We have developed a system in which a foreign antigen replaces nearly all of the surface-exposed region of the fibrillar M protein from Streptococcus pyogenes and is fused to the C-terminal attachment motif of the M molecule. The fusion protein is thus expressed on the surface of Streptococcus gordonii, a commensal organism of the oral cavity. The antigen chosen to be expressed within the context of the M6 molecule was the E7 protein (98 amino acids) of human papillomavirus type 16. Stable recombinant streptococci were obtained by integrating genetic constructs into the chromosome, exploiting in vivo homologous recombination. The M6-E7 fusion protein expressed on the S. gordonii surface was shown to be immunogenic in mice. This is the first step in the construction of recombinant live vaccines in which nonpathogenic streptococci as well as other gram-positive bacteria may be used as vectors to deliver heterologous antigens to the immune system.Gram-negative bacteria, cloned to express foreign antigens either on the surface or within the cell, have been used to deliver these molecules to mammalian hosts for the induction of an immune response (2,21,35). With the exception of mycobacteria (34), gram-positive bacteria have not as yet been exploited for this purpose. In this report, we describe the expression of a heterologous antigen on the surface of a streptococcus. The fibrillar M6 protein, a surface protein from Streptococcus pyogenes (4, 5, 8), was modified to deliver the E7 protein (98 amino acids) of human papillomavirus type 16 (HPV16) to the surface of Streptococcus gordonii. E7 is an oncoprotein (1, 3, 15, 37), antibodies to which are found in patients with cervical cancer (13); it is considered a major candidate antigen for vaccines against HPV-induced malignant neoplasias (19). Its use in this construct emphasizes the ability to deliver a molecule far removed from a bacterial protein to the bacterial cell surface.S. gordonii Challis, formerly classified as Streptococcus sanguis (16), was isolated from the human oral cavity and found to be naturally competent for genetic transformation (23). This strain, in which DNA can be efficiently introduced (17, 28, 30), was chosen as a model host for these experiments. Our approach to genetic manipulation of streptococci is based on the integration of recombinant DNA molecules into the bacterial chromosome, for both transformable and nontransformable species (22,27,29,32). To obtain surface expression of heterologous antigens in streptococci, we developed a host-vector system that allows the construction, chromosomal integration, and expression of translational fusions with the M6 protein gene (emm-6.1) (10). MATERIALS AND METHODSRecombinant DNA techniques. Gene fusions in Escherichia coli vectors were obtained and controlled by standard procedures (18).Streptococcal transformation. Frozen cells of naturally * Corresponding author. competent S. gordonii Challis were prepared and transformed as already described in detail (28). Plating and scoring of transformants o...

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Complete nucleotide sequencing of the RNAs of two unrelated neurovirulent isolates of Sabin-related poliovirus type 2 revealed that two nucleotides and one amino acid (amino acid 143 in the major capsid protein VP1) consistently departed from the sequences of the nonneurovirulent poliovirus type 2 712 and Sabin vaccine strains. This pattern of mutation appeared to be a feature common to all neurovirulent variants of poliovirus type 2.

SUMMARYRetrovirus infection of cultured murine teratocarcinoma cells depends upon the state of differentiation. We have used two cell lines derived from a teratocarcinoma of mouse, strain 129. One, an undifferentiated pluripotential cell line (PCC4), is restrictive to viral infection, while the other, a differentiated myoblast-derived cell line (PCD1), is fully permissive to virus replication. We have shown that no virus RNA expression can be found in PCC4 cells 48 h post-infection and that no nucleic acid sequences can be found in an integrated form in PCC4 cells. However, the kinetics of formation of free proviral intermediates show that the three forms (I, II and III) of free virus DNA are synthesized in both PCC4 and PCD1. Free proviral DNA disappears gradually after 24 h in PCC4 cells while all forms increase in PCD1. These results suggest that the viral multiplication restriction occurs somewhere between the proviral DNA synthesis and integration of DNA in the cellular genome.