Contagious bovine pleuropneumonia (CBPP) is a bovine bacterial disease of major economic importance in sub-Saharan Africa. Vaccination has been recommended to control the disease in endemic areas such as the Maasai ecosystems of Kenya and Tanzania; however, the currently used live attenuated vaccine has been reported to have poor vaccine safety and efficacy. To compare standard (current) and an improved (buffered) version of the live CBPP-vaccine, several epidemiological studies were carried out in Maasai cattle in Kenya between 2006 and 2008. Specifically, the aims were to estimate CBPP seroprevalence at herd and animal level; to identify risk factors for seroprevalence at both levels; to investigate the spatial distribution of seroprevalence; to compare post vaccination adverse events in cattle vaccinated with a standard and a buffered vaccine, and finally to compare efficacy of the two vaccines to induce seroconversion and to prevent development of clinical signs suggestive of CBPP. A cross-sectional study was carried out in 6872 cattle in 175 randomly selected herds from Loita and Mara divisions. A competitive ELISA revealed that 85% of the herds in the area had at least one seropositive animal and that seropositive herds were harbouring 11% seropositive cattle. A complement fixation test revealed that 46% of the herds had at least one seropositive animal and that seropositive herds were harbouring 4% seropositive cattle. A multivariable logistic regression analysis of the seroprevalence indicated that previous vaccination against CBPP, a history of CBPP outbreaks in the herd, animal age and the location of the herd in the division of Mara were positively correlated to seroprevalence. To investigate the observed difference in herd seroprevalence between the two divisions further, a spatial analysis was conducted. A SatScan test revealed clusters in Mara in areas identified by veterinary personnel as CBPP ‘hot spots’. A logistic regression using spatial information identified that location in the midland agro-ecological zone or close to a river and vaccination were positively associated with seroprevalence. To compare safety and efficacy of a standard and a buffered vaccine, two cohorts of approximately 40,000 cattle were used. The study showed that within 100 days post vaccination, 6.2 cattle per 1000 vaccinates developed adverse events, 4.1 of which were specifically attributable to vaccination and ranging from swelling of the tail to the tail sloughing off. This study revealed a slightly higher incidence of adverse events in cattle vaccinated with the buffered vaccine compared to the standard vaccine. A comparison of the efficacy of the two vaccines revealed that cattle vaccinated with the buffered vaccine had higher odds of seroconversion and lower odds of developing symptoms of CBPP, three and twelve months post vaccination respectively. The epidemiological studies conducted clearly show wide spread seroprevalence in the Maasai cattle. Given the (spatial) heterogeneity observed, control measures should probably be targeted in areas of increased risk (clusters). However, positive association of vaccination and seropositivity call for better diagnostics tests that can differentiate vaccinated from infected animals. Vaccination with buffered vaccine resulted in increased seroconversion, decreased clinical signs indicative of CBPP post vaccination and low seroprevalence post ‘outbreak’. Nevertheless, the increase in adverse events related to the buffered vaccine calls for further research into safer CBPP vaccines.

Adult schistosomes depend for growth and development on hormonal signals from the mammalian host, which may include the insulin signalling pathway. In this project, I firstly used microarray analysis to demonstrate that human insulin can be utilised by adult S. japonicum in culture, resulting in the modulation of distinct metabolic effects as reflected in transcriptional levels of parasite genes. The addition of insulin resulted in the differential expression of 1,101 genes with many related to functions corresponding to the biological and metabolic effects of insulin reported for mammalian cells. Those identified genes in male or female S. japonicum worms that were up or down regulated after exposure to insulin were predominantly involved in growth and development, with significant sex-specific responses evident. Insulin appeared to play a similar role in male parasites as those seen in classical mammalian systems including an increase in protein synthesis though gene transcription and the stimulation of mRNA translation and control protein degradation via the ubiquitin proteasome pathway. Microarray analysis indicated that insulin not only leads to increased gene expression of the PI3-K pathway, which enhances parasite growth, but may also play a role in the sexual differentiation and fecundity of female worms by activating the MAPK pathway. As the insulin target proteins, two types of insulin receptors from Schistosoma japonicum were isolated, S. japonicum insulin receptors 1 (SjIR-1) and 2 (SjIR-2), with features similar to insulin receptors from other taxa. The sequences share 70% and 74% identity to S. mansoni insulin receptor 1 and 2 (SmIR-1 and SmIR-2), respectively. SjIR-1 and SjIR-2 are highly conserved in their tyrosine kinase domain to other IRs from Homo, Mus musculus and Drosophila melanogaster. SjIR-2 is located in the parenchyma in males and in the vitelline glands of female worms, which occupy most of male or female tissue and play an important role in growth or fecundity. In contrast, SjIR-1 was located in the tegument and intestinal epithelium of adult worms, representing much smaller cellular regions compared with the voluminous vitelline tissue or parenchyma. This observation was further confirmed by real time PCR showing that SjIR-2 was more abundantly expressed in S. japonicum adult worm than SjIR-1. Phylogenetic analysis showed that SjIR-2 and SmIR-2 are closer to EmIR than to SjIR-1 and SmIR-1, indicating that SjIR-1 and SmIR-1 might perform specific functions in