Immune mechanisms against canine distemper. I. Identification of K cell against canine distemper virus infected target cells in vitro.

Canine peripheral blood lymphocytes, polymorphonuclear leucocytes (PMN) and monocytes (macrophages) were obtained by various cell separation techniques and were tested for their cytotoxic capacity against antibody-sensitized canine distemper virus (CDV) infected Vero cells by an in vitro chromium release assay. Canine lymphocytes were found to destroy CDV infected target cells effectively, while neither PMN nor monocytes (macrophages) could do so. The active lymphocyte was characterized by various rosetting techniques to be a non-T and a non-B lymphocyte. These cells bear no surface immunoglobulin (SIg-) but possessed both Fc receptors (Fc+) and complement receptors (EAC+) suggesting that these cells are neither classical T nor B cells. The possible roles of this K cell in the resistance against canine distemper are discussed.     

Recombinant bovine adenovirus type 3 expressing bovine viral diarrhea virus glycoprotein E2 induces an immune response in cotton rats

Recombinant bovine adenovirus is being developed as a live vector for animal vaccination and for human gene therapy. In this study, two replication-competent bovine adenovirus 3 (BAV-3) recombinants (BAV331 and BAV338) expressing bovine viral diarrhea virus (BVDV) glycoprotein E2 in the early region 3 (E3) of BAV-3 were constructed. Recombinant BAV331 contains chemically synthesized E2 gene (nucleotides modified to remove internal cryptic splice sites) under the control of BAV-3 E3/major late promoter (MLP), while recombinant BAV338 contains original E2 gene under the control of human cytomegalovirus immediate early promoter. Since E2, a class I membrane glycoprotein, does not contain its own signal peptide sequence at the 5' end, the bovine herpesvirus 1 (BHV-1) glycoprotein D signal sequence was fused in frame to the E2 open reading frame (ORF) for proper processing of the E2 glycoprotein in both the recombinant viruses. Recombinant E2 protein expressed by BAV331 and BAV338 recombinant viruses was recognized by E2-specific monoclonal antibodies as a 53-kDa protein, which also formed dimer with an apparent molecular weight of 94 kDa. Insertion of an E2-expression cassette in the E3 region did not effect the replication of recombinant BAV-3s. Intranasal immunization of cotton rats with these recombinant viruses generated E2-specific IgA and IgG responses at the mucosal surfaces and in the serum. In summary, these results show that the pestivirus glycoprotein can be expressed efficiently by BAV-3. In addition, mucosal immunization with replication-competent recombinant bovine adenovirus 3 can induce a specific immune response against the expressed antigen.     

In vitro generation of hydrogen peroxide and of superoxide anion by bovine polymorphonuclear neutrophilic granulocytes,blood monocytes,and alveolar macrophages

Studies were conducted to compare the capacity of bovine blood monocytes, polymorphonuclear granulocytes (PMNs), and alveolar macrophages (AMs) to generate hydrogen peroxide and Superoxide anion. Following stimulation with opsonized zymosan, bovine PMNs respond with an immediate and vigorous liberation of both oxygen species, generating 4.7 ± 0.3 nmol H₂O₂/10⁶ cell and 12.3 ± 1.8 nmol O₂ ^–/10⁶ cell during the initial 15 min. This is more than twice the amount generated by AMs (1.2 nmol H₂O₂/10⁶ cell; 2.5 nmol O₂/10⁶ cell) and blood monocytes (0.5 nmol H₂O₂/10⁶ cell; 2.1 nmolO₂ ^–/10⁶ cell) during the same period. However, AMs continue generating H₂O₂ and O₂ at a steady rate for a longer period and consequently produce amounts equal to those of PMNs when measured over a longer time span. Also, AMs can be stimulated with nonopsonized zymosan in contrast to PMNs. However, the AM population appears to comprise at least two subpopulations, which can be clearly distinguished by their capacity for generation of reactive oxygen species, and which correlate with their tendency for adherence to a plastic surface. In contrast to what has been found in other species, the bovine phagocytes were found to lack receptors for tuftsin and formylated oligopeptides, and thus remained unresponsive to these compounds. The in vitro activity of the three cell types was found to be very dependent on culture conditions, such as cell density and an adherent versus suspended state. In addition, a comparison with macrophages and PMNs elicited into the mammary gland suggest that in vivo factors can significantly influence the in vitro activities. The mammary gland cells have lower activity than blood and alveolar cells, even though they have been primed by chemotactic factors), and this is probably caused by milk components, i.e., the microenvironment. Our observations are discussed with respect to the results obtained from different laboratories, different species, and different cell types; emphasis is placed on the problem of drawing conclusions about in vivo functions of cells from parameters assayed in vitro.     

Effect of anti-herpesvirus drugs on human and bovine lymphoid function in vitro.

