Author Archives: Blue Sky Science

Nobel Prize 2015 – Protecting People against Parasites!

The 2015 Nobel Prize in Physiology or Medicine has been awarded to scientists whose research has led to therapies that have saved hundreds of millions of people around the world from parasitic diseases that can otherwise cause disability and death.

William C. Campbell and Satoshi Ōmura shared one half of the award “for their discoveries concerning a novel therapy against infections caused by roundworm parasites”, while Youyou Tu was awarded the other half “for her discoveries concerning a novel therapy against Malaria”.

Portraits of the winners of the Nobel medicine prize, shown on a screen at the ceremony in Stockholm. Photograph: Jonathan Nackstrand/AFP/Getty Images

Portraits of the winners of the Nobel medicine prize, shown on a screen at the ceremony in Stockholm. Photograph: Jonathan Nackstrand/AFP/Getty Images

In the press release announcing the award the Nobel Assembly at the Karolinska Institute highlighted the impact of the therapies that were developed thanks to the work done by these three scientists, Avermectin in the case of William C. Campbell and Satoshi Ōmura, and Artemisinin in the case of Youyou Tu.

The discoveries of Avermectin and Artemisinin have fundamentally changed the treatment of parasitic diseases. Today the Avermectin-derivative Ivermectin is used in all parts of the world that are plagued by parasitic diseases. Ivermectin is highly effective against a range of parasites, has limited side effects and is freely available across the globe. The importance of Ivermectin for improving the health and wellbeing of millions of individuals with River Blindness and Lymphatic Filariasis, primarily in the poorest regions of the world, is immeasurable. Treatment is so successful that these diseases are on the verge of eradication, which would be a major feat in the medical history of humankind. Malaria infects close to 200 million individuals yearly. Artemisinin is used in all Malaria-ridden parts of the world. When used in combination therapy, it is estimated to reduce mortality from Malaria by more than 20% overall and by more than 30% in children.

For Africa alone, this means that more than 100 000 lives are saved each year.

The discoveries of Avermectin and Artemisinin have revolutionized therapy for patients suffering from devastating parasitic diseases. Campbell, Ōmura and Tu have transformed the treatment of parasitic diseases. The global impact of their discoveries and the resulting benefit to mankind are immeasurable.

Animal research played a key part in the development of these therapies, as the Nobel Prize press release has pointed out.



In the case of Avermectin, after  Satoshi Ōmura had identified a series of bacterial cultures that produced a variety of antimicrobial agents, including the bacteria Streptomyces avermitilis which  showed promise against parasitic roundworm infection in mice, in 1979 William C. Campbell and colleagues identified a particular component produced by S. avermitilis called Avermectin B1a which had a broad efficiency against roundworm infections in a wide range of domesticated animal species, including cattle, sheep, dogs and chickens. Following this the team developed a modified form of Avermectin B1a known as Ivermectin, which was entered into clinical trials following positive tests in animal models of parasitic infection, and has since gone on to become a key treatment for parasitic infections – particularly the nematode worm infections that cause River Blindness and  Lymphatic Filariasis (the extreme manifestation of which is known as elephantitis) – and is on the World Health Organization’s list of Essential Medicines.



Ivermectin, and other members of the Avermectin family of therapies, are also widely used in veterinary practice, and their development and use is a good example of the One Health principle in action. You can learn more about the discovery of the Avermectins and Artemisinins in the advanced material Avermectin and Artemisinin – Revolutionary Therapies against Parasitic Diseases produced by the Nobel Assembly.

In 2011 we took a look at Professor Youyou Tu’s research that led to the development of Artemisinin therapy for malaria, and the key role played by mouse models of malaria infection,  in a post entitled “George is OK: Thank the men who stare down microscopes!”

While the news reports don’t state which drugs Cloony took to beat malaria, It is most likely that he was treated with artemisinin-based combination therapies (ACTs), which became available in the late 1990s and are now in widespread use.  If that is the case, he has benefited from mouse studies done in China the late 1960s and early 1970s when over 100 traditional herbal remedies were screened in a rodent model of malaria for anti-malarial activity (1). Eventually “Project 523” scored a hit when Professor Tu Youyou identified an extract of the plant qinghao, scientific name Artemisia annua, which had good anti-malarial activity, leading to the development of the artemisinin-based anti-malarials which have become the first-line treatment for malaria in the past decade.

We congratulate this years Nobel laureates in Physiology or Medicine, their research has improved the lives of hundreds of millions of people across the world over the past 3 decades, and will continue to do so. We hope that their success continues to inspire scientists around the world to rise to current and future public health challenges!

Speaking of Research

OLAW investigates Primate Products, Inc. and praises staff who care for animals!

This one comes with a helping of déjà vu! On Friday, the National Institutes of Health (NIH) Office of Laboratory Animal Welfare (OLAW), published the results of an investigation into Primate Products, Inc. a facility in Hendry County, South Florida that breeds monkeys for medical research, including NIH funded research. The OLAW is the federal body responsible for monitoring compliance with the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals that regulates animal research funded by US Government Offices and Agencies – including the NIH – and the breeders that supply animals for the research they conduct. The OLAW report (which you can download here) detailed the results on an investigation which was prompted by complaints submitted by the animal rights group PETA after one of their employees infiltrated Primate Products.

In a statement accompanying the publication of OLAW’s findings, as reported by, the federal agency commended Primate Products administration and its:

“dedicated and caring staff for promptly and thoroughly addressing all of the noncompliant items,” wrote the office’s Axel Wolff in an email. “We now find that your program is operating appropriately under monitored self-regulation … We appreciate your forthright communications and prompt responses to all questions and hereby close this investigation.”

The report cleared Primate Products of many allegations made by PeTA.  The report also noted the efforts of Primate Products to address some breaches of regulations and to ensure that care provided to the animals meets the highest standards. Most of these were small technical changes to update training, reporting, or animal handling procedures, but also included the erection of an electric fence to prevent further intrusions following an unprecedented  incident when a wild bear broke into the facility and killed several monkeys.

Cynomolgus monkey, one of several monkey species supplied for scientific research by Primate Products. Image: André Ueberbach

Cynomolgus monkey, one of several monkey species supplied for scientific research by Primate Products. Image: André Ueberbach

PETA’s allegations were reminiscent of the case a couple of years ago during PETA’s campaign against hearing research that involved cats at the University of Wisconsin – Madison, where after a thorough investigation the OLAW cleared the researchers and University of the allegations made by PETA. They also remind us of the incident 5 years ago when another animal rights group (Stop Animal Exploitation Now) used leaked photos of monkeys undergoing appropriate veterinary care following fighting among group housed macaque monkeys (a normal, if infrequent, behaviour for the species) to make lurid allegations against Primate Products. On that occasion the USDA investigated the allegations, and cleared Primate Products of any wrongdoing, with USDA spokesperson Dave Sacks commenting:

It was a clean inspection report…there was nothing found that was against animal welfare regulations…Group housing of primates is allowed in the animal welfare regulations…with the mindset that’s more closely adapted to how they live in the wild. These animals do various fighting among themselves for hierarchy…so that will carry through to how they are housed…But if in those housing situations, if there is a monkey that gets injured, we require the facility to provide adequate care.”

As we’ve noted before, there’s a pattern in this. Animal rights groups have become adept in using infiltrations and Freedom of Information (FOI) record requests as the basis for (often spurious)  complaints to the USDA or OLAW that they then use to gain publicity and organize campaigns against individual researchers, and raise funds for future “investigations.” It’s a tactic that isn’t limited to animal rights activists of course. Not far away from Hendry County, Dr. Kevin Folta, a University of Florida professor who studies plant genetics and who is a dedicated science communicator, has been targeted by a vicious FOI-driven campaign by opponents of genetic modification of crops. It’s a campaign that is clearly aimed at silencing someone who was a strong voice for science, and an illustration of how Freedom of Information can be used to attempt to suppress the Freedom of Speech of scientists at government funded Universities.

