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Journal of Virology, October 2003, p. 10394-10403, Vol. 77, No. 19
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.19.10394-10403.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

An Alphavirus Replicon Particle Chimera Derived from Venezuelan Equine Encephalitis and Sindbis Viruses Is a Potent Gene-Based Vaccine Delivery Vector

Silvia Perri,* Catherine E. Greer, Kent Thudium, Barbara Doe, Harold Legg, Hong Liu, Raul E. Romero, Zequn Tang, Qian Bin, Thomas W. Dubensky Jr., Michael Vajdy, Gillis R. Otten, and John M. Polo

Vaccine Research, Chiron Corporation, Emeryville, California 94608

Received 14 February 2003/ Accepted 2 July 2003


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ABSTRACT
 
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-p55gag replicon RNA packaged within SIN envelope glycoproteins and SIN-p55gag 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.


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INTRODUCTION
 
Alphavirus vectors are being developed as gene-based vaccines for infectious and malignant diseases (38, 42, 45). Alphavirus vectors are known as replicons due to the self-amplification of the vector RNA, which occurs in the cytoplasm of the infected cell. Replicons contain the nonstructural protein genes encoding the viral replicase, the 5'- and 3'-end cis-active replication sequences, and the native subgenomic promoter, which directs expression of the encoded heterologous gene(s). While replicons lack the genes encoding virion structural proteins necessary for packaging and cell-to-cell spread of infectious particles, they may be packaged into virus-like particles by providing the structural proteins in trans, using transient RNA cotransfection systems (2, 29) or stable replicon packaging cell lines (36). Alternatively, the replicon RNA can be introduced into cells as plasmid DNA (10, 18).

Alphavirus replicon particle vectors have been developed using Sindbis virus (SIN) (2, 61), Venezuelan equine encephalitis virus (VEE) (40), and Semliki Forest virus (29). Each has been shown to induce robust cellular, humoral, and mucosal immune responses specific for the replicon-expressed antigen in several animal models (38, 42, 45). A number of features make alphavirus replicon vectors attractive for gene-based vaccines, including high-level expression of the heterologous gene (61), vector amplification through double-stranded RNA intermediates, which stimulates aspects of innate immunity, such as activation of the interferon (IFN) cascade (27), induction of apoptosis in some cell types (28) which may enhance immunogenicity via antigen cross-priming (62), and the overall lack of preexisting immunity in the human population. In addition, alphavirus replicon particles can be used for delivery of antigen to antigen-presenting cells, such as dendritic cells, the most potent antigen-presenting cell population (15, 31).

On the basis of their biological niche, each alphavirus has unique properties, which may be either desirable or undesirable in terms of function, potency, and safety when applied to recombinant vector systems. For example, VEE has a natural lymphotropism (31) that may be ideal for vaccine use, and VEE replicon particles have been shown to induce potent and protective immune responses in primates (6, 19). The pathogenesis of wild-type VEE in humans (20), however, raises safety concerns, some of which have been addressed by identifying and incorporating specific attenuating mutations in the envelope glycoproteins of the replicon particles (40). However, despite these precautions, production of VEE replicon particles must be conducted at biosafety level III, and only after completion of testing for contaminating replication-competent virus can the particles be used at biosafety level II. In contrast, since SIN is not associated with serious human disease (20, 52), SIN-derived replicon particles largely obviate these concerns. In addition, particular engineered SIN variants target lymphoid cells (15), and stable replicon packaging cell lines have been developed (36), which may simplify large-scale production for human testing of vaccine candidates. However, although SIN vectors induce potent immune responses in murine models (38, 42, 46), little is known about whether this translates to robust immune responses in primates.

Since the relative potencies of the different alphavirus vectors have not been characterized yet, we performed initial comparative immunogenicity studies for SIN and VEE replicon particles in this work. Also, we explored the feasibility of combining genetic components from each parent virus toward a goal of developing a chimeric alphavirus replicon particle with optimal potency and safety. For these studies, we generated a panel of SIN, VEE, and chimera replicon particles expressing a human immunodeficiency virus (HIV) p55gag antigen or green fluorescent protein (GFP) reporter. On the basis of in vitro and in vivo studies, we report here the identification of a novel VEE/SIN replicon particle chimera that combines selected desirable qualities of SIN and VEE, which should have utility as a potent gene-based vaccine delivery platform.


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MATERIALS AND METHODS
 
Cell line propagation and infection. BHK-21 cells, mouse fibroblast L929, and SV-BALB (16) were maintained in Dulbecco minimum essential medium (DMEM) supplemented with 10% fetal calf serum (FCS), 10 mM sodium pyruvate, and penicillin and streptomycin at 37°C with 5% CO2. Cell monolayers of approximately 80% confluency were infected with replicon particles for 1 h in DMEM containing 1% FCS at 37°C and then incubated overnight in DMEM containing 10% FCS.

