Comparison of the protective efficacy of DNA and baculovirus-derived protein vaccines for EBOLA virus in guinea pigs

The filoviruses Ebola virus (EBOV) and Marburg virus (MARV) cause severe hemorrhagic fever in humans for which no vaccines are available. Previously, a priming dose of a DNA vaccine expressing the glycoprotein (GP) gene of MARV followed by boosting with recombinant baculovirus-derived GP protein was found to confer protective immunity to guinea pigs (Hevey et al., 2001. Vaccine 20, 568-593). To determine whether a similar prime-boost vaccine approach would be effective for EBOV, we generated and characterized recombinant baculoviruses expressing full-length EBOV GP (GP(1,2)) or a terminally-deleted GP (GPa-) and examined their immunogenicity in guinea pigs. As expected, cells infected with the GPa- recombinant secreted more GP(1) than those infected with the GP(1,2) recombinant. In lectin binding studies, the insect cell culture-derived GPs were found to differ from mammalian cell derived virion GP, in that they had no complex/hybrid N-linked glycans or glycans containing sialic acid. Despite these differences, the baculovirus-derived GPs were able to bind monoclonal antibodies to five distinct epitopes on EBOV GP, indicating that the antigenic structures of the proteins remain intact. As a measure of the ability of the baculovirus-derived proteins to elicit cell-mediated immune responses, we evaluated the T-cell stimulatory capacity of the GPa- protein in cultured human dendritic cells. Increases in cytotoxicity as compared to controls suggest that the baculovirus proteins have the capacity to evoke cell-mediated immune responses. Guinea pigs vaccinated with the baculovirus-derived GPs alone, or in a DNA prime-baculovirus protein boost regimen developed antibody responses as measured by ELISA and plaque reduction neutralization assays; however, incomplete protection was achieved when the proteins were given alone or in combination with DNA vaccines. These data indicate that a vaccine approach that was effective for MARV is not effective for EBOV in guinea pigs.     

A paramyxovirus-vectored intranasal vaccine against Ebola virus is immunogenic in vector-immune animals

Ebola virus (EBOV) causes outbreaks of a highly lethal hemorrhagic fever in humans. The virus can be transmitted by direct contact as well as by aerosol and is considered a potential bioweapon. Because direct immunization of the respiratory tract should be particularly effective against infection of mucosal surfaces, we previously developed an intranasal vaccine based on replication-competent human parainfluenza virus type 3 (HPIV3) expressing EBOV glycoprotein GP (HPIV3/EboGP) and showed that it is immunogenic and protective against a high dose parenteral EBOV challenge. However, because the adult human population has considerable immunity to HPIV3, which is a common human pathogen, replication and immunogenicity of the vaccine in this population might be greatly restricted. Indeed, in the present study, replication of the vaccine in the respiratory tract of HPIV3-immune guinea pigs was found to be restricted to undetectable levels. This restriction appeared to be based on both neutralizing antibodies and cellular or other components of the immunity to HPIV3. Surprisingly, even though replication of HPIV3/EboGP was highly restricted in HPIV3-immune animals, it induced a high level of EBOV-specific antibodies that nearly equaled that obtained in HPIV3-naive animals. We also show that the previously demonstrated presence of functional GP in the vector particle was not associated with increased replication in the respiratory tract nor with spread beyond the respiratory tract of HPIV3-naive guinea pigs, indicating that expression and functional incorporation of the attachment/penetration glycoprotein of this systemic virus did not mediate a change in tissue tropism.     

Chimeric human parainfluenza virus bearing the Ebola virus glycoprotein as the sole surface protein is immunogenic and highly protective against Ebola virus challenge

We generated a new live-attenuated vaccine against Ebola virus (EBOV) based on a chimeric virus HPIV3/ΔF-HN/EboGP that contains the EBOV glycoprotein (GP) as the sole transmembrane envelope protein combined with the internal proteins of human parainfluenza virus type 3 (HPIV3). Electron microscopy analysis of the virus particles showed that they have an envelope and surface spikes resembling those of EBOV and a particle size and shape resembling those of HPIV3. When HPIV3/ΔF-HN/EboGP was inoculated via apical surface of an in vitro model of human ciliated airway epithelium, the virus was released from the apical surface; when applied to basolateral surface, the virus infected basolateral cells but did not spread through the tissue. Following intranasal (IN) inoculation of guinea pigs, scattered infected cells were detected in the lungs by immunohistochemistry, but infectious HPIV3/ΔF-HN/EboGP could not be recovered from the lungs, blood, or other tissues. Despite the attenuation, the virus was highly immunogenic, and a single IN dose completely protected the animals against a highly lethal intraperitoneal challenge of guinea pig-adapted EBOV.     

