Since the discovery of AIDS in 1981, more than 70 million people have been infected with HIV, of whom 36 million died. It is estimated that 40 million people worldwide are currently living with the virus (1). Existing drugs can hinder the onset of AIDS, but do not cure the human immunodeficiency virus (HIV) infection. Only an effective vaccine would be able to control HIV. But even more than 40 years after the discovery of the HIV-caused disease AIDS - and despite countless contrary announcements and promises - such a vaccine is still out of reach. This article explains why HIV research, which is predominantly based on animal experiments, has not yet been able to defeat the global pandemic.

HIV

Schematic presentation of the virus.

The human immunodeficiency virus (HIV) belongs to the family of retroviruses. After infecting a cell, the virus integrates its genetic material into the host cell's own DNA. It is highly likely that HIV is originally a zoonosis in which viruses were transmitted from monkeys to humans and then changed in the human host through adaptation (2). At the end of 2021, about 90,800 people were infected with HIV in Germany, with the number of new infections per year being comparatively low. In 2021, for example, an estimated 1,800 people were infected with HIV in Germany (3). However, about 95% of people with HIV live in developing countries, and in most affected regions, up to 40% of adults carry the virus (2).

The infection often remains asymptomatic for long periods of time. The viruses lead to a decrease of certain white blood cells and thus to an increased susceptibility to infections and cancer. It is only in the late stages of HIV infection that the clinical picture of AIDS (acquired immunodeficiency syndrome) develops, which manifests itself through advanced immunodeficiency and the resulting life-threatening infections.

HIV treatment

There are a number of drugs aiming to prevent the virus from multiplying. These so-called antiretroviral therapies cannot eliminate the viruses and their genetic material integrated into the host cell genome and thus offer no cure. However, they lead to the suppression of the reproduction of the virus and the onset of AIDS (2). In addition, antiretroviral therapy causes almost all patients to become no longer infectious, thereby reducing the spreading of the virus (3). The medication must be taken lifelong, requires careful monitoring, and must be taken regularly to prevent a development of resistance. In Germany, about 96% of those infected with HIV are treated with antiretroviral therapy (3). In addition to antiretroviral therapy to control the viruses, the treatment of opportunistic infections with pathogens, which are harmless to healthy people but can be life-threatening for people with a weakened immune system, plays a crucial role in the treatment of HIV-infected persons.

Since existing drugs offer no cure and a vaccination against HIV is still not available despite decades of research, prevention is and remains the most effective means of combating HIV and AIDS.

HIV research: model viruses and "animal models"

Researchers around the world are trying to investigate HIV infection using various so-called animal models with the aim to develop a vaccine. A major difficulty in vaccine development is that HIV is a human-specific virus that does not cause a disease in any species other than humans. Although the virus can multiply in chimpanzees and gibbons, it does not cause AIDS in them (4). HIV cannot infect other animals such as mice, rats, or macaques. One of the reasons is that the virus attaches itself to the human CD4 receptors and other co-receptors of certain immune cells (especially so-called T cells), which are structured differently in animals than in humans (2).
In order to be able to conduct research in animal models at all, two strategies are pursued: Either an attempt is made to make animals susceptible to the human virus by implanting human immune cells into the animal, or immunodeficiency viruses specific to other animal species are used as so-called model viruses. Most often, non-human primates and mice are used.

Primates in HIV research

Many researchers still assume that monkeys are good "models" because they resemble humans in their anatomy, physiology, and immune system. Nevertheless, these similarities are generally not enough to cause a HIV infection in monkeys. Only chimpanzees can be infected with HIV at all.

Chimpanzees in HIV research

In the 1980s and 1990s, mainly chimpanzees were used for HIV research (5). As mentioned above, chimpanzees can be infected with HIV, but the infection does not usually induce AIDS in the animals. There is one inglorious exception to this, in which the clinical picture of AIDS could be observed in a single chimpanzee that was infected with various virus isolates and then - after more than 10 years - developed AIDS-like symptoms. The fate of this monkey, which was named C499, shows once again how monkeys in animal experimentation have to suffer for years and without any benefit for humans:

• 1985: As one of 12 chimpanzees C499 is infected with an HIV isolate at the age of 2 years. The infection is confirmed.
• 1986: C499 is additionally infected with another HIV isolate. The animal remains clinically healthy.
• 1987: C499 is infected with a third HIV isolate. The animal continues to be clinically healthy.
• 1988: C499 shows a reduction of platelets and lymphocytes, but blood counts return to normal without treatment.
• 1993: As part of an annual routine examination, again a decrease of platelets and lymphocytes is detected, which persists from now on.
• 1995: C499 develops chronic diarrhea and has a suppressed immune system. Since he is the first chimpanzee to develop AIDS, no attempts to treat him are made, instead the course of the disease is observed.
• 1995 – 1996: Diarrhea persists, C499 develops a progressive non-regenerative anemia.
• 1996: In February, C499 is killed at the age of 14 due to the severity of his illness.

