Broadly Neutralizing Immune Responses against Hepatitis C Virus Induced by Vectored Measles Viruses and a Recombinant Envelope Protein Booster

Hepatitis C virus (HCV) infection remains a serious public health problem worldwide. Treatments are limited, and no preventive vaccine is available. Toward developing an HCV vaccine, we engineered two recombinant measles viruses (MVs) expressing structural proteins from the prototypic HCV subtype 1a strain H77. One virus directs the synthesis of the HCV capsid (C) protein and envelope glycoproteins (E1 and E2), which fold properly and form a heterodimer. The other virus expresses the E1 and E2 glycoproteins separately, with each one fused to the cytoplasmic tail of the MV fusion protein. Although these hybrid glycoproteins were transported to the plasma membrane, they were not incorporated into MV particles. Immunization of MV-susceptible, genetically modified mice with either vector induced neutralizing antibodies to MV and HCV. A boost with soluble E2 protein enhanced titers of neutralizing antibody against the homologous HCV envelope. In animals primed with MV expressing properly folded HCV C-E1-E2, boosting also induced cross-neutralizating antibodies against two heterologous HCV strains. These results show that recombinant MVs retain the ability to induce MV-specific humoral immunity while also eliciting HCV neutralizing antibodies, and that anti-HCV immunity can be boosted with a single dose of purified E2 protein. The use of MV vectors could have advantages for pediatric HCV vaccination.     

Measles viruses possessing the polymerase protein genes of the Edmonston vaccine strain exhibit attenuated gene expression and growth in cultured cells and SLAM knock-in mice

Live attenuated vaccines against measles have been developed through adaptation of clinical isolates of measles virus (MV) in various cultured cells. Analyses using recombinant MVs with chimeric genomes between wild-type and Edmonston vaccine strains indicated that viruses possessing the polymerase protein genes of the Edmonston strain exhibited attenuated viral gene expression and growth in cultured cells as well as in mice expressing an MV receptor, signaling lymphocyte activation molecule, regardless of whether the virus genome had the wild-type or vaccine-type promoter sequence. These data demonstrate that the polymerase protein genes of the Edmonston strain contribute to its attenuated phenotype.     

A recombinant measles virus expressing hepatitis B virus surface antigen induces humoral immune responses in genetically modified mice

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.     

Disease model: dissecting the pathogenesis of the measles virus

Host-pathogen interactions of measles virus (MV), a leading cause of childhood mortality worldwide, are still poorly understood. Using transgenic mice that express the human MV receptor CD46, we generated models to study the pathogenesis of MV infection of the central nervous system (CNS) and immune system. CNS infection in CD46 transgenic mice allows replication and spread throughout neurons, inflammation, and ultimately death of the animals. CD46-transgenic mice can also be used to study immunosuppression, a hallmark of measles. Together with mouse knockout technology and a system for generating recombinant MVs, CD46 transgenic mice will ultimately lead to a better understanding of both viral and host factors contributing to disease.     

Role of CD8(+) lymphocytes in control and clearance of measles virus infection of rhesus monkeys

The creation of an improved vaccine for global measles control will require an understanding of the immune mechanisms of measles virus containment. To assess the role of CD8(+) cytotoxic T lymphocytes in measles virus clearance, rhesus monkeys were depleted of CD8(+) lymphocytes by monoclonal anti-CD8 antibody infusion and challenged with wild-type measles virus. The CD8(+) lymphocyte-depleted animals exhibited a more extensive rash, higher viral loads at the peak of virus replication, and a longer duration of viremia than did the control antibody-treated animals. These findings indicate a central role for CD8(+) lymphocytes in the control of measles virus infections and the importance of eliciting a cell-mediated immune response in new measles vaccine strategies.     

Expression of SLAM (CDw150) on Sf9 cell surface using recombinant baculovirus mediates measles virus infection in the nonpermissive cells

Signaling lymphocytic activation molecule (SLAM; also known as CDw150) has been reported as the receptor of measles virus (MV) interacting with MV hemagglutinin (MVH). In this study, we developed a baculovirus-derived vector, the Bacmid-egfp, containing a reporter gene encoding the enhanced green fluorescent protein (EGFP) under the control of the promoter of very late polyhedrin gene from Autographa californica multiple nucleopolyhedrovirus (AcMNPV), and employed the recombinant baculovirus to express SLAM in Sf9 (Spodoptera frugiperda) cells and investigate SLAM function. The result showed that the integration of the EGFP expression cassette in the Bac-to-Bac system facilitated research with the system without introducing compromises due to its use. SLAM protein fused to His-tag was expressed in Sf9 cells through the modified Bac-to-Bac system. The expressed SLAM was identified as approximately 46 kDa, and it presented on the cell surface, as revealed by fluorescent immunochemical staining and confocal microscopic analysis. The pull-down assay proved that SLAM protein expressed in this system could interact with MVH protein. After incubating with MV vaccine strain S191, cell fusion was only observed in the Sf9 cells expressing both EGFP and SLAM from recombinant baculovirus rather than those expressing EGFP only from the modified viral vector. Furthermore, MV replicated and induced apoptosis in the Sf9 cells with SLAM expression.     

