More Monkey Business

Growing attendance at the annual nonhuman primate conference reflects collaboration and the importance of this model for the field

By Andreas von Bubnoff

More and more researchers are becoming interested in monkey business. This year’s 29th Annual Symposium on Nonhuman Primate (NHP) Models for AIDS, which took place October 25-28 in Seattle, had about 40% more registrants than last year and double the number of international participants. Autumn in Seattle can be lovely, but conference chair David Anderson, director of the Washington National Primate Research Center, said part of the reason more researchers attended this year’s meeting is that NHP research is becoming more collaborative.

An example of this collaboration is the NHP reference transcriptome project that uses next generation sequencing (NGS) to sequence the transcriptomes (the totality of all expressed RNAs) of 15 NHP species. In the past several years, NGS has taken the world of biology by storm, making it possible to understand biological systems in unprecedented detail, and NHP researchers are now using this to their advantage. Other research presented at this year’s conference showed how NHPs are used to model almost every aspect of HIV transmission and pathogenesis, allowing researchers to better understand these processes and to address them in humans. 

 

Next generation sequencing in NHPs

Michael Katze, associate director at the Washington National Primate Research Center and a member of the NHP reference transcriptome project’s steering committee, gave an update on the current status of the NHP transcriptome project that is sequencing the transcriptomes from 20 different tissues of 15 NHP species. Most of the species being studied are those used in biomedical experiments, such as the rhesus macaque, although some, such as the gorilla, were included for evolutionary considerations, according to Robert Palermo, a research assistant professor and virologist in Katze’s laboratory at the University of Washington who is also involved in the project.

Just 18 months after the project was launched in April 2010, sequencing of the transcriptomes of five species was completed, Katze said, adding that the company Illumina is doing the sequencing work for free. The transcriptomes, Katze said, will tell researchers what sequences are transcribed and therefore likely have a biological function, and will reveal previously unknown RNAs that do not encode proteins and transcripts of previously unknown genes. They will also allow a better annotation of the genome sequences of some of the species such as the draft rhesus macaque genome that was released in 2006.

Others are using NGS to better understand what happens in the host early after infection. Nicholas Maness, currently a scientist in the laboratory of David Watkins at the University of Wisconsin in Madison, used it to study how simian immunodeficiency virus (SIV)-infected host cells transcribe SIV genes in more detail than ever before. The study is among the first that uses NGS to look at viral transcription itself, Maness said. He and his colleagues sequenced all SIV-related transcripts of SIVmac239-infected CD4+ T cells from Indian rhesus macaques and found several novel protein variants that do not result in any known functional SIV proteins and are even truncated in some cases. It is possible that these “strange” transcripts and proteins do have some still unknown function, but it is also possible that SIV somehow tries to coax the host cell into producing non-functional proteins to elicit an immune response and thereby distract the immune system from responding to functional SIV proteins. In effect, SIV may be putting up a smoke screen for the immune system, Maness said. He and colleagues showed previously that macaques do make CD8+ T cell immune responses to some of these seemingly nonfunctional SIV proteins (1).

In another study using NGS, Palermo studied how host cells respond to infection. In the study, male Indian rhesus macaques were challenged with a single, rectal, high dose of SIVmac251. The researchers then used NGS to sequence and quantify all transcripts in the area of the rectum where the challenge virus was applied both three and 12 days after infection, compared with the same area in three of the animals two weeks before they were infected.

Preliminary data from three rhesus macaques, three days after infection when viral replication is still locally restricted only to the challenge compartment in the rectum, confirmed that there already was low-level active viral replication in the tissue. The host tissue gene expression changes at that time suggested that the infection itself is already beginning to cause an inflammatory response in the gut, Palermo said. “It was really, really surprising that you should see evidence of inflammation so early.”

The other surprising observation at day three, he said, was downregulation of genes that are involved in tight junctions, structures that hold the epithelial cells of the rectum together. Such changes could increase the permeability of the gut epithelium, allowing the virus to spread to the interior gut-associated lymphoid tissue. These changes could also be early indications of the leaky gut that enables gut bacteria in chronically infected animals to leave the gut, which is thought to be one cause for chronic inflammation in SIV-infected animals and HIV-infected people. There was also an increase in collagen gene expression, suggesting the onset of fibrotic tissue damage that is usually caused by long-term inflammation of tissues. Palermo believes these are the earliest observations of changes in collagen or tight junctions in the gut after infection.

