The measles virus vaccine (MVbv) is a clinically certified and well-tolerated vaccine strain that has been given both parenterally and mucosally. It has been extensively used in children and has proven to be safe and effective in eliciting protective immunity. This specific strain was therefore chosen to generate a measles viral vector. The genome of the commercial MVbv vaccine strain was isolated, sequenced and a plasmid, p(+)MVb, enabling transcription of the viral antigenome and rescue of MVb, was constructed. Phylogenic and phenotypic analysis revealed that MVbv and the rescued MVb constitute another evolutionary branch within the hitherto classified measles vaccines. Plasmid p(+)MVb was modified by insertion of artificial MV-type transcription units (ATUs) for the generation of recombinant viruses (rMVb) expressing additional proteins. Replication characteristics and immunogenicity of rMVb vectors were similar to the parental MVbv and to other vaccine strains. The expression of the additional proteins was stable over 10 serial virus transfers, which corresponds to an amplification greater than 1020. The excellent safety record and its efficient application as aerosol may add to the usefulness of the derived vectors.
recombinant measles virus; viral vectors; live-attenuated vaccines
Live attenuated measles virus (MV) vaccines have an impressive record of safety, efficacy and ability to induce life-long immunity against measles infection. Using reverse genetics technology, such negative-strand RNA viruses can now be rescued from cloned DNA. This technology allows the insertion of exogenous genes encoding foreign antigens into the MV genome in such a way that they can be expressed by the MV vaccine strain, without affecting virus structure, propagation and cell targeting. Recombinant viruses rescued from cloned cDNA induce immune responses against both measles virus and the cloned antigens. The tolerability of MV to gene(s) insertion makes it an attractive flexible vector system, especially if broad immune responses are required. The fact that measles replication strictly occurs in the cytoplasm of infected cells without DNA intermediate has important biosafety implications and adds to the attractiveness of MV as a vector. In this article we report the characteristics of reporter gene expression (GFP, LacZ and CAT) and the biochemical, biophysical and immunological properties of recombinant MV expressing heterologous antigens of simian immunogeficiency virus (SIV).
recombinant vaccines; viral vectors; protein expression
Cervical cancer is mainly associated with HPV genotype 16 infection. Recombinant measles virus (rMV) expressing HPV genotype 16 L1 capsid protein was generated by construction of an antigenomic plasmid, followed by rescue using the human “helper” cell line 293-3-46. In cell cultures the recombinant MV-L1 virus replicated practically as efficiently as the standard attenuated MV established as commercial vaccine, devoid of the transgene. The high genetic stability of MVb2-L1 was confirmed by 10 serial viral transfers in cell culture. In transgenic mice expressing the MV receptor CD46 the recombinant induced strong humoral immune responses against both MV and HPV; the antibodies against L1 exhibited mainly neutralizing capacity. Our data suggest that MV is a promising vehicle for development of inexpensive and efficient vaccines protecting from HPV infection.
Recombinant MV; Reverse genetics; HPV-L1
The influence of the highly fluorescent tricyclic cytosine base analogue (tC) on duplex DNA conformation is investigated. The duplex properties are characterized by absorbance and circular dichroism (CD) for all combinations of neighbouring bases to tC, and an NMR structure is determined for one tC-containing sequence. For the oligonucleotides with one tC incorporated instead of cytosine, the melting temperature is increased on average by 2.7°C above that for the unmodified ones. CD spectra are practically identical for modified and unmodified sequences, indicating an unperturbed B-DNA conformation. The NMR structure determination of the self-complementary sequence 5′-CTC(tC)ACGTGGAG shows a DNA conformation consistent with B-form for the whole duplex. The root-mean-square distance for the nucleotides of the eight central base pairs between the 10 structures with lowest CYANA target functions and a mean structure is 0.45 ± 0.17 Å. The NMR data confirm correct base pairing for tC by the observation of both intrastrand and interstrand imino proton NOEs. Altogether, this suggests that tC works well as a cytosine analogue, i.e. it is situated in the base stack, forming hydrogen bonds with G in the complementary strand, without distorting the DNA backbone conformation. This first example of an artificial, highly fluorescent DNA base that does not perturb the DNA conformation could have valuable applications for the study of the structure and dynamics of nucleic acid systems.
