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Display of Domain III from Dengue 2 Envelope Protein on HBsAg Virus-like Particles Vectored by Measles Virus

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Dengue virus infects millions of people every year. Yet there is still no vaccine available to prevent it. Here we use a neutralizing epitope determinant on the dengue envelope (E) protein as an immunogen to be vectored by a measles

Dengue virus infects millions of people every year. Yet there is still no vaccine available to prevent it. Here we use a neutralizing epitope determinant on the dengue envelope (E) protein as an immunogen to be vectored by a measles virus (MV) vaccine. However the domain III (DIII) of the dengue 2 E protein is too small to be immunogenic by itself. In order for it to be displayed on a larger particle, it was inserted into the amino terminus of small hepatitis B surface antigen (HBsAg, S) coding sequence. To generate the recombinant MV vector and verify the efficiency of this concept, a reverse genetics system was used where the MV vectors express one or two additional transcription units to direct the assembly of hybrid HBsAg particles. Two types of recombinant measles virus were produced: pB(+)MVvac2(DIII-S,S)P and pB(+)MVvac2(DIII-S)N. Virus recovered from pB(+)MVvac2(DIII-S,S)P was viable. An ELISA assay was performed to demonstrate the expression and secretion of HBsAg. Supernatant from MVvac2(DIII-S,S)P infected cells confirmed that hybrid HBsAg-domain III particles with a density similar to traditional HBsAg particles were released. Characteristics of the subviral particle have been analyzed for the successful incorporation of domain III. The replication fitness of the recombinant MV was evaluated using multi-step growth kinetics and showed reduced replication fitness when compared to the parental strain MVvac2. This demonstrates that viral replication is hindered by the addition of the two inserts into MV genome. Further analysis of MVvac2(DIII-S)N is needed to justify immune response studies in a small animal model using both of the generated recombinant vectors.

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Date Created
2014-05

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Investigate and characterize the means to reduce the toxicity of genetically engineered cancer therapeutic recombinant attenuated Salmonella

Description

Cancer is one of the leading causes of death in the world and represents a tremendous burden on patients, families and societies. S. Typhimurium strains are specifically attracted to compounds produced by cancer cells and could overcome the traditional therapeutic

Cancer is one of the leading causes of death in the world and represents a tremendous burden on patients, families and societies. S. Typhimurium strains are specifically attracted to compounds produced by cancer cells and could overcome the traditional therapeutic barrier. However, a major problem with using live attenuated Salmonella as anti-cancer agents is their toxicity at the dose required for therapeutic efficacy, but reducing the dose results in diminished efficacy. In this project, we explored novel means to reduce the toxicity of the recombinant attenuated Salmonella by genetically engineering those virulence factors to facilitate maximal colonization of tumor tissues and reduced fitness in normal tissues. We have constructed two sets of Salmonella strains. In the first set, each targeted gene was knocked out by deletion of the gene. In the second set, the predicted promoter region of each gene was replaced with a rhamnose-regulated promoter, which will cease the synthesis of these genes in vivo, a rhamnose-free environment.

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Date Created
2013-05

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Immunogenic subviral particles displaying domain III of dengue 2 envelope protein vectored by measles virus

Description

Vaccines against the arthropod-borne dengue virus (DENV) are still commercially nonexistent. A subunit immunization strategy may be of value, especially if a safe viral vector acts as a biologically active adjuvant. The DENV envelope protein (E), the main target for

Vaccines against the arthropod-borne dengue virus (DENV) are still commercially nonexistent. A subunit immunization strategy may be of value, especially if a safe viral vector acts as a biologically active adjuvant. The DENV envelope protein (E), the main target for neutralizing immune responses, has three conformational domains. The immunoglobulin-like and independently folding domain III (DIII) contains epitopes that elicit highly specific neutralizing antibodies. The hepatitis B small surface antigen (HBsAg, S) was used as a scaffold to display DENV 2 DIII on a virus-like particle (VLP). A measles virus (MV) was engineered to vector HBsAg and the hybrid glycoprotein DIII-HBsAg in two different loci (DIII-S). Despite the relatively deleterious effect on replication caused by the insertion of two transcription cassettes, the recombinant virus MVvac2(DIII-S,S)P induced the secretion of DIII-S hybrid VLP with a similar sucrose density as HBsAg particles (1.10-1.12g/ml) and peaked at 48 h post-infection producing 1.3x106 TCID50/ml infectious MV units in vitro. A second recombinant virus, MVvac2(DIII-S)N, was engineered to vector only the hybrid DIII-S. However, it did not induce the secretion of hybrid HBsAg particles in the supernatant of infected cells. The immunogenicity of the recombinant viruses was tested in a MV-susceptible small animal model, the experimental group which received two 105 TCID50 I.P. doses of MVvac2(DIII-S,S)P in a 28 day interval developed a robust immune response against MV (1:1280), HBsAg (787 mIU/ml) and DENV2 (Log10 neutralization index of 1.2) on average. In summary, it is possible to display DENV E DIII on hybrid HBsAg particles vectored by MV that elicit an immune response. This forms the basis for a potential vaccine platform against DENV.

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Created

Date Created
2015

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Vaccination strategy to protect against flavivirus infection based on recombinant measles vaccine

Description

Despite the approval of a Dengue virus (DV) vaccine in five endemic countries, dengue prevention would benefit from an immunization strategy highly immunogenic in young infants and not curtailed by viral interference. Problematically, infants younger than 9 year of age,

Despite the approval of a Dengue virus (DV) vaccine in five endemic countries, dengue prevention would benefit from an immunization strategy highly immunogenic in young infants and not curtailed by viral interference. Problematically, infants younger than 9 year of age, whom are particularly prone to Dengue severe infection and death, cannot be immunized using current approved DV vaccine. The most important issues documented so far are the lack of efficiency and enhancement of the disease in young seronegative recipients, as well as uneven protection against the four DV serotypes. Based on data from clinical trials that showed enhanced performance of dengue vaccines when the host has previous anti-flaviviral immunity, I proposed here an attractive solution to complement the current vaccine: a recombinant measles vaccine vectoring dengue protective antigens to be administered to young infants. I hypothesized that recombinant measles virus expressing Dengue 2 and 4 antigens would successfully induce neutralizing responses against DV2 and 4 and the vaccine cocktail of this recombinant measles can prime anti-flaviviral neutralizing immunity. For this dissertation, I generated and performed preclinical immune assessment for four novel Measles-Dengue (MV-DV) vaccine candidates. I generated four MVs expressing the pre membrane (prM) and full length or truncated (90%) forms of the major envelope (E) from DV2 and DV4. Two virus, MVvac2-DV2(prME)N and MVvac2-DV4(prME), expressed high levels of membrane associated full-length E, while the other two viruses, MVvac2-DV2(prMEsol)N and MVvac2-DV4(prMEsol)N, expressed and secreted truncated, soluble E protein to its extracellular environment. The last two vectored vaccines proved superior anti-dengue neutralizing responses comparing to its corresponding full length vectors. Remarkably, when MVvac2-DV2/4(prMEsol)N recombinant vaccines were combined, the vaccine cocktail was able to prime cross-neutralizing responses against DV 1 and the relatively distant 17D yellow fever virus attenuated strain. Thus, I identify a promising DV vaccination strategy, MVvac2-DV2/4(prMEsol)N, which can prime broad neutralizing immune responses by using only two of the four available DV serotypes. The current MV immunization scheme can be advantageus to prime broad anti-flaviviral neutralizing immunity status, which will be majorly boosted by subsequent chimeric Dengue vaccine approaches.

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Date Created
2016