Looking for the Pefect Challenge

Researchers are characterizing existing challenge models and tweaking them to better mimic HIV transmission and pathogenesis

By Andreas von Bubnoff

Animal models are the major way to gain insights into human diseases and how to prevent them. In the field of AIDS vaccine research, nonhuman primates (NHPs), especially rhesus macaques, have emerged as the most relevant animal model. While these animals cannot be infected with HIV, they can be infected with simian immunodeficiency virus (SIV), the monkey equivalent of HIV, and SIV-derived challenge stocks have become the major challenge virus used in AIDS vaccine research.

But the quantity of challenge virus stocks is limited, so researchers have to propagate them, often by passaging the existing viruses in cultured cells. This can alter the challenge virus stock, leading to genetic and biological differences. Some challenge stocks are more infectious than others, while some can be neutralized more easily than others.

This has raised concern that even challenge stocks that initially came from the same source—such as SIVmac251—might become different in their biological properties once they have been propagated in different labs. Such differences can make it difficult to compare the outcomes of different NHP studies of vaccine candidates, even if they use the same challenge stock.

The host animals used in challenge studies can also differ from each other. Some respond to viruses with higher viral loads than others, and even animals within the same host species can have genetic differences, including major histocompatibility complex (MHC) type I alleles that protect against the challenge virus.

Researchers have started to characterize both host animals and challenge stocks to more fully understand these differences. “There is an increasing realization within the field that we have to be more exact where we talk about the viruses and the animals that we are using,” says David O’Connor, assistant professor of pathology and laboratory medicine at the University of Wisconsin-Madison, one of the researchers involved in the characterization effort. Eventually, better characterization may help researchers to better compare and interpret the results of vaccine studies.

But just understanding the differences may not be enough. Some researchers are calling for the development of a standardized challenge virus stock for evaluating AIDS vaccine candidates in NHPs. “What would really benefit the field would be to have a standard challenge stock of SIV that had desirable infectivity and pathogenic properties and that also had a neutralization phenotype that resembled primary isolates of HIV,” says David Montefiori, who directs the laboratory for AIDS vaccine research and development at Duke University Medical Center.

The origin of challenge stocks

The origin of most virus challenge stocks that are currently used in AIDS vaccine research is sooty mangabey SIV (see Fig. 1), which is thought to have given rise to HIV-2 in humans. While sooty mangabey SIV typically doesn’t cause disease in its natural hosts, it can infect and cause disease in rhesus macaques. In contrast, chimpanzee SIV, which is thought to have given rise to the HIV-1 epidemic, is unlikely to infect rhesus macaques because it is very similar to HIV-1, which does not replicate in rhesus macaques, says Brandon Keele, a senior scientist at the National Cancer Institute at Frederick.

Figure 1. Origins of Virus Stocks. (A) The challenge stocks SIVmac251, SIVmac239, and SIVmac316 originated from SIV-infected RM number 78 at the New England Primate Research Center (NEPRC). This SIV is thought to initially be derived from SIV-infected SMs at the California Regional Primate Research Center (CRPRC). At CRPRC, the infected SMs are thought to have infected rhesus macaques, some of which were shipped to the NEPRC in 1970. There, they are thought to have infected other RMs and eventually RM 78. (B) The challenge stocks SIVmacE660 and E543-3 also originated from an SIV-infected SM that infected RM F236. References:AIDS 7, S161, 1993Virus Res. 32, 183, 1994J. Med. Primatol. 24, 116, 1995; HIV Sequence Compendium 2001, 182-190;J. Virol. 79, 8991, 2005.

Two of the most commonly used rhesus macaque SIV challenge stocks, SIVmac239 and 251, are derived from rhesus macaques that are thought to have been infected with SIV from sooty mangabeys (see Fig. 1A). About 20 years ago, Ronald Desrosiers’ lab at the New England Primate Research Center isolated SIVmac251 from an infected Indian rhesus macaque (number 251) that had lymphoma. SIVmac251 is a biological isolate or a swarm, which means that it contains many different virus variants or quasispecies, similar to HIV that naturally infects humans. Desrosiers passaged this SIVmac251 from one macaque to another up to animal number 239, from which he derived a stock, and then cloned SIVmac239 from that. As a clone, SIVmac239 consists of genetically identical copies of the same SIV strain or variant, and is therefore much more defined than 251.

