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.     

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.     

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.     

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.     

A chlamydial type III-secreted effector protein (Tarp) is predominantly recognized by antibodies from humans infected with Chlamydia trachomatis and induces protective immunity against upper genital tract pathologies in mice

Chlamydia trachomatis genome is predicted to encode a type III secretion system consisting of more than 40 open reading frames (ORFs). To test whether these ORFs are expressed and immunogenic during chlamydial infection in humans, we expressed 55 chlamydial ORFs covering all putative type III secretion components plus control molecules as fusion proteins and measured the reactivity of these fusion proteins with antibodies from patients infected with C. trachomatis in the urogenital tract (24 antisera) or in the ocular tissue (8 antisera). Forty-five of the 55 proteins were recognized by at least 1 of the 32 human antisera, suggesting that these proteins are both expressed and immunogenic during chlamydial infection in humans. Tarp, a putative type III secretion effector protein, was identified as a novel immunodominant antigen due to its reactivity with the human antisera at high frequency and titer. The expression and immunogenicity of Tarp were confirmed in cell culture and mouse systems. Tarp was mainly associated with the infectious form of chlamydial organisms and became undetectable between 13 and 24 h during the infection cycle in cell culture. Mice intravaginally infected with C. muridarum developed Tarp-specific humoral and cellular immune responses. More importantly, immunization of mice with Tarp induced Th1-dominant immunity that significantly reduced the shedding of live organisms from the lower genital tract and attenuated inflammatory pathologies in the fallopian tube tissues. These observations have demonstrated that Tarp, an immunodominant antigen identified by human antisera, can induce protective immunity against chlamydial infection and pathology in mice.     

Induction of protective immunity against Chlamydia muridarum intracervical infection in DBA/1j mice

We previously reported that intracervical inoculation with Chlamydia muridarum induced hydrosalpinx in DBA/1j mice, but intravaginal inoculation failed to do so. In the current study, we found unexpectedly that intrabursal inoculation of live chlamydial organisms via the oviduct failed to induce significant hydrosalpinx. We further tested whether primary infection via intravaginal or intrabursal inoculation could induce protective immunity against hydrosalpinx following intracervical challenge infection. Mice infected intravaginally with C. muridarum were fully protected from developing hydrosalpinx, while intrabursal inoculation offered partial protection. We then compared immune responses induced by the two genital tract inoculations. Both inoculations induced high IFNγ and IL-17 T cell responses although the ratio of IgG2a versus IgG1 in intravaginally infected mice was significantly higher than in mice infected intrabursally. When the antigen-specificities of antibody responses were compared, both groups of mice dominantly recognized 24 C. muridarum antigens, while each group preferentially recognized unique sets of antigens. Thus, we have demonstrated that intrabursal inoculation is neither effective for causing hydrosalpinx nor efficient in inducing protective immunity in DBA/1j mice. Intravaginal immunization, in combination with intracervical challenge infection in DBA/1j mice, can be a useful model for understanding mechanisms of chlamydial pathogenicity and protective immunity.     

Protective immunity against Chlamydia trachomatis genital infection induced by a vaccine based on the major outer membrane multi-epitope human papillomavirus major capsid protein L1

The administration of an efficacious vaccine is the most effective long-term measure to control the genital tract infection caused by Chlamydia trachomatis (Ct) in humans. The current challenge for Ct vaccine development is to develop an effective delivery vehicle for induction of a high level of mucosal T and complementary B cell responses. We evaluated the immunogenicity and efficacy of a candidate vaccine comprising the major outer membrane protein (MOMP) multiepitope of Ct delivered with the human papillomavirus (HPV) major capsid protein L1 as a vehicle with adjuvant properties, in a murine model of chlamydial genital infection. A recombinant plasmid pcDNA3.1(+) containing mammalian codon-optimization HPV6b L1 gene and Ct MOMP multiepitope was constructed. The Ct MOMP multiepitope containing T- and B-cell epitope-rich peptides was inserted into C-terminal of HPV6b L1-coding sequence. The constructed plasmid after verified by enzyme restriction assay and DNA sequencing was transfected into COS-7 cells. Expression of the chimeric gene in COS-7 cells was confirmed by RT-PCR, Western blot analysis and immunofluorescence assay. Results revealed successful expression of the chimeric HPV6b L1/Ct MOMP multiepitope gene both at the mRNA and protein levels in transfected COS-7 cells. Intramuscular (IM) administration in mice was able to elicit not only antibodies against Ct MOMP, but also Th1 and cytotoxic T lymphocyte activity against the Ct MOMP epitopes. In addition, recipients of IM immunization of HPV6b L1/Ct MOMP multiepitope were highly resistant to infection. Altogether, the results suggested that IM delivery of HPV6b L1-MOMP multiepitope may be a suitable vaccine regimen potentially capable of inducing protective mucosal immunity against Ct 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.     

