Alberto van Olphen

Immunization with DNA, adenovirus or both in biodegradable alginate microspheres: effect of route of inoculation on immune response

To determine the potential for biodegradable alginate microspheres to be used as a delivery vehicle for DNA based vaccines, we constructed a plasmid, pMNe-gal-SV40, containing the bacterial beta-galactosidase (LacZ) gene under the control of the murine cytomegalovirus (MCMV) immediate-early promoter and the simian virus 40 (SV40) polyadenylation signal. The effect of the route of administration and co-administration of adenovirus on systemic and mucosal immune responses were investigated. Mice were inoculated orally, intranasally (i.n.), intramuscularly (i.m.), subcutaneously (s.c.) or intraperitoneally (i.p.) on days 0, 14 and 28 with microspheres containing plasmid DNA, bovine adenovirus type 3 (BAd3) or plasmid DNA + BAd3. Systemic routes of immunization (i.m., s.c. and i.p.) resulted in higher LacZ- or BAd3-specific IgG ELISA titers compared to those obtained by mucosal routes of inoculation (oral and i.n.). Mucosal immunization led to slightly higher titers of LacZ- or BAd3-specific IgA at mucosal sites compared to those obtained by the various systemic routes. All the routes of immunization induced LacZ-specific lymphoproliferation. Co-administration of BAd3 enhanced the LacZ-specific IgG response irrespective of the route of administration.

Epigenetic Tailoring for the Production of Anti-Infective Cytosporones from the Marine Fungus Leucostoma persoonii

Recent genomic studies have demonstrated that fungi can possess gene clusters encoding for the production of previously unobserved secondary metabolites. Activation of these attenuated or silenced genes to obtain either improved titers of known compounds or new ones altogether has been a subject of considerable interest. In our efforts to discover new chemotypes that are effective against infectious diseases, including malaria and methicillin-resistant Staphylococcus aureus (MRSA), we have isolated a strain of marine fungus, Leucostoma persoonii, that produces bioactive cytosporones. Epigenetic modifiers employed to activate secondary metabolite genes resulted in enhanced production of known cytosporones B (1, 360%), C (2, 580%) and E (3, 890%), as well as the production of the previously undescribed cytosporone R (4). Cytosporone E was the most bioactive, displaying an IC90 of 13 µM toward Plasmodium falciparum, with A549 cytotoxicity IC90 of 437 µM, representing a 90% inhibition therapeutic index (TI90 = IC90 A459/IC90 P. falciparum) of 33. In addition, cytosporone E was active against MRSA with a minimal inhibitory concentration (MIC) of 72 µM and inhibition of MRSA biofilm at roughly half that value (minimum biofilm eradication counts, MBEC90, was found to be 39 µM).

Development and characterization of bovine x human hybrid cell lines that efficiently support the replication of both wild-type bovine and human adenoviruses and those with E1 deleted.

ABSTRACT The 293 cell line that was generated by transforming human embryonic kidney cells with human adenovirus type 5 (HAV5) early region 1 (E1) sequences is an excellent host for generating and growing HAV5 recombinants with E1 deleted, but it does not support the replication of bovine adenovirus type 3 (BAV3). Madin-Darby bovine kidney (MDBK), an established bovine cell line, is an excellent host for growing and plaquing BAV3. For the purpose of combining the unique characteristics of these two cell lines (293 and MDBK), we generated a number of bovine × human hybrid (BHH) cell lines. Comparison of three BHH hybrid clones—BHH3, BHH8, and BHH2C—with 293-Puro (puromycin-resistant 293 cells) and MDBK-Neo (G418-resistant MDBK cells) cell lines for total cellular DNA content, species-specific surface markers, isoenzyme analysis, and karyotyping indicate that they are hybrid in nature. BHH clones constitutively expressed the E1 proteins (E1A, E1B-21kDa, and E1B-55kDa) of HAV5 and efficiently supported the replication of both wild-type and replication-incompetent bovine or human adenoviruses. Transient gene expression experiments with a plasmid encoding the bacterial β-galactosidase gene demonstrated that BHH cell hybrids seem to have better transfection efficiencies than either of the parental cell lines. These cell lines will be useful for isolating and growing replication-competent human or bovine adenovirus recombinants with E1 deleted and for the study of cellular or viral factors important for viral replication. The development of somatic cell hybrids appears to be a simple way of combining some of the desirable characteristics present separately in two parental cell lines.

Screening Mangrove Endophytic Fungi for Antimalarial Natural Products

We conducted a screening campaign to investigate fungi as a source for new antimalarial compounds. A subset of our fungal collection comprising Chinese mangrove endophytes provided over 5000 lipophilic extracts. We developed an accelerated discovery program based on small-scale cultivation for crude extract screening and a high-throughput malaria assay. Criteria for hits were developed and high priority hits were subjected to scale-up cultivation. Extracts from large scale cultivation were fractionated and these fractions subjected to both in vitro malaria and cytotoxicity screening. Criteria for advancing fractions to purification were developed, including the introduction of a selectivity index and by dereplication of known metabolites. From the Chinese mangrove endophytes, four new compounds (14–16, 18) were isolated including a new dimeric tetrahydroxanthone, dicerandrol D (14), which was found to display the most favorable bioactivity profile.

