A novel self-replicating chimeric lentivirus-like particle.

Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4⁺ cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation.

Membrane embedded HIV-1 envelope on the surface of a virus-like particle elicits broader immune responses than soluble envelopes.

Virally regulated HIV-1 particles were expressed from DNA plasmids encoding Gag, protease, reverse transcriptase, Vpu, Tat, Rev, and Env. The sequences for integrase, Vpr, Vif, Nef, and the long terminal repeats (LTRs) were deleted. Mutations were engineered into the VLP genome to produce particles deficient in activities associated with viral reverse transcriptase, RNase H, and RNA packaging. Each plasmid efficiently secreted particles from primate cells in vitro and particles were purified from the supernatants and used as immunogens. Mice (BALB/c) were vaccinated intranasally (day 1 and weeks 3 and 6) with purified VLPs and the elicited immunity was compared to particles without Env (Gag(p55)), to soluble monomeric Env(gp120), or to soluble trimerized Env(gp140). Only mice vaccinated with VLPs had robust anti-Env cellular immunity. In contrast, all mice had high titer anti-Env serum antibody (IgG). However, VLP-vaccinated mice had antisera that detected a broader number of linear Env peptides, had anti-Env mucosal IgA and IgG, as well as higher titers of serum neutralizing antibodies. VLPs elicited high titer antibodies that recognized linear regions in V4-C5 and the ectodomain of gp41, but did not recognize V3. These lentiviral VLPs are effective mucosal immunogens that elicit broader immunity against Env determinants in both the systemic and mucosal immune compartments than soluble forms of Env.

Lentivirus-like particles without reverse transcriptase elicit efficient immune responses.

Following infection by HIV or SIV, reverse transcriptase (RT) directs the conversion of the single-stranded RNA genome into a double-stranded DNA molecule that integrates into the host cell genome. RT encodes for several immunogenic epitopes that are desirable for inclusion in a human vaccine for HIV infection, however, issues of safety have dampened enthusiasm for inclusion of an enzymatically-active RT molecule into an AIDS vaccine. In this study, virally-regulated, replication-incompetent lentiviral particles were expressed from DNA plasmids. The sequences for integrase, Vpr, Vif, Nef, and the long terminal repeats (LTRs) were deleted and mutations were engineered into capsid to decreases RNA packaging. Virus-like particles incorporated no RT (HIV-VLP DeltaRT or SHIV-VLP DeltaRT) or contained a full-length enzymatically-inactivated RT molecule (HIV-VLP or SHIV-VLP). Each secreted VLP was enveloped with a lipid bilayer derived from primate cells with embedded, native viral envelopes in similar concentrations as infectious virions. BALB/c mice were vaccinated (weeks 0, 3, and 6) with purified VLPs via intranasal inoculation in the presence of cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs). All VLPs, with or without RT, elicited both robust humoral and cellular immune responses to Gag, Pol, and Env antigens. Therefore, the lack of RT enhances the safety of these VLPs for use in future human clinical trials without a significant reduction in the overall immunogenicity of these VLP immunogens.

Virus-like particles: designing an effective AIDS vaccine.

Viruses that infect eukaryotic organisms have the unique characteristic of self-assembling into particles. The mammalian immune system is highly attuned to recognizing and attacking these viral particles following infection. The use of particle-based immunogens, often delivered as live-attenuated viruses, has been an effective vaccination strategy for a variety of viruses. The development of an effective vaccine against the human immunodeficiency virus (HIV) has proven to be a challenge, since HIV infects cells of the immune system causing severe immunodeficiency resulting in the syndrome known as AIDS. In addition, the ability of the virus to adapt to immune pressure and reside in an integrated form in host cells presents hurdles for vaccinologists to overcome. A particle-based vaccine strategy has promise for eliciting high titer, long-lived, immune responses to a diverse number of viral epitopes against different HIV antigens. Live-attenuated viruses are effective at generating both cellular and humoral immune responses. However, while these vaccines stimulate immunity, challenged animals rarely clear the viral infection and the degree of attenuation directly correlates with protection from disease. Further, a live-attenuated vaccine has the potential to revert to a pathogenic form. Alternatively, virus-like particles (VLPs) mimic the viral particle without causing an immunodeficiency disease. VLPs are self-assembling, non-replicating, non-pathogenic particles that are similar in size and conformation to intact virions. A variety of VLPs for lentiviruses are currently in preclinical and clinical trials. This review focuses on our current status of VLP-based AIDS vaccines, regarding issues of purification and immune design for animal and clinical trials.

Elicitation of immunity to HIV type 1 Gag is determined by Gag structure.

The gag gene of the human immunodeficiency virus type 1 (HIV-1) encodes for viral proteins that self-assemble into viral particles. The primary Gag gene products (capsid, matrix, and nucleocapsid) elicit humoral and cellular immune responses during natural infection, and these proteins are included in many preclinical and clinical HIV/AIDS vaccines. However, the structure (particulate or soluble) of these proteins may influence the immunity elicited during vaccination. In this study, mice were inoculated with four different HIV-1 Gag vaccines to compare the elicitation of immune responses by the same Gag immunogen presented to the immune system in different forms. The immunity elicited by particles produced in vivo by DNA plasmid (pGag) was compared to these same proteins retained intracellularly (pGag(DMyr)). In addition, the elicitation of anti- Gag immunity by Gag(p55) virus-like particles (VLPs) or soluble, nonparticulate Gag(p55) proteins was compared. Enhanced cellular responses, but almost no anti-Gag antibodies, were elicited with intracellularly retained Gag proteins. In contrast, DNA vaccines expressing VLPs elicited both anti-Gag antibodies and cellular responses. Mice vaccinated with purified Gag(p55) VLPs elicited robust humoral and cellular immune responses, which were significantly higher than the immunity elicited by soluble, nonparticulate Gag(p55) protein. Overall, purified particles of Gag effectively elicited the broadest and highest titers of anti-Gag immunity. The structural form of Gag influences the elicited immune responses and should be considered in the design of HIV/AIDS vaccines.

