Academic literature on the topic 'Vaccine candidate genes'

ABSTRACT We constructed chimeric dengue type 2/type 1 (DEN-2/DEN-1) viruses containing the nonstructural genes of DEN-2 16681 virus or its vaccine derivative, strain PDK-53, and the structural genes (encoding capsid protein, premembrane protein, and envelope glycoprotein) of DEN-1 16007 virus or its vaccine derivative, strain PDK-13. We previously reported that attenuation markers of DEN-2 PDK-53 virus were encoded by genetic loci located outside the structural gene region of the PDK-53 virus genome. Chimeric viruses containing the nonstructural genes of DEN-2 PDK-53 virus and the structural genes of the parental DEN-1 16007 virus retained the attenuation markers of small plaque size and temperature sensitivity in LLC-MK2 cells, less efficient replication in C6/36 cells, and attenuation for mice. These chimeric viruses elicited higher mouse neutralizing antibody titers against DEN-1 virus than did the candidate DEN-1 PDK-13 vaccine virus or chimeric DEN-2/DEN-1 viruses containing the structural genes of the PDK-13 virus. Mutations in the envelope protein of DEN-1 PDK-13 virus affected in vitro phenotype and immunogenicity in mice. The current PDK-13 vaccine is the least efficient of the four Mahidol candidate DEN virus vaccines in human trials. The chimeric DEN-2/DEN-1 virus might be a potential DEN-1 virus vaccine candidate. This study indicated that the infectious clones derived from the candidate DEN-2 PDK-53 vaccine are promising attenuated vectors for development of chimeric flavivirus vaccines.

The lack of immunity to the variola virus in the population, increasingly more frequent cases of human orthopoxvirus infection, and increased risk of the use of the variola virus (VARV) as a bioterrorism agent call for the development of modern, safe vaccines against orthopoxvirus infections. We previously developed a polyvalent DNA vaccine based on five VARV antigens and an attenuated variant of the vaccinia virus (VACV) with targeted deletion of six genes (VAC6). Independent experiments demonstrated that triple immunization with a DNA vaccine and double immunization with VAC6 provide protection to mice against a lethal dose (10 LD50) of the ectromelia virus (ECTV), which is highly pathogenic for mice. The present work was aimed at comparing the immunity to smallpox generated by various immunization protocols using the DNA vaccine and VAC6. It has been established that immunization of mice with a polyvalent DNA vaccine, followed by boosting with recombinant VAC6, as well as double immunization with VAC6, induces production of VACV-neutralizing antibodies and provides protection to mice against a 150 LD50 dose of ECTV. The proposed immunization protocols can be used to develop safe vaccination strategies against smallpox and other human orthopoxvirus infections.

Avian coccidiosis is a disease caused by members of the genus Eimeria. Huge economic losses incurred by the global poultry industry due to coccidiosis have increased the need for cost-effective and easily available recombinant vaccines. Microneme protein 2 (MIC2) and surface antigen 1 (SAG1) of E. tenella have been recognised as potential vaccine candidates. However, the genetic diversity of the antigens in field isolates, which affects vaccine efficacy, has yet to be largely investigated. Here, we analysed genetic diversity and natural selection of etmic2 and etsag1 in Korean E. tenella isolates. Both genes exhibited low levels of genetic diversity in Korean isolates. However, the two genes showed different patterns of nucleotide diversity and amino acid polymorphism involving the E. tenella isolates obtained from different countries including China and India. These results underscore the need to investigate the genetic diversity of the vaccine candidate antigens and warrant monitoring of genetic heterogeneity and evolutionary aspects of the genes in larger numbers of E. tenella field isolates from different geographical areas to design effective coccidial vaccines.

Candidate human immunodeficiency virus (HIV) vaccine regimens based on DNA boosted with recombinant modified vaccinia Ankara (MVA) have been in development for some time, and there is evidence for improved immunogenicity of newly developed constructs. This study describes immune responses to candidate DNA and MVA vaccines expressing multiple genes (gag, RT, tat, nef and env) from HIV-1 subtype C in chacma baboons (Papio ursinus). The vaccine regimen induced (i) strong T-cell responses, with a median of 4103 spot forming units per 106 peripheral blood mononuclear cells by gamma interferon (IFN-γ) ELISPOT, (ii) broad T-cell responses targeting all five vaccine-expressed genes, with a median of 12 peptides targeted per animal and without any single protein dominating the response, (iii) balanced CD4+ and CD8+ responses, which produced both IFN-γ and interleukin (IL)-2, including IL-2-only responses not detected by the ELISPOT assay, (iv) vaccine memory, which persisted 1 year after immunization and could be boosted further, despite strong anti-vector responses, and (v) mucosal T-cell responses in iliac and mesenteric lymph nodes in two animals tested. The majority of peptide responses mapped contained epitopes previously identified in human HIV infection, and two high-avidity HIV epitope responses were confirmed, indicating the utility of the baboon model for immunogenicity testing. Together, our data show that a combination of DNA and MVA immunization induced robust, durable, multifunctional CD4+ and CD8+ responses in baboons targeting multiple HIV epitopes that may home to mucosal sites. These candidate vaccines, which are immunogenic in this pre-clinical model, represent an alternative to adenoviral-based vaccines and have been approved for clinical trials.

The burden of human infections with influenza A and B viruses is substantial, and the impact of influenza B virus infections can exceed that of influenza A virus infections in some seasons. Over the past few decades, viruses of two influenza B virus lineages (Victoria and Yamagata) have circulated in humans, and both lineages are now represented in influenza vaccines, as recommended by the World Health Organization. Influenza B virus vaccines for humans have been available for more than half a century, yet no systematic efforts have been undertaken to develop high-yield candidates. Therefore, we screened virus libraries possessing random mutations in the six “internal” influenza B viral RNA segments [i.e., those not encoding the major viral antigens, hemagglutinin (HA) and neuraminidase NA)] for mutants that confer efficient replication. Candidate viruses that supported high yield in cell culture were tested with the HA and NA genes of eight different viruses of the Victoria and Yamagata lineages. We identified combinations of mutations that increased the titers of candidate vaccine viruses in mammalian cells used for human influenza vaccine virus propagation and in embryonated chicken eggs, the most common propagation system for influenza viruses. These influenza B virus vaccine backbones can be used for improved vaccine virus production.

