ABSTRACT While the genomes of a number of Mycoplasma species have been fully determined, there has been limited characterization of which genes are essential. The surface protein (p47) identified by monoclonal antibody B3 is the basis for an enzyme-linked immunosorbent assay for serological detection of Mycoplasma gallisepticum infection and appears to be constitutively expressed. Its gene was cloned, and the DNA sequence was determined. Subsequent analysis of the p47 amino acid sequence and searches of DNA databases found homologous gene sequences in the genomes of M . pneumoniae and M . genitalium and identity with a gene family in Ureaplasma urealyticum and genes in M . agalactiae and M . fermentans . The proteins encoded by these genes were found to belong to a family of basic membrane proteins (BMP) that are found in a wide range of bacteria, including a number of pathogens. Several of the BMP family members, including p47, contain selective lipoprotein-associated motifs that are found in macrophage-activating lipoprotein 404 of M . fermentans and lipoprotein P48 of M . agalactiae . The p47 gene was predicted to encode a 59-kDa peptide, but affinity-purified p47 had a molecular mass of approximately 47 kDa, as determined by polyacrylamide gel analysis. Analysis of native and recombinant p47 by mass peptide fingerprinting revealed the absence of the carboxyl end of the protein encoded by the p47 gene in native p47, which would account for the difference seen in the predicted and measured molecular weights and indicated posttranslational cleavage of the lipoprotein at its carboxyl end. A DNA construct containing the p47 gene interrupted by the gene encoding tetracycline resistance was used to transform M . gallisepticum cells. A tetracycline-resistant mycoplasma clone, P2, contained the construct inserted within the genomic p47 gene, with crossovers occurring between 73 bp upstream and 304 bp downstream of the inserted tetracycline resistance gene. The absence of p47 protein in clone P2 was determined by the lack of reactivity with rabbit anti-p47 sera or monoclonal antibody B3 in Western blots of whole-cell proteins. There was no difference between the p47 − mutant and wild-type M . gallisepticum in pathogenicity in chicken tracheal organ cultures. Thus, p47 , although homologous to genes that occur in many prokaryotes, is not essential for growth in vitro or for attachment and the initial stages of pathogenesis in chickens.

The immune responses to immunostimulating complexes (iscoms) containing recombinant Epstein–Barr virus (EBV) gp340 envelope protein was evaluated in BALB/c (H‐2d) and CBA (H‐2k) mice. Gp340‐iscoms were used either with a low content of Quillaja triterpenoid adjuvant (L‐iscoms) or supplemented with additional Quillaja adjuvant in the form of iscomatrix (S‐iscoms). Class and subclass distribution of anti‐gp340 antibodies, EBV‐neutralizing antibodies, antigen‐specific T cell proliferation and cytokine production were determined and these results compared to those obtained by immunization with non‐adjuvated gp340. The H‐2d and H‐2k mice were characterized as low or high responders in respect to the level of specific anti‐gp340 antibodies, secretion of IgG2a isotype, antigen‐specific lymphoproliferative capacity, interferon‐γ (IFN‐γ) and interleukin‐10 (IL‐10) production in the basic immunizations with gp340. While presentation of the antigen in iscom formulations with low levels of Quillaja triterpenoids induces a moderate enhancement of the immune responses in the low responder H‐2d mice, supplementation with high levels of iscomatrix immunomodulator was required to enhance the immune responses in the high responder H‐2k mice. In both mouse strains subcutaneous immunization with S‐iscoms resulted in a significant increase of IgG1‐ and IgG2a‐specific antibodies, as well as in strong antigen‐specific proliferative response confirmed by the simultaneous cytokine production. The enhanced antigen‐specific secretion of IL‐2 and IFN‐γ together with the abrogation of IL‐10 and the absence of IL‐4 indicates that the responses were driven towards a Th1‐type rather than Th2‐type immune response. The S‐iscom formulations minimized the differences in immune responses between the two mouse strains, but the capacity of immune sera to neutralize EBV transformation in vitro remained completely strain‐dependent. These data indicate that immune responses generated by iscoms can be manipulated by altering the triterpenoid composition of the iscoms and that the levels of triterpenoids can determine whether or not a Th1‐type response is made.

An indirect enzyme-linked immunosorbent assay (ELISA) was evaluated to study the cause of the high level of background reactions which hinders the application of ELISA as a field diagnostic test for Babesia bigemina. Different blockers to improve the specificity of the ELISA were compared. The use of soya milk (25%), gelatine (2.5%) and chicken serum (2%) did not significantly improve the specificity of the test, It was noted that the presence of fibrinogen contributed to the positive ELISA results more than the presence of B. bigemina specific antigen. This conclusion was confirmed by testing bovine fibrinogen as a host protein antigen in ELISA which strongly responded against B. bigemina positive control sera. It is suggested that application of ELISA for B. bigemina is still unreliable until a more purified Babesia-specific antigen or specific monoclonal antibodies are available.