A luciferase-based budding assay for Ebola virus

The VP40 matrix protein of Ebola virus (EBOV) is capable of budding from mammalian cells as a virus-like particle (VLP) and is the major protein involved in virus egress. A functional budding assay has been developed based upon this characteristic of VP40 to assess the contributions of VP40 sequences as well as host proteins to the budding process. This well-defined assay has been modified for potential use in a high-throughput format in which the detection and quantification of firefly luciferase protein in VLPs represents a direct measure of VP40 budding efficiency. Luciferase was found to be incorporated into budding VP40 VLPs. Furthermore, co-expression of EBOV glycoprotein (GP) enhances release of VLPs containing VP40 and luciferase. In contrast, when luciferase is co-expressed with a budding deficient mutant of VP40, luciferase levels in the VLP fraction decrease significantly. This assay represents a promising high-throughput approach to identify inhibitors of EBOV budding.     

Identification of essential and non-essential genes of the guinea pig cytomegalovirus (GPCMV) genome via transposome mutagenesis of an infectious BAC clone

We report application of a transposition methodology that allows the easy characterization and mutation of genes encoded on an infectious bacterial artificial chromosome (BAC) clone. We characterized mutants generated by transposome (Tn) mutagenesis of a BAC clone of guinea pig cytomegalovirus (GPCMV). A pool of Tn mutant GPCMV BACs were screened initially by restriction profile analysis to verify they were full-length, and subsequently GPCMV BAC DNA from individual mutants was transfected onto guinea pig lung fibroblast cells in order to generate virus. Tn GPCMV BAC mutants were classed as either essential or non-essential gene insertions, depending upon their ability to regenerate viable, replication-competent virus. Representative mutants were more fully characterized. Analysis by sequencing the Tn insertion site on the mutated BACs, and by regeneration of virus using transfection of guinea pig fibroblasts (GPL), demonstrated that a recombinant with a Tn insertion in the UL35 homolog gene (GP35) was a non-essential gene for viral replication in tissue culture. A mutant with an insertion in the UL46 homolog (GP46) was nonviable, a phenotype which could be rescued by homologous recombination of BAC DNA with wild-type UL46 sequences, suggesting an essential role of this putative capsid gene in virus replication.     

Ebola virus glycoprotein demonstrates differential cellular localization in infected cell types of nonhuman primates and guinea pigs

BACKGROUND: In vitro studies have previously shown that Ebola virus glycoprotein (GP) is rapidly processed and largely released from infected cells, whereas other viral proteins, such as VP40, accumulate within cells. OBJECTIVE: To determine infected cell types in which Ebola virus GP and VP40, individually, localize in vivo. METHODS: Immunohistochemistry and in situ hybridization using GP- and VP40-specific antibodies and genetic probes were used to analyze archived tissues of experimentally infected nonhuman primates and guinea pigs and Vero E6 and 293 cells infected in vitro. RESULTS: The GP antigen was consistently present in hepatocytes, adrenal cortical cells, fibroblasts, fibroblastic reticular cells, ovarian thecal cells, and several types of epithelial cells, but was not detected in macrophages and blood monocytes of animals, nor in Vero cells and 293 cells. All GP-positive and GP-negative cell types analyzed contained VP40 antigen and both GP and VP40 RNAs. CONCLUSIONS: Ebola virus GP appears to selectively accumulate in many cell types infected in vivo, but not in macrophages and monocytes. This finding suggests that many cell types may have a GP-processing pathway that differs from the pathway described by previous in vitro studies. Differential cellular localization of GP could be relevant to the pathogenesis of Ebola hemorrhagic fever.     

Descriptive analysis of Ebola virus proteins

The virion proteins of two strains of Ebola virus were compared by SDS-polyacrylamide gel electrophoresis (PAGE) and radioimmunoprecipitation (RIP). Seven virion proteins were described; an L (180K), GP (125K), NP (104K), VP40 (40K), VP35 (35K), VP30 (30K), and VP24 (24K). The RNP complex of the virus contained the L, the NP, and VP30, with VP35 in loose association with them. The GP was the major spike protein, with VP40 and VP24 making up the remaining protein content of the multilayered envelope.     

Genome comparison of virulent and avirulent strains of the Pichinde arenavirus

A virulent (P18) strain of the Pichinde arenavirus produces a disease in guinea pigs that somewhat mimics human Lassa fever, whereas an avirulent (P2) strain of this virus is attenuated in infected animals. It has been speculated that the composition of viral genomes may confer the degree of virulence in an infected host; the complete sequence of the viral genomes, however, is not known. Here, we provide for the first time genomic sequences of the S and L segments for both the P2 and P18 strains. Sequence comparisons identify three mutations in the GP1 subunit of the viral glycoprotein, one in the nucleoprotein NP, and five in the viral RNA polymerase L protein. These mutations, alone or in combination, may contribute to the acquired virulence of Pichinde virus infection in animals. The three amino acid changes in the variable region of the GP1 glycoprotein subunit may affect viral entry by altering its receptor-binding activity. While NP has previously been shown to modulate host immune responses to viral infection, we found that the R374 K change in this protein does not affect the NP function of suppressing interferon-beta expression. Four out of the five amino acid changes in the L protein occur in a small region of the protein that may contribute to viral virulence by enhancing its function in viral genomic RNA synthesis.     