The 35 Mb genome of Leishmania should be sequenced by late 2002. It contains approximately 8500 genes that will probably translate into more than 10 000 proteins. In the laboratory we have been piloting strategies to try to harness the power of the genome–proteome for rapid screening of new vaccine candidate. To this end, microarray analysis of 1094 unique genes identified using an EST analysis of 2091 cDNA clones from spliced leader libraries prepared from different developmental stages of Leishmania has been employed. The plan was to identify amastigote–expressed genes that could be used in high–throughput DNA–vaccine screens to identify potential new vaccine candidates. Despite the lack of transcriptional regulation that polycistronic transcription in Leishmania dictates, the data provide evidence for a high level of post–transcriptional regulation of RNA abundance during the developmental cycle of promastigotes in culture and in lesion–derived amastigotes of Leishmania major . This has provided 147 candidates from the 1094 unique genes that are specifically upregulated in amastigotes and are being used in vaccine studies. Using DNA vaccination, it was demonstrated that pooling strategies can work to identify protective vaccines, but it was found that some potentially protective antigens are masked by other disease–exacerbatory antigens in the pool. A total of 100 new vaccine candidates are currently being tested separately and in pools to extend this analysis, and to facilitate retrospective bioinformatic analysis to develop predictive algorithms for sequences that constitute potentially protective antigens. We are also working with other members of the Leishmania Genome Network to determine whether RNA expression determined by microarray analyses parallels expression at the protein level. We believe we are making good progress in developing strategies that will allow rapid translation of the sequence of Leishmania into potential interventions for disease control in humans.

ABSTRACT The aim of this study was to define the effects on antigen-presenting cells of the expression of HIV antigens from an attenuated poxvirus vector. We have analyzed the transcriptional changes in gene expression following infection of human immature monocyte-derived dendritic cells (DC) with recombinant modified vaccinia virus Ankara (MVA) expressing the genes encoding the gp120 and Gag-Pol-Nef antigens of HIV type 1 clade B (referred to as MVA-B) versus parental MVA infection. Using microarray technology and real-time reverse transcription-PCR, we demonstrated that the HIV proteins induced the expression of cytokines, cytokine receptors, chemokines, chemokine receptors, and molecules involved in antigen uptake and processing, including major histocompatibility complex (MHC) genes. Levels of mRNAs for interleukin-1, beta interferon, CCR8, and SCYA20 were higher after HIV antigen production. MVA-B infection also modulated the expression of antigen processing and presentation genes: the gene for MICA was upregulated, whereas those for HLA-DRA and HSPA5 were downregulated. Indeed, the increased expression of the gene for MICA, a glycoprotein related to major histocompatibility complex class I molecules, was shown to enhance the interaction between MVA-B-infected target cells and cytotoxic lymphocytes. The expression profiles of the genes for protein kinases such as JAK1 and IRAK2 were activated after HIV antigen expression. Several genes included in the JAK-STAT and mitogen-activated protein kinase signaling pathways were regulated after HIV antigen expression. Our findings provide the first gene signatures in DC of a candidate MVA-B vaccine expressing four HIV antigens and identified the biological roles of some of the regulatory genes, like that for MICA, which will help in the design of more effective MVA-derived vaccines.

Rotavirus vaccine development has focused on the delivery of live attenuated rotavirus strains by the oral route. The initial "Jennerian" approach involving bovine (RIT4237, WC3) or rhesus (RRV) rotavirus vaccine candidates showed that these vaccines were safe, well tolerated, and immunogenic but induced highly variable rates of protection against rotavirus diarrhea. The goal of a rotavirus vaccine is to prevent severe illness that can lead to dehydration in infants and young children in both developed and developing countries. These studies led to the concept that a multivalent vaccine that represented each of the four epidemiologically important VP7 serotypes might be necessary to induce protection in young infants, the target population for vaccination. Human-animal rotavirus reassortants whose gene encoding VP7 was derived from their human rotavirus parent but whose remaining genes were derived from the animal rotavirus parent were developed as vaccine candidates. The greatest experience with a multivalent vaccine to date has been gained with the quadrivalent preparation containing RRV (VP7 serotype 3) and human-RRV reassortants of VP7 serotype 1, 2, and 4 specificity. Preliminary efficacy trial results in the United States have been promising, whereas a study in Peru has shown only limited protection. Human-bovine reassortant vaccines, including a candidate that contains the VP4 gene of a human rotavirus (VP4 serotype 1A), are also being studied.

ABSTRACT Anthrax, a disease usually associated with herbivores, is caused by the bacterium Bacillus anthracis. The current vaccine licensed for human use requires a six-dose primary series and yearly boosters and causes reactogenicity in up to 30% of vaccine recipients. A minimally reactogenic vaccine requiring fewer inoculations is warranted. Venezuelan equine encephalitis (VEE) virus has been configured for use as a vaccine vector for a wide variety of immunogens. The VEE vaccine vector is composed of a self-replicating RNA (replicon) containing all of the VEE virus nonstructural genes and a multiple-cloning site in place of the VEE structural genes. Four different anthrax vaccines were constructed by cloning the protective antigen (PA) gene from B. anthracis into the VEE vaccine vector. The anthrax vaccines were produced by assembling the vectors into propagation-deficient VEE replicon particles in vitro. A/J mice inoculated subcutaneously with three doses of the mature 83-kDa PA vaccine were completely protected from challenge with the Sterne strain of B. anthracis. Similar results were obtained with vaccines composed of the PA gene fused to either the B. anthracis secretory sequence or to a tissue plasminogen activator secretory sequence in three additional mouse strains. Mice were unprotected from challenge after inoculation with the carboxy-terminal 63-kDa PA vaccine. These results suggest that these VEE-vectored vaccines may be suitable as candidate vaccines against anthrax.

