Intrinsic Control Strategies for Herpesvirus-based Vaccine Vectors

Herpesvirus-based vectors have been used in vaccine strategies to target zoonotic diseases such as Ebola and human monkeypox, through eliciting a highly immunogenic response. We are investigating strategies to attenuate the virus and control its ability to replicate and persist, while maintaining its ability to elicit protective immune responses. In this project we have investigated three distinct and innovative means to provide intrinsic control of herpesvirus-based vectors. The first strategy is based on deletion of a gene essential for viral latency to modify persistence in vivo. Studies by Thirion et al have shown that the bovine herpesvirus 4 (BoHV-4) ORF73 gene product is required for persistence through enabling episome tethering to mitotic chromosomes. The first aim of the project was to investigate the ability of a latency-defective BoHV-4 Δ ORF73 vector expressing heterologous antigens to induce immune responses. Four recombinant defective BoHV-4 Δ ORF73 vectors, each expressing different heterologous target antigens, were constructed. One of these constructs, which expressed a target antigen from Mycobacterium bovis, was tested in vivo. The results showed the attenuated version of BoHV-4 Δ ORF73 was able to induce an immune response against its heterologous antigen. The second attenuation strategy was based on modulation of viral replication fidelity through mutation of the viral DNA polymerase. Based on a substantial body of published work from other laboratories studying the impact of genome replication fidelity on RNA viruses, we hypothesized that the genetic reduction in the fidelity of human cytomegalovirus (HCMV) replication would introduce an unsustainable level of mutations during replication of the virus, and thereby eventually result in 'genomic catastrophe' and demise of the virus. The aim of this second part of the thesis was to characterize a HCMV that has been modified through deletion of an aspartic acid residue 413 of domain II of exonuclease region in CMV DNA polymerase, designated HCMVUL54(ΔD413). Based on the proposed function of this domain in ‘proof-reading’ during virus DNA replication, it was hypothesized that this mutation would result in a decrease in the fidelity of the virus, leading to the accumulation of deleterious mutations and loss of fitness. Data presented in the thesis showed that HCMVUL54(ΔD413) had an accumulation of mutations consistent with an increased mutation rate and lower fidelity. The final attenuation strategy was based on engineering viruses in which the transgenes are flanked by identical regions. It was hypothesized that the presence of these flanking duplications will target intervening regions for genetic deletion. In contrast to the strategy based on fidelity and genomic catastrophic demise of the virus, this strategy aims to restore to recombinant virus to that of the wild type (WT) virus by facilitating excision of the heterologous transgene. Rather than intrinsic control of the CMV virus itself, this strategy is based on providing kinetic control over stability of the heterologous transgene within the recombinant virus. We hypothesized that the presence of a transgene between two homologues flanking regions would lead to its excision from the recombinant virus and restoration of the WT virus. We further hypothesized that the rate of loss of the heterologous transgene would be regulated by the length of the homologous flanking region.Iraqi ministry of higher education and scientific researc

The possible role of high risk HPV infection in Iraqi patients with colorectal cancer

Aims: the aim of this study is RPD3 expression in clinicopathological characteristics in CRC patients and to study the existence of a relationship between CRC and HPV in samples of Iraqi patients. Material and methods: 30 samples of formalin-fixed, paraffin-embedded archival tissues from CRC patients were obtained, together with 10 samples of normal tissue (free cancer), for immunohistochemical analysis of RPD3 and HPV expression. Results: Immunohistochemical (IHC) staining revealed only four cases (15%) with HPV infections while PRD3 was expressed in 19 CRC samples (63.3 percent). The PRD3 expression ratio. It was found in only four (15%) of the CRC samples. At ≤ P 0. 05, there is no significant difference. Conclusion: The results of this study support RPD3 as a supplementary marker to identified histologic screening components; it could possibly be used for the prognosis and targeted therapy. In addition, we discovered a relation with both HPV infection and CRC as oncovirus and tumorigenic factor

Potential oncogenic role of human cytomegalovirus in colorectal cancer

Aims: This study was conducted to investigate whether there is a link between HDAC3 expression and clinical characteristics in colorectal cancer (CRC) patients and to detect the oncopotenial role of HCMV in colorectal cancer (CRC). Material and methods: 30 samples of formalin fixed-Paraffin embedded archival tissues were collected from patient suffering of CRC , In addition to 10 samples of free cancer tissue for determine of expression of HDAC3 and CMV by using immunohistochemistry. Results: HDAC3 expression by Immunohisto-chemical (IHC) staining was found in 19 samples (63. 3%) of CRC. While There is no expression of CMV in all samples of CRC and control groups. Conclusion: This study proved HDAC3 expression pattern in CRC, and the findings support HDAC3 as a supplementary marker to known histopathological diagnostic components; it could potentially be used in prognosis and targeted therapy. We also found that there is no correlation of the CMV infection and CRC as a pro-tumorigenic factor