Bovine rotavirus-cell interactions: effect of virus infection on cellular integrity and macromolecular synthesis

Hamster, rat, and guinea pig cells were transformed by a variant of BK virus, RFV, whose genome consists of two complementary defective molecules, one, R1, with a deletion corresponding to 20–30% of the coding region for capsid proteins VP2 and VP3 and the other, R2, with a deletion corresponding approximately to 50% of the coding region for large T-antigen of BKV. Blot-hybridization of cellular DNA reveals that all three transformed cell types contain viral DNA integrated into high-molecular-weight DNA. Moreover, all the cell lines contain the viral DNA species which has the intact early region. None of the cell lines contains both DNA species. We examined the genome organization of the virus used in these transformation experiments to confirm our previous observations (Pater et al., J. Virol.36, 480–487, 1980a) on the dual genome state of RFV. We found a very similar but not identical map for the plaque-purified RFV DNA used in this study when compared to that for plaque-purified RFV DNA examined previously.     

The properties of Ebola virus proteins

The paper describes the structure and functions of Ebola virus properties. It also presents information on the role of structural (NP, VP40, VP35, GP, VP30, VP24, and L) and secreted (sGP, delta-peptide, GP1, GP(1,2delta), ssGP) proteins in the viral replication cycle and in the pathogenesis of Ebola hemorrhagic fever.     

Adaptive mutations in the H5N1 polymerase complex

Adaptation of the viral polymerase to host factors plays an important role in interspecies transmission of H5N1 viruses. Several adaptive mutations have been identified that, in general, determine not only host range, but also pathogenicity and transmissibility of the virus. The available evidence indicates that most of these mutations are found in the PB2 subunit of the polymerase. Particularly prominent mutations are located in the C-terminal domain of PB2 involving the amino acid exchanges E627K and D701N. Both mutations, that are also responsible for the adaptation of other avian viruses to mammalian hosts, have been described in human H5N1 isolates. In animal models, it could be demonstrated that they enhance pathogenicity in mice and induce contact transmission in guinea pigs. Mutation E627K has also been identified as a determinant of air-borne H5N1 transmission in ferrets. We are only beginning to understand the underlying mechanisms at the molecular level. Thus, mutation D701N promotes importin-α mediated nuclear transport in mammalian cells. Mutation E627K also enhances the replication rate in an importin-α dependent fashion in mammalian cells, yet without affecting nuclear entry of PB2. Numerous other adaptive mutations, some of which compensate for the lack of PB2 E627K, have been observed in PB2 as well as in the polymerase subunit PB1, the nucleoprotein NP, and the nuclear export protein NEP (NS2).     

Detection of antiviral activity of monoclonal antibodies, specific to Marburg virus proteins

Monoclonal antibodies (MAbs) specific to Marburg virus (MBG), Popp strain, have been previously produced and characterized by indirect ELISA. Protein specificity of MAbs was determined by immunoblotting with SDS-PAGE proteins of MBG: one to NP, four to VP40, and protein specificity of one antibody was not detected. The effect of MAb binding to protein epitopes on viral functions was investigated in vitro and in vivo. None of antibodies neutralized the virus in the neutralization test in vitro, but MAb 5G9.G11 and 5G8.H5 specific to MBG VP40 protein were active in antibody-dependent complement mediated lysis of virus-infected cells. In vivo these antibodies (5G9.G11 and 5G8.H5) protected guinea pigs from lethal MBG infection after passive inoculation. Studies of biological activity and analysis of epitope specificity of MAb-antiVP40 by competitive ELISA showed that 2 of 7 epitopes of VP40 protein of MBG induce the production of protective antibodies. Hence, MAbs 5G9.G11 and 5G8.H5 reacting with MBG VP40 protein caused lysis of virus infected cells in the presence of the complement in vitro and protected guinea pigs from MBG infection by passive inoculation.     

