Epitopes involved in antibody-mediated protection from Ebola virus.

To determine the ability of antibodies to provide protection from Ebola viruses, monoclonal antibodies (mAbs) to the Ebola glycoprotein were generated and evaluated for efficacy. We identified several protective mAbs directed toward five unique epitopes on Ebola glycoprotein. One of the epitopes is conserved among all Ebola viruses that are known to be pathogenic for humans. Some protective mAbs were also effective therapeutically when administered to mice 2 days after exposure to lethal Ebola virus. The identification of protective mAbs has important implications for developing vaccines and therapies for Ebola virus.

Host-cell receptors for Sindbis virus.

Sindbis virus has a very wide host range, infecting many species of mosquitoes and other hematophagous insects and infecting many species of higher vertebrates. We have used two approaches to study host cell receptors used by Sindbis virus to enter cells. Anti-idiotype antibodies to neutralizing antibodies directed against glycoprotein E2 of the virus identified a 63-kDa protein as a putative receptor in chicken cells. In a second approach, monoclonal antibodies identified a 67 kDa protein, believed to be a high affinity laminin receptor, as a putative receptor in mammalian cells and in mosquito cells. We conclude that the virus attains its very wide host range by two mechanisms. In one mechanism, the virus is able to use more than one protein as a receptor. In a second mechanism, the virus utilizes proteins as receptors that are highly conserved across the animal kingdom.

Hantaan virus M RNA: coding strategy, nucleotide sequence, and gene order.

The M genome segment of Hantaan virus was molecularly cloned and the nucleotide sequence of cDNA was determined. The virion RNA is 3616 bases long with 3'- and 5'-terminal nucleotide sequences complementary for 18 bases. A single long open reading frame in the viral complementary-sense RNA had the potential to encode 1135 amino acids or a polypeptide of 126,000 Da. Amino-terminal sequences of isolated G1 and G2 envelope glycoproteins were determined, revealing a gene order with respect to message sense RNA of 5'-G1-G2-3'. Mature G1 begins 18 amino acids beyond the first AUG of the open reading frame, preceded by a short, hydrophobic leader sequence. G2 begins at the 649th amino acid of the open reading frame and also follows a hydrophobic sequence. Carboxy termini of G1 and G2 were localized and gene order was verified by immune precipitation of Hantaan proteins with antisera to synthetic peptides generated by using amino acid sequences derived from the cDNA sequence. The antipeptide sera were also reactive by immunoblotting with SDS-denatured G1 and G2. Molecular weights of 64,000 and 53,700 were calculated for the G1 and G2 glycoproteins, respectively, from their predicted amino acid sequences. Five potential asparagine-linked glycosylation sites were contained within the G1 amino acid sequence and two within the G2 sequence. These data are consistent with our previous estimates of the molecular weights and extent of glycosylation of the Hantaan envelope glycoproteins.

Protective monoclonal antibodies define maturational and pH-dependent antigenic changes in Sindbis virus E1 glycoprotein.

Monoclonal (MC) antibodies specific for either the EI or E2 glycoproteins of Sindbis virus (SIN) were used to probe for differences in the surface topography of SIN epitopes between infected cells and mature virions. Employing an enzyme-linked immunosorbent assay (ELISA) in which binding of individual peroxidase-labeled MC antibodies to immobilized (solid-phase) detergent-disrupted SIN was inhibited specifically by one or more unlabeled antibodies, viral epitopes could be grouped into six spatially distinct antigenic sites--five on E1, designated a through e, and one site on E2. All six sites were represented on the surfaces of SIN-infected cells as shown by the complement (C')-dependent lysis mediated by antibodies of the corresponding epitope specificities. In contrast, virus-neutralizing (NT) activity was restricted to antibodies specific for epitopes on E2 and on site c of E1, irrespective of the presence of added C' and an antiserum against mouse immunoglobulins. That E1 sites a, b, d, and e became inaccessible to antibody binding was shown by a competitive-inhibition ELISA. Whereas all MC antibodies were inhibited from binding to solid-phase SIN when premixed with detergent-treated virions, only those having NT activity could be competitively inhibited by intact virions. Sites E1-d and E1-e could be exposed not only by detergent disruption but also by lowering the virion pH from 7.2 to 6.0. These collective results indicate that a majority of immunologically relevant E1 epitopes present on SIN-infected cell surfaces become cryptic during SIN maturation and, except at low pH, remain undetectable on virion surfaces.

