The Lab of Immunoregulation is using molecular, biochemical and cell biological approaches to explore the viral envelope glycoprotein of HIV as a target of immunity. We know that envelope is a target of potent neutralizing antibodies made by people after infection. A mixture of just three human monoclonal antibodies to envelope is capable of protecting monkeys against challenge. Our goal is to develop vaccines that can elicit an immune response as strong as the response to infection. We have two approaches, comparable to the Salk and Sabin vaccines for polio. Our Salk-type vaccine is a particle with a lipid core and an array of HIV envelope glycoprotein on its surface, closely resembling a noninfectious version of HIV virus. These particles have the potential to enhance vaccine potency by up to 1,000-fold, as they have for other successful partulate vaccines. Our Sabin-type vaccine is a live rubella virus containing additional genes coding for the HIV envelope. Each cell infected by this attenuated virus looks to the immune system like a cell infected with HIV. However, the virulence of HIV is gone, only its antigens remain in the vaccine. If these vaccines can elicit a response as strong as to infection, they may be able to prevent or control a subsequent HIV infection. HIV-1 Neutralizing Antibodies from Live Attenuated Vaccines. Many of our most effective viral vaccines are live-attenuated viruses. However, for HIV vaccines, it seems that almost no degree of attenuation would be safe enough for human use. Instead, a number of live recombinant vaccines have been proposed. These have generally been defective viruses, capable of expressing HIV antigens in a single round of infection, but unable to replicate further. In this project, we have begun to make a live viral vector safe enough to express HIV antigens over multiple rounds of viral replication. Like HIV, rubella is an enveloped plus strand RNA virus which infects at mucosal surfaces. A single dose protects for life against mucosal and systemic infection with rubella virus. Live attenuated rubella vaccine is safe for human use. It has been given to millions of children around the world, including asymptomatic HIV+ children. It has no DNA intermediate, so there is no risk of integration and no reservoir of chronic infection. It is minimally pathogenic for adults and children, and its genetic stability is shown by the fact that it has not changed serotype in over 40 years. Ideally, a rubella/HIV hybrid could combine the growth and safety of rubella with the antigenicity of HIV gp120, but without the pathogenicity of HIV. With this vaccine, we may be able to induce a level and duration of immunity to HIV comparable to what has been achieved for rubella. We have used a full length, infectious cDNA clone of rubella, provided by Dr. Teryl Frey. We have identified an acceptor site in rubella where foreign sequences can be inserted without disrupting essential viral functions. For example, we inserted green fluorescent protein as a reporter gene at this site. When Vero cells were transformed with the DNA, they produced GFP in large amount. Significantly, the culture supernatants were infectious with virus that expressed GFP in the next round of infection. These viruses expressed all structural proteins, as measured by western blot, indicating that second strand RNA synthesis and the subgenomic promoter were both active. However, GFP expression was lost over two more passages of virus, although the rubella virus continued to grow without GFP expression. We are working on strategies to stabilize expression of the insert by preventing recombinants from outgrowing the GFP expressing strains. We will then express gp120 in the same manner. The resulting rubella/gp120 hybrid will be tested for growth and gp120 expression in vitro. It will then be tested for propagation in rhesus macaques and for the ability to elicit anti-gp120 antibodies (serum IgG and mucosal IgA), as well as safety and viral shedding and persistence. Macaques are the ideal host for demonstrating protection, since they can be infected with rubella and then challenged with an SHIV or SIV challenge strain expressing envelope glycoproteins of the same type or different from the vaccine strain. Novel Reagents for Making gp120 Conjugate and Particulate Vaccines. HIV envelope glycoprotein gp120 contains epitopes which are targeted by broadly cross-reactive neutralizing antibodies in humans and which depend on the native protein conformation. Monoclonal antibodies to these sites can neutralize a broad range of HIV-1 isolates and have protected monkeys against viral challenge by iv and oral routes. We have found a site on gp120, where foreign protein sequences can be inserted without disrupting the native folding of the neutralizing sites. By linking gp120 to a carrier protein capable of self-assembly, we could enhance the intrinsic vaccine potency of gp120 by assembling virus-like particles, while retaining important conformational sites needed to elicit these antibodies. When hepatitis B surface antigen (HBsAg)was inserted at this site, the resulting HBsAg-gp120 hybrids assembled particles