Downstream processing of a plant-derived malaria transmission-blocking vaccine candidate.

Plants as a platform for recombinant protein expression are now economically comparable to well-established systems, such as microbes and mammalian cells, thanks to advantages such as scalability and product safety. However, downstream processing accounts for the majority of the final product costs because plant extracts contain large quantities of host cell proteins (HCPs) that must be removed using elaborate purification strategies. Heat precipitation in planta (blanching) can remove ∼80% of HCPs and thus simplify further purification steps, but this is only possible if the target protein is thermostable. Here we describe a combination of blanching and chromatography to purify the thermostable transmission-blocking malaria vaccine candidate FQS, which was transiently expressed in Nicotiana benthamiana leaves. If the blanching temperature exceeded a critical threshold of ∼75 °C, FQS was no longer recognized by the malaria transmission-blocking monoclonal antibody 4B7. A design-of-experiments approach revealed that reducing the blanching temperature from 80 °C to 70 °C restored antibody binding while still precipitating most HCPs. We also found that blanching inhibited the degradation of FQS in plant extracts, probably due to the thermal inactivation of proteases. We screened hydrophobic interaction chromatography materials using miniature columns and a liquid-handling station. Octyl Sepharose achieved the highest FQS purity during the primary capture step and led to a final purity of ∼72% with 60% recovery via step elution. We found that 30-75% FQS was lost during ultrafiltration/diafiltration, giving a final yield of 9 mg kg-1 plant material after purification based on an initial yield of ∼49 mg kg-1 biomass after blanching.

Maternal antibody uptake, duration and influence on survival and growth rate in a cohort of indigenous calves in a smallholder farming system in western Kenya.

The passive transfer of antibodies from dams to offspring via colostrum is believed to play an important role in protecting neonatal mammals from infectious disease. The study presented here investigates the uptake of colostrum by 548 calves in western Kenya maintained under smallholder farming, an important agricultural system in eastern Africa. Serum samples collected from the calves and dams at recruitment (within the first week of life) were analysed for the presence of antibodies to four tick-borne haemoparasites: Anaplasma marginale, Babesia bigemina, Theileria mutans and Theileria parva. The analysis showed that at least 89.33% of dams were seropositive for at least one of the parasites, and that 93.08% of calves for which unequivocal results were available showed evidence of having received colostrum. The maternal antibody was detected up until 21 weeks of age in the calves. Surprisingly, there was no discernible difference in mortality or growth rate between calves that had taken colostrum and those that had not. The results are also important for interpretation of serosurveys of young calves following natural infection or vaccination.

Insights into the preclinical treatment of blood-stage malaria by the antibiotic borrelidin.

BACKGROUND AND PURPOSE: Blood-stage Plasmodium parasites cause morbidity and mortality from malaria. Parasite resistance to drugs makes development of new chemotherapies an urgency. Aminoacyl-tRNA synthetases have been validated as antimalarial drug targets. We explored long-term effects of borrelidin and mupirocin in lethal P. yoelii murine malaria. EXPERIMENTAL APPROACH: Long-term (up to 340 days) immunological responses to borrelidin or mupirocin were measured after an initial 4 day suppressive test. Prophylaxis and cure were evaluated and the inhibitory effect on the parasites analysed. KEY RESULTS: Borrelidin protected against lethal malaria at 0.25 mg·kg⁻¹·day⁻¹. Antimalarial activity of borrelidin correlated with accumulation of trophozoites in peripheral blood. All infected mice treated with borrelidin survived and subsequently developed immunity protecting them from re-infection on further challenges, 75 and 340 days after the initial infection. This long-term immunity in borrelidin-treated mice resulted in negligible parasitaemia after re-infections and marked increases in total serum levels of antiparasite IgGs with augmented avidity. Long-term memory IgGs mainly reacted against high and low molecular weight parasite antigens. Immunofluorescence microscopy showed that circulating IgGs bound predominantly to late intracellular stage parasites, mainly schizonts. CONCLUSIONS AND IMPLICATIONS: Low borrelidin doses protected mice from lethal malaria infections and induced protective immune responses after treatment. Development of combination therapies with borrelidin and selective modifications of the borrelidin molecule to specifically inhibit plasmodial threonyl tRNA synthetase should improve therapeutic strategies for malaria.

Pre-clinical assessment of novel multivalent MSP3 malaria vaccine constructs.

