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Kris Huygen

Publications and source records attributed to Kris Huygen.

At least 19 recordsLinked to original sources
PubMed2006

Immunogenicity of eight dormancy regulon-encoded proteins of Mycobacterium tuberculosis in DNA-vaccinated and tuberculosis-infected mice.

Hypoxia and low concentrations of nitric oxide have been reported to upregulate in vitro gene expression of 48 proteins of the dormancy (DosR) regulon of Mycobacterium tuberculosis. These proteins are thought to be essential for the survival of bacteria during persistence in vivo and are targeted by the immune system during latent infection in humans. Here we have analyzed the immunogenicity of eight DosR regulon-encoded antigens by plasmid DNA vaccination of BALB/c and C57BL/6 mice, i.e., Rv1733c, Rv1738, Rv2029c (pfkB), Rv2031c/hspX (acr), Rv2032 (acg), Rv2626c, Rv2627c, and Rv2628. Strong humoral and/or cellular Th1-type (interleukin-2 and gamma interferon) immune responses could be induced against all but one (Rv1738) of these antigens. The strongest Th1 responses were measured following vaccination with DNA encoding Rv2031c and Rv2626c. Using synthetic 20-mer overlapping peptides, 11 immunodominant, predicted major histocompatibility complex class II-restricted epitopes and one K(d)-restricted T-cell epitope could be identified. BALB/c and (B6D2)F(1) mice persistently infected with M. tuberculosis developed immune responses against Rv1733c, Rv2031c, and Rv2626c. These findings have implications for proof-of-concept studies in mice mimicking tuberculosis (TB) latency models and their extrapolation to humans for potential new vaccination strategies against TB.

Animals↗
PubMed2006

Priming but not boosting with plasmid DNA encoding mycolyl-transferase Ag85A from Mycobacterium tuberculosis increases the survival time of Mycobacterium bovis BCG vaccinated mice against low dose intravenous challenge with M. tuberculosis H37Rv.

DNA vaccination is a potent means for inducing strong CD4+ (Th1) and particularly CD8+ mediated immune responses and protective immunity against tuberculosis infection in mice. Here we have analyzed the potential of a DNA vaccine encoding the immunodominant mycolyl-transferase Ag85A for increasing the efficacy of the current tuberculosis vaccine Mycobacterium bovis Bacille Calmette-Guérin (BCG) in three long-term survival experiments. BALB/c mice were vaccinated with BCG either following DNA priming or prior to DNA boosting. Ag85A specific CD4+ and CD8+ mediated IFN-gamma responses were increased in mice primed with DNA prior to BCG, and in BCG vaccinated mice subsequently boosted with DNA. In the latter immunization protocol, antigenic stimulation also induced significant levels of IL-17. Mice were monitored for cachexia and survival following a low dose intravenous challenge with M. tuberculosis H37Rv. Priming with Ag85A but not control DNA increased significantly the protective efficacy of the BCG vaccine as indicated by reduced cachexia and prolonged survival time: 32 weeks versus 23 weeks in one experiment and 33 weeks versus 26 weeks in another experiment (MST in control, TB infected mice: 17 weeks in both experiments). On the other hand, boosting of BCG by subsequent Ag85A DNA in saline or vaxfectin--or recombinant 85A protein or MVA-85A for that matter--did not augment the efficacy of BCG (MST 19-21 weeks in all vaccinated groups versus 11 weeks in control, TB infected mice). Our results demonstrate that Ag85A DNA priming can increase efficacy of BCG and that boosting protocols of BCG may possibly be hampered by the induction of Th(IL-17) cells.

Acyltransferases↗
PubMed2006

Immunogenicity and protective efficacy of tuberculosis DNA vaccines combining mycolyl-transferase Ag85A and phosphate transport receptor PstS-3.

DNA vaccines encoding the 32,000 MW mycolyl-transferase Ag85A and the 40,000 MW phosphate-binding protein PstS-3 can elicit protective immune responses against experimental infection with Mycobacterium tuberculosis in C57BL/6 mice. Here we have analysed the vaccine potential of a combination of both antigens using plasmid vectors expressing either a fusion protein of both antigens or the separate proteins driven by two independent promoters (in pBudCE4.1 vector). Comparable levels of Ag85A specific T helper 1 (Th1) type immune responses could be induced by the two combination vaccines and the single vaccine encoding the mycolyl-transferase, whereas induction of PstS-3 specific Th1-mediated responses was impaired in both combination vaccines. In contrast, magnitude of CD8+ mediated responses against the PstS-3 protein was comparable following combination or single DNA vaccination. Antigenic competition was also observed at the antibody level; PstS-3 specific levels being lower in mice vaccinated with the fusion vector and Ag85A specific levels being lower in mice vaccinated with the combination pBudCE4.1 vector (as compared to levels obtained following single plasmid immunization). Protection against M. tuberculosis was only modestly improved in mice vaccinated with the DNA combinations. It is possible that prior activation of Ag85A specific CD4+ T cells directed against this common mycobacterial antigen leads to cross-competition for major histocompatibility complex class II-restricted peptide complexes of the Pst-3 antigen. This may have implications for future combination vaccines using Ag85.