Replicon vector and defective helper constructs. From the published sequence of VEE Trinidad donkey (TRD) strain (GenBank accession no. L01442) (22), the entire 11,447-nucleotide (nt) genome was synthesized and cloned as 14 separate fragments generated by annealing and ligating overlapping oligonucleotides. Individual fragments were used to assemble VEE-based replicon vector and defective helper constructs under the transcriptional control of a bacteriophage SP6 promoter. A VEE-based replicon (VCR) contains VEE nt 1 to 7561, unique restriction sites for insertion of heterologous genes under the control of the subgenomic promoter, and VEE nt 11330 to 11447 (3'-end region) with an A40 tract and hepatitis delta virus antigenomic ribozyme fused to its 3' end (10).

VCR-Chim2.1 was derived from VCR by (i) inserting a PCR-amplified SIN packaging sequence (nt 945 to 1076 of the SIN genome [59]) as an in-frame fusion within the VEE nonstructural protein gene 3 (nsP3) gene between the XhoI sites at nt 5493 and 5595 (22) and (ii) replacing the VEE 3' untranslated region (3'UTR) with the SIN 3'UTR from the previously published SIN-derived replicon vector, SINCR (15).

The CAP GFP reporter gene (Clontech) or the sequence-modified HIV type 1 (HIV-1) p55gag gene (63) was inserted into the SINCR, VCR, and VCR-Chim2.1 replicon vectors, resulting in constructs SINCR-GFP, SINCR-p55gag, VCR-GFP, VCR-p55gag, VCR-Chim2.1-GFP, and VCR-Chim2.1-p55gag.

Sequences encoding either capsid or envelope glycoproteins from SIN or VEE were inserted into the VEE-based defective helper backbone (VCR-DH), similar to VCR but with VEE nt 662 to 6961 deleted. Similarly, capsid or envelope glycoproteins from SIN or VEE were cloned into SIN defective helper backbones tDH or tDH-VUTR. These helpers are similar to the SIN-dl backbone (15, 36) but with the native SIN 5'-end sequences replaced by a tRNA-based SIN defective interfering 5'-end sequence (11). In addition, tDH-VUTR has the VEE subgenomic 5'UTR (nt 7536 to 7561 of the VEE-TRD strain [22]) in place of the SIN subgenomic 5'UTR. VCR-DH-Vgly and tDH-Vgly contain the VEE TRD-derived envelope glycoproteins, with a single amino acid substitution at E2 residue 120, analogous to the TC83 vaccine strain (22). VCR-DH-Sgly and tDH-VUTR-Sgly contain the envelope glycoproteins from a dendritic cell-tropic SIN strain with the E2 amino acid 160 deleted (15). VCR-DH-Vcap and tDH-Vcap contain the capsid sequence from VEE TRD. SIN-dl-cap (36), DH-BB-CSIN (11), and VCR-DH-Scap contain a capsid sequence from the SIN HR strain (51). Finally, tDH-S113Vcap contains a hybrid capsid protein gene with the amino terminus from SIN capsid (nt 7647 to 7986 [51]) and the carboxy terminus from VEE capsid (nt 7938 to 8386 of the VEE TRD strain [22]).

Production of alphavirus replicon particles. Replicon particles were generated by coelectroporation of in vitro-transcribed RNAs corresponding to a replicon and two defective helpers, one expressing capsid protein and the other expressing envelope glycoproteins, as previously described (36) (Table 1).


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  		TABLE 1. Replicon and defective helper RNA combinations used to generate the various replicon particles

Replicon particles expressing GFP were harvested as culture supernatants 24 h postelectroporation and clarified by centrifugation. Titers of replicon particles on BHK-21 cells or mouse fibroblast cells L929 and SV-BALB were determined as previously described (35).

Replicon particles expressing HIV-1 p55gag were harvested as culture supernatants at 20 and 30 h postelectroporation, clarified by filtration, and purified by cation exchange chromatography. Replicon particle titers were determined by intracellular staining of expressed Gag, following overnight infection of BHK-21 cells with serial dilutions of particles. Infected cells were permeabilized and fixed by using a Cytofix/Cytoperm kit (Pharmingen) and then stained with fluorescein isothiocyanate-conjugated antibodies to HIV-1 core antigen (Coulter). Using flow cytometry analysis, the percentage of Gag-positive cells was determined and used to calculate titers. The absence of contaminating replication-competent virus was determined by five consecutive infections of naive BHK-21 cell and determination of titers. Finally, endotoxin levels were measured for all replicon particle samples and shown to be <0.5 endotoxin unit/ml.