A comparison of the antigenicity and immunogenicity of ISCOM vaccines prepared from equine influenza viruses H3N8 and H7N7 was made with inactivated whole-virus vaccines containing equivalent amounts of virus haemagglutinin. ISCOMs stimulated superior antibody responses in terms of both amount and duration. As with conventional whole-virus vaccines, the levels of antibody to virus haemagglutinin induced by ISCOMs correlated with protection.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for determining Mycoplasma gallisepticum antibodies in chicken sera. The M. gallisepticum antigen was detergent extracted and incorporated into ISCOMs. Sediment of broth medium treated with sarcosyl was used as control antigen. Sera were tested before and after absorption with broth medium components and ELISA titres are expressed as optical density (OD) at 492 nm. Sera from experimentally or naturally infected chickens, those vaccinated with Salsbury Mg bacterin or both vaccinated and experimentally infected were compared with sera from M. gallisepticum free or SPF chickens. A high OD was observed when unabsorbed sera (even from SPF chickens) were tested with control antigen. The non-specific binding of M. gallisepticum negative sera could be removed by absorbing the sera with broth media components before the ELISA was performed. In contrast, ELISA titres obtained with sera from M. gallisepticum positive birds did not decrease significantly after absorption, except in the vaccinated and experimentally infected group. When the OD obtained with control antigen was subtracted from that obtained with ISCOM antigen, the mean value for M. gallisepticum free chickens was 0.083. Higher values were obtained with absorbed sera from experimentally or naturally infected (0.248-0.526), vaccinated (0.506), or vaccinated and infected (0.276-0.930) birds. The use of the ISCOM antigen presentation system in the indirect ELISA, combined with absorption of sera with broth components was demonstrated to be a useful diagnostic assay for M. gallisepticum antibodies.

A monoclonal blocking enzyme-linked immunosorbent assay (blocking-ELISA) was developed to detect antibodies to Mycoplasma gallisepticum (MG) in poultry sera with the help of a peroxidase-labeled monoclonal antibody (MAb) recognizing an epitope of a 56-kilodalton polypeptide (p56) of MG. Immunoglobulins from undiluted MG-positive sera prevent the MAb conjugate from attaching to its specific binding site on p56, which results in no color development. The opposite result-a strong color reaction-was obtained after incubation with MG-negative sera (or when no serum was added before the MAb conjugate). Results were expressed in percent inhibition or ELISA titers.The blocking-ELISA detected 84.7% positive chickens in an experimentally infected flock and 72.6% of chickens in naturally infected flocks, whereas the hemagglutination-inhibition (HI) test was positive only with 68.4% and 48.6% of these serum samples, respectively. All HI-positive serum samples reacted positively in blocking-ELISA. Of sera negative by the HI test, 51.6% and 46.8% proved to be positive when examined with the blocking-ELISA. Overall agreement between the ELISA and HI test was 76.8%. Infection with closely related M. synoviae did not induce any false-positive reactions in blocking-ELISA. There was a strong positive correlation between HI and blocking-ELISA titers (r = 0.83).

Dogs and mice were immunized with either a rabies glycoprotein subunit vaccine incorporated into an immune stimulating complex (ISCOM) or a commercial human diploid cell vaccine (HDCV) prepared from a Pitman Moore (PM) rabies vaccine strain. Pre-exposure vaccination of mice with two intraperitoneal (i.p.) doses of 360 ng ISCOM or 0.5 ml HDCV protected 95% (3840) and 90% (3640) of mice, respectively, against a lethal intracerebral (i.c.) dose with challenge virus strain (CVS). One 360 ng i.p. dose of ISCOM protected 87.5% (3540) of mice against i.c. challenge with CVS. Three groups of five dogs were vaccinated intramuscularly (i.m.) with 730 ng of rabies ISCOM prepared from either the PM or the CVS rabies strains, and they resisted lethal street rabies challenge. Postexposure treatment of mice with three or four 120 ng i.m. doses of ISCOM protected 90% (2730) and 94% (4548), respectively, of mice inoculated in the footpad with street rabies virus, but three doses of HDCV conferred no protection. When four doses of HDCV were administered postexposure, 78% (3241) of the mice died of anaphylactic shock; 21% (1152) of mice had already died of rabies 4 days after the third vaccine dose was administered.

