Robert Drillien

A matrix-less measles virus is infectious and elicits extensive cell fusion: consequences for propagation in the brain

Measles viruses (MV) can be isolated from the brains of deceased subacute sclerosing panencephalitis patients only in a cell‐associated form. These viruses are often defective in the matrix (M) protein and always seem to have an altered fusion protein cytoplasmic tail. We reconstituted a cell‐free, infectious M‐less MV (MV‐ΔM) from cDNA. In comparison with standard MV, MV‐ΔM was considerably more efficient at inducing cell‐to‐cell fusion but virus titres were reduced ∼250‐fold. In MV‐ΔM‐induced syncytia the ribonucleocapsids and glycoproteins largely lost co‐localization, confirming the role of M protein as the virus assembly organizer. Genetically modified mice were inoculated with MV‐ΔM or with another highly fusogenic virus bearing glycoproteins with shortened cytoplasmic tails (MV‐Δtails). MV‐ΔM and MV‐Δtails lost acute pathogenicity but penetrated more deeply into the brain parenchyma than standard MV. We suggest that enhanced cell fusion may also favour the propagation of mutated, assembly‐defective MV in human brains.

Construction of vaccinia virus recombinants expressing several measles virus proteins and analysis of their efficacy in vaccination of mice

Measles virus genes encoding the haemagglutinin (HA), fusion protein (F) or nucleoprotein (NP) have been inserted into the vaccinia virus genome either alone or in various combinations. In each case the measles virus genes were expressed from the 7.5K promoter and were incorporated into the thymidine kinase (tk) or K1L loci of the Copenhagen strain of vaccinia virus. Cells infected by the recombinants synthesized measles virus proteins indistinguishable from those induced in measles virus-infected cells. However, in some instances the level of expression in cells infected by recombinants expressing more than one measles virus gene was reduced when compared to those encoding a single gene. The sera from mice immunized with recombinants containing either HA, HA.F, HA.NP or HA.F.NP had similar levels of measles virus neutralizing antibodies which remained constant throughout a 7 month period. Analysis of these sera by immunoprecipitation of radiolabelled measles virus confirmed the presence of specific antibody to each of the antigens where appropriate. The introduction of the measles virus genes into the K1L and the tk sites despite attenuating the virus for mice by 10-fold and 1000-fold respectively did not affect the vaccination efficiency, i.e. ability to induce measles virus antibody and protect mice. Vaccination of BALB/c (H2d) mice with HA and F, but not NP, recombinants completely protected the animals against a lethal measles virus challenge. In contrast, although the HA recombinant protected CBA (H2k) mice, the F recombinant did so poorly. However, by immunizing CBA mice with a recombinant expressing both F and NP, protection was increased to more than 75%. Our findings demonstrate the ability of three measles virus antigens expressed from the vaccinia virus genome alone or in combination to contribute to protective immunity against measles virus infection of mice. They also suggest that the association of measles virus antigens in a single recombinant DNA vaccine could be beneficial to overcome host-related restriction of the immune response to particular antigens.

Host range deletion mutant of vaccinia virus defective in human cells

Abstract A vaccinia virus host range (hr) mutant unable to multiply in most human cell lines assayed has been isolated after nitrous acid mutagenesis. This mutant also displayed various alterations in plaque morphology and cytopathic effect on permissive cell lines. The block in multiplication in human cells was at an early stage of infection. Only early cytoplasmic RNA and early viral-induced polypeptides could be detected and there was no evidence of morphological events within viroplasms. Protein synthesis constantly declined as infection proceeded suggesting either that early viral mRNA was unstable or that the mutant virus was defective in a step necessary for the maintenance of translation. Restriction enzyme digestion of DNA from the hr mutant revealed that it was deleted of about 12.6 × 10 6 daltons in the left-hand end of the genome leaving intact a fragment containing the terminal crosslink. In both permissive and nonpermissive cells the mutant failed to induce the synthesis of an early 42K polypeptide which could thus be encoded within a region of the deleted sequence. The failure to segregate the various phenotypic and biochemical properties of the hr mutant from one another through recombination with a temperature sensitive mutant indicated that the pleiotropic characteristics of the mutant virus were due to the deletion.

Heat shock proteins 70 and 60 share common receptors which are expressed on human monocyte-derived but not epidermal dendritic cells

Priming of CTL by means of heat shock proteins (hsp) is dependent on antigen‐presenting cells (APC), which present the hsp‐associated peptides, via their cell surface MHC class I molecules, toCD8+ T cells. It has not yet been established how human (hu) hsp70 interacts with the major (hu)APC, the dendritic cells (DC). Here we show that (hu)hsp70 is specifically internalized intoCD14–, Toll‐like receptor 4– monocyte‐derived (hu)DC by receptor‐mediated endocytosis. We further demonstrate that (hu)hsp70 and (hu)hsp60 share the same receptors on (hu)monocyte‐derived DC. Both molecules as well as MHC class I molecules are spontaneously internalized and reach the MHC class II‐enriched compartments. Finally, freshly isolated (hu) epidermal Langerhans cells (LC), the DC of the skin, as well as CD34+‐derived LC do not bind hsp60 or hsp70. Given the likely importance of the internalization of hsp70 by APC in the induction of the immune responses, the finding that hsp60 and hsp70 are internalized through the same receptor(s) may explain why microbial hsp60 represents a major T cell antigen. This may rationalize the use of microbial hsp60 to primeimmune responses against microbes. The lack of hsp60/70 receptors on epidermal LC raises the crucial question as to whether absence of priming of the skin and mucosal immune systems by hsp‐polypeptide complexes could account for some tissue‐specific diseases. This work also points to a potential advantage of using monocyte‐derived DC in human immunotherapeutic applications of hsp60/70.

