Search

Many AIDS vaccine candidates under development may elicit immune responses similar to those observed in and used to screen human immunodeficiency virus type 1 (HIV-1)-infected individuals. Therefore, it is important to develop vaccine candidates that incorporate antigenic markers and allow vaccinees to be distinguished from HIV-1 infectees. To this end, we introduced a series of mutations into and in the vicinity of the major immunodominant region (MIR) of gp41 (residues 598–609), a domain recognized by almost all HIV-1 infectees, and evaluated whether HIV-1-like particles incorporating such mutant glycoproteins could be expressed in mammalian cells. Results indicated that although up to three consecutive amino acids could be replaced within MIR without significantly affecting particle formation or gp160 processing, deletions within MIR impaired envelope processing. Replacement of HIV-1 MIR by part or most of the corresponding domain from other lentiviruses markedly decreased or abolished gp160 processing. Synthetic peptides corresponding to a mutated MIR incorporating three amino acid replacements were not recognized by a panel of sera from HIV-1 infectees, suggesting that HIV-1-like particles with this type of mutation represent potential candidate vaccines that could allow vaccinees to be distinguished from HIV-1 infectees.

Retroviral Gag proteins, in the absence of any other viral products, induce budding and release of spherical, virus-like particles from the plasma membrane. Gag-produced particles, like those of authentic retrovirions, are not uniform in diameter but nevertheless fall within a fairly narrow distribution of sizes. For the human immunodeficiency virus type 1 (HIV-1) Gag protein, we recently reported that elements important for controlling particle size are contained within the C-terminal region of Gag, especially within the p6 sequence (L. Garnier, L. Ratner, B. Rovinski, S.-X. Cao, and J. W. Wills, J. Virol. 72:4667–4677, 1998). Deletions and substitutions throughout this sequence result in the release of very large particles. Because the size determinant could not be mapped to any one of the previously defined functions within p6, it seemed likely that its activity requires the overall proper folding of this region of Gag. This left open the possibility of the size determinant residing in a subdomain of p6, and in this study, we examined whether the late domain (the region of Gag that is critical for the virus-cell separation step) is involved in controlling particle size. We found that particles of normal size are produced when p6 is replaced with the totally unrelated late domain sequences from Rous sarcoma virus (contained in its p2b sequence) or equine infectious anemia virus (contained in p9). In addition, we found that the large particles released in the absence of p6 require the entire CA and adjacent spacer peptide sequences, whereas these internal sequences of HIV-1 Gag are not needed for budding (or proper size) when a late domain is present. Thus, it appears the requirements for budding are very different in the presence and absence of p6.

The retroviral Gag protein plays the central role in the assembly process and can form membrane-enclosed, virus-like particles in the absence of any other viral products. These particles are similar to authentic virions in density and size. Three small domains of the human immunodeficiency virus type 1 (HIV-1) Gag protein have been previously identified as being important for budding. Regions that lie outside these domains can be deleted without any effect on particle release or density. However, the regions of Gag that control the size of HIV-1 particles are less well understood. In the case of Rous sarcoma virus (RSV), the size determinant maps to the CA (capsid) and adjacent spacer sequences within Gag, but systematic mapping of the HIV Gag protein has not been reported. To locate the size determinants of HIV-1, we analyzed a large collection of Gag mutants. To our surprise, all mutants with defects in the MA (matrix), CA, and the N-terminal part of NC (nucleocapsid) sequences produced dense particles of normal size, suggesting that oncoviruses (RSV) and lentiviruses (HIV-1) have different size-controlling elements. The most important region found to be critical for determining HIV-1 particle size is the p6 sequence. Particles lacking all or small parts of p6 were uniform in size distribution but very large as measured by rate zonal gradients. Further evidence for this novel function of p6 was obtained by placing this sequence at the C terminus of RSV CA mutants that produce heterogeneously sized particles. We found that the RSV-p6 chimeras produced normally sized particles. Thus, we present evidence that the entire p6 sequence plays a role in determining the size of a retroviral particle.

Noninfectious human immunodeficiency virus type 1 (HIV-1) viruslike particles containing chimeric envelope glycoproteins were expressed in mammalian cells by using inducible promoters. We engineered four expression vectors in which a synthetic oligomer encoding gp120 residues 306 to 328 (amino acids YNKRKRIHIGP GRAFYTTKNIIG) from the V3 loop of the MN viral isolate was inserted at various positions within the endogenous HIV-1LAI env gene. Expression studies revealed that insertion of the heterologous V3(MN) loop segment at two different locations within the conserved region 2 (C2) of gp120, either 173 or 242 residues away from the N terminus of the mature subunit, resulted in the secretion of fully assembled HIV-like particles containing chimeric LAI/MN envelope glycoproteins. Both V3 loop epitopes were recognized by loop-specific neutralizing antibodies. However, insertion of the V3(MN) loop segment into other regions of gp120 led to the production of envelope-deficient viruslike particles. Immunization with HIV-like particles containing chimeric envelope proteins induced specific antibody responses against both the autologous and heterologous V3 loop epitopes, including cross-neutralizing antibodies against the HIV-1LAI and HIV-1MN isolates. This study, therefore, demonstrates the feasibility of genetically engineering optimized HIV-like particles capable of eliciting cross-neutralizing antibodies.