Thesis (MSc)--University of Stellenbosch, 2006.
ENGLISH ABSTRACT: Ostrich farming is of significant economical importance in South Africa. Three ostrich mycoplasmas, Ms01, Ms02 and Ms03 have been identified previously, and were provisionally named ‘Mycoplasma struthiolus’ (Ms) after their host Struthio camelus. Ostrich mycoplasmas are the major causative organisms of respiratory diseases, and they cause stock losses, reduced production and hatchability, and downgrading of carcasses and therefore lead to large economic losses to the industry. In order to be pathogenic to their host, they need to attach through an attachment organelle, the so-called tip structure. This structure has been identified in the poultry mycoplasma, M. gallisepticum, and is made up of the adhesin GapA and adhesin-related CrmA. Currently, no ostrich mycoplasma vaccine is commercially available and for this reason the need to develop one has arisen. Therefore the first part of this study was dedicated to the identification and isolation of vaccine candidate genes in the three ostrich mycoplasmas. Four primer approaches for polymerase chain reactions (PCR’s), cloning and sequencing, were used for the identification of adhesin or adhesin-related genes from Ms01, Ms02 and Ms03. The primer approaches revealed that the target genes could not be identified due to the high diversity of sequences that were generated. Therefore sequences were also compared with those of other mycoplasma species in BLAST searches. Results showed that the most significant hit was with the human pathogen M. hominis oppD, which is located in the same operon as the membrane protein P100 involved in adhesion. Other hits were with ABC transporters which may also play a role in cytadhesion. The second part of this study was aimed at testing whether two poultry mycoplasma vaccines, M. synoviae and M. gallisepticum, can be used in ostriches to elicit immune responses until an ostrich mycoplasma vaccine has been developed. Ostriches on three farms of different age groups in the Oudsthoorn district were therefore vaccinated with these vaccines in a vaccine trial. The enzymelinked immunosorbent assay (ELISA) was used to test the level of antibody response. Results showed that both vaccines elicited an immune response in all three age groups. A high percentage of the ostriches reacted positively, which indicates that both vaccines elicit antibody responses and may therefore give protection against ostrich mycoplasma infections.
AFRIKAANSE OPSOMMING: Volstruisboerdery is ‘n belangrike ekonomiese sektor in Suid-Afrika. Drie volstruismikoplasmas, Ms01, Ms02 en Ms03, is voorheen geïdentifiseer en voorlopig ‘Mycoplasma struthiolus’ (Ms) benaam na aanleiding van hul gasheer, Struthio camelus. Volstruismikoplasmas is die grootste oorsaaklike organismes van respiratoriese siektes, kudde verliese en die afgradering van karkasse wat lei tot groot ekonomiese verliese in die volstruisbedryf. Ten einde patogenies vir die gasheer te wees, moet mikoplasmas deur middel van ‘n aanhegtingsmeganisme vasheg – die sogenaamde puntvormige struktuur. Hierdie struktuur is in die pluimvee mikoplasma M. gallisepticum geïdentifiseer, en bestaan uit aanhegting proteïen GapA en die aanhegting verwante proteïen CrmA. Tans is geen volstruismikoplasma entstof kommersieel beskikbaar nie, en derhalwe het die behoefte ontstaan om so ‘n entstof te ontwikkel. Die eerste gedeelte van hierdie studie is dus gewy aan die identifisering en isolering van entstof kandidaat gene in al drie volstruismikoplasmas. Vier inleier benaderings vir polimerase ketting reaksies (PKR), klonering asook geenopeenvolging bepalings vir die identifisering van aanhegting of aanhegting verwante gene vanuit Ms01, Ms02 en Ms03 is gebruik. Die inleier benaderings het getoon dat die teikengene nie geïdentifiseer kon word nie as gevolg van hoë variasie in die gegenereerde geenopeenvolgings. Derhalwe is geenopeenvolgings met ander mikoplasma spesies deur middel van BLAST soektogte vergelyk. Resultate het getoon dat die betekenisvolste ooreenstemming dié met die menslike patogeen M. hominis oppD was, wat deel vorm van die membraan proteïen P100 operon wat betrokke is by aanhegting. Ander ooreenstemmings sluit ABC transporters in wat moontlik betrokke kan wees by aanhegting. Die tweede gedeelte van hierdie studie het ten doel gehad om te toets of twee pluimvee mikoplasma entstowwe, M. synoviae en M. gallisepticum, gebruik kan word in volstruise om immuunresponse te ontlok tot tyd en wyl ‘n volstruismikoplasma entstof ontwikkel is. Volstruise vanaf drie plase in verskillende ouderdomsgroepe in die Oudtshoorn distrik was ingeënt met hierdie entstowwe in ‘n entstof proefneming. Die ensiem-afhanklike immuno-absorpsie essaï (ELISA) was gebruik om antiliggaam response te toets. Die resultate het getoon dat beide entstowwe immuunresponse ontlok het in al drie ouderdomsgroepe. ‘n Groot persentasie van die volstruise het positief gereageer wat ‘n aanduiding is dat beide entstowwe immuunresponse ontlok het en kan dus beskerming bied teen volstruismikoplasma infeksies.

