Standardization of natural mycolic acid antigen composition and production for use in biomarker antibody detection to diagnose active tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is characterized by the abundance of species specific, antigenic cell wall lipids called mycolic acids. These wax-like molecules all share an identical, amphiphilic mycolic motif, but have different functional groups in a long hydrophobic hydrocarbon mero-chain that divide them into three main classes: alpha-, keto- and methoxy-mycolic acids. Whereas alpha-mycolic acids constitutively maintain an abundance of around 50%, the ratio of methoxy- to keto-mycolic acid types may vary depending on, among other things, the growth stage of M. tuberculosis. In human patients, antibodies to mycolic acids have shown potential as diagnostic serum biomarkers for active TB. Variations in mycolic acid composition affect the antigenic properties and can potentially compromise the precision of detection of anti-mycolic acids antibodies in patient sera to natural mixtures. We demonstrate this here with combinations of synthetic mycolic acid antigens, tested against TB patient and control sera. Combinations of methoxy- and α-mycolic acids are more antigenic than combinations of keto- and α-mycolic acids, showing the former to give a more sensitive test for TB biomarker antibodies. Natural mixtures of mycolic acids isolated from mature cultures of M. tuberculosis H37Rv give the same sensitivity as that with synthetic methoxy- and α-mycolic acids in combination, in a surface plasmon resonance inhibition biosensor test. To ensure that the antigenic activity of isolates of natural mycolic acids is reproducible, we cultured M. tuberculosis H37Rv on Middlebrook 7H10 solid agar plates to stationary growth phase in a standardized, optimal way. The proportions of mycolic acid classes in various batches of the isolates prepared from these cultures were compared to a commercially available natural mycolic acid isolate. LC-MS/MS and NMR data for quantitation of mycolic acids class compositions show that the variation in batches is small, suggesting that the quality of the results for anti-mycolic acid antibody detection in the TB patients should not be affected by different batches of natural mycolic acid antigens if prepared in a standard way.

In vivo uptake and acute immune response to orally administered chitosan and PEG coated PLGA nanoparticles.

Nanoparticulate drug delivery systems offer great promise in addressing challenges of drug toxicity, poor bioavailability and non-specificity for a number of drugs. Much progress has been reported for nano drug delivery systems for intravenous administration, however very little is known about the effects of orally administered nanoparticles. Furthermore, the development of nanoparticulate systems necessitates a thorough understanding of the biological response post exposure. This study aimed to elucidate the in vivo uptake of chitosan and polyethylene glycol (PEG) coated Poly, DL, lactic-co-glycolic Acid (PLGA) nanoparticles and the immunological response within 24 h of oral and peritoneal administration. These PLGA nanoparticles were administered orally and peritoneally to female Balb/C mice, they were taken up by macrophages of the peritoneum. When these particles were fluorescently labelled, intracellular localisation was observed. The expression of pro-inflammatory cytokines IL-2, IL-6, IL-12p70 and TNF-α in plasma and peritoneal lavage was found to remain at low concentration in PLGA nanoparticles treated mice as well as ZnO nanoparticles during the 24 hour period. However, these were significantly increased in lipopolysaccharide (LPS) treated mice. Of these pro-inflammatory cytokines, IL-6 and IL-12p70 were produced at the highest concentration in the positive control group. The anti-inflammatory cytokines IL-10 and chemokines INF-γ, IL-4, IL-5 remained at normal levels in PLGA treated mice. IL-10 and INF-γ were significantly increased in LPS treated mice. MCP-1 was found to be significantly produced in all groups in the first hours, except the saline treated mice. These results provide the first report to detail the induction of cytokine production by PLGA nanoparticles engineered for oral applications.

Chapter 5 - Detection of antimycolic acid antibodies by liposomal biosensors.

