Jean-François Viret

Virosomal adjuvanted antigen delivery systems

The development of novel and increasingly safer vaccines frequently utilizeswell-characterized antigens, in particular highly purified proteins or synthetic peptides. In spite of some achievements, this approach is frequently impeded by the fact that such antigens are often poor immunogens when administered alone. This fact has necessitated the development of suitable adjuvants that possess the ability to enhance the immunogenicity of a given antigen, preferably with little or no side effects. This paper discusses one of the successes of vaccinology of the past decade: virosomal vaccines. The principles of the concept, immunoadjuvant action and application of virosomes in two currently licensed vaccines are detailed, with specific reference to the induction of both humoral and cellular immunity.

Experience with registered mucosal vaccines

Most pathogens gain access to their host through mucosal surfaces. It is therefore desirable to develop vaccination strategies that lead to mucosal immune responses. Ideally, a vaccine should be administered mucosally in order to elicit mucosal protection. Several attenuated live viral and bacterial pathogens are registered as oral vaccines for human use, including the oral polio vaccine (Sabin) as well as attenuated strains of Salmonella typhi and Vibrio cholerae. These attenuated bacterial live vaccines-S. typhi Ty21a as well as V. cholerae CVD 103-HgR-are employed as vaccines against typhoid and cholera, respectively. In this manuscript, we review the immune responses that are induced by these vaccines, with a focus on mucosal immunity.

Replication and incompatibility properties of plasmid pUB110 in Bacillus subtilis.

SummaryWithin plasmid pUB110 we have identified a 1.2 kb segment necessary and sufficient for driving autonomous replication in Rec+ cells at a wild-type copy number. This region can be divided into three functionally discrete segments: a 24 base pair (bp) region that acts as an origin, a 949 bp determinant of an essential replication protein, repU, and a 358 bp incompatibility region, incA, overlapping with the repU gene. The synthesis of the IncA determinant/s proceeds in the direction opposite to that of RepU. The positively (RepU) and negatively (IncA) trans-acting products seem to be involved in the control of plasmid replication. The RepU product has an Mr of 39 kDa, could be overproduced in Escherichia coli, and binds to the pUB110 origin region. Outside the minimal replicon a cis-acting, orientation dependent, 516 bp determinant is required (i) to compete with a coexisting incompatible plasmid and (ii) for segregational stability.

Molecular cloning and characterization of the genetic determinants that express the complete Shigella serotype D (Shigella sonnei) lipopolysaccharide in heterologous live attenuated vaccine strains

The genetic determinants for the complete Shigella sonnei lipopolysaccharide (LPS) have been cloned, characterized by restriction mapping, and expressed in heterologous genetic backgrounds, including Salmonella typhi and Vibrio cholerae live attenuated vaccine strains. The rfb/rfc locus encoding the polymerized serotype‐specific O polysaccharide was mapped within 23 kb of DNA isolated from S. sonnei virulence plasmid pWR105. A highly similar chromosomal DNA sequence was identified by Southern hybridization analysis in Plesiomonas shigelloides known to have the same O serotype specificity as S. sonnei. Expression studies of the rfb/rfc locus have shown that S. sonnei. O polysaccharide is covalently bound to LPS cores of both the K‐12 and RI types, but neither to Salmonella (Ra‐type) nor to V. cholerae O1 cores. In order to express a compatible core structure in the latter organisms, chromosomal rfa loci encoding R1‐type LPS were isolated from both an Escherichia coli R1 strain (rfaR1) and from S. sonnei (rfdsonnei). Restriction mapping and functional analysis of cloned DNA allowed us to localize the rfaR1 locus and to orient it with respect to the neighbouring cysE chromosomal marker. A high degree of sequence similarity was found at the DNA level between rfa loci of enterobacterial species characterized by Ri‐type LPS. Co‐expression studies involving S. sonnei rfb/rfc and rfa loci propagated on compatible plasmids have shown that, at most, 13 to 14 kb of r/api DNA are required for the expression of complete phase‐l‐like S. sonnei LPS in E. coli K‐12 and S. typhi, whereas an adjacent region of about 3.5 kb is needed in the more stringent host, V. cholerae, S. sonnei O antigen expressed in a V. eholerae recombinant vaccine strain is present on the cell surface in a form suitable for the induction of a specific antibody response in vaccinated rabbits.

Mycobacterium bovis BCG-based vaccines against tuberculosis: novel developments.

Mycobacterium bovis Bacille Calmette-Guérin (BCG) is one of the most widely used vaccines. Modern techniques in genome manipulation allow the construction of recombinant (r)-BCG strains that can be employed as highly immunogenic vaccines against tuberculosis (TB) with an enhanced safety profile. In addition, the development of novel procedures to cultivate BCG will allow the large-scale production of future BCG-based vaccines.

