Mucosal immune responses following oral immunization with rotavirus antigens encapsulated in alginate microspheres.

Availability of effective oral vaccine delivery vehicles should contribute to the success of oral immunization in domestic animals. To achieve this goal, we evaluated alginate microspheres for their capacity to induce mucosal immune responses following oral and enteric immunizations. Mice were immunized with either live porcine rotavirus (PRV) or its recombinant VP6 protein, encapsulated in alginate microspheres or unencapsulated. VP6-specific IgG (but no IgA) antibodies were detected in the sera of mice after a single intraperitoneal (i.p.) immunization with either VP6 in Incomplete Freund's adjuvant (VP6-IFA), VP6 in alginate microspheres (VP6-MS) or with live PRV in incomplete Freund's adjuvant (PRV-IFA). In contrast, VP6-specific IgA (but no IgG) was detected in culture supernatants of mesenteric lymph nodes from mice immunized i.p. with either VP6-IFA or with PRV-IFA. Oral immunization with VP6-MS induced the highest level of VP6-specific fecal IgA antibody, similar to responses induced by oral immunization with live PRV. Furthermore, the VP6-specific fecal IgA could be boosted by a secondary i.p. immunization with VP6. Further experiments were performed in a sheep intestinal 'loop' model to evaluate uptake of microspheres by Peyer's patches. Microspheres containing colloidal carbon were specifically bound and transported by follicle-associated epithelium of Peyer's patches. Additionally, mucosal immune responses were detected following enteric immunization with porcine serum albumin (PSA) encapsulated in alginate microspheres. Our results confirm that alginate microspheres are an effective oral delivery vehicle for protein antigens and intestinal IgA antibody responses are induced by antigens encapsulated in alginate microspheres without any additional mucosal adjuvant. These investigations confirm that alginate microspheres have the potential as an effective delivery vehicle for oral immunization of ruminants.

Dermal and transdermal delivery of protein pharmaceuticals: lipid-based delivery systems for interferon alpha.

The dermal and transdermal delivery of protein pharmaceuticals faces enormous challenges, and at the same time has very significant potential for the non-invasive treatment of both localized and systemic diseases. In this article we review the various approaches used to enhance and control the delivery of protein therapeutic agents through the dermal barrier. We show results of the delivery of interferon (IFN) alpha, an antiviral agent used in the treatment of condylomata acuminata (genital warts), using lipid-based delivery systems (LBDS). In the general category of LBDS, we investigated the use of liposomes and fatty acylation as ways to increase IFNalpha delivery into human skin.

Palmitoyl derivatives of interferon alpha: potential for cutaneous delivery.

Palmitoyl derivatives of interferon alpha2b (p-IFNalpha) were prepared by covalent attachment of the fatty acid to lysine residues in the protein through a reaction with N-hydroxysuccinimide palmitate ester. The p-IFNalpha was characterized by capillary electrophoresis (CE), mass spectrometry (MS), SDS-PAGE, and antiviral assay. Flow-through diffusion cells and human breast skins were used to measure cutaneous and percutaneous absorption. Formation of p-IFNalpha derivatives was demonstrated by CE to be dependent on reaction time and reagent: protein ratio. Electrospray MS of the crude p-IFNalpha mixture indicated three populations of IFNalpha derivatives with 10, 11, and 12 palmitoyl substitutions. The addition of palmitoyl residues to IFNalpha under the conditions described reduced the antiviral specific activity by 50%. However, the cutaneous absorption of p-IFNalpha was about 5-6 times greater than the parent protein. The amount of p-IFNalpha and IFN alpha in whole skin after 24 h of treatment was 2.106 +/- 1.216 microg/cm2 and 0.407 +/- 0.108 microg/cm2, respectively. Approximately two times higher flux was detected for p-IFNalpha compared to the nonfatty acylated IFNalpha. The total amount of drug diffused in 24 h was also approximately two times higher for the p-IFNalpha. The results indicate a potential for using fatty acylated derivatives of IFN alpha for dermal and transdermal delivery.

Inhibition of rotavirus infection in vitro and in vivo by a synthetic peptide from VP4.