A third stock currently in use is SIVsmE660, a swarm virus that also originated from a sooty mangabey SIV that infected a rhesus macaque (see Fig. 1B). It was then passaged through an additional Indian rhesus macaque before it was eventually isolated from macaque number E660, according to Vanessa Hirsch, a senior investigator at the National Institute of Allergy and Infectious Diseases, who isolated this stock along with Philip Johnson in the late 1980s (1,2).

The fact that most challenge stocks are derived from the SIVmac239/251 and the SIVsmE660 lines (see Fig. 1) is a problem, says Cristian Apetrei, an associate professor at the University of Pittsburgh, because it doesn’t reflect the genetic diversity of HIV-1. “When we have only two strains in order to develop vaccine studies, [we] will be handicapped by the lack of virus divergence in the animal model,” he says. To add genetic diversity to the available challenge stocks, he has been deriving new SIV stocks from rhesus macaques infected with plasma from SIV-infected sooty mangabeys (3,4). Another advantage of the new strains is that they have not been changed by passage in animals or in vitro, he says. “[These are] unadapted strains directly derived from sooty mangabeys.”

In the mid-1990s, researchers also started to develop hybrid viruses called SHIVs that are clones of SIVmac239 combined with HIV Env protein. SHIVs are useful for studies of HIV-specific antibody responses. More recently, researchers developed SHIVs that contain the reverse transcriptase (RT) from HIV to test drugs that inhibit this enzyme. Some SHIVs, for example 89.6, have been made more pathogenic by passaging them through animals, says Adrian McDermott, director of immunology and vaccine discovery at IAVI. Passaging between animals can make a stock “hotter” or more virulent, perhaps because it selects for virus variants that replicate more quickly, adds Alan Schultz, a project director of the NHP program at IAVI.

Researchers also create “boutique” challenge viruses to answer specific research questions, O’Connor says.

“To culture is to disturb”

The different ways labs maintain and propagate challenge stocks has some researchers concerned that it could be difficult to compare the results of different studies. There is less concern with stocks that are clones like SIVmac239, than for stocks like 251 that are swarms composed of many different virus variants or quasispecies, O’Connor says, adding that there are at least three or four different 251 stocks, and different virus variants within these stocks could predominate depending on who prepared them. “Multiple people have virus stocks that they refer to as SIVmac251, but in fact these viruses, while closely related, are distinct,” adds R. Paul Johnson of the New England Primate Research Center.

Differences between virus stocks can arise when labs propagate them by passaging them in animals or in tissue culture, R. Paul Johnson says. “To culture is to disturb,” he says, quoting the French virologist Simon Wain-Hobson. “Even very seemingly minor changes in how the virus is propagated can perturb its characteristics,” R. Paul Johnson adds. Passaging the 251 stock in tissue culture can increase its susceptibility to neutralizing antibodies. Preston Marx, a professor of tropical medicine and microbiology at Tulane University, says his 251 stock has been passaged in tissue culture, which made it a little less pathogenic and a little more susceptible to antibody than the Desrosiers stock.

But despite the concern that challenge stocks might be different from each other, little is known about the extent of these differences. “Really the only way to understand those differences is to sequence the entire genome of the infecting viruses,” adds O’Connor, who is one of several researchers who have started to characterize different challenge stocks. In a collaboration initiated by R. Paul Johnson, O’Connor and colleagues are examining possible differences in the sequences of the 251 stocks from Desrosiers and Chris Miller at the University of California in Davis.

Keele and colleagues have found differences when they recently used single genome amplification (SGA) to characterize env gene sequences from different 251 stocks. SGA allows researchers to determine the proportion of virus strains with a certain sequence of the env gene within a challenge stock. The analysis found differences between the 251 stock from the lab of Norm Letvin, a professor of medicine at Harvard Medical School, and the Desrosiers stock, Keele says. While both stocks are still very similar, they have accumulated some unique changes due to independent propagation in different labs over time. “There aren’t any viruses in [Letvin’s] stock that are identical to the viruses in [Desrosiers’] stock,” Keele says. “It’s like taking a monkey on an island and then leaving it for thousands of years.”