efficiently. The particles sedimented at large size and banded at light density (1.22-1.25), consistent with a lipoprotein composition. On electron microscopy, they were 20 to 30 nm in diameter, similar to the spherical particles formed by native HBsAg. By analogy with native HBsAg particles, these contain about 100 to 200 copies of HBsAg-gp120, arrayed at a lipid/water interface. This recreates the natural milieu of gp120 on the surface of virions. Gp120 in the hybrids showed normal glycosylation, high affinity binding of the natural receptor CD4, and bound a panel of broadly reactive human neutralizing monoclonals, indicating that it was folded correctly in the native conformation. This is the first multimeric gp120 vaccine, and we anticipate that it may elicit a strong immune response in mice, rabbits and monkeys. We have now tested the immunogenicity of gp120 particles in mice and rabbits. Both species showed comparable antibody ELISA titers for particles as for monomeric gp120. However, the virus neutralizing activity of the particle-immune sera was much greater after two doses than for the monomer-immune sera. In some rabbits, the neutralizing titer was nearly as great as the ELISA titer, indicating efficient induction of neutralizing antibodies. The ratio of neutralization over ELISA titer for the rabbits was the same as for high-titered HIV-Ig from infected humans and was 30-fold greater than for animals immunized with gp120 monomers. If monkeys also produce high titered neutralizing anti-bodies, they will be challenged with titered stocks of SHIV virus of the same or different envelope type. We have expressed three envelope types: IIIB, 89.6, and SIV453 as particles. The first two correspond to SHIV challenge strains. The last one could be used to protect monkeys from a potent SIV challenge, which may more closely represent a natural HIV infection with a highly adapted virus. Through potent carrier and multimer effects, particulate forms of gp120 and other viral antigens may have a profound effect on HIV immunology. This project is funded by an intramural NIH grant. Transmembrane glycoprotein gp41 is another important target for HIV neutralizing antibodies. Human monoclonals such as 2F5 bind a site on gp41 close to the lipid bilayer. They can neutralize a broad range of HIV isolates, including fresh isolates. So far, vaccines have been unable to elicit similar antibodies. We hypothesize that, because they lack lipid, they cannot fold or display the 2F5 site correctly. We have expressed HBsAg-gp41 hybrids, which form gp41-rich particles. In these particles, gp41 is displayed at a lipid-water interface, and its transmembrane domain may span the lipid layer, just as it does on virus. We have produced sufficient quantities for immunological studies. These particles may be the first immunogen to recreate gp41 as it exists on the virus. Since the neutralizing sites on gp41 are conserved among diverse HIV isolates, antibodies elicited by these antigens have the potential to be broadly neutralizing. Antibodies to functional sites on viruses and prions. We have recently found that a variety of viral antigens, when expressed in tandem with HBsAg, will spontaneously assemble into 20-30 nm virus-like particles. The particles contain about 20% lipid, based on their density. The proteins are displayed on the surface of a lipid micelle. This milieu resembles the surface of an enveloped virus, as well as the local environment of a bacterial toxin bound to the surface of a cell. The HBsAg carrier is quite flexible in accepting other viral envelope proteins, while still forming particles. For example, the E2 and E1 envelope glycoproteins of VEE virus, as well as both E2E1 together, were expressed as HBsAg hybrids. All three forms assembled into particles. When expressed together, the E2E1 precursor was processed correctly into E2 and E1, and both proteins remained attached to the particles. This may be an ideal immunogen, since envelope proteins on the surface of a lipid particle resemble the same proteins on the viral surface. Immunogenicity will be tested in rabbits, and the resulting antibodies will be tested for the ability to protect cells in vitro from infection with VEE. Similarly, we have created hybrid particles containing protective antigen PA of anthrax. Antibodies to this protein are known to protect against anthrax. We anticipate that the particulate form of PA may greatly increase immunogenicity, allowing us to immunize with fewer doses and to maintain immunity with less frequent boosting than the current anthrax vaccine. In addition, PA was expressed in its active, cleaved form, which may elicit antibodies to sites involved in critical functions such as toxin subunit binding and translocation into the cell. PA particles will be tested for immunogenicity, and the resulting antibodies will be tested for the ability to block toxin-mediated killing of cell lines in culture. If successful, we will immunize mice and challenge them with anthrax spores, in collaboration with NIH scientists. This project incorporates FY2002 projects 1Z01BK009002-11, 1Z01BK009003-11, and 1Z01BK009008-04.
