Crystal structure of the oligomerization domain of NSP4 from rotavirus reveals a core metal-binding site.

During the maturation of rotaviral particles, non-structural protein 4 (NSP4) plays a critical role in the translocation of the immature capsid into the lumen of the endoplasmic reticulum. Full-length NSP4 and a 22 amino acid peptide (NSP4(114-135)) derived from this protein have been shown to induce diarrhea in young mice in an age-dependent manner, and may therefore be the agent responsible for rotavirally-induced symptoms. We have determined the crystal structure of the oligomerization domain of NSP4 which spans residues 95 to 137 (NSP4(95-137)). NSP4(95-137) self-associates into a parallel, tetrameric coiled-coil, with the hydrophobic core interrupted by three polar layers occupying a and d-heptad positions. Side-chains from two consecutive polar layers, consisting of four Gln123 and two of the four Glu120 residues, coordinate a divalent cation. Two independent structures built from MAD-phased data indicated the presence of a strontium and calcium ion bound at this site, respectively. This metal-binding site appears to play an important role in stabilizing the homo-tetramer, which has implications for the engagement of NSP4 as an enterotoxin.

The authentic sequence of rotavirus SA11 nonstructural protein NSP4.

Recent studies demonstrate that the rotavirus nonstructural protein NSP4 functions as an enterotoxin and plays an important role in viral pathogenesis. Previous in vitro studies of NSP4 have used a cDNA clone of gene 10 derived from the prototypic rotavirus strain, SA11. We recently compared the sequence of the commonly used NSP4 cDNA with the sequence obtained from several SA11 isolates by direct sequencing of reverse transcription polymerase chain reaction products. One codon difference was identified between the cDNA clone and the SA11 virus isolates, and this resulted in a predicted amino acid substitution at position 47. The cDNA sequence specifies an asparagine at position 47, and the SA11 virus gene 10 encodes a hisitidine. To determine if this amino acid substitution altered the function of NSP4, we analyzed the ability of both NSP4-Asn47 and NSP4-His47 to regulate intracellular calcium levels and exhibit cell cytotoxicity. Our results indicate that the expression of NSP4-His47 from a recombinant baculovirus displays enhanced cytotoxicity and calcium flux.

Controlled release microparticles as a single dose diphtheria toxoid vaccine: immunogenicity in small animal models.

Diphtheria toxoid (DT) was encapsulated in microparticles prepared from polylactide-co-glycolide (PLG) polymers using a solvent evaporation technique. Combinations of small and large sized microparticles with controlled release characteristics were used to immunize Sprague Dawley rats and the antibody responses were monitored for one year. For comparison, control groups of rats were immunized at 0, 1 and 2 months with DT adsorbed to alum. The antibody responses generated by the microparticles were comparable to the alum immunized control groups from 32 weeks. Microparticles with a single entrapped antigen (DT) induced better antibody responses than microparticles with two antigens entrapped simultaneously (DT + TT). Microparticles prepared from a single polymer were less effective for long term antibody induction than a combination of microparticles prepared from three different polymers. A combination vaccine consisting of antigen absorbed to alum and also entrapped in microparticles gave the best response. In an inhibition assay designed to determine the relative binding of antisera to the antigen, the sera from the microparticle and the alum immunized animals showed comparable binding. An intradermal challenge study was performed in rabbits, which showed similar levels for the alum and the microparticle immunized animals at 4, 12 and 32 weeks after immunization.

Biodegradable microparticles with an entrapped branched octameric peptide as a controlled-release HIV-1 vaccine.

Polyactide-co-glycolide microparticles, with an entrapped branched octameric peptide from human immunodeficiency virus (HIV-1), were prepared by a solvent evaporation method. The microparticles were characterized for size distribution, antigen loading level, and integrity. Mice in one group were each immunized with a single dose of a controlled-release microparticle formulation containing 300 micrograms of peptide and the serum IgG responses to the antigen were compared with those of mice from a second group that were immunized at 0, 4, and 26 weeks with 100-microgram doses of the same peptide immunogen adsorbed to alum. The controlled-release microparticles induced an antibody response comparable to that from the alum-immunized group. The subcutaneous and the intramuscular routes of administration were compared in additional groups of mice for the microparticles, and both routes induced similar responses. A suspending vehicle for the microparticles was also evaluated and did not affect the immunogenicity of the controlled-release formulation containing both small and large microparticles, although the immunogenicity of smaller microparticles immunized alone was affected.