A synthetic peptide corresponding to bovine rotavirus C486 (BRV) VP4 amino acid sequence 232-255 (VP4-peptide) was studied with the objective of defining the origin of the protective immune response reported previously by Ijaz et al. (J. Virol. 1991, 65, 3106-3113). Pretreatment of MA-104 cells with the VP4-peptide before infection with rotavirus prevented both the attachment of 35S-labelled virus and plaque formation in vitro. In vivo studies using a murine rotavirus model demonstrated that intragastric administration of VP4-peptide protected subjects from challenge with virulent rotavirus. These results clearly indicate the importance of this epitope in virus-cell interactions and their potential as a rotavirus vaccine candidate.

Characterization of the interaction between VP8 of bovine rotavirus C486 and cellular components on MA-104 cells and erythrocytes.

Rotavirus VP8*, the N-terminal trypsin cleavage product of VP4, has been shown to bind to MA-104 cells and human O type erythrocytes. To examine whether bacterially expressed VP8* binds to cellular components of MA-104 cells, the VP8* (aa 1-247) was expressed in E. coli and radiolabelled with 35S-methionine. The radiolabelled rVP8* was immunoprecipitated with antiserum to bovine rotavirus C486 (BRV). The rVP8* was found to bind to MA-104 cells and its binding was competed by BRV. To study the interaction between VP8* and receptors of erythrocytes, hemagglutination (HA) and hemagglutination inhibition (HI) assays were carried out using solubilized rVP8*. rVP8* showed HA which could be inhibited by antiserum to BRV. This interaction was also inhibited by gangliosides, demonstrating a sialic acid dependent interaction. To study the contribution of the C-terminal region of VP8* to HA, a number of approaches were used. First, a peptide spanning aa 230-247 was synthesized and antisera was raised against the peptide to see whether it could inhibit HA of rVP8*. Second, a truncated form of VP8* (tVP8*: aa 1-229) was expressed to examine its hemagglutinating activity. Third, the dimerization of rVP8* and tVP8* was compared by Western-blotting following electrophoresis using native SDS-PAGE. The results indicated that antibody to aa 230-247 inhibits hemagglutination by preventing dimerization of VP8* which in turn allows the molecule to cause HA. To characterize the interaction between the HA domain and sialic acid receptors, erythrocytes were treated with sialidases of different specificities. Arthrobacter ureafaciens, Clostridium perfringens and alpha 2-8 linkage-specific neuraminidase destroyed the ability of sialic acid of erythrocytes to interact with rVP8*, indicating that bovine rotavirus C486 binding requires an alpha 2-8 linkage but acetylation of the sialic acid is not necessary.

Serum haptoglobin as an indicator of the acute phase response in bovine respiratory disease.

The early stages of the host response to infectious agents include a number of physiologic changes, collectively known as the acute phase response. The acute phase response is comprised of reactions localized at the site of infection, as well as the initiation of systemic responses, which include a rapid increase in the serum concentration of some proteins, known as acute phase proteins (APP). Using polyacrylamide gel electrophoresis, we detected two APP of approximately 22 and 37 kDa molecular weight in sera obtained from cattle with bovine respiratory disease (BRD). Based on their presence in the sera of sick, but not normal animals, the molecular weights, N-terminal amino acid sequence analysis, and the ability to bind hemoglobin, we identified these proteins as the alpha and beta subunits of haptoglobin. The haptoglobin molecule and the alpha subunit were isolated from serum, purified, and used to produce monoclonal and polyclonal antibodies. With these reagents, an enzyme linked immunosorbent assay was developed to measure the concentration of haptoglobin in bovine serum. Using an experimental model of BRD induced by a sequential challenge of calves with bovine herpesvirus type-1 and Pasteurella haemolytica, we observed a temporal relationship between the increase in haptoglobin concentration in serum and the onset of bacterial infection. The haptoglobin concentration ranged from undetectable in the serum of most calves prior to challenge, to greater than 1 mg ml(-1) in over one-third of the calves at the height of disease. Furthermore, the concentration of haptoglobin was associated significantly with other measures of the severity of disease. Together, these results indicate that quantification of acute phase proteins in animals with BRD could be a valuable diagnostic and prognostic aid.

Identification and characterization of a bovine herpesvirus-1 (BHV-1) glycoprotein gL which is required for proper antigenicity, processing, and transport of BHV-1 glycoprotein gH.

