally with 10⁴ inclusion-forming units (IFU) of C. muridarum. Animals were weighed daily and at 10 days post-challenge, they were euthanized, their lungs harvested, weighed and the number of chlamydial IFU counted.ResultsFollowing vaccination the nine Pmps elicited immune responses. Based on body weight changes, or number of IFU recovered from lungs, mice vaccinated with Pmp C, G or H were the best protected. For example, over the 10-day period, the negative control group vaccinated with PBS lost significantly more body weight than mice immunized with PmpC or G (P < 0.05). C. muridarum MOMP vaccinated mice were better protected against body weight losses than any group immunized with Pmps. Also, the median number of IFU recovered from the lungs of mice vaccinated with PmpC (72 × 10⁶) or PmpH (61 × 10⁶) was significantly less than from mice immunized with PBS (620 × 10⁶; P < 0.05). As determined by the number of IFU, all Pmps elicited less protection than C. muridarum MOMP (0.078 × 10⁶ IFU; P < 0.05).ConclusionsThis is the first time PmpC has been shown to elicit cross-species protection against a respiratory challenge. Additional work with Pmps C, G and H is recommended to determine their ability to protect animal models against genital and ocular challenges.     

The cationic liposomal adjuvants CAF01 and CAF09 formulated with the major outer membrane protein elicit robust protection in mice against a Chlamydia muridarum respiratory challenge

Two cationic liposomal adjuvants CAF01 and CAF09 were formulated with the native or the recombinant Chlamydia muridarum major outer membrane protein (nMOMP and rMOMP). BALB/c mice were immunized with the four vaccine formulations using the subcutaneous followed by the intranasal (i.n.) routes. As positive controls mice were inoculated i.n. with live C. muridarum and negative controls received i.n. minimal essential medium (MEM). Four weeks after the last immunization mice were challenged i.n. with 10⁴ inclusion forming units (IFU) of C. muridarum. Following the challenge the mice were weighed daily. At 10 days post-challenge the mice were euthanized, their lungs weighed and the number of C. muridarum IFU determined. Serum collected the day before the challenge showed that all four groups of mice immunized with CAF01, or CAF09 and MOMP had significant C. muridarum-specific antibody titers. As determined by a T-cell lymphoproliferative assay, these four groups of mice also mounted robust cell mediated immune responses with high production of IFN-γ and IL17 and low levels of IL-4. Following the challenge the four groups of mice lost significantly less body weight than the MEM-immunized group. Lungs of mice vaccinated with CAF01, or CAF09, and nMOMP were significantly lighter than those from mice immunized using rMOMP. The number of IFU recovered from the lungs of mice vaccinated with CAF01, or CAF09, and nMOMP was similar to the number of IFU recovered from mice immunized with live EB. Mice that received rMOMP had significantly higher numbers of IFU than other groups. In conclusion, CAF01 and CAF09 elicited very robust protective humoral and cellular immune responses and were equally effective at adjuntavizing the C. muridarum MOMP. Mice vaccinated with nMOMP were significantly better protected than those immunized with rMOMP, indicative of the importance of the structural conformation of this antigen in protection.     

Induction of immune memory by a multisubunit chlamydial vaccine

We tested the hypothesis that intramuscular immunization with a multisubunit chlamydial vaccine candidate will induce long lasting immune responses in mice. Accordingly, groups of female C57BL/6 mice were immunized intramuscularly with Vibrio cholerae ghosts (VCG) expressing the Poring B and polymorphic membrane protein-D proteins of Chlamydia trachomatis or a control antigen. Humoral and cell-mediated immune responses were evaluated following immunization and after live chlamydial infection. Immunization induced an anamnestic response characterized by chlamydial-specific IgG2a and IgA antibodies in sera and vaginal lavage as well as specific genital and splenic T cell responses. The results also revealed that the local mucosal and systemic cellular and humoral immune effectors induced in mice following immunization with the vaccine candidate are long lasting. Vaccinated mice cleared intravaginal challenge with 10⁵ chlamydial inclusion forming units within 12 days compared to control mice, which shed up to 2 × 10³ IFUs at this time point. Moreover, rechallenge of mice 98 days after resolution of the primary infection resulted in the recall and retention of a relatively high frequency of chlamydial-specific Th1 cells and IgG2a in the genital mucosa. These results provide the first evidence that a VCG-based multisubunit chlamydial vaccine is capable of effectively stimulating anamnestic systemic and mucosal immune responses in mice. The data support further vaccine evaluation and testing for induction of long-term protective immunity.     