Complete nucleotide sequences of the M and S segments of two hantavirus isolates from California: evidence for reassortment in nature among viruses related to hantavirus pulmonary syndrome.

We report the complete nucleotide sequence of the M and the S genome segments and a portion of the L segments of two hantavirus isolates from Peromyscus maniculatus trapped in eastern California. The isolates, Convict Creek 107 and 74 (CC107 and CC74) are genetically similar to viruses known to cause hantavirus pulmonary syndrome in New Mexico. CC107 and CC74 each have an M segment consisting of 3696 nucleotides with a coding potential of 1140 amino acids in the virus complementary-sense RNA (cRNA). The S segments of CC107 and CC74 are 2083 and 2047 nucleotides long, respectively, and each has an ORF in the cRNA capable of encoding a protein of 428 amino acids. Unusually long 3' noncoding regions of 757 and 721 nucleotides follow the S segment ORF of CC107 and CC74, respectively, and include numerous imperfect repetitive sequences. Whereas the M and S segments of any given hantavirus typically appear to diverge at comparable rates from homologous genes of any other hantavirus, CC107 and CC74 have M segments that differ by only 1% from one another but S segments that differ by 13%. After trivial explanations are rendered improbable, i.e., by consideration of the genetics of closely and distantly related hantaviruses, the most likely explanation for our data is that hantavirus genome segment reassortment occurred within rodent populations in California.

Antiidiotypic antibodies as probes for the Sindbis virus receptor.

Rabbit polyclonal antiidiotypic antibodies were made to mouse monoclonal antibodies that neutralize the infectivity of Sindbis virus. One of the antiidiotypic antisera obtained has properties characteristic of an antireceptor antiserum. It binds to the surface of chicken cells as shown by immunofluorescence and partially blocks virus binding to these cells as determined by binding of radiolabeled virus or by a plaque reduction assay. It also immunoprecipitates a protein with a molecular weight of 63,000 from chicken cells. From the fact that the antiserum will only partially block virus uptake, and that it does not block uptake of a variant of Sindbis virus resistant to the monoclonal antibody used to produce the antiidiotypic antiserum, we propose that at least two distinguishable receptors can be used by Sindbis virus to enter chicken cells. Furthermore, the receptors used by Sindbis to enter BHK cells appear to be different from those on chicken cells, at least in part, in that the antiidiotypic antiserum does not recognize the BHK counterpart of the chicken cell receptor. We suggest that the alphaviruses use a number of distinguishable receptors which differ depending on the host and the tissue. In chicken cells the 63,000 molecular weight protein may be one of them. The diversity of such multiple receptors could account for the very wide host range of the alphaviruses, which infect mosquitoes, birds, and mammals.

Characterization of Hantaan virus envelope glycoprotein antigenic determinants defined by monoclonal antibodies.

A panel of 24 monoclonal antibodies (MAbs) to the G1 or G2 envelope glycoproteins of Hantaan virus were used to determine the surface topography and functional properties of antigenic sites. Nine distinct, partially overlapping antigenic sites, two on G1 and seven on G2, were demonstrated by competitive binding assays. Analyses of the antigenic sites by haemagglutination (HA) inhibition and plaque-reduction neutralization tests showed that all of the sites, except one on G1, were related to viral HA. Only one of the G1 antigenic sites and two of the G2 sites were involved in virus neutralization. These results suggest that certain epitopes related to HA were not critical for virus neutralization. The nine antigenic sites could be further divided into 13 based upon the serological cross-reactivity of MAbs with viruses representative of each of the four known antigenic groups within the Hantavirus genus of Bunyaviridae, i.e. Hantaan, Seoul, Puumala and Prospect Hill viruses.