Data from three animals at day 12, when the virus has already spread to blood and all of the lymphoid tissue in the rectum and when the animals are probably close to their peak plasma viremia, showed a very different picture. These animals had a strong type I and II interferon antiviral innate immune response. Next, Palermo wants to analyze animals just one or two days after infection to see if the immune response already provides some local control over the virus this early after infection.

Characterizing the host

Because host factors that affect viral replication can affect the outcome of challenge experiments, researchers are trying to better characterize such factors in different NHP species that are used for such experiments.

For example, the restriction factor TRIM5α can restrict SIV replication in host cells. It recognizes and binds the viral capsid as it enters the cell and thereby restricts infection in cells from many monkey species including rhesus macaques. TRIM5α could therefore affect the outcome of challenge experiments because it might give the impression that the animals were protected.

Another restriction factor, TRIMCyp, is related to TRIM5α but has cyclophilin A as the capsid binding domain. TRIMCyp has been found in four species of Asian macaques—it was first described in cynomolgus, pigtail, and rhesus macaques in 2008, and in northern pigtailed macaques in 2009, according to Elizabeth Dietrich, a graduate student in the laboratory of Shiu-Lok Hu at the University of Washington. Not much is known about prevalence, diversity, and restriction activity among the cynomolgus macaque population, but Dietrich and her colleagues are studying which versions of TRIMCyp can be found in cynomolgus macaques from different geographical areas, and how different versions of TRIMCyp can affect the susceptibility of cultured cells to infection with HIV-1 and HIV-2 (2).

She found that TRIMCyp is quite common in cynomolgus macaques from Indonesia and the Philippines, but is relatively rare in animals from mainland Indochina, and apparently absent in animals from Mauritius.

Three different TRIMCyp variants were identified in cynomolgus macaques. Expressing each of these variants in a cultured cat cell line (which does not express its own TRIM5α or TRIMCyp) either restricts HIV-1 infection, HIV-2 infection, or infection with both HIV-1 and HIV-2, depending on changes of the amino acids in just two positions of the cyclophilin A domain. Preliminary results suggest that changes in these same two amino acids also affect susceptibility to infection with SIVsmE041, suggesting that differences in TRIMCyp may also affect how NHP species such as cynomolgus macaques behave in SIV challenge experiments.

While all three variants are found in cynomolgus macaques, only one has so far been found in rhesus and pigtail macaques, Dietrich said, suggesting that cynomolgus macaques are the most diverse macaque species described so far with respect to the variability of their TRIMCyp restriction factors.

Modeling human transmission

One big advantage of NHPs is that they can be used as a model for HIV transmission. Thomas Hope, professor of cell and molecular biology at Northwestern University, is using NHPs to study how fluorescently labeled HIV particles enter the mucosal barrier of the female macaque reproductive tract, and how this varies during the menstrual cycle. “[This is] becoming an interesting and important topic,” Hope said.

He studied HIV entry into the vagina of macaques that had been treated with Depo-Provera, a contraceptive that mimics an extreme version of the luteal phase of the menstrual cycle by thinning the vaginal mucosa and has recently been shown to double the susceptibility to HIV infection in women (3). He found that in Depo-Provera treated animals, more T cells came close to the surface of the vaginal mucosa, possibly explaining why Depo-Provera increases transmission (see cover image).

Hope has also been studying virus penetration in pigtail macaques at different points of the menstrual cycle. While the study is still blinded, there is considerable variation, similar to the difference between animals that were treated with Depo-Provera and untreated animals, he said.

Different stages of the menstrual cycle may also influence the antiviral activity of vaginal gels containing the antiretroviral (ARV) tenofovir, according to Charles Dobard of the US Centers for Disease Control and Prevention (CDC). Dobard reported that in pigtail macaques, the concentration of the active metabolite of tenofovir in vaginal lymphocytes was about five times higher during the luteal phase (when the vaginal epithelium is thinnest and the susceptibility to HIV infection is highest) than in the follicular phase of the menstrual cycle. The tenofovir levels in plasma were also higher in the luteal phase.