BACKGROUND: MSP3 has been shown to induce protection against malaria in African children. The characterization of a family of Plasmodium falciparum merozoite surface protein 3 (MSP3) antigens sharing a similar structural organization, simultaneously expressed on the merozoite surface and targeted by a cross-reactive network of protective antibodies, is intriguing and offers new perspectives for the development of subunit vaccines against malaria. METHODS: Eight recombinant polyproteins containing carefully selected regions of this family covalently linked in different combinations were all efficiently produced in Escherichia coli. The polyproteins consisted of one monovalent, one bivalent, one trivalent, two tetravalents, one hexavalent construct, and two tetravalents incorporating coiled-coil repeats regions from LSA3 and p27 vaccine candidates. RESULTS: All eight polyproteins induced a strong and homogeneous antibody response in mice of three distinct genotypes, with a dominance of cytophilic IgG subclasses, lasting up to six months after the last immunization. Vaccine-induced antibodies exerted a strong monocyte-mediated in vitro inhibition of P. falciparum growth. Naturally acquired antibodies from individuals living in an endemic area of Senegal recognized the polyproteins with a reactivity mainly constituted of cytophilic IgG subclasses. CONCLUSIONS: Combination of genetically conserved and antigenically related MSP3 proteins provides promising subunit vaccine constructs, with improved features as compared to the first generation construct employed in clinical trials (MSP3-LSP). These multivalent MSP3 vaccine constructs expand the epitope display of MSP3 family proteins, and lead to the efficient induction of a wider range of antibody subclasses, even in genetically different mice. These findings are promising for future immunization of genetically diverse human populations.

Localization of ferrochelatase in Plasmodium falciparum.

Our previous studies have demonstrated de novo haem biosynthesis in the malarial parasite (Plasmodium falciparum and P. berghei). It has also been shown that the first enzyme of the pathway is the parasite genome-coded ALA (delta-aminolaevulinate) synthase localized in the parasite mitochondrion, whereas the second enzyme, ALAD (ALA dehydratase), is accounted for by two species: one species imported from the host red blood cell into the parasite cytosol and another parasite genome-coded species in the apicoplast. In the present study, specific antibodies have been raised to PfFC (parasite genome-coded ferrochelatase), the terminal enzyme of the haem-biosynthetic pathway, using recombinant truncated protein. With the use of these antibodies as well as those against the hFC (host red cell ferrochelatase) and other marker proteins, immunofluorescence studies were performed. The results reveal that P. falciparum in culture manifests a broad distribution of hFC and a localized distribution of PfFC in the parasite. However, PfFC is not localized to the parasite mitochondrion. Immunoelectron-microscopy studies reveal that PfFC is indeed localized to the apicoplast, whereas hFC is distributed in the parasite cytoplasm. These results on the localization of PfFC are unexpected and are at variance with theoretical predictions based on leader sequence analysis. Biochemical studies using the parasite cytosolic and organellar fractions reveal that the cytosol containing hFC accounts for 80% of FC enzymic activity, whereas the organellar fraction containing PfFC accounts for the remaining 20%. Interestingly, both the isolated cytosolic and organellar fractions are capable of independent haem synthesis in vitro from [4-14C]ALA, with the cytosol being three times more efficient compared with the organellar fraction. With [2-14C]glycine, most of the haem is synthesized in the organellar fraction. Thus haem is synthesized in two independent compartments: in the cytosol, using the imported host enzymes, and in the organellar fractions, using the parasite genome-coded enzymes.

Passive immunity and serological immune response in dairy calves associated with natural Giardia duodenalis infections.

In a previous study, Giardia infection patterns were studied in newborn dairy calves over a 4-month period. Chronic Giardia infections were observed in all calves with initial cyst excretion occurring at approximately 1 month of age. In the work presented here, the passive immunity and serological immune response associated with these Giardia infections were examined. Colostrum and milk samples were collected from the dams of these calves, and monthly serum samples were collected from each calf. The colostrum, milk and sera samples were analyzed by ELISA and Western blot for the presence of anti-Giardia IgG antibodies. In addition, the in vitro anti-Giardia activity of milk and colostrum was examined using a miniculture adherence assay. When examined by ELISA, mean anti-Giardia antibody titres were found to be significantly higher in colostrum compared to milk. The monthly mean serum antibody titres in the calves were not found to differ significantly at any time point during the study. Western blot analysis revealed that colostrum from the dams reacted strongly with many different Giardia antigens between 205 and 7.5kDa, while milk reacted with few antigens in the same size range. Sera collected from the calves when 30 and 60 days of age reacted with few Giardia antigens, but as the calves aged, IgG antibodies in their sera began to react with antigens of 21, 50, 65, 73 and 79kDa. The miniculture adherence assay demonstrated that colostrum had significantly more anti-Giardia activity in vitro compared to milk. These results suggest that the calves in this dairy did not mount a significant humoral immune response against Giardia following infection. However, colostrum contained a high level of anti-Giardia antibodies and exhibited anti-Giardia activity in vitro. Therefore, colostrum may have the potential to provide initial protection against Giardia infections in calves, but the lack of a strong, specific humoral immune response by these calves could account for the high prevalence and chronic duration of the infections.

