Research with monkeys
Monkeys are used in research only when it is not possible to use other species. They are employed primarily in studies into the disorders, structure and function of the brain, and in research to produce a vaccine for HIV/AIDS. Monkeys are needed for this work because they are so closely related to humans.
In studies of the safety of drug candidates before being used in humans documentation according to GLP (Good Laboratory Practice) is required. Monkeys are used only in cases where the drug authorities require that these tests are performed on monkeys.
The Astrid Fagræus Laboratory (AFL)
The Astrid Fagraeus Laboratory (AFL), where experiments with monkeys are performed, includes one of the few facilities in Sweden with authorization to conduct animal testing under GLP. AFL is also the first in Sweden to become accredited by AAALAC International Organization for its high quality of animal welfare.
HIV vaccine research
HIV/AIDS – a global threat without cure
Since the 1980s, about 35 million people have died of HIV/AIDS, and the epidemic is still regarded a global health threat. In 2015, there were near to 37 million HIV infected individuals in the world, of which a majority in sub-Sahara Africa, according to the World Health Organization (WHO). It is primarily people of reproductive age who are affected, though children also account for a high percentage of those infected. This epidemic is the world's largest yet, more deadly than either the Black Death or Spanish Flu. The disease is a major drain on countries' productivity and an entire continent is on its knees.
Without treatment, HIV infected individuals are at risk of dying within ten years. However, treatments available today do not cure the HIV infection but can, in a best-case scenario, slow its progression and prevent AIDS from developing. These medicines are expensive and can cause serious side-effects, are difficult to administer and require regular healthcare visits.
Furthermore, they often become ineffective over time as the virus changes and develops resistance to them. They are not therefore a realistic alternative for the vast majority of HIV carriers living in low-income countries. An effective and accessible vaccine could be a solution, as a complement to other preventive strategies.
Only monkeys and humans susceptible to HIV
There are two types of HIV that can lead to AIDS: HIV-1 and HIV-2. HIV-1 has spread virtually worldwide, while HIV-2 is to be found predominantly in West Africa. The HIV-like simian immunodeficiency virus (SIV) occurs in African monkeys without causing disease. However, SIV can induce an immunodeficiency disease similar to AIDS in experimentally infected Asian macaques such as rhesus and crab-eating macaques.
Animal research is necessary in the development of a vaccine for HIV/AIDS. It is primarily macaques that are used in studies involving experimental infection with HIV or SIV. Smaller experimental animals such as mice, guinea pigs and rabbits are not susceptible to these infections.
Chimpanzees and gibbons have also been used in some cases as they are the animals that are most susceptible to HIV-1 infection, but ethical and legal considerations have meant that the use of these animals for HIV vaccine trials is very limited. For some years now, it has been prohibited in the EU to use so-called great apes (chimpanzees, gorillas, orangutans, and bonobos) in research.
Asian monkeys are not susceptible to HIV-1. This being the case, the most regularly used animal models for AIDS research involve the experimental infection of macaques with SIV or HIV-2, or with a hybrid virus of SIV and HIV-1 known as SHIV.
This happens in a HIV vaccine study
It is possible to investigate the protective effect of HIV or SIV vaccines using a relatively small number of monkeys for each study. The animals are divided into different groups: some will be given the vaccines to be tested (candidate vaccines), while others will make up an unvaccinated control group. The animals will typically be vaccinated three to four times at intervals of a couple of months. After the final vaccination the monkeys are given an infectious virus to determine whether the vaccine offers protection against HIV/SIV infection. Researchers will often compare two or more candidate vaccines in the same trial, with six monkeys in each vaccine group and in the control group.
We do not yet fully understand how the body's immune system reacts during infection with HIV or SIV. It is therefore important to take blood samples during the trial to monitor the development and function of some cell types. These results are then compared with how the vaccination protects against infection. Only small volumes of blood are taken from the monkeys during the trial.
In order to get an idea of the level of protection afforded against the virus (HIV or SIV) in the vaccinated monkeys, researchers measure the concentration of the virus in the blood during the first three to six months. Investigating the level of protection afforded against the full-blown disease (AIDS) would require the animals to be monitored for a longer period. But trials are never allowed to proceed to the point where the disease has developed fully. As soon as the monkeys show signs of the disease they are removed from the study and euthanized in a pain-free manner with an overdose of anaesthetic.