Measles virus (MV) can infect the central nervous system and, in rare cases, causes subacute sclerosing panencephalitis, characterized by a progressive degeneration of neurons. The route of MV transmission in neurons was investigated in cultured rat hippocampal slices by using MV expressing green fluorescent protein. MV infected hippocampal neurons and spread unidirectionally, in a retrograde manner, from CA1 to CA3 pyramidal cells and from there to the dentate gyrus. Spreading of infection depended on cell-to-cell contact and occurred without any detectable release of infectious particles. The role of the viral proteins in the retrograde MV transmission was determined by investigating their sorting in infected pyramidal cells. MV glycoproteins, the fusion protein (F) and hemagglutinin (H), the matrix protein (M), and the phosphoprotein (P), which is part of the viral ribonucleoprotein complex, were all sorted to the dendrites. While M, P, and H proteins remained more intracellular, the F protein localized to prominent, spine-type domains at the surface of infected cells. The detected localization of MV proteins suggests that local microfusion events may be mediated by the F protein at sites of synaptic contacts and is consistent with a mechanism of retrograde transmission of MV infection.
Knowledge of the mechanisms of virus dissemination in acute measles is cursory, but cells of the monocyte/macrophage (MM) lineage appear to be early targets. We characterized the dissemination of the Edmonston B vaccine strain of measles virus (MV-Ed) in peripheral blood mononuclear cells (PBMC) of two mouse strains expressing the human MV-Ed receptor CD46 with human-like tissue specificity and efficiency. In one strain the alpha/beta interferon receptor is defective, allowing for efficient MV-Ed systemic spread. In both mouse strains the PBMC most efficiently infected were F4/80-positive MMs, regardless of the inoculation route used. Circulating B lymphocytes and CD4-positive T lymphocytes were infected at lower levels, but no infected CD8-positive T lymphocytes were detected. To elucidate the roles of MMs in infection, we depleted these cells by clodronate liposome treatment in vivo. MV-Ed infection of splenic MM-depleted mice caused strong activation and infection of splenic dendritic cells (DC), followed by enhanced virus replication in the spleen. Similarly, depletion of lung macrophages resulted in strong activation and infection of lung DC. Thus, in MV infections of genetically modified mice, blood monocytes and tissue macrophages provide functions beneficial for both the virus and the host: they support virus replication early after infection, but they also contribute to protecting other immune cells from infection. Human MM may have similar roles in acute measles.
The dissemination of the Edmonston measles virus (Ed-MV) vaccine strain was studied with genetically modified mice defective for the alpha/beta interferon receptor and expressing human CD46 with human-like tissue specificity and efficiency. A few days after intranasal infection, macrophages expressing Ed-MV RNA were detected in the lungs, in draining lymph nodes, and in the thymus. In lymph nodes, large syncytia which stained positive for viral RNA and for macrophage surface marker proteins were found and apoptotic cell death was monitored. In the thymus, smaller syncytia which stained positive for macrophage and dendritic cell markers were detected. Thus, macrophages appear to be the main vectors for dissemination of MV infection in these mice; human macrophages may have a similar function in the natural host. We then compared the pathogenicities of two recombinant viruses lacking the C or V nonstructural proteins to that of the parental strain, Ed-MV. These viruses were less effective in spreading through the lymphatic system and, unlike Ed-MV, were not detected in the liver. After intracerebral inoculation the recombinant viruses caused lethal disease less often than Ed-MV and induced distinctive patterns of gliosis and inflammation. Ed-MV was reisolated from brain tissue, but its derivatives were not. C- and V-defective viruses should be considered as more-attenuated MV vaccine candidates.
A recombinant measles virus (MV) which expresses enhanced green fluorescent protein (EGFP) has been rescued. This virus, MVeGFP, expresses the reporter gene from an additional transcription unit which is located prior to the gene encoding the measles virus nucleocapsid protein. The recombinant virus was used to infect human astrocytoma cells (GCCM). Immunocytochemistry (ICC) together with EGFP autofluorescence showed that EGFP is both an early and very sensitive indicator of cell infection. Cells that were EGFP-positive and ICC-negative were frequently observed. Confocal microscopy was used to indirectly visualize MV infection of GCCM cells and to subsequently follow cell-to-cell spread in real time. These astrocytoma cells have extended processes, which in many cases are intimately associated. The processes appear to have an important role in cell-to-cell spread, and MVeGFP was observed to utilize them in the infection of surrounding cells. Heterogeneity was seen in cell-to-cell spread in what was expected to be a homogeneous monolayer. In tissue culture, physical constraints govern the integrity of the syncytia which are formed upon extensive cell fusion. When around 50 cells were fused, the syncytia rapidly disintegrated and many of the infected cells detached. Residual adherent EGFP-positive cells were seen to either continue to be involved in the infection of surrounding cells or to remain EGFP positive but no longer participate in the transmission of MV infection to neighboring cells.