Eimeria infections in cows in the periparturient phase and their calves: oocyst excretion and levels of specific serum and colostrum antibodies.

Faecal Eimeria oocyst excretion and levels of antibodies to first generation merozoite antigen of E. bovis in sera and colostra were followed in 86 and 70 cow-calf pairs in northern (group EF) and central Germany (group H), respectively, over periods of 3 weeks before to 3 weeks after calving in cows and from birth to an age of 63 days in calves. Oocysts were found in 30 and 7.7% of cows in groups EF and H, respectively. They belonged to 10 (group EF) and four Eimeria spp. (group H) with E. bovis, E. ellipsoidalis, E. auburnensis and E. zuerni as the most frequently occurring species. Prevalence and intensity of oocyst excretion varied with time resulting in peak values around the date of parturition, particularly in the case of E. bovis. Peak values at the time of parturition were also seen in case of strongyle egg excretion. Seven (group H) and nine Eimeria spp. (group EF) were found in the calves. The predominant species E. ellipsoidalis, E. zuerni, E. bovis and E. auburnensis were detected for the first time earlier after birth (3-5 weeks) than the others. The prevalence of Eimeria infections increased to 67.1% (group EF) and 50.1% (group H) 9 weeks after birth. Specific IgM and IgA antibody levels (the latter only determined in group EF) in cow sera remained almost constant throughout the observation period, whereas IgG(1) and IgG(2) levels were reduced at the time of parturition. Levels of specific antibodies in sera and colostra were significantly correlated. Except IgM antibodies, significant inverse correlations were found in cows between intensity of infection with E. bovis and specific serum IgG (group H) and IgG(2) (group EF) antibodies. Antibodies to E. bovis were detected in calves sera only after colostrum intake with significant correlations between levels in calves sera and colostra. Levels decreased, starting within the first week of life (most conspicuously in case of IgM and IgA) until the third week. Subsequently, but except IgG(1) antibody concentrations increased until the end of the observation period. Interrelations between antibody levels and the total amount of E. bovis oocysts excreted by the calves until the ninth week of life varied with the age of the animals. Inverse relationships in the first 3 weeks of life as suggested by negative correlation coefficients could not be proven statistically. Thus, there is no unambiguous proof for immunoprotection of calves against E. bovis via maternal immunity. Considering antibody levels in the 3-9 weeks old calves significant direct correlations with E. bovis oocyst excretion were found in case of IgM, IgG(2) and IgA, reflecting an active immune response of young calves to coccidial infection.

Treatment of experimental ovine cryptosporidiosis with ovine or bovine hyperimmune colostrum.

Ovine or bovine colostrums with different antibody titers were tested for their ability to prevent cryptosporidiosis in five groups of neonatal lambs experimentally infected with 10(6) Cryptosporidium parvum oocysts 2 days after birth (Day 0). In a control group (Group 1), six lambs were deprived of ewe colostrum and exclusively fed with milk replacer. Two groups of six lambs were allowed to suckle their non-hyperimmunized (Group 2) or hyperimmunized (Group 3) dams throughout the experiment. Two groups of seven lambs were separated from their dams at birth before suckling and fed with non-hyperimmune (Group 4) or hyperimmune (Group 5) bovine colostrum; for 7 days they received 50 ml of colostrum completed by milk replacer twice a day, then they were fed with milk replacer exclusively. Control lambs became infected and developed clinical cryptosporidiosis with diarrhea on Days 4-9 post inoculation, oocyst shedding and mortality (2/6). In all the treated groups, the colostrum prevented mortality and clinical cryptosporidiosis. The mortality (5/7) observed in Group 5 was not due to cryptosporidiosis but anemia. In treated groups, specific antibodies were detected on Day 0 after 2 days of colostrum intake and varied little in time for IgM and IgG in spite of the parasite development, whereas they appeared later in the control group, on Day 4 for IgM, Day 11 for IgA and Day 14 for IgG. In all groups, the response which was the most consistent was the IgA response which evolved from Days 11 to 18 in association with the decline of oocyst shedding. Our results show that whatever the serum antibody titers were, the specific C. parvum circulating antibodies have no influence on the control of cryptosporidiosis. The prophylaxis or the treatment of cryptosporidiosis require high titers of specific C. parvum antibodies in the gut lumen during a sufficiently long period.