• The bacterial second messenger cdiGMP exhibits promising activity as a mucosal adjuvant.

      Ebensen, Thomas; Schulze, Kai; Riese, Peggy; Morr, Michael; Guzmán, Carlos A; Department of Vaccinology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. (2007-08)
      The development of mucosal adjuvants is still a critical need in vaccinology. In the present work, we show that bis(3',5')-cyclic dimeric GMP (cdiGMP), a second messenger that modulates cell surface properties of several microorganisms, exerts potent activity as a mucosal adjuvant. BALB/c mice were immunized intranasally with the model antigen beta-galactosidase (beta-Gal) coadministered with cdiGMP. Animals receiving cdiGMP as an adjuvant showed significantly higher anti-beta-Gal immunoglobulin G (IgG) titers in sera than controls (i.e., 512-fold [P < 0.05]). Coadministration of cdiGMP also stimulated efficient beta-Gal-specific secretory IgA production in the lung (P < 0.016) and vagina (P < 0.036). Cellular immune responses were observed in response to both the beta-Gal protein and a peptide encompassing its major histocompatibility complex class I-restricted epitope. The IgG1-to-IgG2a ratio of anti-beta-Gal antibodies and the observed profiles of secreted cytokines suggest that a dominant Th1 response pattern is promoted by mucosal coadministration of cdiGMP. Finally, the use of cdiGMP as a mucosal adjuvant also led to the stimulation of in vivo cytotoxic T-lymphocyte responses in C57BL/6 mice intranasally immunized with ovalbumin and cdiGMP (up to 30% of specific lysis). The results obtained indicate that cdiGMP is a promising tool for the development of mucosal vaccines.
    • Cyclic di-nucleotides: new era for small molecules as adjuvants.

      Libanova, Rimma; Becker, Pablo D; Guzmán, Carlos A; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2012-03)
      The implementation of vaccination as an empiric strategy to protect against infectious diseases was introduced even before the advent of hygiene and antimicrobials in the medical practice. Nevertheless, it was not until a few decades ago that we really started understanding the underlying mechanisms of protection triggered by vaccination. Vaccines were initially based on attenuated or inactivated organisms. Subunit vaccines were then introduced as more refined formulations, exhibiting improved safety profiles. However, purified antigens tend to be poorly immunogenic and often require the use of adjuvants to achieve adequate stimulation of the immune system. Vaccination strategies, such as mucosal administration, also require potent adjuvants to improve performance. In the 1990s, immunologists found that pathogens could be sensed as 'danger signals' by receptors recognizing conserved motifs. Although our knowledge is still limited, tremendous advances were made in the understanding of host defence mechanisms regulated by these evolutionary conserved receptors, and the molecular structures which are recognized by them. This opened a new era in adjuvant development. Some of the latest players arrived to this field are the cyclic di-nucleotides, which are ubiquitous prokaryotic intracellular signalling molecules. This review is focused on their potential for the development of vaccines and immunotherapies.
    • Evaluation of the sublingual route for administration of influenza H5N1 virosomes in combination with the bacterial second messenger c-di-GMP.