HIV-1 retrovirus-like particles can be produced in VERO cells that have been transfected with an expression construct encoding HIV-1 structural proteins. The particles are entirely non-infectious although structurally they resemble infectious virus particles. This makes them a promising candidate for use as an HIV-1 vaccine. In order to ensure their safety and enhance their immunogenicity, the retrovirus-like particles were modified in a number of ways. A large deletion in the HIV-1 pol gene has eliminated reverse transcriptase and integrase activities. Deletion of RNA packaging signals in the RNA untranslated leader sequence and in Gag reduced packaged RNA to 5% of that in HIV-1 virus. Replacement of the existing HIV-1LAI envelope protein with that of HIV-1MN has ensured that immune responses to the particles are relevant to those against the majority of HIV-1 clade B isolates. In addition to these changes in particle composition, yields of the modified particles were increased using a superior method of inducing the expression construct promoter, and an effective scheme for particle purification was developed. Immunization of non-human primates demonstrated that the particles were capable of generating anti-HIV-1 neutralizing antibodies. The technological refinements reported here will permit retrovirus-like particles to be tested safely in humans, and the change in envelope proteins should allow a more realistic evaluation of the immunogenicity of these particles. Experience gained in engineering these refinements will greatly facilitate other modifications that may be required to achieve maximum efficacy as a vaccine against HIV-1.

Genetically engineered, noninfectious HIV-1-like particles containing processed envelope glycoproteins represent potential candidate immunogens for a vaccine against HIV-1. However, since the gp120 glycoprotein is known to be rapidly lost from the surface of infected cells and purified virions as a result of its low-affinity interaction with gp41, shedding of this extracellular subunit could compromise the immunogenic potential of particle-based HIV-1 vaccine candidates. In this study, we demonstrate for the first time the feasibility of producing fully assembled HIV-1-like particles containing only unprocessed gp160 glycoproteins. Monkey kidney Vero cells were transfected with an inducible, human metallothionein-based expression vector containing most of the HIV-1LAI coding sequences that were genetically modified to introduce safety mutations and destroy the major cleavage site of the HIV-1 envelope glycoprotein. A stably-transfected cell line was isolated and shown to secrete HIV-1-like particles containing unprocessed gp160. Immunization with these particles induced HIV-1 cross-neutralizing, syncytium-inhibiting and env-CD4 blocking antibodies. Thus, these novel HIV-1-like particles represent alternative candidate immunogens for the development of a particle-based AIDS vaccine.

The production of genetically-engineered, noninfectious virions of human immunodeficiency virus (HIV) represents a novel approach to the development of a safe and effective vaccine for the acquired immune deficiency syndromes (AIDS). Insofar as preparations of inactivated simian immunodeficiency virus (SIV) are now demonstrating protection in immunization-challenge studies in rhesus monkeys, a safe preparation of noninfectious HIV virions produced in a genetically-engineered cell line becomes a logical candidate vaccine for studies in humans. These particles, or pseudovirions, offer distinct advantages over the use of inactivated HIV for human AIDS vaccines. Guarantees of safety without the requirement for inactivation and their potential for structural modification for the modulation of immunogenicity are compelling reasons for the acceptance of HIV pseudovirions as a candidate vaccine in humans.

A proviral fragment from human immunodeficiency virus type 1 (HIV-1) (LAV-1BRU) containing only protein-coding information, was expressed in COS cells using constitutive promoters in transient and stable transfection experiments. The presence of viruslike particles in cell supernatants was verified by Western blot analysis, density gradient centrifugation, and electron microscopy. Transfection of Vero cells with a similar construct employing the human metallothionein promoter led to the isolation of stable cell lines exhibiting inducible viruslike particle expression in response to cadmium chloride treatment. Induction ratios for viruslike particle expression were in excess of 1000-fold with production levels of p24 core antigen as high as 0.6 mg/L per 24 h. HIV-1 viruslike particles were immunogenic in mice, leading to strong envelope and core-specific humoral responses after two immunizations. The development of stable cell lines expressing significant quantities of HIV-1 viruslike particles offers an alternative to the use of live virus vectors for the production and evaluation of particle-based AIDS vaccines.