11 Jul 2019

This is Part 1 of a two-part story.

In a rare meeting of two worlds, experts on human herpesviruses and Alzheimer’s disease shared the stage at a special workshop at the 11th International Conference on HHV-6 and HHV-7, held June 23 to 26 in Quebec City, Canada. The virologists hosted the gathering because recent studies from within the AD field implicating these exact viruses in the disease had piqued their interest. The meeting was a mix of review and some new data on viruses in the AD brain and other tissues, and what they might be doing there. After a day of discussion, the virologists and the Alzheimerologists reached a consensus: Herpesviruses do not cause AD. And yet, because they seed, and speed, amyloid plaque deposition and inflame the immune system, they likely press on the gas pedal to accelerate the disease process. The scientists also agreed that much more work needs to be done on how viruses affect the brain. Most importantly: Could targeting viral infection slow the onslaught of dementia? This is an entirely open question.

Recent studies highlight potential role of viruses in AD.

At Quebec meeting, virologists and AD researchers reviewed the evidence.

Their conclusion? More work needed.

This confluence of fields signals a growing interest in how microbes might contribute to AD. This was for decades a fringe topic in the AD field but is now being taken more seriously.

In the 1990s, Ruth Itzhaki, now emeritus at the University of Manchester, U.K., first identified brain infection with herpes simplex virus (HSV-1), best known for causing cold sores, as a risk factor for AD (Itzhaki et al., 1997). Subsequent evidence suggested that recurrent reactivation of a latent infection, or low-grade persistent viral activity in the brain, could over time inflame and damage neurons, leading to neurodegeneration and dementia. The idea is being tested in a Phase 2 trial. It started in 2018 at Columbia University, and evaluates whether 18 months of daily valacyclovir (brand name Valtrex) slows cognitive decline in 130 people with mild AD who tested positive for HSV-1 or 2 (Devanand, 2018). A Phase 2 trial of the antiviral drug pleconaril is ongoing in Poland and the Czech Republic (Nov 2018 conference news).

A Roseolovirus. Human Herpesvirus 6. [Courtesy of Bernard Kramarsky, National Cancer Institute.]

There were hints of a role for other herpesviruses in AD, too. PCR analysis of postmortem brain found a higher prevalence of HHV-6 RNA and DNA in AD tissue than healthy brain (Lin et al., 2002), but serological studies produced mixed results (Carbone et al., 2014; Agostini et al., 2015).

Then, in 2018 a trio of papers grabbed the attention of a wide group of virologists. A multi-omics analysis of AD data sets from Joel Dudley, Icahn School of Medicine at Mount Sinai, New York, reported higher expression of HHV-6A and HHV-7 RNAs in AD brain samples, and that HSV-1, HSV-2, and HHV-6A regulated the expression of AD risk genes (Jun 2018 news). Around the same time, Robert Moir and Rudolph Tanzi, Massachusetts General Hospital, linked viruses to amyloid pathology. They showed that HSV-1 and HHV-6 could initiate and accelerate deposition of Aβ plaques, which sequester the viruses and render them less infectious in mice (Jun 2018 news). Lastly, an epidemiological study indicated that old people taking antivirals for an HSV-1 outbreak were protected against developing dementia (Tzeng et al., 2018; for commentary, see Itzhaki and Lathe, 2018).

Network News: A multidimensional analysis of human brain tissue found HHV-6A presence correlated with changes in expression of multiple genes related to AD. [Courtesy of Readhead et al., Neuron, 2018.]

In Quebec, Philip Pellett, Wayne State University School of Medicine, Detroit, opened the meeting by welcoming the newcomers, i.e. the handful of neurobiologists in the crowd of virologists. Himself a herpesvirus expert, Pellett described what’s known about HHV-6A, -6B, and 7, collectively called roseoloviruses, and disease. The viruses primarily infect, and persist in, lymphoid cells, though they are common inhabitants of brain tissue and CSF. It’s established that HHV-6B causes roseola in infants and toddlers, marked by a transient fever and characteristic rash. But proving causality for other roseolovirus diseases has been more difficult because they occur in adults who are already seropositive from earlier infection, and who carry different numbers of infected cells whose viral activity waxes and wanes over time.

Several neurologic inflammatory diseases have been associated with HHV-6B. In immunocompromised patients, reactivation of the virus causes encephalitis. Multiple lines of evidence support a role for HHV-6A and -6B in some cases of multiple sclerosis. The association of HSV-1 with dementia meets some, but not all of the criteria for causation, Pellett said. To his mind, the epidemiological data tying antiviral treatment to a lower risk of dementia provides the most compelling argument for the link.

Remarking on Dudley’s bioinformatics study, Pellett stressed the need for independent confirmation, as well as analysis of viral activity in individual patients and in single cells. Pellet asked many questions of the nascent virus-in-Alzheimer’s evidence. Among them:

What fraction of the study population had the HHV-6A profiles?

In those patients, how many cells harbor viral genomes, and how does that relate to abundance of viral transcripts?

What viral proteins do the cells express?

How is viral activity linked to alterations of host transcription and do both happen in infected cells?

Do infected cells release cytokines or exosomes that modulate surrounding cells?

Finally, Pellet cautioned investigators in the AD field not to assume that what goes for one herpesvirus goes for all, because different viruses interact in unique ways with the host immune system and cellular machinery.

Taking the stage for his plenary, Dudley welcomed Pellett’s questions and agreed it was important to maintain a healthy skepticism about the results. Recapping his group’s bioinformatics analysis linking viral RNAs to AD-related gene expression, pathology, and cognition, Dudley emphasized that the results are entirely correlative. They say nothing about whether the viruses cause or accelerate AD pathogenesis, or opportunistically grow as a consequence of disease.