The blood of C499 has been injected into other chimpanzees in the hope to obtain a so-called animal model of AIDS. None of the animals to which the blood has been administered became ill or died from AIDS.

Macaques and other monkeys

Macaques that are not susceptible to HIV are instead infected with so-called model viruses

Other monkey species cannot be infected with HIV. Instead, they are infected with immunodeficiency viruses specific to their species, which are called SIV (simian immunodeficiency viruses) (6).

So, researchers are working with model viruses in model organisms. Furthermore, the simian viruses differ significantly from the human virus. Therefore, chimeric viruses were also generated, which usually consist of the basic SIV structure, but contain envelope proteins of the human virus. However, this means that the human-specific viral proteins are no longer able to dock onto the monkey's cells and the viruses then have to be further adapted in order to be able to infect the monkey, thereby in turn becoming more dissimilar to the human virus (7). The use of model viruses results in an irresolvable conflict between the need to modify the virus in such a way that it can infect monkeys, but at the same time it still needs to be similar enough to the human virus to be able to generate supposedly useful knowledge of human HIV infection at all. But even if this conflict would be solved successfully, the chimeric virus would still be confronted with a non-human organism and immune system, which behaves very differently than those of humans.

It is thus hardly surprising that, conversely, knowledge derived from humans cannot be transferred to monkeys. One option to cure HIV, which has already been achieved in humans in a few individual cases, is stem cell transplantation after whole-body irradiation. This experimental treatment was carried out on individual HIV-infected patients who were suffering from cancer. In order to be able to investigate this possible treatment strategy in so-called animal models, rhesus monkeys were infected with a chimeric virus, treated with antiretroviral therapy, and finally, after whole-body irradiation, transplanted with stem cells taken from them before infection. After discontinuation of the medication, viruses were quickly detectable again in 2 out of 3 animals; the successes achieved in humans could not be transferred to monkeys (6).

Mice in HIV research

While for monkeys it is tried to adapt the monkey-specific virus to make it more similar to the human virus, the procedure is different for so-called mouse models. Here, the "model" is made more similar to humans to allow replication of HIV in mice. For this purpose, parts of a human immune system are implanted in mice (8). Some of these humanized mouse models are described below.

• SCID-hu mice: Mice with a severe combined immune deficiency (SCID) lack important immune cells, the B and T cells. Pieces of thymus and liver tissue obtained from human embryos are implanted into the kidney capsule of the SCID mouse. The liver contains stem cells from which human T cells can develop. However, these T cells are mainly limited to the implantation site and are hardly found in the circulation of the animals. In addition, other important human immune cells are missing (9).
• hu-HSC mice: Mice are subjected to irradiation to weaken their immune system. Then they are implanted with human hematopoietic stem cells (HSCs), which are stem cells that are mainly found in the bone marrow in humans and from which various blood cells - including immune cells - emerge. As a result, different human immune cell types can form in the animals, but only few of the T cells, that are particularly crucial for HIV research, are formed (9).
• BLT mice: Is a combination of SCID-hu and hu-HSC model. Here, the mice are irradiated and implanted with human fetal liver and thymus tissue, then HSCs derived from the same embryo are injected. The lifespan of these animals is limited to about 6 months after implantation, as the human immune cells turn against the cells of the mice (graft-versus host disease, GvHD).

In all these mouse models, at its best, only individual aspects of HIV infection can be imitated and examined, and this is done in the wrong organism, which also has a limited lifespan that makes long-term studies impossible. It is known that HIV in humans affects not only T cells, but also other cells, for example brain cells. Those represent an important reservoir of the virus, which lies dormant in the cells in the brain and can spread again from there. It is hardly surprising that HIV research on so-called animal models, which cannot depict such crucial characteristics of human infection, has not yet led to a cure or an effective vaccine.

Other animals in HIV research

Cats are also affected by an immunodeficiency virus, the feline immunodeficiency virus (FIV). The so-called FIV model is the only naturally occurring model of immunodeficiency in animals and the course of the disease in cats has parallels to that in humans (10). For these reasons, cats have also been used in HIV research. However, FIV binds to a different receptor than HIV (CD134 instead of CD4) and can therefore infect B cells as well as T cells. Thus, the target cells of HIV and FIV differ and the use of cats in the search for a cure or vaccine for HIV does not seem very promising (2).

