The Great Barrier

Understanding mucosal immune responses is critical to developing effective AIDS vaccines, but progress has been slow

By Andreas von Bubnoff

HIV is primarily a mucosal infection—about 85% of transmissions occur at the mucosal surfaces of the genitals or rectum and most of the target cells necessary for HIV replication are found in mucosal tissues.

The gut mucosa is also a critical battleground during acute HIV infection (see Beast in the belly, IAVI Report, March-April 2006). Just two weeks after initial infection is established, 70% of T cells in the gut are depleted. In the blood, perhaps 30% to 40% fewer T cells are observed one or two months after HIV infection, says Jiri Mestecky of the University of Alabama at Birmingham.

For these reasons, understanding mucosal immunity is important in preventing HIV transmission, and also in controlling infection. Bolstering immune responses at the mucosa during transmission may make it even harder for the virus to gain an initial foothold, and in the early stages of infection (see Figure 2), mucosal immune responses play an important role in limiting the depletion of T cells in mucosal tissues, averting permanent damage to the immune system. “There are lots of antiretroviral drugs [that] can partially restore CD4+ T cells in the blood,” Mestecky says, “but so far you cannot fully restore them in the mucosal tissues.” Satya Dandekar’s lab at the University of California in Davis has found that to restore CD4+ T-cell levels in mucosal tissues of rhesus macaques, antiretrovirals must be given within days or, at most, a few weeks after infection, a largely impractical time frame in human infection.

Figure 2: HIV Infecting a Mucosal Site

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Despite its perceived importance, only a few research groups study HIV infection at the mucosal level, says Lucia Lopalco of the San Raffaele Scientific Institute in Milan, Italy. “This is a huge gap,” Lopalco says. “We need more scientists who study mucosal immunity. But we are late because we should have started 20 years ago.”

Despite its perceived importance, only a few research groups study HIV infection at the mucosal level, says Lucia Lopalco of the San Raffaele Scientific Institute in Milan, Italy. “This is a huge gap,” Lopalco says. “We need more scientists who study mucosal immunity. But we are late because we should have started 20 years ago.”

The understanding of mucosal immunity in HIV infection is hampered by the difficulty of studying these types of immune responses in humans. Currently, AIDS vaccine clinical trials are not designed to systematically look for mucosal antibodies in external secretions. “[It] is a terrible mistake,” Mestecky says. “Everybody measures serum antibodies, which is fine, but it’s a mucosal disease after all.”

Measuring mucosal immune responses is difficult and, Lopalco says, it is also much harder to come up with an in vitro model for mucosal tissues because they harbor many kinds of cells in a specific arrangement. Even when mucosal responses are assessed, measurements are often not standardized, leading to contradictory results.

Despite these difficulties, researchers have gained important insights over the past several years into mucosal immune responses in HIV infection. But they are just beginning to understand their role.

Mechanism of protection

Researchers are using many different models to help determine the role of mucosal immune responses in protection against HIV. One involves challenge studies in nonhuman primates with live-attenuated simian immunodeficiency virus (SIV) vaccines. Researchers attenuate SIV by deleting the virus’s nef gene. Such studies have shown that vaccines containing this live-attenuated version of SIV can protect rhesus macaques from subsequent SIV infection (Science 258, 1938, 1992), however the mechanism of this protection is still unknown. Ashley Haase’s laboratory at the University of Minnesota, in collaboration with Paul Johnson of Harvard Medical School and IAVI’s Live-Attenuated Consortium, is studying this model to garner clues about how this vaccination strategy works and whether or not it involves mucosal immunity.

Researchers have also been studying individuals who remain HIV uninfected despite repeat exposure to the virus, a group known as exposed seronegatives (ESNs), to mine for clues about a possible role of mucosal immunity. Several explanations have already been used to explain this phenomenon in cohorts of sex workers and discordant couples, but studies that looked specifically at the presence of Immunoglobulin A (IgA) antibodies—the major type of antibody response in most human secretions—have led to contradictory results, according to Mestecky. Some studies suggest that HIV-uninfected partners in discordant couples have high levels of HIV-specific IgA antibodies in vaginal or urine samples (Nature Medicine 3, 1250, 1997). Additional studies showed that some of these IgA antibodies were directed against the coiled-coil pocket region of the gp41 part of HIV’s Env protein (AIDS 16, 1731, 2002). However, Mestecky says that other labs could not reproduce these results. Recently, samples from 70 women were sent to six different labs and the results still could not be confirmed. “[The uninfected women] have protection by some other mechanism, but not mucosal antibodies,” concludes Mestecky.

