Syftet med denna valideringsstudie är att värdera lämpligheten att överföra den manuella analysen av aktuell infektion av Varicella Zoster Virus (aVZV IgM) och Herpes Simplex Virus (aHSV IgM) med SIEMENS Enzygnost® till en av de automatiserade analysinstrumenten EUROIMMUN Analyzer I (ELISA) eller DiaSorin LIAISON® XL. Arbetet utfördes på Klinisk Mikrobiologi i Lund. Konsekutiva serumprover för VZV IgM (n=108) och för HSV IgM (n=116) från det vardagliga flödet analyserades, tillsammans med 10 PCR- eller serokonversion-konfirmerade positiva serumprover av primär infektion VZV och HSV samt 10 positiva för reaktiverad infektion av VZV och HSV. Utöver det användes 10 serumprover konfirmerade positiva för Cytomegalovirus (CMV) respektive 10 för Epstein-Barr Virus (EBV) för att testa korsreaktionen metoderna emellan. Resultatet från VZV-valideringen i Analyzer I samt LIAISON® XL gav en överensstämmelse på 93% respektive 94% av de konsekutiva proverna, 71% respektive 86% av de primärinfekterade proverna och 75% respektive 58% av de reaktiverade proverna, samt en korsreaktivitet (positiva och gränsvärden) på totalt 33% respektive 20% av proverna. Resultatet från HSV-valideringen i Analyzer I samt LIAISON® XL gav en överensstämmelse på 84% respektive 87% av de konsekutiva proverna, 82% respektive 18% av de primärinfekterade proverna och 40% respektive 10% av de reaktiverade proverna, samt en korsreaktivitet (positiva och gränsvärden) på totalt 67% respektive 47% av proverna. Enligt rekommendation efter utförandet av denna studie så bör analysen av HSV IgM uteslutas från båda automatiserade metoder medan VZV IgM bör kontrolleras något ytterligare i Analyzer I, med förhoppning om att denna metod kan vara känsligare. / The approach of this validation study is to evaluate the adequacy for transferring the manual analysis method of ongoing infection of Varicella Zoster Virus (aVZV IgM) and Herpes Simplex Virus (aHSV IgM) with SIEMENS Enzygnost® to one of the automated instruments EUROIMMUN Analyzer I (ELISA) or DiaSorin LIAISON® XL. The study was carried out at Clinical Microbiology in Lund. Consecutive serum samples for VZV IgM (n=108) and HSV IgM (n=116) from the daily local flow of tests were analyzed, along with 10 positive for primary infection of VZV and HSV, confirmed by PCR or seroconversion, and 10 with reactivated infection of VZV and HSV. Beyond those, 10 serum samples confirmed positive for Cytomegalovirus (CMV) respectively 10 for Epstein-Barr Virus (EBV) to test the cross-reaction between the three methods. The results from the validation of VZV in Analyzer I and LIAISON® XL gave an agreement of 93% and 94% respectively in the consecutive tests, 71% and 86% respectively in the primary infected tests and 75% and 58% respectively in the reactivated tests, and also a cross-reactivity (both positive and in between-values) at a total of 33% respectively 20% of the tests. The results from the validation of HSV in Analyzer I and LIAISON® XL gave an agreement of 84% and 87% respectively in the consecutive tests, 82% and 18% respectively in the primary infected tests and 40% and 10% respectively in the reactivated tests, and also a cross-reactivity (both positive and in between-values) at a total of 67% respectively 47% of the tests. According recommendations after the performance of this study, the analysis of HSV IgM should be excluded from both of the automated methods while VZV IgM should be controlled further in Analyzer I, with hopes that this new method could be more sensitive.