Role of Toll-like receptors in host responses to a virulence antigen of Streptococcus mutans expressed by a recombinant,attenuated Salmonella vector vaccine

In the present study, we investigated the role of Toll-like receptors (TLRs) in host responses to the saliva-binding region (SBR) of Streptococcus mutans expressed by a recombinant, attenuated Salmonella vaccine. C57BL/6 wild type (wt), TLR2^−/−, TLR4^−/− and MyD88^−/− mice were immunized by the intranasal route on days 0, 18 and boosted on day 98 with Salmonella typhimurium BRD 509 containing a plasmid encoding SBR. Serum and saliva samples were collected throughout the experiment and assessed for antibody activity by ELISA. Evidence is provided that the induction of a serum IgG2a (Th1-type) anti-SBR antibody response involved TLR2 signaling, whereas the anti-Salmonella response involved signaling through TLR4. The adaptor molecule MyD88 was not essential for the induction of a primary Th1-type response to SBR or Salmonella, but was necessary for a secondary response to SBR. Furthermore, the absence of TLR2, TLR4 or MyD88 resulted in enhanced Th2-type serum IgG1 anti-SBR and anti-Salmonella responses. Mucosal IgA responses to SBR were TLR2-, TLR4- and MyD88-dependent, while IgA responses to Salmonella were TLR4- and MyD88-dependent.     

Immunization with the Chlamydia trachomatis major outer membrane protein, using adjuvants developed for human vaccines, can induce partial protection in a mouse model against a genital challenge

To test several vaccines for Chlamydia trachomatis we vaccinated BALB/c and C3H/HeN female mice with a purified preparation of the native mouse pneumonitis (MoPn) major outer membrane protein (MOMP). The MOMP was formulated with anyone of three different adjuvants MF59, LT-K63 or LT-R72. As a negative control the animals were immunized with ovalbumin. Positive controls were inoculated intranasally (i.n.) with 10(4) inclusion-forming units (IFU) of C. trachomatis MoPn. High levels of Chlamydia-specific antibodies were detected in the serum and vaginal washes of the mice immunized with MOMP. Using a lymphoproliferative assay (LPA) a significant response was obtained in splenocytes from most of the groups of mice vaccinated with MOMP. Two weeks after the last immunization the mice were challenged in the left ovarian bursa with 10(5) IFU of C. trachomatis MoPn and vaginal cultures were collected for a period of 6 weeks. Overall, BALB/c and C3H/HeN mice immunized with MOMP showed a decrease in the severity and length of the infection but the difference with the controls was not statistically significant. Following mating the percentage of mice with bilateral fertility was not significantly different between mice vaccinated with MOMP and their respective ovalbumin-immunized controls. However, the C3H/HeN mice immunized with MOMP using MF59 or LTR72 as adjuvants had significantly more embryos per mouse than the control groups. In conclusion, mice immunized with native MOMP and adjuvants developed for human vaccines showed significant Chlamydia-specific immune response and a limited protection against infection and long-term sequelae.     

Induction of protection in mice against a respiratory challenge by a vaccine formulated with the Chlamydia major outer membrane protein adjuvanted with IC31®