Infectious enveloped RNA virus antigenic chimeras.

Random insertion mutagenesis has been used to construct infectious Sindbis virus structural protein chimeras containing a neutralization epitope from a heterologous virus, Rift Valley fever virus. Insertion sites, permissive for recovery of chimeric viruses with growth properties similar to the parental virus, were found in the virion E2 glycoprotein and the secreted E3 glycoprotein. For the E2 chimeras, the epitope was expressed on the virion surface and stimulated a partially protective immune response to Rift Valley fever virus infection in vivo. Besides providing a possible approach for developing live attenuated vaccine viruses, insertion of peptide ligands into virion surface proteins may ultimately allow targeting of virus infection to specific cell types.

Baculovirus expression of the small genome segment of Hantaan virus and potential use of the expressed nucleocapsid protein as a diagnostic antigen.

Autographa californica nuclear polyhedrosis virus (AcNPV) was used as an expression vector for the nucleocapsid protein gene of Hantaan virus. Two different cDNA clones representing the small genome segment of Hantaan virus were inserted into the transfer vector pAcYM1, and recombinants were generated by replacement of a portion of the baculovirus polyhedrin gene with the foreign, Hantaan virus gene. Recombinants containing both the first and second ATG initiation codons of the Hantaan virus gene produced nucleocapsid protein, while those containing only the second codon did not. The expressed nucleocapsid protein was evaluated as a potential diagnostic antigen with a variety of hantavirus-immune sera. The high levels of expression obtained, specific serological reactivity with immune sera and the low level of biological containment required for production of this protein all suggest a significant advantage over authentic viral antigen for diagnosis of hantavirus infection.

DNA vaccination with vaccinia virus L1R and A33R genes protects mice against a lethal poxvirus challenge.

Previously we found that passive transfer of monoclonal antibodies (MAbs) specific to either the vaccinia virus (VACV) L1R or A33R gene product protected mice from challenge with VACV. The L1R-specific MAbs, which bind the intracellular mature virion (IMV), neutralized virus in cell culture, whereas the A33R-specific MAbs, which bind extracellular enveloped virions (EEV), did not. To investigate whether a protective response could be generated by vaccination with these genes, we constructed and evaluated DNA vaccines expressing the VACV L1R and/or A33R genes under control of a cytomegalovirus promoter. Mice were vaccinated with DNA-coated gold beads by using a gene gun and then challenged with VACV (strain WR) intraperitoneally. Mice vaccinated with L1R alone developed neutralizing antibodies and were partially protected. Mice vaccinated with a combination of both genes loaded on the same gold beads developed a robust anti-A33R response; however, no neutralizing antibody response was detected, and the mice were not protected. In contrast, when mice were vaccinated with L1R and A33R loaded on different gold beads, neutralizing (presumably anti-L1R) and anti-A33R antibody responses were detected, and protection was markedly improved. Our results indicated that vaccination with both L1R and A33R proteins, intended to evoke mechanistically distinct and complementary forms of protection, was more effective than vaccination with either protein by itself.

Use of telemetry to assess vaccine-induced protection against parenteral and aerosol infections of Venezuelan equine encephalitis virus in non-human primates.

