• Neutral Lipopolyplexes for In Vivo Delivery of Conventional and Replicative RNA Vaccine.

      Perche, Federico; Clemençon, Rudy; Schulze, Kai; Ebensen, Thomas; Guzmán, Carlos A; Pichon, Chantal; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier (Cell Press), 2019-09-06)
      Nucleic acid vaccination relies on injecting DNA or RNA coding antigen(s) to induce a protective immune response. RNA vaccination is being increasingly used in preclinical and clinical studies. However, few delivery systems have been reported for in vivo delivery of RNA of different sizes. Using a tripartite formulation with RNA, cationic polymer, and anionic liposomes, we were able to encapsulate RNA into neutral lipopolyplexes (LPPs). LPPs were stable in vitro and successfully delivered conventional RNA and replicative RNA to dendritic cells in cellulo. Their injection led to reporter gene expression in mice. Finally, administration of LPP-Replicon RNA (RepRNA) led to an adaptive immune response against the antigen coded by the RepRNA. Accordingly, LPPs may represent a universal formulation for RNA delivery.
    • A new adjuvanted nanoparticle-based H1N1 influenza vaccine induced antigen-specific local mucosal and systemic immune responses after administration into the lung.

      Neuhaus, Vanessa; Chichester, Jessica A; Ebensen, Thomas; Schwarz, Katharina; Hartman, Caitlin E; Shoji, Yoko; Guzmán, Carlos A; Yusibov, Vidadi; Sewald, Katherina; Braun, Armin (2014-05-30)
      Annually influenza virus infections are responsible for hospitalization and mortality, especially in high risk groups. Constant antigenic changes in seasonal influenza viruses resulted from antigenic shifts and antigenic drifts, enable emerging of novel virus subtypes that may reduce current vaccine efficacy and impose the continuous revision of vaccine component. Currently available vaccines are usually limited by their production processes in terms of rapid adaptation to new circulating subtypes in high quantities meeting the global demand. Thus, new approaches to rapidly manufacture high yields of influenza vaccines are required. New technologies to reach maximal protection with minimal vaccine doses also need to be developed. In this study, we evaluated the systemic and local immunogenicity of a new double-adjuvanted influenza vaccine administered at the site of infection, the respiratory tract. This vaccine combines a plant-produced H1N1 influenza hemagglutinin antigen (HAC1), a silica nanoparticle-based (SiO₂) drug delivery system and the mucosal adjuvant candidate bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP). Mice were vaccinated by intratracheal route with HAC1/SiO₂ or HAC1/c-di-GMP (single-adjuvanted vaccine) or HAC1/SiO₂/c-di-GMP (double-adjuvanted vaccine) and evaluated for target-specific immune responses, such as hemagglutination inhibition and hemagglutinin-specific IgG titers, as well as local antibody (IgG and IgA) titers in the bronchoalveolar lavage (BAL). Furthermore, the HAC1-specific T-cell re-stimulation potential was assessed using precision-cut lung slices (PCLS) of vaccinated mice. The double-adjuvanted vaccine induced high systemic antibody responses comparable to the systemic vaccination control. In addition, it induced local IgG and IgA responses in the BAL. Furthermore, HAC1 induced a local T-cell response demonstrated by elevated IL-2 and IFN-γ levels in PCLS of c-di-GMP-vaccinated mice upon re-stimulation. Overall, the present study showed the potential of the double-adjuvanted vaccine to induce systemic humoral immune responses in intratracheally vaccinated mice. Furthermore, it induced a strong mucosal immune response, with evidence of antigen-primed T-cells in the lung.
    • Next Generation Influenza Vaccines: Looking into the Crystal Ball.

      Guzmán, Carlos Alberto; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-08-21)
      Influenza infections are responsible for significant number of deaths and overwhelming costs worldwide every year. Vaccination represents the only cost-efficient alternative to address this major problem in human health. However, current vaccines are fraught by many limitations, being far from optimal. Among them, the need to upgrade vaccines every year through a time-consuming process open to different caveats, and the critical fact that they exhibit poorer efficacy in individuals who are at high risk for severe infections. Where are we? How can knowledge and technologies contribute towards removing current roadblocks? What does the future offer in terms of next generation vaccines?
    • The NKT cell ligand αgalactosylceramide suppresses allergic airway inflammation by induction of a Th1 response.

