Monthly Archives: March 2012

Epidemics, Immunology

HIV Microbicides and the Risks of Clinical Trials

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Contributed by guest blogger: Julia Ding ’12

Once preliminary studies suggest that a drug is safe for human use, clinical trials are conducted in order to further investigate the effects and possible adverse reactions of the drug. The example of HIV microbicides has shown that caution and careful scrutiny is highly important for these trials. HIV microbicides are chemical entities which, when applied before vaginal or rectal intercourse, prevent the transmission of the virus. Of the potential microbicide agents that have been studied, two compounds classified as polyanions were thought to be promising for inhibiting HIV-1 transmission: carrageenan and cellulose sulfate (CS). However, these compounds were deemed in phase III clinical trials to be ineffective as microbicides.

In addition to that discovery, the more surprising and disturbing result of these trials was that the HIV microbicides appeared to actually enhance the rates of HIV infection. Pirrone and colleagues examined the validity of this claim in a study reassessing the in vitro activities of the compounds. Cells were infected with different strains of HIV-1 in the presence of three different polyanions: CS, λ-carrageenan (LC), and destran sulfate (DS). Resulting assays showed that all of these compounds exhibited antiviral activity against both R5 and X4 HIV-1 strains. However, further experiments also discovered that application and removal of polyanion microbicides prior to HIV exposure enhanced and increased the rates of HIV-1 infection. The compounds were added to cell cultures and washed out prior to HIV-1 infection to simulate the natural loss of the compound after vaginal application. In both HIV-susceptible cells and regular human cells, the results indicated an increase in the percentage of cells infected, unrelated to any change in cell viability. The level of enhancement was found to be dependent on the target cell, its co-receptor phenotype, the compound identity and concentration, and the timing of the viral challenge. While the mechanism through which HIV-1 transmission increased in the in vitro experiments is still unclear, these factors suggest that the nature of the host cell also plays a role in polyanion-dependent HIV-1 infection.  This data provides a discouraging outlook on the use of these compounds as effective microbicides, while introducing new questions about its mechanisms of action.

This study provides us with many valuable insights about not only the microbicide technology itself, but also the risks and complications associated with clinical trials. The data suggested a significant increase in HIV-1 infection after the application and removal of the two microbicides. Furthermore, it emphasized the need for intense scrutiny of compounds prior to clinical trials, considering the dangers they may pose on human subjects. While previous studies supported the use of polyanion microbicides as a safe and possibly effective means of preventing HIV-1 transmission in women, the effects of the leakage and loss of the product over time was not taken into consideration, and significantly more women on the drug were found to have contracted HIV than if they had not taken it. The study also provides us with an example of the vital role clinical trials play in the testing of a drug, and how certain adverse effects may be missed through in vitro studies that only become apparent when applied to real world uses.

Links:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3295645/

http://www.nejm.org/doi/full/10.1056/NEJMoa0707957#t=abstract

 

Julia Ding is a senior at Vassar College, with a major in Science, Technology and Society.

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Guest Blogger, Immunology, Molecular Virology

Polydnavirus: Good for the Parasitic Wasp, Bad for the Host Caterpillar

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Contributed by guest blogger: Jason Adler 

An endoparasitoid wasp would disagree with the popular perception of viruses as malevolent. Parasitoids are organisms that spend a substantial portion of their life cycle in the host; unlike a parasite, a parasitoid usually kills or sterilizes the host. Endoparasitoid wasp oviposit into the body cavity of caterpillars. When the wasp larvae emerges, it then consumes the host as it develops.

Polydnaviruses (PDV), a family of double stranded DNA insect viruses, are symbiotic to some endoparasitoid wasps. Of two PDV genera, genus Ichnovirus is specific to ichneumoid wasps and Bracovirus to braconid wasps. The PDV genome is located on host wasp chromosomes in a segmented, proviral form. However, the integrated PDV genome is not fully functional as it cannot replicate independent of the wasp and capsid proteins are non-existent.  It is unknown if PDV is derived from wasp genes or if ancestral wasps integrated a beneficial PDV into their genome with resulting loss of the genes responsible for capsid formation and virus replication.

As such, PDV only replicates at specific ovarian cells during the late pupal phase, where it acquires two viral envelopes. PDV integration does not occur in the viral life cycle; instead, the viral genome is vertically transmitted to wasp offspring during meiosis. When the female wasp injects her eggs into the lepidopteran host, virions are co-injected and result in infection. Although, PDV does not replicate in the host caterpillar, it does result in immunosuppression and alters the host development (i.e. prevents metamorphosis) and metabolism to favor the parasitoid larva. The normal response of lepidopteran larvae to small foreign material is phagocytosis, but larger pathogens must be encapsulated. This is accomplished through melanization, where certain hemocytes, invertebrate immune cells found in the hemolymph, secrete melanin, which surrounds the pathogen so that anti-microbial peptides can destroy it. When immune suppressed, host hemocytes do not destroy the wasp egg by forming hemocyte nodules. Thus, PDV and the wasp share a mutualistic relationship.