Antibodies to mycolic acid (MA) antigens can be detected as surrogate markers of active tuberculosis (TB) with evanescent field biosensors where the lipid antigens are encapsulated in liposomes. Standard immunoassay such as ELISA, where the lipid antigen is not encapsulated, but directly adsorbed to the well-bottoms of microtiter plates, does not yield the required sensitivity and specificity for accurate diagnosis of TB. One reason for this is the cross-reactivity of natural anticholesterol antibodies with MAs. MAs are the major cell wall lipids of mycobacteria. Mycobacterial MA has immunomodulatory properties and elicits specific antibodies in TB patients. Liposomes were optimized for their use as carriers both for the presentation of immobilized purified mycobacterial MA on sensor surfaces, and as a soluble inhibitor of antibody binding in inhibition assays. By using an inhibition assay in the biosensor, the interference by anticholesterol antibodies is reduced. Here, we describe the MA carrying capacity of liposomes with and without cholesterol as a stabilizing agent, optimized concentration and size of liposomes for use in the biosensor assay, comparison of the methods for wave-guide and surface plasmon resonance biosensors and how the cholesteroid nature of MA can be demonstrated by the biosensor when Amphotericin B is allowed to bind to MA in liposomes.

The protective efficacy of a recombinant VP2-based African horsesickness subunit vaccine candidate is determined by adjuvant.

We previously demonstrated that soluble baculovirus-expressed African horsesickness virus (AHSV) serotype 5 VP2 protein (AHSV5 rVP2) elicits neutralising antibodies in guinea pigs. We have now determined the immunogenicity of soluble AHSV5 rVP2 in horses when administered in three different adjuvant types, ISA-50, aluminium phosphate and different saponin preparations. Doses of 10 and 50microg of rVP2 administered with saponin induced full protection to a lethal challenge, albeit with dose-related side effects. The results establish that soluble rVP2 is the biologically active form and that it can induce complete protection when it is delivered with saponin adjuvants. We conclude that the use of the soluble biologically active form of AHSV rVP2 and the choice of adjuvant will be crucial factors in determining efficacy, safety and the production cost of recombinant AHSV subunit vaccines.

Mycolic acids from Mycobacterium tuberculosis: purification by countercurrent distribution and T-cell stimulation.

Bacterial cell wall lipids are recognized as immunostimulatory molecules which make an important component of vaccines against bacterial diseases. Even mycolic acids, forming the waxy outer layer of the bacilli which cause tuberculosis, have been shown to stimulate human CD4/8 double negative T-cells. The role of these cells in resistance to tuberculosis is currently still debated. In this work, a method is described to purify mycolic acids from bacterial crude extracts in a single step using countercurrent distribution. Mycolic acids obtained in this way approach 100% purity and stimulate both double negative and CD4 positive T-cells in peripheral blood leucocytes obtained from healthy human donors. Stimulation of CD4 cells by mycolic acid antigens has not been reported before, emphasizing the potential importance of mycolic acids in the context of the fight against tuberculosis.

Detection of zooplankton prey in squid paralarvae with immunoassay.

Sustainable management of economically important squid requires monitoring of changes in their abundance, which are related inter alia, to their success in the food chain. The highest mortality is expected in the paralarval stages, which are prone to starvation. Causes of starvation may be linked to the lack of suitable prey. A multiple detection system was developed for the simultaneous identification of five putative zooplankton prey in the guts of paralarval Chokka squid, Loligo vulgaris reynaudii, by employing polyclonal rabbit antisera in conjunction with solid phase immunoassays. Specificities of antisera were validated by ELISA screening against different zooplankton taxa. Cross-reactions observed with ELISA were minimized through manipulation of antibody and antigen concentrations resulting in more specific detection of target prey antigens when used in an immunodot assay. Application of this optimised immunoassay detected multiple predation in paralarval squid samples collected from diverse areas in the Agulhas Bank ecosystem on the south coast of South Africa.

Effects of growth conditions and incubation times on the expression of antigens of Haemophilus paragallinarum which are detected by monoclonal antibodies.

Haemophilus paragallinarum causes infectious coryza in poultry, and a panel of monoclonal antibodies (Mabs) were established, which detect surface antigens of this bacterium. It was postulated that these Mabs could be used to detect antigenic differences between strains of H. paragallinarum used in infectious coryza (IC) vaccines, and isolates made from the field, from poultry vaccinated against IC. It has previously been reported that in South Africa there are three different Mab patterns that have been common to H. paragallinarum isolates for the past three decades. The effects of different growth conditions such as duration of incubation, inoculum size, levels of NAD or NaCl in the medium, and the pH of the medium on these Mab patterns were investigated. It was found that many different factors appear to influence the expression of the antigens detected by the panel of Mabs. It was found that at different stages during the growth cycles, the isolates could be classified into different Mab groups. It was also found that alteration of the inoculum size resulted in Mab-pattern switches. Addition of extra NaCl to the medium, in order to slow the growth rate, was found to result in Mab-pattern switches. pH was found to have significant effects on the levels of expression of the antigens detected by the Mabs, although these changes did not result in Mab-pattern switches. The effects of pH were also found to be highly strain dependent. The use of NAD, rather than sterile chicken serum, in the medium did not significantly alter the levels of expression of these antigens. Alterations of the growth conditions greatly affected the levels of expression of the antigens detected by the Mabs, and were highly strain dependent. It was not possible to predict the effects of a particular growth condition on a particular strain or isolate of H. paragallinarum.