IC31^®, a novel adjuvant, has been shown to be effective by increasing the levels of IFN-γ in animal models when delivered with several antigens. Here, we tested the ability of IC31^®, to enhance the protective ability of the Chlamydia trachomatis native major outer membrane protein (nMOMP). BALB/c mice were immunized by the intramuscular (i.m.) and subcutaneous (s.c.) routes with nMOMP + IC31^®. Another group of animals was immunized with nMOMP + Alum and as a negative control mice were immunized with ovalbumin (Ova) + IC31^®. Animals immunized with nMOMP + IC31^® developed high Chlamydia-specific IgG titers. The serum levels of IgG1 were higher than those of the IgG2a. T cells, from the spleens of mice immunized with IC31^®-adjuvanted nMOMP demonstrated a strong lymphoproliferative reaction to Chlamydia elementary bodies (EB) compared with the groups immunized with nMOMP + Alum or Ova + IC31^®. A similar comparison between these groups of mice revealed that the levels of IFN-γ in the supernatants from stimulated T-cells were significantly higher in animals immunized with nMOMP + IC31^®. Following an intranasal challenge with C. trachomatis, the mice immunized with IC31^®-adjuvanted nMOMP showed significant protection. The change in body weight, an indication of the severity of the infection, was significantly less reduced in mice immunized with nMOMP + IC31^®. Furthermore, the weight of the lungs, as well as the pulmonary Chlamydia burden, was significantly lower in the animals immunized with nMOMP + IC31^® when compared with the groups immunized with nMOMP + Alum or with Ova + IC31^®. In conclusion, these results provide the rationale for further preclinical testing of IC31^® using other chlamydial antigens, and support the potential evaluation of this adjuvant in human vaccines against Chlamydia.     

Nasal vaccination with attenuated Salmonella expressing VapA: TLR2 activation is not essential for protection against R. equi infection

Virulent strains of Rhodococcus equi have a large plasmid of 80–90 kb, which encodes several virulence-associated proteins (Vap), including VapA, a lipoprotein highly associated with disease. We have previously demonstrated that oral immunisation with attenuated Salmonella enterica Typhimurium strain expressing the antigen VapA (STM VapA+) induces specific and long-term humoral and cellular immunity against R. equi. It was shown that VapA activates Toll-like receptor 2 (TLR2) on macrophages by establishing an interaction that ultimately favours immunity against R. equi infection. The purpose of this study was to evaluate the immune response triggered by nasal immunisation with STM VapA+ and to determine whether TLR2 supports the vaccine effect. We developed an optimised protocol for a single nasal immunisation that conferred protection against R. equi infection in mice, which was manifested by efficient R. equi clearance in challenged animals. Nasal vaccination with STM VapA+ has also induced protection in Tlr2^−/− mice and mice with non-functional TLR4. Moreover, spleen cells of vaccinated mice augmented T-bet expression, as well as the production of IL-12, IFN-γ, nitric oxide and hydrogen peroxide. Notably, the population of CD4⁺ T cells with memory phenotype significantly increased in the spleens of vaccinated mice challenged 1 or 5 months after immunisation. In these animals, the spleen bacterial burden was also reduced. When similar experimental procedures were performed in TLR2 knockout mice, an increase in CD4⁺ T cells with memory phenotype was not observed. Consequently, we conclude that nasal vaccination with attenuated Salmonella expressing the R. equi virulence factor VapA confers long-lasting protection against experimental rhodoccocosis and that TLR2 engagement was not crucial to induce this protection but may be required for a long-term immune response.     

Vaccination of newborn mice induces a strong protective immune response against respiratory and genital challenges with Chlamydia trachomatis

Chlamydia trachomatis infections can occur early in life and may result in long-term sequelae. To assess the feasibility of implementing a vaccine in newborns, groups of 2-day-old BALB/c mice were immunized intranasally (i.n.) with 1x10(4) inclusion forming units (IFU) of C. trachomatis mouse pneumonitis (MoPn). As a control, newborn mice were sham-immunized i.n. with minimal essential medium. In the vaccinated animals, strong Chlamydia-specific humoral and cell-mediated immune responses were observed. Six weeks after immunization, mice were challenged with MoPn i.n. or intravaginally (i.vag.). For the i.n. challenge, mice were inoculated with 10(4) or 10(5)IFU of MoPn per mouse, and in the case of the i.vag. challenge, each animal received 10(6)IFU. By day 10 post-infection (p.i.), the vaccinated mice challenged i.n. with 10(4)IFU, had gained an average of 6.7+/-1% of their body weight. In contrast, the sham-immunized mice had lost 14.9+/-1% of their weight (P<0.05). The mean number of IFU/lungs in the vaccinated animals was 800+/-300, while for the sham-immunized mice was 211+/-49x10(6) (P<0.05). Significant differences between the Chlamydia-vaccinated and the sham-immunized mice were also found in the groups challenged with 10(5)IFU. In the mice challenged i.vag., a significant decrease in the number of mice with positive cultures, and the intensity and duration of vaginal shedding was noted in the vaccinated mice compared to the sham-immunized mice (P<0.05). In conclusion, these results indicate that vaccination of neonatal mice can result in a protective response against a subsequent pulmonary or genital challenge with Chlamydia.     