      Pedersen, Gabriel Kristian; Ebensen, Thomas; Gjeraker, Ingrid Hjetland; Svindland, Signe; Bredholt, Geir; Guzmán, Carlos Alberto; Cox, Rebecca Jane; The Gade Institute, University of Bergen, Norway. gabriel.pedersen@gades.uib.no (2011)
      Avian influenza A H5N1 is a virus with pandemic potential. Mucosal vaccines are attractive as they have the potential to block viruses at the site of entry, thereby preventing both disease and further transmission. The intranasal route is safe for the administration of seasonal live-attenuated influenza vaccines, but may be less suitable for administration of pandemic vaccines. Research into novel mucosal routes is therefore needed. In this study, a murine model was used to compare sublingual administration with intranasal and intramuscular administration of influenza H5N1 virosomes (2 µg haemagglutinin; HA) in combination with the mucosal adjuvant (3',5')-cyclic dimeric guanylic acid (c-di-GMP). We found that sublingual immunisation effectively induced local and systemic H5N1-specific humoral and cellular immune responses but that the magnitude of response was lower than after intranasal administration. However, both the mucosal routes were superior to intramuscular immunisation for induction of local humoral and systemic cellular immune responses including high frequencies of splenic H5N1-specific multifunctional (IL-2+TNF-α+) CD4+ T cells. The c-di-GMP adjuvanted vaccine elicited systemic haemagglutination inhibition (HI) antibody responses (geometric mean titres ≥ 40) both when administered sublingually, intranasally and inramuscularly. In addition, salivary HI antibodies were elicited by mucosal, but not intramuscular vaccination. We conclude that the sublingual route is an attractive alternative for administration of pandemic influenza vaccines.
    • Immune modulator adamantylamide dipeptide stimulates efficient major histocompatibility complex class I-restricted responses in mice.

      Becker, Pablo D; Nörder, Miriam; Guzmán, Carlos Alberto; Grinstein, Saul; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2007-05)
      Adamantylamide L-alanyl-D-isoglutamine (AdDP) is a synthetic adjuvant which belongs to the family of the desmuramyl peptides. AdDP exerts its adjuvant properties when it is administered either by the parenteral or by the mucosal route, leading to the elicitation of strong humoral responses at both the systemic and the mucosal levels. However, very little is known about the effect of AdDP on cellular immunity. Here we demonstrate that AdDP is able to stimulate cellular responses, which are characterized by the release of gamma interferon by CD8+ T cells when they are restimulated with a major histocompatibility complex class I-restricted peptide and strong in vivo lymphocyte-mediated cytotoxic activity. The capacity of AdDP to stimulate the elicitation of both cellular and humoral adaptive responses makes this adjuvant a promising tool for the development of mucosal vaccine formulations.
    • Immune modulators with defined molecular targets: cornerstone to optimize rational vaccine design.

      Ebensen, Thomas; Guzmán, Carlos A; Department of Vaccinology, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2011-03-02)
      Vaccination remains the most valuable tool for preventing infectious diseases. However, the performance of many existing vaccines should be improved and there are diseases for which vaccines are still not available. The use of well-defined antigens for the generation of subunit vaccines has led to products with an improved safety profile. However, purified antigens are usually poorly immunogenic, making essential the use of adjuvants. Despite the fact that adjuvants have been used to increase the immunogenicity of vaccines for more than 70 years, only a handful has been licensed for human use (e.g., aluminium salts, the micro-fluidized squalene-in-water emulsion MF59 and monophosphoryl lipid A). Thus, the development of new adjuvants which are able to promote broad and sustained immune responses at systemic and mucosal levels still remains as a major challenge in vaccinology. Recent advances in our understanding of the immune system have facilitated the identification of new biological targets for screening programs aimed at the discovery of novel immune stimulators. This resulted in the identification of new candidate adjuvants, which made possible the modulation of the immune responses elicited according to specific needs. A number of promising adjuvants which are currently under preclinical or clinical development will be described in this review.
    • Intramammary application of non-methylated-CpG oligodeoxynucleotides (CpG) inhibits both local and systemic mammary carcinogenesis in female BALB/c Her-2/neu transgenic mice.