Comparison of the protective effects of killed Burkholderia pseudomallei and CpG oligodeoxynucleotide against live challenge

Melioidosis is a fatal disease caused by Burkholderia pseudomallei. Currently there is no vaccine available. Synthetic oligodeoxynucleotides with unmethylated CpG dinucleotide motifs (CpG ODN) can stimulate vertebrate immune cells and clear certain pathogens that are susceptible to a strong Th1 response. In our previous study, pretreatment with CpG ODN alone or CpG-ODN with cationic liposomes for 2–10 or 30 days before B. pseudomallei infection in mice conferred 80–100% protection. In the present study we investigated the protective effect of CpG-ODN together with heat-killed (HK) or paraformaldehyde-killed B. pseudomallei (PP). HK or PP were used to immunize BALB/c mice twice at 15-day intervals before intra-peritoneal challenge with 5LD50 of B. pseudomallei and observed for 30 days. We found that PP could significantly protect mice (60%) with an increased survival time (24.8 ± 11.63 days) while in the HK and PBS groups, all infected mice died within 6 days. Although either CpG ODN or PP conferred significant protection, giving them in combination did not enhance it further. Serum IFN-γ levels on day-5 (before challenge) of the PP and PP + CpG ODN groups were significantly higher than those of the PBS control group. The results further support the importance of IFN-γ in host protection against B. pseudomallei and suggest further study on paraformaldehyde-killed bacteria as a component of a future B. pseudomallei vaccine.     

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.     

Novel Chlamydia pneumoniae vaccine candidates confirmed by Th1-enhanced genetic immunization

Identification of highly immunogenic antigens is critical for the construction of an efficacious subunit vaccine against Chlamydia pneumoniae infections. A previous project used a genome-wide screen to identify 12 protective C. pneumoniae candidate genes in an A/J mouse lung disease model (Li et al. [14]). Due to insufficient induction of Th1 immunity, these genes elicited only modest protection. Here, we used the Escherichia coli heat-labile enterotoxin as a Th1-enhancing genetic adjuvant, and re-tested these 12 genes, in parallel with six genes identified by other investigators. Vaccine candidate genes cutE and Cpn0420 conferred significant protection by all criteria evaluated (prevention of C. pneumoniae-induced death, reduction of lung disease, elimination of C. pneumoniae). Gene oppA_2 was protective by disease reduction and C. pneumoniae elimination. Four other genes were protective by a single criterion. None of the six genes reported elsewhere protected by reduction of lung disease or elimination of C. pneumoniae, but three protected by increasing survival.     

Heat denatured enzymatically inactive recombinant chlamydial protease-like activity factor induces robust protective immunity against genital chlamydial challenge

We have shown previously that vaccination with recombinant chlamydial protease-like activity factor (rCPAF) plus interleukin-12 as an adjuvant induces robust protective immunity against primary genital Chlamydia muridarum challenge in mice. Since CPAF is a protease, we compared the effects of enzymatically active and inactive (heat denatured) rCPAF to determine whether proteolytic activity is expendable for the induction of protective immunity against chlamydial challenge. Active, but not inactive, rCPAF immunization induced high levels of anti-active CPAF antibody, whereas both induced robust splenic CPAF-specific IFN-γ production. Vaccination with active or inactive rCPAF induced enhanced vaginal chlamydial clearance as early as day 6 with complete resolution of infection by day 18, compared to day 30 in mock-vaccinated and challenged animals. Importantly, significant and comparable reductions in oviduct pathology were observed in active and inactive rCPAF-vaccinated mice compared to mock-vaccinated animals. Thus, rCPAF induced anti-chlamydial immunity is largely independent of enzymatic activity and secondary or higher order protein conformation.     

Plasma fibrinogen levels after vaccination with a native outer membrane vesicle vaccine for Neisseria meningitidis

Several studies have shown plasma fibrinogen increases following some vaccinations, but the specific triggers and the kinetics of this response are not well understood. We conducted a phase I trial of an outer membrane vesicle vaccine for Neisseria meningitidis. Plasma fibrinogen was measured on days 0, 2 and 14 following each of 3 doses. The highest dose of vaccine was associated with the greatest increase in fibrinogen at day 2, which decreased by day 14. The first vaccination caused a greater increase than either subsequent vaccination. These transient increases in fibrinogen are comparable to what occurs with upper respiratory infections and have not been demonstrated to represent an increased risk of adverse vascular events.     

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.     

