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K C McCullough

Publications and source records attributed to K C McCullough.

At least 19 recordsLinked to original sources
PubMed1992

The immune response against foot-and-mouth disease virus: influence of the T lymphocyte growth factors IL-1 and IL-2 on the murine humoral response in vivo.

Recombinant and pure "natural" IL-1 and IL-2 were compared with the muramyl dipeptide (MDP) component of Freund's adjuvant for their capacity to enhance the humoral immune response against foot-and-mouth disease (FMD) virus antigen. Using a dose of this antigen which alone did not give a detectable immune response, anti-FMD virus antibody was measured at 14 and 28 days post-vaccination. Although IL-1 could enhance the response against the virus antigen, in particular when administered 24 h before the vaccine, this was not as strong as that obtained when MDP was adjuvant. In contrast, IL-2 was at least as efficient as MDP when applied concomitantly with the antigen. If the IL-2 treatment preceded the vaccination by 24 h, a diminution in the magnitude of the response was seen; however, this was countered by the fact that 10 times less IL-2 was required, compared with concomitant cytokine/vaccine administration, in order to have the maximum effect. When both IL-1 and IL-2 were used together, an even greater enhancement of the immune response against FMD virus antigen was observed, but only when given concomitantly with the antigen. These results demonstrate the relevance of T lymphocyte growth factors to the immune response against FMD virus, and how current immunological and biotechnological knowledge could be applied to the improvement of adjuvant systems in a chemically and biologically defined manner.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1992

Relationship between the anti-FMD virus antibody reaction as measured by different assays, and protection in vivo against challenge infection.

The antibody response of cattle after vaccination against foot-and-mouth disease (FMD) virus was monitored using the serum neutralization test (SNT), the sandwich ELISA, liquid-phase ELISA, sandwich competition ELISA, liquid-phase competition ELISA, and the liquid-phase sandwich competition (blocking) ELISA. The competition ELISAs (in particular the "blocking" ELISA) were the most effective at detecting reactivity in these cattle sera. However, 95% of negative sera also competed in the most sensitive ELISA (the "blocking" ELISA) to titres of 1:32 (4% of the sera competed to a titre of 1:128). Comparisons between the different ELISAs, and between these ELISAs and the SNT, demonstrated that the tests were not measuring exactly the same reaction of antibody with FMD virus. With respect to the capacity of animals to resist FMD virus challenge, neither the SNT nor the competition ELISAs were consistently able to identify such animals. The anti-FMD virus antibody titres obtained could be classified into three zones; the "white zone" wherein antibody titres were high and donor animals likely to be protected; the "black zone" wherein antibody titres were low and donor animals likely to be susceptible to infection; the "grey zone" wherein the antibody titres were intermediary and no interpretation could be made with respect to protection. Assays such as ELISA and SNT cannot and do not measure immunological protection; they are a measure of antibody responses and nothing more, and should be interpreted in terms of the "three zone" phenomenon.

Animals↗
PubMed1992

Protective immune response against foot-and-mouth disease.

The causative agents of foot-and-mouth disease (FMD) are small icosahedral viruses of the Aphthovirus group within the Picornaviridae family. There is no evidence that these viruses infect cells of the immune system or otherwise interfere detrimentally with their function; additionally, it has not been possible to relate cytotoxicity reactions against virus-infected cells to the efficacy of the immune response against FMD virus infection. In contrast, there is a close association between FMD virus antibody and the protective immune response (10, 14, 15, 20, 24, 25, 29-32). Induction of this antibody is dependent on the structure of the viral antigenic sites (7-9, 11, 18) and on the concomitant presence of Th-lymphocyte epitopes (4, 5, 7, 8), although a Th-lymphocyte-independent response has been reported (2). Recent work by Piatti et al. (26) showed that the immune response induced by FMD virus was only Th-lymphocyte dependent when low doses of antigen were used. This latter work was performed in mice, and it is not certain that a similar situation would be found in cattle. As for the major effector immune defense, this relies on the interaction between antibody-virus complexes and the phagocytic cells of the reticuloendothelial system (17, 19).

Animals↗
PubMed1991

Production of monoclonal antibodies specific for African swine fever virus following in vitro primary immunization of mouse splenocytes in the presence of stimulated T lymphocyte supernatants.