A shotgun genome sequencing project was undertaken in the expectation that access to the entire protein coding potential of E. Ruminantium (Welgevonden) will facilitate the identification of vaccine candidate genes against heartwater. The 1,516,355 bp sequence is predicted to encode 888 proteins and 41 stable RNA species. The most prominent feature is the large number of tandemly repeated and duplicated sequences, some of continuously variable copy number. These repeats have mediated numerous translocation and inversion events and seem to be responsible for the generation of both new full and partial protein coding sequences. There are 32 predicted pseudogenes, most of which are truncated fragments of genes associated with repeats. Of the 13 members of the order Rickettsiales compared in this study, E. Ruminantium has the lowest coding capacity (62%), lowest GC content (27.5%), but the highest proportion of repetitive sequences, which comprise 8.5% of the genome. Metabolic reconstruction of E. Ruminantium revealed the metabolic and biosynthetic capabilities typical of an obligate intracellular organism. We identified a number of genes unique to E. Ruminantium, most of which are not functionally characterised in any organism, and those shared with 12 other members of the Rickettsiales. Bioinformatic tools were used to identify possible vaccine candidates from the annotated genome sequence. The protective properties of seven open reading frames (ORFs), which induced cellular immune responses in vitro, were tested in vivo Only 20% survival was obtained in sheep immunised with a DNA formulation consisting of three ORFs. We found that the levels of peripheral blood mononuclear cell proliferation and interferon-gamma (IFN-γ) production did not correlate with each other, nor with the levels of protection, suggesting that the current assays are just not reliable and that IFN-γ expression alone is not an indicator of protection. Therefore more cytokines and different assays will have to be investigated to define in detail what constitutes a protective immune response against E. Ruminantium infection. However, the data generated from the genome sequence will continue to facilitate novel approaches to study the organism and to develop an efficacious vaccine against heartwater.
Thesis (PhD)--University of Pretoria, 2010.
Veterinary Tropical Diseases
unrestricted

Conselho Nacional de Desenvolvimento Científico e Tecnológico
Bovine herpesvirus 5 (BoHV-5) the agent of meningoencephalitis in cattle - is an important pathogen of cattle in South America and efforts have been made to produce safer and more effective vaccines. This dissertation relates an investigation of the virulence in rabbits of three BoHV-5 recombinants, vaccine candidates, defective in the glycoprotein E (BoHV-5gEΔ), thymidine kinase (BoHV-5TKΔ) and both genes (BoHV-5gEΔTKΔ). To this, four groups of eight rabbits each were inoculated intranasally with each recombinant or the parental strain (SV507/99) and monitored thereafter. At day 42 post inoculation (pi) the inoculated animals were submitted to dexamethasone (Dx) administration to reactivate latent infection. At day 70 pi, all animals were euthanized and the brain was collected for investigation of latent viral DNA by PCR. Rabbits inoculated with the parental virus shed virus between days 2 and 8 pi and all rabbits (n=8) developed neurological disease and died or were euthanized in extremis, between days 7 and 13 pi. Among the animals inoculated with the recombinants, viral shedding was detected between days 2 and 10 pi, in 7 out of 8 rabbits of the BoHV-5gEΔ group, in 6 out of 8 rabbits of the BoHV-5TKΔ group and in 3 out of 8 of the BoHV-5gEΔTKΔ group. In spite of variable levels of virus shedding, all rabbits inoculated with the recombinants seroconverted, developing virus-neutralizing antibodies in titers from 2 to 256 at day 42 pi. The rabbits inoculated with the parental virus showed a wide distribution of the virus in their brains, including the olfactory bulbs, cortices, medulla oblongata, pons, midbrain and thalamus. Three out of eight rabbits inoculated with the recombinant BoHV-5gEΔ developed neurological signs at days 10 and 15pi. A more restricted virus distribution, confined mainly to cerebral cortices and thalamus was detected in the brain of these animals. Rabbits inoculated with the recombinants BoHV-5TKΔ (n=8) or BoHV-5gEΔTKΔ (n=8) remained healthy during the experiment. Dx administration to rabbits inoculated with the three recombinants at day 42 pi did not result in viral reactivation, as demonstrated by lack of seroconversion or virus shedding. Nevertheless, viral DNA was detected in the trigeminal ganglia or olfactory bulbs of all these animals at day 28pDx, demonstrating they were latently infected. These results showed that the three recombinants were able to establish latent infection yet they were not easily reactivated by Dx administration. In summary, the recombinants BoHV-5TKΔ and BoHV-5gEΔTKΔ are attenuated for rabbits and constitute potential vaccine candidates.
O herpesvírus bovino tipo 5 (BoHV-5), agente da meningoencefalite herpética bovina, possui grande importância na América do Sul e tem motivado pesquisas para o desenvolvimento de vacinas eficazes e seguras. Essa dissertação relata a investigação da virulência em coelhos de três recombinantes do BoHV-5, candidatos vacinais, contendo deleções nos genes da glicoproteína E (gE) (BoHV-5gEΔ), da enzima timidina quinase (TK) (BoHV-5TKΔ) e deleção dupla nos genes da gE e TK (BoHV-5gEΔTKΔ). Para isso, quatro grupos de oito coelhos cada foram inoculados pela via intranasal com um dos recombinantes ou com a cepa parental (SV507/99) e monitorados nos dias seguintes à inoculação. No dia 42 pós-inoculação (pi) realizou-se a administração de dexametasona (Dx) para reativar a infecção latente e no dia 70 pi a eutanásia para a coleta do encéfalo para a pesquisa de DNA latente por PCR. Os coelhos inoculados com o vírus parental (SV507/99) excretaram vírus nas secreções nasais entre os dias 2 e 8 pós-inoculação (pi), e todos (n=8) desenvolveram doença neurológica e morreram ou foram submetidos à eutanásia in extremis entre os dias 7 e 13 pi. Excreção viral entre os dias 2 e 10 pi também foi detectada em 7 de 8 coelhos inoculados com o BoHV-5gEΔ; 6 de 8 inoculados com BoHV-5TKΔ e 3 de 8 inoculados com BoHV-5gEΔTKΔ. Apesar dos níveis variáveis de excreção viral, os animais inoculados com os três recombinantes soroconverteram, apresentando anticorpos neutralizantes em títulos entre 2 e 256 no dia 42 pi. Nos animais inoculados com o vírus parental, o vírus foi detectado amplamente disseminado no encéfalo, incluindo o bulbo olfatório, córtices, bulbo, ponte, mesencéfalo e tálamo. Dentre os animais inoculados com o recombinante BoHV-5gEΔ, três desenvolveram doença neurológica, nos dias 10 e 15 pi. Uma distribuição viral restrita aos córtices e tálamo foi detectada no encéfalo desses animais. Os coelhos inoculados com os recombinantes BoHV-5TKΔ (n=8) e BoHV-5gEΔTKΔ (n=8) permaneceram saudáveis. A administração de dexametasona (Dx) no dia 42 pi não resultou em reativação da infecção por nenhum dos recombinantes, demonstrada por ausência de soroconversão ou excreção viral em secreções. Entretanto, o DNA viral latente foi detectado no gânglio trigêmeo ou no bulbo olfatório de todos esses animais no dia 28 pDx (70dpi), demonstrando o estabelecimento da infecção latente. Esses resultados demonstram que os recombinantes são capazes de estabelecer a infecção latente, mas não são facilmente reativados pela administração de Dx. Em resumo, os recombinantes BoHV-5TKΔ e BoHV-5gEΔTKΔ são atenuados para coelhos constituindo-se, assim, em candidatos vacinais em potencial.

