Efforts to develop a vaccine to prevent infections with Human Immunodeficiency Virus Type 1 (HIV-1) have been complicated by resistance of the virus to the effects of antibodies. Specifically, efforts to develop vaccines that induce antibodies that neutralize the infectivity of diverse strains of HIV-1 have had limited success. Neutralizing antibodies are likely to be critical for vaccine success, since they are the only immunological mechanism that may completely prevent infection. Neutralizing antibodies are the principal mechanism for effectiveness of most or all proven viral vaccines (Galasso (1997) Antiviral Agents and Diseases of Man, Raven Press, 791-833). Even natural infections with HIV-1 are not associated with robust neutralizing antibody responses. In most patients, infection progresses for a number of years before antibodies develop that neutralize a variety of HIV strains (Quinnan et al. (1999) AIDS Res. Hum. Retroviruses 15, 561-70). Even after such an extended period, it is rare that an individual will develop antibodies that neutralize most strains of HIV-1.
The component of HIV-1 that is the target of neutralizing antibodies is the envelope protein spike. The essential unit comprising the spike is a dimer composed of the 120 kd surface protein (gp120) and the 41 kd transmembrane protein (gp41). The spike is believed to be a trimer of such heterodimers. The gp41 molecules anchor the complex to the viral membrane, and the gp120 molecules are associated with the gp41 molecules in such a way that they mediate the interaction of the virus with receptors on target cells. The epitopes that induce neutralizing antibodies and interact with them are in the gp120 and gp41 molecules. Infection of target cells by HIV-1 is a multi-step process, which begins when the viral gp120 molecules bind to the principal viral receptor on target cells, CD4. Binding to CD4 induces conformational change in the envelope protein spike, such that it is then competent to bind to the viral coreceptor, a chemokine receptor molecule that is usually either CCR5 or CXCR4. It is believed that the binding of the envelope proteins to the coreceptor results in further conformational change that results in the membranes of the virus and target cell being drawn together and undergoing fusion. Once membrane fusion occurs, the viral core may enter the target cell and initiate subsequent steps in the infection process. Neutralizing epitopes in envelope proteins are highly conformation-dependent, and many of them may only be formed during the conformational transitions that occur subsequent to CD4 or coreceptor interaction. Vaccines that are intended to induce antibodies that neutralize HIV-1 are designed using forms of envelope protein that are prepared in ways that may result in presentation to the immune system of epitopes that will induce broadly cross-reactive neutralizing antibodies.
An extraordinary variety of approaches to preparation of HIV-1 envelope protein-based vaccines has been tried for induction of broadly cross-reactive neutralizing antibodies with limited success. The approaches used have included the administration of envelope protein prepared using various recombinant DNA techniques, synthetic peptides representative of particular structures in the envelope protein complex, live viral vectors that express envelope proteins in vivo, covalently linked complexes of envelope proteins and CD4, and other materials. Previous research utilized a unique HIV-1 envelope proteins as immunogen, and two methods of presentation of envelope proteins as vaccine, both of which were designed to present the HIV-1 envelope proteins in a form that closely resembled the conformation it assumes on the surface of the virus (Dong et al. (2003) J. Virol. 77, 3119-3130).
The unique envelope protein that was used in those studies is designated R2 (Quinnan et al. (1999) AIDS Res. Hum. Retroviruses 15, 561-570; Quinnan et al. (1998) AIDS Res. Hum. Retroviruses 14, 939-949; Trkola et al. (1995) J. Virol. 69, 6609-6617; Zhang et al. (2002) J. Virol. 76, 644-655). The gene encoding this envelope protein was recovered from cells from an HIV-1-infected donor, who had antibodies that neutralized many different primary isolates of HIV-1. Primary isolates are notoriously difficult to neutralize, and sera from infected humans generally neutralize few, or a limited subset of strains of HIV-1. The envelope protein gene from the donor was cloned and the envelope protein that it encodes has been characterized extensively. When the envelope protein is expressed on the surface of HIV-1, using a method known as pseudotyping, the virus displays unique characteristics. It is able to infect cells that express the HIV-1 coreceptor, CCR5, in the absence of the primary receptor, CD4. All other naturally occurring strains of HIV-1 require CD4 for infection. Other characteristics of the virus suggest that the envelope protein is in a conformation that most envelope protein do not assume until after binding to CD4. The R2 envelope protein is sensitive to neutralization by monoclonal antibodies (Mabs) that do not neutralize most strains of HIV-1 unless they are first bound to CD4. These Mabs are said to be directed against CD4-induced (CD4i) epitopes. Since these epitopes are required for coreceptor binding, they are highly conserved among strains of HIV-1. A rare mutation in variable region 3 (V3) of the R2 envelope protein is necessary for its CD4-independent infectivity as well as its sensitivity to CD4i Mabs. This mutation has similar, but variable effects on other strains of HIV-1, indicating that its effects depend to a certain extent on other sequences in the R2 envelope protein. The mutation involves a proline substitution near the tip of the V3 loop structure. This proline undoubtedly has significant effects on conformation of the V3 loop, and apparently has significant effects on the conformation of the entire Env. It is this Env, which is apparently triggered to express cross-reactive CD4i epitopes, which has been used to induce broadly cross-reactive neutralization.