Splenocytes from non-immune mice were stimulated in vitro using a kit of cytokine preparations (obtained from murine MLR and EL-4 cell cultures), and concomitantly immunized with African swine fever (ASF) virus antigen. In addition, fusions were performed at 5 days after primary or secondary stimulation/immunization. The detection of specific antibodies in the culture supernatants was not successful. In contrast, specific antibody-producing hybridomas could be generated, and this was at least comparable to a standard in vivo immunization regime, even though the optimum fusion ratio employed with these in vitro immunized splenocytes was one which is not optimum when in vivo immunized lymphocytes are used. Consequently, it would appear that hybridoma generation is a more sensitive method than the direct measurement of antibody at detecting in vitro primary immune responses. After primary in vitro immunization, the majority of immunoglobulins produced were apparently of the IgG isotype, with only 8-17% clearly IgM. These antibodies were mainly against VP73 (the major viral envelope protein) as expressed on viral antigen extracted from infected cells, although other specificities were also found. This demonstrated, by in vitro means, that the VP73 carried dominant immunogenic epitopes on ASF virus. Such observations show that the in vitro responses were closely related to those which have been detected in vivo.

African Swine Fever Virus↗
PubMed1991

Hybridoma antibody production in vitro in type II serum-free medium using Nutridoma-SP supplements. Comparisons with in vivo methods.

Six hybridoma clones (three producing anti-bovine viral diarrhoea antibodies and three producing anti-hog cholera virus antibodies) were studied with respect to their growth in type II serum-free medium. This was found to be successful in all cases when the serum-free medium was supplemented with 1% v/v Nutridoma, but only after adaptation over a small number of subcultures. Adapted hybridoma cultures could be grown in 250 ml spinner flasks, and the antibody-containing supernatants concentrated by tangential flow filtration. These were compared both with conventional cultures of the clones grown in serum-containing media and with ascites fluids generated using the latter type of culture. Adaptation to growth in the Nutridoma-based serum-free medium did not impair the capacity of the cells to produce and secrete antibodies, although their growth rate was slightly reduced when 250 ml spinner cultures were employed. The titre obtained from the ascites fluids were, as expected, higher than those from the tissue cultures, but contamination by other proteins, a significant problem in ascites fluids, did not present itself with the serum-free cultures. Consequently, 'serum-free' hybridoma antibodies could easily be concentrated in a relatively pure form, with respect to immunoglobulins present, having no need for affinity purification procedures. In addition, the acute in vivo problems of solid tumours and haemorrhage production could be avoided through the use of in vitro methods.

Animals↗
PubMed1991

Identification of epitope(s) on the internal virion proteins of rinderpest virus which are absent from peste des petits ruminants virus.

Monoclonal antibodies (MAb) raised against the RBOK vaccine strain of rinderpest virus were characterized by radio-immunoprecipitation (RIPA) and in the indirect ELISA using measles (MV), distemper (CDV), rinderpest (RPV) and peste des petits ruminants viruses (PPRV). Those found to be specific for the matrix (M) protein and the nucleocapsid (N) protein could be classified into different groups on the basis of the anti-morbillivirus MAb classification scheme; a number of these MAb showed a selective recognition of RPV, measles virus and distemper virus, or of different isolates of rinderpest virus, demonstrating that greater inter-isolate variation occurs than was apparent from analyses using polyclonal antisera. One group of anti-F protein MAb (group F1) reacted with all isolates of both RPV and PPRV. A second group of anti-N protein MAb (group N1/A) reacted with all RPV isolates, but not with the PPRV isolates. Furthermore, these group N1/A antibodies reacted strongly with RPV isolates which were upon original isolation of high pathogenicity, but had a weaker reaction against the isolates of this virus which were of low pathogenicity. Thus, MAb against RPV, in particular those against the N protein offered a potential superior to that of molecular analyses for "isolate fingerprinting", the differentiation of RPV from PPRV and the discrimination between rinderpest viruses which had been, upon isolation, of either high or low pathogenicity.

Animals↗
PubMed1991

Function of idiotypic networks in vivo: immunisation with idiotype-bearing (Id1) antibody induces further production of Id1 antibody.

Injection of BALB/c mice with an anti-foot-and-mouth disease virus (FMDV) monoclonal antibody (mAb) apparently induced the idiotype network to produce more anti-FMDV (idiotype-bearing) antibody, as determined by hybridoma production. Anti-idiotype antibodies were also induced, detected by binding directly to the mAb used for the immunizations (the "immunising" antibody). Many of the anti-idiotype antibodies were directed against regions in or near the paratope of the immunising mAb, since they competed for the binding of the latter mAb to 146S antigen. The induced idiotype-bearing (anti-FMDV) antibodies also competed for the binding of the immunizing mAb to 146S antigen, demonstrating that both antibodies were of similar epitope specificity. Consequently, it would appear that an idiotype-bearing (Id1) antibody can induce the idiotypic networks to produce more Id1 antibody of the same specificity as that used for the initial stimulation, demonstrating the in vivo functioning of the idiotype network.

