Antibodies Protect Against HIV in Mice - Vectored immunoprophylaxis

Researchers have devised a gene transfer technique in mice that, with a single injection, protects the immune cells that HIV targets. With further development, the approach may prove effective at helping to prevent HIV infection in people. Most vaccines work by triggering the immune system to produce antibodies to help beat back infections. But a vaccine for HIV has been elusive. Proteins on the surface of HIV mutate rapidly, changing shape and preventing most antibodies from latching onto the virus. Scientists have discovered several antibodies that can neutralize HIV. They've gained important insights into how they bind to the virus and why they're effective. But designing a vaccine that prompts the human immune system to generate such antibodies and mount an effective attack remains a difficult challenge. A team of researchers led by Drs. Alejandro Balazs decided to pursue a different strategy—one that doesn't require the immune system to generate antibodies. They described the approach, called vectored immunoprophylaxis, in the November 30, 2011, advance online edition of Nature.

The scientists began with a virus capable of expressing high levels of full-length human antibodies when injected into muscle. They modified the virus by inserting the genes that code for an HIV-neutralizing antibody called b12. When the virus was injected into mouse leg muscle, the mice produced high levels of antibodies for at least a year. The researchers next tested whether the technique could protect against HIV. Mice aren't susceptible to HIV, so the researchers used specialized mice with human CD4 cells, the immune cells that HIV targets and infects. After exposure to the virus, mice expressing b12 antibodies showed none of the CD4 cell loss that control animals did. The researchers tested other antibodies known to neutralize a broad range of HIV strains. Another antibody called VRC01, which was identified by scientists at NIH, produced results similar to b12. Both antibodies protected CD4 cells against HIV doses 100-fold higher than the levels that would infect most animals. This protection would be well beyond that needed to prevent HIV infection in humans.

“Normally, you put an antigen or killed bacteria or something into the body, and the immune system figures out how to make an antibody against it,” Balazs explains. “We've taken that whole part out of the equation.”

The team is now developing a plan to test the method in human clinical trials. “If humans are like mice, then we have devised a way to protect against the transmission of HIV from person to person,” says Baltimore. “But that is a huge if, and so the next step is to try to find out whether humans behave like mice.”

Looking Inside Viruses - Bubblegram imaging.

Since the discovery of the microscope, scientists have tried to visualize smaller and smallerstructures to provide insights into the inner workings of human cells, bacteria and viruses. Now, researchers have developed a new way to see tiny structures within viruses.

Conventional cryo-electron microscopy (cryo-EM) has allowed researchers to image the surface of viruses in great detail. But scientists hadn't been able to clearly visualize structures inside viruses. Cryo-EM procedures use radiation, and higher doses damage viruses, destroying the very structures researchers would like to view. A team led by Dr. Alasdair Steven of NIH's National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) and Dr. Lindsay Black at the University of Maryland Medical School was studying a type of virus that infects bacteria and so might one day be used to combat pathogens. Past studies showed that the virus, called ϕKZ, contains a cylindrical protein structure called the inner body. Those studies, however, used disrupted viruses. The inner body can't be distinguished from the DNA that surrounds it in intact viruses using conventional cryo-EM.

In the January 13, 2012, issue of Science, the researchers described how they were able to turn the problem of radiation damage into an asset. They realized that the proteins inside the virus are more sensitive than DNA to radiation damage. After recording images of the virus with low doses of radiation, they used higher doses. As the inner structure deteriorated, it appeared as a cylinder of bubbles. The team was able to superimpose the images and, using 3-D computer reconstruction, clearly visualize the viral structure. The investigators call their technique bubblegram imaging.

Based on the shape and position of the inner body, the researchers believe that it helps organize DNA into its compact structure. In the future, bubblegram imaging may yield further insights into the inner workings of viruses and suggest strategies for developing novel therapies.

The scientists anticipate other uses for bubblegram imaging as well. For example, it could be used to visualize the interactions of proteins with DNA in human cells.

“This new cryo-EM procedure renders previously invisible proteins visible and, thus, will provide new understanding of cell biology,” Steven says.