Trying to parse cause and effect, Benjamin Readhead, Arizona State University, Tempe, the first author on Dudley’s paper, is delving deeper into their data set. He presented new work on how ApoE might affect the virus-AD relationship. The ApoE4 allele is the strongest genetic risk for late-onset AD, and reduces the disease’s age of onset. ApoE4 has pleotropic effects on the immune system and increases susceptibility to viral, bacterial and parasitic infections. People with two copies of the E4 allele have 15 times the risk of AD compared to people without one, whereas ApoE2 protects against AD.

Readhead found two instances where ApoE alleles correlated with virus abundance. First, people with one copy of E4 had more of the HSV-1 latency-associated transcripts than those with none, and those with two had higher still. Second, E2 carriers had lower expression of the HHV-6B transactivator gene than non-carriers.

The number of E4 alleles also signaled higher neurofibrillary tangle density. In ongoing work, causal modeling was consistent with HSV-1 mediating at least part of ApoE4’s effect on tangles, meaning that ApoE4’s association with higher tangle abundance may be due to its influence on viral infection. That is consistent with Itzhaki’ s work showing that HSV-1 infection boosts risk of AD in ApoE4 carriers, and with Moir’s data on the amyloidogenic effects of virus particles. Readhead suggested that lack of HHV-6B virus in ApoE2 carriers might explain some part of the protective effects of that allele.

Echoing Pellet’s remarks, the virologists in the audience wanted to know a lot more about the infection. Do Dudley and Readhead know if the viral RNA comes from blood? Where was the organism in the brain? What cells are infected? Readhead said the virally regulated gene profiles gave some hints. HHV6A and 6B tended to be linked to neuronally expressed genes, as well as oligodendrocytic and microglial genes, while HSV 1 was associated with microglia gene expression. “That’s indirect evidence, and comes from tissue homogenates. We really need to go down to the single-cell level for the expression data. We also need more comprehensive immune profiling in the periphery," he said.

In the meantime, the group is studying viral infection in three-dimensional cerebral organoids, which enable them to directly measure how viruses change gene expression in single cells, and how that affects plaque and tangle formation. Readhead believes the problem of how viruses interact with each other also needs to be studied. Almost all people are infected with HHV-6B and approximately half have HSV-1, as well. “In isolation, one might not have an effect, but jointly, they could become a different beast,” he said.

The story could get even more complicated. “There are probably a dozen viruses implicated in some corner of the analysis,” Readhead told Alzforum. The existing analysis is limited in that the available RNA-expression data is skewed toward mammalian sequences; the microbiome profile is sparse. “The data was optimized to characterize human gene expression, so in looking for microbial sequences we are rifling through the junk drawer,” he said. “We need to expand the profile to look for viruses, bacteria, anything we can pick up.”

Steven Jacobson is chief of the Viral Immunology section at the National Institute of Neurological Disorders and Stroke, Bethesda, Maryland. He has studied HHV-6 extensively as a trigger for multiple sclerosis. At the NIH, Jacobson was tasked with exploring the possible connection of HHV-6 with AD, which he did by analyzing tissue from some 800 postmortem brains. He tapped the same cohorts Dudley had analyzed, but used a different computational technique to detect viral RNA. Jacobson applied the PathSeq algorithm, optimized to detect human pathogens, on the RNA-Seq data from the Mount Sinai Brain Bank and the Religious Orders Study/Memory and Aging Project (ROSMAP).

Jacobson detected HHV-6A and -6B sequences in 1.3 percent of the samples, and the frequency was the same in AD and healthy controls. Likewise, he detected no correlation between viral load and plaque pathology or cognition. In addition, Jacobson performed a sensitive PCR for HHV-6 DNA, developed to study MS, on 346 samples from the Johns Hopkins Brain Resource Center (Blauwendraat et al., 2019), and 87 saliva samples from a Swiss AD cohort. Again, his team did not find much. Overall, about 3 percent of the samples tested positive, with no difference between control and AD. For comparison, these scientists find the virus in about 10 percent of brains samples from people with MS. “We don’t see that overrepresentation in brains from people with AD,” he said.

These results contradict the Dudley study, in which Readhead found HHV-6 RNA in about 30 percent of people in both groups, and higher levels of viral transcripts in the AD group, Readhead told Alzforum. An earlier study reported HHV-6 in 70 percent of samples of AD brain, and 30 percent of unaffected (Lin et al., 2002). Other studies report baseline infection rates of 25 to 35 percent (Chan et al., 2001; Prusty et al., 2018). Readhead told Alzforum he found it difficult to explain the discrepancies, without looking more closely at the different methodologies used, but said, “I’m glad someone else is starting to look at this.”

Jacobson emphasized that his results do not rule out a role for the virus. “It’s fascinating that pathogens can nucleate amyloid plaques, but that doesn’t mean you have to find the virus in the brain at the time of disease,” Jacobson said. Also, HHV-6 is tricky to track, he added. Its copy number is low, and it forms focal infections that may be missed when sampling tissue. Besides, HHV-6 may be but one of many viruses that rile up the immune system as people age. “Maybe it’s one of multiple triggers for AD. It may be related, but it’s not likely to be the cause of disease,” Jacobson said.

Jacobson would like to see additional bioinformatics analyses of more samples, and mechanistic studies. “There is much more work to be done on Alzheimer’s. The collective knowledge of the virology field should be brought to bear on the problem,” he said.—Pat McCaffrey