Onchocerciasis is a severely debilitating yet neglected tropical disease (NTD) currently affecting approximately 15.5 million people, including 12.2 million people with skin disease and 1.025 million with vision loss. The disease causes social stigma, generates and perpetuates poverty, and leads ultimately to irreversible unilateral or bilateral blindness if untreated. Consequently, onchocerciasis is a major impediment to socioeconomic development in addition to being a major public health concern in some African countries. Many control programs have been launched against the disease with moderate successes achieved in Africa. These limited outcomes are partially due to the unavailability of reliable, non-invasive and easily applicable diagnostic tools for mapping endemic regions, monitoring control program successes, determining treatment endpoints and post-elimination surveillance. The current WHO recommendations for certification of elimination include the use of the Ov16 antibody detection ELISA which is flawed by an intrinsic systematic error as 15-25% of infected populations may not produce antibodies against this antigen due to genetic restrictions. With the recent shift in the global health goal of onchocerciasis from control to elimination, there is a need for the development of novel appropriate tools. These tools include amongst others, drugs, diagnostics, and vaccines. In this work, bioinformatics analyses combined with immunological assays were applied in a bid to develop potential tools for the current elimination programs. With regards to chemotherapy, Ivermectin which has been the sole drug for onchocerciasis treatment for over 30 years kills only the microfilariae (mf) leaving the adult worms intact which continue to produce the mf. Moreover, there is a recent problem of development of parasite resistance to this drug. In addition, moxidectin which was recently approved for treatment is contra-indicated in pregnant women and children under 12 who could continue to serve as reservoirs for infection. There is therefore a need to develop new treatment strategies, preferably for macrofilaricidal drugs. For a total eradication of onchocerciasis, diagnosis and treatment must be complemented with vaccine development. The aim of this work was therefore (i) to characterise an O. volvulus antigen, Ov58GPCR and (ii) to design an epitope-based chimeric antigen, which we designated, Ov-DKR-1, within the framework of the development of onchocerciasis control tools. In order to achieve the first goal, towards diagnosis, synthetic peptides representing linear B-epitopes and the recombinant extracellular domain of a G-protein coupled receptor (GPCR) with diagnostic potential were tested for their immune responses using serum from onchocerciasis-infected individuals and various controls. The results obtained indicate that (i) the O. volvulus antigen, Ov58GPCR is a G-protein coupled receptor (GPCR) conserved in related nematodes, (ii) synthetic peptides predicted to be in the extracellular domain (ECD) of Ov58GPCR are indeed immunogenic epitopes in onchocerciasis-infected individuals, (iii) synthetic peptide cocktails discriminate between actively-infected individuals, treated non-infected individuals and healthy African controls, (iv) polyclonal antibodies against one of the peptides or against the bacterially-expressed ECD reacted specifically with the native antigen in O. volvulus total and surface extracts, (v) Ov58GPCR is transcribed in both larvae and adult parasite stages, (vi) IgG and IgE responses to the recombinant ECD decline with Ivermectin treatment. All these findings suggest that the recombinant extracellular domain and synthetic peptides of Ov58GPCR, as well as the specific immune responses generated, could be harnessed in the context of disease diagnosis and surveillance. To assess the potential role of Ov58GPCR in drug or vaccine target development, preliminary examination on the essentiality of the Ov58GPCR for parasite survival was evaluated through RNA interference. A short-interfering RNA (siRNA) sequence targeting the gene designed and tested by soaking with O. volvulus male worms resulted in a reduction in motility. Results indicated that the gene may be involved primarily in motility. Further investigations are recommended in this light. With regards to the second goal, towards the development of a potential immune-protective tool, many indicators reveal the possibility of the development of protective tools against onchocerciasis. Consequently, an immuno-informatics approach was applied to design a filarial-conserved multi-epitope subunit vaccine candidate consisting of B-and T-cell epitopes of proteins reported to be potential novel vaccine candidates. Conservation of the selected proteins in other nematode parasitic species and predicted epitopes suggests that the generated chimera protein (Ov-DKR-1) could be vital in cross-protection. The 3D structure was predicted, refined and validated bioinformatically. Protein-protein docking of the chimeric vaccine candidate with the TLR4 receptor predicted efficient favourable binding. Immune simulation predicted significantly high levels of IgG1, T-helper, T-cytotoxic cells, INF-γ, and IL-2 responses. Overall, the designed chimeric peptide demonstrated antigenicity superior to the current vaccine candidates.
L’onchocercose est une maladie tropicale sévèrement débilitante mais négligée qui touche actuellement environ 15,5 millions de personnes, dont 12,2 millions de souffrant de maladies de la peau et 1,025 millions de souffrant de perte de vision. La maladie provoque une stigmatisation sociale, génère et perpétue la pauvreté et finit par conduire à une cécité unilatérale ou bilatérale irréversible si elle n'est pas traitée. En conséquence, l’onchocercose est un obstacle majeur au développement socioéconomique en plus d’être une préoccupation majeure pour la santé publique. De nombreux programmes de lutte ont été lancés contre la maladie, avec quelques succès en Afrique. Ces résultats sous-optimaux (limités) sont en partie dus à l’absence d’outils fiables, non invasifs et facilement applicables pour la cartographie des régions endémiques, le suivi des succès des programmes de contrôle, la détermination des paramètres de traitement et la surveillance post-élimination. Les recommandations actuelles de l’OMS pour la certification de l’élimination incluent l’utilisation du test ELISA de détection d’anticorps Ov16, entaché d’une erreur systématique intrinsèque puisque 15 à 25% des populations infectées peuvent ne pas produire d’anticorps contre cet antigène en raison de restrictions génétiques. Avec l’évolution récente de l’objectif de santé mondial de l’onchocercose de passé de la lutte à l’élimination, il est donc nécessaire de mettre au point de nouveaux outils appropriés. Ces outils nécessaires incluent, entre autres, des médicaments, des diagnostics et des vaccins. Dans