      Knothe, S; Mutschler, V; Rochlitzer, S; Winkler, C; Ebensen, T; Guzman, C A; Hohlfeld, J; Braun, A; Muller, M; Fraunhofer Institute for Toxicology and Experimental Medicine, Department of Immunology, Allergology and Immunotoxicology, Nikolai-Fuchs-Str. 1, 30625 Hannover, Germany. (2011-06-06)
      One experimental approach for the treatment of allergic reactions is the stimulation of immunoregulatory NKT cells with the synthetic glycolipid αgalactosylceramide. For a first evaluation of the immunomodulatory potential of αGalCerMPEG a human in vitro allergy model was exploited. Acting as an adjuvant, the glycolipid induced an enhanced Th1-biased allergen-specific immune response of autologous lymphocytes. In a mouse model of allergic airway inflammation, αGalCerMPEG-activated NKT cells promoted a cytokine environment in the spleen, leading to priming of Th1 cells. The shift towards a Th1-dominated allergen-specific immune response thus might mediate the abrogation of allergic airway inflammation and thereby might provide a valid option for therapeutic intervention.
    • NKT cell stimulation with α-galactosylceramide results in a block of Th17 differentiation after intranasal immunization in mice.

      Zygmunt, Beata M; Weissmann, Sebastian F; Guzman, Carlos A; Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany. beata.zygmunt@bbsrc.ac.uk (2012)
      In a previous study we demonstrated that intranasal (i.n.) vaccination promotes a Th17 biased immune response. Here, we show that co-administration of a pegylated derivative of α-galactosylceramide (αGCPEG) with an antigen, even in the presence of Th17-polarizing compounds, results in a strong blocking of Th17 differentiation. Additional studies demonstrated that this phenomenon is specifically dependent on soluble factors, like IL-4 and IFNγ, which are produced by NKT cells. Even NK1.1 negative NKT cells, which by themselves produce IL-17A, are able to block Th17 differentiation. It follows that the use of αGCPEG as adjuvant would enable to tailor Th17 responses, according to the specific clinical needs. This knowledge expands our understanding of the role played by NKT cells in overall control of the cytokine microenvironment, as well as in the overall shaping of adaptive immune responses.
    • NKT cell stimulation with α-galactosylceramide results in a block of Th17 differentiation after intranasal immunization in mice.

      Zygmunt, Beata M; Weissmann, Sebastian F; Guzman, Carlos A; Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany. beata.zygmunt@bbsrc.ac.uk (2012)
      In a previous study we demonstrated that intranasal (i.n.) vaccination promotes a Th17 biased immune response. Here, we show that co-administration of a pegylated derivative of α-galactosylceramide (αGCPEG) with an antigen, even in the presence of Th17-polarizing compounds, results in a strong blocking of Th17 differentiation. Additional studies demonstrated that this phenomenon is specifically dependent on soluble factors, like IL-4 and IFNγ, which are produced by NKT cells. Even NK1.1 negative NKT cells, which by themselves produce IL-17A, are able to block Th17 differentiation. It follows that the use of αGCPEG as adjuvant would enable to tailor Th17 responses, according to the specific clinical needs. This knowledge expands our understanding of the role played by NKT cells in overall control of the cytokine microenvironment, as well as in the overall shaping of adaptive immune responses.
    • Oral vaccination with Salmonella enterica as a cruzipain-DNA delivery system confers protective immunity against Trypanosoma cruzi.