Vaccination with profilin encapsulated in oligomannose-coated liposomes induces significant protective immunity against Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that can infect a variety of mammals and birds, causing toxoplasmosis. Several types of vaccines against T. gondii have been developed, but these have limitations in terms of their safety and inadequate efficacy. T. gondii profilin (TgPF) is a potential immunodominant antigen for a candidate vaccine. In this study, we encapsulated TgPF in oligomannose-coated liposomes (OMLs) to evaluate the immune response induced by this vaccine. C57BL/6 mice were immunized with TgPF-OML three times at 14-day intervals and challenged with T. gondii. TgPF-OML increased the survival of the mice and reduced the parasite burden in their brains after T. gondii infection. Immunization with TgPF-OML also induced TgPF-specific interferon-γ production and IgG antibodies in mice. Our results demonstrate that OML-encapsulated TgPF triggers strong humoral and cellular responses against T. gondii, and that TgPF-OML is a candidate vaccine that warrants further development.     

Design and evaluation in mice of a broadly protective meningococcal group B native outer membrane vesicle vaccine

A vaccine based on native outer membrane vesicles (NOMV) that has potential to provide safe, broad based protection against group B strains of Neisseria meningitidis has been developed. Three antigenically diverse group B strains of N. meningitidis were chosen and genetically modified to improve safety and expression of desirable antigens. Safety was enhanced by disabling three genes: synX, lpxL1, and lgtA. The vaccine strains were genetically configured to have three sets of antigens each with potential to induce protective antibodies against a wide range of group B strains. Preliminary immunogenicity studies with combined NOMV from the three strains confirmed the capacity of the vaccine to induce a broad based bactericidal antibody response. Analysis of the bactericidal activity indicated that antibodies to the LOS were responsible for a major portion of the bactericidal activity and that these antibodies may enhance the bactericidal activity of anti-protein antibodies.     

A TLR2 agonist is a more effective adjuvant for a Chlamydia major outer membrane protein vaccine than ligands to other TLR and NOD receptors

Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterial pathogen in the World and there is an urgent need for a vaccine to prevent these infections. To determine what type of adjuvant can better enhance the immunogenicity of a Chlamydia vaccine, we formulated the recombinant major outer membrane protein (Ct-rMOMP) with several ligands for Toll-like receptors (TLR) and the nucleotide-binding oligomerization domain (NOD) including Pam₂CSK₄ (TLR2/TLR6), Poly (I:C) (TLR3), monophosphoryl lipid A (TLR4), flagellin (TLR5), imiquimod R837 (TLR7), imidazoquinoline R848 (TRL7/8), CpG-1826 (TLR9), M-Tri-_DAP (NOD1/NOD2) and muramyldipeptide (NOD2). Groups of female BALB/c mice were immunized intramuscularly (i.m.) three times with the Ct-rMOMP and each one of those adjuvants. Four weeks after the last immunization the mice were challenged intranasally (i.n.) with 10⁴C. trachomatis mouse pneumonitis (MoPn) inclusion forming units (IFU). As negative antigen control, mice were immunized with the Neisseria gonorrhoeae recombinant porin B (Ng-rPorB) and the same adjuvants. As a positive vaccine control, mice were inoculated i.n. with 10⁴ IFU of MoPn. The humoral and cell mediated immune responses were determined the day before the challenge. Following the challenge the mice were weighed daily and, at 10 days post-challenge (p.c.), they were euthanized, their lungs weighted and the number of IFU in the lungs counted. As determined by the IgG2a/IgG1 ratio in the sera, mice immunized with Ct-rMOMP + Pam₂CSK₄ showed a strong Th2 biased humoral immune response. Furthermore, these mice developed a robust cellular immune response with high Chlamydia-specific T cell proliferation and levels of IFN-γ production. In addition, based on changes in body weight, weight of the lungs and number of IFU recovered from the lungs, the mice immunized with Ct-rMOMP + Pam₂CSK₄, were better protected against the i.n. challenge than any group of mice immunized with Ct-rMOMP and the other adjuvants. In conclusion, Pam₂CSK₄ should be evaluated as a candidate adjuvant for a C. trachomatis vaccine.     

Toward the development of a stable,freeze-dried formulation of Helicobacter pylori killed whole cell vaccine adjuvanted with a novel mutant of Escherichia coli heat-labile toxin