Lymphocyte proliferative responses to phytohemagglutinin, pokeweed mitogen, and herpesvirus (herpes simplex virus and infectious bovine rhinotracheitis virus) antigens were evaluated in the presence of various concentrations of the anti-herpesvirus drugs, methylmethoxydeoxyuridine (OCH3CH2UdR), cytosine arabinoside was inhibitory at 25 mug/ml and cytosine arabinoside was inhibitory OCH3CH2UdR per ml, lymphocyte proliferative responses were unaffected and were not abolished even by concentrations of 2,500 mug/ml. In contrast, adenine arabinocide was inhibitory at 25 mug/ml and cytosine was inhibitory at 5 mug/ml. Both direct and antibody-dependent lymphocyte cytotoxicity of herpesvirus-infected target cells were highly resistant to OCH3CH2UdR; concentrations of 1,000 mug/ml were only marginally inhibitory. The anti-herpesvirus activity of all three drugs is similar (0.5 to 8 mug/ml) (L.A. Babiuk, B. Meldrum, V.S. Gupta, and B. T. Rouse, submitted for publication). The drug OCH3CH2UdR should be given a careful, well-controlled evaluation of its effectiveness in the treatment of herpes simplex infections in animals, perhaps including humans, since our results using in vitro models show that cellular responses important for recovery from herpesvirus infections are unaffected by doses far in excess of those found be be antiviral.     

Bovine coronavirus nonstructural protein ns2 is a phosphoprotein.

To investigate the nature of the bovine coronavirus (BCV) ns2 protein, the gene encoding this protein was cloned and was expressed as a beta-galactosidase fusion protein. Antiserum raised against this protein reacted specifically with BCV-infected fixed cells in indirect immunofluorescence microscopy and precipitated an in vitro synthesized product approximately 32-kDa in molecular weight and an equivalent protein from BCV-infected cells. The synthesis of ns2 was found to be similar to the structural proteins of BCV and pulse-chase experiments indicated that ns2 protein was stable and that it accumulated in BCV-infected cells. Synthesis of ns2 in the presence of [32P] orthophosphate revealed that it is a phosphoprotein. Phosphoamino acid analysis confirmed the phosphorylated nature of ns2 and identified serine and threonine as its phosphorylated amino acid residues. This is the first demonstration of a phosphorylated nonstructural protein in coronavirus-infected cells.     

Maternal immunity provides protection against pertussis in newborn piglets

Pertussis continues to be a significant cause of morbidity and mortality in infants and young children worldwide. Methods to control the disease are based on vaccination with either whole-cell or acellular vaccines or treatment with antibiotics. However, despite worldwide vaccination infants are still at the highest risk for the disease. Here we used our newly developed newborn-piglet model to investigate whether transfer of maternal immunity can protect newborn piglets against infection with Bordetella pertussis. Pregnant sows were vaccinated with heat-inactivated B. pertussis or treated with saline (controls). Newborn piglets were allowed to suckle colostrum and milk for 4 to 5 days before they were challenged with 5 x 10(9) CFU of bacteria intrapulmonarily. Elevated levels of B. pertussis-specific secretory immunoglobulin A (S-IgA) and IgG antibodies were found in the colostrum and serum of vaccinated sows but not in those of control sows. Subsequently, significant levels of specific IgG and S-IgA were detected in the serum and bronchoalveolar lavage fluid of piglets born to vaccinated sows. Following infection with 5 x 10(9) CFU of B. pertussis, clinical symptoms, pathological alterations, and bacterial shedding were significantly reduced in piglets that had received passively transferred immunity. Thus, our results demonstrate that maternal immunization might represent an alternative approach to provide protection against pertussis in young infants.     

Design and delivery of a cryptic PrP epitope for induction of PrP-specific antibody responses

Transmissible spongiform encephalopathies (TSEs) depend on misfolding of a normal cellular protein (PrP^C) to an infectious conformation (PrP^Sc). Targeting PrP^Sc may represent an effective strategy for immunotherapy while avoiding consequences associated with immune responses to self-proteins. A weakly immunogenic epitope of PrP^C (YYR), which induces PrP^Sc-specific antibodies, is used as a starting point for vaccine development. Through optimization of epitope, as well as formulation/delivery, we enhance immunogenicity while retaining PrP^Sc specificity. In particular, QVYYRPVDQYSNQN, presented by a leukotoxin carrier protein, emerges as a strong vaccine candidate. A vaccine representing this construct induces consistent and sustained serum PrP^Sc-specific IgG antibody responses following two vaccinations. Antigen specific antibodies are also present within cerebral spinal fluid and mucosal secretions. These characteristics provide a foundation for development of a TSE vaccine.     

Strategies for induction of protective immunity to bovine herpesvirus-1 in newborn calves with maternal antibodies

The objective of this study was to evaluate Th1 promoting strategies for vaccination of neonates against bovine herpesvirus-1 (BHV-1). A plasmid encoding a secreted truncated version of glycoprotein D (tgD) and tgD protein formulated with CpG oligodeoxynucleotide (ODN) effectively primed the immune system of newborn lambs, whereas without CpG ODN the tgD protein was less effective. Furthermore, a heterologous DNA prime-protein/CpG boost induced stronger and more balanced immune responses than either the DNA vaccine or a protein/CpG prime-DNA boost. Three of these strategies were compared as an approach to induce protective immunity in newborn calves with BHV-1-specific maternal antibodies. Whereas the DNA vaccine induced minimal protection, the DNA prime-protein boost resulted in reduced temperature response, weight loss and virus shedding in comparison to the placebo group. Close to complete protection against BHV-1 challenge was elicited in the calves immunized with the protein/CpG formulation, as these animals lost very little weight, had only slightly elevated temperatures and shed almost no virus.