Expression of Shigella sonnei lipopolysaccharide in Vibrio cholerae

Making use of a newly designed mobilizable suicide vector, the genetic determinants encoding Shigella sonnei lipopolysaccharide (LPS) were stably integrated into the chromosome of the live attenuated Vibrio cholerae vaccine strain CVD103‐HgR. Expression studies showed that the production of complete S. sonnei O‐polysaccharide (O‐PS)‐bearing LPS was limited in bivalent recombinant strains that were also proficient in the synthesis of the host‐encoded Inaba O‐PS. Conversely, high amounts of LPS carrying S. sonnei O‐PS are produced in monovalent Inaba‐deficient derivatives, even in those strains which do not co‐express the compatible R1 LPS core. Thus, the non‐enterobacterial V. cholerae LPS core efficiently acts as a receptor for covalent binding of S. sonnei O‐PS provided that competition with the host O‐PS is avoided. Expression of the R1 core interferes with cell division in recombinant V. cholerae without affecting other physiological properties of vaccine strain CVD103‐HgR. Both monovalent and bivalent strains stimulated high serum‐antibody titres specific for their respective O‐serotype(s) when administered to rabbits. The potential of V. cholerae as an expression carrier for heterologous O‐serotypes is discussed.

Functional analysis of the dna (Ts) mutants of Bacillus subtilis: Plasmid pUB110 replication as a model system

SummaryWe determined the effect of various Bacillus subtilis dna(Ts) mutations on pUB110 and chromosomal replication. Leading strand DNA synthesis of pUB110, starting by a nick at the plasmid replication origin (oriU), is performed by DNA polymerase III, since replication is blocked at non-permissive temperature in thermosensitive mutants dnaD, dnaF, dnaH and dnaN known to cause thermosensitivity of the various subunits of DNA polymerase III. When the lagging strand origin (oriL) is exposed, the DnaG protein (DNA primase) alone, or in association with unknown protein(s) binds asymmetrically to oriL to form a primer that is also extended by DNA polymerase III. In oriL- plasmids like pBT32, leading and lagging strand DNA syntheses are decoupled from each other. The DnaB protein, that is not required for pUB110 replication, may be associated with priming at a second unidentified lagging strand origin on pBT32. At non-permissive temperature, the dnaC30 and dnaI2 mutations affect both pUB110 and chromosomal DNA synthesis.

Novel vaccination strategies based on recombinant Mycobacterium bovis BCG

In this manuscript, we will review the utilization of Mycobacterium bovis Bacille Calmette-Guerin (BCG) as a vaccine against tuberculosis (TB) and as a carrier system for heterologous antigens. BCG is one of the most widely used vaccines. Novel techniques in genome manipulation allow the construction of virulence-attenuated recombinant (r)-BCG strains that can be employed as homologous vaccines, or as heterologous antigen delivery systems, for priming pathogen-specific immunity against infectious diseases, including TB. Several approaches are available for heterologous antigen expression and compartmentalization in BCG and recent findings show the potential to modulate and direct the immune responses induced by r-BCG strains as desired. Recent achievements in complete genome analysis of various target pathogens, combined with a better understanding of protective pathogen-specific immune responses, form the basis for the rational design of a new generation of recombinant mycobacterial vaccines against a multitude of infectious diseases.

Further molecular characterization and stability of the live oral attenuated cholera vaccine strain CVD103-HgR

Vibrio cholerae CVD103-HgR, the first live attenuated vaccine licensed for human use produced by recombinant DNA technology, was genetically compared to its parent strains 569B and CVD103. The genetic stability for both lyophilized vaccine in final container form and for viable organisms shed from vaccinees was determined. Results obtained lead us to conclude: (i) the genetic composition of the examined genes in CVD103-HgR is identical to that of the parent strains except for the alterations induced; (ii) the level of mercury resistance depends on the orientation of the mer operon within hlyA, with the highest level being observed for the orientation found in CVD103-HgR; (iii) no DNA sequences from plasmids used in construction remain in the genome; (iv) the strain is genetically stable; and (v) both CVD103-HgR and its parent strains contain defective lysogenic prophages. We have further confirmed that a certain amount of restriction fragment length polymorphism (RFLP) exists around the chromosomal ctx locus within V. cholerae strains of the classical biotype (detectable on chromosomal DNA restricted by either HindIII or EcoRI, but not PstI).

A new mutator strain of Bacillus subtilis

SummaryBacillus subtilis strain SB1207, widely used in our laboratory, was found to be highly temperature-sensitive and to exhibit a strong SOS-independent mutator phenotype at elevated temperatures. Both chromosomal and plasmid-borne genes were affected by the mutator. Lethality and mutator phenotype could not be attributed to a replication shut off or to thymine starvation. Due to the high frequency of base misincorporation, the mutator phenotype probably results from an editing defect rather than from a post-replication defect (mismatch repair).