Animals↗
PubMed1991

Rapid isolation of monoclonal hybridoma cultures by a 'fusion-cloning' method: the requirement for aminopterin.

Hybridomas were generated by fusing the Balb/c SP2/0 myeloma-like cell line with either: (i) splenocytes from Balb/c mice immunized with foot-and-mouth disease virus (FMDV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV), African swine fever virus (ASFV) or pig thymocytes; or (ii) lymph node cells from cattle immunized with FMDV. If the fusion mixtures were plated in cloning medium of methyl cellulose and HAT medium, small hybridoma colonies developed which rarely survived. Fusion mixtures were then plated in liquid HT medium on to 3T3/A31 feeder layers in 75 cm2 flasks, incubated at 37 degrees C for 24 h before adding aminopterin, and incubated for a further 2 to 4 days before cloning in methyl cellulose/HT medium. Without the aminopterin in the cloning medium, colonies of hybridomas, which could be cultured, developed from the majority of fusions. These colonies were isolated in HT medium over feeder layers and given two subcultures in HAT medium as a precaution against any reversion to aminopterin sensitivity during the cloning. No evidence of such reversions were seen, and recloning results suggested that the initial cloning was highly efficient at generating monoclonal cultures.

Aminopterin↗
PubMed1990

The production of peste des petits ruminants hyperimmune sera in rabbits and their application in virus diagnosis.

Hyperimmune sera were produced by serial inoculation of rabbits with Vero cell-adapted, sucrose gradient-purified Nigerian peste des petits ruminants virus (PPRV) isolate. Two antisera produced, neutralized the homologous PPRV but not the heterologous rinderpest Kabette "O" virus. The antisera gave strong precipitin lines with purified PPRV antigens and were used to detect PPRV and rinderpest virus antigens from ante-mortem secretions and post-mortem tissue homogenates from PPR and rinderpest virus infected goats and cattle by the agar gel precipitation tests (AGPT). The hyperimmune sera gave good titration curves with both purified Nigerian goat and the United Arab Emirate wildlife PPRV isolates in the indirect enzyme linked immunosorbent assay (ELISA). Results of indirect ELISA showed that although there were some cross reactions with the rinderpest, canine-distemper and measles viruses, at 1:100 dilution, the antisera would give a positive signal with only the homologous PPR virus.

Animals↗
PubMed1990

Inactivation of foot and mouth disease virus in skimmed milk with propionic acid, citric acid and hydrogen peroxide.

In order to protect farm animals from infections such as foot and mouth disease (FMD) and tuberculosis, the pasteurisation of milk and milk products designated for the feeding of animals is compulsory in Switzerland. Nowadays, milk products are often treated chemically with acids or with hydrogen peroxide in order to keep bacterial contamination low. The capacity of these chemical treatments to inactivate FMD virus in skimmed milk within 6 h at 5 degrees C was tested in this study. The results indicated that the addition of 0.1%-0.3% of consumable acids, such as citric acid or propionic acid, could not guarantee the complete inactivation of FMD virus in skimmed milk. Similar results were obtained both with FMD virus deliberately added to skimmed milk and with skimmed milk obtained from naturally infected cows. Hydrogen peroxide in concentrations of 0.1%-0.3% was also an ineffective means of controlling the risk of FMD virus transmission from contaminated milk.

Animals↗
PubMed1988

Mitomycin C-treated 3T3/B (3T3/A31) cell feeder layers in hybridoma technology.

Several methods were compared for their efficiency at supporting the growth of hybridoma cells at low cell densities. Soluble growth factors from different sources gave poor results, whereas actively metabolising syngeneic cells proved to be effective feeder systems. Of the latter, the transformed subline of BALB/c embryo fibroblast 3T3/B cells - 3T3/A31 - were best. Macrophage (peritoneal exudate) feeder layers could be as effective as the 3T3/A31 cells, but were much more variable, probably reflecting the physiological state of the donor mice, the degree of sensitisation of the cells in vitro and the rate of recovery after isolation from the peritoneal cavity. Since the 3T3/A31 cells could replicate more rapidly than the hybridoma cells, division of the feeder cell chromosomes was inhibited using mitomycin C; 1-2 X 10(4) 3T3/A31 cells/ml were treated with 1 microgram/ml mitomycin C for 8-16 h at 37 degrees C, washed, and incubated for 3-7 days at 37 degrees C. The latter incubation was to permit the metabolism and breakdown of unreacted but intracytoplasmic drug, and the establishment of an active feeder layer before use with the hybridoma cells.