Molecular determinants of neuropathogenesis have been shown to be present in the hemagglutinin (H) protein of measles virus (MV). An H gene insertion vector has been generated from the Edmonston B vaccine full-length infectious clone of MV. Using this vector, it is possible to insert complete H open reading frames into the parental (Edtag) background. The H gene from a rodent brain-adapted MV strain (CAM/RB) was inserted into this vector, and a recombinant virus (EdtagCAMH) was rescued by using a modified vaccinia virus which expresses T7 RNA polymerase (MVA-T7). The recombinant virus grew at an equivalent rate and to similar titers as the CAM/RB and Edtag parental viruses. Neurovirulence was assayed in a mouse model for MV encephalitis. Viruses were injected intracerebrally into the right cortex of C57/BL/6 suckling mice. After infection mice inoculated with the CAM/RB strain developed hind limb paralysis and ataxia. Clinical symptoms were never observed with an equivalent dose of Edtag virus or in sham infections. Immunohistochemistry (IHC) was used to detect viral antigen in formalin-fixed brain sections. Measles antigen was observed in neurons and neuronal processes of the hippocampus, frontal, temporal, and olfactory cortices and neostriatum on both sides of symmetrical structures. Viral antigen was not detected in mice infected with Edtag virus. Mice infected with the recombinant virus, EdtagCAMH, became clinically ill, and virus was detected by IHC in regions of the brain similar to those in which it was detected in animals infected with CAM/RB. The EdtagCAMH infection had, however, progressed much less than the CAM/RB virus at 4 days postinfection. It therefore appears that additional determinants are encoded in other regions of the MV genome which are required for full neurovirulence equivalent to CAM/RB. Nevertheless, replacement of the H gene alone is sufficient to cause neuropathology.
It has been shown previously that measles virus (MV) can be successfully used to express foreign proteins (M. Singh and M. A. Billeter, J. Gen. Virol. 80:101–106, 1998). To develop an inexpensive MV-based vaccine, we generated recombinant MVs that produce structural proteins of hepatitis B virus (HBV). A recombinant virus that expressed the HBV small surface antigen (HBsAg) was analyzed in terms of its replication characteristics, its genetic stability in cell culture, and its immunogenic potential in genetically modified mice. Although this virus showed a progression of replication slightly slower than that of the parental MV, it appeared to stably maintain the added genetic information; it uniformly expressed the appropriately glycosylated HBsAg after 10 serial passages. Genetically modified mice inoculated with this recombinant MV produced humoral immune responses against both HBsAg and MV proteins.
The P gene of measles virus (MV) encodes the phosphoprotein, a component of the virus ribonucleoprotein complex, and two nonstructural proteins, C and V, with unknown functions. Growth of recombinant MV, defective in C or V expression, was explored in human peripheral blood mononuclear cells (PBMC). The production of infectious recombinant MV V− was comparable to that of parental MV tag in simian Vero fibroblasts and in PBMC. In contrast, MV C− progeny was strongly reduced in PBMC but not in Vero cells. Consistently, the expression of both hemagglutinin and fusion proteins, as well as that of nucleoprotein mRNA, was lower in MV C−-infected PBMC. Thus, efficient replication of MV in natural host cells requires the expression of the nonstructural C protein. The immunosuppression that accompanies MV infection is associated with a decrease in the in vitro lymphoproliferative response to mitogens. MV C− was as potent as MV tag or MV V− in inhibiting the phytohemagglutinin-induced proliferation of PBMC, indicating that neither the C protein nor the V protein is directly involved in this effect.
Nonstructural proteins encoded by measles virus (MV) include the V protein which is translated from an edited P mRNA. V protein is not associated with intracellular or released viral particles and has recently been found to be dispensable for MV propagation in cell culture (H. Schneider, K. Kaelin, and M. A. Billeter, Virology 227:314–322, 1997). Using recombinant MVs (strain Edmonston [ED]) genetically engineered to overexpress V protein (ED-V+) or to be deficient for V protein (ED-V−), we found that in the absence of V both MV-specific proteins and RNAs accumulated to levels higher than those in the parental MV molecular clone (ED-tag), whereas MV-specific gene expression was strongly attenuated in human U-87 glioblastomas cells after infection with ED-V+. The titers of virus released from these cells 48 h after infection with either V mutant virus were lower than those from cells infected with ED-tag. Similarly, significantly reduced titers of infectious virus were reisolated from lung tissue of cotton rats (Sigmodon hispidus) after intranasal infection with both editing mutants compared to titers isolated from ED-tag-infected animals. In cell culture, expression of V protein led to a redistribution of MV N protein in doubly transfected Cos-7 cells, indicating that these proteins form heterologous complexes. This interaction was further confirmed by using a two-hybrid approach with both proteins expressed as Gal4 or VP16 fusion products. Moreover, V protein efficiently competed complexes formed between MV N and P proteins. These findings indicate that V protein acts to balance accumulation of viral gene products in cell culture, and this may be dependent on its interaction with MV N protein. Furthermore, expression of V protein may contribute to viral pathogenicity in vivo.
