Exploring mucosal protection for a mucosal virus

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 (see Primer, this issue). The virus is transmitted most often through the mucosal tissues of the genitals or rectum. And although the progression of HIV infection is tracked by measuring the number of CD4+ T cells (a subset of immune cells) in the blood, most of the havoc wreaked by HIV is in the moist mucosal surfaces that line several of the body’s internal cavities. Understanding immune responses against HIV at these mucosal surfaces is therefore important in designing AIDS vaccine candidates capable of preventing HIV transmission or in controlling infection once it occurs.

Only a few research groups are currently studying 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. Lopalco also points out that researchers are getting a late start at studying the types of immune responses that could effectively block HIV at mucosal sites. We should have started 20 years ago, she adds.

Progress in studying mucosal immunity and HIV infection has been slow, in part, because measuring mucosal immune responses is much more complicated than measuring those produced systemically. While systemic immune responses can be measured with a simple blood test, measuring mucosal immune responses requires taking tissue samples or collecting secretions from these sites. It is also more difficult to deliver a vaccine directly at mucosal tissues.

Still, over the past several years, researchers have gained some important insights into the role of mucosal immune responses in HIV infection that could contribute to the development of an AIDS vaccine candidate that stimulates mucosal immunity against the virus.

Measure for measure

Measuring immune responses in mucosal tissues can be difficult, especially within the context of an AIDS vaccine clinical trial. The major type of antibody in most mucosal secretions is known as immunoglobulin A or IgA, according to Jiri Mestecky of the University of Alabama at Birmingham. But there is often inconsistency in the measurement of IgA levels in human secretions, depending on where the laboratory tests were conducted. This makes it more difficult to interpret and compare findings from different studies.

Collecting secretions from mucosal tissues, such as the vagina or the rectum, is not easy. One method called lavage involves washing the mucosal surfaces with a salt solution and then collecting the liquid for analysis. But Pam Kozlowski of Louisiana State University has found that this approach often dilutes the secretions too much, making it hard to detect antibodies.

Instead, she has developed a method that uses an absorbent sponge to obtain vaginal and rectal secretions. This sponge method can be used in both animal studies and in human volunteers and causes very little discomfort. It is also less intrusive than lavage methods. It’s only in for ten minutes at most, Kozlowski says, adding that it would be much easier to use in clinical trials. Also lavages require immediate processing, while the sponges can simply be frozen after collection and analyzed later, making them more practical.

While this sponge method may help researchers get around some of the problems with collecting mucosal antibodies, measuring cellular mucosal immune responses at these sites is still challenging. Isolating cellular immune responses in the rectum requires collecting a tissue sample by biopsy, according to Julie McElrath of the University of Washington. This is a much riskier and more invasive medical procedure than a lavage or blood test. If it is not done properly, the colon can be perforated, which results in a condition called peritonitis. To collect vaginal cells, some researchers use a cytobrush, a small brush-like device that is inserted into the cervix and rotated.

But even when mucosal tissue samples are properly collected, the number of cells that can be analyzed is often much smaller than in blood samples. This drastically limits the type of immune responses that can be measured, says Robin Shattock of the University of London. These samples must also be analyzed within a few hours of collection, which requires researchers to have a lab available at the same site where samples are obtained, McElrath says, which is not always feasible during clinical trials that are conducted in developing countries.

Together, these limitations are part of the reason why mucosal immune responses are not frequently measured during clinical trials. McElrath says that in AIDS vaccine trials, mucosal tissue 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. For example, in the recently conducted STEP trial with Merck’s AIDS vaccine candidate MRKAd5, mucosal samples were only collected in about 20 of the 3,000 total volunteers. These samples were analyzed in McElrath’s laboratory.