      Cazorla, Silvia I; Becker, Pablo D; Frank, Fernanda M; Ebensen, Thomas; Sartori, María J; Corral, Ricardo S; Malchiodi, Emilio L; Guzmán, Carlos A; Instituto de Estudios de la Inmunidad Humoral, CONICET-UBA, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina. (2008-01)
      To stimulate both local and systemic immune responses against Trypanosoma cruzi, Salmonella enterica serovar Typhimurium aroA was exploited as a DNA delivery system for cruzipain (SCz). In a murine model we compared SCz alone (GI) or coadministered with Salmonella carrying a plasmid encoding granulocyte-macrophage colony-stimulating factor (GII), as well as protocols in which SCz priming was followed by boosting with recombinant cruzipain (rCz) admixed with either CpG-ODN (GIII) or MALP-2, a synthetic derivative of a macrophage-activating lipopeptide of 2 kDa from Mycoplasma fermentans (GIV). The results showed that protocols that included four oral doses of SCz (GI) elicited mainly a mucosal response characterized by immunoglobulin A (IgA) secretion and proliferation of gut-associated lymphoid tissue cells, with weak systemic responses. In contrast, the protocol that included a boost with rCz plus CpG (GIII) triggered stronger systemic responses in terms of Cz-specific serum IgG titers, splenocyte proliferation, gamma interferon (IFN-gamma) secretion, and delayed-type hypersensitivity response. Trypomastigote challenge of vaccinated mice resulted in significantly lower levels of parasitemia compared to controls. Protection was abolished by depletion of either CD4+ or CD8+ T cells. Parasite control was also evident from the reduction of tissue damage, as revealed by histopathologic studies and serum levels of enzymes that are markers of muscle injury in chronic Chagas' disease (i.e., creatine kinase, aspartate aminotransferase, and lactate dehydrogenase). Enhanced release of IFN-gamma and interleukin-2 was observed in GI and GII upon restimulation of splenocytes in the nonparasitic phase of infection. Our results indicate that Salmonella-mediated delivery of Cz-DNA by itself promotes the elicitation of an immune response that controls T. cruzi infection, thereby reducing parasite loads and subsequent damage to muscle tissues.
    • PD-1 expression affects cytokine production by ILC2 and is influenced by peroxisome proliferator-activated receptor-γ.

      Batyrova, Banu; Luwaert, Fien; Maravelia, Panagiota; Miyabayashi, Yuria; Vashist, Neha; Stark, Julian M; Soori, Sara Y; Tibbitt, Christopher A; Riese, Peggy; Coquet, Jonathan M; et al. (Wiley, 2019-11-19)
      Innate lymphoid cells (ILCs) can provide early cytokine help against a variety of pathogens in the lungs and gastrointestinal tract. Type 2 ILC (ILC2) are comparable to T helper 2 cells found in the adaptive immune system, which secrete cytokines such as interleukin 5 (IL-5) and IL-13 and have been found to play roles in host defense against helminth infections and in allergic responses. Recent studies have identified that programmed cell death protein 1 (PD-1) and peroxisome proliferator activated receptor-γ (PPAR-γ) are highly expressed by ILC2. We examined whether PD-1 plays a role in ILC2 function and whether there was any connection between PD-1 and PPAR-γ METHODS: To ensure that only innate immune cells were present, ILC2 cells were examined from RAG1-/- and PD-1-/- xRAG1-/- mice under steady-state or following inoculation with IL-33. We also tested ILC2 generated from bone marrow of RAG1-/- and PD-1-/- xRAG1-/- mice for their production of cytokines. These in vitro-derived ILC2 were also exposed to agonist and antagonist of PPAR-γ.
    • Peptidoglycan-treated tumor antigen-pulsed dendritic cells impart complete resistance against tumor rechallenge.

      Patidar, A; Selvaraj, S; Chauhan, P; Guzman, C A; Ebensen, T; Sarkar, A; Chattopadhyay, D; Saha, B (2020-06-26)
    • Peritoneal Cavity Is Dominated by IFNγ-Secreting CXCR3 Th1 Cells.

      Zygmunt, Beata M; Groebe, Lothar; Guzman, Carlos A; Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2011)
      The chemokine receptor CXCR3, which was shown to take part in many inflammatory processes, is considered as a Th1 specific marker. Here, we show in a mouse model that CXCR3 expressing CD4(+) cells preferentially migrate to the peritoneal cavity under steady-state conditions. The peritoneal cavity milieu leads to an up-regulated expression of CXCR3. However, blocking of known ligands of this chemokine receptor did not alter the preferential migration. The peritoneal cavity environment also results in an increased percentage of memory cells producing cytokines. Up-regulation of IFNγ production occurs mostly in CXCR3(+) cells considered as Th1, whereas the up-regulation of IL-4 affects mostly in CXCR3(-) cells which are considered as Th2. We conclude that the peritoneal cavity does not change the Th-lineage of the cells, but that domination of this anatomic niche by Th1 cells rather results from preferential migration to this compartment.
    • Peritoneal cavity is dominated by IFNγ-secreting CXCR3+ Th1 cells.