Why HIV is not eliminated from the body by Antibodies?

The latent provirus is invisible to the immune system.The virus undergoes rapid mutational changes in antigens during replication which eventually overwhelms the immune system.The population of helper T-cells eventually declines to the point where cell-mediated immunity collapses. Researchers are not entirely sure why this occurs. Secondary infections characteristic of HIV infection develop (Pneumocystis pneumonia and Kaposi's sarcoma).

Human Immunodeficiency Viruses

In 1981, the first cases of a new disease now called acquired immune deficiency syndrome (AIDS) were recognized. The victims died of rare infections because their immune systems were crippled. The cause was identified two years later by Luc Montagnier and coworkers. AIDS is produced by human immunodeficiency virus (HIV), of which two major classes are known: HIV-1 and the much less common HIV-2. Like other retroviruses, HIV contains a single-stranded RNA genome that is replicated through a double-stranded DNA intermediate. This viral DNA becomes integrated into the genome of the host cell. In fact, viral genes are transcribed only when they are integrated into the host DNA. The HIV virion is enveloped by a lipid bilayer membrane containing two glycoproteins: gp41 spans the membrane and is associated with gp120, which is located on the external face. The core of the virus contains two copies of the RNA genome and associated transfer RNAs, and several molecules of reverse transcriptase. They are surrounded by many copies of two proteins called p18 and p24. The host cell for HIV is the helper T cell. The gp120 molecules on the membrane of HIV bind to CD4 molecules on the surface of the helper T cell.This interaction allows the associated viral gp41 to insert its amino-terminal head into the host-cell membrane. The viral membrane and the helper cell membrane fuse, and the viral core is released directly into the cytosol. Infection by HIV leads to the destruction of helper T cells because the permeability of the host plasma membrane is markedly increased by the insertion of viral glycoproteins and the budding of virus particles. The influx of ions and water disrupts the ionic balance, causing osmotic lysis. Indeed, the mutation rate of HIV is more than 65 times as high as that of influenza virus. A major aim now is to define relatively conserved sequences in these HIV proteins and use them as immunogens.

Figure

What happens once the DNA from the HIV particle enters the cell nucleus?

When physicians in Los Angeles and other cities noted an unusually large number of opportunistic microbial infections. Destruction of T lymphocytes of the immune system cells were associated with these infections, and it soon became obvious that an epidemic of disease was in progress. By 1984 the responsible virus had been identified, and in 1986 it was given the name human immunodeficiency virus (HIV). HIV is a very fragile virus, and for this reason it does not survive long periods of exposure outside the body.
In infected individuals, HIV infects T lymphocytes by combining its spike glycoproteins with the CD4 receptor sites of T lymphocytes. The nucleocapsid enters the cytoplasm of the T lymphocyte, and the viral enzyme reverse transcriptase synthesizes a DNA molecule using the RNA of HIV as a template (for this reason, the virus is called a retrovirus). The DNA molecule migrates to the cell nucleus and becomes part of a chromosome in the T lymphocyte nucleus.
The DNA molecule, known as a provirus, assumes a relationship with the DNA of the T lymphocyte, and the provirus enters the state of lysogeny. From this point in the nucleus, the provirus encodes new HIV particles, which acquire their envelope by budding through the membrane of the T lymphocyte. The human body attempts to keep up with the mass of new viral particles, but eventually the newly emerging strains of HIV overwhelm the body defenses and the T lymphocyte count begins to drop. Normally, it is approximately 800 T lymphocytes per cubic millimeter of blood, but as the disease progresses, the count drops into the low hundreds and tens. This drop may occur as soon as weeks after infection or as long as 20 years or more after infection. Thus far, vaccines are not available against HIV. Two glycoproteins called gp120 and gp160 from the envelope are being investigated as possible vaccines. Vaccine development is hampered however, since it is difficult to find volunteers who would become antibody- positive and could suffer discrimination as a result of antibody presence. Nevertheless, candidate vaccines have been prepared with gp120 and gp160. Many candidate vaccines are now in the testing stage