Cotesia plutellae, a braconid wasp, possesses a PDV – C. plutellae bracovirus (CpBV) – and parasitizes larvae of the diamond-back moth Plutella xylotsella. Recent research has found that CpBV encodes a viral histone H4 that shares high sequence homology with histone H4 on P. xylostella, except for the last 38 residues comprising the N-terminal tail. Additionally, this viral histone H4 N-terminal tail have been observed in other Cotesia-associated PDVs. It has been suggested that the N-terminal tail is altering gene expression regulation as viral H4 histones less easily detach from DNA than host H4 histones, thereby inhibiting transcription. Is the N-terminal tail of CpBV-H4 causing immunosuppression? The researchers hypothesized that the N-terminal tail is causing the suppression of antimicrobial peptide (AMP) genes.

To examine the effects of CpBV-H4, the researchers constructed two viral recombinants: a WT CpBV-H4 and a truncated CpBV-H4 that lacks the N-terminal tail. After injection of the viral vector into the host caterpillar, RT-PCR was used to look at the expression of putative AMP genes. Although basal expression levels were unchanged, when E. coli was introduced to the host to present an immune challenge CpBV-H4 inhibited inducible expression, while truncated CpBV-H4 did not. Additionally, by counting the number of melanized black nodules on the host caterpillar after injection of E. coli and the viral vector, the researchers assessed the immune response. While the larvae show hemocyte nodule formation in response to E. coli infection, transient expression of CpBV-H4 significantly suppressed the immune response by decreasing nodule formation, while truncated CpBV-H4 had no effect. Finally, the researchers examined a possible synergistic effect of CpBV-H4 and the entomopathogenic bacterium X. nematophila. Without CpBV-H4, X. nematophila infection resulted in low mortality; however, with CpBV-H4, there was significantly increased mortality with this synergistic effect lost if CpBV-H4 was truncated.

Based on these results, the researchers concluded that the N-terminal tail appears to be responsible for immunosuppression by inhibiting inducible expression of AMP genes, possibly by altering a normal epigenetic control. CpBV-H4 containing nucleosomes may less easily detach from DNA during transcription due to the increased positive charge resulting from the increased number of lysine residues in the N-terminal tail. By introducing a virus that expresses a viral H4 histone with a N-terminal tail, the parasitoid wasp is able to suppress the host immune system. This is important as without the immune suppression, the host hemocytes would encapsulate and destroy the wasp egg.

With 157 putative genes, CpBV is likely to have more than this one mechanism to suppress host immunity. Are there other mechanisms of CpBV immune suppression?  How else is the complex ecological relationship of wasp, virus, and caterpillar host mediated at the molecular level?

Link:

http://www.sciencedirect.com/science/journal/0006291X/415/2

Jason Adler is a senior at Vassar College, majoring in biology.

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Founding editor of JVI, Lloyd Kozloff, dies

The Journal of Virology was founded in 1967 by three scientists, including Lloyd Kozloff, who passed away this week.

Sarah Kozloff, Lloyd’s daughter, is a professor in the Film Department at Vassar College.  She told me of her scientist father about a year ago and I did a little digging to find out more about him and his career.   Kozloff began his career as a scientist in a very exciting time.  He was a part of a group of bacteriophage biologists at Cold Spring Harbor in the late 1940s and early 1950s.  Members of this group revolutionized biology by ushering in a new discipline called molecular biology.  Very little was known about viruses or the molecular mechanisms that make cells work.  Other members of this group would go on to demonstrate that DNA was the genetic material (A.D. hershey) and discover the structure of DNA (James Watson).  Sarah mentioned to me that she remembered the party that the Kozloff’s threw to celebrate Watson’s Nobel prize.  The names of scientists that are legendary to most of us were, to the Kozloffs,  part of everyday dinner table conversation about friends and colleages.

Lloyd Kozloff made some important contributions to phage biology and basic understanding of viruses.  He was one of few people using a new technique in biology: radioisotope labeling.  In a 1948 paper he used phosporus isotopes to determine that phages obtain their phosphorus primarily from the media, though he presumed it must be vis a cellular metabolic pathway.  That paper in Science, has a single table, the result of what appears to be a single experiment.  The phosphorus was all in the DNA component of the phage, something that would be important later when Hershey and Chase showed in 1952 that DNA was the genetic material.   In fact, the authors are careful to define the acronymn DNA, the macromolecule perhaps being something not too familiar to many at the time.    In a later 1956 paper, Kozloff demonstrated that the bacteriophage does something to the bacterial cell wall to allow the genetic amterial to enter, and that activity was conferred by some kind of protein.  We now know that what he was seeing was the action of lysozyme, an enzyme at the base of the bacteiophage tail that degrades the bacterial peptidoglycan wall to allow the DNA to enter the cell.

I rarely go so far back in the literature, although it is always interesting to see the foundational papers upon which our current knowledge is based, and to see the style of experiments, the difference in the style of scientific writing and presentation, and to imagine what it was like to explore virology without really understanding yet what viruses really are or how they work.

The journal of virology will be publishing an obituary and I will add a link once it is available.

The university of San francisco, where Kozloff spent the last part of his career, has a brief biography.

A scholarship fund to support graduate students has been created, to which donations can be made in his name.  (The family has specifically requested donations to this fund in lieu of flowers.  A card will be sent to the family.)

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