Principal Investigator: Ira Berkower, MD, PhD
Office / Division / Lab: OVRR / DVP / LI
Our laboratory is working to solve some of the difficult problems and answer some of the pressing questions facing scientists who are trying to develop safe and effective vaccines against HIV, the virus that causes AIDS.
One of the biggest challenges facing AIDS vaccine developers is the difficulty in designing a vaccine that triggers the immune system to produce "broadly neutralizing" antibodies. Such antibodies are produced by the immune system in response to HIV infection; but they have never been made in response to an AIDS vaccine.
In order to neutralize the virus, the immune system must produce antibodies that target structures on the virus that perform essential viral functions and therefore more likely to have identical or similar structures in different strains. Our lab showed that on the envelope protein, or outer most layer of HIV, there is a structure called the CD4 binding site that is found on all HIV strains identified to date. The CD4 binding site enables HIV to bind to the cells that it subsequently infects., and therefore meets this criterion. Therefore, we are trying to expose the CD4 binding site to the immune system in order to trigger antibody production against it. If an AIDS vaccine could elicit antibodies to this type of site, it might be able to protect people from the many different existing varieties of HIV.
In the native structure found on the virus particle, the CD4 binding site is not accessible to antibody binding. Therefore, we are exploring two potential strategies to expose the CD4 binding site. These strategies involve two envelope proteins that cover HIV: gp120 and gp41.
First, we identified surface features on gp120 that interfere with antibody binding to the CD4 binding site. By removing these structures, we made a form of gp120 that antibodies can bind more effectively.
Second, we identified sites on gp120 that appear to control the ability of this protein to fold into different shapes, concealing or partially disrupting parts of this protein. By changing these sites, we could induce gp120 to assume the more open and accessible form that enables the immune system to recognize that protein and produce antibodies that neutralize it.
We are also studying ways to adapt a technique already successfully used in the development of other vaccines: presenting antigens as part of a virus-sized particle. (Antigens are molecules that stimulate the immune system to make antibodies.) A protein on the surface of the hepatitis B virus called hepatitis B surface antigen (HBsAg) readily binds to other HbsAg proteins to form particles. In fact, HBsAg itself is already a successful vaccine against hepatitis B, and the potency of this vaccine depends on the ability of HBsAg to assemble itself into virus-like particles. Our laboratory developed a way to incorporate the gp41 HIV antigen into virus-like particles by linking this HIV protein to HBsAg.
The HBsAg particles resemble the outer lipid (fatty) envelope of HIV. This allows the gp41 protein to insert itself into the particle, just like it does in HIV viruses. Having gp41 in the lipid environment of HBsAg makes this protein more available to stimulate the immune system. The combination of gp41 and lipid is an important target of neutralizing antibodies against HIV. We are currently studying ways to improve this strategy so it reliably triggers production of broadly neutralizing antibodies that recognize and bind gp41 on many different varieties of HIV.
Some of the most successful vaccines currently available against other viruses are live attenuated (weakened) viruses. Since using live, attenuated HIV as a vaccine would be unsafe, researchers have proposed genetically modifying non-AIDS viruses so they can make HIV antigens. We are the first laboratory to successfully engineer the virus that causes rubella (German measles) so it carries a foreign gene, continues to replicate like a normal rubella virus, and uses the gene to make the protein it codes for. We are studying the use of this strategy to produce HIV proteins that could act like a vaccine.
These studies will provide new scientific information and tools for developing and evaluating vaccines for HIV/AIDS. Only through hands-on experience with HIV vaccine antigens will CBER gain the expertise it needs to evaluate proposed HIV vaccines and anticipate their likely side effects.
For many vaccine antigens, such as polio, rabies, and hepatitis B, vaccine potency depends on particle formation. To enhance the immune response to biologically important targets on HIV, we have incorporated them into virus-like particles. Despite many advances in recombinant DNA technology, only two recombinant vaccines have been made so far, and both depend on the formation of virus-like particles. One of these, hepatitis B surface antigen (HBsAg) assembles into 22 nm particles that display HBsAg protein on the surface of a lipid droplet.