Two methods were used for immunization of mice and monkeys with the R2 envelope protein (Dong et al. (2003) J. Virol. 77, 3119-3130). One of the methods involved use of a viral expression vector for in vivo expression, and the other involved administration a form of the envelope protein that had been engineered to be missing part of the gp41 molecule (Broder et al. (1994) Proc. Natl. Acad. Sci. USA 91, 11699-11703; Earl et al. (1994) J. Virol. 68, 3015-3026). This protein is referred to as gp140, and is similar to the intact protein spike, but is produced by cells engineered to express the protein as a soluble trimeric molecule. The gp140 protein retains its conformation in potent adjuvant. The two immunization methods have been used separately and sequentially.
Immunization of mice and monkeys with R2 Env induced neutralizing antibodies with cross-reactivity patterns similar to each other and to the cross-reactivity of the serum from the donor of the R2 envelope protein. The serum from the donor of R2 neutralizes strains of all HIV-1 subtypes that have been tested, but neutralizes strains of the A, B, C, and F subtypes much better than the D and E subtypes. The sera from the immunized mice and monkeys neutralize HIV-1 strains of the A, B, C, and F subtypes, but not of the D or E subtypes. It is speculated that this pattern of cross-reactivity reflects the cross-reactivity of the CD4i neutralization epitopes expressed on R2 envelope protein. It is noteworthy that the responses induced in monkeys neutralized one of three strains tested of recombinant Simian-Human Immunodeficiency virus (SHIV); the two strains that were not neutralized are sensitive to neutralization by a Mab directed against a cross-reactive epitope in gp41, 2F5. An implication of this finding is that the R2 envelope protein may not be an effective inducer of antibodies that recognize the 2F5 epitope. Since 2F5 is a human Mab, envelope protein from other donors with cross-reactive neutralizing antibodies may express epitopes that would be better inducers than R2 of antibodies that recognize the 2F5 epitope.
The 2F5 Mab is of particular interest, since it is one of the three most highly cross-reactive neutralizing human Mabs that have been discovered (Trkola et al. (1995) J. Virol. 69, 6609-6617). Its importance is documented in studies, which demonstrated that combinations of 2F5 and the other two highly cross-reactive Mabs could protect monkeys from infection with SHIV (Mascola et al. (1999) J. Virol. 73, 4009-4018; Mascola et al. (2000) Nat. Med. 6, 207-210). The core epitope recognized by 2F5 has been localized by epitope mapping studies to a region of the gp41 ectodomain near the viral membrane. The amino acid sequence of the core epitope is the sequence ELDKWAS (SEQ ID NO: 1). However, there have been no reports of successful induction of neutralizing antibodies using as immunogens synthetic peptides comprising either this sequence or this sequence plus additional flanking sequences. It is likely, therefore, that the capacity of HIV-1 Envelope protein to induce neutralizing antibodies directed against the 2F5 epitope depends upon additional, not yet identified sequences, or is dependent upon conformation of this region of the molecule. It is thought that this region of gp41 undergoes conformational changes during the process of viral attachment to target cells and fusion of the virus and cell membranes. It is reasonably possible that the actual 2F5 neutralization epitope of most strains of HIV-1 does not actually form until fusion-related conformational changes have occurred. HIV-1 Envelope protein which expressed the epitope in its neutralization-active form in the absence of target cell interaction would be particularly good candidates for use in vaccination regimens for induction of 2F5-like antibodies.
Previously, Applicants have demonstrated the isolation of a unique HIV-1 envelope protein gene from an individual with BCN antibodies (see, for example, WO 00/07631). The Envelope protein encoded by this gene was designated R2, and is unique with respect to its amino acid sequence and its ability to infect target cells in the absence of CD4. The R2 Envelope protein is unusual with respect to its sensitivity to neutralization by Mabs against epitopes that are usually neutralization sensitive only in the presence of CD4. The CD4-independence and sensitivity of the R2 Envelope protein to neutralization by these Mabs are both dependent upon an unusual sequence in V3 of the protein. The R2 Envelope protein has been used to immunized mice and monkeys, and induced BCN antibodies in each species. Envelope proteins that induce antibodies against neutralization epitopes distinct from those targeted by R2 could be important components of an immunogen that approached universal effectiveness in prevention of HIV-1 infection.