Researchers are also studying long-term nonprogressors (LTNPs)—people who are HIV infected but do not progress to AIDS within the typical time frame—to unlock potential clues about the role of mucosal immunity. Some studies have found mucosal antibodies directed not toward parts of HIV, but to the CCR5 coreceptor that HIV uses to infect T cells. Lopalco’s group has found that mucosal secretions from LTNPs contain CCR5-specific IgA and Immunoglobulin G (IgG) antibodies and follow-up studies suggest that loss of these antibody responses is associated with the development of AIDS. One possible mechanism is that such antibodies cause the CCR5 receptors to disappear from the surface of T cells because they are internalized (Blood 107, 4825, 2006).

In addition to antibodies, LTNPs may also have higher levels of cellular mucosal immune responses. Some LTNPs have a higher CD8+ T-cell response in the rectal mucosa than people who progress normally, according to studies from Barbara Shacklett’s lab at the University of California in Davis.

Researchers have also started to use in vitro models of mucosal tissue to see if antibodies can inhibit transmission of HIV. Lopalco’s group showed that anti-CCR5 antibodies can block transcytosis of HIV through a cultured monolayer of human epithelial cells, which is one way HIV is thought to enter the body through mucosal tissues (AIDS 21, 13, 2007). But Pam Kozlowski of Louisiana State University cautions that the transcytosis assay is very difficult to reproduce because the epithelial cells grown in vitro must form a really tight layer.

Measure for measure

To better understand mucosal immunity in humans, researchers must collect samples, but collecting secretions from mucosal tissues in the vagina or rectum can be difficult. One method, called lavage, involves washing the mucosal surfaces with a buffer solution and then collecting the liquid for analysis, but this approach can often dilute the secretions too much, making it hard to detect antibodies, according to Kozlowski. This is problematic because antibodies in mucosal secretions are already more diluted than in blood, according to Morgane Bomsel of the Institute Cochin in Paris.

An alternative approach developed by Kozlowski for use in both humans and nonhuman primates involves using an absorbent sponge called Weck-Cel to obtain vaginal and rectal secretions. She says it should be more acceptable to volunteers in clinical trials because it causes very little discomfort and is only in place for 10 minutes, at most. And unlike rectal washes, which require immediate processing, the sponge can simply be frozen after collection. Robin Shattock of St George’s, University of London will use the sponge method to sample vaginal fluids in a clinical trial he has initiated.

Measuring cellular immune responses in mucosal tissues is even more complicated. For colorectal tissues, a biopsy is needed to collect tissue samples, according to Julie McElrath of the University of Washington. This is an invasive and somewhat risky procedure. One possible complication is peritonitis, which occurs when the colon gets perforated. “If it’s only for research and not for the patient’s benefit, it makes it hard to convince people to do it,” says Jay Berzofsky of the National Cancer Institute (NCI).

To assess cervical cellular responses, researchers like Shattock use a small brushlike device called a cytobrush that is inserted into the cervix and rotated 360 degrees. The number of T cells collected in mucosal samples is usually much smaller than in blood—vaginal samples from a cytobrush contain only about 1,000 lymphocytes, Shattock says, much fewer than the 10 million obtained from a typical 10 ml blood sample. This drastically limits the assays that can be conducted, especially when researchers are interested in multiparameter analysis using flow cytometry. “That becomes almost undoable with those types of samples,” Shattock says.

It is also important to have fresh cells and to measure cellular immune responses in the samples within a few hours after collection, which requires having a laboratory available at the same site where samples are obtained. And while blood samples can be put directly into cytokine assays, mucosal tissue samples from rectal biopsies must first be run through a series of enzymatic digests to release the lymphocytes from the collagen matrix, Shacklett says, which is a time-consuming process.

Together, these limitations are part of the reason why mucosal immune responses are not routinely measured in clinical trials. McElrath says that in AIDS vaccine trials, mucosal samples are usually only taken from a subset of volunteers. “We wouldn’t do it in all people,” she says. “It’s just an amazing amount of work technically.” In the STEP trial, cellular immune responses were measured in the semen of only about 20 of the 3,000 volunteers, who were primarily men who have sex with men. Analysis of these samples is still ongoing.

Even when human samples are collected, there is inconsistency in the measurement of mucosal antibodies that can make it difficult to compare results. “Most of the data in the literature is really meaningless because people don’t know how to measure IgA responses in secretions,” says Kozlowski. Few labs have been able to accurately detect and quantitate anti-HIV IgA in clinical samples, according to a 2002 study (AIDS Res. Hum. Retroviruses18, 1291, 2002).

Given the inconsistencies in measuring mucosal immune responses, the US National Institutes of Health (NIH) has formed a group called the Mucosal Immunity Discovery Team chaired by Johnson that will try to standardize the measurement of cellular mucosal immune responses, according to Ronald Veazey of Tulane University, who is the group’s co-chair. A similar effort at the Center for HIV-AIDS Vaccine immunology (CHAVI) is trying to standardize measuring humoral mucosal immune responses, Veazey says.

