Research Reports

By Philip Cohen and Simon Noble

Vaccine Helps Chimp Cells Fight HCV

About 3% of people are chronically infected with hepatitis C virus (HCV), putting them at elevated risk of liver disease and cancer. There is currently no vaccine to protect against HCV infection and developing one has been challenging, partly because antibodies that neutralize one HCV strain don't work on the highly diverse range of viruses found in infected individuals.

Studies of spontaneous viral clearance in humans and chimpanzees suggest that cellular responses can also control HCV infection. Now a new report finds that an adenovirus vaccine that elicits a robust HCV-specific cellular response in chimps reduces viral replication, protects against liver damage, may improve viral clearance—and can work against a virus with significant genetic differences from the strain used to construct the vaccine (Nat. Med. 12, 190, 2006). This is encouraging news for adenovirus-based AIDS vaccines attempting to elicit protective cellular responses.

The prime-boost-boost vaccination regimen used three vaccine components sequentially: injections of human adenovirus serotypes 6 and 24 (Ad6 and Ad24) and electroporated plasmid DNA. The five vaccinated chimpanzees received doses of the vectors containing a segment of the HCV genome and five control animals received the same vaccination schedule but with vectors containing the HIV-1 gag gene.

In vaccinated animals, researchers saw an increase in the number of IFN-g secreting HCV-specific CD4+ and CD8+ T cells after just the Ad6 prime. After the Ad24 boost, there was an additional increase on CD8+ T cells, but not CD4+. The DNA boost resulted in up to a 4-fold increase increase in the frequency IFN-g secreting CD8+ T cells and a 2 to 10-fold boost in CD4+ T cell responses. None of the control animals developed cell-mediated immunity to HCV in response to vaccination with the gag gene vectors.

All the animals were then challenged with H77, an HCV strain that is 13% different at the protein level from the virus used to create the vaccine. In the vaccinated animals, the average peak of viral blood titers was 100 times lower than in the control group. All control animals developed acute hepatitis as judged by increases in the blood levels of two liver enzymes, while no such increase was found in the vaccinated animals. It was not possible, however, to demonstrate a statistically significant improvement in viral control given the high rate of spontaneous clearance: 4 of 5 vaccinated chimpanzees cleared the virus (one developed a relatively weak T cell response), as well as 3 of 5 controls.

Poliovirus Quasispecies Cooperate to Cloud the Brain

No RNA viral genome, whether it is HIV or poliovirus, is really alone during a natural infection. A hallmark of RNA viruses is a high rate of replication errors which rapidly generate a mutational cloud of related genomes known as a quasispecies.

Because of their rapid mutation rate, RNA viruses can evolve rapidly to, for instance, develop resistance to antiviral drugs. But laboratory experiments and mathematical models suggest that the actual error rate must be balanced between two extremes. If too many mutations develop too quickly the virus population is driven to extinction. Too slow a rate of evolution leaves the virus less able to generate beneficial mutations to deal with adverse conditions.

And according to a new report from researchers in California and Pennsylvania, a quasispecies is not simply the sum of the abilities of its individual members. Instead they find evidence in a mouse model of poliovirus infection that the viruses of a quasispecies population cooperate to invade the central nervous system (CNS), suggesting that evolution of such viruses can take place at the population level, rather than the individual level (Nature 439, 344, 2006).

The researchers began by identifying an unusually accurate mutant viral polymerase, G64S, which generates six times fewer mutations than wild type, while still replicating the virus at the same rate. During replication this mutant poliovirus generates a less diverse quasispecies. To test the consequences of increased accuracy, mice were given intramuscular injections of wild-type and mutant viruses, a mode of delivery known to lead to rapid CNS infection. Animals injected with G64S developed paralysis later and only at much higher viral doses than those receiving the wild type. The dose at which 50% of the animals died was 300 times higher for G64S. When the mutant was delivered intravenously, it was unable to establish infection in spinal cord and brain.

The researchers showed that the limited ability of the mutant to invade the CNS was related to the lack of quasispecies diversity by treating stocks of the G64S with chemical mutagens, bringing its frequency of mutation to wild type levels. Even though this population still contained the same accurate polymerase, its ability to invade the CNS was improved and the lethal dose dropped to the same level as wild-type virus. However, samples of the formerly chemically-mutated G64S isolated from brain tissue were not able to invade the CNS again when re-inoculated intravenously, suggesting the ability to invade the CNS was the result of diversity and not the result of the selection of particular neurotropic viruses.

As a final test of the theory that quasispecies diversity per se determined pathologic behavior such as CNS invasion, the researchers used a version of the G64S virus marked with a SacI restriction site as a genetic barcode. As expected, this virus was not able to invade and replicate in the brain when inoculated alone intravenously. But when it was co-inoculated with wild type or chemically-mutated G64S it was found in brain. The researchers conclude that a diverse population of viruses in a quasispecies can evolve as a group to accomplish certain tasks cooperatively such as gut colonization, immunological evasion, or penetration of the blood-brain barrier.

