Donata Medaglini

Surface Display of Recombinant Proteins on Bacillus subtilis Spores

We developed a novel surface display system based on the use of bacterial spores. A protein of the Bacillus subtilis spore coat, CotB, was found to be located on the spore surface and used as fusion partner to express the 459-amino-acid C-terminal fragment of the tetanus toxin (TTFC). Western, dot blot and fluorescent-activated cell sorting analyses were used to monitor TTFC surface expression on purified spores. We estimated that more than 1.5 x 10(3) TTFC molecules were exposed on the surface of each spore and recognized by TTFC-specific antibodies. The efficient surface presentation of the heterologous protein, together with the simple purification procedure and the high stability and safety record of B. subtilis spores, makes this spore-based display system a potentially powerful approach for surface expression of bioactive molecules.

Commensal bacteria as vectors for mucosal vaccines against sexually transmitted diseases: vaginal colonization with recombinant streptococci induces local and systemic antibodies in mice

There is a need to develop vaccines to control the spread of sexually transmitted diseases (STDs). Novel immunization strategies that elicit a mucosal immune response in the genital tract, may show improved protection by preventing or at least limiting entry of the pathogenic micro-organism. However, it has proven difficult to obtain a local immune response in the vaginal mucosa. Our approach is based on the use of recombinant bacteria capable of colonizing mucosal surfaces as live vaccine vectors. The human commensal Streptococcus gordonii, engineered to express the E7 protein of human papillomavirus type 16, was used for intravaginal immunization of mice. A single inoculum of recombinant bacteria was sufficient to establish colonization of the murine vagina and therefore induce papillomavirus-specific vaginal IgA and serum IgG. Evidence that mucosal colonization with recombinant commensal bacteria can induce a local immune response in the female genital tract represents a significant step toward the development of new vaccines against STDs.

Immunization with recombinant Streptococcus gordonii expressing tetanus toxin fragment C confers protection from lethal challenge in mice.

Tetanus toxin fragment C (TTFC) was expressed on the surface of the vaccine vector Streptococcus gordonii, a Gram-positive commensal bacterium of the human oral cavity. The immunogenicity of recombinant S. gordonii expressing TTFC was assayed in mice immunized by the parenteral and mucosal routes. High serum TTFC-specific IgG responses were induced in both BALB/c and C57BL/6 mice immunized subcutaneously. A total of 82% of vaccinated BALB/c mice were protected from the lethal challenge with 50 LD(50) of tetanus toxin (TT) and a direct correlation between the serum TTFC-specific IgG concentration and survival time of unprotected animals was observed. Intranasal immunization of BALB/c mice was also effective in inducing TTFC-specific serum IgG and local IgA in lung washes. Furthermore, 38% of animals immunized intranasally were protected from the lethal challenge with 10 LD(50) of TT while all control animals died within 24 h. Analysis of the serum IgG subclasses showed that the IgG1 subclass was predominant after parenteral immunization in BALB/c mice (IgG1/IgG2a ratio congruent with6) while following mucosal immunization a mixed IgG1 and IgG2a pattern (IgG1/IgG2a ratio congruent with1) was observed. These data show that TTFC expressed on the surface of S. gordonii is immunogenic by the subcutaneous and mucosal routes and the immune response induced is capable of conferring protection from the lethal challenge with TT.

Vaginal immunization of cynomolgus monkeys with Streptococcus gordonii expressing HIV-1 and HPV 16 antigens

Cynomolgus monkeys (Macaca fascicularis) were immunized by intravaginal administration of live recombinant Streptococcus gordonii. The vaccine strains of S. gordonii expressed the V3 domain of the gpl20 of human immunodeficiency virus type 1 (HIV-1), and the E7 protein of human papillomavirus type 16 (HPV 16). Multiple inocula of recombinant bacteria were used, since S. gordonii could persist for no longer than 3 days in the monkey vagina. Vaginal immunization was found to induce a local and systemic immune response specific for the heterologous antigen expressed by the bacteria. This antigen-specific immune response consisted of vaginal IgA, serum IgG, and a T-cell proliferative response measured on PBMCs. Vaginal IgG and serum IgA were not detected.

Vaccine adjuvants: a priority for vaccine research.

The workshop on vaccine adjuvants was held in July of 2009 at the European Commission in Brussels, with the goal of identifying key scientific priorities as they pertain to the development of effective vaccines against life-threatening diseases especially those associated with poverty, including HIV/AIDS, malaria and tuberculosis as well as neglected infectious diseases. On the basis of new advances in adjuvant research and related technology as well as potential challenges and roadblocks, six priorities were identified to accelerate development of improved or novel vaccine adjuvants for human use.