Given the risk that those targeted by campaigns such as PETA’s against Primate Products might decide to stay quiet, it’s reassuring to note that Dr. Jeff Rowell, a veterinarian and President of Primate Products, has a great record of explaining the work done by Primate Products and how it supports important research in Universities and other biomedical research institutions across the USA. An interview he gave to a local journalist earlier this year is a good example of this willingness to engage with the public.

Primate products

These latest  PETA allegations are unlikely to be the last made by animal rights groups against Primate Products, but it is heartening that the regulators are willing to take an honest and objective look at the evidence. In the meantime we hope that Jeff Rowell and his colleagues at Primate Products will continue to be vocal advocates for science, just as their work supports crucial medical research across the nation.

Speaking of Research

Truvada prevents HIV infection in high-risk individuals! A clinical success built on animal research

In the past two weeks we’ve learned of a major advance in ongoing efforts to halt the spread of  HIV, two separate clinical studies have reported that a daily regimen of a pill called Truvada as a pre-exposure prophylaxis (PrEP) is highly effective in preventing infection in high risk groups. This success is a result not just of the dedication of the clinicians who conducted these trials, but also of a series of pivotal studies conducted in non-human primates more than a decade ago that laid the scientific foundations for them.

In the first study of more than 600 high-risk individuals conducted at Kaiser Permanente in San Francisco, which was published in the journal Clinical Infectious Diseases, researchers found that Truvada – a combination of the anti-viral drugs tenofovir and emtricitabine – was 100% effective in preventing infection.  In the 2nd  study, called the PROUD study and published online this week in the Lancet, of more than 500 high-risk men undertaken in 13 sexual health clinics in England Truvada reduced infections by 86%.

Truvada prevents HIV transmission in high-risk individuals. Image: AFP / Kerry Sheridan

Truvada prevents HIV transmission in high-risk individuals. Image: AFP / Kerry Sheridan

These results have been greeted with enthusiasm in media reports, with headlines such as “Aids vanquished: A costly new pill promises to prevent HIV infection” , “A pill designed to prevent HIV is working even better than people thought” and  “Truvada Protected 100 Percent Of Study Participants From HIV: This is exciting!”. It’s worth noting that these are not the only trials to show the potential for Truvada to block HIV infection, earlier trials in Kenya, Uganda and Botswana also showed that it could substantially reduce infection rates, including in heterosexual couples where one partner was HIV positive and the other was not. There has been some concern that those taking Truvada would be less likely to take other safe sex measures – such as using condoms – but the results of the PROUD study showed no difference in acquisition of other sexually transmitted infections between those who started Truvada treatment immediately and those who delayed for 1 year, suggesting that they did not engage in riskier behavior as a consequence of taking Truvada.

Thanks to a multi-pronged approach to preventing HIV infection, combining barrier methods such as condoms,  Highly Active Antiretroviral Therapy (HAART) to lower viral load in infected individuals, and the use of antiviral medications to prevent mother-to-child transmission, the spread of HIV infection has slowed dramatically in many regions of the world, and pre-exposure prophylaxis with Truvada certainly has the potential to help reduce it further.

As we applaud the researchers who conducted these first real-world evaluations of Tenofovir in high-risk populations, it is also a good opportunity to remember the researchers whose work led us to this point. One of those pioneers is Dr. Koen Van Rompay, a virologist at the University of California at Davis who played a key role in the early development of Tenofovir and  its evaluation in pre- and post- exposure phophylaxis in macaque models of HIV infection. In 2009 Dr Van Rompay wrote an article for Speaking of Research explaining how important animal research was to the early development of such HIV prophylaxis regimes, and how important it continues to be as scientists develop ever better treatments, which we share again today:

Contributions of nonhuman primate studies to the use of HIV drugs to prevent infection – Koen van Rompay

Since the early days of the HIV pandemic, as soon as it was clear that an effective HIV vaccine would still be years away, there has been considerable interest in using anti-HIV drugs to reduce the risk of infection following exposure to HIV (so-called prophylaxis). Animal models of HIV infection, especially the rhesus macaque, have played a major role in developing and testing these treatments.

The development of HIV drugs to treat HIV-infected persons has shown that many compounds that are effective in vitro (i.e., in tissue culture assays) fail to hold their promise when tested in humans, because of unfavorable pharmacokinetics, toxicity or insufficient antiviral efficacy. The same principles apply to the development of drugs to prevent HIV infection. The outcome of drug administration is determined by many complex interactions in vivo between the virus, the antiviral drug(s) and the host; with current knowledge, these interactions cannot be mimicked and predicted sufficiently by in vitro studies or computer models.

Testing different compounds in human clinical trials is logistically difficult, time-consuming and expensive, so only a very limited number of candidates can be explored in a given time. Fortunately, the development of antiviral strategies can be accelerated by efficient and predictive animal models capable of screening and selecting the most promising compounds. No animal model is perfect and each model has its limitations, but the simian immunodeficiency virus (SIV) of macaques is currently considered the best animal model for HIV infection because of the many similarities of the host, the virus and the disease. Non-human primates are phylogenetically the closest to humans, and have similar immunology and physiology (including drug metabolism, placenta formation, fetal and infant development). In addition, SIV, a virus closely related to HIV-1, can infect macaques and causes a disease that resembles HIV infection and AIDS in humans, and the same markers are used to monitor the disease course. For these reasons, SIV infection of macaques has become an important animal model to test antiviral drugs to prevent or treat infection.

Studies in rhesus macaques first indicated that Tenofovir could block HIV infection. Photo: Understanding Animal Research

Studies in rhesus macaques first indicated that Tenofovir could block HIV infection. Photo: Understanding Animal Research

Different nonhuman primate models have been developed based on the selection of the macaque species, the particular SIV strain and the inoculation route (e.g. IV injection, vaginal exposure) used (reviewed in (33)). These models have been improved and refined during the past two decades. For example, SIV-HIV chimeric viruses have been engineered to contain portions of HIV-1, such as the enzyme reverse transcriptase (“RT-SHIV”) that the virus requires in order to multiply or the envelope protein (“env-SHIV”) that the virus needs if it is to escape from a cell and infect other cells, to allow these models to also test drugs that are specific for HIV-1 reverse transcriptase or envelope (28, 35).

Many studies in non-human primates have investigated whether the administration of anti-HIV drugs prior to or just after exposure to virus can prevent infection. The earliest studies indicated that drugs such as the reverse transcriptase inhibitor zidovudine (AZT), the first approved drug treatment for HIV, were not very effective in preventing infection, but a likely reason for this was the combination of a high-dose viral inoculums used, the direct intravenous route of virus inoculation, and the relative weak potency of drugs at that time (2, 4, 13, 19, 20, 36). The proof-of-concept that HIV drugs can prevent infection was demonstrated in 1992 when a 6-weeks zidovudine regimen, started 2 hours before an intravenous low-dose virus inoculation that more accurately represented HIV infection in humans, protected infant macaques against infection (29). These results were predictive of a subsequent clinical trial (Pediatric AIDS Clinical Trials Group Protocol 076), which demonstrated that zidovudine administration to HIV-infected pregnant women beginning at 14 to 34 weeks of gestation, and continuing to their newborns during the first 6 weeks of life reduced the rate of viral transmission by two-thirds (10).

