Efficacy of an inactivated feline leukemia virus vaccine.

An inactivated whole-virus FeLV vaccine, developed from a molecularly cloned FeLV isolate (FeLV-61E-A), was assessed for its ability to protect cats against homologous and heterologous virulent viral challenge. The fractions of cats that resisted the induction of persistent viremia after FeLV challenge were as follows: FeLV-61E-A vaccine, 95%; adjuvant controls, 26%; and established commercial control FeLV vaccine, 35%. The prechallenge mean neutralizing antibody titers for each group were as follows: FeLV-61E-A vaccine, 1:43; adjuvant controls, < 1:8; and commercial control FeLV vaccine, 1:12. The prototype FeLV-61E-A vaccine was developed commercially for immunization of pet cats by substitution of a proprietary adjuvant and development of stable, high antigen production cell lines. This vaccine (Fel-O-Vax) has been studied extensively, alone and in multivalent combination with other feline virus vaccines, in seven efficacy trials involving a total of 150 immunized cats. These studies yielded an FeLV-resistant fraction of 87% in vaccinated cats as compared with 8% in adjuvant controls. The duration of immunity induced by an FeLV-61E-A commercial vaccine (Fel-O-Vax-LvK IV) was also assessed. One year after vaccination, 100% of challenged vaccinated cats and none of challenged controls resisted induction of persistent viremia. The results of these studies demonstrate that an inactivated FeLV vaccine prepared from a molecularly cloned subgroup A FeLV produces a high level of protective immunity against heterologous and homologous FeLV infection. This vaccine-induced immunity is durable for at least 1 year without intervening booster immunization or exposure to virus.

Development and testing of an inactivated feline leukemia virus vaccine.

We assessed an inactivated whole virus feline leukemia virus (FeLV) vaccine developed from a molecularly cloned feline leukemia virus isolate (FeLV-61E-A) for its ability to protect cats against homologous and heterologous virulent virus challenge. The fractions of cats that resisted the induction of persistent viremia after FeLV challenge were the following: (1) FeLV-61E-A vaccine, 95%; (2) adjuvant controls, 26%; and (3) established commercial control FeLV vaccine, 35%. The pre-challenge mean neutralizing antibody titers for each group were (1) FeLV-61E-A vaccine, 1:43; (2) adjuvant controls, <1:8; and (3) established commercial control FeLV vaccine: 1:12. The commercial version of the prototype FeLV-61E-A vaccine (Fel-O-Vax, Fort Dodge Laboratories, Fort Dodge, IA) was developed through use of a proprietary adjuvant and a stable high antigen production cell lines. The efficacy and duration of immunity produced by Fel-O-Vax was studied alone and in multivalent combination with other feline virus vaccines in seven subsequent efficacy trials conducted in over 150 immunized cats. The overall FeLV-resistant fraction in these trials was 87% in vaccinated cats versus 8% in adjuvant controls. The duration of protective immunity induced by the multivalent Fel-O-Vax-LvK IV at 1 year postvaccination was 100% in challenged vaccinees versus 0% in challenged controls. The results of these studies show that an inactivated FeLV vaccine prepared from a molecularly cloned subgroup A FeLV can produce high level protective immunity against FeLV infection. This immunity is durable for at least 1 year without intervening booster immunization or virus exposure.

Protection of cats from infectious peritonitis by vaccination with a recombinant raccoon poxvirus expressing the nucleocapsid gene of feline infectious peritonitis virus.

Feline Infectious Peritonitis Virus (FIPV) is a coronavirus that induces an often fatal, systemic infection in cats. Various vaccines designed to prevent FIPV infection have been shown to exacerbate the disease, probably due to immune enhancement mediated by virus-specific immunoglobulins against the outer envelope (S) protein. An effective vaccine would be one that induces cell-mediated immunity without disease enhancing antibodies. In this report, we describe the use of a recombinant raccoon poxvirus that expresses the gene encoding the nucleocapsid protein of FIPV (rRCNV-FIPV N) as an effective vaccine against FIPV-induced disease. Cats were parenterally or orally vaccinated twice, three weeks apart. Cats were then orally challenged with Feline Enteric Coronavirus (FECV), which induces a subclinical infection that can cause enhancement of subsequent FIPV infection. Three weeks later, cats were orally challenged with FIPV. The FIPV challenge induced a fatal infection in 4/5 (80%) of the controls. On the other hand, all five cats vaccinated subcutaneously with rRCNV-FIPV N showed no signs of disease after challenge with FIPV. Four of the five subcutaneous vaccinates survived an additional FIPV challenge. Vaccination with rRCNV-FIPV N induced serum IgG antibody responses to FIPV nucleocapsid protein, but few, if any, FIPV neutralizing antibodies. In contrast to the controls, protected vaccinates maintained low FIPV serum neutralizing antibody titers after FIPV challenge. This suggests that the protective immune response involves a mechanism other than humoral immunity consisting of FIPV neutralizing antibodies.