Revealing HIV Core Values

The formation of a proteinaceous bullet-like core that encases HIV's RNA genome is an important step in the maturation of the virus after it buds from an infected cell. A British and German team of researchers has now determined the three-dimensional structure of the core in mature virions, providing a new model for a core assembly mechanism (Structure 14, 15, 2006).

As HIV buds from a cell, it forms a particle with an outer viral membrane and inner proteinaceous core of capsid proteins. Typical of retroviruses, the membrane and core can vary considerably in size and shape, although the membranes are generally spherical and most cores are conical with rounded edges. In a crucial step in the viral replication cycle, upon infection the core disassembles. For reasons that are unclear, this uncoating of the genome must be precisely timed. At least one host factor with antiviral activity, TRIM5a, may work by corrupting the uncoating process (see Making a monkey out of HIV, IAVI Report 9, 3, 2005). One key question the heterogeneity of the particles raises is how capsid core proteins manage to create a well-defined structure that can accommodate a wide variation in size.

To derive core structures, the researchers used cryo-electron microscopy, which allowed them to view individual particles at high resolution in nearly physiological conditions. Electron densities of 75 particles were recorded at 2 degree tilt increments through a range of 132 degrees and the resulting data assembled by computer into a 3-D image. As expected, the virions were roughly spherical with diameters ranging from 106 to 183 nanometers (nm).

The paper focuses on 40 of these virions that contained a single, complete core of conical morphology. Others contained incomplete, indistinct cores or, in the case of four large virions, two cores. One advantage of cryo-electron microscopy is that it allows examination of the membrane and core simultaneously. The diameter of the broad end of the core was found to strongly correlate with virion diameter and mirrored the curve of the membrane about 12 nm away. In contrast, the average diameter of the narrow end and the angle of the cone did not correlate with virion size.

The researchers say the data argue for formation of the cone starting at the narrow end, with the diameter and core angle being determined by intrinsic properties of the capsid proteins and perhaps other molecules, including the viral genome. The core then continues its growth until constrained by the viral membrane. Ultimately, a better understanding of core dynamics could lead to the design of drugs that disrupt this important viral structure.

Immune Cell Chimera Identified

The link between innate and adaptive immunity has been further strengthened. In two reports researchers describe the hybrid features of a new distinct immunological cell type in mice, the interferon-producing killer dendritic cell (IKDC).

Like natural killer (NK) cells, activated IKDCs can destroy cells lacking self MHC molecules. Like plasmacytoid DCs (PDCs), they can secrete relatively large amounts of interferon (IFN)-a. And typical of conventional DCs (cDCs), they can process and present antigens to T cells, altogether providing another bridge between innate and adaptive immune responses.

In the first study, researchers studied the morphological, phenotypic, and developmental characteristics of the new cell population (Nat. Med. 12, 207, 2006). The IKDCs displayed a set of surface markers that overlap those seen on NK cells, PDCs, and cDCs, but with some clear differences that set them apart. Gene expression profiles were also reminiscent of the other cell types, with all IKDCs expressing the NK-activating receptor NKG2D. Under the transmission electron microscope IKDCs were morphologically distinct from cDCs, PDCs, and NK cells. IKDCs were sensitive to the TLR9 ligand, unmethylated CpG oligodeoxynucleotide, and in response developed the dendrites and veils typical of DCs.

Functional characterization showed that spleen- (but not lymph node-) derived IKDCs could lyse target cells through either NKG2D- or Ly49h-dependent pathways, but only after activation with CpG. In a recombinantListeria monocytogenes in vivo infection model, the researchers looked for direct evidence of antigen-presenting cell (APC) activity, and found that lymph node (but not spleen) IKDCs could present antigen to naïve T cells. The authors contend this is consistent with a model where IKDCs lose their cytolytic NK activity as they migrate to draining lymph nodes and develop APC activity there. They propose that IKDCs extend the DC family and constitute a cell type with dual innate effector functions and antigen-presenting capacity.

In the second study, researchers investigated the role of these cells in tumor immunosurveillance. In a mouse melanoma model the research team showed that IKDCs rather than NK cells recognized and lysed tumor cells in a TRAIL-dependent manner. TRAIL is an apoptosis-inducing ligand and its expression is induced by IFN-g; this study shows that TRAIL expression on IKDCs was induced by activated IKDCs' own IFN-g expression.

This second, more functional study also perhaps suggests why such a hybrid cell that combines the function of two other cell types might be beneficial; perhaps sometimes it's better to have all these functions coordinated by a single cell.

Researchers will now be fervently searching for a human equivalent to get an idea of the medical relevance of this new cell type. Future studies will investigate just how common and important this cell type is and its role in specific aspects of different infections. Researchers will also have to ask to what extent previously described activities are actually due to IKDCs rather than NK or other cell types.