Two investigational vaccines, TC-83 (live-attenuated) and C-84 (formalin-inactivated), are currently available to immunize at-risk individuals against Venezuelan equine encephalitis virus (VEE). Ideally, such vaccines should protect against both the natural mosquito-borne route of infection and from aerosol, the most common route of laboratory infection. Whereas considerable data on vaccine efficacy following parenteral challenge are available, the efficacy of these vaccines against disease caused by aerosol exposure is not well established in primates. We compared the immunogenicity and protective capacity of TC-83 and C-84 against either subcutaneous or aerosol routes of infection in cynomolgus monkeys implanted with temperature-monitoring radiotelemetry devices. A single s.c. dose of TC-83, or three s.c. doses (days 0, 7, 28) of C-84, elicited similar serum virus-neutralizing antibody responses. Animals immunized with either TC-83 or C-84 were protected against s.c. infection. In contrast, after aerosol infection, 40% of the animals vaccinated with either TC-83 or C-84 developed signs nearly as severe as those seen in unvaccinated animals. Protection was not entirely consistent with the measured preinfection immune responses: unprotected animals had serum virus-neutralizing antibody titers and lymphoproliferative responses similar to those seen in protected animals. In this study, C-84 (three doses) protected monkeys as well as TC-83 (one dose) against either a s.c. or aerosol VEE challenge.

Identification of antigenically important domains in the glycoproteins of Sindbis virus by analysis of antibody escape variants.

To study important epitopes on glycoprotein E2 of Sindbis virus, eight variants selected to be singly or multiply resistant to six neutralizing monoclonal antibodies reactive against E2, as well as four revertants which had regained sensitivity to neutralization, were sequenced throughout the E2 region. To study antigenic determinants in glycoprotein E1, four variants selected for resistance to a neutralizing monoclonal antibody reactive with E1 were sequenced throughout the E2 and E1 regions. All of the salient changes in E2 occurred within a relatively small region between amino acids 181 and 216, a domain that encompasses a glycosylation site at residue 196 and that is rich in charged amino acids. Almost all variants had a change in charge, suggesting that the charged nature of this domain is important for interaction with antibodies. Variants independently isolated for resistance to the same antibody were usually altered in the same amino acid, and reversion to sensitivity occurred at the sites of the original mutations, but did not always restore the parental amino acid. The characteristics of this region suggest that this domain is found on the surface of E2 and constitutes a prominent antigenic domain that interacts directly with neutralizing antibodies. Previous studies have shown that this domain is also important for penetration of cells and for virulence of the virus. Resistance to the single E1-specific neutralizing monoclonal antibody resulted from changes of Gly-132 of E1 to either Arg or Glu. Analogous to the findings with E2, these changes result in a change in charge and are found near a glycosylation site at residue 139. This domain of E1 may therefore be found near the 181 to 216 domain of E2 on the surface of the E1-E2 heterodimer; together, they could form a domain important in virus penetration and neutralization.

Antigenic subunits of Hantaan virus expressed by baculovirus and vaccinia virus recombinants.

Baculovirus and vaccinia virus vectors were used to express the small (S) and medium (M) genome segments of Hantaan virus. Expression of the complete S or M segments yielded proteins electrophoretically indistinguishable from Hantaan virus nucleocapsid protein or envelope glycoproteins (G1 and G2), and expression of portions of the M segment, encoding either G1 or G2 alone, similarly yielded proteins which closely resembled authentic Hantaan virus proteins. The expressed envelope proteins retained all antigenic sites defined by a panel of monoclonal antibodies to Hantaan virus G1 and G2 and elicited antibodies in animals which reacted with authentic viral proteins. A Hantaan virus infectivity challenge model in hamsters was used to assay induction of protective immunity by the recombinant-expressed proteins. Recombinants expressing both G1 and G2 induced higher titer antibody responses than those expressing only G1 or G2 and protected most animals from infection with Hantaan virus. Baculovirus recombinants expressing only nucleocapsid protein also appeared to protect some animals from challenge. Passively transferred neutralizing monoclonal antibodies similarly prevented infection, suggesting that an antibody response alone is sufficient for immunity to Hantaan virus.

Antibody-selected variation and reversion in Sindbis virus neutralization epitopes.