ce travail, des analyses bio-informatiques combinées à des tests immunologiques ont été appliquées dans le but de développer des outils potentiels pour les programmes d'élimination actuels. En ce qui concerne la chimiothérapie, l’ivermectine, qui est le seul médicament utilisé depuis plus de 30 ans pour le traitement de l’onchocercose, ne tue que les microfilaires (mf) laissant intacts les vers adultes qui continuent à produire le mf. A ceci joint, il y a le problème récent du développement de la résistance des parasites à ce médicament. En outre, un traitement récemment approuvé, la moxidectine, est contre-indiquée chez les femmes enceintes et les enfants de moins de 12 ans qui pourraient continuer à servir de réservoirs d’infection. Il est donc absolument nécessaire d’élaborer de nouvelles stratégies de traitement, de préférence pour les médicaments macrofilaricides. Pour une éradication totale de l’onchocercose, le développement du vaccin doit être complété par le diagnostic et le traitement. Le but de ce travail est donc de caractériser un antigène d'O. volvulus, Ov58GPCR et de concevoir un antigène chimérique à base d'épitope, que nous avons appelé Ov-DKR-1, dans le cadre du développement d'outils de contrôle de l'onchocercose.Concernant le premier objectif, des peptides synthétiques représentant des épitopes B linéaires et le domaine extracellulaire (DEC) recombinant d'un récepteur couplé à la protéine G (GPCR) présentant un potentiel diagnostique ont été testés pour déterminer leur réponse immunitaire en utilisant du sérum d'individus infectés par l'onchocercose et divers témoins. Les résultats obtenus indiquent que (i) l'antigène d'O. volvulus, Ov58GPCR est un récepteur couplé à la protéine-G (GPCR) conservé dans les nématodes apparentés (ii) les peptides synthétiques prédits comme localisés dans le domaine extracellulaire de Ov58GPCR sont bien des epitopes immunogéniques chez les individus infectés par l’onchocercose, (iii) les cocktails de peptides synthétiques établissent une distinction entre les individus activement infectés avec l’onchocercose, les individus non-infectés traités et les témoins africains en bonne santé, (iv) les anticorps polyclonaux contre un des peptides ou le domaine extracellulaire exprimé au bactéries réagisent spécifiquement avec l'antigène natif dans les extraits total et de surface d'O. volvulus, (v) Ov58GPCR est transcrit aux stades larvaire et adulte, (vi) les niveaux détectés d’IgG et IgE grâce à le DEC recombinant diminuent au cours du traitement par l'ivermectine. Toutes ces découvertes suggèrent que le domaine extracellulaire recombinant et les peptides synthétiques de Ov58GPCR, ainsi que les réponses immunitaires spécifiques générées, pourraient être exploités dans le contexte du diagnostic et de la surveillance de la maladie. Pour évaluer le rôle potentiel d’Ov58GPCR dans le développement de médicaments ou de vaccins cible, un examen préliminaire de l’indispensabilité du gène Ov58GPCR pour la survie du parasite a été évalué par interférence d’ARN. Une séquence d'ARN interférant court (ARNic) ciblant le gène conçu et testé par trempage avec des vers mâles d'O. volvulus a entraîné une réduction de la motilité. Les résultats ont indiqué que le gène pourrait être impliqué principalement dans la motilité. Des investigations complémentaires sont recommandées dans cette optique.Concernant le deuxième objectif, de nombreux indicateurs révèlent la possibilité de développer des outils de protection contre l’onchocercose. En conséquence, une approche immuno-informatique a été appliquée pour concevoir un candidat-vaccin des sous-unités de multi-épitopes conservées-filarienne consistant des épitopes de cellules B et T de protéines qui seraient de nouveaux candidats vaccins. La conservation des protéines sélectionnées chez d'autres espèces parasitaires de nématodes et d'épitopes prédits suggère que la protéine chimère générée (Ov-DKR-1) pourrait être vitale pour la protection croisée. La structure 3D a été prédite, raffinée et validée bioinformatiquement. La fixation protéine-protéine du candidat vaccin chimère au récepteur TLR4 prédit une liaison favorable efficace. La simulation immunitaire prédit des niveaux significativement élevés d'IgG1, de réponses T-helper, de cellules T-cytotoxiques, de INF-γ et d'IL-2. Globalement, le peptide chimère conçu a démontré une antigénicité supérieure aux candidats vaccins actuels.
Option Biologie moléculaire du Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Rabies is a neurological disease caused by viruses of the genus Lyssavirus belonging to the family Rhabdoviridae and order Mononegavirales. The Lyssavirus genus consists of eleven species namely rabies virus; Lagos bat virus; Mokola virus , Duvenhage virus; European bat lyssavirus virus type 1; European bat lyssavirus type 2; Australian bat lyssavirus; Aravan; Khujand; Irkut; West Caucasian bat viruses and recently isolated Shimoni bat virus. The prototype virus of the Lyssavirus genus is represented by rabies virus. The remaining lyssaviruses are collectively denoted as rabies-related viruses. It has been proposed that these lyssavirus genotypes/species could be divided into 2 and possibly 3 phylogroups, based on diversity and shared biological/pathogenicity properties. Currently available vaccines have proven efficacy against viruses that would make up phylogroup I, but fail to cross-protect against those lyssaviruses belonging to phylogroup II and III. The potential public health burden associated with mortality due to rabies-related virus infection has prompted several vaccine studies to focus on the pan-lyssavirus vaccine cross-protection. The studies demonstrated that lyssaviruses glycoprotein domains (antigenic sites) can be exchanged to generate chimeric vaccine constructs and that combining antigenic sites II and III of different lyssaviruses raised virus neutralizing antibodies against different lyssaviruses. The objective of our study was to determine the cross protective capacity of similar constructs in a vaccinia virus Copenhagen strain based recombinant in proof of concept to ascertain cross protection in a murine model. The current study substantiates findings of preceding studies that chimeric lyssavirus glycoprotein vaccine constructs conferred protection against homologous and heterologous lyssaviruses. The replicating vaccinia virus based vector demonstrated the benefit of a replicating recombinant vaccine vector as illustrated by the high protective neutralizing antibody titers obtained. The value of administering a booster dose was also highlighted in the higher antibody titers obtained upon a vaccine boost. The study clearly demonstrated that glycoprotein antigenic site II and III are not equal in inducing protection and that site II confers better protection against a homologous virus.
Dissertation (MSc)--University of Pretoria, 2011.
Microbiology and Plant Pathology
Unrestricted