CTL induction using synthetic peptides delivered in emulsions--critical role of the formulation procedure.

Emulsions have been used with variable degrees of success to deliver antigen to stimulate immune responses. We have investigated three different ways of incorporating peptide antigen into soybean emulsions to induce CTL responses in mice. Two of these emulsions (oil-in-water, o/w, and water-in-oil-in-water, w/o/w) had peptide incorporated at the formulation stage, while the third had peptide added to a pre-formed o/w emulsion. High levels of CTL activity were induced when peptide was dispersed into the o/w or w/o/w emulsions, in contrast to addition of peptide to the pre-formed o/w emulsion, which did not stimulate a CTL response. Induction of CTL activity was independent of emulsion globule size but was correlated with a negative zeta potential and dispersion of peptide in the oil phase. The ability of peptide in soybean oil emulsion to induce CTL is critically dependent on dispersion of peptide at the time of emulsion formation.

Immunogenicity and protection in small-animal models with controlled-release tetanus toxoid microparticles as a single-dose vaccine.

Tetanus toxoid (TT) was encapsulated in microparticles prepared from polylactide-co-glycolide polymers by a solvent-evaporation technique. Combinations of small- and large-sized microparticles with controlled-release characteristics were used to immunize Sprague-Dawley rats, and the antibody responses were monitored for 1 year. For comparison, control groups of rats were immunized at 0, 1, and 2 months with TT adsorbed to alum. The antibody responses generated by the TT entrapped in microparticles were comparable to those generated by TT adsorbed to alum in control groups from 32 weeks onwards. Microparticles with a single entrapped antigen (TT) induced better antibody responses than microparticles with two antigens (TT and diphtheria toxoid) entrapped simultaneously. A combination vaccine consisting of TT adsorbed to alum and also entrapped in microparticles gave the best antibody responses. In an inhibition assay designed to determine the relative levels of binding of antisera to the antigens, the sera from the microparticle- and the alum-immunized animals showed comparable levels of binding. In addition, in a passive-challenge study with mice, TT adsorbed to alum and TT entrapped in microparticles provided equal levels of protection against a lethal challenge with tetanus toxin. An intradermal-challenge study was also performed with rabbits, which showed similar levels of protection in sera from alum- and microparticle-immunized animals at 4, 12, and 32 weeks after immunization.

Controlled release microparticles as a single dose hepatitis B vaccine: evaluation of immunogenicity in mice.

Hepatitis B surface antigen (HBsAg) was encapsulated in microparticles prepared from polylactide-co-glycolide (PLG) and polylactide (PLA) polymers using a solvent evaporation process. The immunoreactivity of the entrapped antigen was investigated by SDS-PAGE and Western blot. The microencapsulation process was modified to obtain both small (< 10 microns) and large microparticles (10-100 < microns). 80% of the antigen was encapsulated. Various combinations of small and large microparticles with controlled release characteristics were investigated in CD1 mice. Groups of animals were immunized with 30 micrograms equivalent of HBsAg in microparticles per animals. The control group received, three injections of 10 micrograms of HBsAg on alum at 0, 1 and 6 months. Results indicated that a single injection of HBsAg in microparticles could maintain the antibody response at a level comparable to the three-injection alum schedule for at least 1 year. An in vitro inhibition assay was developed to demonstrate that antigen-antibody reactivity were comparable for the microparticle immunized mice and the alum immunized mice. A competition assay with a monoclonal antibody specific for the neutralizing epitope of HBsAg demonstrated comparable binding for the sera from the microparticle and alum immunized mice.

Synthetic peptides entrapped in microparticles can elicit cytotoxic T cell activity.