At around a year, the follow-up period is considerably shorter in trials with monkeys than with humans (three to five years). This means that using monkeys ensures that the best strategies for vaccination against HIV benefit humans more quickly.There are also other advantages to using monkeys when carrying out the initial studies. Studies with humans have to include far more people as it is not possible to control who will become infected. Furthermore, anyone contracting HIV/AIDS during a vaccine study will have to undergo treatment for the rest of their life if they are to avoid dying of it.
Achieved research results
Researchers at Karolinska Institutet (KI) and the former Swedish Institute for Communicable Disease Control (SMI) were among the first in the world to use studies with monkeys, mainly macaques, to show that an HIV/SIV vaccine can offer protection against AIDS even if it does not confer full protection against the virus itself. This finding is important for future investigations into the protective effect of HIV vaccines in humans.
Together with research groups in Europe and the US, the Swedish researchers have investigated different types of vaccine and found that live attenuated virus vaccines offer the best protection. However, it is unlikely that this type of HIV vaccine will ever be used for humans because of the risk of it causing the disease. Research is instead focusing on other types of vaccine and combinations of different types of vaccine produced using new techniques such as viral DNA plus live recombinant vaccines.
An HIV vaccine trial on monkeys at the AFL used a combination of two vaccines: HIV DNA, and HIV MVA. Promising results from the trial led to this vaccine combination being tested on humans in a study in Sweden and then in Tanzania. These studies showed that nearly all of the vaccinated participants developed a strong immune response to HIV.
Overall, there has been tremendous progress in HIV vaccine research through the use of macaques. We now know a lot more than ten years ago about the building blocks needed to put together a safe and effective vaccine against the disease. But HIV is an extremely variable virus, and far from all research in the field has been successful. It is probably a long way to go before there is a fully functional vaccine in use in the regular health care.
Malaria vaccine research
Positive development – but difficult to reach all the way
In recent years there has been a positive development in the world as regard to malaria incidence, and the number of infected individuals has declined by about 20 percent since 2010. However, malaria still pose a huge global health burden. In December 2016, there were about 212 million malaria infected people in the world, most of them in southern Africa, according to the WHO. The number of deaths from the disease in 2015 was estimated to 429 000 people, of which about 30 percent were children under the age of five. Research from KI aims to help the development of a vaccine that could save the lives of hundreds of thousands of children.
Malaria is caused by parasites which are far more complex than the viruses and bacteria that we have vaccines for today. The malaria parasite has multiple stages in their life cycle where in each stage they express different antigens (proteins that are able create an immune response). This makes it very challenging for the development of an effective vaccine. Another challenge is that different types of vaccine responses (antibodies or cytotoxic T cell responses) are required for protection depending on where the parasite is in its life cycle. An intimate understanding of the interaction between the host immune system and the vaccine and how sustained and protective immunity can be generated is therefore critical.
To get a complete picture of the interactions between the immune system and the vaccine and to evaluate whether a protective and enduring immunity develops, it is necessary to use primates, such as rhesus macaques, in research. Primate immune system is very similar to the human, which is different in several respects to the rodent's for example. The kinetics, functions and levels of antibody response and T-cells are also very similar.
The ability to test the relevant doses of vaccine and the anatomical similarities between monkey and human is a great advantage when to evaluate the results and draw conclusions to proceed to clinical trials in humans. It is, for example, not possible to evaluate intramuscular administration of the vaccine, which is by far the most common method to give the vaccine, in mice. The target group for a vaccine is usually healthy people, including children. This adds an extra safety aspect, as the vaccine needs to be proven both exceptionally safe to use and protect against the disease before administered to humans.
Research on rhesus macaques at AFL
During 2017, researchers at the Astrid Fagraeus Laboratory (AFL) completed a malaria vaccine trial, including a total of 24 rhesus macaques which were brought in from the US via Germany. The ethical permit for this research covers the use of 120 rhesus macaques, but the current plan is to use no more than 24 animals. The development of the current vaccine is the result of decades of research in which several research groups from different countries have contributed. The vaccine has been refined in several stages and has undergone extensive laboratory testing and then tested on mice. It has now reached the last stage before it will begin to be tested in humans.
The malaria vaccine tested at the AFL is based on a unique conserved antigen that has been selected for its low variability. Previous studies have shown that vaccination with this antigen prevents mosquitoes to reproduce and transmit the malaria parasite. This makes it potentially possible to eradicate malaria from a geographical area. However, the antigen must be displayed in such a way that it stimulates the immune system to induce a protective response. Vaccines based on the same technology have already been tested against other diseases in humans with good results.