      Mastini, Cristina; Becker, Pablo D; Iezzi, Manuela; Curcio, Claudia; Musiani, Piero; Forni, Guido; Cavallo, Federica; Guzmán, Carlos A; Molecular Biotechnology Center, Department of Clinical and Biological Sciences, University of Torino, 10126 Torino, Italy. (2008-05)
      CpG are powerful drugs activating the innate immune system. In this study, the ability of their intramammary administration in impeding the devastating progression of carcinogenesis in all the mammary glands of female BALB/c mice transgenic for the rat neu transforming oncogene was assessed. Starting when in situ carcinomas were scattered over all their mammary glands (week 10), mice received CpG injections in the stroma of the fourth left gland. Local neoplastic progression was inhibited by six monthly administrations. CpG not only delayed the onset of carcinomas in the injected gland, but also hampered their progression. Extended latency was observed for tumors in glands both close to and far from the injection site. When the experiment ended (week 45), no tumors were palpable in 67% of the injected glands and a markedly impaired tumor growth was evident in the others. An impressive local infiltrate of CD11b(+) cells with the morphologic features of macrophages, plasma cells, B220(+) B cells, and CD4(+) and CD8(+) T cells was quickly recruited to the CpG-treated glands. High quantities of IFN-gamma producing cells were only present in the ipsilateral axillary draining lymph nodes of the treated glands. Enhanced natural killer (NK) lytic activity was also detected in the spleens. Inhibition of progression was weaker when only four injections were given, and abolished by in vivo depletion of NK cells. CpG monotherapy is thus effective in an aggressive model of autochthonous cancer. The results strongly support the administration of CpG as a local monotherapy of multiple invasive microscopic lesions.
    • Intranasal vaccination with recombinant outer membrane protein CD and adamantylamide dipeptide as the mucosal adjuvant enhances pulmonary clearance of Moraxella catarrhalis in an experimental murine model.

      Becker, Pablo D; Bertot, Gustavo M; Souss, David; Ebensen, Thomas; Guzmán, Carlos A; Grinstein, Saúl; Virology Laboratory, Ricardo Gutiérrez Children's Hospital, Gallo 1330, 1425 Buenos Aires, Argentina. (2007-04)
      Moraxella catarrhalis causes acute otitis media in children and lower respiratory tract infections in adults and elderly. In children the presence of antibodies against the highly conserved outer membrane protein CD correlates with protection against infection, suggesting that this protein may be useful as a vaccine antigen. However, native CD is difficult to purify, and it is still unclear if recombinant CD (rCD) is a valid alternative. We performed a side-by-side comparison of the immunogenicities and efficacies of vaccine formulations containing native CD and rCD with adamantylamide dipeptide as the mucosal adjuvant. Intranasal vaccination of mice stimulated the production of high CD-specific antibody titers in sera and of secretory immunoglobulin A in mucosal lavages, which cross-recognized both antigens. While vaccination with native CD increased the number of interleukin-2 (IL-2)- and gamma interferon-producing cells, rCD mainly stimulated IL-4-secreting cells. Nevertheless, efficient bacterial clearance was observed in the lungs of challenged mice receiving native CD and in the lungs of challenged mice receiving rCD (96% and 99%, respectively). Thus, rCD is a promising candidate for incorporation in vaccine formulations for use against M. catarrhalis.
    • Modified vaccinia virus Ankara exerts potent immune modulatory activities in a murine model.

      Nörder, Miriam; Becker, Pablo D; Drexler, Ingo; Link, Claudia; Erfle, Volker; Guzmán, Carlos A; Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2010)
      BACKGROUND: Modified vaccinia virus Ankara (MVA), a highly attenuated strain of vaccinia virus, has been used as vaccine delivery vector in preclinical and clinical studies against infectious diseases and malignancies. Here, we investigated whether an MVA which does not encode any antigen (Ag) could be exploited as adjuvant per se. METHODOLOGY/PRINCIPAL FINDINGS: We showed that dendritic cells infected in vitro with non-recombinant (nr) MVA expressed maturation and activation markers and were able to efficiently present exogenously pulsed Ag to T cells. In contrast to the dominant T helper (Th) 1 biased responses elicited against Ags produced by recombinant MVA vectors, the use of nrMVA as adjuvant for the co-administered soluble Ags resulted in a long lasting mixed Th1/Th2 responses. CONCLUSIONS/SIGNIFICANCE: These findings open new ways to potentiate and modulate the immune responses to vaccine Ags depending on whether they are co-administered with MVA or encoded by recombinant viruses.
    • 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.
    • 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.