ATP-Binding Cassette Transporters↗
PubMed2006

Development of luminescent Mycobacterium avium subsp. paratuberculosis for rapid screening of vaccine candidates in mice.

Mycobacterium avium subsp. paratuberculosis is a slowly growing mycobacterial species, requiring 6 to 8 weeks of culture before colonies can be counted visually. Here, we describe the development of luminescent M. avium subsp. paratuberculosis expressing luxAB genes of Vibrio harveyi and its use for vaccine testing in an experimental mouse model, replacing fastidious CFU counting by rapid luminometry.

Animals↗
PubMed2006

Members of the 30- to 32-kilodalton mycolyl transferase family (Ag85) from culture filtrate of Mycobacterium avium subsp. paratuberculosis are immunodominant Th1-type antigens recognized early upon infection in mice and cattle.

The characterization of protective antigens is essential for the development of an effective, subunit-based vaccine against paratuberculosis. Surface-exposed and secreted antigens, present abundantly in mycobacterial culture filtrate (CF), are among the well-known protective antigens of Mycobacterium tuberculosis and Mycobacterium bovis. Culture filtrate, prepared from Mycobacterium avium subsp. paratuberculosis ATCC 19698 grown as a surface pellicle on synthetic Sauton medium, was strongly and early recognized in experimentally infected B6 bg/bg beige mice and cattle, as indicated by elevated spleen cell gamma interferon (IFN-gamma) secretion and lymphoproliferative responses of peripheral blood mononuclear cells, respectively. Strong proliferative and ex vivo IFN-gamma responses against antigen 85 (Ag85) complex (a major protein component from M. bovis BCG culture filtrate) could be detected in cattle as early as 10 weeks after oral M. avium subsp. paratuberculosis infection. Synthetic peptides from the Ag85A and Ag85B components of this complex were strongly recognized, whereas T-cell responses were weaker against peptides from the Ag85C protein. A promiscuous T-cell epitope spanning amino acids 145 to 162 of Ag85B (identical sequence in M. bovis and M. avium subsp. paratuberculosis) was identified in experimentally infected cattle. Finally, young calves, born from cows with confirmed paratuberculosis, demonstrated proliferative responses to purified, recombinant Ag85A and Ag85B from M. avium subsp. paratuberculosis. These results indicate that the M. avium subsp. paratuberculosis Ag85 homologues are immunodominant T-cell antigens that are recognized early in experimental and natural infection of cattle.

Acyltransferases↗
PubMed2006

Immune responses induced in cattle by vaccination with a recombinant adenovirus expressing Mycobacterial antigen 85A and Mycobacterium bovis BCG.

Cattle were vaccinated with an adenovirus expressing the mycobacterial antigen 85A (rAd85A), with Mycobacterium bovis BCG followed by rAd85A heterologous boosting, or with rAd85A followed by BCG boosting. BCG/rAd85A resulted in the highest direct gamma interferon responses. Cultured enzyme-linked immunospot assay analysis demonstrated that memory responses were induced by all three protocols but were strongest after BCG/rAd85A and rAd85A/BCG vaccination.

Adenoviridae↗
PubMed2006

Partial reconstitution of the CD4+-T-cell compartment in CD4 gene knockout mice restores responses to tuberculosis DNA vaccines.