Since then, a growing number of studies have been performed in macaques to identify more effective and simpler prophylactic drug regimens. These studies generally used lower virus doses, sometimes combined with a mucosal route of virus inoculation that mimics vaginal or anal exposure responsible for the majority of human HIV infections. These studies demonstrated that administration of some newer anti-HIV drugs, including the reverse transcriptase inhibitors adefovir (PMEA), tenofovir (PMPA), and emtricitabine (FTC) that prevent the virus from multiplying in the infected cell, and the CCR5 inhibitor CMPD167 that stops the virus from binding the CCR5 receptor on the cell surface and entering a cell in the first place, starting prior to, or at the time of virus inoculation, was able to prevent infection, though with varying success rates (3, 4, 16, 24, 25, 31, 34, 35). Only very few compounds such as the reverse transcriptase inhibitors tenofovir, BEA-005 and GW420867, and the CCR5 inhibitor CMPD167, were able to reduce infection rates when treatment was started after virus inoculation. For those drugs that were successful in post-exposure prophylaxis studies, a combination of the timing and duration of drug administration was found to determine the success rate, because a delay in the start, a shorter duration, or interruption of the treatment regimen all reduced the prophylactic efficacy (5, 11, 21, 22, 26, 27, 31) , information that has guided the design of subsequent clinical trials.

While some of the compounds such as GW420867 that showed prophylactic efficacy in the macaque model are no longer in clinical development (e.g., due to toxicity or pharmacokinetic problems discovered later in pre-clinical testing), the very promising results achieved with tenofovir have sparked further studies aimed at simplifying the prophylactic regimen. Several studies in infant and adult macaques have demonstrated that short or intermittent regimens of tenofovir (with or without coadministration of emtricitabine) consisting of one dose before and one dose after each virus inoculation were highly effective in reducing SIV infection rates (15, 30, 32).

The demonstration at the beginning of the 1990’s that anti-HIV drugs can prevent infection in macaques has provided the rationale to administer these compounds to humans to reduce the likelihood of infection in several clinical settings. Antiviral drugs are now recommended, usually as a combination of several drugs, to reduce the risk of HIV infection after occupational exposure (e.g., needle-stick accidents of health care workers) and non-occupational exposure (e.g. sex or injection-drug use) (6, 7). As mentioned previously, drug regimens containing zidovudine and more recently also more potent drugs such as nevirapine have proven to be highly effective in reducing the rate of mother-to-infant transmission of HIV, including in developing countries (10, 14, 17), and save many thousands of lives every year . Because the short nevirapine regimen that is given to pregnant HIV-infected women at the onset of labor frequently induces drug resistance mutations in the mother that may compromise future treatment (12), tenofovir’s high prophylactic success in the infant macaque model has sparked clinical trials in which a short tenofovir-containing regimen was added to existing perinatal drug regimens to reduce the occurrence of resistance mutations and/or further lower the transmission rate (8, 9, 18, 30, 32).

Scanning electron micrograph of HIV-1, colored green, budding from a cultured lymphocyte. Photo: C. Goldsmith Content Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus

Scanning electron micrograph of HIV-1, colored green, budding from a cultured lymphocyte. Photo: C. Goldsmith Content Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus

Because an efficacious HIV vaccine has so far not been identified, the concept of using pre-exposure prophylaxis also as a possible HIV prevention strategy in adults has gained rapid momentum in recent years. The promising prophylactic data of tenofovir (with or without emtricitabine) in the macaque model (23, 32, 35, 37) combined with the favorable pharmacokinetics, safety profile, drug resistance pattern and therapeutic efficacy of these drugs in HIV-infected people, have pushed these compounds into front-runner position in ongoing clinical trials that investigate whether uninfected adults who engage in high-risk behavior will have a lower infection rate by taking a once daily tablet of tenofovir or tenofovir plus emtricitabine. The results of these ongoing trials are highly anticipated. An overview of the design, status and challenges of these trials which are currently underway at several international sites and target different high-risk populations can be found on the website of the AIDS Vaccine Advicacy Coalition (1, 23).

In conclusion, nonhuman primate models of HIV infection have played an important role in guiding the development of pre- and post-exposure prophylaxis strategies. Ongoing comparison of results obtained in these models with those observed in human studies will allow further validation and refinement of these animal models so they can continue to provide a solid foundation to advance our scientific knowledge and to guide clinical trials.

Koen van Rompay DVM Ph.D. is a research virologist at the California National Primate Research Center at UC Davis.