No perfect model in sight

None of the mentioned animal models is capable of replicating the human HIV infection in animals. In view of the generally poor translation from animals to humans, the use of model viruses in model organisms makes the efforts seem hopeless. However, instead of turning away from the - obviously failed - animal models, the researchers continue to work on a "refinement" of the models and/or the viruses, and individual aspects of the infection are investigated in different models (11). Thus, new humanized mouse models are constantly being developed (11).

It seems almost naive to assume that the knowledge gained in different animal models with different model viruses could be put together to form a complete picture of human infection with human-specific viruses and the associated disease.

Searching for a vaccine

According to a study published in 2008, there were already 197 clinical trials of 85 different potential HIV vaccines at that time. Only seven of the clinical trials were continued up to phase 3, which means that a large proportion of the studies (96.5%) were terminated prematurely, presumably mainly due to serious side effects or the lack of efficacy in humans (5). A lot of time, human and scientific resources, vast amounts of research funding, and countless animal lives have fallen victim to the search for a vaccine and have led to dozens of vaccines that are effective and safe – but only for monkeys with artificially induced HIV-like infections (5).

Vaccines that were effective in chimpanzees failed in humans. Even worse, several vaccine candidates, including Merck's V520, even increased the risk of contracting HIV or developing AIDS more quickly compared to placebo (1).

Testing drugs directly on humans?

It is often claimed that not testing on animals would result in unsafe drugs being tested directly on humans. This is already the case today, because as soon as preclinical studies - predominantly animal experiments - classify a drug as safe and effective, the first testing on humans takes place in clinical trials. It should be emphasized that the previous animal experiments only provide apparent safety and efficacy for humans, as shown, for example, by the HIV vaccine candidates, which increased the risk of infection in humans. In fact, the use of unsuitable animal models entails imponderable risks for human subjects.

Human-based HIV research

The development of HIV vaccines is difficult for a number of reasons. These include the high variability of the virus and the mechanisms by which HIV bypasses the immune system. In view of these challenges, it is even more important to use the best possible models and not to generate further imponderables and a lack of translatability through the use of so-called animal models and model viruses. Instead, the models should be as close to humans and the human infection as possible (1). Human cells are particularly suitable for this purpose. In the following, some publications describing the use of human-relevant models are briefly described.

Human cells in HIV research

HIV uses the machinery of infected human cells for its own reproduction. This provides a variety of targets for possible therapies. Insights into the processes involved are needed for a deeper understanding of the infection, which is the basis of developing a cure. Since HIV mainly infects CD4-positive T cells, which also form an important reservoir for the virus, these cells in particular are used in research of the infection.

For example, researchers from San Francisco and Chicago used T cells obtained from blood donations and systematically switched off 426 different genes in the cells using the CRISPR-Cas method, also known as “genetic scissors”. These knock-out cells were then infected with HIV and then analyzed. The researchers found 86 genes that may play a role in HIV replication. This provides insights into how HIV integrates into human DNA and establishes a chronic infection, which can ultimately lead to new treatments (12). Researchers from Munich used a similar approach, in which several genetic knock-outs and also knock-ins were generated, which made it possible to study the interaction of cellular and viral components with single-cell resolution (13).

Scientists from San Francisco studied T cells derived from HIV-infected patients and were able to identify the cell types that are particularly susceptible to HIV, and to observe changes in cellular receptor proteins induced by HIV (14).

Human organoids in HIV research

HIV can cause an inflammation of the brain and lead to cognitive impairment through neurodegenerative processes. Up to 50% of HIV-infected people suffer from such impairments to a greater or lesser extent, even if they are treated with antiretroviral drugs (16). In order to analyze the underlying processes and to thereby enable new therapeutic strategies, human brain organoids have been developed, which consist of different neural cells and specialized immune cells found in the brain. Infection of these brain organoids with HIV led to inflammation and damage to nerve cells. The system can be used to investigate the development and course of HIV-induced nerve damage and to develop new therapies (17).

Since the infected nerve cells in the brain represent a virus reservoir that has so far been neglected in HIV research, it is expected that research on brain organoids can make a significant contribution to the development of a cure (18). Further developments of organoids such as the integration of blood vessels, the size and complexity of the organoids achievable as a result, as well as the inclusion of the blood-brain barrier will further contribute to enabling even more realistic and, above all, human-relevant investigations (19).