Similar genetic organization between a region of fowlpox virus DNA and the vaccinia virus HindIII J fragment despite divergent location of the thymidine kinase gene

DNA from Fowlpox virus, a member of the Avipoxvirus genus, has been found to hybridize to DNA from vaccinia virus, a member of the Orthopoxvirus genus. The greatest homology detected was around the region containing the vaccinia virus thymidine kinase locus. A 3.1-kbp fowlpox virus fragment that hybridizes to the vaccinia virus HindIII J fragment has been cloned and its sequence determined. Comparison of the nucleotide and deduced amino acid sequence to the cross hybridizing vaccinia fragment revealed extensive conservation of six open reading frames as well as a similar organization along the genome. Nevertheless a fowlpox virus gene corresponding to the vaccinia virus thymidine kinase gene was apparently lacking within the region studied and is probably located elsewhere in the genome. Despite this intriguing divergence, our results indicate that the Avipoxviruses are more closely related to the Orthopoxviruses than previously suspected.

Short- and long-term immunogenicity and protection induced by non-replicating smallpox vaccine candidates in mice and comparison with the traditional 1st generation vaccine

This study assessed three non-replicating smallpox vaccine candidates (modified vaccinia Ankara (MVA), NYVAC and HR) for their immunogenicity and ability to protect mice against an intranasal cowpox virus challenge and compared them with the traditional replicating vaccine. A single immunisation with the non-replicating vaccines induced a complete protection from death at short-term, but was not fully protective when mice were challenged 150 days post-vaccination with protection correlated with the specific neutralizing antibodies and CD4(+) T-cells responses. Prime-boost vaccination enabled effective long-term protection from death for mice vaccinated with MVA, but protection from disease and CD4(+) T-cell level were lower than the ones induced by the traditional vaccine over the long-term period. Further investigations are necessary with MVA to determine the optimal conditions of immunisation to induce at long-term immunogenicity and protection observed with the 1st generation smallpox vaccine.

Detection of a protein encoded by the vaccinia virus C7L open reading frame and study of its effect on virus multiplication in different cell lines

Vaccinia virus encodes several proteins, the activity of which is essential for multiplication in different cell types. Both the C7L and K1L open reading frames (ORFs) have been characterized as viral determinants for multiplication in human cells. To confirm and extend these findings we inserted the C7L ORF into the genome of a mutant virus unable to multiply in human cells and showed that this virus recovered its ability to replicate. Deletion of C7L from a wild-type viral genome did not adversely affect virus multiplication in human cells but it did reduce replication in hamster Dede cells. When both C7L and K1L were deleted from the vaccinia virus genome only poor or no viral yields were obtained from various human cell lines. Recombinant viruses were also constructed to facilitate the study of C7L protein synthesis during infection. One virus in which the lacZ ORF was fused downstream and in-frame with the C7L ORF enabled us to characterize the C7L protein as an early gene product. Another recombinant virus was constructed so that the carboxy terminus of the C7L ORF product contained an additional 28 amino acids from the carboxy terminus of K1L. Tagging of C7L in this way allowed us to detect the fusion protein by immunoprecipitation with antibodies against the K1L protein. Furthermore, the hybrid protein retained its biological properties. The recombinant viruses constructed in this work should be useful for studies of the molecular basis of the activity of viral host range proteins.

Fowlpox virus recombinant encoding the measles virus fusion protein: protection of mice against fatal measles encephalitis

A recombinant Fowlpox virus engineered to encode the measles virus fusion protein was shown to protect mice against a challenge measles infection. A vaccine dose of about 10(6) p.f.u. was needed to protect nearly 100% of the animals. Mice failed to develop a significant level of antibodies directed against measles virus suggesting that other components of the immune system may be involved.

Vaccination of mice against canine distemper virus-induced encephalitis with vaccinia virus recombinants encoding measles or canine distemper virus antigens

Measles and canine distemper are caused by serologically related viruses. Although dogs immunized with measles virus (MV) do not elicit canine distemper virus (CDV) neutralizing antibodies, they are protected against the fatal disease. To investigate the potential role of the MV antigens in protection against CDV, we have immunized mice with vaccinia virus (VV) recombinants expressing the MV haemagglutinin (HA), fusion (F), nucleoprotein (NP) and matrix (M) antigens and challenged them with CDV. A partial protection was observed with the VV recombinants expressing the F, NP and M antigens, but not the HA. In contrast, immunization with a VV recombinant expressing the CDV F protein completely protected mice from CDV.

Complementation and genetic linkage between vaccinia virus temperature-sensitive mutants

Abstract Vaccinia virus temperature-sensitive (ts) mutants were isolated after nitrosoguanidine mutagenesis. A number of these mutants exhibited host range temperature sensitivity in that the efficiency of plaque formation at the nonpermissive temperature was poorer on chick cells than on hamster or human cells. Forty-two mutants were assigned to 23 different complementation groups on the basis of complementation and the efficiency of apparent recombination at the nonpermissive temperature. Recombination frequencies were also determined from mixed infections carried out at the permissive temperature and it was confirmed that mutants within the same complementation group recombined less efficiently with each other than mutants belonging to different groups. Mutants from two of the largest groups could be tentatively ordered on linear intragenic maps that spanned 0.8 and 2 recombination units. Moreover, from intergenic crosses between mutants in 14 different complementation groups, a linkage map spanning 66.3 recombination units, was derived. This study illustrates the feasibility of two-factor recombination mapping of poxvirus mutations and provides genetic data that should be of relevance in further analysis of the is mutations.