schistosomes, while SjIR-2, SmIR-2 and EmIR might share similar roles in parasite growth and development in the three parasitic flatworms. Structure modelling recovered the conserved structure between the SjIRs and Homo sapiens IR (HIR) implying a common predicted binding mechanism in the ligand domain and the same downstream signal transduction processing in the tyrosine kinase domain as in HIR. Two-hybrid analysis was used to confirm that the ligand domains of SjIR-1 and SjIR-2 contain the insulin binding site. Incubation of adult worms in vitro, both with a specific insulin receptor inhibitor and anti-SjIRs antibodies, resulted in a significant decrease in worm glucose levels, suggesting again the same function for SjIRs in regulating glucose uptake as described for mammalian cells. Adult worms of S. japonicum possess insulin receptors that can specifically bind to insulin, indicating that the parasite can utilize host insulin for development and growth by sharing the same pathway as mammalian cells in regulating glucose uptake. In vaccination/challenge trials, there was no significant reduction in adult worm burdens with either of the SjLD vaccines. However, there were significant reductions in mean lengths of adult worms ranging from 22-25% in the SjLD1 vaccinated group to 37-42% in the SjLD2 vaccinated groups, significant reductions in faecal eggs in both the SjLD1 (66%) and SjLD2 (68%) vaccinated groups, and a reduction in liver egg numbers in the SjLD1(33%) vaccinated group. These results show that although the SjLDs vaccines were unable to reduce adult worm numbers by clearing them from the vaccinated mice, nevertheless, they significantly depressed the growth of male and female adult worms and affected female egg production. The protective efficacy obtained in terms of the substantial decrease in faecal eggs exceeded that of many of the recently available schistosome antigens and prototype vaccine formulations, which, at best, elicit 40–50% protection in animals using the standard readouts of reduced worm burden or egg production and viability. Overall, disruption of this insulin pathway leading to parasite starvation through the prevention of glucose uptake thereby affecting parasite growth, development and female fecundity, provides a new intervention target and transmission blocking approach to combat schistosomiasis and may be applicable for the control of other debilitating parasitic infections as well.

A major objective in Theileria parva subunit vaccine development is to induce a vaccine antigen specific response mediated by cytotoxic CD8+ T cells (CTL). Therefore it is essential to be able to measure the frequency of the responding CD8+ T cells after vaccination and correlate it with a clinical outcome on challenge. Recently concluded immunogenicity and efficacy studies of T. parva specific CTL antigens showed successful induction of CTL responses in some animals, which correlated with reduced disease severity after challenge. To provide correlates of immunity antigen-specific CD8+ T cell mediated IFN-γ responses and CTL lytic responses were measured over the course of the experiments. Several challenges presented in these trials aimed at optimising vaccine efficacy. While the IFN-γ ELISPOT is a sensitive and reliable assay widely used in vaccine research, the use of chromium/indium release assay remains to be the only assay in use that measures T. parva-specific CTL activity. Hence the overall goal of the study was to develop novel reagents and novel assays to identify parasite-specific CD8+ T lymphocytes with lytic potential. To address this objective, bovine perforin, granzymes A and B, as specific effector proteins expressed in activated CTL were cloned and expressed using a baculovirus expression system. Sequence analysis of the cloned cDNAs showed the isolated cDNA belonged to the perforin and granzyme sub-families respectively. Perforin cDNA demonstrated 85% homology to human perforin with presence of conserved regions resembling calcium binding motif, membrane attack complex component as well complement protein. The sequences encoded by the cloned granzyme A and B cDNAs have the features of a trypsin like serine protease and demonstrates over 70% homology to the human cDNA over the active enzyme region as well catalytic residues characteristic of serine proteases. The expressed polypeptides of all three proteins were used to produce specific antibodies for use as reagents in immunoassays including ELISpot and intracellular staining for flow cytometric analysis. While the antibodies showed reactivity to the recombinant proteins, these reagents displayed different functionality in the recognition of the native protein. Peptide-major histocompatibility complexes (MHC) class I tetrameric complexes (tetramers) are proving invaluable as fluorescent reagents for enumeration, characterisation and isolation of peptide-specific CD8+ T cells and have afforded advantages to phenotype antigen-specific T cells with minimal in vitro manipulation. Fluorescent bovine tetramers were shown to specifically stain antigen-specific CTL by directly binding the T cell receptor (TCR). Analyses of CD8 T-cell responses in live-vaccine immunised cattle also showed that this method is robust and demonstrates changes in the kinetics and specificity of the CD8+ T cell response in primary and secondary infections with T. parva. On average, results of functional assays and tetramer staining followed parallel trends, measured roughly the same populations and allowed for surface and intracellular staining for CD8 T cell marker and perforin, respectively, demonstrating a method that reliably quantifies the frequency, phenotype and function of specific CD8+ T cells. The technical simplicity, rapidity and ability of the flow cytometric technique described in this thesis to measure low frequency antigen-specific responses suggests that tetramer staining, combined with functional assays could be broadly applicable to the valuation of vaccination efficacy to determine which protocols are most successful in inducing CTL responses.