Maternal floor infarction: relationship to X cells, major basic protein, and adverse perinatal outcome.

OBJECTIVE: Maternal floor infarction of the placenta is characterized by gross placental abnormalities and histologic evidence of X-cell proliferation. Previously, pregnancy-associated major basic protein has been localized to the placental X cell and identified at elevated levels in serum and amniotic fluid in all normal pregnancies. Here we test the hypothesis that pregnancy-associated major basic protein is localized to the X cells in maternal floor infarction and that it contributes to the pathophysiologic features of pregnancies complicated by maternal floor infarction. STUDY DESIGN: Seven patients with eight pregnancies complicated by maternal floor infarction were evaluated. We analyzed placental tissue, serum, amniotic fluid, and placental cyst fluid for pregnancy-associated major basic protein. RESULTS: Placental tissue from pregnancies complicated by maternal floor infarction had increased numbers of X cells and fibrinoid material that occupied or surrounded degenerating villi and that stained intensely for pregnancy-associated major basic protein. Serum pregnancy-associated major basic protein levels were variable and likely cannot be used to predict the occurrence of maternal floor infarction. CONCLUSION: Pregnancy-associated major basic protein, a potent cytotoxin, is localized to X cells and is deposited in close proximity to chorionic villi in maternal floor infarction and may contribute to the pathophysiology of this disorder.

Examination of Koch's postulates for Borrelia burgdorferi as the causative agent of limb/joint dysfunction in dogs with borreliosis.

Borrelia burgdorferi has been implicated as the causative agent of borreliosis in dogs, which is characteristically a limb/joint disorder, but can be associated with multiple-organ dysfunction. Attempts to reproduce this disease by inoculating dogs with B burgdorferi have not been successful. In the study of this report, B burgdorferi from Ixodes dammini ticks was used to induce signs of limb/joint dysfunction, fever, anorexia, depression, and systemic infection in dogs. A pure culture of this bacterium from the blood of an infected dog has been used to fulfill Koch's postulates for B burgdorferi as the causative agent of limb/joint dysfunction associated with borreliosis in dogs.

Immunogenicity and efficacy study of a commercial Borrelia burgdorferi bacterin.

The immunogenicity and efficacy of a commercial Borrelia burgdorferi bacterin was evaluated for stimulation of the host immune response and protection against clinical disease associated with experimentally induced borreliosis in dogs. A total of 30 vaccinated and 24 control dogs were used in 3 separate studies. The vaccine was given IM as two 1-ml doses separated by a 3-week interval. Two weeks or 5 months following the last vaccination, the dogs were challenge inoculated with 7 daily doses of a virulent preparation of a B burgdorferi field isolate through intraperitoneal, subcutaneous, and intradermal routes with or without glucocorticoid administration at the same time. The development of B burgdorferi spirochetemia and clinical disease in the dogs after challenge exposure was studied. Serum samples were obtained from the dogs at various times during the study for serum neutralizing antibody determination and protein immunoblot antibody assay against various geographic isolates of B burgdorferi. Challenge exposure induced limb/joint disorder, fever, anorexia, signs of depression, and B burgdorferi spirochetemia in the nonvaccinated control dogs. The vaccine was found to elicit cross-reactive serum neutralizing and protein immunoblot antibody responses in dogs to various isolates of B burgdorferi and to protect the vaccinated dogs against experimentally induced borreliosis.

Plasma levels of eosinophil granule major basic protein in pregnant cynomolgus monkeys.