Isolation and identification of NAD-independent bacteria from chickens with symptoms of infectious coryza.

Since 1990, NAD-independent bacteria have been isolated in South Africa from poultry showing respiratory manifestations similar to infectious coryza. A total of 126 isolates was examined biochemically and serologically, using polyclonal as well as monoclonal antibodies. Forty isolates were identified as Ornithobacterium rhinotra-cheale, some of which agglutinated glutaraldehyde-fixed red blood cells. Furthermore, fourteen Pasteurella avium isolates, five P. volantium and three Pasteurella species A were isolated for the first time. The remaining 64 isolates were biochemically identified as NAD-independent H. paragallinarum. Of these, 37 were Page serovar A, while no Page serovar B isolates were found. The remaining 25 isolates were typed as Page serovar C. Two different haemagglutination inhibition reaction patterns were found among the Page serovar C isolates, i.e. isolates which reacted with a 1 in 100 dilution of the Page serovar C antiserum, and another larger group which did not react with this dilution of the serum, but did react with rabbit raised antiserum prepared against other Kume serogroup C isolates. This is the first recorded isolation of Page serovar C NAD-independent H. paragallinarum.

Monoclonal antibody characterization of reference isolates of different serogroups of Haemophilus paragallinarum.

Previously, a panel of five monoclonal antibodies (Mabs) was used to study the antigens of strains 0083, 0222 and Modesto of Haemophilus paragallinarum and marked antigenic differences were noted. To establish if these differences were serogroup specific, more reference strains were examined with these Mabs. It was not possible to detect any relationship between the antigens recognized by the Mabs and the serogroup of the reference strain. None of the Mabs produced reacted with the haemagglutinins of the reference strains. The F1 Mab detected an outer membrane protein of 39 kDa, while the V1 Mab detected a lipopolysaccharide of between 13.8 to 14 kDa. Mabs VF1 and VF2 both recognized antigens of 39 kDa of unknown chemical nature and with extremely low frequency of occurrence among strains and isolates. The VF3 Mab detected a lipopolysaccharide with multiple bands at 37 to 39 kDa, which broke down after freezing and thawing to multiple bands of 29 to 32 kDa. These results imply that the haemagglutinins, which are the major typing and protective antigens remain undetected by this panel of Mabs.

Changes in the incidences of the different serovars of Haemophilus paragallinarum in South Africa: a possible explanation for vaccination failures.

Infectious coryza remains an important disease in the poultry industry despite the long-term and widespread use of vaccines against its causative agent, Haemophilus paragallinarum, in South Africa. In order to detect antigenic changes between populations of H. paragallinarum isolated before the use of vaccines against infectious coryza in this country, and field isolates obtained after the introduction of infectious coryza vaccines, 106 different NAD-dependent isolates (of which 93 were identified as H. paragallinarum) from 63 different farms, and dating from 1972 to March 1995, were identified by means of rabbit antisera against serogroups A, B and C. Serogroup C isolates show weaker cross-protection, requiring the further subdivision of this serogroup into its four different serovars. The percentages of the different serovars obtained in the 1970s, confirmed previously published data on South African isolates. A tendency towards a decrease in the number of serogroup A and serovar C-2 isolates, and an increase in the percentage of serovar C-3 isolates, was noted among isolates of the 1980s. These changes were markedly enhanced in the isolates obtained from 1990 to March 1995. The percentage of serogroup A isolates decreased significantly from 34% in the 1970s to only 5% in the 1990s, and that of serovar C-2 isolates, from 31-18%, while the abundance of serovar C-3 isolates increased significantly from 31% in the 1970s to 73% in the 1990s. Serogroup B remained more or less constant and never reached more than 10% of the population. These results indicate the need for the incorporation of serovar C-3 in a vaccine for use in South Africa, particularly in those areas of the country from which isolates were collected during this study. Some of the NAD-dependent isolates obtained from poultry in South Africa between 1970 and 1995, were biochemically identified as Pasteurella avium and P. volantium. As H. avium has been subdivided and reclassified into the genus Pasteurella, this represents the first report of the identification of P. avium and P. volantium in South Africa.