“The good thing is that we see more absorption when there is more thinning [of the epithelium], so with the higher susceptibility to infection there is also more drug going in,” said Walid Heneine of the CDC, who led the study, adding that knowing how absorption of ARVs changes during the menstrual cycle is important when interpreting the results of clinical trials, such as the recent CAPRISA 004 trial, which showed for the first time that a microbicide gel containing 1% tenofovir was able to reduce HIV incidence by 39% in a cohort of South African women. Most of the women in that trial were on Depo-Provera, Heneine said, so “you would think that they were absorbing the drug very well vaginally.” Still, researchers don’t know if the variation in absorption of ARVs during the menstrual cycle in women is the same as in NHPs. “We have to see to what extent this happens in women,” he added.

Reducing viral reservoirs

While highly active antiretroviral therapy (HAART) can lead to a drop in viral loads to undetectable levels in a majority of HIV-infected individuals, eliminating the remaining viral reservoirs is still one of the biggest challenges to curing HIV. Deborah Fuller, an associate professor in the department of microbiology at the University of Washington, reported that a therapeutic vaccine can further reduce residual virus levels in ARV-treated rhesus macaques and also suppress the virus even after ARV treatment is stopped.

In the study, Fuller and colleagues intravenously infected rhesus macaques with SIVdeltaB670. Six weeks later, when the animals had high viral loads, they were treated with the ARVs tenofovir and Kaletra, resulting in viral loads of about 10,000 copies per ml of blood. Because the animals in the study were not given HAART, there wasn’t as strong a reduction in viral load as seen in humans on HAART. Rather, the study design was more similar to humans who are not responding very well to drug therapy. Animals that didn’t show at least a 10-fold reduction in viral load in response to therapy were excluded, Fuller said.

Twelve weeks after starting ARV treatment, animals that responded to the drugs were vaccinated with six monthly doses of an SIV DNA vaccine that expressed Gag, Reverse Transcriptase, Nef, and Env that were almost identical to SIVmac239. The vaccine was administered into the epidermis of the skin with a gene gun. This type of delivery elicits a mucosal immune response in the gut. To further augment mucosal immune responses in the gut, the vaccine also contained DNA encoding the heat labile enterotoxin from E. coli (LT), a mucosal adjuvant. Induction of mucosal immune responses in the gut was important because the gut harbors a viral reservoir that cannot be eliminated with drug therapy, Fuller said.

While the animals were on ARVs, the LT-adjuvanted vaccine further lowered the average viral load of the monkeys to about 100 copies per ml of blood. When ARV treatment was stopped eight weeks after the final vaccination, the mean viral load did not rebound in the group immunized with the LT-adjuvanted vaccine, whereas in the unvaccinated animals, the mean viral load rebounded. As a result, six months after stopping ARVs, the vaccinated animals had an almost five log lower mean viral load compared with the unvaccinated animals. “Nobody has ever been able to achieve that level of virus suppression with a therapeutic vaccine before,” Fuller said. Ten and a half months after stopping the ARVs, all seven vaccinated animals still showed no CD4+ T cell decline and were healthy, whereas five of the six unvaccinated animals had progressed to AIDS.

Fuller said the study also showed for the first time that compared with the unvaccinated controls, the therapeutic vaccine was able to achieve a significant reduction in the amount of virus in the viral reservoirs in the gut and other tissues.

Steven Deeks, a professor of medicine at the University of California in San Francisco, said that one reason that even people on HAART still have about a ten year shorter life expectancy than uninfected people is that they show residual inflammation, probably because of remaining virus in tissue reservoirs. He said Fuller’s study shows that it might be possible to use a therapeutic vaccine to treat this residual inflammation in people who are on HAART. “It’s one of the few studies I have seen in which you can give an intervention that affects the size of the reservoir,” Deeks said.

How HSV-2 Infection Increases HIV-1 Infection Risk

Herpes simplex virus (HSV)-2 infection, which is the cause of genital herpes, is known to increase the risk of HIV infection, in part because the lesions and inflammation it causes increase the number of HIV target cells. This suggests that treating HSV-2 infection should reduce the increased risk of HIV infection, but a randomized trial called HPTN 039 found that a twice-daily 400 mg dose of acyclovir to treat HSV-2 infection did not reduce HIV infection risk (see Clues from CROI, IAVI Report, Jan.-Feb. 2008). At the Annual Symposium on Nonhuman Primate Models for AIDS, Elena Martinelli, a scientist at the Population Council in New York City, reported on experiments that suggest a possible explanation of how HSV-2 infection could increase susceptibility to HIV infection, even in the absence of the lesions or inflammation caused by HSV-2. Her study was based on previous findings that HIV and SIV bind to a receptor called α4ß7 on CD4+ T cells and that CD4+ T cells that express very high levels of this receptor are more susceptible to HIV and SIV infection and produce more virus. Martinelli found that rectally infecting macaques with HSV-2 leads to an increased percentage of CD4+ T cells with high α4ß7 expression in their blood and rectal mucosa, suggesting that they might be more susceptible to SIV infection. She and her colleagues then showed that infecting dendritic cells with HSV-2 in vitro caused the dendritic cells to secrete the vitamin A derivative retinoic acid, which in turn led to increased expression of the α4ß7 receptor on CD4+ T cells and a concomitant increase in HIV replication in the CD4+ T cells. The work suggests that blocking the α4ß7 receptor might reduce the increased susceptibility to HIV infection in patients that are coinfected with HSV-2 (4). —AvB