      Zygmunt, Beata M; Groebe, Lothar; Guzman, Carlos A; Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2011)
      The chemokine receptor CXCR3, which was shown to take part in many inflammatory processes, is considered as a Th1 specific marker. Here, we show in a mouse model that CXCR3 expressing CD4(+) cells preferentially migrate to the peritoneal cavity under steady-state conditions. The peritoneal cavity milieu leads to an up-regulated expression of CXCR3. However, blocking of known ligands of this chemokine receptor did not alter the preferential migration. The peritoneal cavity environment also results in an increased percentage of memory cells producing cytokines. Up-regulation of IFNγ production occurs mostly in CXCR3(+) cells considered as Th1, whereas the up-regulation of IL-4 affects mostly in CXCR3(-) cells which are considered as Th2. We conclude that the peritoneal cavity does not change the Th-lineage of the cells, but that domination of this anatomic niche by Th1 cells rather results from preferential migration to this compartment.
    • Phylogenetic Analysis of Previously Nontypeable Hepatitis C Virus Isolates from Argentina†

      Gismondi, María Inés; Becker, Pablo Daniel; Valva, Pamela; Guzmán, Carlos Alberto; Preciado, María Victoria (American Society for Microbiology, 2006-06)
    • Pidotimod promotes functional maturation of dendritic cells and displays adjuvant properties at the nasal mucosa level.

      Giagulli, Cinzia; Noerder, Miriam; Avolio, Manuela; Becker, Pablo D; Fiorentini, Simona; Guzman, Carlos A; Caruso, Arnaldo; Department of Experimental and Applied Medicine, Section of Microbiology, University of Brescia, Medical School, Brescia, Italy. (2009-11)
      Mucosal dendritic cells (DCs) are very important in the process of antigen presentation to T cells, playing a key role in the induction of primary and secondary immune responses. Pidotimod is a synthetic substance capable of modulating immune cell functions, but the effect of pidotimod on human DCs has not been investigated yet. Here we demonstrate the ability of pidotimod to induce DC maturation and up-regulate the expression of HLA-DR and co-stimulatory molecules CD83 and CD86, which are fundamental for communication with adaptative immunity cells. Pidotimod also stimulated DCs to release high amounts of pro-inflammatory molecules such as MCP-1 and TNF-alpha cytokines and to drive T cell proliferation and differentiation towards a Th1 phenotype. Moreover, we demonstrate that pidotimod in vivo promotes strong and specific humoral and cellular immune response when co-administered intranasally with a model antigen. Taken together our data suggest the possibility to use pidotimod as adjuvant molecule to facilitate the activation of the innate immune system as well as to promote an effective mucosal and systemic immune response.
    • Porcine pulmonary angiotensin I-converting enzyme--biochemical characterization and spatial arrangement of the N- and C-domains by three-dimensional electron microscopic reconstruction.

      Chen, Hui-Ling; Lünsdorf, Heinrich; Hecht, Hans-Jürgen; Tsai, Hsin; Development Center for Biotechnology, Taipei County 221, Taiwan, ROC. (2010-08)
      The somatic angiotensin I-converting enzyme (sACE; peptidyl-dipeptidase A; EC 3.4.15.1) was isolated from pig lung and purified to homogeneity. The purified enzyme has a molecular mass of about 180 kDa. Upon proteolytic cleavage, two approximately 90 kDa fragments were obtained and identified by amino-terminal sequence analysis as the N- and C-domains of sACE. Both purified domains were shown to be catalytically active. A 2.3 nm resolution model of sACE was obtained by three-dimensional electron microscopic reconstruction of negatively stained sACE particles, based on atomic X-ray data fitting. Our model shows for the first time the relative orientation of the sACE catalytically active domains and their spatial distance.
    • Prime-boost immunization with cruzipain co-administered with MALP-2 triggers a protective immune response able to decrease parasite burden and tissue injury in an experimental Trypanosoma cruzi infection model.