Effects of transformation on the hemagglutinins of Haemophilus paragallinarum.

Strain 0083 and two field isolates of H. paragallinarum were previously converted into NAD-independent organisms by the use of crude DNA extractions from naturally occurring NAD-independent H. paragallinarum isolates. Two of these transformed isolates [0083(T) and A745(T)] were used as DNA donors in another transformation experiment in which another field isolate (M85) was used as the DNA recipient. Transformation was confirmed by lack of NAD requirement for growth, by carbohydrate fermentation patterns and by a comparison of the monoclonal antibody patterns of the isolates before and after transformation. Previously, antigenic differences were observed when DNA from an NAD-independent isolate was introduced into strain 0083. Antigenic differences were also seen in the transformed M85 organisms prepared in this work, and these differences were dependent on the antigenic patterns of the DNA donors. It was established by haemagglutination (HA) and haemagglutination inhibition (HI) that the hemagglutinins of 0083, A745/92 and M85 were not affected by transformation. The use of strains transformed to NAD independence for vaccine production appears to be a valid approach, as the transformation appears not to affect the hemagglutinins of the transformed organisms The major advantage would be the alleviation of the requirement for chicken serum or NAD in the bacterial growth medium used for infectious-coryza-vaccine production.

Production of monoclonal antibodies against Xanthomonas campestris pv. mangiferaeindicae and their use to investigate differences in virulence.

Four Xanthomonas campestris pv. mangiferaeindicae isolates from mango black spot lesions were grouped according to differences in virulence and used to raise monoclonal antibodies (mAbs). Two immunization approaches were followed. In the first, four groups of mice were immunized, each with a different isolate and the spleens from each group homogenized together for cell fusion. The second approach entailed immunization of a single group of mice with bacteria pooled from all four isolates. The resultant mAbs were characterized with regard to the antigen binding specificity and antibody class. A relationship between mAb binding specificity and virulence of the bacteria was shown by Western blot analysis.

Monoclonal antibody characterization of South African field isolates of Haemophilus paragallinarum.

A total of 27 different isolates of Haemophilus paragallinarum were made from chickens between June 1991 and December 1992. All of these isolates were examined by ELISA, by means of a locally produced panel of three monoclonal antibodies (denoted F1, V1 and VF3). The isolates were all of the F1 antigenic type. Three of them showed a weak reaction with the F1 monoclonal antibody, while three other isolates reacted strongly with the F1 as well as with the VF3 Mab. A selection of stored Haemophilus isolates, dating from 1984 to 1985, were also examined with the Mabs and found to be of the F1 antigenic type. Fifteen isolates were collected before 1974, i.e. before the use of Haemophilus vaccines in this country. The majority of them were of the F1 antigenic grouping. Some showed a weak reaction with the F1 Mab; others showed a strong reaction with both the F1 and VF3 Mabs; and a few showed no significant reaction with any of the Mabs used. Strains used for the production of infectious coryza vaccine were also examined with the Mabs. Strain 0083 showed a stronger reaction with the V1 Mab than with the F1 Mab, whereas strain 0222 showed no reaction with any of the Mabs. None of the SA field isolates collected since the use of vaccines exhibits the V1 antigenicity, which is the prevalent antigen of strain 0083. Most (80%) of the SA field isolates showed a stronger reaction with the F1 Mab than did strain 0083. Antigenically silent isolates similar to 0222 (Page's serotype B) were isolated before the use of vaccines, but not since.

Plasmid-encoded NAD independence in some South African isolates of Haemophilus paragallinarum.