It is proposed that the envelope glycoprotein, gp 51, is the protective antigen of bovine leukemia virus (BLV). An experimental iscom vaccine has been prepared from immunoaffinity purified gp 51. To overcome the problem of integrating a nonamphipathic protein, gp 51 was partially denatured at pH 2.4 before integration into the iscom. The recovery of gp 51 into the iscom was calculated to be 85%. The gp 51 incorporated into iscom retained its physicochemical properties and the neutralizing epitopes F, G and H were found to be intact. The iscom preparation was shown to induce a specific immune response to gp 51 after inoculation into mice and calves, as tested by ELISA and Western blotting. Sera from the immunized calves specifically inhibited the VSV-(BLV) pseudotypes. Thus the gp 51-iscom preparations appear to be highly immunogenic and to induce a gp 51 specific response.

The outer envelope glycoprotein gp51 and the core protein p24 of bovine leukemia virus (BLV), were purified from culture media of FLK-BLV cells by a single-step procedure, using immunoaffinity chromatography based on monoclonal antibodies to the respective proteins. About 90% of the envelope glycoprotein in the culture medium was recovered as a highly purified product. Both purified protein (gp51 and p24) preparations, were found to be highly specific antigens by ELISA, and did not cross-react with sera raised against the other antigen. The conformational epitopes on the purified gp51 were preserved as judged by their reactions with the corresponding monoclonal antibodies. The p24 ELISA reacted only with sera from naturally infected animals and not with sera from animals immunized with an experimental gp51-iscom vaccine. The p24 antigen is therefore useful for discriminating between BLV-infected animals and those immunized with a gp51 subunit vaccine.

The outer envelope glycoprotein gp51 and the core protein p24 of bovine leukemia virus (BLV), were purified from culture media of FLK-BLV cells by a single-step procedure, using immunoaffinity chromatography based on monoclonal antibodies to the respective proteins. About 90% of the envelope glycoprotein in the culture medium was recovered as a highly purified product. Both purified protein (gp51 and p24) preparations, were found to be highly specific antigens by ELISA, and did not cross-react with sera raised against the other antigen. The conformational epitopes on the purified gp51 were preserved as judged by their reactions with the corresponding monoclonal antibodies.The p24 ELISA reacted only with sera from naturally infected animals and not with sera from animals immunized with an experimental gp51-iscom vaccine. The p24 antigen is therefore useful for discriminating between BLV-infected animals and those immunized with a gp51 subunit vaccine.

Fifteen pregnant ewes were vaccinated twice with an experimental immunostimulating complex (ISCOM) subunit vaccine designed to contain the envelope proteins of a Danish cytopathic bovine virus diarrhoea virus (BVDV). The serological responses were measured in ELISA and virus neutralization (VN) tests. All ISCOM-vaccinated ewes developed high VN antibody titres to BVDV in contrast to the 14 non-vaccinated ewes. Both groups of ewes were challenged parenterally when 48–65 days pregnant with a Swedish cytopathic BVDV isolate. In the vaccinated group 26 fetuses out of 29 detected by ultrasound were liveborn, whereas only six out of 26 were liveborn in the non-vaccinated group. It is concluded that the ISCOM vaccine had the potential of eliciting high VN titres as well as protecting fetuses against transplacental infection after challenge with a virulent BVDV isolate.

An experimental ISCOM vaccine has been prepared from gradient purified equine herpes virus type 1 (EHV-1). Radiolabelling studies demonstrated that this vaccine contained all the major viral glycoproteins in relative amounts similar to those found in non-detergent disrupted viral preparations. This EHV-1 ISCOM vaccine generated fully protective responses in hamsters challenged with an otherwise lethal dose of the hamster-adapted EHV-1 strain RACH.

With the dissection of microorganisms followed by biochemical and immunological characterization, antigens inducing protective immunity became recognized. Early attempts to use these isolated antigens as vaccines, i.e. subunit vaccines, showed that although immunogenic in situ as part of the microorganism, they were not immunogenic as purified antigens. Subsequent studies showed that the formation of antigen into defined multimeric forms such as protein micelles or into liposomes made them considerably more immunogenic. In a way, micelles and liposomes mimic a submicroscopic particle of a microorganism with several copies of surface antigens. By contrast, monomeric forms of antigens, e.g., envelope proteins of parainfluenza-3 virus or Semliki forest virus, not only had a low immunogenicity but had a specific suppressive effect on the immune response as well; this was shown when the monomers were given simultaneously with the same antigen in a micelle (Morein et al., 1982, 1983; Morein and Simons, 1985; Jennings, 1987).