An understanding of the determinants of measles virus (MV) virulence has been hampered by the lack of an experimental model of infection. We have previously demonstrated that virulence phenotypes in human infections are faithfully reproduced by infection of human thymus/liver (thy/liv) implants engrafted into SCID mice, where the virus grows primarily in stromal cells but induces thymocyte apoptosis (P. G. Auwaerter et al., J. Virol. 70:3734–3740, 1996). To begin to elucidate the roles of the C protein, V protein, and the 5′ untranslated region of the F gene (F 5′UTR) in MV infection in vivo, the replication of strains bearing mutations of these genes was compared to that of the parent sequence-tagged Edmonston strain (EdTag). Growth curves show that mutants fall into two phenotypic classes. One class of mutants demonstrated kinetics of growth similar to that of EdTag, with decreased peak titers. The second class of mutants manifested peak titers similar to that of EdTag but had different replication kinetics. Abrogation of V expression led to delayed and markedly prolonged replication. Additionally, thymocyte survival was prolonged and implant architecture was preserved throughout the course of infection. In contrast, massive bystander thymocyte death occurred after infection with EdTag and all other mutants. A mutant which overexpressed V in Vero cells (V+) had the opposite phenotype of the A mutant not expressing V (V−). V+ grew more rapidly than EdTag with 100-fold-greater levels of virus production 3 days after infection. These results suggest that C, V, and the F 5′UTR are accessory factors required for efficient virus replication in vivo. In addition, thymocyte survival after V− infection suggests this protein may play multiple roles in pathogenesis of MV infection of thymus. Since these recombinant mutant viruses grew identically to the parent virus in Vero cells, the data show that thy/liv implants are an excellent model for investigating the determinants of MV virulence.
Measles virus (MV) and vesicular stomatitis virus (VSV) are both members of the Mononegavirales but are only distantly related. We generated two genetically stable chimeric viruses. In MGV, the reading frames of the MV envelope glycoproteins H and F were substituted by a single reading frame encoding the VSV G glycoprotein; MG/FV is similar but encodes a G/F hybrid in which the VSV G cytoplasmic tail was replaced by that of MV F. In contrast to MG/FV, MGV virions do not contain the MV matrix (M) protein. This demonstrates that virus assembly is possible in the absence of M; conversely, the cytoplasmic domain of F allows incorporation of M and enhances assembly. The formation of chimeric viruses was substantially delayed and the titers obtained were reduced about 50-fold in comparison to standard MV. In the novel chimeras, transcription and replication are mediated by the MV ribonucleoproteins but the envelope glycoproteins dictate the host range. Mice immunized with the chimeric viruses were protected against lethal doses of wild-type VSV. These findings suggest that it is feasible to construct MV variants bearing a variety of different envelopes for use as vaccines or for gene therapeutic purposes.
Binuclear polypyridine ruthenium compounds have been shown to slowly intercalate into DNA, following a fast initial binding on the DNA surface. For these compounds, intercalation requires threading of a bulky substituent, containing one RuII, through the DNA base-pair stack, and the accompanying DNA duplex distortions are much more severe than with intercalation of mononuclear compounds. Structural understanding of the process of intercalation may greatly gain from a characterisation of the initial interactions between binuclear RuII compounds and DNA. We report a structural NMR study on the binuclear RuII intercalator Λ,Λ-B (Λ,Λ-[μ-bidppz(bipy)4Ru2]4+; bidppz=11,11′-bis(dipyrido[3,2-a:2′,3′-c]phenazinyl, bipy = 2,2′-bipyridine) mixed with the palindromic DNA [d(CGCGAATTCGCG)]2. Threading of Λ,Λ-B depends on the presence and length of AT stretches in the DNA. Therefore, the latter was selected to promote initial binding, but due to the short stretch of AT base pairs, final intercalation is prevented. Structural calculations provide a model for the interaction: Λ,Λ-B is trapped in a well-defined surface-bound state consisting of an eccentric minor-groove binding. Most of the interaction enthalpy originates from electrostatic and van der Waals contacts, whereas intermolecular hydrogen bonds may help to define a unique position of Λ,Λ-B. Molecular dynamics simulations show that this minor-groove binding mode is stable on a nanosecond scale. To the best of our knowledge, this is the first structural study by NMR spectroscopy on a binuclear Ru compound bound to DNA. In the calculated structure, one of the positively charged Ru2+ moieties is near the central AATT region; this is favourable in view of potential intercalation as observed by optical methods for DNA with longer AT stretches. Circular dichroism (CD) spectroscopy suggests that a similar binding geometry is formed in mixtures of Λ,Λ-B with natural calf thymus DNA. The present minor-groove binding mode is proposed to represent the initial surface interactions of binuclear RuII compounds prior to intercalation into AT-rich DNA.
DNA; intercalation; minor-groove binding; NMR spectroscopy; ruthenium