Single-chain antibody displayed on a recombinant measles virus confers entry through the tumor-associated carcinoembryonic antigen

To redirect the tropism of the vaccine strain of measles virus (MV), Edmonston B, to a targeted cell population, we displayed on the viral hemagglutinin (H) a single-chain antibody (scAb) specific for the tumor-associated carcinoembryonic antigen (CEA). We generated H fusion proteins with three forms of the scAb appended, differing in the lengths of the linkers separating the VH and VL domains and thus in the oligomerization states of the scAbs. All proteins were stable, appeared properly folded, and were transported to the cell surface, but only H displaying the long-linker form of scAb was functional in supporting cell-cell fusion. This protein induced extensive syncytia in cells expressing the normal virus receptor CD46 and also in CD46-negative cells expressing the targeted receptor, human CEA. Replication-competent MV with H replaced by H displaying the long-linker form of scAb was recovered and replicated efficiently in both CD46-positive and CD46-negative, CEA-positive cells. Thus, MV not only tolerates the addition of a scAb on its H protein but also infects cells via a novel interaction between the scAb and its targeted receptor.     

Subacute sclerosing panencephalitis virus dominantly interferes with replication of wild-type measles virus in a mixed infection: implication for viral persistence.

Interaction between the Edmonston or Nagahata strain of acute measles virus (MV) and the defective Biken strain of MV isolated from a patient with subacute sclerosing panencephalitis (SSPE) was examined by a cell fusion protocol. Biken-CV-1 cells nonproductively infected with Biken strain SSPE virus were fused with neomycin-resistant CV-1 cells. All the fused cells selected with the neomycin analog G418 expressed Biken viral proteins, as determined by an immunofluorescence assay. This procedure enabled the transfer of Biken viral genomes into cells previously infected with MV. In the fused cells coinfected by Biken strain SSPE virus and Edmonston or Nagahata strain MV, early MV gene expression was suppressed, as determined by immunoprecipitation with strain-specific antibodies. Maturation of Edmonston strain MV was also suppressed. When the coinfected fused cells were selected with G418, Biken viral proteins remained at a constant level for up to 7 weeks. Wild-type MV proteins gradually decreased to a barely detectable level after 4 weeks and became undetectable after 7 weeks. Immunofluorescence studies showed a steady decline in cells expressing wild-type MV proteins in the coinfected cultures. These results suggest that Biken strain SSPE virus dominantly interferes with the replication of wild-type MV. The possible mechanisms of dominant interference and the implication for evolution of a persistent MV infection are discussed.     

SLAM (CD150)-independent measles virus entry as revealed by recombinant virus expressing green fluorescent protein

Wild-type measles virus (MV) strains use human signaling lymphocyte activation molecule (SLAM) as a cellular receptor, while vaccine strains such as the Edmonston strain can use both SLAM and CD46 as receptors. Although the expression of SLAM is restricted to cells of the immune system (lymphocytes, dendritic cells, and monocytes), histopathological studies with humans and experimentally infected monkeys have shown that MV also infects SLAM-negative cells, including epithelial, endothelial, and neuronal cells. In an attempt to explain these findings, we produced the enhanced green fluorescent protein (EGFP)-expressing recombinant MV (IC323-EGFP) based on the wild-type IC-B strain. IC323-EGFP showed almost the same growth kinetics as the parental recombinant MV and produced large syncytia exhibiting green autofluorescence in SLAM-positive cells. Interestingly, all SLAM-negative cell lines examined also showed green autofluorescence after infection with IC323-EGFP, although the virus hardly spread from the originally infected individual cells and thus did not induce syncytia. When the number of EGFP-expressing cells after infection was taken as an indicator, the infectivities of IC323-EGFP for SLAM-negative cells were 2 to 3 logs lower than those for SLAM-positive cells. Anti-MV hemagglutinin antibody or fusion block peptide, but not anti-CD46 antibody, blocked IC323-EGFP infection of SLAM-negative cells. This infection occurred under conditions in which entry via endocytosis was inhibited. These results indicate that MV can infect a variety of cells, albeit with a low efficiency, by using an as yet unidentified receptor(s) other than SLAM or CD46, in part explaining the observed MV infection of SLAM-negative cells in vivo.     