Interaction of Herpes Simplex Virus (HSV) glycoprotein D (gD) with the host cell surface during Chlamydia trachomatis/HSV co-infection stimulates chlamydiae to become persistent. During viral entry, gD interacts with one of 4 host co-receptors: HVEM (herpes virus entry mediator), nectin-1, nectin-2 and 3-O-sulfated heparan sulfate. HVEM and nectin-1 are high-affinity entry receptors for both HSV-1 and HSV-2. Nectin-2 mediates HSV-2 entry but is inactive for HSV-1, while 3-O-sulfated heparan sulfate facilitates HSV-1, but not HSV-2, entry. Western blot and RT-PCR analyses demonstrate that HeLa and HEC-1B cells express nectin-1 and nectin-2, but not HVEM. Because both HSV-1 and HSV-2 trigger persistence, these data suggest that nectin-1 is the most likely co-receptor involved. Co-infections with nectin-1 specific HSV-1 mutants stimulate chlamydial persistence, as evidenced by aberrant body (AB) formation and decreased production of elementary bodies (EBs). These data indicate that nectin-1 is involved in viral-induced chlamydial persistence. However, inhibition of signal transduction molecules associated with HSV attachment and entry does not rescue EB production during C. trachomatis/HSV-2 co-infection. HSV attachment also does not activate Cdc42 in HeLa cells, as would be expected with viral stimulated activation of nectin-1 signaling. Additionally, immunofluorescence assays confirm that HSV infection decreases nectin-1 expression. Together, these observations suggest that gD binding-induced loss of nectin-1 signaling negatively influences chlamydial growth. Chlamydial infection studies in nectin-1 knockdown (NKD) HeLa cell lines support this hypothesis. In NKD cells, chlamydial inclusions are smaller in size, contain ABs, and produce significantly fewer infectious EBs compared to C. trachomatis infection in control HeLa cells. Overall, the current study indicates that the actions of host molecule, nectin-1, are required for successful C. trachomatis development.

The recent discovery that T cells recognize different sets of herpes simplex virus type 1 and type 2 epitopes from seropositive symptomatic and asymptomatic individuals might lead to a fundamental immunologic advance in vaccine development against herpes infection and diseases. The newly introduced needle-free mucosal (i.e., topical ocular and intravaginal) lipopeptide vaccines provide a novel strategy that might target ocular and genital herpes and possibly provide 'heterologous protection' from HIV-1. Indeed, mucosal self-adjuvanting lipopeptide vaccines are easy to manufacture, simple to characterize, extremely pure, cost-effective, highly immunogenic and safe. In this review, we bring together recent published and unpublished data that illuminates the status of epitope-based herpes vaccine development and present an overview of our recent approach to an 'asymptomatic epitope'-based lipopeptide vaccine.

Gene therapy is a fast growing technology that offers treatments for genetic diseases. The method is based on introducing genetic material into a patient to replace the disease-causing gene, using a vector. This report examines the potential of some viral vectors for gene therapy, to give Bio-Works Technologies a recommendation on what the future market demands. Oncolytic viruses, vaccines and gene editing are not treated in the report as a delimitation.  Viral vectors have different biological properties and require different purification methods, making them suitable for different applications in gene therapy. In the purification of the viruses it can be challenging to obtain a high purity and large-scale manufacturing. One major drawback with most purification methods is that they are not specific to just one virus, which leads to contaminants in the solution and lower purity. The viral vectors handled in the report are the adenovirus, adeno-associated virus, gammaretrovirus, lentivirus, alpharetrovirus, foamy virus, herpes simplex virus and baculovirus. These were chosen as they are relevant vectors for gene therapy and stay within the scope of the report. Lentiviral vectors (LVs) and adeno-associated viral vectors (AAVs) will dominate the gene therapy field in the coming years. This is based on the information that the use of AAVs and LVs in clinical trials have increased in recent years, while the other vectors mentioned above have slightly decreased or show no apparent change. However, challenges still remain in the purification processes. Ligands used in affinity chromatography for purification of AAVs are effective at removing most contaminants, but cannot distinguish between empty and loaded capsids, which can induce immune response when used clinically. This is the main challenge when purifying AAVs. The empty capsids can be removed with ion exchange chromatography, which results in higher purity but also lower recovery. There is no specific purifying method for LVs, therefore a lentivirus-specific affinity ligand, such as an antibody ligand, would be beneficial for the purification and manufacturing procedure.  In addition to AAVs and LVs, baculoviral vectors and foamy viral vectors show great potential in a long-term perspective but they only have been researched in preclinical studies. Moreover, herpes simplex viral vectors and adenoviral vectors show potential in cancer treatments or as vaccines rather than in augmentation gene therapy.