Cited literature
1. AIDS Vaccine Advocacy Coalition. August 2008, posting date. Anticipating the results of PrEP trials.
2. Black, R. J. 1997. Animal studies of prophylaxis. Am. J. Med. 102 (5B):39-43.
3. Böttiger, D., P. Putkonen, and B. Öberg. 1992. Prevention of HIV-2 and SIV infections in cynomolgus macaques by prophylactic treatment with 3′-fluorothymidine. AIDS Res. Hum. Retrovir. 8:1235-1238.
4. Böttiger, D., L. Vrang, and B. Öberg. 1992. Influence of the infectious dose of simian immunodeficiency virus on the acute infection in cynomolgus monkeys and on the effect of treatment with 3′-fluorothymidine. Antivir. Chem. Chemother. 3:267-271.
5. Böttiger, D., N. G. Johansson, B. Samuelsson, H. Zhang, P. Putkonen, L. Vrang, and B. Öberg. 1997. Prevention of simian immunodeficiency virus, SIVsm, or HIV-2 infection in cynomolgus monkeys by pre- and postexposure administration of BEA-005. AIDS 11:157-162.
6. Centers for Disease Control and Prevention. 1996. Update: provisional Public Health Service recommendations for chemoprophylaxis after occupational exposure to HIV. MMRW 45:468-472.
7. Centers for Disease Control and Prevention. 2005. Antiretroviral postexposure prophylaxis after sexual, injection-drug use, or other nonoccupational exposure to HIV in the United States: recommendations from the U.S. Department of Health and Human Services. MMWR 54:1-19.
8. Chi, B. H., M. Sinkala, F. Mbewe, R. A. Cantrell, G. Kruse, N. Chintu, G. M. Aldrovandi, E. M. Stringer, C. Kankasa, J. T. Safrit, and J. S. Stringer. 2007. Single-dose tenofovir and emtricitabine for reduction of viral resistance to non-nucleoside reverse transcriptase inhibitor drugs in women given intrapartum nevirapine for perinatal HIV prevention: an open-label randomised trial. Lancet 370:1698-705.
9. Chi, B. H., N. Chintu, R. A. Cantrell, C. Kankasa, G. Kruse, F. Mbewe, M. Sinkala, P. J. Smith, E. M. Stringer, and J. S. Stringer. 2008. Addition of single-dose tenofovir and emtricitabine to intrapartum nevirapine to reduce perinatal HIV transmission. J. Acquir. Immune Defic. Syndr. 48:220-3.
10. Connor, E. M., R. S. Sperling, R. Gelber, P. Kiselev, G. Scott, M. J. O’Sullivan, R. VanDyke, M. Bey, W. Shearer, R. L. Jacobson, E. Jiminez, E. O’Neill, B. Bazin, J.-F. Delfraissy, M. Culnane, R. Coombs, M. Elkins, J. Moye, P. Stratton, J. Balsley, and for the Pediatric AIDS Clinical Trials Group Protocol 076 Study Group. 1994. Reduction of maternal-infant transmission of human immunodeficiency virus type 1 with zidovudine treatment. N. Engl. J. Med. 331:1173-1180.
11. Emau, P., Y. Jiang, M. B. Agy, B. Tian, G. Bekele, and C. C. Tsai. 2006. Post-exposure prophylaxis for SIV revisited: Animal model for HIV prevention. AIDS Res. Ther. 3:29.
12. Eshleman, S. H., M. Mracna, L. A. Guay, M. Deseyve, S. Cunningham, M. Mirochnick, P. Musoke, T. Fleming, M. G. Fowler, L. M. Mofenson, F. Mmiro, and J. B. Jackson. 2001. Selection and fading of resistance mutations in women and infants receiving nevirapine to prevent HIV-1 vertical transmission (HIVNET012). AIDS 15:1951-1957.
13. Fazely, F., W. A. Haseltine, R. F. Rodger, and R. M. Ruprecht. 1991. Postexposure chemoprophylaxis with ZDV or ZDV combined with interferon-a: failure after inoculating rhesus monkeys with a high dose of SIV. J. Acquir. Immune Defic. Syndr. 4:1093-1097.
14. Gaillard, P., M.-G. Fowler, F. Dabis, H. Coovadia, C. van der Horst, K. Van Rompay, A. Ruff, T. Taha, T. Thomas, I. de Vicenzi, M.-L. Newell, and for the Ghent IAS Working Group on HIV in Women and Children. 2004. Use of antiretroviral drugs to prevent HIV-1 transmission through breastfeeding: from animal studies to randomized clinical trials. J. Acquired Immune Defic. Syndr. 35:178-187.
15. Garcia-Lerma, J. G., R. A. Otten, S. H. Qari, E. Jackson, M. E. Cong, S. Masciotra, W. Luo, C. Kim, D. R. Adams, M. Monsour, J. Lipscomb, J. A. Johnson, D. Delinsky, R. F. Schinazi, R. Janssen, T. M. Folks, and W. Heneine. 2008. Prevention of rectal SHIV transmission in macaques by daily or intermittent prophylaxis with emtricitabine and tenofovir. PLoS Med. 5:e28.
16. Grob, P. M., Y. Cao, E. Muchmore, D. D. Ho, S. Norris, J. W. Pav, C.-K. Shih, and J. Adams. 1997. Prophylaxis against HIV-1 infection in chimpanzees by nevirapine, a nonnucleoside inhibitor of reverse transcriptase. Nature Med. 3:665-670.
17. Guay, L. A., P. Musoke, T. Fleming, D. Bagenda, M. Allen, C. Nakabiito, J. Sherman, P. Bakaki, C. Ducar, M. Deseyve, L. Emel, M. Mirochnick, M. G. Fowler, L. Mofenson, P. Miotti, K. Dransfield, D. Bray, F. Mmiro, and J. B. Jackson. 1999. Intrapartum and neonatal single-dose nevirapine compared with zidovudine for prevention of mother-to-child transmission of HIV-1 in Kampala, Uganda: HIVNET 012 randomized trial. Lancet 354:795-802.
18. Hirt, D., S. Urien, D. K. Ekouevi, E. Rey, E. Arrive, S. Blanche, C. Amani-Bosse, E. Nerrienet, G. Gray, M. Kone, S. K. Leang, J. McIntyre, F. Dabis, and J. M. Treluyer. 2009. Population pharmacokinetics of tenofovir in HIV-1-infected pregnant women and their neonates (ANRS 12109). Clin. Pharmacol. Ther. 85:182-9.
19. Lundgren, B., D. Bottiger, E. Ljungdahl-Ståhle, E. Norrby, L. Ståhle, B. Wahren, and B. Öberg. 1991. Antiviral effects of 3′-fluorothymidine and 3′-azidothymidine in cynomolgus monkeys infected with simian immunodeficiency virus. J. Acquir. Immune Defic. Syndr. 4:489-498.
20. McClure, H. M., D. C. Anderson, A. A. Ansari, P. N. Fultz, S. A. Klumpp, and R. F. Schinazi. 1990. Nonhuman primate models for evaluation of AIDS therapy. Ann. N. Y. Acad. Sci. 616:287-298.
21. Mori, K., Y. Yasumoti, S. Sawada, F. Villinger, K. Sugama, B. Rosenwirth, J. L. Heeney, K. Überla, S. Yamazaki, A. A. Ansari, and H. Rübsammen-Waigmann. 2000. Suppression of acute viremia by short-term postexposure prophylaxis of simian/human immunodeficiency virus SHIV-RT-infected monkeys with a novel reverse transcriptase inhibitor (GW420867) allows for development of potent antiviral immune responses resulting in efficient containment of infection. J. Virol. 74:5747-5753.
22. Otten, R. A., D. K. Smith, D. R. Adams, J. K. Pullium, E. Jackson, C. N. Kim, H. Jaffe, R. Janssen, S. Butera, and T. M. Folks. 2000. Efficacy of postexposure prophylaxis after intravaginal exposure of pig-tailed macaques to a human-derived retrovirus (human immunodeficiency virus type 2). J Virol 74:9771-5.
23. PrEP Watch,
24. Subbarao, S., R. A. Otten, A. Ramos, C. Kim, E. Jackson, M. Monsour, D. R. Adams, S. Bashirian, J. Johnson, V. Soriano, A. Rendon, M. G. Hudgens, S. Butera, R. Janssen, L. Paxton, A. E. Greenberg, and T. M. Folks. 2006. Chemoprophylaxis with Tenofovir Disoproxil Fumarate Provided Partial Protection against Infection with Simian Human Immunodeficiency Virus in Macaques Given Multiple Virus Challenges. J. Infect. Dis. 194:904-11.
25. Tsai, C.-C., K. E. Follis, A. Sabo, R. F. Grant, C. Bartz, R. E. Nolte, R. E. Benveniste, and N. Bischofberger. 1994. Preexposure prophylaxis with 9-(-2-phosphonylmethoxyethyl)adenine against simian immunodeficiency virus infection in macaques. J. Infect. Dis. 169:260-266.
26. Tsai, C.-C., K. E. Follis, T. W. Beck, A. Sabo, R. F. Grant, N. Bischofberger, and R. E. Benveniste. 1995. Prevention of simian immunodeficiency virus infection in macaques by 9-(2-phosphonylmethoxypropyl)adenine (PMPA). Science 270:1197-1199.
27. Tsai, C.-C., P. Emau, K. E. Follis, T. W. Beck, R. E. Benveniste, N. Bischofberger, J. D. Lifson, and W. R. Morton. 1998. Effectiveness of postinoculation (R)-9-(2-phosphonylmethoxypropyl)adenine treatment for prevention of persistent simian immunodeficiency virus SIVmne infection depends critically on timing of initiation and duration of treatment. J. Virol. 72:4265-4273.
28. Uberla, K., C. Stahl-Hennig, D. Böttiger, K. Mätz-Rensing, F. J. Kaup, J. Li, W. A. Haseltine, B. Fleckenstein, G. Hunsmann, B. Öberg, and J. Sodroski. 1995. Animal model for the therapy of acquired immunodefiency syndrome with reverse transcriptase inhibitors. Proc. Natl. Acad. Sci. U.S.A. 92:8210-8214.
29. Van Rompay, K. K. A., M. L. Marthas, R. A. Ramos, C. P. Mandell, E. K. McGowan, S. M. Joye, and N. C. Pedersen. 1992. Simian immunodeficiency virus (SIV) infection of infant rhesus macaques as a model to test antiretroviral drug prophylaxis and therapy: oral 3′-azido-3′-deoxythymidine prevents SIV infection. Antimicrob. Agents Chemother. 36:2381-2386.
30. Van Rompay, K. K. A., C. J. Berardi, N. L. Aguirre, N. Bischofberger, P. S. Lietman, N. C. Pedersen, and M. L. Marthas. 1998. Two doses of PMPA protect newborn macaques against oral simian immunodeficiency virus infection. AIDS 12:F79-F83.
31. Van Rompay, K. K. A., M. L. Marthas, J. D. Lifson, C. J. Berardi, G. M. Vasquez, E. Agatep, Z. A. Dehqanzada, K. C. Cundy, N. Bischofberger, and N. C. Pedersen. 1998. Administration of 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) for prevention of perinatal simian immunodeficiency virus infection in rhesus macaques. AIDS Res. Hum. Retroviruses 14:761-773.
32. Van Rompay, K. K. A., M. B. McChesney, N. L. Aguirre, K. A. Schmidt, N. Bischofberger, and M. L. Marthas. 2001. Two low doses of tenofovir protect newborn macaques against oral simian immunodeficiency virus infection. J. Infect. Dis. 184:429-438.
33. Van Rompay, K. K. A. 2005. Antiretroviral drug studies in non-human primates: a valid animal model for innovative drug efficacy and pathogenesis studies. AIDS Reviews 7:67-83.
34. Van Rompay, K. K. A., B. P. Kearney, J. J. Sexton, R. Colón, J. R. Lawson, E. J. Blackwood, W. A. Lee, N. Bischofberger, and M. L. Marthas. 2006. Evaluation of oral tenofovir disoproxyl fumarate and topical tenofovir GS-7340 to protect infant macaques against repeated oral challenges with virulent simian immunodeficiency virus. J. Acquir. Immune Defic. Syndr. 43:6-14.
35. Veazey, R. S., M. S. Springer, P. A. Marx, J. Dufour, P. J. Klasse, and J. P. Moore. 2005. Protection of macaques from vaginal SHIV challenge by an orally delivered CCR5 inhibitor. Nat Med.
36. Wyand, M. S. 1992. The use of SIV-infected rhesus monkeys for the preclinical evaluation of AIDS drugs and vaccines. AIDS Res. Hum. Retrovir. 8:349-356.
37. García-Lerma J. G., Otten R. A., Qari S. H., Jackson E., Cong M. E., Masciotra S., Luo W., Kim C., Adams D. R., Monsour M., Lipscomb J., Johnson J. A., Delinsky D., Schinazi R. F., Janssen R , Folks T. M., Heneine W. Prevention of rectal SHIV transmission in macaques by daily or intermittent prophylaxis with emtricitabine and tenofovir. PLoS Med. 2008 Feb;5(2):e28