Altered interaction of the matrix protein with the cytoplasmic tail of hemagglutinin modulates measles virus growth by affecting virus assembly and cell-cell fusion

Clinical isolates of measles virus (MV) use signaling lymphocyte activation molecule (SLAM) as a cellular receptor, whereas vaccine and laboratory strains may utilize the ubiquitously expressed CD46 as an additional receptor. MVs also infect, albeit inefficiently, SLAM(-) cells, via a SLAM- and CD46-independent pathway. Our previous study with recombinant chimeric viruses revealed that not only the receptor-binding hemagglutinin (H) but also the matrix (M) protein of the Edmonston vaccine strain can confer on an MV clinical isolate the ability to grow well in SLAM(-) Vero cells. Two substitutions (P64S and E89K) in the M protein which are present in many vaccine strains were found to be responsible for the efficient growth of recombinant virus in Vero cells. Here we show that the P64S and E89K substitutions allow a strong interaction of the M protein with the cytoplasmic tail of the H protein, thereby enhancing the assembly of infectious particles in Vero cells. These substitutions, however, are not necessarily advantageous for MVs, as they inhibit SLAM-dependent cell-cell fusion, thus reducing virus growth in SLAM(+) B-lymphoblastoid B95a cells. When the cytoplasmic tail of the H protein is deleted, a virus with an M protein possessing the P64S and E89K substitutions no longer grows well in Vero cells yet causes cell-cell fusion and replicates efficiently in B95a cells. These results reveal a novel mechanism of adaptation and attenuation of MV in which the altered interaction of the M protein with the cytoplasmic tail of the H protein modulates MV growth in different cell types.     

Rescue of measles viruses from cloned DNA.

A system has been established allowing the rescue of replicating measles viruses (MVs) from cloned DNA. On one hand, plasmids were constructed from which MV antigenomic RNAs with the correct termini are transcribed by phage T7 RNA polymerase. On the other hand, helper cells derived from the human embryonic kidney 293 cell line were generated constitutively expressing T7 RNA polymerase together with MV nucleocapsid protein and phosphoprotein. Simultaneous transfection of the helper cells with the MV antigenomic plasmid and with a plasmid encoding the MV polymerase under direction of a T7 promoter led to formation of syncytia from which MVs were easily recovered. A genetic tag comprising three nucleotide changes was present in the progeny virus. As a first application of reverse genetics, a segment of 504 nucleotides from the 5' non-coding region of the fusion gene was deleted, leading to an MV variant whose replication behaviour in Vero cells was indistinguishable from that of the laboratory Edmonston B strain. Since no helper virus is involved, this system, in principle, should be applicable to the rescue of any member of the large virus order Mononegavirales, i.e. viruses with a nonsegmented negative-strand RNA genome.     

A Recombinant Measles Vaccine Virus Expressing Wild-Type Glycoproteins: Consequences for Viral Spread and Cell Tropism

Wild-type, lymphotropic strains of measles virus (MV) and tissue culture-adapted MV vaccine strains possess different cell tropisms. This observation has led to attempts to identify the viral receptors and to characterize the functions of the MV glycoproteins. We have functionally analyzed the interactions of MV hemagglutinin (H) and fusion (F) proteins of vaccine (Edmonston) and wild-type (WTF) strains in different combinations in transfected cells. Cell-cell fusion occurs when both Edmonston F and H proteins are expressed in HeLa or Vero cells. The expression of WTF glycoproteins in HeLa cells did not result in syncytia, yet they fused efficiently with cells of lymphocytic origin. To further investigate the role of the MV glycoproteins in virus cell entry and also the role of other viral proteins in cell tropism, we generated recombinant vaccine MVs containing one or both glycoproteins from WTF. These viruses were viable and grew similarly in lymphocytic cells. Recombinant viruses expressing the WTFH protein showed a restricted spread in HeLa cells but spread efficiently in Vero cells. Parental WTF remained restricted in both cell types. Therefore, not only differential receptor usage but also other cell-specific factors are important in determining MV cell tropism.     

Human membrane cofactor protein (CD46) acts as a cellular receptor for measles virus.