Animals↗
PubMed1988

Opsonization-enhanced phagocytosis of foot-and-mouth disease virus.

Using isolated peritoneal adherent cells, in which monocytes and macrophages dominate, the uptake and destruction of foot-and-mouth disease virus (FMDV) was enhanced by the opsonization with mAb of particular epitope specificity. This was seen under conditions in which virus infectivity was not neutralized, as determined by in vitro assay. Activation of macrophages in vivo further enhanced the uptake of opsonized virus, presumably by increasing the percentage of phagocytosing cells. The enhanced phagocytosis required opsonization and apparently made use of FcR+ cells, because pepsin-treated antibodies and separated F(ab')2 fragments did not enhance the capacity of the peritoneal cells to react with the virus. The reaction also relied on active phagocytosis, because inhibition of phagocytosis using silica interfered with the binding of both virus alone and virus/antibody complexes. This evidence shows that the previous in vivo observations (McCullough et al., 1986b) of enhanced protection by the mAb can be related to active phagocytosis of virus and virus/antibody complexes. The reaction is not passive adsorption to the monocyte surface, but an active phagocytosis of the virus or the complex.

Animals↗
PubMed1987

Epitopes on foot-and-mouth disease virus particles. I. Topology.

Monoclonal antibodies (MAb) against an O1 Suisse isolate of FMDV were used to identify epitopes on the virus particle and to determine their relative function. Six major antigenic sites containing one or more epitopes were identified using competition ELISA. An epitope relationship is proposed consisting of a trypsin-sensitive sequential site, termed B2/D9, from the codings for the MAb which reacted with it, which was associated with virus infectivity and is probably at or near to the cell-binding site of the virion; a trypsin-resistant, conformational site 1C6/4C9 MAb reaction at which also resulted in neutralization of virus infectivity; a second trypsin-resistant, conformational site 3C8, where again MAb reaction neutralised virus infectivity; a third trypsin-resistant, conformational site 6C3/2G5, at which MAb-dependent neutralisation of virus infectivity was inefficient; a site 3G4, the expression of which was impaired but not destroyed by trypsin treatment, and was not related to virus infectivity; an internal site A8, which appears to be a "12S subunit-specific" site. This work clearly demonstrates for the first time that both trypsin-sensitive and trypsin-resistant neutralisable (infectivity-associated) sites exist on the FMDV particle, and only one of these can be related to the sequential site used to formulate current FMDV peptide vaccines.

Antibodies, Monoclonal↗
PubMed1987

Neutralization of foot-and-mouth disease virus can be mediated through any of at least three separate antigenic sites.

Seven neutralizing monoclonal antibodies were used to characterize 30 escape mutants of a type O foot-and-mouth disease (FMD) virus (O1 Kaufbeuren) selected with the five most active antibodies. Three non-overlapping antigenic sites were found by ELISA and cross-neutralization studies. Within two of the sites the epitopes of two or more monoclonal antibodies overlapped. Two of the sites were conformation-dependent and could not be detected on virus subunits or isolated denatured polypeptides. The third site was less conformation-dependent since the appropriate monoclonal antibodies were able to bind to 12S subunits, isolated VP1 protein and a synthetic peptide containing residues 141 to 160 of VP1 in ELISA. Electrofocusing of mutants of that site showed a high frequency of electrophoretic alterations in VP1. The sequence of most or all of the VP1 coding region of 10 escape mutants of that site plus three parental isolates was determined by primer extension sequencing. At least five amino acids were found to be involved but in only one case (residue 148 of VP1) did a change at that residue produce complete resistance to neutralization. Partial resistance was produced by changes at residues 144, 154 or 208 of VP1 or another residue(s), as yet undefined, that is probably in one of the other capsid polypeptides. Thus the site defined by these mutants was made up of at least three regions, the region involving residues 144 to 154 of VP1, the region encompassing residue 208 from the COOH terminus of VP1, plus a region, probably of VP2 or VP3, encompassing the undefined residue(s).