Behind the Scenes of Zebrafish Research

Today we have the 2nd in a series of articles by Jan Botthof, a PhD Student at the Cambridge University Department of Haematology and the world renowned Wellcome Trust Sanger Institute. Following his first article “Zebrafish: the rising star of animal models”, Jan discusses here how Zebrafish used in scientific research are housed, cared for and bred.

Today I am going to look at some of the things that have to happen in the background to allow scientists to carry out their research. These things include the rules and regulations covering zebrafish use in research, general care and daily work in the fish facility.

Zebrafish research in the UK is covered by the same laws that govern research on all other vertebrates, as outlined in the Animals (Scientific Procedures) Act (ASPA), originally instated in 1986 and recently revised to implement the provisions of the new EU directive. This means that the standard of care is just as high for fish as for mammals. All institutes housing fish need a licence ensuring that standards are met, every research project is evaluated for possible harm to the animals and all of the people involved in research or care for the fish receive mandatory training in order to ensure that the fish are treated correctly. Everyone takes utmost care to ensure that the fish lead a comfortable life in the zebrafish facility.

Zebrafish: Wellcome Trust Sanger Institute

Zebrafish: Wellcome Trust Sanger Institute

Zebrafish housing
Now that we have covered the basic legislation, let’s talk about essential zebrafish care. Nowadays, fish are usually housed in special rooms, unlike the beginning of their use in research back in the 1970’s, when they were commonly just kept in a few tanks on a shelf in the lab. These rooms are designed to keep a constant temperature (between 24 and 28°C depending on the institution) and the lights are programmed to give a constant light-dark cycle to simulate the sun (usually around 16 hours of light and 8 hours of darkness).

Various commercial fish housing systems exist, but most of their features are very similar. The basic components of such a system are the fish tanks, racks to hold them, an integrated water supply, as well as water filtration and monitoring components.

Typical tank used for long-term housing with holes for water to flow in/out and to allow easy access for feeding.

Typical tank used for long-term housing with holes for water to flow in/out and to allow easy access for feeding.

The tanks are designed to allow a constant inflow of fresh water, easy removal from the rack and convenient access for feeding. These tanks used to be made of glass, but currently different kinds of plastics are much more popular due to the lower weight, making it much easier to handle them. Unless a procedure requires identification or separation of a specific fish, they are always kept in groups, not only for practical reasons, but also because zebrafish are very social animals and need interaction with other fish.

The water filtration and monitoring system ensures that the water is free of contaminants, has the right pH, salinity, hardness, enough dissolved oxygen and does not contain too much nitrite and nitrate stemming from waste products (i.e. fish excretions, excess food). Apart from the constant flow of fresh water, tanks are cleaned regularly to prevent the accumulation of waste products, as well as microbial and algal growth.

Zebrafish tanks at Dalhousie University Medical School. Image: Cory Burris

Zebrafish tanks at Dalhousie University Medical School. Image: Cory Burris

A separate quarantine room is also very important. This is where incoming fish from outside facilities are kept on a separate water system to prevent the introduction of parasites and diseases into the main facility. These fish are preferably received as early embryos, which are disinfected before shipping to kill any germs.

Just like there are different housing systems, there are different possible food sources, ranging from commercially available dry fish flakes to adult or larval brine shrimp. This diet is often supplemented with paramecia (small single celled organisms) to achieve optimal growth and survival rates when the fish are raised to adulthood. Exactly which diet is chosen depends on the individual facility. At the Sanger Institute, fish are fed adult brine shrimp, which are very rich in protein, soft and easily digestible (especially compared to brine shrimp cysts) and they are able to survive and swim even in fresh water. This is much closer to the natural diet that the fish would obtain in the wild than most commercially available diets.

Brine Shrimp. Image: Hans Hillewaert

Brine Shrimp. Image: Hans Hillewaert

Disease prevention
During the daily cleaning and feeding tasks, all tanks are monitored for diseased or injured fish, which are then humanely euthanized to minimize suffering. Euthanasia is usually carried out using an overdose of a common fish anaesthetic followed by destruction of the brain to ensure the death of the animal. Detailed records are kept to identify recurrent problems, such as potential parasitic infections. Dead fish are also removed from the tanks and their data recorded. This monitoring is especially important for fish that have been treated with drugs or that carry mutations likely to cause disease.

One essential component of working with fish is setting up matings between them. This is essential if you want to obtain embryos for studying them, or when crossing different genetically modified lines and many other procedures. Fish are placed in small mating tanks in the late afternoon before the actual mating, as zebrafish begin to mate right after sunrise in the wild. These mating tanks have a removable insert between the fish and the floor of the tank, so the fish cannot consume their own eggs, which they would otherwise do.

It is also possible to tilt the separation between the floor of the tank and the fish to further stimulate the fish, as they prefer shallow water for egg laying. If you need the embryos at a specific stage you can use tanks with a separator between the male and female, so you can control the time of the mating. An occasion when you would need to do this is when using the gene editing technique CRISPR to modify zebrafish genes, a process which requires injections of the Cas9 enzyme and appropriate guide RNAs during the first stage of development. Matings can be done in small groups or in pairs. It is very important to be able to correctly identify the sex of the animals – not only do you obviously need a male and a female to have a successful mating, but you also need to know this when you combine different transgenic lines. Here you would take a male from one line and a female from another, so you can put them back in the correct tank after the mating, as it is otherwise nearly impossible to identify individual fish.