The sera of all pregnant women contain increased amounts of a protein biochemically and immunologically similar to the eosinophil granule major basic protein (MBP). Immunofluorescence shows that the pregnancy-associated MBP is localized to placental trophoblastic cells. This information raises important questions about the function of pregnancy-associated MBP because of the potential biological functions attributed to its eosinophil counterpart (namely, its potent toxic and cytostimulatory activities). Previous studies demonstrated the presence of an immunologically cross-reactive protein in the placentae and plasma of pregnant non-human primates. Here, plasma MBP levels were measured throughout gestation in cynomolgus monkeys. In early pregnancy, only modest increases in MBP were found in contrast to the sharp rise observed in the first 20 weeks of human pregnancy. During the final one-third of gestation, striking increases in plasma MBP occurred in the monkeys. This parallels the late rise in MBP seen in humans in the third trimester. Thus, the cynomolgus monkey may serve as a model to clarify the role of the MBP in the biochemical events that culminate in parturition.

Association of immunoreactive eosinophil major basic protein with placental septa and cysts.

A protein that is immunochemically indistinguishable from the major basic protein of the eosinophil granule is present at elevated concentrations in the plasma of pregnant women. Major basic protein has been localized to placental trophoblasts known as X cells. Because X cells are located in placental septa and septal cysts, we tested whether the numbers of these structures are correlated with plasma levels of major basic protein. Data analysis revealed strong positive correlations between maternal plasma major basic protein concentrations and the number of placental septa, septal cysts, and subchorial cysts and the presence of subchorial fibrin deposits. No significant correlation was found between plasma major basic protein levels and other variables examined, such as placental weight. Thus major basic protein is a specific marker for septa and cysts, presumably because it is an X-cell product. The study of major basic protein may aid in delineating the function and ontogeny of X cells, as well as their role in fibrin deposition and in septa and cyst formation.

Evidence of eosinophil granule major basic protein in human placenta.

A protein immunochemically related to the eosinophil granule major basic protein (gMBP) is found in increased concentration in the plasma of pregnant women and has been localized to placental trophoblasts by immunofluorescence. Pregnancy MBP (pMBP) is indistinguishable from gMBP in its reactivity with polyclonal antisera and a panel of 14 mouse mAbs. We report the purification of pMBP from human placenta by: (a) affinity chromatography over mAb immobilized on Sepharose, (b) gel filtration in 6 M guanidine.HCl buffer, and (c) reversed-phase HPLC. Purified pMBP and gMBP are biochemically indistinguishable in that both: (a) bind to DNA, (b) polymerize and bind to carrier proteins via disulfide linkages, (c) have a molecular weight of 14,000, (d) have isoelectric points greater than 10.6, (e) comigrate in two-dimensional gels, (f) coelute during reversed-phase HPLC on C18 columns, (g) have identical peptide maps after three different digestions, and (h) have partial amino acid sequence identity. This physicochemical identity has important implications as to the role of pMBP in human placentation.

Biochemical and amino acid sequence analysis of human eosinophil granule major basic protein.

Eosinophil granule major basic protein (MBP) is a relatively low molecular weight cationic (pI greater than 10) protein present in the crystalloid core of the eosinophil granule. Amino acid sequence analysis of this protein was undertaken as part of an analysis of the structural basis of the potent cytotoxic activities of MBP on parasites and mammalian cells. Many conventional sequencing strategies were unworkable because of the unusual amino acid composition of MBP and its insolubility in solutions buffered at neutral pH. Less conventional chemical reactions, including cyanogen bromide-induced cleavage at tryptophan and acid-induced cleavage at aspartic acid, were used successfully to obtain peptides which allowed definition of the amino acid sequence of MBP. Characterization of MBP by reverse-phase high pressure liquid chromatography and two-dimensional gel analysis showed no microheterogeneity that might be attributed to post-translational modifications. Comparison of the MBP sequence with a protein sequence data base showed that MBP has no significant sequence homology with other characterized proteins. The basicity (pI 10.9) and hydrophobicity predicted from the MBP sequence are likely responsible for the observed affinity of this cytotoxic molecule for cell surfaces and some serum proteins.

Rift Valley fever virus M segment: cellular localization of M segment-encoded proteins.

