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.

Marburg virus vaccines: comparing classical and new approaches.

An effort to develop a safe and effective vaccine for Marburg virus (MBGV), one of the filoviruses known to cause high mortality rates in humans, led us to compare directly some of the merits of modern versus classical vaccine approaches for this agent. Prior work had established the MBGV-glycoprotein (GP), the only known virion surface antigen, as a candidate for inclusion in a vaccine. In this study, we vaccinated groups of Hartley guinea pigs with killed MBGV, live attenuated MBGV, soluble MBGV-GP expressed by baculovirus recombinants, MBGV-GP delivered as a DNA vaccine, or MBGV-GP delivered via an alphavirus RNA replicon. Serological responses were evaluated, and animals were challenged with a lethal dose of MBGV given either subcutaneously or via aerosol. Killed MBGV and replicon-delivered MBGV-GP were notably immunogenic and protective against MBGV, but results did not exclude any approach and suggested a role for DNA vaccines in immunological priming.

Folate receptor-alpha is a cofactor for cellular entry by Marburg and Ebola viruses.

Human infections by Marburg (MBG) and Ebola (EBO) viruses result in lethal hemorrhagic fever. To identify cellular entry factors employed by MBG virus, noninfectible cells transduced with an expression library were challenged with a selectable pseudotype virus packaged by MBG glycoproteins (GP). A cDNA encoding the folate receptor-alpha (FR-alpha) was recovered from cells exhibiting reconstitution of viral entry. A FR-alpha cDNA was recovered in a similar strategy employing EBO pseudotypes. FR-alpha expression in Jurkat cells facilitated MBG or EBO entry, and FR-blocking reagents inhibited infection by MBG or EBO. Finally, FR-alpha bound cells expressing MBG or EBO GP and mediated syncytia formation triggered by MBG GP. Thus, FR-alpha is a significant cofactor for cellular entry for MBG and EBO viruses.

Recombinant RNA replicons derived from attenuated Venezuelan equine encephalitis virus protect guinea pigs and mice from Ebola hemorrhagic fever virus.

RNA replicons derived from an attenuated strain of Venezuelan equine encephalitis virus (VEE), an alphavirus, were configured as candidate vaccines for Ebola hemorrhagic fever. The Ebola nucleoprotein (NP) or glycoprotein (GP) genes were introduced into the VEE RNA downstream from the VEE 26S promoter in place of the VEE structural protein genes. The resulting recombinant replicons, expressing the NP or GP genes, were packaged into VEE replicon particles (NP-VRP and GP-VRP, respectively) using a bipartite helper system that provided the VEE structural proteins in trans and prevented the regeneration of replication-competent VEE during packaging. The immunogenicity of NP-VRP and GP-VRP and their ability to protect against lethal Ebola infection were evaluated in BALB/c mice and in two strains of guinea pigs. The GP-VRP alone, or in combination with NP-VRP, protected both strains of guinea pigs and BALB/c mice, while immunization with NP-VRP alone protected BALB/c mice, but neither strain of guinea pig. Passive transfer of sera from VRP-immunized animals did not confer protection against lethal challenge. However, the complete protection achieved with active immunization with VRP, as well as the unique characteristics of the VEE replicon vector, warrant further testing of the safety and efficacy of NP-VRP and GP-VRP in primates as candidate vaccines against Ebola hemorrhagic fever.

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.

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.

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.

Induction of Epstein-Barr virus-specific cytotoxic T-lymphocyte responses using dendritic cells pulsed with EBNA-3A peptides or UV-inactivated, recombinant EBNA-3A vaccinia virus.

Cell-mediated immunity, especially the cytotoxic T lymphocyte (CTL), provides resistance to Epstein-Barr virus (EBV), as is demonstrated by the occurrence of posttransplant lymphoproliferative disease in immunosuppressed patients. We set out to use dendritic cells (DCs) to elicit anti-EBV-specific CTLs in culture. In unselected, HLA-B8(+) donors, monocyte-derived mature DCs were pulsed with the HLA-B8-restricted EBNA-3A peptide, FLRGRAYGL, and added to autologous T cells for 7 days at a DC:T ratio of 1:5 to 1:60. The cultured cells specifically lysed EBNA-3A peptide-pulsed, HLA-B8(+), B-lymphoblastoid cell lines in a 5-hour (51)Cr-release assay. The generation of CTLs did not require the addition of interleukin-2. In comparison, monocytes were weak antigen-presenting cells. DCs were then infected with recombinant vaccinia-EBNA-3A. Vaccinia infection significantly decreased the viability of immature DCs after 3 days of culture (to 25% to 45%) but had a smaller effect on mature DC recovery (40% to 70%). To decrease these cytopathic effects and to expand the potential use of vaccinia vectors for DC therapy in immunocompromised patients, we successfully used psoralen and UV-inactivated virus. Mature DCs pulsed with either live or inactivated vaccinia EBNA-3A virus could elicit strong EBNA-3A-specific CTLs. Therefore, mature DCs are powerful stimulators of EBV-specific CTLs and their major histocompatibility complex class I products can even be charged with UV-inactivated recombinant vaccinia.

Marburg virus vaccines based upon alphavirus replicons protect guinea pigs and nonhuman primates.