Peptides from Plasmodium berghei circumsporozoite protein (CS) and influenza A virus nucleoprotein (NP) were entrapped in microparticles prepared from poly (lactide-co-glycolide) polymers, and the microparticles were administered parenterally to mice. After immunization with single or multiple doses, splenocytes were tested for a cytotoxic T cell (CTL) response and high levels of CTL activity were detected. The CTL induced were CD8+, MHC class I restricted, and could recognize virus infected cells. Peptide entrapped in microparticles of mean size < 500 nm were better inducers of CTL than larger microparticles (mean > 2 microns and above). Microparticles could also be used to deliver lipid modified peptides (lipopeptides) and elicited higher levels of cytolytic activity than either free peptide in microparticles or lipopeptide alone. Microparticles provide a novel way of inducing a CTL response using synthetic peptides.

Comparison of adjuvant formulations for cytotoxic T cell induction using synthetic peptides.

We have investigated the capacity of synthetic peptides delivered in different adjuvant formulations to induce cytotoxic T lymphocyte (CTL) responses to a class I H-2Kd-restricted Plasmodium berghei circumsporozoite epitope, CS 252-260. Using three immunogen formulations: soybean emulsion; Montanide ISA720; and lipopeptide (P3-CS), we first evaluated the effects of immunization routes on CTL induction. No CTL response was induced in mice immunized s.c. or i.p. with CS peptide formulated in soybean emulsion. In contrast, immunization with lipopeptide P3-CS either s.c. or i.p. effectively primed for CTL. Interestingly, CS peptide emulsified in Montanide ISA720 induced a CTL response only when delivered s.c. and not i.p., indicating the critical influence of immunization routes on CTL induction. We then compared the effectiveness of eight adjuvant formulations to induce CTL response following a single s.c. immunization. Notably, lipopeptide P3-CS and CS peptide admixed with P3 or POE lipid molecules stimulated a vigorous CTL response. However, only mice immunized with P3-CS and CS peptide admixed with P3 molecule generated long-lived CTL which persisted in vivo for 5 months. Thus, based on a simultaneous comparison of the different adjuvant formulations, we demonstrated that the conjugated and unconjugated P3 lipopeptides were the most effective immunogens for eliciting primary and memory CTL in mice.

A subunit gIV vaccine, produced by transfected mammalian cells in culture, induces mucosal immunity against bovine herpesvirus-1 in cattle.

A truncated version of bovine herpesvirus-1 (BHV-1) glycoprotein IV (tgIV) was produced in a novel, non-destructive expression system based upon regulation of gene expression by the bovine heat-shock protein 70A (hsp70) gene promoter in Madin Darby bovine kidney (MDBK) cells. In this system, up to 20 micrograms ml-1 of secreted tgIV, which is equivalent to the yield from 4 x 10(6) cells, was produced daily over a period of up to 18 days. Different doses of tgIV were injected intramuscularly into seronegative calves. Virus-neutralizing antibodies were induced by all doses of tgIV, both in the serum and in the nasal superficial mucosa. However, the low dose (2.3 micrograms) induced significantly (p < 0.05) lower antibody titres than the medium (7 micrograms) and high (21 micrograms) doses. The medium and high doses of tgIV conferred protection from BHV-1 infection, as demonstrated by a significant (p < 0.05) reduction in clinical signs of respiratory disease and virus shedding in the nasal secretions postchallenge. However, the 2.3 micrograms group, although partially protected, was not significantly (p > 0.05) different from the placebo group. This study demonstrated the potential of an intramuscularly administered tgIV subunit vaccine to induce mucosal immunity to BHV-1 using an economic protein production system and an acceptable vaccine formulation. In addition, a strong correlation was observed between neutralizing antibodies in the serum and nasal superficial mucosa, virus shedding and clinical disease. Thus, serum neutralizing antibody levels in tgIV-immunized animals may be a good prognosticator of protection from BHV-1 infection and disease.

Identification of a putative cellular receptor for bovine herpesvirus 1.