Keele says it is unclear if such differences can result in biological differences that could affect the evaluation of candidate vaccines in animals challenged with the two strains. He says experiments are underway to see if there are differences in how the two stocks are transmitted in macaques.

O’Connor predicts there could be a wake-up call for the field when studies with different 251 stocks show different outcomes. “I think the field needs to have a clear-cut example of a case where viruses that were called the same thing had different outcomes,” he says. “I think that we will probably pick up such an example with SIVmac251 if we look hard enough. That will create a moment of action that will motivate the field to establish uniform standards for applying these viruses.”

But not everyone is convinced that differences between stocks matter that much. Letvin acknowledges differences between 251 stocks, but says that they should not matter once a vaccine candidate clearly works. “My working assumption is that these differences won’t obscure our ability to see a dramatically better vaccine approach than what we currently have available,” Letvin says.

Escaping variability

One way to improve the comparability of results would be to have a standardized challenge stock everyone in the field uses, says Montefiori. Ideally, such a standardized challenge stock should be a clone, Montefiori adds, to better allow researchers to find the immune correlate of protection. This is because in in vitro neutralization assays, they can then use the exact same virus strain that was used for the challenge to test if the animal’s serum has antibodies that can neutralize the challenge strain.

If, however, researchers use a swarm like uncloned SIVmac251 for a challenge, a different virus variant establishes infection in different animals, Montefiori says. As a result, the virus used in an in vitro neutralization assay of the animal’s serum after challenge will likely be different than the virus that established infection in the challenged animal. In many cases, researchers are measuring neutralizing antibodies as an immune correlate of protection with a different quasispecies or a different clone than what is transmitted, Montefiori says. He suggests that this complexity could be eliminated by using a molecularly cloned virus for the challenge and the assays.

Better reproducibility is also the reason why an IAVI project that compares different vectors for candidate vaccines uses SIVmac239, a clone, as a challenge virus. “Because it’s a clone, we will get the same virus loads in those animals every single time,” McDermott says. In addition, the type and time of escape mutations from the host’s T-cell response are very well characterized with 239, which means that any deviation from that pattern will give researchers clues as to whether there is a vector-specific effect.

The drawback of using a clone, however, is that it’s not very realistic (see box, below). Humans are not usually exposed to a single virus variant but rather a swarm. To combine the more realistic features of a swarm with the reproducibility of a clone, Desrosiers is trying to create a challenge stock that is a mixture of cloned viruses. It consists of SIVmac239 as a backbone with different versions of the envelope sequences present in the original 251 stock he generated in his lab. “I think to provide greater standardization and better control I would definitely like to see the field move toward the use of cloned viruses or even mixtures of cloned viruses,” Desrosiers says. “I just think it’s much better defined. You can have total control of what you are looking at.”

Clones or Swarms? 

In addition to developing challenge stocks that better mimic the biological properties of HIV, researchers are also trying to better mimic HIV transmission in animal models by using swarm viruses because humans are exposed to swarms, not clones. A recent study by Brandon Keele, of the National Cancer Institute at Frederick, and colleagues found that the swarm challenge stocks 251 and E660 actually mimic HIV transmission quite well in rhesus macaques. They used single genome amplification (SGA) to characterize env gene sequences of an SIVmac251 stock and an SIVsmE660 stock from a different lab (5). The study found that within each stock, the virus strains differed from each other by about 1%-2% at most, similar to the average HIV diversity in the blood of an HIV-infected person during the first few years of infection.