Unique V3 loop sequence derived from the R2 strain of HIV-type 1 elicits broad neutralizing antibodies.

DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies. In this study, DNA vaccines were constructed to express the gp120 subunit of Env from the isolate HIV-1(R2) using both wild-type and codon-optimized gene sequences. Three copies of the murine C3d were added to the carboxyl terminus to enhance the immunogenicity of the expressed fusion protein. Mice (BALB/c) vaccinated with DNA plasmid expressing the gp120(R2) using codon-optimized Env sequences elicited high-titer anti-Env antibodies regardless of conjugation to C3d. In contrast, only mice vaccinated with DNA using wild-type gp120(R2) sequences fused to mC3d(3), had detectable anti-Env antibodies. Interestingly, mice vaccinated with DNA expressing gp120(R2) from codon-optimized sequences elicited antibodies that neutralized both homologous and heterologous HIV-1 isolates. To determine if the unique sequence found in the crown of the V3 loop of the Env(R2) was responsible for the elicitation of the cross-clade neutralizing antibodies, the codons encoding for the Pro-Met (amino acids 313-314) were introduced into the sequences encoding the gp120(ADA) (R5) or gp120(89.6) (R5X4). Mice vaccinated with gp120(ADA)-mC3d(3)-DNA with the Pro-Met mutation had antibodies that neutralized HIV-1 infection, but not the gp120(89.6)-mC3d(3)-DNA. Therefore, the use of the unique sequences in the Env(R2) introduced into an R5 tropic envelope, in conjunction with C3d fusion, was effective at broadening the number of viruses that could be neutralized. However, the introduction of this same sequence into an R5X4-tropic envelope was ineffective in eliciting improved cross-clade neutralizing antibodies.

Characterization of a DNA vaccine expressing a human immunodeficiency virus-like particle.

An ideal human immunodeficiency virus type-1 (HIV-1) vaccine will most likely need to elicit cross-reactive neutralizing antibodies and a strong cell-mediated immune response against multiple HIV-1 antigens to confer protection against challenge. In this study, DNA vaccines were constructed to express virally regulated human immunodeficiency virus-like particles (VLP) to elicit broad-spectrum immune responses to multiple HIV-1 antigens. VLPs were efficiently produced using sequences encoding gag and pol gene products from an X4 isolate and sequences encoding for tat, rev, vpu, and env from R5 or R5X4 isolates. The integrase, vpr, vif, and nef genes were deleted. In addition, the long terminal repeats (LTRs) were removed and transcription of the VLP insert was driven by the addition of the cytomegalovirus immediate-early (CMV-IE) promoter. A second generation of VLP vaccine plasmids was constructed with mutations engineered into the VLP DNA to produce particles deficient in activities associated with viral reverse transcriptase and protease. Primate cell lines, transiently transfected with DNA, efficiently secreted VLP into the supernatant that banded within a sucrose gradient at densities similar to infectious virions. In addition, these particles incorporated Env on the particle surface that bound soluble human CD4. These VLPs provide a safe and efficient strategy for presenting multiple HIV-1 antigens, expressed from a single insert, to the immune system in a structure that mimics the infectious virion.

Particle-based vaccines for HIV-1 infection.

The use of live-attenuated viruses as vaccines has been successful for the control of viral infections. However, the development of an effective vaccine against the human immunodeficiency virus (HIV) has proven to be a challenge. HIV infects cells of the immune system and results in a severe immunodeficiency. In addition, the ability of the virus to adapt to immune pressure and the ability to reside in an integrated form in host cells present hurdles for vaccinologists to overcome. A particle-based vaccine strategy has promise for eliciting high titer, long-lived, immune responses to a diverse number of viral epitopes from different HIV antigens. Live-attenuated viruses are effective at generating both cellular and humoral immunity, however, a live-attenuated vaccine for HIV is problematic. The possibility of a live-attenuated vaccine to revert to a pathogenic form or recombine with a wild-type or defective virus in an infected individual is a drawback to this approach. Therefore, these vaccines are currently only being tested in non-human primate models. Live-attenuated vaccines are effective in stimulating immunity, however challenged animals rarely clear viral infection and the degree of attenuation directly correlates with the protection of animals from disease. Another particle-based vaccine approach for HIV involves the use of virus-like particles (VLPs). VLPs mimic the viral particle without causing an immunodeficiency disease. HIV-like particles (HIV-LP) are defined as self-assembling, non-replicating, nonpathogenic, genomeless particles that are similar in size and conformation to intact virions. A variety of VLPs for both HIV and SIV are currently in pre-clinical and clinical trials. This review focuses on the current knowledge regarding the immunogenicity and safety of particle-based vaccine strategies for HIV-1.