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: The ostrich industry in South Africa is currently threatened by respiratory disease in feedlot ostriches which causes a dramatic loss in production. Ms01, Ms02 and Ms03 were identified as the three ostrichspecific mycoplasmas to be associated with this respiratory disease in ostriches of South Africa. The ostrich-specific mycoplasmas have a major impact on ostrich production and for this reason there is a serious need for treatment for these infections. For this reason, the ostrich industry has undertaken an investigation into the development of vaccines against mycoplasma infections. In this study, an approach to DNA vaccine development will be investigated and applied, specifically for the ostrich mycoplasma Ms02. Firstly, the whole genome of Ms02 was sequenced using GS FLX sequencing technology. The contiguous sequences obtained from the whole-genome sequencing were analysed bioinformatically which included the annotation of the contiguous sequences and the subsequent search for a vaccine candidate gene for the development of a DNA vaccine. The P100 gene of Ms02, which showed a high degree of homology with the P100 gene of the human pathogen M. hominis, was chosen as a vaccine candidate gene for the development of a DNA vaccine. The P100 gene was successfully cloned and subsequently modified by means of site-directed mutagenesis to correct for alternative codon usage, where after the modified P100 gene was subcloned into the mammalian expression vector, pCI-neo for vaccination trials in the near future.
AFRIKAANSE OPSOMMING: Die volstruisbedryf van Suid-Afrika is tans bedreig deur 'n respiratoriese siekte in voerkraal volstruise wat lei tot aansienlike verliese in volstruisproduksie. Ms01, Ms02 en Ms03 is geïdentifiseer as die drie volstruis-spesifieke mikoplasmas wat 'n rol speel in hierdie respiratoriese siektes van volstruise in Suid- Afrika. Die drie volstruis-spesifieke mikoplasmas het 'n groot impak op die produksie van volstruise en om hierdie rede is daar 'n ernstige behoefte aan 'n behandeling van hierdie infeksies. Ten einde mikoplasma infeksies in volstruise te voorkom, het die Suid-Afrikaanse volstruisbedryf 'n ondersoek geloods na moontlike strategieë vir entstof ontwikkeling. In hierdie studie, is 'n benadering van DNA entstof ontwikkeling ondersoek en toegepas, spesifiek teen die volstruis mikoplasma Ms02. Eerstens, is die volledige Ms02 genoomvolgorde bepaal deur gebruik te maak van GS FLX volgordebepalingstegnologie. Die gedeeltelike volgordes verkry vanaf die heelgenoom volgordebepaling is bioinformaties geanaliseer wat die annotering van die gedeeltelike volgordes asook die soektog vir 'n kandidaat entstof geen vir die ontwikkeling van 'n DNA entstof ingesluit het. Die P100 geen van Ms02, wat hoë homologie met die P100 geen van die menslike patogeen M. hominis getoon het, is gekies as die kandidaat entstof geen. Die P100 geen is suksesvol gekloneer en gemodifiseer deur middel van setelgerigte mutagenese om die P100 geen geskik te maak vir die invoeging in die soogdier ekspressie vektor, pCI-neo vir toekomstige entstofproewe.