Distinct mutant hepatitis B virus genomes, with alterations in all four open reading frames, in a single South African hepatocellular carcinoma patient

Sequence variation of hepatitis B virus (HBV) can influence the replication, antigen expression and pathogenicity of the virus. We report on the mutational analysis of HBV performed in a 28-year-old Black South African female diagnosed with HBV-induced hepatocellular carcinoma. Full-genome amplification and DNA sequencing of HBV was carried out. Five distinct complete genomic clones were described with extensive genomic and intragenic variation. Phylogenetic analysis revealed that all five clones belonged to subgenotype A1 and that there were at least four virus populations with genomes of different lengths ranging from 3194 to 3253 base pairs. In this particular patient, four major characteristic features, not previously reported to occur simultaneously in HBV isolated from a single patient, were observed. Firstly, all the clones harboured a 13 base pair deletion and a 45 base pair insertion in the basic core promoter (BCP). Secondly, a 37 base pair insertion in the core gene with three adjacent single nucleotide deletions were observed. Thirdly, premature S gene stop codons were observed in some clones and lastly X gene initiation codon mutations were also observed. The complex nature of the mutations in the HBV isolated from this single patient may have contributed to the early onset of hepatocarcinogenesis.     

The amino acid sequence of the outer coat protein VP2 of neutralizing monoclonal antibody-resistant, virulent and attenuated bluetongue viruses.

Monoclonal antibodies which reacted with four different epitopes were used to select neutralization-resistant variants of Australian bluetongue virus serotype 1 (BTV1AUS; isolate CS156). Nucleotide sequencing of the VP2 outer coat protein gene of these variants showed that two of them contained alterations within the previously defined neutralization site at amino acids 328 to 335 (Gould et al., 1988). Comparison of VP2 sequences of several BTV serotypes, in addition to nucleotide sequence changes in a number of variants, suggested that this neutralization site was larger and contained 19 amino acids, the conformation of which could be affected by other regions of the VP2 protein. Nucleotide sequencing of neutralization-resistant variants revealed a total of four other regions of VP2 affecting the ability of monoclonal antibodies to neutralize the virus and these results support the notion that the neutralization site in VP2 was conformation dependent. The complete nucleotide sequence of the VP2 gene of virulent BTV1AUS (C5156) was determined directly from viral nucleic acid isolated from the blood of a sheep suffering clinical bluetongue disease. Comparison of the VP2 sequence of this virulent virus with that previously published for an avirulent, laboratory strain (Gould, 1988), indicated that the passage of virulent virus approximately 20 times in tissue culture over the last decade, not only led to attenuation but resulted in the appearance of ten nucleotide changes in the VP2 gene. Six of these nucleotide changes were silent, two resulted in conservative amino acid substitutions and two generated radical amino acid changes. However, in a separate experiment, a single passage of the virulent virus in tissue culture while leading to attenuation did not result in a nucleotide change in the VP2 outer coat protein gene.     

Analysis of influenza A virus nucleoproteins for the assessment of molecular genetic mechanisms leading to new phylogenetic virus lineages

Summary The nucleoprotein (NP) gene of influenza A viruses is decisive for separating two large individually evolving reservoirs in birds and humans. A phylogenetic analysis of the NP gene revealed that all mammalian influenza viruses originated — directly or indirectly — from an avian ancestor. The stable introduction of an avian influenza A virus into a mammalian species seems to be a relatively rare event, the latest one occurred in 1979 when such an avian virus was introduced into pigs in Northern Europe which gave rise to a new lineage. At least two concomitant events are required for such a new and stable introduction: (1) The new species has to become infected, and (2) a mutation in the polymerase complex has to establish a labile variant, which is prone to provide a large number of different variants, from which some can adapt rapidly to the new host (or to any unusual environments). Since such mutator mutations might be advantageous only during stress periods, variants with a less error prone polymerase might emerge again after adaptation. Examples for such fluctuations in terms of mutational and evolutionary rates are discussed in this brief review.     

Production and characterization of monoclonal antibodies against different epitopes of Ebola virus antigens

Ebola virus (EBOV) causes hemorrhagic fever in humans and nonhuman primates with up to 90% mortality rate. In this study, Ebola virus like particles (EVLPs) and the aglycosyl subfragment of glycoprotein (GP(1) subfragment D) were used to generate monoclonal antibodies (MAbs) against different epitopes of the viral antigens. Such MAbs could be useful in diagnostics and potential therapeutics of viral infection and its hemorrhagic symptoms. Hybridoma cell fusion technology was used for production of MAbs. The MAbs were characterized using ELISA and Western blot analysis. Furthermore, five recombinant sub-domains of GP(1) subfragment D were produced, which were used as antigen in Western blot analysis for epitope mapping. Seventeen MAbs of different epitope specificities against EBOV antigens [virion protein (VP40), secreted glycoprotein (sGP), and GP(1) subfragment D] were developed. Based on epitope mapping studies, the anti-GP MAbs were categorized into six groups. The binding of the three anti-sGP MAbs with different epitope specificities were mostly between aa 157 and 221. The two anti-VP40 MAbs with the same or overlapping epitopes are potentially good candidates for developing antigen detection assays for early diagnosis of EBOV infection. The anti-GP MAbs with different epitope specificities as an oligoclonal cocktail could be tested for therapy.