The study also determined a dose where rectal infection of rhesus macaques with these stocks leads to infection by just one or a few transmitted founder viruses, which is similar to what has been observed in HIV infection. The virus strains from the challenge stock that established infection were different in each infected animal, suggesting that just like in humans, the initial selection of the virus strains that established infection appears to be random, Keele says. Recently, David Watkins of the University of Wisconsin-Madison collaborated with Keele and George Shaw of the University of Alabama, to use SGA to determine a dose at which only a few transmitted founder viruses of SIVsmE660 establish infection in a repeat rectal challenge study (see Capsules from Keystone, IAVI Report, March-April, 2009; 6). This study is also an example of efforts to make challenge models more realistic by using heterologous challenge strains that differ from the sequence used for the construction of candidate vaccines. Proponents of this approach believe it better simulates the huge variation in HIV strains that infect humans than homologous challenge.

With all these different choices, getting the field to agree on one challenge stock as a standard could be difficult, Montefiori says. “You just cannot get the people at the primate facilities to agree—they all have their favorite challenge stocks, and since many of them have been using those challenge viruses for many years, it’s hard to get them to change.” Others see advantages to having an array of challenge virus stocks available. “I don’t think there is going to be one model that fits all,” Letvin says. “It depends on exactly what experiment one is doing.”

Looking to better mimic HIV

Challenge stocks also differ in how closely they mimic the biological properties of HIV. SIVmac239, for example, is harder to neutralize than HIV, which means that testing candidate vaccines with 239 could underestimate their ability to induce protective neutralizing antibody responses, Montefiori says. Also, SHIV89.6P infects target cells via the CXCR4 coreceptor in primates, unlike most mucosally transmitted strains of HIV, which infect target cells with the CCR5 coreceptor (R5), says Ruth Ruprecht, a professor of medicine at Harvard Medical School. As a result, 89.6P eliminates naive CD4+ T cells, unlike recently transmitted HIV which targets CD4+CCR5+ memory cells. Most monkeys infected with 89.6P lose their entire CD4+ T-cell population irreversibly within two weeks after virus challenge. This is not what happens to HIV-infected humans during acute infection. “People don’t die of AIDS in a year,” Ruprecht says. “So too pathogenic is not a good thing.”

Researchers are therefore looking for new challenge strains with more similar biological properties to HIV. Hirsch is experimenting with a clone called E543-3 that can be neutralized more easily than 239. It was derived from the same animal which was the source of the virus stock that gave rise, after passage in an additional macaque, to SIVsmE660 (7). Another alternative is SIVmac316, which was cloned from macaques infected with 239. SIVmac316 is macrophage tropic like most HIV strains and not T-cell tropic like SIVmac239, according to Schultz. It is also more neutralization sensitive and not as pathogenic as 239.

Researchers have also been developing R5-tropic SHIV challenge viruses. Ruprecht has developed neutralization-sensitive, R5-tropic SHIV strains that contain an HIV clade C envelope glycoprotein from a recently transmitted HIV strain isolated from an infected child, who was part of a mother-infant cohort in Lusaka, Zambia (8). These SHIV strains cause disease much more slowly than other SHIVs and pathogenic SIVs, making them more similar to HIV, Ruprecht says. “AIDS vaccines should focus on the transmitted forms of HIV-1,” she adds, “not on the highly aggressive, highly pathogenic end stage forms that are not typically transmitted among humans.”

Paul Bieniasz and Theodora Hatziioannou, of the Aaron Diamond AIDS Research Center in New York City, and colleagues recently developed another cloned challenge virus that is an engineered version of HIV called simian tropic HIV-1 (stHIV-1). It differs from HIV-1 only in that it contains an SIVmac239 version of its vif gene. This SIV Vif can destroy the APOBEC3 defense proteins of pigtail macaques, and can therefore infect them, although it doesn’t make them sick (9). The acute virus levels in the stHIV-1 infected pigtail macaques are almost as high as in acutely infected humans, but taper off later toward low or undetectable levels, similar to what is observed in human long-term nonprogressors.

However, the current stHIV-1 virus has an Env protein that is primarily X4 tropic, which means that it tends to infect different target cells than most HIV-1 strains currently circulating in humans. Bieniasz is currently developing versions of stHIV-1 with an R5 tropic Env protein, and hopes that future versions of stHIV-1 will eventually even cause disease in the pigtail macaques.

What about the host?

