Robert E. Shope

Antigenic Relationships between Flaviviruses as Determined by Cross-neutralization Tests with Polyclonal Antisera

The recently established virus family Flaviviridae contains at least 68 recognized members. Sixty-six of these viruses were tested by cross-neutralization in cell cultures. Flaviviruses were separated into eight complexes [tick-borne encephalitis (12 viruses), Rio Bravo (six), Japanese encephalitis (10), Tyuleniy (three), Ntaya (five), Uganda S (four), dengue (four) and Modoc (five)] containing 49 viruses; 17 other viruses were not sufficiently related to warrant inclusion in any of these complexes.

Japanese encephalitis virus-vaccinia recombinants produce particulate forms of the structural membrane proteins and induce high levels of protection against lethal JEV infection

Four recombinant vaccinia viruses were engineered for expression of different portions of the Japanese encephalitis virus (JEV) open reading frame. All four recombinant vaccinias contained the NS1 and NS2A genes, and each of these viruses specified the synthesis, glycosylation, and secretion of the nonstructural glycoprotein (NS1). All four recombinants also contained the E gene, and each virus correctly directed the synthesis and glycosylation of the envelope glycoprotein (E). Interestingly, two of these viruses (vP555 and vP650), which expressed the prM gene in addition to E and NS1, produced an extracellular hemagglutinin containing M and E that migrated in sucrose gradients similarly to the slowly-sedimenting hemagglutinin found in the culture fluid of JEV-infected cells. Immunization of 3-week-old mice with the recombinant viruses vP555 and vP658 resulted in immune responses to NS1, whereas only the virus that directed the synthesis of extracellular forms of E (vP555) induced an immune response to E. Both viruses provided protection against lethal challenge with JEV. Animals given two inoculations with vP555 were fully protected from greater than 10,000 LD50 of JEV. This high level of protection was correlated with the production of high titers of neutralizing and hemagglutination-inhibiting antibodies.

Mice immunized with a subviral particle containing the Japanese encephalitis virus prM/M and E proteins are protected from lethal JEV infection.

Extracellular subviral particles produced by HeLa cells infected with a recombinant vaccinia virus encoding the prM and E genes of Japanese encephalitis virus (JEV) were purified and characterized. These particles contained the JEV prM/M and E proteins embedded in a lipid bilayer, and RNA was not detected in particles using the polymerase chain reaction and primers recognizing a part of the JEV E gene. The particles were uniformly spherical with a 20-nm diameter and had 5-nm projections on their surface. Mice that received a single inoculation of the purified extracellular particles emulsified with Freunds complete adjuvant were fully protected against 4.9 x 10(5) LD50 of JEV. Comparison of the neutralizing and hemagglutination-inhibiting antibody titers and radioimmunoprecipitation data showed that immunization with the particles induced an immune response similar to that following inoculation with the recombinant vaccinia virus.

Biophysical Characterization and Vector-Specific Antagonist Activity of Domain III of the Tick-Borne Flavivirus Envelope Protein

ABSTRACT The molecular determinants responsible for flavivirus host cell binding and tissue tropism are largely unknown, although domain III of the envelope protein has been implicated in these functions. We examined the solution properties and antagonist activity of Langat virus domain III. Our results suggest that domain III adopts a stably folded structure that can mediate binding of tick-borne flaviviruses but not mosquito-borne flaviviruses to their target cells. Three clusters of phylogenetically conserved residues are identified that may be responsible for the vector-specific antagonist activity of domain III.

Recombinant vaccinia virus producing the prM and E proteins of yellow fever virus protects mice from lethal yellow fever encephalitis

Four recombinant vaccinia viruses were constructed for expression of different portions of the 17D yellow fever virus (YFV-17D) open reading frame. A recombinant, vP869, expressing prM and E induced high titers of neutralizing and hemagglutination inhibiting antibodies in mice and was protective against intracranial challenge with the French neurotropic strain of YFV. Levels of protection were equivalent to those achieved by immunization with the YFV-17D vaccine virus. Recombinant vaccinia viruses expressing E and NS1, C prM, E, NS1, or only NS1 failed to protect mice against challenge with YFV despite eliciting antibodies to NS1. The vP869-infected HeLa cells produced a particulate extracellular hemagglutinin (HA) similar to that produced by YFV-infected cells, supporting previous studies with Japanese encephalitis virus (Mason et al., 1991), suggesting that the ability of recombinant vaccinia virus to produce extracellular HA particles is important for effective flavivirus immunity.