Zebrafish mating tank with removable separation before and after assembly.

Zebrafish mating tank with removable separation before and after assembly.

Once the eggs have been laid and fertilized, you can collect them in a sieve, and place them in petri dishes containing water with some salts and minerals essential for development. Embryos are then raised at 28.5°C. Here at the Sanger, the zebrafish larvae are placed in nursery tanks when they are five days old and the yolk that feeds them during early development has run out. These fry reach sexual maturity within three months, from which point on they are considered adults and housed in the main facility. Zebrafish in the lab can live about two to three years, but usually we use younger fish for breeding, as they  lay more eggs.

In summary, a lot of work needs to be done before any actual research can be carried out. Moreover, a lot of effort is put into ensuring the health and welfare of all laboratory animals. The next time you read about some exciting new discovery made using animal research, try to picture how much effort was needed before any actual science was done!

Jan Botthof

Cotton Rats, Calves and Clinical Trials: New RSV vaccine shows great promise.

Respiratory syncytial virus (RSV) affects almost two-thirds of babies in their first year of life, and is a leading cause of bronchiolitis and severe respiratory disease in infants, young children, immunocompromised individuals, and the elderly throughout the world. It is a major cause of hospital admission for infants, and results in up to 200,000 deaths per year in children under the age of 5 years worldwide. Development of an effective vaccine is a public health priority, but has proven difficult, in part due to fact that RSV infection cannot be easily studied in the standard mouse and rat species that are most commonly used in laboratory research.

Oxford University researchers have announced the successful completion of the first trial of a vaccine against RSV in adult humans, which indicated that the vaccine was safe and could induce a robust immune response (though this Phase 1 study did not evaluate its ability to protect against RSV).

In winter RSV accounts for more than 10% of UK infant hospitan admissions.

In winter RSV accounts for more than 10% of UK infant hospital admissions.

In two papers published back-to-back in the journal Science Translational Medicine this week, the University of Oxford team and their colleagues at The Pirbright Institute and the Italian biotechnology firm Okairos (now Reithera Srl) report on the successful Phase 1 clinical trial in adult human volunteers, and the animal studies that led to the trial (1,2).

The basis for their vaccine was a vector derived from the chimpanzee adenovirus PanAd3, which was modified to express several highly conserved human RSV (HRSV) proteins, which they had shown could provide a good level of protection against RSV infection in cotton rats, which are one of the less commonly used laboratory animals, but a very useful model of viral infection of the respiratory system. The cotton rat immune system more closely resembles that of humans at a genetic level than that of more commonly used laboratory mouse and rat species, and because of this similarity cotton rats have been successfully used to evaluate novel therapies for RSV prior to clinical trials, including predicting the efficacy of these drugs in children. As we discussed in an earlier post on the development of gene therapy for Hemophilia B, choosing the right adenoviral vector for the task is critical, and the chimpanzee adenovirus was chosen because there is no preexisting immunity to it in the human population that would compromise its effectiveness as a vaccine vector.

Because studies with other virus-vectored vaccines had shown that heterologous prime/boost with Chimpanzee adenovirus based vector followed several weeks later by a modified vaccinia Ankara (MVA) – a vector used in several experimental vaccines, including HIV vaccines – generates a stronger  immune response than with chimpanzee adenoviral vector alone, the researchers next examined this strategy in cotton rats, demonstrating that intranasal prime/boost immunization was effective in protecting against infection, and did not lead to adverse effects.

The cotton rat - a valuable model for studying lung disease. Image: J.N. Stuart

The cotton rat – a valuable model for studying lung disease. Image: J.N. Stuart

While the cotton rat is a valuable model for the study of respiratory infections, it does not demonstrate all the clinical features of RSV infection, the infection tends to be less severe and to be cleared more quickly in cotton rats than in human infants, and  the extent to which vaccine efficacy in the cotton rat model of HRSV can predict efficacy in humans  is unclear. The scientists therefor evaluated the prime/boost vaccine in a more stringent model, calves, which are the natural hosts of the bovine form of RSV – called bovine RSV (BRSV). The disease course and epidemiology of BRSV infection in calves is very similar to that of HRSV in children, and the very high degree of similarity in sequences of the BRSV proteins to their HRSV equivalents used to develop the vaccine  suggested that the calf would be a valuable preclinical animal model to evaluate the safety and efficacy of their prime/boost vaccine strategies (2).

The results of this evaluation indicated that the different vaccination strategies they assessed were safe and did not cause any adverse immune responses, and showed that heterologous vaccination strategies that used an intranasal administration of the  PanAd3-based vector followed by intramuscular administration of the MVA-vased vector provided superior protection against BRSV infection compared to the PanAd3-based vector alone, or repeated doses of the PanAD3-based vector. As the authors described in the article reporting on the successful phase 1 trial (1) noted in their introduction, these studies paved the way for the evaluation of this vaccine strategy in 42 human volunteers:

In developing this approach toward an RSV vaccine in humans, homologous and heterologous combinations of PanAd3-RSV, including IN vaccination route, and MVA-RSV were tested in preclinical models. The genetic vaccines elicited RSV-specific neutralizing antibodies and T cell immunity in nonhuman primates and protective efficacy in challenge experiments in rodents with human RSV and in young seronegative calves with bovine RSV (32, 33). Of critical importance in both rodent and bovine challenge models was the absence of immunopathology associated with ERD after vaccination, with the calf model acting as a translational model for the development of a vaccine for the pediatric population. All regimens fully protected the lower respiratory tract from bovine RSV infection in the calf, and heterologous combinations resulted in sterilizing immunity in both upper and lower respiratory tracts (33).

The demonstration that the prime/boost vaccine strategy developed by the Oxford University team can safely induce a strong immune response in adult humans, and protect against RSV infection in both the cotton rat and calf models, is very promising, and pave the way for further clinical trials.  Professor Andrew Pollard of the Oxford Vaccine Group, who lead the clinical trial, is keen to now move the development of this much needed vaccine forward:

Both components of the vaccine were found to be safe and to create an immune response.

‘While I am delighted with these results, this was just a first trial. We need this vaccine for children and the elderly and that is where the efforts in vaccine development will now focus.’

Paul Browne

  1. Christopher A. Green et al. “Chimpanzee adenovirus– and MVA-vectored respiratory syncytial virus vaccine is safe and immunogenic in adults” Science Translational Medicine, Vol 7, Issue 300, 300ra126, 12 August 2015 Link
  2. Taylor G. et al.”Efficacy of a virus-vectored vaccine against human and bovine respiratory syncytial virus infections” Science Translational Medicine, Vol 7, Issue 300, 300ra127, 12 August 2015 Link

Animal models are essential to biological research: issues and perspectives

The following article by Françoise Barré-Sinoussi and Xavier Montagutelli was published on 31 July 2015 in the journal Future Science OA, and is reproduced here under a Creative Commons Attribution 4.0 License

Françoise Barré-Sinoussi leads the Regulation of Retroviral Infections Division at the Institut Pasteur in Paris, and was awarded the Nobel Prize in Physiology or Medicine in 2008 for her role in the discovery of HIV, and Xavier Montagutelli is head of the Central Animal Facility of the Institut Pasteur. This article follows the recent decision by the European Commission to reject the Stop Vivisection Initiative that sought to repeal European Directive 2010/63/EU on the protection of animals used for scientific purposes and ban animal research in the EU.