Another factor that differs in challenge models is the animal host. The vast majority of researchers in the field use rhesus macaques of Indian origin, in part because they were used in the polio vaccine effort, says O’Connor. “That’s what people had access to,” he adds. Other host animals being used for HIV vaccine research are rhesus macaques of Chinese or Burmese origin and cynomolgus macaques.

These host animals differ in many ways, including their susceptibility to infection. Indian rhesus macaques tend to be highly susceptible to the commonly used SIV challenge strains SIVmac239 and 251 because these strains were initially adapted to Indian rhesus macaques. “If you want to work with a highly pathogenic infection, then Indian rhesus would be the best,” Marx says. McDermott agrees, noting that SIVmac239 or 251 will cause higher viral load in these animals than HIV in humans. In contrast, Chinese rhesus macaques infected with these same strains die more slowly and show similar peak viral loads to HIV-infected humans, Schultz says.

However, the genetics of the immune system in Chinese rhesus macaques are largely undefined, whereas for Indian rhesus macaques, MHC alleles like Mamu-B*08 and -B*17 are known to be associated with protection. This enables researchers to exclude them from studies for a more rigorous evaluation of candidate vaccines. “When we do vaccine challenge studies in our laboratory, we always MHC class I type the animals so that we don’t include some animals that have those protective class I genes and others that don’t,” Letvin says.

Such genetic differences may be the reason that even if the same host species is used, the old adage that mice lie, and monkeys don’t always tell the truth may be correct. O’Connor’s group found that genetic differences in the MHC could explain why in a study of an adenovirus-based vaccine, both the vaccinated and the control group controlled the challenge virus (10).

Indian rhesus macaque on Cayo Santiago, an island off the coast of Puerto Rico that houses a colony of almost 1,000 Indian rhesus macaques. The island, also known as "monkey island," is a facility of the Caribbean Primate Research Center and one of the largest providers of Indian rhesus macaques for AIDS vaccine research in the US. The animal in the picture is an adult eight-year-old male, a resident of the smallest of the six social groups on the island. He likes walks on the beach and bananas. Photo courtesy of Andreas von Bubnoff.

In light of such findings, O’Connor says, it is worth revisiting past studies because perhaps what was considered an effective vaccine candidate was really more attributable to the monkeys in the study. “If you look back through the literature,” he says, “there are lots of animals that have been spontaneous controllers of virus in vaccine studies or animals that fared particularly poorly that often get written off in the process of writing a paper or get aggregated with the other animals in the study. If the fact that they are outliers has nothing to do with their treatment and has everything to do with their genetics, then that changes the way we may have to interpret a lot of those studies.”

O’Connor uses Mauritian cynomolgus macaques for his studies of the role of cellular immune responses in protection. They are genetically very similar to each other because they come from a small isolated population on the island Mauritius. “[They are] almost like a monkey mouse,” says McDermott, referring to inbred mouse strains. This may allow researchers to better study the effect of T cells on protection. “You are trying to keep the animal constant, keep the virus constant, and see what’s involved in protection,” says McDermott. O’Connor is characterizing cynomolgus macaques to better understand their protective MHC alleles and other factors that could affect the outcome of vaccine studies.

Biological differences are not the only factor that determines which animals researchers use for their studies. Sometimes, the reasons are more practical such as cost and availability. Because females are often used as breeder animals, fewer of them tend to be available for actual experiments, according to Letvin. As a result, mucosal challenge experiments in monkeys more often involve rectal than vaginal challenges. “There is no perfect way to model heterosexual transmission of the virus in monkeys because of simple animal availability,” Letvin says. Cost is a factor as well, says Ruprecht. “If someone tells me you can have a free Chinese origin rhesus monkey, would I say no? Of course not. I’ll take it.”

1. AIDS 7, S161, 1993 
2. Virus Res. 32, 183, 1994
3. J. Virol. 79, 8991, 2005 
4. Virology 362, 257, 2007
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6. J. Virol. 83, 6508, 2009
7. Virus Res. 32, 183, 1994
8. AIDS 21, 1841, 2007
9. Proc. Natl. Acad. Sci. 106, 4425, 2009
10. Vaccine 26, 3312, 2008