Comparison of protective immunity elicited by recombinant vaccinia viruses that synthesize E or NS1 of Japanese encephalitis virus

Immunization with recombinant vaccinia viruses that specified the synthesis of Japanese encephalitis virus (JEV) glycoproteins protected mice from a lethal intraperitoneal challenge with JEV. Recombinants which coexpressed the genes for the structural glycoproteins, prM and E, elicited high levels of neutralizing (NEUT) and hemagglutination inhibiting (HAI) antibodies in mice and protected mice from a lethal challenge by JEV. Recombinants expressing only the gene for the nonstructural glycoprotein, NS1, induced antibodies to NS1 but provided low levels of protection from a similar challenge dose of JEV. Antibodies to the NS3 protein in postchallenge sera, representing the degree of infection with challenge virus, were inversely correlated to NEUT and HAI titers and levels of protection. These results indicate that although vaccinia recombinants expressing NS1 can provide some protection from lethal JEV infection, recombinants expressing prM and E elicited higher levels of protective immunity.

A highly attenuated host range-restricted vaccinia virus strain, NYVAC, encoding the prM, E, and NS1 genes of Japanese encephalitis virus prevents JEV viremia in swine

A highly attenuated strain of vaccinia virus (NYVAC) was engineered to express the Japanese encephalitis virus (JEV) prM, E, and NS1 genes or the prM and E genes. The recombinant viruses were tested as vaccine candidates in pigs, a natural host of JEV. JEV-neutralizing and hemagglutination-inhibiting antibodies appeared in swine sera 7 days after immunization with 10(8) PFU of the recombinant viruses and increased after a second dose at 28 days. The JEV levels detected in the serum after JEV challenge (d56) of the swine with 2 x 10(5) PFU of JEV were significantly reduced in animals inoculated with the recombinant viruses. These results demonstrate the ability of these NYVAC-vectored recombinants to protect pigs from JEV viremia.

Physicochemical and morphological relationships of some arthropod-borne viruses to bluetongue virus--a new taxonomic group. Electron microscopic studies.

Summary The morphology and mode of maturation of a number of relatively solvent resistant arboviruses were examined by thin-section and negative-stain electron microscopy of infected mouse brain and cell culture specimens. These viruses, which have physicochemical properties distinct from other arboviruses, included Colorado tick fever, Tribec, Wad Medani, Chenuda, Irituia, Palyam, Lebombo, epizootic haemorrhagic disease of deer and bluetongue. They were 65 to 80 nm. in diameter and matured in the cytoplasm as unenveloped particles with an electron-dense core. Virus development occurred in association with a cytoplasmic granular matrix and was accompanied by formation of regularly substructured filaments and tubules. Surface architecture was compatible with icosahedral symmetry with T = 3 (32 capsomeres). The combination of taxonomic parameters, morphologic and morphogenetic as well as physicochemical, was distinct from that of any presently recognized virus group. The independent classification of these viruses with bluetongue as the type virus is thus proposed.

Recombinant vaccinia viruses co-expressing dengue-1 glycoproteins prM and E induce neutralizing antibodies in mice

Four recombinant vaccinia viruses expressing different portions of the dengue type 1 virus (DEN-1) genome (C-prM-E-NS1-NS2A-NS2B; prM-E; prM-E-NS1-NS2A-NS2B; or NS1-NS2A) were constructed in order to establish the most immunogenic configuration of DEN-1 proteins. Both recombinants producing prM and E in the absence of C induced the synthesis of extracellular forms of E in vitro. Mice inoculated with these two recombinants produced DEN-1 neutralizing (NEUT) and haemagglutination inhibiting (HAI) antibodies. The other two recombinant vaccinia viruses, which did not induce the production of extracellular forms of E, did not induce E-specific immune responses. These results support our previous studies on the design of flavivirus-vaccinia vaccine candidates by showing the importance of co-expressing prM and E in order to induce the synthesis of extracellular E and to elicit NEUT and HAI antibodies.

Safety and immunogenicity of NYVAC-JEV and ALVAC-JEV attenuated recombinant Japanese encephalitis virus — poxvirus vaccines in vaccinia-nonimmune and vaccinia-immune humans

A controlled, randomized, double-blind clinical trial evaluated whether two attenuated recombinant poxviruses with identical Japanese encephalitis virus (JEV) gene insertions, NYVAC-JEV and ALVAC-JEV, were safe and immunogenic in volunteers. Groups of 10 volunteers distinguished by vaccinia immune status received two doses of each vaccine. The vaccines appeared to be equally safe and well tolerated in volunteers, but more reactogenic than licensed formalin-inactivated JE and placebo vaccines given as controls. NYVAC-JEV and ALVAC-JEV vaccine recipients had frequent occurrence of local warmth, erythema, tenderness, and/or arm pain after vaccination. There was no apparent effect of vaccinia immune status on frequency or magnitude of local and systemic reactions. NYVAC-JEV elicited antibody responses to JEV antigens in recipients but ALVAC-JEV vaccine poorly induced antibody responses. However, NYVAC-JEV vaccine induced neutralizing antibody responses only in vaccinia-nonimmune recipients while vaccinia-immune volunteers failed to develop protective antibodies (5/5 vs. 0/5 seroconversion, p<0.01). These data suggest that preexisting immunity to poxvirus vector may suppress antibody responses to recombinant gene products.