Mucosal homing pigeons

To get around these limitations, some researchers have proposed a shortcut. Instead of directly measuring mucosal T cells, researchers could identify T cells from blood samples that are heading for mucosal sites. T cells that traffic to mucosal tissues express specific homing receptors on their surface. Antibody-secreting cells in the mucosal tissues have been shown to express such homing receptors three weeks after intranasal immunization (J. Clin. Invest. 99, 1281, 1997). “If you had a marker on cells that allowed you to purify them from peripheral blood,” Shattock says, “it would really change the number of parameters that we could assess in terms of mucosal immune responses.”

The two most well-known markers are the α4β7 receptor, which seems to be associated with trafficking to the gastrointestinal (GI) tract, and cutaneous lymphocyte antigen (CLA), which is associated with cells homing to skin tissues, Shattock says. But there are no receptors that have been specifically identified for homing to genital sites or the colorectum, he adds. “It’s definitely an important priority for the scientific community to start trying to address.”

Another problem is that one can never be certain that cells that express such a homing receptor will actually reach the desired mucosal tissues, Mestecky says. He compares it to a letter with an address. “Whether it will actually get there and have its effect is unknown,” he says. There is also limited time for measuring the cells that express such homing receptors in blood before they enter their target tissues, according to Marianne Neutra of Harvard University.

Routes of delivery

There are several ways to administer a vaccine to induce mucosal immune responses. Often a vaccine has to be delivered to mucosal tissues, for example intranasally, to induce mucosal immune responses, although systemic immunizations can also induce mucosal immunity. The company Mymetics is developing a mucosal vaccine using gp41-derived antigen, which is fused to a lipid anchor that is inserted into the membrane of a virosome, a stripped down version of the flu virus. Researchers at Mymetics have found that intranasal, as well as intramuscular, immunizations of the virosome-based vaccine can induce vaginal and intestinal mucosal IgA antibodies in rabbits and macaques, says Sylvain Fleury, the company’s chief scientific officer. “People believe that the classical route of administration which is intramuscular or intraperitoneal does not work for mucosal [immunity],” he says. “In our case [it] can trigger vaginal and intestinal [IgA] antibodies.” Mymetics plans to initiate a Phase I trial of this vaccine candidate later this year.

Similarly, a recent study has shown that intramuscular immunization alone or combined with intranasal immunization can protect rhesus macaques against a vaginal SHIV challenge (AIDS 22, 339, 2008).

Still, the strongest mucosal responses would be expected after mucosal immunization, Neutra says. Berzofsky’s lab showed that applying a vaccine rectally in rhesus macaques leads to a better mucosal cellular CD8+ T-cell immune response and better protection against a rectal challenge than subcutaneous administration of the same vaccine (Nature Medicine 7, 1320, 2001; J. Immunol. 178, 7211, 2007). “You can get mucosal T cells by systemic immunization [as well],” Berzofsky says, “but you don’t have the same level or the same quality.” Berzofsky’s lab also showed that the presence of mucosal CD8+ T cells in monkeys delayed the appearance of the virus in the blood (Blood 107, 3258, 2006).

Researchers have also learned in recent years that mucosal antibody responses are more localized than previously thought, according to Shattock. Previously researchers thought that there was a common mucosal immune system, meaning that if an immune response is induced at one mucosal site, induction also occurs at other sites. This may not be entirely true. “There are links between mucosal sites, but they are not universal,” Shattock says. For example, oral vaccination will give an immune response in the GI tract and in breast milk, but little vaginal response, while nasal immunization will promote strong vaginal responses but poor GI tract responses (see Figure 3).

Figure 3. Routes of Immunization

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Kozlowski’s lab has found mostly localized responses when using a strong immunogen—cholera toxin B subunit—to compare different mucosal delivery routes in women. After applying it orally, rectally, vaginally, and nasally, and measuring mucosal IgA antibodies in saliva, rectal, and vaginal secretions, she found that often the responses were observed at the site of vaccination. Only the nasal immunization route generated IgA responses in both the female genital tract and rectum (J. Immunol. 169, 566, 2002). Kozlowski says she was surprised to see such a distal response. “The female reproductive tract is pretty far away [from the nose].” Based on these results, Kozlowski is now working on intranasally-administered vaccine candidates.

But nasal immunizations could be risky if they involve viral vectors, which could migrate to the central nervous system, according to experiments in mice (J. Virol. 77, 10078, 2003). Shattock has chosen instead to explore vaginal vaccination to induce mucosal immune responses against HIV. He has started both nonhuman primate and human studies simultaneously to evaluate a trimeric gp140 clade C protein vaccine candidate using a gel that is applied vaginally. The plan is to apply nine doses of the gel over a month-long menstrual cycle. “Our approach is to maintain high levels of mucosal immune responses,” he says. Memory cells generated by conventional vaccinations take three to five days to become activated and that may be too slow to provide sterilizing immunity at mucosal surfaces, since it only takes up to three days for a localized HIV infection to occur. In the vagina, there is a window of opportunity of three days after initial exposure to keep HIV from spreading systemically.