Vaccines represent one of the most cost-effective ways to prevent and treat diseases. The use of vaccines in the control of viral diseases represents an important milestone in the history of medicine. The genomic revolution brought us the possibility to scan genomes in the search of new and more effective vaccine candidates and the advancement of bioinformatics provided the framework for the application of strategies that were focused not only on antigen discovery but also on comparative genomics, and pathogenic factor identification and data mining. In addition, the progress in post-genomic technologies including gene expression technologies such as microarray and proteomics gave us the opportunity to explore the host responses to vaccines leading to a better understanding of immune responses to pathogens and/or to vaccines, assisting in the development of new and better vaccines and adjuvants. This chapter will review how systems biology-based approaches including genomics, gene expression technologies, and bioinformatics have changed the way of thinking about antigen discovery and vaccine development. In addition, the chapter will discuss how the study of the host responses in combination with “in silico” approaches could help predict immunogenicity and improve the efficacy of vaccines.

Vaccines represent one of the most cost-effective ways to prevent and treat diseases. The use of vaccines in the control of viral diseases represents an important milestone in the history of medicine. The genomic revolution brought us the possibility to scan genomes in the search of new and more effective vaccine candidates and the advancement of bioinformatics provided the framework for the application of strategies that were focused not only on antigen discovery but also on comparative genomics, and pathogenic factor identification and data mining. In addition, the progress in post-genomic technologies including gene expression technologies such as microarray and proteomics gave us the opportunity to explore the host responses to vaccines leading to a better understanding of immune responses to pathogens and/or to vaccines, assisting in the development of new and better vaccines and adjuvants. This chapter will review how systems biology-based approaches including genomics, gene expression technologies, and bioinformatics have changed the way of thinking about antigen discovery and vaccine development. In addition, the chapter will discuss how the study of the host responses in combination with “in silico” approaches could help predict immunogenicity and improve the efficacy of vaccines.

Fascioliasis is a food-borne neglected disease caused by digenetic trematodes in the genus Fasciola. There is a significant increase in the global prevalence of human fascioliasis with a strong correlation with a high infection rate among ruminant definitive hosts. Fasciola is a liver fluke with complex life cycle. Fascioliasis is endemic in every continent of the world with the exception of Antarctica. Discharge of the metabolites of liver flukes into the circulatory system of hosts has pathological consequences. Fascioliasis has been diagnosed by parasitological, immunological, and molecular means, and it is being reliably treated chemotherapeutically. The emerging drug-resistant strains of liver flukes have led to the need for vaccine development. Most vaccine candidates were first isolated as native proteins from adult worms. Several of the early antigens, including cathepsin L proteases, Glutathione S-transferase (GST), and fatty acid binding protein (FABP), significantly reduced worm burden, egg output, and liver pathology in cattle and sheep. Climate change, emerging drug resistance, and the development of new parasite strains through hybridization are the current challenges that could potentially alter the epidemiology of fascioliasis soon. Therefore, researchers need to produce promising vaccines that offer maximum protection to farm animals and humans.

Herpesviruses are large, spherical, enveloped viral particles with linear double-stranded DNA genome. Herpesvirus virion consists of an icosahedral capsid containing viral DNA, surrounded by a protein layer called tegument, and enclosed by an envelope consisting of a lipid bilayer with various glycoproteins. Herpesviruses persist lifelong in their hosts after primary infection by establishing a latent infection interrupted recurrently by reactivations. The Herpesviridae family is divided into three subfamilies; α-herpesviruses, β-herpesviruses, and γ-herpesviruses based on the genome organization, sequence homology, and biological properties. There are eight human herpes viruses: Herpes simplex virus type 1 and 2 (HSV-1, −2) andVaricella-zoster virus (VZV), which belong to the α-herpesvirus subfamily; Human cytomegalovirus (HCMV), and Human herpesvirus type 6 and 7 (HHV-6,HHV-7), which belong to the β-herpesvirus subfamily; and Epstein–Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV) or Human herpesvirus 8 (HHV-8), which belong to the γ-herpesvirus subfamily. Within this chapter, we summarize the current knowledge about EBV and CMV, regarding their genome organization, structural characteristics, mehanisms of latency, types of infections, mechanisms of immune escape and prevention. Epstein–Barr Virus (EBV) genome encodes over 100 proteins, of which only (30) proteins are well characterized, including the proteins expressed during latent infection and lytic cycle proteins. Based on major variation in the EBNA-2 gene sequence, two types of EBV are recognized, EBV type 1 and 2. Epstein–Barr virus types occur worldwide and differ in their geographic distribution depending on the type of virus. EBV spreads most commonly through bodily fluids, especially saliva. However, EBV can also spread through blood, blood transfusions, and organ transplantations. The EBV is associated with many malignant diseases such as lymphomas, carcinomas, and also more benign such as infectious mononucleosis, chronic active infection. The EBV has also been suggested as a trigger/cofactor for some autoimmune diseases. Overall, 1–1.5% of the cancer burden worldwide is estimated to be attributable to EBV The latently infected human cancer cells express the most powerful monogenic proteins, LMP-1 and LMP-2(Latent Membrane Protein-1,-2), as well as Epstein–Barr Nuclear Antigens (EBNA) and two small RNAs called Epstein–Barr Encoded Small RNAs (EBERs). The EBV can evade the immune system by its gene products that interfering with both innate and adaptive immunity, these include EBV-encoded proteins as well as small noncoding RNAs with immune-evasive properties. Currently no vaccine is available, although there are few candidates under evaluation. Human cytomegalovirus (HCMV) is a ubiquitous beta herpesvirus type 5 with seroprevalence ranges between 60 to 100% in developing countries. CMV is spread from one person to another, usually by direct and prolonged contact with bodily fluids, mainly saliva, but it can be transmitted by genital secretions, blood transfusion and organ transplantation. In addition, CMV can be transmitted vertically from mother to child. CMV infection can result in severe disease for babies, people who receive solid organ transplants or bone marrow/stem cell transplants and people with severe immune suppression such as advanced human immunodeficiency virus (HIV) infection. The HCMV has several mechanisms of immune system evasion. It interferes with the initiation of adaptive immune responses, as well as prevent CD8+ and CD4+ T cell recognition interfering with the normal cellular MHC Class I and MHC Class II processing and presentation pathways. Challenges in developing a vaccine include adeptness of CMV in evading the immune system. Though several vaccine candidates are under investigation.