Recognition of a host cell receptor by a virus is the first and perhaps the most crucial step in initiating the disease process. This study was undertaken to identify the cellular receptor(s) for bovine herpesvirus 1 (BHV-1). Previously, we reported the development and characterization of bovine anti-idiotypic antibodies (anti-ids) that induce neutralizing antibodies to BHV-1. These anti-ids inhibit BHV-1 penetration of permissive cells. We have used these anti-ids, which mimic an epitope on the virus glycoprotein IV (gIV), and gradient-purified virus in immunoprecipitation (IP) as well as photoaffinity labelling (PAL) assays. In the IP assays, both bovine anti-ids and BHV-1 virions coupled to Sepharose precipitated a 60K protein from 125I-labelled BHV-1 permissive cell membrane extracts. Normal bovine IgG or an irrelevant virus, transmissible gastroenteritis virus (TGEV), used as negative controls failed to precipitate this protein. Similarly, in the PAL assays, the 60K cell surface protein was identified on cells permissive for BHV-1 infection, but not on non-permissive cells when 125I-labelled ligands, the anti-ids or BHV-1 were used as probes. The iodinated ligands failed to identify the 60K protein if they had been pretreated with the antibody 1. Pretreatment of the iodinated ligands with an isotype-matched control antibody had no effect on the identification of the 60K protein present on cells permissive for BHV-1 infection. The negative controls, i.e. normal bovine IgG and TGEV, failed to identify this 60K protein on permissive or non-permissive cells. These results suggest that the 60K protein is a cellular receptor recognized by BHV-1 during the infection process.

Bovine herpesvirus 1: immune responses in mice and cattle injected with plasmid DNA.

Mice and cattle injected with plasmids encoding bovine herpesvirus 1 (BHV-1) glycoproteins developed gene-specific antibody responses capable of neutralizing BHV-1. The ability of animals to respond serologically to DNA injections was in part dependent on the quantity of DNA injected and was also negatively affected by carrier DNA. Calves injected with a plasmid encoding BHV-1 gIV developed significant antibody titers to gIV and shed less virus than did the control calf after challenge. This report indicates the potential of DNA injection as a method of vaccination.

Expression of glycoprotein gIII-human decay-accelerating factor chimera on the bovine herpesvirus 1 virion via a glycosyl phosphatidylinositol-based membrane anchor.

Mutants of bovine herpesvirus 1 that express a truncated envelope glycoprotein gIII or a gIII-human decay-accelerating factor (hDAF) chimeric protein (gIII.hDAF) were employed to evaluate the function of the transmembrane and cytoplasmic domains of the gIII molecule. Truncated gIII (i.e., lacking the transmembrane and cytoplasmic region) was readily released from infected cells and was not detected on mature virus particles. In contrast, replacement of the transmembrane and cytoplasmic domains with the carboxyl-terminal portion of hDAF restored the expression of gIII on the membranes of infected cells as well as on virion surfaces. The presence of the gIII.hDAF chimera on virus particles was also associated with normal gIII function, i.e., the mediation of virus attachment and penetration. The gIII-hDAF chimera, which is present on both infected cell surfaces and virions, could be cleaved by a phosphatidylinositol-specific phospholipase C, indicating that it was anchored in the membrane via glycosyl phosphatidylinositol. Our results from this study suggest that the transmembrane and cytoplasmic regions of the gIII molecule serve as a general membrane anchor, but they do not contain structural signals required for the specific assembly of envelope proteins into mature virions.

Identification and deletion mutagenesis of the bovine herpesvirus 1 dUTPase gene and a gene homologous to herpes simplex virus UL49.5.

The gene encoding bovine herpesvirus 1 (BHV 1) deoxyuridine triphosphatase (dUTPase) was isolated by a PCR procedure using degenerate oligonucleotide primers whose sequences were based upon conserved motifs commonly present in dUTPase genes. This gene was found to reside between 0.059 and 0.066 map units in the BHV 1 Cooper strain. DNA sequence analysis of this region revealed an open reading frame of 975 base pairs capable of encoding 325 amino acids. The deduced amino acid sequence of the open reading frame exhibits significant homology with dUTPases of other herpesviruses (including human herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus), and it contains five conserved amino acid motifs characteristics of all dUTPases identified to date. A mutant virus carrying a partial deletion of the putative dUTPase gene was made and was found to lack virus-encoded dUTPase activity. This further confirmed that we have identified the BHV 1 dUTPase gene. In addition, a further analysis of the genomic fragment which contains the dUTPase coding sequence revealed an additional 288-base-pair open reading frame which appears to be colinear with the HSV 1 UL49.5 gene. The deduced amino acid sequence of this open reading frame is significantly homologous to the HSV 1 UL49.5 gene product, and as with UL49.5, it contains a potential signal sequence and transmembrane domain characteristic of membrane-associated proteins. These results suggest that this open reading frame represents the BHV 1 homolog of the HSV 1 UL49.5 gene. Since our dUTPase negative mutant was fully viable and since the mutant was constructed such that the UL49.5 gene was also deleted, both the dUTPase and the UL49.5 gene homolog are not required for virus growth in cell culture.