Atypical Haemophilus paragallinarum have been isolated from both laying hens and broilers suffering from typical symptoms of infectious coryza in South Africa. Re-inoculation of these bacteria into SPF chickens resulted in similar pathology. The bacteria could be successfully re-isolated from the experimentally infected chickens. Four of the isolates from layers and 3 of those from broilers were found to be closely related to H. paragallinarum serotype A (0083 strain) when tested by the use of a panel of locally developed monoclonal antibodies in the enzyme linked immunosorbent assay (ELISA). A total of 15 isolates from layers and 19 from broilers were found to be more typical of previously collected South African field isolates of H. paragallinarum. A 3rd group, consisting of 5 isolates from layers and 15 from broilers, showed no reaction with the panel of monoclonal antibodies. All the isolates were regarded as atypical because they no longer required V factor (NAD) for growth, whereas strain 0083 and previously collected field isolates M 85 and SB 86 did require it. Crude plasmid extractions from an isolate serologically related to 0083 was used to convert reference strains of H. paragallinarum into NAD-independent isolates, thus indicating that NAD independence is carried on a plasmid.

The localization of a paralysis toxin in granules and nuclei of prefed female Rhipicephalus evertsi evertsi tick salivary gland cells.

A monoclonal antibody directed against a paralysis toxin of Rhipicephalus evertsi evertsi ticks was used to localize the toxin in cytoplasmic granules and, surprisingly, chromatin of the nuclei of cells which resemble the "b" cell type in the salivary glands of Rhipicephalus appendiculatus, Boophilus microplus and Ixodes holocyclus. The association of toxin with chromatin indicates that the toxin may have a regulatory function. Evidence is provided to support the view that the toxin is made up of three identical sub-units, with only the trimeric form being toxic.

The identification of a shared immunogen present in the salivary glands and gut of ixodid and argasid ticks.

The identification of a 70-kDa immunogen present in salivary gland extracts of several ixodid species, namely Hyalomma truncatum (sweating-sickness-inducing (SS+) and non-inducing (SS-) strains), Hyalomma marginatum rufipes and Rhipicephalus evertsi evertsi, is reported. The immunogen was identified by Western blots using a monoclonal antibody of the IgM isotype directed against a 70-kDa immunogen present in the salivary glands of (SS-) female H. truncatum ticks. Cross-reactivity with the gut of unfed adult ixodid ticks, Amblyomma hebraeum, Rhipicephalus simus simus, R. evertsi evertsi, Rhipicentor nuttali, H.m. rufipes, and salivary glands of adult argasid species, Ornithodoros savignyi and Ornithodoros moubata, was demonstrated using ELISA.

Production and properties of monoclonal antibodies against African horsesickness virus, serotype 3.

Four polyethylene glycol-mediated cell fusions yielded a total of 23 monoclonal antibodies (McAbs) specific for African horsesickness virus (AHSV). Two recognised the major core structural polypeptide, VP7, while one each was specific for the outer capsid proteins, VP2 and VP5. The remainder co-precipitated both VP2 and VP7. An inhibition ELISA and radio-immunoprecipitation revealed two types of co-precipitating McAbs, distinguishable from each other by the different relative amounts of the two proteins they precipitated. Only co-precipitating McAbs reduced the size and number of plaques formed by AHSV on VERO cell monolayers, but even at low dilution did not completely abolish virus infectivity. A McAb specific for VP7 showed potential as a group-reactive diagnostic reagent since guinea pig antisera to all nine serotypes of AHSV, as well as an anti-serotype 4 horse serum and an anti-serotype 3 rabbit serum, inhibited its binding in ELISA to AHSV serotype 3.

Spontaneous hybridoma formation induced by immunization with Haemophilus paragallinarum: evidence for a lipopolysaccharide fusion inducer.

The phenomenon of spontaneous fusion between myeloma cells and splenocytes from mice immunized with formalin-inactivated Haemophilus paragallinarum cells, has been reported on recently (1). The identity and properties of the bacterial inducer of fusogenicity of splenocytes have been further investigated with the aid of a monoclonal antibody VF3 against H. paragallinarum (2), which has a bacterial strain specificity correlating with the ability of the strains to induce spontaneous fusion between splenocytes of immunized mice and myeloma cells. It was shown that the lipopolysaccharide fraction of the bacteria was required for the induction of fusogenicity. LPS involvement was clearly indicated by the parallel effects on VF3 antigenicity and fusogenic inductivity of various treatments such as proteolytic digestion, periodate oxidation and sensitivity towards alkali, acid or freezing.