Animal models are essential to biological research: issues and perspectives

Françoise Barré-Sinoussi (1) & Xavier Montagutelli*,(2)

The use of animals for scientific purposes is both a longstanding practice in biological research and medicine, and a frequent matter of debate in our societies. The remarkable anatomical and physiological similarities between humans and animals, particularly mammals, have prompted researchers to investigate a large range of mechanisms and assess novel therapies in animal models before applying their discoveries to humans. However, not all results obtained on animals can be directly translated to humans, and this observation is emphasized by those who refute any value to animal research. At the same time, the place of the animals in our modern societies is often debated, particularly the right to use animals to benefit human purposes, with the possibility that animals are harmed. These two aspects are often mixed in confusing arguments, which does not help citizens and politicians to get a clear picture of the issues. This has been the case in particular during the evaluation of the European Citizen Initiative (ECI) ‘Stop Vivisection’ recently presented to the European Commission [1].


Humans and other mammals are very complex organisms in which organs achieve distinct physiological functions in a highly integrated and regulated fashion. Relationships involve a complex network of hormones, circulating factors and cells and cross-talk between cells in all the compartments. Biologists interrogate organisms at multiple levels: molecules, cells, organs and physiological functions, in healthy or diseased conditions. All levels of investigations are required to get a full description and understanding of the mechanisms. The first two, and in some instances three, levels of organization can be studied using in vitro approaches (e.g., cell culture). These techniques have become very sophisticated to mimic the 3D and complex structures of tissues. They represent major scientific advances and they have replaced the use of animals. On the other hand, the exploration of physiological functions and systemic interactions between organs requires a whole organism. It is, for example, the case for most hormonal regulations, for the dissemination of microorganisms during infectious diseases or for the influence of the intestinal microorganisms on immune defense or on the development of brain functions. In these many cases, no in vitro model is currently available to fully recapitulate these interactions, and investigations on humans and animals are still necessary. Hypotheses and models can emerge from in vitro studies but they must be tested and validated in a whole organism, otherwise they remain speculative. Scientists are very far from being able to predict the functioning of a complex organism from the study of separate cells, tissues and organs. Therefore, despite arguments put forward by the promotors of the ECI, studies on animals cannot be fully replaced by in vitro methods, and it is still a long way before they can.

Animal models have been used to address a variety of scientific questions, from basic science to the development and assessment of novel vaccines, or therapies. The use of animals is not only based on the vast commonalities in the biology of most mammals, but also on the fact that human diseases often affect other animal species. It is particularly the case for most infectious diseases but also for very common conditions such as Type I diabetes, hypertension, allergies, cancer, epilepsy, myopathies and so on. Not only are these diseases shared but the mechanisms are often also so similar that 90% of the veterinary drugs used to treat animals are identical or very similar to those used to treat humans. A number of major breakthroughs in basic science and medical research have been possible because of observations and testing on animal models. Most vaccines, which save millions of human and animal lives every year, have been successfully developed using animal models. The treatment of Type I diabetes by insulin was first established in the dog by Banting and McLeod who received the Nobel Prize in 1921 [2]. Cellular therapies for tissue regeneration using stem cells have been engineered and tested in animals [3]. Many surgical techniques have been designed and improved in various animal species before being applied to humans. The discoveries in which animal models played a critical role are indeed numerous and led to many Nobel Prizes.

It is, however, noticeable that the results obtained on animals are not necessarily confirmed in further human studies. Various reasons can be evoked. First, despite large similarities, there are differences between a given animal species and humans. For example, over 95% of the genes are homologous between mice and humans but there are also differences for example in the members of genes families, in gene redundancies and in the fine regulation of gene-expression level. These genetic differences translate into physiological differences which are increasingly better described and understood. While some people like the ECI promotors use these differences to refute the value of animal models, many including ourselves strongly advocate for further improving our knowledge and understanding of these differences and for taking them into account in experimental designs and interpretation of observations [4]. Moreover, these differences may provide opportunities to unravel novel mechanisms and imagine innovative therapies.

Research in mice has led to many medical advances - most recently the development of PD-1 inhibitors for treating cancers

Research in mice has led to many medical advances – most recently the development of PD-1 inhibitors for treating cancers

The second reason is due to genetic and physiological variations within each species or between closely related species. Laboratory mice have been developed as inbred strains which have highly homogeneous genetic composition to increase the reproducibility of results and the statistical power of experiments. Reports on animal models of human conditions often speak of ‘the mouse model of…’, referring in fact to observations made in a given genetic background. However, the clinical presentation often varies if another mouse strain is considered. A striking example is provided by a study published in November 2014 in Science by a team who reported that some mouse strains are fully resistant to Ebola virus, others die without specific symptoms and others develop fatal hemorrhagic fever [5]. Another example is the difference of responses to SIV, the monkey homolog to human HIV, between Rhesus macaques which develop simian AIDS and sooty mangabeys which do not develop symptoms despite high levels of circulating virus [6]. This range of responses reflects in fact the variety of clinical observations among human patients. These examples illustrate how animal models must be considered: no single animal model is able to mimic a given human disease which is itself polymorphic between patients, but the differences between strains or species provide unmatched opportunity to understand disease development and differential host response, and to eventually find new cures.

The second issue regarding the use of animals for scientific purposes is animal protection and welfare. This is the scope of the European Directive 2010/63/EU, which has set the regulatory framework for all animal research. Scientists have recognized for decades the importance of giving full consideration to three fundamental principles [7], which have become the backbone of the European Directive. First, animals must not be used whenever other, non-animal-based, experimental approaches are available, with similar relevance and reliability. Second, the number of animals used must be adjusted to the minimum needed to reach a conclusion. Third, all provisions must be taken throughout the procedures to minimize any harm inflicted to the animals. These principles, known as ‘the three Rs rules’, for replacement, reduction and refinement, have become the standard to which every project involving the use of animals is evaluated.

Animal research is conducted in compliance with regulatory provisions which cover the inspection and licensing of animal premises, the training and competence of all personal designing projects, performing animal procedures and taking care of animals and the mandatory authorization of every project by a competent authority upon ethical evaluation by an Animal Ethics Committee. The criteria for evaluation are based on the 3Rs rules and a cost–benefit analysis to evaluate if the potential harm to the animals, which must be reduced to the lowest possible level, is outweighed by significant progress in terms of knowledge on human or animal health. Regulation imposes that ethics committees include members concerned by animal protection and not involved in animal research. In response to the ECI, the European Commission has underlined, in a statement issued on 3 June 2015 [8], that animal experimentation remains important for improving human and animal health. At the same time, it is committed to promoting the development and validation of non-animal-based approaches, and to enforcing the application of the 3Rs rules by all players, including the research community. Europe has therefore implemented one of the strictest regulatory frameworks for the protection of animals used in research.

21st century medical research is highly interdisciplinary, a fact that is reflected in the design of new research institutions such as the Francis Crick Institute in London

21st century medical research is highly interdisciplinary, a fact that is reflected in the design of new research institutions such as the Francis Crick Institute in London

The greatest challenges faced by modern biomedical research concern complex, multifactorial, diseases such as cancer, cardiovascular diseases, infectious diseases, neurodegenerative disorders, pathological consequences of aging among others, for which all experimental approaches are indispensable because of their complementarity: biochemistry, genomics, cell culture, computer modeling, animal model and clinical studies. Research on relevant, carefully designed, well-characterized and controlled animal models will remain for a long time an essential step for fundamental discoveries, for testing hypotheses at the organism level and for the validation of human data. Animal models must be constantly improved to be more reliable and informative. Likewise, animal protection requires permanent consideration. These two objectives, far from being antagonistic, must be anchored in high-quality science.


