Alphavirus replicon particle-based vaccine vectors derived from Sindbis virus (SIN), Semliki Forest virus,and Venezuelan equine encephalitis virus (VEE) have been shown to induce robust antigen-specific cellular, humoral, and mucosal immune responses in many animal models of infectious disease and cancer. However, since little is known about the relative potencies among these different vectors, we compared the immunogenicity of replicon particle vectors derived from two very different parental alphaviruses, VEE and SIN, expressing a human immunodeficiency virus type 1 p55Gag antigen. Moreover, to explore the potential benefits of combining elements from different alphaviruses, we generated replicon particle chimeras of SIN and VEE. Two distinct strategies were used to produce particles with VEE-p55 gag replicon RNA packaged within SIN envelope glycoproteins and SIN-p55 gag replicon RNA within VEE envelope glycoproteins. Each replicon particle configuration induced Gag-specific CD8 ؉ T-cell responses in murine models when administered alone or after priming with DNA. However, Gag-specific responses varied dramatically, with the strongest responses to this particular antigen correlating with the VEE replicon RNA, irrespective of the source of envelope glycoproteins. Comparing the replicons with respect to heterologous gene expression levels and sensitivity to alpha/ beta interferon in cultured cells indicated that each might contribute to potency differences. This work shows that combining desirable elements from VEE and SIN into a replicon particle chimera may be a valuable approach toward the goal of developing vaccine vectors with optimal in vivo potency, ease of production, and safety.
Striated muscle is the predominant site of gene expression after i.m. immunization of plasmid DNA, but it is not clear if myocytes or professional antigen-presenting cells (APCs) of hematopoietic origin present the encoded antigens to class I major histocompatibility complex (MHC)-restricted cytotoxic T Iymphocytes (CTL). To address this issue, CTL responses were assessed in mice engrafted with immune systems that were partially MHC matched with antigen-producing muscle cells. Spleen cells (sc) from immunocompetent F1 H-2bxd mice were infused' into H-2" or H-2d mice carrying the severe combined immunodeficiency (scid) mutation, creating Flc -k H-2b and F1lc -> H-2d chimeras, respectively. Immunization with DNA plasmids encoding the herpes simplex virus gB or the human immunodeficiency virus gpl20 glycoproteins elicited antiviral CTL activity. Flsc ' H-2d chimeras responded to an H-2d-restricted gp120 epitope but not an H-2b-restricted gB epitope, whereas Flsc -> H-2b chimeras responded to the H-2" but not the H-2d-restricted epitope. This pattern of epitope recognition by the sc chimeras indicated that APCs of recipient (scid) origin were involved ip initiation of CTL responses. Significantly, CTL responses against epitopes presented by the mismatched donor class I molecules were elicited if F1 bone marrow cells and sc were transferred into scid recipients before or several days to weeks after DNA immunization. Thus, bone marrow-derived APCs are sufficient for class I MHC presentation of viral antigens after i.m. immunization with plasmid DNA. Expression of plasmid DNA by these APCs is probably not a requirement for CTL priming. Instead, they appear to present proteins synthesized by other host cells.
A major challenge for the next generation of human immunodeficiency virus (HIV) vaccines is the induction of potent, broad, and durable cellular immune responses. The structural protein Gag is highly conserved among the HIV type 1 (HIV-1) gene products and is believed to be an important target for the host cellmediated immune control of the virus during natural infection. Expression of Gag proteins for vaccines has been hampered by the fact that its expression is dependent on the HIV Rev protein and the Rev-responsive element, the latter located on the env transcript. Moreover, the HIV genome employs suboptimal codon usage, which further contributes to the low expression efficiency of viral proteins. In order to achieve high-level Rev-independent expression of the Gag protein, the sequences encoding HIV-1 SF2 p55Gag were modified extensively. First, the viral codons were changed to conform to the codon usage of highly expressed human genes, and second, the residual inhibitory sequences were removed. The resulting modified gag gene showed increases in p55Gag protein expression to levels that ranged from 322-to 966-fold greater than that for the native gene after transient expression of 293 cells. Additional constructs that contained the modified gag in combination with modified protease coding sequences were made, and these showed high-level Rev-independent expression of p55Gag and its cleavage products. Density gradient analysis and electron microscopy further demonstrated that the modified gag and gagprotease genes efficiently expressed particles with the density and morphology expected for HIV virus-like particles. Mice immunized with DNA plasmids containing the modified gag showed Gag-specific antibody and CD8 ؉ cytotoxic T-lymphocyte (CTL) responses that were inducible at doses of input DNA 100-fold lower than those associated with plasmids containing the native gag gene. Most importantly, four of four rhesus monkeys that received two or three immunizations with modified gag plasmid DNA demonstrated substantial Gag-specific CTL responses. These results highlight the useful application of modified gag expression cassettes for increasing the potency of DNA and other gene delivery vaccine approaches against HIV.