Marburg virus (MBGV), for which no vaccines or treatments currently exist, causes an acute hemorrhagic fever with a high mortality rate in humans. We previously showed that immunization with either killed MBGV or a glycoprotein (GP) subunit prevented lethal infection in guinea pigs. In the studies reported here, an RNA replicon, based upon Venezuelan equine encephalitis (VEE) virus, was used as a vaccine vector; the VEE structural genes were replaced by genes for MBGV GP, nucleoprotein (NP), VP40, VP35, VP30, or VP24. Guinea pigs were vaccinated with recombinant VEE replicons (packaged into VEE-like particles), inoculated with MBGV, and evaluated for viremia and survival. Results indicated that either GP or NP were protective antigens while VP35 afforded incomplete protection. As a more definitive test of vaccine efficacy, nonhuman primates (cynomolgus macaques) were inoculated with VEE replicons expressing MBGV GP and/or NP. Three monkeys received packaged control replicons (influenza HA); these died 9 or 10 days after challenge, with typical MBGV disease. MBGV NP afforded incomplete protection, sufficient to prevent death but not disease in two of three macaques. Three monkeys vaccinated with replicons which expressed MBGV GP, and three others vaccinated with both replicons that expressed GP or NP, remained aviremic and were completely protected from disease.

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.

DNA vaccines expressing either the GP or NP genes of Ebola virus protect mice from lethal challenge.

DNA vaccines expressing the envelope glycoprotein (GP) or nucleocapsid protein (NP) genes of Ebola virus were evaluated in adult, immunocompetent mice. The vaccines were delivered into the skin by particle bombardment of DNA-coated gold beads with the Powderject-XR gene gun. Both vaccines elicited antibody responses as measured by ELISA and elicited cytotoxic T cell responses as measured by chromium release assays. From one to four vaccinations with 0.5 microgram of the GP DNA vaccine resulted in a dose-dependent protection from Ebola virus challenge. Maximal protection (78% survival) was achieved after four vaccinations. Mice were completely protected with a priming dose of 0.5 microgram of GP DNA followed by three or four subsequent vaccinations with 1.5 micrograms of DNA. Partial protection could be observed for at least 9 months after three immunizations with 0.5 microgram of the GP DNA vaccine. Comparing the GP and NP vaccines indicated that approximately the same level of protection could be achieved with either vaccine.

Quantitative studies of heteropolymer-mediated binding of inactivated Marburg virus to the complement receptor on primate erythrocytes.

Previous in vitro and in vivo experiments in our laboratory have demonstrated that cross-linked bispecific monoclonal antibody (mAb) complexes (Heteropolymers, HP) facilitate binding of prototype pathogens to primate erythrocytes (E) via the E complement receptor, CR1. These E-bound immune complexes are safely and rapidly cleared from the bloodstream. In order to generate a robust bispecific system for HP-mediated clearance of real pathogens such as Filoviruses, we have developed the necessary methodologies and reagents using both inactivated Marburg virus (iMV) and a recombinant form of its surface envelope glycoprotein (rGP). We identified mAbs which bind rGP in solution phase immunoprecipitation experiments. HP were prepared by chemically cross-linking an anti-CR1 mAb with several of these anti-Marburg virus mAbs and used to facilitate binding of iMV and rGP to monkey and human E. These HP mediate specific and quantitative binding (> or = 90%) of both antigens to monkey and human E. Binding was also demonstrable in an indirect RIA. E with bound Marburg virus were probed with 125I labeled mAbs to the Marburg surface glycoprotein and more than 100 mAbs are bound per E. It should be possible to adapt this general approach to other pathogens, and experiments underway should lead to an in vivo test of HP-mediated clearance of Marburg virus.

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.

Antigenicity and vaccine potential of Marburg virus glycoprotein expressed by baculovirus recombinants.

There is no effective vaccine for Marburg virus (MBGV) or any other filovirus, nor enough pertinent information to expedite rational vaccine development. To ascertain some of the minimal requirements for a MBGV vaccine, we determined whether whole inactivated MBGV, or a baculovirus-expressed virion subunit, could be used to immunize guinea pigs against a lethal infection. Baculovirus recombinants were made to express the MBGV glycoprotein (GP) either as a full-length, cell-associated molecule or a slightly truncated (5.4%) product secreted into medium; the latter, for its far greater ease in manipulation, was tested for its vaccine potential. Like MBGV GP, both the full-length and truncated GP expressed by baculovirus recombinants were abundantly glycosylated with both N- and O-linked glycans; differences in glycosylation were detectable, but these could not be shown to affect antigenicity with respect to available antibodies. The recombinant truncated glycoprotein elicited protection against lethal challenge with the MBGV isolate from which it was constructed and less effectively against an antigenically disparate MBGV isolate. Killed (irradiated) MBGV antigen was protective, in a reciprocal fashion, against both MBGV types. In a preliminary assessment of possible protective mechanisms, serum antibodies from immune animals were shown to be sufficient for protecting naive guinea pigs from lethal MBGV infections

Cross-neutralization of hantaviruses with immune sera from experimentally infected animals and from hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome patients.

Plaque-reduction neutralization tests were done with eight of nine known representative hantaviruses and immune sera from experimentally infected animals and from patients with hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). Results obtained with animal sera demonstrated each virus to be antigenically unique. Neutralization with the HPS patient sera was highest with Sin Nombre (SN) virus and to a lesser extent with Black Creek Canal (BCC) virus. Sera from Korean HFRS patients reacted best with Hantaan virus, but cross-reactivity with all other viruses except Thottapalayam (TPM) virus was also observed. Sera from Swedish HFRS patients reacted best with Puumala virus but cross-reacted with Prospect Hill, SN, and BCC viruses and to a lesser extent with all of the other viruses except TPM virus.

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.

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.

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.

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.

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.