Sindbis virus variants evidencing a complex and bidirectional tendency toward spontaneous antigenic change were isolated and characterized. Variants were selected on the basis of their escape from neutralization by individual monoclonal antibodies to either of the two envelope glycoproteins, E2 and E1. Multisite variants, including one altered in three neutralization sites, were obtained by selecting mutants consecutively in the presence of different neutralizing monoclonal antibodies. Two phenotypic revertants, each of which reacquired prototype antigenicity, were back-selected on the basis of their reactivity with a neutralizing monoclonal antibody. An incidental oligonucleotide marker distinguished these and the variant from which they arose from parental Sindbis virus and other mutants, thereby confirming that the revertants were true progeny of the antigenic variant. Prototype Sindbis virus and variants derived from it were compared on the basis of their reactivities with each of a panel of monoclonal antibodies; patterns revealed a minimum of five independently mutable Sindbis virus neutralization epitopes, segregating as three antigenic sites (two E2 and one E1).

Alternative forms of a strain-specific neutralizing antigenic site on the Sindbis virus E2 glycoprotein.

Experiments with monoclonal antibodies raised against two laboratory strains of Sindbis virus, SB and SIN, suggested the existence of a strain-specific neutralizing antigenic site (E2-b) on the E2 glycoprotein. A comparison of monoclonal antibody binding patterns and E2 glycoprotein gene sequences of six laboratory strains distinguished three different configurations of E2-b that correlated with specific amino acid substitutions at position 216 of the E2 glycoprotein. Further study of neutralization escape mutants selected with E2-b-specific antibodies confirmed that amino acid 216 is a major determinant of the E2-b antigenic site. Eight of nine mutants showed a coding change at position 216. One neutralization escape mutation created a new glycosylation site at position 213 and resulted in an E2 protein with an altered migration rate in SDS-PAGE. The neutralization escape mutants studied included amino acid substitutions not found in the laboratory strains that revealed differing binding requirements for two E2-b-specific monoclonal antibodies. The E2-b site is contrasted with the E2-c neutralizing antigenic site described previously (R.A. Olmsted, W.J. Meyer, and R.E. Johnston, 1986, Virology 148, 245-254).

Clinical evaluation of a vaccinia-vectored Hantaan virus vaccine.

We evaluated a vaccinia-vectored vaccine for hemorrhagic fever with renal syndrome in clinical trials. A Phase I dose-escalation study in 16 volunteers divided into four groups demonstrated that subcutaneous inoculation of approximately 10(7) plaque-forming units of the recombinant virus was safe and immunogenic. Vaccination of a fifth group of 12 volunteers indicated that neutralizing antibody titers to both vaccinia virus and Hantaan virus were enhanced after a second inoculation. Comparing two routes of vaccination showed that scarification effectively induced neutralizing antibodies in vaccinia virus-naive volunteers but that subcutaneous inoculation was superior to scarification in vaccinia virus-immune individuals. A Phase II, double-blinded, placebo-controlled clinical trial was conducted among 142 volunteers. Two subcutaneous vaccinations were administered at 4-week intervals. Neutralizing antibodies to Hantaan virus or to vaccinia virus were detected in 72% or 98% of vaccinia virus-naive volunteers, respectively. In contrast, only 26% of the vaccinia virus-immune volunteers developed neutralizing antibody responses to Hantaan virus. J. Med. Virol. 60:77-85, 2000. Published 2000 Wiley-Liss, Inc.

Isolation and initial characterization of a newfound hantavirus from California.

A fatal case of hantavirus pulmonary syndrome (HPS) in northern California prompted our attempt to isolate viruses from local rodents. From tissues of two deer mice, Peromyscus maniculatus, two hantaviruses (Convict Creek virus 107 and 74, CC107 and CC74) were established in cell culture. Viral antigens, proteins, and RNAs of the first and archetypical isolate (CC107) were examined, and portions of the medium (M) and small (S) genome segments of both isolates were sequenced. Antigenically, CC107 virus and the second isolate, CC74 virus, were more closely related to Puumala virus than Hantaan (HTN) virus, though distinct from both. Northern blots of viral RNAs showed the large and M segments of CC107 to be the same size as those of HTN virus, whereas the S segment was larger. Protein gels did not reveal CC107 to have a substantially larger nucleocapsid protein than HTN virus. Partial nucleotide sequence comparisons of CC107 and CC74 viruses revealed their M segments to be highly similar to one another, while their S segments differed by more than 10%. Nucleotide and deduced amino acid sequence comparisons showed the California isolates to be closely related to the newfound hantaviruses first detected in the Four Corners area and since incriminated in HPS through wide areas of the United States.