A monoclonal antibody (MCI20.6) which inhibited measles virus (MV) binding to host cells was previously used to characterize a 57- to 67-kDa cell surface glycoprotein as a potential MV receptor. In the present work, this glycoprotein (gp57/67) was immunopurified, and N-terminal amino acid sequencing identified it as human membrane cofactor protein (CD46), a member of the regulators of complement activation gene cluster. Transfection of nonpermissive murine cells with a recombinant expression vector containing CD46 cDNA conferred three major properties expected of cells permissive to MV infection. First, expression of CD46 enabled MV to bind to murine cells. Second, the CD46-expressing murine cells were able to undergo cell-cell fusion when both MV hemagglutinin and MV fusion glycoproteins were expressed after infection with a vaccinia virus recombinant encoding both MV glycoproteins. Third, M12.CD46 murine B cells were able to support MV replication, as shown by production of infectious virus and by cell biosynthesis of viral hemagglutinin after metabolic labeling of infected cells with [35S]methionine. These results show that the human CD46 molecule serves as an MV receptor allowing virus-cell binding, fusion, and viral replication and open new perspectives in the study of MV pathogenesis.     

Neonatal immunization with a Sindbis virus-DNA measles vaccine induces adult-like neutralizing antibodies and cell-mediated immunity in the presence of maternal antibodies

Infants younger than age 9 mo do not respond reliably to the live attenuated measles vaccine due the immaturity of their immune system and the presence of maternal Abs that interfere with successful immunization. We evaluated the immune responses elicited by Sindbis virus replicon-based DNA vaccines encoding measles virus (MV) hemagglutinin (H, pMSIN-H) or both hemagglutinin and fusion (F, pMSINH-FdU) glycoproteins in neonatal mice born to naive and measles-immune mothers. Despite the presence of high levels of maternal Abs, neonatal immunization with pMSIN-H induced long-lasting, high-avidity MV plaque reduction neutralization (PRN) Abs, mainly IgG2a, that also inhibited syncytium formation in CD150(+) B95-8 cells. IgG secreting plasma cells were detected in spleen and bone marrow. Newborns vaccinated with pMSINH-FdU elicited PRN titers that surpassed the protective level (200 mIU/ml) but were short-lived, had low syncytium inhibition capacity, and lacked avidity maturation. This vaccine failed to induce significant PRN titers in the presence of placentally transferred Abs. Both pMSIN-H and pMSINH-FdU elicited strong Th1 type cell-mediated immunity, measured by T cell proliferation and IFN-gamma production, that was unaffected by maternal Abs. Newborns responded to measles DNA vaccines with similar or even higher PRN titers and cell-mediated immunity than adult mice. This study is the first demonstration that a Sindbis virus-based measles DNA vaccine can elicit robust MV immunity in neonates bypassing maternal Abs. Such a vaccine could be followed by the current live attenuated MV vaccine in a heterologous prime-boost to protect against measles early in life.     

Immunoglobulin g antibody-mediated enhancement of measles virus infection can bypass the protective antiviral immune response

Antibodies to viral surface glycoproteins play a crucial role in immunity to measles by blocking both virus attachment and subsequent fusion with the host cell membrane. Here, we demonstrate that certain immunoglobulin G (IgG) antibodies can also enhance the entry of measles virus (MV) into monocytes and macrophages. Antibody-dependent enhancement of infectivity was observed in mouse and human macrophages using virions opsonized by a murine monoclonal antibody against the MV hemagglutinin (H) glycoprotein, polyclonal mouse anti-MV IgG, or diluted measles-immune human sera. Neither H-specific Fab fragments nor H-specific IgM could enhance MV entry in monocytes or macrophages, indicating involvement of a Fc γ receptor (FcγR)-mediated mechanism. Preincubation with an anti-fusion protein (anti-F) monoclonal antibody or a fusion-inhibitory peptide blocked infection, indicating that a functional F protein was required for viral internalization. Classical complement pathway activation did not promote infection through complement receptors and inhibited anti-H IgG-mediated enhancement. In vivo, antibody-enhanced infection allowed MV to overcome a highly protective systemic immune response in preimmunized IfnarKo-Ge46 transgenic mice. These data demonstrate a previously unidentified mechanism that may contribute to morbillivirus pathogenesis where H-specific IgG antibodies promote the spread of MV infection among FcγR-expressing host cells. The findings point to a new model for the pathogenesis of atypical MV infection observed after immunization with formalin-inactivated MV vaccine and underscore the importance of the anti-F response after vaccination.     

Lymphatic dissemination and comparative pathology of recombinant measles viruses in genetically modified mice

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.