The effectiveness of cationic microparticles with adsorbed DNA at inducing immune responses was investigated in mice, guinea pigs, and rhesus macaques. Plasmid DNA vaccines encoding human immunodeficiency virus (HIV) Gag and Env adsorbed onto the surface of cationic poly(lactide-coglycolide) (PLG) microparticles were shown to be substantially more potent than corresponding naked DNA vaccines. In mice immunized with HIV gag DNA, adsorption onto PLG increased CD8 ؉ T-cell and antibody responses by ϳ100-and ϳ1,000-fold, respectively. In guinea pigs immunized with HIV env DNA adsorbed onto PLG, antibody responses showed a more rapid onset and achieved markedly higher enzyme-linked immunosorbent assay and neutralizing titers than in animals immunized with naked DNA. DNA vaccines have been shown to induce immune responses and protective immunity in many animal models of infectious disease (for a review, see reference 11). In mice, such responses can often be achieved with low doses (Ͻ1 g) of naked DNA. However, the immunogenicity of DNA vaccines in larger animals (e.g., guinea pigs, rabbits, and nonhuman primates) has been much lower than that observed in mice, even at higher doses of DNA. In human clinical trials, certain DNA vaccines have been shown to induce immune responses (5,19,30), but multiple immunizations of high doses of DNA were required. Therefore, in order to provide protective efficacy in humans, the potency of DNA vaccines needs to be increased. So far, it appears that DNA vaccines are more effective at priming T-cell responses than antibodies, as exemplified by induction of cytotoxic T lymphocytes (CTL) but no antibodies against malaria circumsporozoite protein in humans (30). Similarly, we show here that in mice, human immunodeficiency virus (HIV) gag DNA primed CD8 ϩ T-cell responses at doses of DNA 10-to 100-fold lower than that required for priming of antibody responses. Therefore, technologies aimed at increasing the potency of DNA vaccines need to be especially effective at boosting humoral responses.The technology described herein, formulation of DNA onto cationic poly(lactide-coglycolide) (PLG) microparticles, has been developed as a means to better target DNA to antigen-presenting cells (APCs). PLG microparticles are an attractive approach for vaccine delivery, since the polymer is biodegradable and biocompatible and has been used to develop several drug delivery systems (21). In addition, PLG microparticles have also been used for a number of years as delivery systems for entrapped vaccine antigens (24). More recently, PLG microparticles have been described as a delivery system for entrapped DNA vaccines (15,18). Nevertheless, recent observations have shown that DNA is damaged during microencapsulation, leading to a significant reduction in supercoiled DNA (2, 29). Moreover, the encapsulation efficiency is often low. Therefore, we developed a novel approach of adsorbing DNA onto the surface of PLG microparticles to avoid the problems associated with microencapsulation of DNA. This approach, ...
Dendritic cells (DC) play a key role in antigen presentation and activation of specific immunity. Much current research focuses on harnessing the potency of DC for vaccines, gene therapy, and cancer immunotherapy applications. However, DC are not readily transfected in vitro by traditional nonviral techniques. A novel DNA vaccine formulation was used to determine if DC are transfected in vitro. The formulation consists of plasmid DNA adsorbed on to cationic microparticles composed of the biodegradable polymer polylactide-co-glycolide (PLG) and the cationic surfactant, cetyltrimethylammonium bromide (CTAB). Using preparations of fluorescentlabeled plasmid DNA formulated on PLG-CTAB microparticles to study internalization by macrophages and dendritic cells in vitro and in vivo, we found that most, but not all, of
DNA vaccines have been used widely in experimental primate models of human immunodeficiency virus (HIV), but their effectiveness has been limited. In this study, we evaluated three technologies for increasing the potency of DNA vaccines in rhesus macaques. These included DNA encoding Sindbis virus RNA replicons (pSINCP), cationic poly(lactide-co-glycolide) (PLG) microparticles for DNA delivery, and recombinant protein boosting. The DNA-based pSINCP replicon vaccines encoding HIV Gag and Env were approximately equal in potency to human cytomegalovirus (CMV) promoter-driven conventional DNA vaccines (pCMV). The PLG microparticle DNA delivery system was particularly effective at enhancing antibody responses induced by both pCMV and pSINCP vaccines and had less effect on T cells. Recombinant Gag and Env protein boosting elicited rapid and strong recall responses, in some cases to levels exceeding those seen after DNA or DNA/PLG priming. Of note, Env protein boosting induced serum-neutralizing antibodies and increased frequencies of gamma interferon-producing CD4 T cells severalfold. Thus, PLG microparticles are an effective means of delivering DNA vaccines in nonhuman primates, as demonstrated for two different types of DNA vaccines encoding two different antigens, and are compatible for use with DNA prime-protein boost regimens.Both neutralizing antibodies and cell-mediated immunity (CMI) likely will be required to protect against viruses that can establish chronic infections, such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV). Moreover, in animal models for HIV, both neutralizing antibodies against Env (9, 35-37) and cytotoxic T lymphocytes (CTL) that target multiple viral antigens (3-5, 17, 18, 21, 26, 30, 32, 38, 39, 45, 48, 51-53, 59) can contribute to protection through prevention of infection and clearance of virus-infected cells, respectively. Vaccines consisting of inactivated pathogens or recombinant proteins generally are not effective at inducing CTL and typically are used to induce protective antibodies. In contrast, viruses and intracellular bacteria can induce CTL responses, in part due to neoexpression of the antigens during infection.Plasmid DNA vaccines were born out of the need for inducing both antibody and CMI responses, including CTL, without the problems associated with live organism-based vaccines, such as potential reversion to virulence and antivector immunity that can limit boosting. Indeed, DNA vaccines that express antigens from strong viral promoters have been used to elicit protective antibodies and CMI in many animal models (14, 23). However, naked DNA vaccines, i.e., plasmid DNA in saline, have proven to be only modestly potent in humans, thereby limiting their utility. Many approaches have been explored to improve DNA vaccine potency, including better expression vectors, DNA formulation and delivery systems, adjuvants, and the use of booster vaccines.We developed an alternative DNA vector that launches a self-amplifying Sindbis virus (alphavirus) RNA replicon (1...
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