Amino Acid Sequence↗
PubMed1987

Conformational alteration in foot-and-mouth disease virus virion capsid structure after complexing with monospecific antibody.

A mechanism of neutralization of virus infectivity by antibody is described and related to the immune defences in vivo. The interaction of a particular monoclonal antibody with homologous foot-and-mouth disease virus alters the conformation of the virions to permit penetration of staining reagents. A consequence of this structural alteration is that the RNA genome becomes susceptible to dissociation from the capsid proteins. This mechanism of virus neutralization is irreversible and therefore provides an effective in vivo defence measure against virus attack, complementing the enhanced phagocytosis effected through opsonization of virions. With viruses that can replicate in phagocytes, such a mechanism of virus neutralization could provide a major 'specific' immunological defence against virus invasion.

Antibodies, Monoclonal↗
PubMed1986

The antigenic relationship between measles, canine distemper and rinderpest viruses studied with monoclonal antibodies.

Monoclonal antibodies (MAbs) were used to delineate the antigenic relationship between the three morbillivirus types: measles virus (MV), canine distemper virus (CDV) and rinderpest virus (RPV). Panels of six to 31 MAbs against the haemagglutinin (H), fusion (F), nucleocapsid protein (NP), phosphoprotein (P) and matrix (M) proteins of MV and the H, F, NP and P proteins of CDV were employed. Nine strains of MV, three strains of CDV and four strains of RPV were examined by radioimmunoprecipitation assay and immune fluorescence for reactivity with the heterologous MAbs. Overall, the NP and in particular the F proteins of the morbilliviruses showed a high degree of epitopic homology; the P and M proteins showed a partial epitopic homology, with the greatest variation between the M proteins of CDV and MV; the H proteins showed a low degree of epitopic homology and then only between MV and RPV. These data indicate that the major cross-protecting antigen in heterotypic vaccination amongst morbilliviruses is the F antigen. The epitopic relationships found between morbilliviruses as identified by the MAbs were classified as follows. (i) Group-specific epitopes were present on all strains of the three morbillivirus types. (ii) Group-cross-reactive epitopes were present on only some of the strains from each morbillivirus type (these epitopes identified the presence of intratypic strain variation in all proteins of all three virus types). (iii) Type-specific epitopes, i.e. MV unique or CDV unique, were found only on the homologous morbillivirus type. (iv) CDV-RPV intertypic and MV-RPV intertypic epitopes were, respectively, epitopes shared by CDV and RPV but not with any MV strain, and epitopes shared by MV and RPV but not with any CDV strain. These cross-reactivities and type-specific reactions were obtained with the internal viral proteins (M, P and NP). The epitopes of the F proteins were mainly group-specific and no CDV-RPV or MV-RPV intertypic epitopes were found. The epitopes of the H protein were either type-specific or MV-RPV intertypic. These data support the proposed evolutionary relationship between the morbilliviruses.

Antibodies, Monoclonal↗
PubMed1986

Immune protection against foot-and-mouth disease virus studied using virus-neutralizing and non-neutralizing concentrations of monoclonal antibodies.

Monoclonal antibodies (MAb) against sequential or conformational epitopes on foot-and-mouth disease virus (FMDV) passively protected neonatal syngeneic (BALB/c) mice at dilutions at which they could not neutralize virus infectivity in vitro. The B2, D9, 1C6 and 4C9 MAb, against the Group 1 (sequential) and Group 2 (conformational) epitopes, protected the mice at an antibody:virion molar ratio of between 38:1 and 84:1 (12-18 times lower than that required for neutralization of virus infectivity in vitro). The 3C8 (Group 3) and 6C3 (Group 4) MAb were, respectively, between 5 and 12 times, and between 18 and 40 times, less efficient at protection. There was no consistent correlation between the efficiency of neutralization of virus infectivity in vitro and the protection of neonatal mice against the virus pathogen. Thus, immune protection against FMDV must use mechanisms other than the direct neutralization of virus infectivity by antibody. Complement did not increase the virus neutralization titre of the MAb, but pepsin digestion of the MAb abrogated the enhanced in vivo protection over in vitro neutralization, with little effect on their capacity to neutralize virus infectivity. It is therefore likely that opsonization to a minimum affinity, and subsequent rapid phagocytosis, play a major role in the immune defence against FMDV. This is discussed in terms of the natural host for FMDV and the induction of immunological protection.

Animals↗