DNA sequence analysis of the bovine herpesvirus-1 (BHV-1) genome revealed the presence of an open reading frame named UL1 which exhibited limited homology to glycoprotein gL of herpes simplex virus-1 (S. K. Khattar, S. van Drunen Littel-van den Hurk, L. A. Babiuk, and S. K. Tikoo, Virology 213, 28-37). To identify the BHV-1 UL1 protein, rabbit antisera were prepared against two synthetic peptides that were predicted by computer analysis to encompass antigenic epitopes. Sera against both peptides immunoprecipitated a 16- to 17-kDa protein from in vitro translated in vitro transcribed mRNA, BHV-1-infected MDBK cells, and purified virions. Enzymatic deglycosylation and lectin binding assays confirmed that the BHV-1 UL1 protein contains only O-linked oligosaccharides and was named glycoprotein gL. Sera against UL22 protein immunoprecipitated a protein of 108 kDa from BHV-1-infected MDBK cells and purified virions, which was modified only by N-linked oligosaccharides and was named glycoprotein gH. Glycoprotein gL expressed by recombinant vaccinia virus was properly processed and secreted into the medium. In contrast glycoprotein gH expressed by recombinant vaccinia virus was found to be retained in the rough endoplasmic reticulum. However, gH coexpressed with gL by recombinant vaccinia viruses was properly processed and transported to the cell surface, suggesting that complex formation between gH and gL is necessary for the proper processing and transport of gH but not gL. In addition gH--gL complex formation is also required for induction of neutralizing antibody response and anchoring of gL to the plasma membrane.

Glycoprotein Bb, the N-terminal subunit of bovine herpesvirus 1 gB, can bind to heparan sulfate on the surfaces of Madin-Darby bovine kidney cells.

The present study confirms our previous findings made by using heparin affinity chromatography that bovine herpesvirus 1 gB can bind to heparin-like structures. In order to locate the functional domain for heparin binding, we expressed the extracellular portion of gB (gBt) and the large subunit of gB (gBb) in Madin Darby bovine kidney (MDBK) cells under the control of the bovine heat shock protein 70A gene promoter. The recombinant gBt and gBb were both efficiently secreted from the transfected cells. They were shown to have structural and antigenic properties similar to those of authentic gB. Like authentic gB, both gBt and gBb were able to bind heparin-Sepharose as well as heparan sulfates on MDBK cells. Thus, we suggest that at least one heparin-binding domain is localized in gBb, the N-terminal portion of gB, which agrees with the presence of clusters of prolines and basic residues, thought to be essential for heparin binding.

Characterization of bovine herpesvirus 1 UL49 homolog gene and product: bovine herpesvirus 1 UL49 homolog is dispensable for virus growth.

The sequence of the bovine herpesvirus 1 (BHV-1) gene that is homologous to the herpes simplex virus UL49 gene was determined. The BHV-1 UL49 homolog open reading frame consists of 774 bp and is capable of encoding 258 amino acids. Northern (RNA) blot analysis showed that the BHV-1 UL49 homolog is transcribed into a 1.1-kb RNA which is coterminal with the transcripts of an upstream UL49.5 homolog gene. Rabbit antisera produced against synthetic peptides of the predicted UL49 homolog gene product recognized a polypeptide of 33 to 35 kDa in both virus-infected cells and isolated virions. Further analysis by unionic-detergent partition of isolated virions suggested that the UL49 homolog gene product is a virion tegument protein. Indirect immunofluorescence assay revealed that the UL49 homolog gene product was predominantly localized in the nuclei of BHV-1-infected cells. A mutant virus with the UL49 homolog gene deleted was produced, and it was able to replicate in noncomplementing cells. Nevertheless, the yield of mutant virus was significantly reduced. The results from this study suggest that the BHV-1 UL49 homolog gene encodes a nuclear protein which constitutes a tegument component in mature virions and that it is dispensable for virus growth in cell culture.

Production of hybrid bispecific antibody recognizing human colorectal carcinoma and CD3 antigen.

The present study reports on the use of gene transfer by vector DNA in the generation of hybrid hybridoma, the quadroma secreting the hybrid bispecific antibody. A quadroma B72.3neo/OKT3gpt was simply derived from the fusion of two hybridoma cell lines, B72.3 and OKT3, tagged with vector DNA mpSV2neo and mpSV2gpt, respectively, and selected in the media containing both G418 and mycophenolic acid. The hybrid bispecific antibody B72.3/OKT3 was purified from the quadroma ascites by the use of hydroxylapatite column on high-pressure liquid chromatography. This bispecific antibody contained one binding site for the TAG72 antigen on OVCAR3 tumor cells and the other binding site for the CD3 molecule on human T cells. It was able to target human T lymphocytes to significantly lyse the human ovarian cancer cells and may therefore be useful in immunotherapy of cancer.