Reactivation tuberculosis (TB) is a serious problem in immunocompromised individuals, especially those with human immunodeficiency virus (HIV) coinfection. The adaptive immune response mediated by CD4+ and CD8+ T cells is known to confer protection against TB. Hence, vaccines against TB are designed to activate these two components of the immune system. Anti-TB DNA vaccines encoding the immunodominant proteins Ag85A, Ag85B, and PstS-3 from Mycobacterium tuberculosis are ineffective in mice lacking CD4+ T cells (CD4-/- mice). In this study, we demonstrate that reconstitution of the T-cell compartment in CD4-/- mice restores vaccine-specific antibody and gamma interferon (IFN-gamma) responses to these DNA vaccines. The magnitude of the immune responses correlated with the extent of reconstitution of the CD4+-T-cell compartment. Reconstituted mice vaccinated with DNA encoding PstS-3, known to encode a dominant D(b)-restricted CD8+-T-cell epitope, displayed CD8+-T-cell responses not observed in CD4-/- mice. M. tuberculosis challenge in reconstituted mice led to the extravasation of IFN-gamma-producing CD4+ and CD8+ T cells into lungs, the primary site of bacterial replication. Importantly, a reconstitution of 12 to 15% of the CD4+-T-cell compartment resulted in Ag85B plasmid DNA-mediated protection against a challenge M. tuberculosis infection. Our findings provide evidence that anti-TB DNA vaccines could be effective in immunodeficient individuals after CD4+-T-lymphocyte reconstitution, as may occur following antiretroviral therapy in HIV+ patients.

Acyltransferases↗
PubMed2005

Evaluation of the immunogenicity of pBudCE4.1 plasmids encoding mycolyl-transferase Ag85A and phosphate transport receptor PstS-3 from Mycobacterium tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb), remains a major health problem. The only currently available vaccine, BCG, confers only variable protection and an improved vaccine is urgently needed. Administration of DNA vaccines encoding the secreted mycolyl-transferase Ag85A and the surface-exposed phosphate transport receptor PstS-3 elicit an immune response capable of protecting mice challenged with Mtb. In order to combine the protection against Mtb infection induced by these two DNA vaccines, we have cloned Ag85A and PstS-3 in pBudCE4.1 vector, in which antigenic expression is controlled by two independent promoters. (BALB/cxC57BL/6)F1 mice were vaccinated with this combination vaccine and immune responses were compared to those induced by vaccination with plasmids encoding the single antigens on pBudCE4.1 or pV1J.ns-tPA backbone. Antibody and Th1 type cytokine responses against Ag85A were comparable, whereas responses against PstS-3 were clearly lower in mice vaccinated with the combination plasmid, suggesting antigenic competition with the mycolyl-transferase being the dominant antigen.

ATP-Binding Cassette Transporters↗
PubMed2005

DNA vaccination for the priming of neutralizing antibodies against non-immunogenic STa enterotoxin from enterotoxigenic Escherichia coli.

In order to test the use of DNA vaccination for its capacity to induce antibodies against the non-immunogenic heat-stable enterotoxin STa from Escherichia coli, BALB/c mice were immunized with plasmid DNA encoding hybrid proteins made by the insertion of wild type STa or insertion of the Cys6Ala, Cys17Ala and Cys6Ala-Cys17Ala STa mutants at positions 195 or 216 of the TEM-1 beta-lactamase. No STa specific antibodies could be detected after three plasmid injections, but a subsequent boost with native STa peptide was capable of inducing low levels of neutralizing antibodies, as tested in the suckling mouse assay. Highest STa specific responses were found in mice primed with the double mutated STa inserted in position 195. This plasmid induced highest T-cell responses to the TEM-1 protein, indicating that priming of helper T-cell responses to the carrier protein was essential. Mixed IgG1/IgG2a isotypes also reflected this T helper 1 type priming. Moreover, insertion into loop A of the TEM-1 carrier may be more suitable than insertion into loop B, because of reduced competition between carrier and hapten B cell responses.

Amino Acid Sequence↗
PubMed2005

Plasmid DNA vaccination.

Plasmid DNA vaccination against tuberculosis is a very powerful and easy method for the induction of strong humoral responses, CD4+ mediated secretion of Th1 cytokines and CD8+ mediated CTL activity in mice. Tuberculosis DNA vaccines have not been assessed so far in humans, and clinical trials with DNA in general have been somewhat disappointing. However, numerous studies have reported on the potent priming capacity of plasmid DNA for Th1 and CD8+ mediated immune responses, which can be boosted subsequently by recombinant protein or recombinant pox-viruses. With respect to tuberculosis, prime/boost regimens with Mycobacterium bovis BCG vaccine are particularly promising and warrant further analysis.

Animals↗
PubMed2005

Evaluation of vaccines in the EU TB Vaccine Cluster using a guinea pig aerosol infection model of tuberculosis.