Mapping of heparin-binding structures on bovine herpesvirus 1 and pseudorabies virus gIII glycoproteins.

The gIII glycoproteins of both bovine herpesvirus 1 (BHV 1) and pseudorabies virus (PrV) mediate the initial and dominant interactions between virus and permissive host cells. By studying virus binding to wild-type and heparin-deficient CHO cells, we demonstrated that the cellular heparin-like moieties play an essential role in BHV 1 and PrV gIII-mediated virus attachment. Subsequent studies were carried out to map the gIII structures that are responsible for heparin binding. First, based on the observation that BHV 1 and PrV are differentially sensitive to heparin inhibition of gIII-mediated attachment to cells, we conducted a gIII domain shuffling experiment. This involved the construction of a set of recombinant BHV 1 expressing BHV 1 and PrV gIII chimeras and then using the sensitivity to heparin inhibition as a means of mapping the potential heparin-binding regions on the gIII molecules. Next, we synthesized panels of partially overlapping BHV 1 and PrV gIII peptides and examined their reactivity to heparin. The results from these experiments demonstrated five heparin-binding sites between amino acid 129 and 310 of BHV 1 gIII and four heparin-binding sites between amino acid 90 and 275 of PrV gIII.

Analysis of bovine herpesvirus 1 glycoprotein gIV truncations and deletions expressed by recombinant vaccinia viruses.

Glycoprotein gIV is an envelope component of bovine herpesvirus type 1 and appears to be involved in attachment, penetration, and cell fusion. Four antigenic domains which include both continuous and discontinuous epitopes have been previously defined by competition binding assays using gIV-specific monoclonal antibodies (MAbs). Here we describe the construction of C-terminal truncations and internal deletions in the gIV-encoding gene and analyses of the effects of these mutations on the synthesis, processing, transport, and antigenicity of glycoprotein gIV as expressed by recombinant vaccinia viruses. Wild-type gIV expressed by recombinant vaccinia virus STgIV was indistinguishable from authentic gIV produced in bovine herpesvirus 1-infected cells with respect to molecular weight, processing, transport, and antigenicity. Analysis of the mutant proteins showed that the binding sites for MAbs 9D6 and 3D9S, which recognize linear epitopes, lie between amino acids 164 and 216 and amino acids 320 and 355, respectively. Discontinuous epitopes recognized by MAbs 3E7, 4C1, 2C8, and 3C1 were located between amino acids 19 and 320, whereas amino acids 320 to 355 were critical for binding of MAb 136. All mutant proteins containing amino acids 245 to 320 were processed, possess endo-beta-N-acetylglucosaminidase H-resistant oligosaccharides, and were transported to the cell surface or secreted into the medium. In contrast, mutant proteins missing amino acids 245 to 320 were retained in the rough endoplasmic reticulum. These findings suggest that residues 245 to 320 are important for proper processing and transport of gIV to the cell surface.

Role of N-linked glycans in antigenicity, processing, and cell surface expression of bovine herpesvirus 1 glycoprotein gIV.

Glycoprotein gIV, a structural component of bovine herpesvirus type 1, stimulates high titers of virus-neutralizing antibody. The protein contains three potential sites for the addition of N-linked carbohydrates. Three mutants were constructed by oligonucleotide-directed mutagenesis, in each case changing one N-linked glycosylation site from Asn-X-Thr/Ser to Ser-X-Thr/Ser. A fourth mutant was altered at two sites. The altered forms of the gIV gene were cloned into a vaccinia virus transfer vector to generate recombinant vaccinia viruses expressing mutant proteins. Analysis of these mutants revealed that only two (residues 41 and 102) of the three (residues 41, 102, and 411) potential sites for the addition of N-linked glycans are actually utilized. Absence of glycans at residue 41 (gN1) showed no significant effect on the conformation of the protein or induction of a serum neutralizing antibody response. However, mutant proteins lacking glycans at residue 102 (gN2) or residues 41 and 102 (gN1N2) showed altered reactivity with conformation-dependent gIV-specific monoclonal antibodies. These mutants also induced significantly lower serum neutralizing antibody responses than wild-type gIV. Nonetheless, each of the mutant proteins were modified by the addition of O-glycans and transported to the cell surface. Our results demonstrate that absence of N-linked glycans at one (residue 102) or both (residues 41 and 102) utilized N-linked glycosylation sites alters the conformation but does not prevent processing and transport of gIV to the cell surface.