      Cazorla, SI; Frank, FM; Becker, PD; Corral, RS; Guzmán, CA; Malchiodi, EL; Cátedra de Inmunología and Instituto de Estudios de la Inmunidad Humoral (IDEHU), CONICET-UBA, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junin 956 4to P, 1113 Buenos Aires, Argentina; Departamento de Microbiología, Parasitología e Inmunología, Facultad de Medicina, Universidad de Buenos Aires, Argentina; Department of Vaccinology, Helmholtz Centre for Infection Research, Inhoffenstraße 7, D-38124 Braunschweig, Germany. (2008-04-07)
      Cruzipain (Cz), a key Trypanosoma cruzi enzyme, is a main candidate antigen for vaccines against Chagas' disease. We evaluated a vaccination protocol based on intradermal priming with recombinant Cz and intranasal boosting with rCz co-administered with a derivative of the TLR2/6 agonist MALP-2. Vaccination triggered strong systemic and mucosal antibody responses, and a vigorous cell-mediated immunity characterized by lymphoproliferation, DTH reactivity and IFN-gamma production. The immune responses protected against a lethal trypomastigote challenge and, upon sub-lethal infection, immunized mice showed reduction of tissue damage and normal enzymatic markers of muscle injury. This prime-boost regimen appears promising for further development, since warranted survival, provided efficient control of parasite load and restricted inflammatory myopathy.
    • A prime-boost vaccination protocol optimizes immune responses against the nucleocapsid protein of the SARS coronavirus.

      Schulze, Kai; Staib, Caroline; Schätzl, Hermann M; Ebensen, Thomas; Erfle, Volker; Guzman, Carlos A; Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. (2008-12-02)
      Severe acute respiratory syndrome (SARS) is a serious infectious disease caused by the SARS coronavirus. We assessed the potential of prime-boost vaccination protocols based on the nucleocapsid (NC) protein co-administered with a derivative of the mucosal adjuvant MALP-2 or expressed by modified Vaccinia virus Ankara (MVA-NC) to stimulate humoral and cellular immune responses at systemic and mucosal levels. The obtained results demonstrated that strong immune responses can be elicited both at systemic and mucosal levels following a heterologous prime-boost vaccination protocol consisting in priming with NC protein add-mixed with MALP-2 by intranasal route and boosting with MVA-NC by intramuscular route.
    • The proneurotrophin receptor sortilin is required for Mycobacterium tuberculosis control by macrophages.

      Vázquez, Cristina L; Rodgers, Angela; Herbst, Susanne; Coade, Stephen; Gronow, Achim; Guzman, Carlos A; Wilson, Mark S; Kanzaki, Makoto; Nykjaer, Anders; Gutierrez, Maximiliano G; et al. (2016)
      Sorting of luminal and membrane proteins into phagosomes is critical for the immune function of this organelle. However, little is known about the mechanisms that contribute to the spatiotemporal regulation of this process. Here, we investigated the role of the proneurotrophin receptor sortilin during phagosome maturation and mycobacterial killing. We show that this receptor is acquired by mycobacteria-containing phagosomes via interactions with the adaptor proteins AP-1 and GGAs. Interestingly, the phagosomal association of sortilin is critical for the delivery of acid sphingomyelinase (ASMase) and required for efficient phagosome maturation. Macrophages from Sort1(-/-) mice are less efficient in restricting the growth of Mycobacterium bovis BCG and M. tuberculosis. In vivo, Sort1(-/-) mice showed a substantial increase in cellular infiltration of neutrophils in their lungs and higher bacterial burden after infection with M. tuberculosis. Altogether, sortilin defines a pathway required for optimal intracellular mycobacteria control and lung inflammation in vivo.
    • Prophylactic Multi-Subunit Vaccine against Chlamydia trachomatis In Vivo Evaluation in Mice.