A study was conducted to determine if the purification of Parafilaria bovicola antigens can increase the specificity of serodiagnosis of parafilariasis in enzyme-linked immunosorbent assay. Antigens released from adult worms of P. bovicola were separated by chromatofocusing on a polybuffer exchanger of the pH range 7.3−4.0 Polypeptide analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis showed the presence of four major polypeptides with MWs of 41, 36, 24 and 20 kDa. Additional biochemical characterization identified the 24- and 20-kDa polypeptides as hydrophobic glycoproteins. The chromatofocusing purification procedures were also applied for separation of a whole-worm extract. Again, the 41- and 36-kDa antigens were identified in separate peak fractions. Using ELISA, it was shown that the 41- and 35-kDa antigens were recognized by bovine antibodies specific for P. bovicola, but not by other sera collected from cattle infected by Onchocerca gutturosa, Onchocerca lienalis, Ostertagia ostertagi and Dictyocaulus viviparus. The serological evaluation strongly suggests that the 41- and 36-kDa antigens are P. bovicola specific.

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in bovine sera against Parafilaria bovicola nematodes was developed and its sensitivity was compared with the immunodiffusion (ID) method. An exoantigen of P. bovicola which was shown to contain four major polypeptides was used in these procedures. The serological reactivity of the antigen polypeptides was defined by using the enzyme-linked immunoelectrotransfer blot technique (EITB) and whole-worm extract proteins. It identified only four serologically reactive polypeptides with sera from one experimentally infected calf and a verified field case. These two positive sera reacted mainly with four major antigens which coincided in molecular weights of the polypeptides of the exoantigenic preparation, namely, 43, 39, 28 and 25 KDa.

A monovalent experimental ISCOM vaccine has been prepared with the envelope glycoproteins haemagglutinin and neuraminidase of the equine virus strain A/Solvalla/79 (H3N8). In vaccination trials on BALB/c mice the ISCOM vaccine induced more than ten times higher serum antibody titres measured in ELISA than a corresponding experimental micelle vaccine. Similarly, in guinea-pigs the ISCOMs induced about tenfold higher haemagglutination inhibition (HI) and neuraminidase inhibition (NI) titres than a micelle vaccine or a conventional killed influenza whole virus vaccine. Horses vaccinated with a divalent experimental ISCOM vaccine, containing the equine strains A/Prague/56 (H7N7) and A/Solvalla/79 (H3N8), responded with ELISA antibody titres against haemagglutinin which were higher and lasted considerably longer than those in horses vaccinated with conventional whole virus vaccine. ISCOMs induced complete immunoprotection in mice vaccinated with a dose of 1 μg envelope glycoproteins of the mouse pathogenic strain A/PR/8/34 (H1N1).

Immunostimulating complexes (ISCOMs) have been prepared from influenza A virus envelope glycoproteins, i.e. haemagglutinin (HA) and neuraminidase (NA). An ISCOM consists of a matrix, which is the micellar form of the glycoside, Quil A, in hydrophobic interaction with both the envelope glycoproteins (HA/NA). The Quil A bound to the ISCOM amounted to 50 μg mg−1 (5%) of ISCOM protein. ISCOMs were morphologically identified as symmetrical cage-like structures of ≈40 nm in diameter with hexagonal or pentagonal subunits of ≈12 nm. The sedimentation coefficient was ≈19 S as compared to 30 S for the glycoprotein micelles. The biological activities of the HA and NA are preserved in both ISCOMs and micelles.

Immune-stimulating complexes (iscoms), which have recently been shown to be highly effective for the antigenic presentation of membrane proteins of viruses, were prepared with affinity-purified fusion (F) protein of measles virus (MV), using an adaptation of the standard method for iscom preparation. Immunization of monkeys with the F iscom preparation induced biologically active anti-F protein antibodies as was shown in haemolysis inhibition and cell-cell fusion inhibition tests. A whole MV iscom preparation, which also contained the haemagglutinin protein, induced not only also haemolysis-inhibiting antibodies, but, in contrast to the F iscom preparation, also haemagglutination-inhibiting and virus-neutralizing antibodies. In addition the F iscom preparation was shown to activate measles virus-specific T cells in mice. This was demonstrated by the generation of an MV-specific delayed type hypersensitivity response in F iscom-immunized animals and by the isolation of T cell clones specific for MV F protein with the T helper phenotype. Vaccination of mice with MV iscom or F iscom protected them from MV-induced fatal encephalopathy. The data concerning the immunogenicity of MV proteins presented in iscoms are discussed in relation to their potential for the development of an inactivated measles vaccine.