The TB Vaccine Cluster project funded by the EU Fifth Framework programme aims to provide novel vaccines against tuberculosis that are suitable for evaluation in humans. This paper describes the studies of the protective efficacy of vaccines in a guinea pig aerosol-infection model of primary tuberculosis. The objective was to conduct comparative evaluations of vaccines that had previously demonstrated efficacy in other animal models. Groups of 6 guinea pigs were immunized with vaccines provided by the relevant EU Vaccine Cluster partners. Survival over 17 or 26 weeks was used as the principal measure of vaccine efficacy following aerosol challenge with H37Rv. Counts of mycobacteria in lungs and spleens, and histopathological changes in the lungs, were also used to provide evidence of protection. A total of 24 vaccines were evaluated in 4 experiments each of a different design. A heterologous prime-boost strategy of DNA and MVA, each expressing Ag85A and a fusion protein of ESAT-6 and Ag85B in adjuvant, protected the guinea pigs to the same extent as BCG. Genetically modified BCG vaccines and boosted BCG strategies also protected guinea pigs to the same extent as BCG but not statistically significantly better. A relatively high aerosol-challenge dose and evaluation over a protracted time post-challenge allowed superior protection over BCG to be demonstrated by BCG boosted with MVA and fowl pox vectors expressing Ag85A.

Aerosols↗
PubMed2005

Mycobacterium tuberculosis with disruption in genes encoding the phosphate binding proteins PstS1 and PstS2 is deficient in phosphate uptake and demonstrates reduced in vivo virulence.

By measuring phosphate uptake by Mycobacterium tuberculosis strains with the pstS1 and pstS2 genes genetically inactivated, we showed that these pstS genes encode high-affinity phosphate binding proteins. In a mouse infection model, both mutants were attenuated in virulence, suggesting that M. tuberculosis encounters limiting phosphate concentrations during its intracellular life span.

Animals↗
PubMed2005

Novel bacterial delivery system with attenuated Salmonella typhimurium carrying plasmid encoding Mtb antigen 85A for mucosal immunization: establishment of proof of principle in TB mouse model.

Tuberculosis (TB), the leading killer of young adults worldwide, newly affects one person every second and kills one in every 15 seconds. The recent increase of TB in developing countries has been exacerbated by many causes including pandemic HIV, war and political instability, drug resistance, and increasing poverty. Genetic immunization has emerged with tremendous potential in vaccination against TB with success in animal models with naked DNA encoding different genes such as Ag85A, Pst3, and hsp65. However, there are shortcomings in translating this success into reality in human clinical trials due to limitations at the level of delivery, quality, and quantity of DNA to be administered, which can be circumvented by using an attenuated bacteria delivery system for transgene vaccination for mucosal immunization targeting the inductive sites of the immune system. We compare this novel delivery system using an attenuated Salmonella delta aroA strain through a mucosal route with classic intramuscular DNA delivery using a potential protective antigen, Ag85A, of Mycobacterium tuberculosis in a mouse infection virulent challenge model. We show an immune response and superior protection in the mice at the level of the lungs as well as the spleen against a virulent challenge after intranasal immunization by recombinant Salmonella typhimurium carrying a eukaryotic expression plasmid encoding Ag85A rather than the classic DNA immunization and at par with the protection conferred by BCG. This study establishes the proof of principle of this system for further exploitation of this platform for vaccine development, which is being pursued for postexposure vaccine development for TB.

Acyltransferases↗
PubMed2004

Single mucosal, but not parenteral, immunization with recombinant adenoviral-based vaccine provides potent protection from pulmonary tuberculosis.

Bacillus Calmette-Guerin (BCG) vaccine has failed to control the global tuberculosis (TB) epidemic, and there is a lack of safe and effective mucosal vaccines capable of potent protection against pulmonary TB. A recombinant replication-deficient adenoviral-based vaccine expressing an immunogenic Mycobacterium tuberculosis Ag Ag85A (AdAg85A) was engineered and evaluated for its potential to be used as a respiratory mucosal TB vaccine in a murine model of pulmonary TB. A single intranasal, but not i.m., immunization with AdAg85A provided potent protection against airway Mycobacterium tuberculosis challenge at an improved level over that by cutaneous BCG vaccination. Systemic priming with an Ag85A DNA vaccine and mucosal boosting with AdAg85A conferred a further enhanced immune protection which was remarkably better than BCG vaccination. Such superior protection triggered by AdAg85 mucosal immunization was correlated with much greater retention of Ag-specific T cells, particularly CD4 T cells, in the lung and was shown to be mediated by both CD4 and CD8 T cells. Thus, adenoviral TB vaccine represents a promising novel vaccine platform capable of potent mucosal immune protection against TB. Our study also lends strong evidence that respiratory mucosal vaccination is critically advantageous over systemic routes of vaccination against TB.

Acyltransferases↗
PubMed2004

Comparative analysis of different vaccine constructs expressing defined antigens from Mycobacterium tuberculosis.