Bakgrund: Malaria är en av de allvarligaste infektionssjukdomarna i världen. De allvarligaste malariafallen orsakas främst av Plasmodium falciparum som sprids av Anopheles-myggor. Ett vaccin med långvarigt och potent skydd skulle kunna minska dödligheten, men också minska behovet av kontrollåtgärder och problemet med läkemedelsresistens. Subenhetsvaccin är den vaccintyp som kommit längst i kliniska studier men dessa uppvisar begränsad effekt. Helparasitvaccin ger en bredare immunitet vilket kan ge ett mer fullständigt skydd. Syfte: Syftet med denna litteraturstudie var att jämföra effekt och varaktighet i skydd mot P. falciparum hos de två P. falciparum sporozoit (PfSPZ)-helparasitvaccinkandidaterna: RAS (Radiation-attenuated sporozoites) och CPS (Chemoprophylaxis and sporozoites), samt att undersöka betydelsen av vaccindos och administreringssätt. Metod: Arbetet är en litteraturstudie baserat på 14 vetenskapliga studier vilka har erhållits via sökning i PubMed. De aspekter som avhandlas är: vaccineffekt och dess varaktighet, immunsvar och dess korrelation till vaccineffekt, betydelsen av dos och administreringssätt samt vaccinens säkerhetsprofil. Resultat: Litteraturstudien visade att RAS-vaccin och CPS-vaccin kan ge ett potent samt säkert kort- och långvarigt skydd mot homolog kontrollerad human malaria-infektion (CHMI) vid immunisering via myggor eller venös inokulation. Dosen har stor betydelse för vaccineffekten och CPS-vaccin kan uppnå potent skydd vid mycket lägre doser än RAS-vaccin. En del immunmekanismer har visat sig korrelera med skydd men CD8+ T-celler i levern verkar ha störst betydelse för långvarigt sterilt skydd. Det långvariga skyddet mot heterolog kontra homolog CHMI är bristfälligt för både RAS-vaccin och CPS-vaccin. Slutsats: En potent vaccineffekt uppnås med PfSPZ-vaccin mot homolog CHMI vid tillräckligt hög dos, men inte ett långvarigt skydd mot heterolog CHMI vilket begränsar användningen i endemiska områden. / Background: Malaria is still one of the most common infectious diseases in the world and there is an overwhelming threat to the development of resistance to different control methods such as drugs and insecticides. A durable vaccine with sterile protection would reduce and maybe eradicate the disease. The most serious cases of malaria are caused by Plasmodium falciparum that is transmitted through the bites of infected Anopheles mosquitoes. The life cycle of malaria is extremely complex and different vaccine candidates have effects at different stages. Naturally acquired immunity develops gradually after many years of clinical episodes but never becomes sterile. RTS,S is the only vaccine candidate who has been in phase III clinical trials. Unfortunately this vaccine has limited efficacy, like many other subunit vaccines, due to rapidly diminishing antibody titers. Whole parasite vaccines have the ability to generate a greater quantity and breadth of antigenic exposure within both the humoral and cellular immunity. This results in stronger immune response and can provide sterile protection. The development of whole parasite vaccines has mainly focused on the pre-erythrocytic stage and the most tested vaccine candidates that are in early clinical trial are radiation-attenuated sporozoites (RAS), chemoprophylaxis and sporozoites (CPS) and genetically attenuated parasites (GAP). Aim: The purpose of this literature study is to examine and compare the vaccine efficacy and durability towards P. falciparum of the two whole parasite vaccine candidates: RAS and CPS and to examine the importance of dose and different routes of administration. Methods: Fourteen different clinical studies were selected from PubMed to be included in this literature study. Different variables were selected for study: the vaccine efficacy and it´s durability after controlled human malaria infection (CHMI) using P. falciparum parasites homologous or heterologous to the vaccine strain, the correlation between the immunogenicity and protection, the importance of the dose and different kinds of administration and vaccine safety. Results: According to the findings in the literature study, direct venous inoculation of RAS-vaccine and CPS-vaccine have the ability to give short and longlasting protection against CHMI using P. falciparum parasites homologous to the vaccine strain. The dose is of great importance to the vaccine efficacy and CPS-vaccine has the ability to give potent protection with much lower doses than RAS-vaccine. Some immune mechanisms in the blood correlate with protection but it seems to be the number of CD8+ T-cells in the liver that are of greatest importance for longlasting and steril protection. Whole parasite vaccines are safe but transient parasitemia is common when using CPS-vaccine. Unfortunately, vaccines with longlasting protection against CHMI using P. falciparum parasites heterologous to the vaccine strain has limited efficacy. Conclusion: RAS-vaccine and CPS-vaccine have the ability to give a potent vaccine efficacy against CHMI using P. falciparum parasites homologous to the vaccine strain when used in sufficiently high doses. Longterm protection against CHMI using P. falciparum parasites heterologous to the vaccine strain is limited and this in turn affects the use in endemic areas. In the future, the vaccine effect can be improved by higher doses, more infectious vaccine strains or vaccine cocktails. An alternative to RAS-vaccine and CPS-vaccine could be direct venous inoculation of late arresting GAP.