Heat-shock promoter-driven synthesis of secreted bovine herpesvirus glycoproteins in transfected cells.

The bovine hsp70A heat-shock gene promoter was isolated and used to direct the heat-regulated synthesis of bovine herpesvirus glycoproteins gIII and gIV in transfected cultured bovine cells. Sequences encoding the viral glycoproteins incorporated mutations that deleted the transmembrane anchors. Both proteins were efficiently secreted from transfected cells in a temperature-dependent manner and the gIV so produced was found to be antigenically similar to the authentic molecule. Stable cell lines with regulated expression of these proteins were obtained and repeated thermal cycling of the cultures enabled high-yield production of these subunit vaccine antigens. The continuous production demonstrated by this system is highly relevant to the efficient and economic manufacture of vaccines and other protein biopharmaceuticals.

Protection of cattle from BHV-1 infection by immunization with recombinant glycoprotein gIV.

High levels of recombinant bovine herpesvirus-1 (BHV-1) glycoprotein IV were produced in baculovirus, adenovirus, vaccinia virus and Escherichia coli expression systems. The different recombinant forms as well as authentic gIV were injected intramuscularly into seronegative calves. With the exception of E. coli-produced gIV, all forms of gIV induced high levels of neutralizing antibodies both in the serum and in the nasal superficial mucosa. Animals immunized with gIV produced in insect or mammalian cells were completely protected from infection with BHV-1, as demonstrated by the absence of temperature responses, clinical signs or detectable virus in the nasal secretions after challenge exposure. The E. coli-derived gIV induced partial protection from clinical disease, even though it was not glycosylated and did not induce appreciable levels of neutralizing antibodies. This study demonstrated that all forms of glycosylated gIV, whether authentic or recombinant, confer protection from BHV-1 infection and thus may be useful as an effective subunit vaccine.

The effect of subunit or modified live bovine herpesvirus-1 vaccines on the efficacy of a recombinant Pasteurella haemolytica vaccine for the prevention of respiratory disease in feedlot calves.

The efficacy of a Pasteurella haemolytica vaccine (PhV) administered once to calves within 24 hours of arrival at a feedlot was tested for the ability to prevent morbidity and mortality from all bovine respiratory disease (BRD) and specifically from fibrinous pneumonia mortality. The PhV consisted of two immunizing ingredients: outer membrane proteins extracted from P. haemolytica, plus genetically attenuated leukotoxin produced by recombinant DNA technology. This double blind study was conducted at a large Saskatchewan feedlot using 2,324 high-risk calves purchased at auction markets and kept under typical commercial feedlot conditions. The trial design included four vaccine test groups: 1) PhV and a bovine herpesvirus type-1 (BHV-1) subunit vaccine comprised only of the virus glycoprotein IV (gIV); 2) PhV and a commercial modified live vaccine (MLV) containing BHV-1 and parainfluenza-3 viruses; 3) gIV alone; and 4) MLV alone. Calves were assigned to vaccine groups in a random systematic manner, individually identified, and monitored for 90 days after vaccination. The vaccines were given once, on arrival, to reflect common feedlot practice, although vaccination prior to expected risk would be more appropriate.The PhV in combination with gIV reduced BRD morbidity by 20% (p < 0.05) compared to gIV alone and 24% (p < 0.05) compared to MLV alone, and reduced BRD mortality by 88% (p < 0.05) and fibrinous pneumonia mortality by 100% (p < 0.05) when compared to either gIV or MLV alone. Vaccination with PhV in combination with MLV significantly reduced the efficacy of the PhV in preventing BRD morbidity, BRD mortality, and fibrinous pneumonia mortality and also reduced the antibody response to P. haemolytica leukotoxin. These results suggest that the MLV interfered with the protective capacity of the PhV.