Smallpox DNA vaccine protects nonhuman primates against lethal monkeypox.

Two decades after a worldwide vaccination campaign was used to successfully eradicate naturally occurring smallpox, the threat of bioterrorism has led to renewed vaccination programs. In addition, sporadic outbreaks of human monkeypox in Africa and a recent outbreak of human monkeypox in the U.S. have made it clear that naturally occurring zoonotic orthopoxvirus diseases remain a public health concern. Much of the threat posed by orthopoxviruses could be eliminated by vaccination; however, because the smallpox vaccine is a live orthopoxvirus vaccine (vaccinia virus) administered to the skin, the vaccine itself can pose a serious health risk. Here, we demonstrate that rhesus macaques vaccinated with a DNA vaccine consisting of four vaccinia virus genes (L1R, A27L, A33R, and B5R) were protected from severe disease after an otherwise lethal challenge with monkeypox virus. Animals vaccinated with a single gene (L1R) which encodes a target of neutralizing antibodies developed severe disease but survived. This is the first demonstration that a subunit vaccine approach to smallpox-monkeypox immunization is feasible.

Immunologic interference from sequential administration of live attenuated alphavirus vaccines.

Two different human vaccine trials examined interference arising from sequential administration of vaccines against heterologous alphaviruses. The first trial indicated that persons previously vaccinated against Venezuelan equine encephalitis virus (VEEV) exhibited poor neutralizing antibody responses to a live attenuated chikungunya virus (CHIKV) vaccine (46% response rate). The second trial prospectively examined neutralizing antibody responses to live attenuated VEEV vaccine in persons previously inoculated with either CHIKV vaccine or placebo. Following seroconversion to CHIKV, CHIKV vaccine recipients' geometric mean titers (GMTs) to VEEV by 80% plaque-reduction neutralization titration never exceeded 10, compared with a peak GMT of 95 after VEEV vaccination for alphavirus-naive volunteers who initially received placebo (P < .003). ELISA antibody responses demonstrated cross-reactive IgG to VEEV after primary CHIKV immunization and then an anamnestic response upon subsequent VEEV vaccination. These data indicate that preexisting alphavirus immunity in humans interferes with subsequent neutralizing antibody response to a live attenuated, heterologous vaccine.

Baculovirus expression of the M genome segment of Rift Valley fever virus and examination of antigenic and immunogenic properties of the expressed proteins.

Autographa californica nuclear polyhedrosis viral recombinants containing coding information for the Rift Valley fever virus (RVFV) envelope glycoproteins (G1 and G2) and varying amounts of preglycoprotein coding sequences were prepared by using transfer vectors pAc373 or pAcYM1. Expression products were processed to yield proteins indistinguishable from authentic G1 and G2 by gel electrophoresis. The immunogenic properties of the expressed proteins were assessed by immunizing mice and challenging with RVFV. A single inoculation with lysates of cells infected with recombinants expressing both G1 and G2 induced neutralizing antibody responses in mice and protected them from an otherwise lethal challenge with RVFV. Lysates of cells infected with a recombinant expressing only G2 also induced a protective response after two immunizations. Survivors displayed elevated antibody titers to G1 and G2 and also developed antibodies to the RVFV nucleocapsid protein, the latter allowing discrimination from vaccinated mice and indicating that animals had survived infection. Nonimmune mice were protected from lethal RVFV infection by passive transfer of sera from animals immunized with recombinant antigens, indicating that a humoral immune response is sufficient to protect against RVFV.