Bioinformatics approaches in HIV research

What could be more obvious than to orient the development of a vaccine to the reaction of the human immune system to the virus? For example, so-called broadly neutralizing antibodies (bnAbs) have been known for some time. These are antibodies that the immune system produces and that are able to fight and protect against a wide range of different variants of the virus. Infusion of bnAbs has shown to be a promising alternative to antiretroviral therapy against HIV. A key challenge in the use of bnAbs is to prevent the development of resistance to treatment. This can be achieved with a combination of several bnAbs. In this context, researchers used a bioinformatics approach to optimize the effectiveness of bnAb therapy. For this purpose, data obtained from clinical studies on the change of the virus over time was used to establish models of virus dynamics and the influence of individual mutations on the development of resistance were obtained. It has been shown that a mixture of three bnAbs is necessary to effectively suppress the virus in the long term (20).

bnAbs not only represent a promising therapeutic option, they could also be the key to the development of a vaccine. For this purpose, it is necessary to identify the structural components of the virus that stimulate the immune system to produce bnAbs in order to obtain an effective immunogen that could be used as a vaccine. Using ultra-deep sequencing data from 14 non-HIV-exposed donors, the researchers identified the most promising bnAbs and the human genes needed to produce them and developed vaccine candidates (15).

The role of human-relevant models

While much of HIV research is still focused on "animal models" that have not led to the development of the long-awaited vaccine for decades, animal-free research is finally showing promising results on the way to a vaccine. Since research is carried out directly on human cells and patient material, these methods generate human-relevant results and will also contribute to accelerating HIV research, as the time-consuming and ineffective "refinement" of so-called animal models will no longer be necessary and the resulting lack of transferability of the results to humans will be eliminated.

It is also indicative of the failure of HIV research based on animal experimentation, which has been struggling for decades to infect monkeys and other animals with HIV, that essential aspects of HIV infection are only being uncovered today and thanks to modern human-based methods. Without an in-depth understanding of the processes by which the virus interacts with human cells – which will certainly not be elucidated in any animal model, no matter how elaborately refined – neither a cure nor an effective vaccine can be developed.

Prevention

HIV is transmitted through blood and other infectious body fluids (semen, vaginal secretions, and the film on the intestinal mucosa). Most often, transmission occurs through unprotected sexual intercourse (2). In addition, HIV can also be transmitted through the use of joint syringes when consuming injectable drugs. The risk of infection through blood products is low in Germany nowadays due to the testing of donated blood with effective procedures, resulting in HIV being transmitted in less than one in 3 million blood transfusions (2). The infection can also be transmitted from mother to child at birth or during breastfeeding. Here, the risk of infection of the child can be significantly reduced by retroviral therapy, by caesarean section, and by not breastfeeding (2).

HIV is mainly transmitted by people whose HIV infection is still undetected. It is estimated that about 8,600 infections were still undiagnosed in 2021. Easily accessible testing services and better information about infection risks are important to obtain early diagnosis and to prevent infections (3).

Various preventive measures have also been developed. Pre-exposure prophylaxis (PrEP) is a tool for preventing infections. Since September 2019, the statutory health insurance companies cover the costs for people with a substantial risk of HIV (3). HIV medication is taken prophylactically by healthy people as a preventive measure if they have an increased risk of becoming infected with HIV. In the case of so-called post-exposure prophylaxis, which should be carried out after potential contact with the virus, medication must be started within hours after the possible infection.

Despite the availability of preventive drugs, all of which are associated with side effects, the renunciation of unprotected sexual intercourse is and remains the most important option for prevention. Protected sexual intercourse using condoms effectively helps to avoid infection with HIV and other sexually transmitted pathogens. This protection should be a matter of course for frequently changing or new sexual partners and before dispensing with condoms in long-term partnerships, both partners should be tested for HIV. If one of the partners is infected, sex without condoms should only be considered after successful retroviral treatment, which has been proven to minimize the viral load.

Conclusion

HIV research, which is based on animal experiments, has so far mainly produced new "animal models" in which animals are infected with HIV or similar "model viruses" at great expense. To this day, more than 40 years after the discovery of AIDS, there is no cure or vaccine available. On the contrary, the obsession with animal models has led to the fact that many important aspects of the infection have not yet been understood and are thus completely ignored in the development of drugs and vaccines (1). The results of HIV research to date are correspondingly sobering: although existing drugs help to delay the onset of AIDS, they cannot cure HIV.

In order to finally gain a comprehensive understanding of the human HIV infection - which will enable the development of effective drugs and vaccines in the first place - the use of human-based methods and models is indispensable. In addition, effective education and prevention play a crucial role to impede the spreading of the disease.

References

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