1. The European Citizens ‘Initiative – Stop vivisection.
2. Nobelprize.Org – The discovery of insulin.
3. Klug MG, Soonpaa MH, Koh GY, Field LJ. Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts. J. Clin. Invest. 98(1), 216–224 (1996). [CrossRef] [Medline] [CAS]
4. Ergorul C, Levin LA. An example on glaucoma research: solving the lost in translation problem: improving the effectiveness of translational research. Curr. Opin. Pharmacol. 13(1), 108–114 (2013). [CrossRef] [Medline] [CAS]
5. Rasmussen AL, Okumura A, Ferris MT et al. Host genetic diversity enables ebola hemorrhagic fever pathogenesis and resistance. Science 346(6212), 987–991 (2014). [CrossRef] [Medline] [CAS]
6. Liovat AS, Jacquelin B, Ploquin MJ, Barre-Sinoussi F, Muller-Trutwin MC. African non human primates infected by SIV – why don’t they get sick? Lessons from studies on the early phase of non-pathogenic siv infection. Curr. HIV Res. 7(1), 39–50 (2009). [CrossRef] [Medline] [CAS]
7. Russell WMS, Burch RL. The Principles of Human Experimental Technique. Methuen, London, UK (1959).
8. European Commission – Annex to the communication from the commission on the European Citizen’s Initiative, ‘Stop Vivisection’.


Françoise Barré-Sinoussi
1. INSERM & Unité de Régulation des Infections Rétrovirales, Institut Pasteur, 75724 Paris, France
Xavier Montagutelli
2. Animalerie Centrale, Institut Pasteur, 75724 Paris, France

Clinical trial success for Cystic Fibrosis gene therapy: built on animal research

This morning the Cystic Fibrosis Gene Therapy Consortium (GTC) announced the results of clinical trial in 140 patients with cystic fibrosis, which demonstrate the potential for gene therapy to slow – and potentially halt – the decline of lung function in people with the disorder. It is a success that is built on 25 years of research, in which studies in animals have played a crucial role.

Cystic fibrosis is one of the most commonly inherited diseases, affecting about one in every four thousand children born in the USA, and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR gene produces a channel that allows the transport of chloride ions across membranes in the body, and the many mutations identified in cystic fibrosis sufferers either reduce the activity of the channel or eliminate it entirely. This defect in chloride ion transport leads to defects in several major organs including the lungs, digestive system, pancreas, and liver. While the severity of the disease and the number of organs affected varies considerably, cystic fibrosis patients often ultimately require lung transplant s, and too many still die early in their 20’s and 30’s as the disease progresses.

In a paper published in Lancet Respiratory Medicine today (1), the GTG members led by Professor Eric Alton of Imperial College London compared monthly delivery to the airway of a non-viral plasmid vector containing the CFTR gene in the liposome complex pGM160/GL67A using a nebuliser with a placebo group who received saline solution via the nebuliser. They reported stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group after a year. This is the first time that gene therapy has been shown to safely stabilise the disease, and while the difference between the treated and control group was modest, and the therapy is not yet ready to go into clinical use, it provides a sound bases for further development and improvement.


The Chief Executive of the Cystic Fibrosis Trust, which is one of the main funders of the GTC, has welcomed the results, saying:

Further clinical studies are needed before we can say that gene therapy is a viable clinical treatment. But this is an encouraging development which demonstrates proof of concept.

“We continue to support the GTC’s ground-breaking work as well as research in other areas of transformational activity as part of our mission to fight for a life unlimited by cystic fibrosis.”

So how did animal research pave the way for this trial?

Following the identification of the CFTR gene in 1989 scientists sought to create animal models of cystic fibrosis with which to study the disease, and since the early 1990’s more than a dozen mouse models of cystic fibrosis have been created. In some of these the CFTR gene has been “knocked out”, in other words completely removed, but in others the mutations found in human cystic fibrosis that result in a defective channel have been introduced. These mouse models show many of the defects seen in human cystic fibrosis patients and over the past few years have yielded important new information about cystic fibrosis, and in 1993 Professor Alton and colleagues demonstrated that it is possible to deliver a working copy of the CFTR gene using liposomes to the lungs of CFTR knockout mice and correct some of the deficiencies observed.

To get a working copy of the CFTR gene to the lungs of cystic fibrosis patients Professor Alton and colleagues needed three things:

• A DNA vector containing the working CFTR gene that is safe and  can express sufficient amounts of the CFTR channel protein in the lungs to correct the disease

• A lipid-like carrier that can form a fatty sphere around the DNA vector to so that it can cross the lipid membrane of cells in the lung, as “naked” DNA will not do this efficiently.

• A nebuliser device that produces an aerosol of the gene transfer agent so that it can be inhaled into the lungs of the patient.

Several early attempts to use gene therapy using viral vectors to deliver the working copy of the CFTR gene to patients failed because the immune response rapidly neutralised the adenoviral vector (see this post for more information on challenges using adenoviral vectors), and while attempts to use non-viral vectors were more promising, it was found that they caused a mild inflammation in most patients, which would make then unsuitable for long term use. As reported in a paper published in 2008 the GTC members developed and assessed in mice a series of non-viral DNA vectors, repeatedly modifying them and testing their ability to both drive CFTR gene expression in the lungs and avoid inducing inflammation. They finally hit on a vector – named pGM169 – which fulfilled both key criteria.

Earlier the consortium had undertaken a study to determine which carrier molecule to use in their non-viral gene transfer agent (GTA). To do this they assessed 3 GTA’s, each consisting of a lipid like molecule that could form a sphere around the non-viral DNA vector; either the 25 kDa-branched polyethyleneimine (PEI), the cationic liposome GL67A, or as a compacted DNA nanoparticle formulated with polyethylene glycol-substituted lysine 30-mer. Because there are significant differences in airway physiology between mouse and human they carried out this study in sheep, whose lung physiology more closely matches that of humans. The study identified the cationic liposome GL67A as the most promising candidate, resulting in robust expression of the CFTR transgene in the sheep lungs.

Studies in sheep play a key role in the development of gene therapy for cystic fibrosis

Studies in sheep play a key role in the development of gene therapy for cystic fibrosis

It now remained to bring the DNA vector and carrier together. In a 2013 publication the consortium reported that repeated aerosol doses of pGM169/GL67A to sheep over a 32 week period were safe and induced expression of the CFTR transgene in the sheep lungs, although the level of expression varied between individuals (this variation was also observed in human CF patients in the clinical trial reported today). A final study, this time in mice, assessed the suitability of the Trudell AeroEclipse II nebuliser as a device to create stable pGM169/GL67A aerosols, finding that it did so in a reproducible fashion. When aerosolized to the mouse lung, the new pGM169/GL67A formulation was capable of directing persistent CFTR transgene expression for at least 2 months, with minimal inflammation. These studies provided the evidence to support the gene delivery system and dosage strategy used in the clinical trial reported today.

The trial results announced today are an important accomplishment, but they mark a beginning rather than the end for Cystic Fibrosis gene therapy. It will be necessary to improve the efficiency of the therapy before it can enter widespread clinical use. Animal research will certainly play an important part in this work, notably the observation that the efficiency of CFTR gene delivery using this strategy was varied between individuals in both sheep and humans indicates that sheep are a good model in which to assess changes to improve the consistency and effectiveness of the gene therapy.

If you would like to know more about this cystic fibrosis gene therapy clinical trial you can watch two videos recorded at a meeting for cystic fibrosis patients at ICL on the  Cystic Fibrosis Trust website.

Paul Browne

1) Alton E.W.F.W. et al. “Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: a randomised, double-blind, placebo-controlled, phase 2b trial” Lancet Respiratory Medicine Published online July 3, 2015