The microbicide cyanovirin-N expressed on the surface of commensal bacterium Streptococcus gordonii captures HIV-1

ObjectiveTo explore the feasibility of expressing the potent HIV-inactivating protein, cyanovirin-N (CV-N), in the human commensal bacterium Streptococcus gordonii, as a possible approach for local delivery of CV-N to prevent sexual transmission of HIV-1. Design and methodsTo express CV-N in S. gordonii, we used the host-vector system we had previously developed. CV-N was expressed as a fusion protein both attached to the bacterial surface and secreted in soluble form in the supernatant of liquid cultures. The soluble form of recombinant CV-N was tested for gp120-binding activity in an enzyme-linked immunosorbent assay, whereas S. gordonii strain expressing CV-N on the surface was analyzed in an in vitro HIV capturing assay. ResultsTwo recombinant S. gordonii strains secreting or displaying CV-N on the bacterial surface were constructed and the expression of CV-N was confirmed by immunoblot and flow-cytometric analysis. The secreted form of recombinant CV-N exhibited a concentration-dependent binding to the envelope glycoprotein gp120 of HIV-1, whereas CV-N displayed on the bacterial surface was able to capture HIV virions efficiently. ConclusionThe anti-HIV protein CV-N in S. gordonii was expressed in a biologically active form. This represents a first step in the development of a system to deliver and maintain an effective concentration of a microbicide in the vaginal mucosa.

Human T-helper cell recognition of an immunodominant epitope of HIV-1 gp120 expressed on the surface of Streptococcus gordonii

Our genetic system for expression of heterologous proteins on the surface of the Gram-positive bacterium Streptococcus gordonii was used to express a human T-helper epitope of HIV-1 envelope glycoprotein gp120. In previous work on the naive repertoire of human T-helper cells, it was shown that a 15-amino acid synthetic peptide of the HIV-1 gp120 sequence contained an immunodominant T-helper epitope. Synthetic DNA coding for this peptide was cloned in frame within the gene for the streptococcal surface protein M6, and the gene fusion was integrated by transformation into the chromosome of S. gordonii. The expected M6-gp120 fusion protein was found to be expressed on the surface of the recombinant streptococci. To test whether the T epitope could be recognized by T cells when expressed on the bacterial surface within the context of M6, recombinant bacteria were used as antigen in proliferation assays to stimulate the 15-amino acid-specific human T-helper clone, in the presence of autologous antigen-presenting cells. Bacteria expressing the T epitope were efficiently recognized by the T cells in culture. In proliferation assays, 10(6)-10(7) bacteria induced responses comparable to those obtained by standard amounts of synthetic peptide (0.02-0.2 micrograms). Recombinant S. gordonii, a candidate for a live vaccine vector, appeared suitable for delivering T epitopes to the immune system.

Vaginal immunization with recombinant gram-positive bacteria.

PROBLEM: Many viral and bacterial pathogens enter the body through the genital mucosa. Therefore, one of the major goals of a vaccine against sexually transmitted diseases (STDs) should be to induce an immune response in the genital mucosa capable of controlling the entry of the pathogen. Our approach for the development of vaccines against STDs is based on the use of nonpathogenic Gram‐positive bacteria as live vaccine vectors.

Gram-positive commensal bacteria for mucosal vaccine delivery

To avoid the use of engineered pathogens for vaccine delivery, systems have been developed that allow the expression of heterologous antigens in commensal Gram-positive bacteria. In some cases, both a serum IgG and secretory IgA response are induced to the recombinant protein after vaccination, verifying the validity of the approach. These live recombinant bacteria may be used in the future to introduce a protective immune response to pathogenic microorganisms after mucosal colonization.

Immunogenicity of the B Monomer of Escherichia coli Heat- Labile Toxin Expressed on the Surface of Streptococcus gordonii

ABSTRACT The B monomer of the Escherichia coli heat-labile toxin (LTB) was expressed on the surface of the human oral commensal bacterium Streptococcus gordonii. Recombinant bacteria expressing LTB were used to immunize BALB/c mice subcutaneously and intragastrically. The LTB monomer expressed on the streptococcal surface proved to be highly immunogenic, as LTB-specific immunoglobulin G (IgG) serum titers of 140,000 were induced after systemic immunization. Most significantly, these antibodies were capable of neutralizing the enterotoxin in a cell neutralization assay. Following mucosal delivery, antigen-specific IgA antibodies were found in feces and antigen-specific IgG antibodies were found in sera. Analysis of serum IgG subclasses showed a clear predominance of IgG1 when recombinant bacteria were inoculated subcutaneously, while a prevalence of IgG2a was observed upon intragastric delivery, suggesting, in this case, the recruitment of a Th1 type of immune response.