No vaccine exists for the prevention of infection with the ubiquitous gastric pathogen Helicobacter pylori, and drug therapy for the infection is complicated by poor patient compliance, the high cost of treatment, and ineffectiveness against drug-resistant strains. A new medical advancement is required to reduce the incidence of peptic ulcer disease and stomach cancer, two conditions caused by infection with H. pylori. Clinical trials have been performed with a formalin-inactivated H. pylori whole cell (HWC) vaccine, given orally in combination with the mucosal adjuvant mLT(R192G), a mutant of Escherichia coli heat-labile toxin. Following the initial dose of this vaccine, some subjects experienced gastrointestinal side effects. To reduce side effects and potentially further increase the amount of adjuvant that can safely be administered with the HWC vaccine, experiments were performed with a form of LT that carried two mutations in the A subunit, a substitution of G for R at position 192, and A for L at position 211. The double mutant LT (dmLT) adjuvant stimulated immune responses as effectively as the single mutant LT in mice. Additionally, following a challenge infection, the dmLT-adjuvanted vaccine was as effective as single mutant LT in reducing gastric urease levels (diagnostic for H. pylori infection), and H. pylori colonization in the stomach as assessed by quantitative analysis of stomach homogenates. A lyophilized formulation of HWC was developed to improve stability and to potentially reduce reliance on cold chain maintenance. It was observed that a dmLT-adjuvanted lyophilized vaccine was equally as protective in the mouse model as the liquid formulation as assessed by gastric urease analysis and analysis of stomach homogenates for viable H. pylori. No readily detectable effect of tonicity or moisture content was observed for the lyophilized vaccine within the formulation limits evaluated. In an accelerated stability study performed at 37 °C the lyophilized vaccine remained equally as protective as vaccine stored at 2–8 °C. The formulation selected for clinical development consisted of 2.5 × 10¹⁰ formalin-inactivated cells per ml in 6.5% trehalose, 0.5% mannitol, and 10 mM citrate buffer at pH 6.8.     

Heat shock protein 70 from Trichinella spiralis induces protective immunity in BALB/c mice by activating dendritic cells

Trichinella spiralis heat shock protein 70 (Ts-Hsp70) is a protective antigen that induces partial protective immunity against T. spiralis infection in mice. To determine whether dendritic cells are involved in the mechanism responsible for the protection induced by Ts-Hsp70, mouse bone marrow-derived dendritic cells (DCs) were incubated with recombinant Ts-Hsp70 (rTs-Hsp70), and the DC-secreted cytokines and expressed surface markers were measured. The results demonstrated that rTs-Hsp70 activated DC maturation that was characterized by the secretion of IL-1β, IL-12p70, TNF-α, and IL-6 and the increased surface expression of CD11c, MHC II, CD40, CD80, and CD86. The rTs-Hsp70-activated DCs enabled the stimulation, proliferation and secretion of Th1/2 cytokines (i.e., INF-γ, IL-2, IL-4 and IL-6) in CD4⁺ T cells from T. spiralis-infected mice. The mice that received rTs-Hsp70-activated DCs exhibited a 38.4% reduction in muscle larvae upon larval challenge with T. spiralis compared to the group that received PBS-incubated DCs. This partial protection was correlated with Th1 and Th2 mixed anti-Ts-Hsp70-specific immune responses that included high titers of total IgG, IgG1 and IgG2a and increased levels of Th1/2 cytokines (i.e., IFN-γ, IL-2, IL-4, IL-6). These results indicate that the rTs-Hsp70-induced protective immunity was mediated by the activation of the DCs and that rTs-Hsp70-loaded DCs could be an alternative vaccine approach against trichinellosis.     

Immunization with recombinant Chaperonin60 (Chp60) outer membrane protein induces a bactericidal antibody response against Neisseria meningitidis

Sera from individuals colonized with Neisseria meningitidis and from patients with meningococcal disease contain antibodies specific for the neisserial heat-shock/chaperonin (Chp)60 protein. In this study, immunization of mice with recombinant (r)Chp60 in saline; adsorbed to aluminium hydroxide; in liposomes and detergent micelles, with and without the adjuvant MonoPhosphoryl Lipid A (MPLA), induced high and similar (p > 0.05) levels of antibodies that recognized Chp60 in outer membranes (OM). FACS analysis and immuno-fluorescence experiments demonstrated that Chp60 was surface-expressed on meningococci. By western blotting, murine anti-rChp60 sera recognized a protein of Mr 60 kDa in meningococcal cell lysates. However, cross-reactivity with human HSP60 protein was also observed. By comparing translated protein sequences of strains, 40 different alleles were found in meningococci in the Bacterial Isolate Genome Sequence database with an additional 5 new alleles found in our selection of 13 other strains from colonized individuals and patients. Comparison of the non-redundant translated amino acid sequences from all the strains revealed ≥97% identity between meningococcal Chp60 proteins, and in our 13 strains the protein was expressed to high and similar levels. Bactericidal antibodies (median reciprocal titres of 32–64) against the homologous strain MC58 were induced by immunization with rChp60 in liposomes, detergent micelles and on Al(OH)₃. Bactericidal activity was influenced by the addition of MPLA and the delivery formulation used. Moreover, the biological activity of anti-Chp60 antisera did not extend significantly to heterologous meningococcal strains. Thus, in order to provide broad coverage, vaccines based on Chp60 would require multiple proteins and specific bactericidal epitope identification.