The level of response to the malaria vaccine that is needed to inhibit the parasite's reproduction in mosquitoes and further transmission to humans has already been established in previous studies. Researchers at AFL can therefore compere their results with these known levels, and there is no need to infect the monkeys in the present AFL study with live malaria parasites.
The malaria vaccine trial at AFL has shown very promising results, with vaccine responses that are above what have been detected in previous studies. Samples from the animals have been analyzed by several different methods in mulitple laboratories, to measure the strength, quality and durability of the vaccine responses, and the study results will be published in a scientific journal. A team of researchers and malaria experts from academia, the vaccine industry, and funders are discussing the progress into clinical trials in humans. Our hope is that this vaccine has the potential to be the first to protect against malaria.
In the recently completed study, the animals received the malaria vaccine three times, and their immune responses were carefully monitored by collecting regular blood samples (approx. 1 - 2 per month) for about a year. In the last procedure, a superficial lymph node and a bone marrow biopsy collected for a more detailed analysis of the immunological memory of malaria being built by the vaccine. The animals were not exposed to malaria parasites, as the ability to block parasite reproduction by the vaccine responses can be evaluated using a laboratory assay of the blood samples.
None of the monkeys developed fever, showed altered behavior or had symptoms e.g. at the injection area. Standard testing of liver and kidney enzymes after immunization showed a transient but mild effect similar to those found by known vaccines. Weight gain, growth and activity were normal in all animals throughout the study.
Since the monkeys were not exposed to malaria nor developed disease during the malaria study, the animals could eventually be included in tests of other vaccines. In this way, researchers could avoid the import of new monkeys, keeping the total number of experimental animals as low as possible. The new studies were conducted to investigate the direct and local response to different types of vaccines, using a method developed at Karolinska Institutet. In these studies, extensive sampling from injection areas and local lymph nodes were performed, and in order to avoid suffering, the monkeys were euthanized with an overdose of anesthetics in connection to the sampling procedure.
Animal housing at AFL
During their time at AFL, the monkeys have been living in groups of two or three individuals who grew up together and get along with each other. Monkeys at AFL live in an enriched environment that gives them the ability to express their natural behaviours. At AFL, significant focus is directed towards creating harmonic and social milieus for the animals. All animals at the facility have established relationships with their personal care-taker who trains and offers daily intellectual and physical exercises, as well as access to water games.
Research into the brain's function
PET technology in brain research
Positron emission tomography (PET) has enabled major progress in biomedical research, particularly in research into the structure and function of the brain. It is also important for detecting tumours and developing new medications. PET involves labelling organic molecules with a radioactive isotope and then injecting these tracers into one of the patient's blood vessels. The positron camera is then used to monitor how the molecules are absorbed into the brain. Before a tracer can be used on patients, it must first be tested on research animals, normally monkeys as they are closest to human beings in terms of brain structure and function.
The PET research carried out on the AFL monkeys is intended to develop new tracers so that PET can be used to investigate signal systems in the living human brain. These systems are vital for diagnosing and treating neurological and neuropsychiatric disorders such as Alzheimer's, Parkinson's, schizophrenia, depression and ADHD.
The close correlation between the results from monkey experiments and subsequent studies with humans has meant that researchers at Karolinska Institutet (KI) have been able to confirm the value of the monkey studies for developing suitable tracers. This work has included producing markers to investigate subtypes of dopamine, serotonin, noradrenaline and GABA receptors.
Researchers at KI have developed a tracer that has made it possible for the first time to investigate the serotonin transporter (SERT). They have shown in studies using monkeys that the marker binds selectively to SERT, paving the way for studies with humans. This method will be used in investigations into depressive disorders and into the mode of action and clinical guidelines for antidepressants.
What happens with monkeys in a PET trial?
The monkeys used for PET trials are totally healthy and live together for many years in compatible groups of four to ten. In this type of experiment a monkey is used roughly once a month, though often less frequently. It is anaesthetised in its cage at the AFL and remains anaesthetised throughout the experiment. Several hours later once it has fully regained consciousness it is returned to its cage. By way of preparation, a cannula is inserted into one of the monkey's legs at the AFL so that researchers can administer the tracer safely and take blood samples. Blood samples are taken during the experiment to enable analysis of the tracer in the blood plasma.
Each day will generally involve two experiments. The first sees the tracer being injected so that researchers can study its distribution in the brain. The second PET experiment is identical to the first except that a known reference substance is injected 10-20 minutes before the tracer. The reference substance binds to the receptor in question, thus preventing the tracer from doing so. These experiments demonstrate the degree to which the tracer binds specifically to the receptor, and are extremely important for subsequent studies on humans.