The obligate intracellular bacterium Chlamydia trachomatis is the most common bacterial STD agent in the US. This bacterium has a unique biphasic developmental cycle in which the infectious elementary body (EB) infects a host mucosal epithelial cell and differentiates into the replicative form (the reticulate body or RB) within a modified vacuole called an inclusion. The RB later divides and develops back into an EB and is released, perpetuating the infectious cycle. When developing chlamydiae are exposed to unfavorable environmental conditions, they deviate from the normal developmental cycle into a non-infectious but viable state termed persistence. Previous data from our laboratory indicate that i) during C. trachomatis/HSV co-infection, the chlamydiae become persistent and ii) HSV gD interaction with host cell surface is sufficient to induce this response. During viral entry, HSV gD interacts with one of four host co-receptors, one of which is the host adhesion molecule nectin-1. Interestingly, Western blotting demonstrated that nectin-1 is significantly decreased in C. trachomatis-infected HeLa cells. Additional studies indicated that active C. trachomatis replication is required for nectin-1 down-regulation and nectin-1 is likely down-regulated post-translationally. CPAF, a chlamydia-secreted protease, is responsible for degrading several host proteins. Both in vivo experiments using CPAF-specific chemical inhibitors and cell-free cleavage assays using recombinant CPAF indicate that nectin-1 is degraded by CPAF in C. trachomatis-infected cells. Further studies suggest that nectin-1 is the most likely candidate involved in triggering HSV-induced chlamydial persistence. Co-infection experiments using nectin-1-specific HSV-1 mutants suggest that nectin-1 is, indeed, required for persistence induction. Additional studies in single co-receptor-expressing CHO cells demonstrate that, despite the fact that HSV-1 enters both HVEM- and nectin-1-expressing cells, viral co-infection reduces chlamydial infectivity only in the CHO-nectin-1 cell line. These data confirm that HSV/nectin-1 interaction is sufficient for chlamydial persistence induction. Although nectin-1 ligation is known to activate Cdc42, pull-down assays indicate that Cdc42 is not activated in co-infected HeLa cells. Taken together, these data suggest that: i) HSV gD-nectin-1 binding activates a novel host epithelial cell pathway that restricts chlamydial development and ii) the chlamydiae may degrade nectin-1 to evade this inhibitory host response.

Previous studies have established that ozone-oxygen mixtures can be used to inactivate a variety of microorganisms including bacteria, fungi and viruses. Ozone is a potent reactive oxygen species (ROS) that rapidly decays into a variety of additional short half-life ROS which have been shown to cause oxidative damage to biological molecules. I hypothesize that controlled ozone exposure and the subsequent generation of additional ROS would reduce viral infectivity by lipid and/or protein peroxidation. A proprietary ozone-oxygen delivery system was used to inactivate a series of enveloped [herpes simplex virus type-1 strain McIntyre (HSV-1), vaccinia strain Elstree (VAC), vesicular stomatitis virus strain Indiana (VSV), and influenza A strain (H1N1) A/WS/33] and non-enveloped [human adenovirus type2 (Ad2)] viruses. Plaque reduction and suspension-infection viral antigen assays were used to determine inactivation kinetics. After ozonation, HSV-1 and VSV lost up to 6 log10 infectious particles in 15 min, while VAC and influenza A lost up to 5 log10 in 40 min and 30 min, respectively. In comparison, the non-enveloped Ad2 lost up to 5 log10 in 60 min. Increasing amounts of serum supplementation in the ozone treated virus suspensions slowed the rate of inactivation in both enveloped and non-enveloped viruses, suggesting the protective effect of serum against ozone. Lipid peroxidation was determined through a chromogenic assay for malondialdehyde (MDA), a byproduct of peroxidation events. MDA concentrations were inversely correlated with virus infectivity, as MDA concentrations elevated with virus exposure time to ozone. Transmission electron microscopy images of Ad2, HSV-1, VAC and VSV confirmed the drastic morphological changes that resulted from ozone treatment. The ROS-mediated attack compromised the integrity of the lipid envelopes and protein shells of the viruses. These data suggest that a wide range of viruses can be inactivated through use of an innovative ozone delivery system, thus validating my hypothesis.

Experiments were carried out to determine if pretreatment of cells with N-Methyl-N'-Nitro-N-Nitrosoguanidine (a potent alkylating agent known to induce DNA repair) would affect Herpes Simplex Virus Replication. The data demonstrated a 1.5 fold increase in virus yield, a 2 fold increase in HSV specific TK activity and no change in HSV specific DNA polymerase activity in MNNG treated cultures. The effects of MNNG treatment on virus replication and enzyme expression are discussed. / Thesis / Master of Science (MSc)

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