Generation of infectious genome of bovine adenovirus type 3 by homologous recombination in bacteria

The widely used technique of generating adenovirus vectors by homologous recombination in mammalian cells is usually not very efficient. This communication describes a simple method of generating a plasmid containing the full-length genome of an adenovirus by homologous recombination in bacteria. Following transfection of a suitable mammalian cell line with the full-length adenovirus genome, infectious virus progeny could easily be generated. Using this technique the generation of adenovirus recombinants would be efficient and straightforward.

CNS and antimalarial activity of synthetic meridianin and psammopemmin analogs.

The marine invertebrate-derived meridianin A, the originally proposed structure for psammopemmin A, and several related 3-pyrimidylindole analogs were synthesized and subsequently investigated for central nervous system, antimalarial, and cytotoxic activity. A Suzuki coupling of an indoleborate ester to the pyrimidine electrophile was utilized to form the natural product and derivatives thereof. The 3-pyrimidineindoles were found to prevent radioligand binding to several CNS receptors and transporters, most notably, serotonin receptors (<0.2 μM K(i) for 5HT(2B)). Two compounds also inhibited the human malaria parasite Plasmodium falciparum (IC(50) <50 μM). Only the natural product was cytotoxic toward A549 cells (IC(50)=15 μM).

A 72-bp Internal Deletion in the Left Inverted Terminal Repeat of the Bovine Adenovirus Type 3 Genome Does Not Affect Virus Replication

The genome of bovine adenovirus type 3 (BAV3) is flanked by 195-base pair (bp) inverted terminal repeats (ITR). We isolated a BAV3 mutant (BAV3c29) having an internal deletion within the left ITR. The deletion eliminated 72 bp between nucleotides (nt) 89 and 162, including most of the GC-rich sequences located close to the end of the ITR sequences. This deletion did not seem to have any affect on the virus plaque size or morphology and the kinetics of viral replication compared to wild-type (wt) BAV3. The nt sequence of the right ITR of BAV3c29 remained identical to the right or left ITR of wt BAV3. These results indicate that the cis-acting sequences present within the 72 bp between nt 89 and 162 of the left ITR are not essential for BAV3 DNA replication in cultured cells.

Miniaturized Cultivation of Microbiota for Antimalarial Drug Discovery.

The ongoing search for effective antiplasmodial agents remains essential in the fight against malaria worldwide. Emerging parasitic drug resistance places an urgent need to explore chemotherapies with novel structures and mechanisms of action. Natural products have historically provided effective antimalarial drug scaffolds. In an effort to search natures chemical potential for antiplasmodial agents, unconventionally sourced organisms coupled with innovative cultivation techniques were utilized. Approximately 60,000 niche microbes from various habitats (slow‐growing terrestrial fungi, Antarctic microbes, and mangrove endophytes) were cultivated on a small‐scale, extracted, and used in high‐throughput screening to determine antimalarial activity. About 1% of crude extracts were considered active and 6% partially active (≥67% inhibition at 5 and 50 μg/mL, respectively). Active extracts (685) were cultivated on a large‐scale, fractionated, and screened for both antimalarial activity and cytotoxicity. High interest fractions (397) with an IC50 < 1.11 μg/mL were identified and subjected to chromatographic separation for compound characterization and dereplication. Identifying active compounds with nanomolar antimalarial activity coupled with a selectivity index tenfold higher was accomplished with two of the 52 compounds isolated. This microscale, high‐throughput screening project for antiplasmodial agents is discussed in the context of current natural product drug discovery efforts.

The Pursuit of Potent Anti-influenza Activity from the Antarctic Red Marine Alga Gigartina skottsbergii

With an estimated 3–5 million infections and as many as 500,000 deaths from the complications of influenza infections each year, there is a critical need to identify novel drug classes and structures that can be exploited for antiviral development. A bioassay-guided fractionation of extracts from the red marine algae Gigartina skottsbergii, collected near Anvers Island, Antarctica, has yielded fractions that significantly inhibited the replication of influenza viruses A/Wyoming/3/2003 (H3N2) and A/WSN/1933 in vitro with an IC50 value of 4–8 μg/mL. The antiviral effect was dose dependent, strain specific, and selective as the virus-induced cytopathogenic effect (CPE) was reduced at nontoxic concentrations of the extract. SDS-gel electrophoresis and sequencing of the active fraction suggested homology with lectins. Insight into the mechanism of action via hemagglutination and plaque assay indicates that the antiviral effect occurs early during viral infection by interfering with virus-receptor interactions. This study suggests that activity from current over-the-counter anti-influenza vitamin supplements prepared from the same algal species may not completely arise from the previously reported antiviral sulfated polysaccharides.