Modeling STEP

NHP models are also being used to better understand the results of recent clinical trials of HIV vaccine candidates. Irene Bukh, a graduate student in the lab of Michael Betts at the University of Pennsylvania School of Medicine, is using NHPs to try to address why in the STEP trial, Merck’s MRKAd5 vaccine candidate not only failed to protect from HIV infection but in some vaccinees with previous adenovirus serotype 5 (Ad5) immunity actually increased the risk of HIV infection. One hypothesis has been that the vaccine may have increased HIV infection risk by increasing the number of activated CD4+ T cells, targets for HIV infection, in the gut or rectum.

To test this hypothesis, Bukh and colleagues tried to replicate the STEP trial in rhesus macaques. However, because human Ad5 doesn’t naturally infect rhesus macaques, researchers instead used a replication incompetent simian Ad7 (SAd7) vector derived from a naturally occurring adenovirus that infects rhesus macaques. About half of the rhesus macaques used in the experiments had antibodies to SAd7 in their blood, which is similar to the percentage of pre-existing immunity to Ad5 in humans in the US, one of the areas where the STEP trial was conducted, Bukh said. Also, unlike the vaccine used in the STEP trial, the SAd7 vector carried the spike protein from the human SARS virus, not SIV proteins. “We were just looking for the responsiveness to adenovirus,” she said.

Bukh and colleagues vaccinated 12 rhesus macaques three times intramuscularly with this SAd7 vector, with a prime at week 0 and two boosts at week 17 and 31. This too was different from the STEP trial where the three intramuscular vaccinations were done at weeks 0, 4, and 26. They also included five animals vaccinated with human Ad5 as controls.

Researchers extracted CD4+ T cells from peripheral blood and rectal biopsies before and several times after each of the three vaccinations. They then stimulated them in vitro by adding the same replication incompetent SAd7 vector they used for vaccination to reactivate any adenovirus-specific CD4+ T cells that were still around from previous exposures to SAd7, turning them into activated CD4+ T cells, which are the targets for SIV and HIV.

They found that this treatment could stimulate a greater fraction of CD4+ T cells taken from the rectum than from blood of the vaccinated animals. The animals vaccinated with human Ad5 and animals without previous SAd7 antibodies in their blood also showed a larger fraction of Ad7 CD4+ T cells that could be activated in their rectum than in blood after SAd7 vaccination, suggesting that even vaccination with, or exposure to, adenoviruses other than SAd7 might increase the animal’s fraction of CD4+ cells in the gut that can be activated by SAd7 vaccination.

Bukh said the SAd7 vaccination likely reactivates memory CD4+ T cells from previous adenovirus infections that then home to the place where they got activated during the initial infection. Because many adenovirus infections target the gut, the memory cells will typically home to the gut, Bukh said, explaining why the Ad7 vaccinated animals had a higher percentage of activated Ad7-specific CD4+ T cells in the rectum than in blood.

This means that as a result of the vaccination, “there might be a redistribution of the Ad-specific CD4+ T cells from the peripheral blood to the rectal lamina propria,” Bukh said. “If you are exposed [to] HIV or SIV at that point, you could have a higher likelihood of infection.”

To see if animals with a larger fraction of Ad7-specific CD4+ T cells in the rectum are indeed more likely to get infected, Bukh and colleagues next plan to vaccinate another group of macaques in a similar way and then intra-rectally challenge them with SIVmac239 or 251.

1. J. Virol. 84, 11569, 2010
2. J. Virol. 85, 9956, 2011
3. Lancet Infect. Dis. 12, 19, 2012
4. PloS Pathog. 7, e1002109, 2011