Heterotypic passive protection induced by synthetic peptides corresponding to VP7 and VP4 of bovine rotavirus.

We have evaluated the potential of two peptides derived from highly conserved regions of rotavirus outer capsid proteins (VP7 and VP4) to act as a rotavirus vaccine. The capacity of peptides coupled to rotavirus VP6 spherical particles to provide passive protection in a murine model was compared with the protection induced by peptide-keyhole limpet hemocyanin (KLH) conjugates. Female mice were immunized a total of three times before and during pregnancy. Suckling mouse pups were challenged at 7 days of age with either homologous or heterologous rotavirus serotypes. The efficacy of vaccination was determined by analyzing the clinical symptoms and measuring xylose adsorption in the intestine. In this model the VP4 peptide-VP6 conjugate provided protection equal to that obtained using bovine rotavirus (BRV) as the immunogen. The VP7 peptide-VP6 conjugate provided slightly less protection than the VP4 peptide-VP6 conjugate. A mixture of the VP4 peptide-VP6 and VP7 peptide-VP6 conjugates provided better heterologous protection than immunization with BRV. In contrast, KLH-conjugated peptides provided only partial protection. The significance of a synthetic-peptide-based rotavirus vaccine in the prevention of rotavirus infections is discussed.

Mapping of 10 epitopes on bovine herpesvirus type 1 glycoproteins gI and gIII.

In order to map some of the immunologically important sites on bovine herpesvirus type 1 (BHV-1), deleted, truncated, and hybrid forms of glycoproteins gI and gIII were expressed in transfected murine LMTK- cells. The cells were tested for reactivity with a panel of 16 gI- or gIII-specific monoclonal antibodies (MAbs) possessing conformation-independent antigen binding properties. This panel represented five epitopes on gI and five epitopes on gIII. For gI, two epitopes were mapped between residues 68 and 119, one epitope was mapped between residues 370 and 440, one epitope was mapped to the vicinity of residue 487, and one epitope was mapped between residues 744 and 763. For gIII, three epitopes were mapped between residues 22 and 150, one epitope was mapped between residues 140 and 240, and one epitope was mapped between residues 230 and 287. The location of the gI epitope in the vicinity of residue 487, which was recognized by a virus-neutralizing MAb, was verified by synthetic peptide binding studies. The epitope locations were consistent with proposed models for the structure of gI and gIII, and comparable to some of the epitope locations reported for the homologous glycoproteins of herpes simplex virus type 1. The implications of these results for development of a subunit vaccine against BHV-1 are discussed.

Synthetic beta-turn peptides as substrates for a tyrosine protein kinase.

An attempt has been made at defining the secondary structural requirement for phosphorylation of substrates of a protein tyrosine kinase from the leukemia virus-transformed LSTRA cell line. An examination of the sites of phosphorylation of substrates of protein tyrosine kinases indicated a relatively high probability of the beta-turn as the secondary structural feature at these sites. We have, therefore, synthesized three tyrosine peptides: Ala-Pro-Tyr-Gly-NHCH3, Leu-Pro-Tyr-Ala-NHCH3, and Pro-Gly-Ala-Tyr-NH2, of which the first two peptides, but not the third, would be expected to contain the tyrosine residue in a beta-turn. Circular dichroism and infrared spectral data on the peptides confirmed this expectation. Phosphorylation data on the peptides by the tyrosine kinase showed that the two beta-turn peptides were phosphorylated with Vmax and Km values comparable to those of the 13-residue-long arginine-containing synthetic peptide substrate having a sequence homologous to the autophosphorylation site of the LSTRA kinase. The peptides used here contain the shortest sequence length among the reported synthetic peptide substrates for protein tyrosine kinases. Their preference for the beta-turn indicated that this conformation may serve as the recognition site for tyrosine phosphorylation.

Acyclic model peptides with ionophoretic activity. Pr3+ transport by N-tBoc-Pro-Xxx-Ala-NHCH3.

Two linear synthetic peptides, N-tBoc-Pro-Gly-Ala-NHCH3 and N-tBoc-Pro-D-Ala-Ala-NHCH3, have previously been shown by us to complex with Ca2+ and form 2:1 (peptide:calcium) complexes. Here we report their binding to Pr3+ and demonstrate, by 1H NMR, the peptide-mediated transport of Pr3+ across dimyristoylphosphatidylcholine unilamellar vesicles via a 2:1 ion-sandwich complex.