Others are trying oral vaccinations, which is best for induction of immune responses in the gut. “Oral immunization is the best way to generate an immune response in the small intestine where HIV wants to live,” says Kozlowski. Currently several groups are exploring this approach. Gary Nabel’s lab at the Vaccine Research Center, part of the NIH, is investigating oral administration of an adenovirus serotype 41 (Ad41) vector-based vaccine candidate to see if it can elicit mucosal immune responses in the gut. Marjorie Robert-Guroff at the NCI is planning a Phase I safety trial of an orally-administered tablet containing a replicating Ad4 vector encoding HIV clade C Env. An oral, replicating Ad4 vaccine was shown to be safe and effective during 25 years of use protecting American soldiers against acute respiratory disease, Robert-Guroff says. She later plans to follow the oral vaccination with an Env protein boost injected intramuscularly. She says similar oral vaccinations with a replicating Ad5 vector in rhesus macaques followed by a protein boost resulted in mucosal immunity and protected from a rectal challenge with SIVmac251 (Vaccine 25, 8021, 2007).

Others are looking for new routes to induce mucosal immune responses. For example, a recent study has shown in mice that sublingual immunization induces mucosal responses in the respiratory and genital mucosa, as well as the GI tract (Vaccine 25, 8598, 2007).

Another study found that spraying adenoviral vector vaccines onto the tonsils can elicit both cellular and humoral immune responses, and spraying a combination of different vectors provides similar protection from an SIV challenge as systemic immunization with the same vaccine (J. Virol. 81, 13180, 2007). Another good route might be application through the skin, perhaps like a nicotine patch, Kozlowski says, adding that there is some evidence that it may generate mucosal IgA immune responses in humans.

Researchers are also looking for adjuvants that will make it possible to get mucosal immune responses without having to deliver the vaccines directly to mucosal tissues. The hope is to change immune cells so they will go to mucosal tissues. “We could continue to administer vaccines intramuscularly if an adjuvant is identified that targets the immune cells that are activated to go to the mucosa,” Kozlowski says.

For example, one recent study by Harriet Robinson’s group at Emory University in Atlanta found that in macaques, intramuscular injection of DNA encoding Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) together with the DNA prime for a DNA-MVA prime-boost regimen results in increased IgA antibody response in rectal secretions (Virology 369, 153, 2007).

Another possible adjuvant is retinoic acid (RA). J. Rodrigo Mora’s lab at Harvard and others have found that dendritic cells in the gut-associated lymphoid tissue can secrete RA, which induces T and B cell migration to the gut mucosa. But delivering RA could be a challenge because it is not soluble in water and would have to be administered in a solvent like dimethyl sulphoxide (DMSO), which could have side effects. Therefore, Mora’s lab is working on methods to “teach” dendritic cells to make RA and to “pack” RA in dendritic cells so that dendritic cells loaded with RA (and not soluble RA) can be used to immunize and induce T- and B-cell mucosal immune responses. In this way, the effect of RA will be restricted to the site of immunization, thus eliminating potential systemic effects, Mora says. However, RA only appears to be relevant to immune responses in the upper GI tract and not to other relevant mucosal areas like the colorectum or genital mucosal surfaces, says Shattock. Nonetheless, Mora says, it might be possible to make B cells that migrate to the small bowel using RA. The resulting antibody-secreting cells would then make protective IgA antibodies, which can potentially be transported to, and protect, the colon mucosa—similar to what happens with maternal antibodies in breast milk.

Despite all of these efforts to study different routes to induce mucosal immune responses, what is needed and what has been lacking in the field is the systematic comparison of the same immunogen using various routes of administration in nonhuman primate SIV challenge studies, says Wayne Koff of IAVI. “IAVI is in the preliminary stage of developing plans to do such studies,” adds Koff.

So given what is now known about mucosal immunity, what should future AIDS vaccine candidates look like? Some researchers say that inducing a combination of mucosal and systemic immune responses is the goal. Still, it remains an open question as to whether mucosal immunity can actually prevent HIV infection, Shacklett says. “Exposure at a mucosal site might bypass the mucosal immune system entirely if the mucosal surface is breached,” she says. “It’s easy to see how this might happen in the context of sexual intercourse.” Shattock agrees. “If you look at women after intercourse, in 60% of them you can visually identify microabrasions.”

But that does not mean inducing mucosal immune responses is not important, says Shacklett. “Even if we can’t prevent the initial infection,” she says, “we may be able to limit viral replication and dissemination, and lower the total amount of replicating virus by arming effector cells that are able to localize to tissues.”