To get around these limitations, some researchers have proposed a way to measure mucosal immune responses using a blood sample. Immune cells that are headed for mucosal tissues can be identified by a molecule known as a receptor on their outer surface, which acts like a tag that shows where the cell is going. If these tagged cells can be detected in a blood sample, it could provide a rough estimate of the quantity of immune cells that will end up at mucosal tissues. But this model is not perfect. These tag-like receptors are only known for a few specific mucosal sites. And even though a cell may be headed for mucosal tissues, detecting its presence in the blood does not show whether or not it will actually arrive there. Mestecky compares it to a letter with an address. Whether it will actually get there and have its effect is unknown, he says.

Mucosal protection?

Researchers have also been trying to find out if mucosal immunity could, in part, explain why some individuals, called exposed or highly-exposed seronegatives (ESNs), remain HIV uninfected despite repeated exposure to the virus (seeVAX March 2007 Primer on Undestanding Why an Effective Vaccine is Feasible). Researchers are closely studying ESNs and are exploring many different hypotheses for their apparent immunity to HIV. Some studies have investigated if mucosal antibodies, like IgA, are responsible for protection. But these have led to contradictory results, according to Mestecky.

Other studies have focused on characterizing the mucosal immune responses in long-term nonprogressors (LTNPs) people who are HIV infected but don’t develop AIDS within the typical time frame. It is still unclear if mucosal antibodies detected in ESNs or LTNPs are actually playing a protective role. To find out, researchers are creating models of mucosal tissue in the lab using actual human cells, which simulate the mucosal tissue barrier, and running experiments to see if antibodies isolated from LTNPs or ESNs can effectively block HIV. Indeed some studies show that such antibodies can keep HIV from crossing these tissues in the laboratory. However, not everyone is convinced that these observations are meaningful because the model is not foolproof.

Routes of Delivery

Delivering vaccines in a way that will induce mucosal immune responses is another challenge for AIDS vaccine researchers. Often systemic immunizations, by intramuscular injection for example, are not sufficient to induce immunity at all mucosal tissues, says Mestecky. Antibodies from the blood may protect the genital tract but probably not the intestinal tract, he says.

Instead, studies suggest that the strongest mucosal immune responses would be expected when a vaccine is administered directly at a mucosal surface. Researchers have learned in recent years that mucosal antibody responses are often local and restricted to the sites where they are first induced. Kozlowski’s lab, for instance, has found mostly localized mucosal immune responses when comparing different mucosal delivery routes in women. An exception was nasal immunization, which generates responses in both the rectum and the female genital tract. Because of these results, Kozlowski is now conducting nonhuman primate studies with intranasally-administered vaccine candidates. Shattock’s group has initiated studies in both nonhuman primates and human volunteers to evaluate a vaccine that is applied vaginally, with the hope of inducing vaginal immune responses against HIV.

Others are trying oral vaccinations, which are best for induction of immune responses in the intestine or gut, says Kozlowski. The gut is a critical site for HIV infection. Gary Nabel’s group at the Vaccine Research Center, part of the US National Institute of Allergy and Infectious Diseases (NIAID), is investigating oral administration of adenovirus serotype 41 (Ad41) as a vector for a mucosal AIDS vaccine candidate because of its tendency to travel to intestinal tissues.

Still others are evaluating new routes such as applying a vaccine candidate directly onto the skin, under the tongue, or onto the tonsils. Researchers are also looking for substances called adjuvants that, they hope, can augment mucosal immune responses (see VAX December 2005 Primer on Understanding Mucosal Immunity).

Although it is clear that understanding mucosal immunity is important, it remains an open question as to whether a mucosal immune response will be sufficient to prevent HIV infection. Still that doesn’t mean it is not important to induce mucosal immune responses, says Barbara Shacklett of the University of California, Davis. Even if we can’t prevent the initial infection, we may be able to limit viral replication and dissemination, she adds.

Given what is now known about mucosal immunity, some researchers say that inducing a combination of mucosal and systemic immune responses is the ideal goal for future AIDS vaccine candidates.