      Lanfermann, Christian; Wintgens, Sebastian; Ebensen, Thomas; Kohn, Martin; Laudeley, Robert; Schulze, Kai; Rheinheimer, Claudia; Hegemann, Johannes H; Guzman, Carlos Alberto; Klos, Andreas; et al. (MDPI, 2021-06-06)
      Chlamydia trachomatis is the most frequent sexually-transmitted disease-causing bacterium. Urogenital serovars of this intracellular pathogen lead to urethritis and cervicitis. Ascending infections result in pelvic inflammatory disease, salpingitis, and oophoritis. One of 200 urogenital infections leads to tubal infertility. Serovars A-C cause trachoma with visual impairment. There is an urgent need for a vaccine. We characterized a new five-component subunit vaccine in a mouse vaccination-lung challenge infection model. Four recombinant Pmp family-members and Ctad1 from C. trachomatis serovar E, all of which participate in adhesion and binding of chlamydial elementary bodies to host cells, were combined with the mucosal adjuvant cyclic-di-adenosine monophosphate. Intranasal application led to a high degree of cross-serovar protection against urogenital and ocular strains of C. trachomatis, which lasted at least five months. Critical evaluated parameters were body weight, clinical score, chlamydial load, a granulocyte marker and the cytokines IFN-γ/TNF-α in lung homogenate. Vaccine antigen-specific antibodies and a mixed Th1/Th2/Th17 T cell response with multi-functional CD4+ and CD8+ T cells correlate with protection. However, serum-transfer did not protect the recipients suggesting that circulating antibodies play only a minor role. In the long run, our new vaccine might help to prevent the feared consequences of human C. trachomatis infections.
    • PspA can form large scaffolds in Escherichia coli.

      Standar, Kerstin; Mehner, Denise; Osadnik, Hendrik; Berthelmann, Felix; Hause, Gerd; Lünsdorf, Heinrich; Brüser, Thomas; Institute of Biology/Microbiology, University of Halle-Wittenberg, Kurt-Mothes-Strasse 3, D-06120 Halle, Germany. (2008-10-29)
      The phage shock protein A (PspA) of Escherichia coli stabilizes the cytoplasmic membrane under stress conditions. Here we demonstrate that PspA can form hollow spherical or prolate spheroidal particles of about 30-40nm diameter with a scaffold-like arrangement of protein subunits at the surface. The 'PspA-scaffold' is the basic structure that is common to all particles. The PspA-scaffold may be of fundamental importance, as it could allow PspA to stabilize the integrity of membranes through numerous contact points over a large surface area.
    • Quantitative reverse transcription polymerase chain reaction analysis of Vibrio cholerae cells entering the viable but non-culturable state and starvation in response to cold shock.

      González-Escalona, Narjol; Fey, Axel; Höfle, Manfred G; Espejo, Romilio T; A Guzmán, Carlos; Vaccine Research Group, Division of Microbiology, GBF-German Research Centre for Biotechnology, Braunschweig, Germany. (2006-04)
      We performed a comparative analysis of the Vibrio cholerae strain El Tor 3083 entering the viable but non-culturable (VBNC) state and starvation after incubation in artificial seawater (ASW) at 4 and 15 degrees C respectively. To this end, we determined bacterial culturability and membrane integrity, as well as the cellular levels of 16S rRNA and mRNA for the tuf, rpoS and relA genes, which were assessed by real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR). Bacterial cells entering the VBNC state showed a 154, 5.1 x 10(3), 24- and 23-fold reduction in the number of copies of 16S rRNA and mRNA for tuf, rpoS and relA, in comparison to exponentially growing cells. The differences were less striking between cells in the VBNC and starvation states. The mRNA for relA was selectively increased in VBNC cells (3.2-folds), whereas a 3.9-fold reduction was observed for 16S rRNA. The obtained results confirmed that key activities of the cellular metabolism (i.e. tuf representing protein synthesis, and relA or rpoS stress response) were still detected in bacteria entering the VBNC state and starvation. These data suggest that the new Q-RT-PCR methodology, based on the selected RNA targets, could be successfully exploited for the identification (rRNA) of V. cholerae and assessment of its metabolic activity (tuf, rpoS, relA mRNA) in environmental samples.