The immune enhancement of a novel soy lecithin/β-glucans based adjuvant on native Neospora caninum tachyzoite extract vaccine in mice

Efficient, cost-effective and safe Th1-immunity-inducing vaccine formulations are paramount for achieving protection against Neospora caninum. In this study, a new adjuvant (Providean-AVEC^®) was used in the development of a N. caninum vaccine and evaluated in a mouse model. Soluble N. caninum tachyzoite native protein extract (sNcAg) was selected as vaccine antigen based on its capacity to activate production of pro-inflammatory cytokines on dendritic cells. Vaccines containing 4 and 0.4 μg of sNcAg, and Providean-AVEC^®, ISCOM-Matrix or aluminum hydroxide (Alum) were tested in BALB/c mice. While mice vaccinated with 4 μg of sNcAg + Providean-AVEC^® developed specific antibodies shortly after the first dose, the rest of the high antigen payload formulations only induced seroconversion after the booster. Mice immunized with the high payload ISCOM vaccine (4 μg sNcAg) or with either low or high payload Providean-AVEC^® formulations (0.4 μg and 4 μg sNcAg, respectively) elicited higher IgG2a than IgG1 serum levels, and IFN-γ anamnestic responses with a Th1-cytokine biased profile. These animals had no histological signs of cerebral lesions and parasite burden assessed by quantitative real-time PCR was not detected. Vaccine preparations including Providean-AVEC^® as adjuvant limited N. canimum multiplication even with only a tenth of antigen payload compared to vaccines containing other adjuvants. Using adjuvants to specifically activate dendritic cells, combined with a careful antigen selection can enhance cellular responses to inert N. caninum vaccines.     

Improved protection against Chlamydia muridarum using the native major outer membrane protein trapped in Resiquimod-carrying amphipols and effects in protection with addition of a Th1 (CpG-1826) and a Th2 (Montanide ISA 720) adjuvant

A new vaccine formulated with the Chlamydia muridarum native major outer membrane protein (nMOMP) and amphipols was assessed in an intranasal (i.n.) challenge mouse model. nMOMP was trapped either in amphipol A8-35 (nMOMP/A8-35) or in A8-35 conjugated with Resiquimod (nMOMP/Resiq-A8-35), a TLR7/8 agonist added as adjuvant. The effects of free Resiquimod and/or additional adjuvants, Montanide ISA 720 (TLR independent) and CpG-1826 (TLR9 agonist), were also evaluated. Immunization with nMOMP/A8-35 alone administered i.n. was used as negative adjuvant-control group, whereas immunizations with C. muridarum elementary bodies (EBs) and MEM buffer, administered i.n., were used as positive and negative controls, respectively. Vaccinated mice were challenged i.n. with C. muridarum and changes in body weight, lungs weight and recovery of Chlamydia from the lungs were evaluated. All the experimental groups showed protection when compared with the negative control group. Resiquimod alone produced weak humoral and cellular immune responses, but both Montanide and CpG-1826 showed significant increases in both responses. The addition of CpG-1826 alone switched immune responses to be Th1-biased. The most robust protection was elicited in mice immunized with the three adjuvants and conjugated Resiquimod. Increased protection induced by the Resiquimod covalently linked to A8-35, in the presence of Montanide and CpG-1826 was established based on a set of parameters: (1) the ability of the antibodies to neutralize C. muridarum; (2) the increased proliferation of T-cells in vitro accompanied by higher production of IFN-γ, IL-6 and IL-17; (3) the decreased body weight loss over the 10 days after challenge; and (4) the number of IFUs recovered from the lungs at day 10 post challenge. In conclusion, a vaccine formulated with the C. muridarum nMOMP bound to amphipols conjugated with Resiquimod enhances protective immune responses that can be further improved by the addition of Montanide and CpG-1826.