The Phlebovirus Rift Valley fever virus (RVFV), like other members of the Bunyaviridae family, matures intracellularly at the smooth-surfaced vesicles in the Golgi region of infected cells. Here we show that in cultured cells the RVFV glycoproteins G2 and G1 accumulate and are retained at this site. To investigate the parameters governing this subcellular localization, we have engineered portions of the cloned RVFV M segment (which encodes a 14- and a 78-kDa protein, in addition to glycoproteins G2 and G1) into vaccinia virus. Immunofluorescent analysis of cells infected with a vaccinia virus recombinant containing the entire open reading frame of the RVFV M segment revealed Golgi localization for glycoproteins G2, G1, the 78-kDa protein, and Golgi as well as some reticular distribution for the 14-kDa protein. These distributions paralleled those seen in authentic RVFV-infected cells. RVFV-vaccinia virus recombinants possessing progressive deletions within the 152 amino acid preglycoprotein sequence of the M segment were analyzed for possible effects on the cellular distribution of G2 and G1. Removal of the first 130 amino acids of the open reading frame amino-terminal to the mature glycoprotein coding sequences, while abolishing production of the 78- and 14-kDa proteins, did not alter the Golgi location of G2 and G1. The data suggest that Golgi-specific signals reside within the G2 and/or G1 glycoprotein sequences. The use of vaccinia virus recombinants provides a genetically manipulable expression system with which to further investigate the sequences involved in the intracellular localization of these Phlebovirus proteins.

Rift Valley fever virus M segment: use of recombinant vaccinia viruses to study Phlebovirus gene expression.

Recombinant vaccinia viruses were constructed and used in conjunction with site-specific antisera to study the coding capacity and detailed expression strategy of the M segment of the Phlebovirus Rift Valley fever virus (RVFV). The M segment could be completely and faithfully expressed in recombinant RVFV-vaccinia virus-infected cells, the gene products apparently being correctly processed and modified in the absence of the RVFV L and S genomic segments. The proteins encoded by the RVFV M segment included, in addition to the viral glycoproteins G2 and G1, two previously uncharacterized polypeptides of 78 and 14 kilodaltons (kDa). By manipulation of RVFV sequences present in the recombinant vaccinia viruses and use of specific antibody reagents, it was found that the 78-kDa protein initiated at the first initiation codon of the open reading frame and encompassed the entire preglycoprotein and glycoprotein G2 coding sequences. The 14-kDa protein appeared to begin from the second in-phase ATG and was composed of only the preglycoprotein sequences. Both viral glycoproteins G2 and G1 could be synthesized and correctly processed in the absence of the 78- and 14-kDa proteins, as well as a large portion of the preglycoprotein sequences. However, the hydrophobic amino acid sequence immediately preceding the mature glycoprotein coding sequences was required for authentic glycoprotein production. The M-segment expression strategy involving aspects of translational initiation and protein processing are discussed. The functional roles of the 78- and 14-kDa proteins remain unclear.

Increases of plasma eosinophil major basic protein levels late in pregnancy predict onset of labor.

Major basic protein (MBP), the core of the eosinophil granule, is a potent toxin for parasites and mammalian cells; it also causes histamine release from mast cells and basophils. Plasma levels of MBP are increased in all pregnant women, and MBP has been localized by immunofluorescence to placental X cells and placental-site giant cells. Analyses of plasma MBP levels during pregnancy showed a striking correlation between increases late in the third trimester and spontaneous onset of labor. In one case with premature labor, the plasma MBP level also was increased coincidentally with labor. In contrast, in cases of prolonged gestation in which labor was induced, there were no late increases in MBP levels. Also, uterine contraction during spontaneous labor or oxytocin-induced labor did not cause elevations of plasma MBP. Measurement of plasma MBP levels in pregnancy may be useful for the prediction of labor and for the development of new means of intervention for premature labor and prolonged gestation.

Demonstration of immunoreactive eosinophil granule major basic protein in the plasma and placentae of non-human primates.

A protein immunologically identical to the major basic protein (MBP) of eosinophil granules is present in the plasma of pregnant women and has been localized to human placental X cells using immunofluorescence. This study demonstrates the presence of similar immunoreactive molecules in the placental X cells from several non-human primates. We also describe the presence of increased plasma levels of MBP in the pregnant gorilla and in pregnant cynomolgus and rhesus monkeys as compared to non-pregnant counterparts. Such findings suggest that non-human primates are a suitable animal model for the study of the X cell function and in particular the physiological importance of MBP in pregnancy.