We demonstrated that HBsAg can be used as a carrier protein: when linked to another viral protein, it assembles into particles and incorporates the other protein as well. This effectively displays viral envelope proteins, such as HIV gp120, since they are normally expressed on a viral surface that includes lipid. In addition, the transmembrane domain of HIV gp41 can be incorporated into the HBsAg structure, allowing gp41 to be anchored to the lipid layer by its own transmembrane domain.
These two proteins, gp120 and gp41, contain important antigenic determinants that are targeted by human neutralizing antibodies that cross react to 35-100% of HIV isolates around the world. For gp120, we modified the protein to further expose neutralizing sites and enhance antibody binding. These findings were published in a cover story in Virology (2008), patented by the US government, confirmed by other labs, and incorporated into new vaccine constructs made by the Vaccine Research Center at NIH for further testing. For gp41, we found that increasing the number of antigenic determinants per particle greatly increased antibody binding and antibody induction against neutralizing determinants. We patented this work last year and have a paper ready for submission.
Another effective way to enhance the immune response to viral antigens is to present them in the context of an ongoing infection with a live attenuated viral vector. We are using rubella as a vector because of its safety and potency: one dose protects against rubella for life. We expressed a foreign reporter gene in rubella and demonstrated stable expression for at least 12 generations. These results have been published and patented.
We recently incorporated HIV viral antigens, such as the conserved neutralizing determinant from gp41, into this vector and demonstrated protein expression. These vectors will be tested for growth and immunogenicity in macaques. If successful in expressing gp41 antigens, rubella has many advantages, including its ability to elicit mucosal immunity, genetic stability, ease of production, and ability to immunize at a very low dose.
These two approaches can be combined in a prime/boost strategy to give the strong T cell and B cell responses needed to prevent HIV infection. They each address important issues concerning antigenicity, antigen presentation, and vaccine potency.
Retrovirology 2013 Sep 16;10(1):99
Live attenuated rubella vectors expressing SIV and HIV vaccine antigens replicate and elicit durable immune responses in rhesus macaques.
Virnik K, Hockenbury M, Ni Y, Beren J, Pavlakis GN, Felber BK, Berkower I
Vaccine 2013 Apr 19;31(17):2119-25
Enhanced expression of HIV and SIV vaccine antigens in the structural gene region of live attenuated rubella viral vectors and their incorporation into virions.
Virnik K, Ni Y, Berkower I
Vaccine 2012 Aug 10;30(37):5453-8
Live attenuated rubella viral vectors stably express HIV and SIV vaccine antigens while reaching high titers.
Virnik K, Ni Y, Berkower I
J Virol 2011 Mar;85(5):2439-48
Hepatitis B virus surface antigen assembly function persists when entire transmembrane domains 1 and 3 are replaced by a heterologous transmembrane sequence.
Berkower I, Spadaccini A, Chen H, Al-Awadi D, Muller J, Gao Y, Feigelstock D, Virnik K, Ni Y
Vaccine 2010 Feb 3;28(5):1181-7
Stable expression of a foreign protein by a replication-competent rubella viral vector.
Spadaccini A, Virnik K, Ni Y, Prutzman K, Berkower I
Virology 2008 Aug 1;377(2):330-8
Targeted deletion in the beta20-beta21 loop of HIV envelope glycoprotein gp120 exposes the CD4 binding site for antibody binding.
Berkower I, Patel C, Ni Y, Virnik K, Xiang Z, Spadaccini A
Virology 2008 Mar 30;373(1):72-84
Analysis of the human immunodeficiency virus type 1 gp41 membrane proximal external region arrayed on hepatitis B surface antigen particles.
Phogat S, Svehla K, Tang M, Spadaccini A, Muller J, Mascola J, Berkower I, Wyatt R
J Infect Dis 2007 Oct 1;196(7):1026-32
Antibodies to the A27 Protein of Vaccinia Virus Neutralize and Protect against Infection but Represent a Minor Component of Dryvax Vaccine-Induced Immunity.
He Y, Manischewitz J, Meseda CA, Merchlinsky M, Vassell RA, Sirota L, Berkower I, Golding H, Weiss CD
Virology 2004 Mar 30;321(1):75-86
Assembly, structure, and antigenic properties of virus-like particles rich in HIV-1 envelope gp120.
Berkower I, Raymond M, Muller J, Spadaccini A, Aberdeen A