BACKGROUND: Studies of different vaccine constructs have demonstrated variable efficacy against Mycobacterium tuberculosis in animal models. Despite the fact that these vaccines have used one or another of a very small number of immunodominant antigens, a direct comparison of the relative efficacy of the antigens and delivery systems has been difficult, because the studies have used different parameters for assessment. METHODS: We compared the efficacies of the most commonly used vaccine constructs--adjuvanted protein, plasmid DNA, and live bacterial vectors--bearing the immunodominant secreted antigens early secreted antigen target-6 and antigen 85B, either alone or as a fusion protein. Mice were vaccinated with these constructs, and the effects of different delivery systems on protective efficacy (as assessed by survival studies and by monitoring bacterial load) and antigen-specific responses (including the contribution of CD4 and CD8 T cells to these responses) were assayed by various methods. RESULTS: The relative efficacy of different vaccines is dependent on the delivery system, the antigen, and the animal model. Likewise, the relative immunodominance of individual antigens in the fusion molecule is altered by the choice of delivery system. CONCLUSION: These results clearly demonstrate the importance of assessing vaccine function by use of multiple parameters and indicate which parameters are most reliable for assessing vaccine efficacy.

Acyltransferases↗
PubMed2004

Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and naturally acquired antimycobacterial immunity in humans.

Protective immunity against Mycobacterium tuberculosis depends on the generation of a T(H)1-type cellular immune response, characterized by the secretion of interferon-gamma (IFN-gamma) from antigen-specific T cells. The induction of potent cellular immune responses by vaccination in humans has proven difficult. Recombinant viral vectors, especially poxviruses and adenoviruses, are particularly effective at boosting previously primed CD4(+) and CD8(+) T-cell responses against a number of intracellular pathogens in animal studies. In the first phase 1 study of any candidate subunit vaccine against tuberculosis, recombinant modified vaccinia virus Ankara (MVA) expressing antigen 85A (MVA85A) was found to induce high levels of antigen-specific IFN-gamma-secreting T cells when used alone in bacille Calmette-Guerin (BCG)-naive healthy volunteers. In volunteers who had been vaccinated 0.5-38 years previously with BCG, substantially higher levels of antigen-specific IFN-gamma-secreting T cells were induced, and at 24 weeks after vaccination these levels were 5-30 times greater than in vaccinees administered a single BCG vaccination. Boosting vaccinations with MVA85A could offer a practical and efficient strategy for enhancing and prolonging antimycobacterial immunity in tuberculosis-endemic areas.

Acyltransferases↗
PubMed2004

Induction of in vivo functional Db-restricted cytolytic T cell activity against a putative phosphate transport receptor of Mycobacterium tuberculosis.

Using plasmid vaccination with DNA encoding the putative phosphate transport receptor PstS-3 from Mycobacterium tuberculosis and 36 overlapping 20-mer peptides spanning the entire PstS-3 sequence, we determined the immunodominant Th1-type CD4(+) T cell epitopes in C57BL/10 mice, as measured by spleen cell IL-2 and IFN-gamma production. Furthermore, a potent IFN-gamma-inducing, D(b)-restricted CD8(+) epitope was identified using MHC class I mutant B6.C-H-2(bm13) mice and intracellular IFN-gamma and whole blood CD8(+) T cell tetramer staining. Using adoptive transfer of CFSE-labeled, peptide-pulsed syngeneic spleen cells from naive animals into DNA vaccinated or M. tuberculosis-infected recipients, we demonstrated a functional in vivo CTL activity against this D(b)-restricted PstS-3 epitope. IFN-gamma ELISPOT responses to this epitope were also detected in tuberculosis-infected mice. The CD4(+) and CD8(+) T cell epitopes defined for PstS-3 were completely specific and not recognized in mice vaccinated with either PstS-1 or PstS-2 DNA. The H-2 haplotype exerted a strong influence on immune reactivity to the PstS-3 Ag, and mice of the H-2(b, p, and f) haplotype produced significant Ab and Th1-type cytokine levels, whereas mice of H-2(d, k, r, s, and q) haplotype were completely unreactive. Low responsiveness against PstS-3 in MHC class II mutant B6.C-H-2(bm12) mice could be overcome by DNA vaccination. IFN-gamma-producing CD8(+) T cells could also be detected against the D(b)-restricted epitope in H-2(p) haplotype mice. These results highlight the potential of DNA vaccination for the induction and characterization of CD4(+) and particularly CD8(+) T cell responses against mycobacterial Ags.

ATP-Binding Cassette Transporters↗