Semiquantitative reverse transcription-PCR (RT-PCR) for RNA replication. BHK-21 cells were electroporated with equivalent amounts of in vitro-transcribed replicon RNA. Each electroporated cell sample was seeded in quadruplicate, with individual samples harvested at 4, 10, and 22 h postelectroporation for extraction of total RNA and for flow cytometry analysis at 22 h to verify that similar numbers of cells had been transfected. Oligonucleotides complementary to either plus- or minus-strand RNA were used for strand-specific cDNA synthesis, after which a 415-bp GFP fragment was PCR amplified. Each PCR mixture was divided in multiple aliquots, and one aliquot was analyzed every five amplification cycles. As an internal standard, a fragment of the housekeeping gene BHKp23 (43) was also synthesized from each electroporated sample. The PCR products were analyzed by agarose gel electrophoresis, followed by staining and destaining with ethidium bromide and water. Photographic exposure times were adjusted so that all the signals were below saturation levels of the film. The intensity of the bands was also measured by densitometry.

HIV Gag-specific CD8+ T-cell responses in mice. For the replicon particle immunizations, groups of four BALB/c or CB6F1 mice (Charles River) were immunized two times (day 0 and day 14) with 106 or 107 infectious units (IU) of HIV p55gag-encoding replicon particles in a total volume of 100 µl, split equally between two intramuscular (tibialis anterior) sites. As a control, plasmid pCMVKm2.GagModSF2, expressing HIV p55gag (63), was used for intramuscular immunization, with 10 µg in 100 µl, also split equally between the two sites. Spleens were removed 2 weeks after the final immunization, pooled spleen cell suspensions were prepared, and samples were analyzed by flow cytometry for gamma interferon (IFN-{gamma})-secreting CD8+ T cells following 3 to 5 h of stimulation with Gag peptides as described previously (63). For the DNA prime-replicon particle boost model, groups of four BALB/c or CB6F1 mice were primed with 10 µg of the pCMVKm2.GagModSF2 plasmid and then given booster doses 28 days later with 106 IU of replicon particles. Five days after the booster doses, spleens were removed and analyzed for IFN-{gamma}-secreting CD8+ T cells (as described above) or positive staining of phycoerythrin-coupled tetrameric H-2Kd (Immunomics, San Diego, Calif.) containing the immunodominant HIVSF2 Gag peptide, AMQMLKETI (B. Doe and C. M. Walker, Letter, AIDS 10:793-794, 1996). Each study was done twice.

IFN-{alpha}/ß sensitivity. Within five passages after the cells were thawed, mouse fibroblasts L929 and SV-BALB were treated for 24 h with twofold serial dilutions of mouse IFN-{alpha}/ß (Biosource International) ranging from 500 to 0 U/ml and then infected with replicon particles at a multiplicity of infection (MOI) of 0.5. The cells were infected in triplicate in the absence and presence of anti-mouse IFN-{alpha} monoclonal antibody (350 neutralizing units/well; HyCult Biotechnology) and rabbit anti-mouse IFN-ß polyclonal antibody (500 neutralizing units/well; Biomedical Laboratories). At 24 h postinfection, cells were analyzed by flow cytometry, and the vector viability was evaluated as the ratio of the number of cells expressing GFP in the presence of IFN-{alpha}/ß to the control number of cells expressing GFP in the absence of IFN-{alpha}/ß. Each assay was performed three times.


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RESULTS
 
Construction of alphavirus replicon particle chimeras. To evaluate the possibility of combining genetic components from multiple parental alphaviruses into new chimeric vectors with optimal potency and safety, we chose two alphaviruses with distinct biological properties, SIN and VEE. First, we generated DNA clones for the entire genome of the VEE TRD strain (22) to construct VEE-derived replicon and defective helper packaging components in which the 5' VEE nt 1 is preceded by a SP6 bacteriophage promoter and the 3'-terminal region is followed by a synthetic 40-nt poly(A) tract and hepatitis delta virus antigenomic ribozyme (10, 36).

From the above VEE replicon vectors, as well as the corresponding SIN-derived vectors described previously (15, 36), two strategies were undertaken to produce SIN/VEE replicon particle chimeras. In the first instance, a hybrid capsid protein was engineered to exploit the functionally separable RNA-binding and glycoprotein interaction domains of the protein (Fig. 1). This hybrid capsid protein, designated S113V, contains a SIN-derived amino-terminal portion (SIN amino acids 1 to 113), which has been shown to be important for RNA binding (34, 59), and a VEE-derived carboxy-terminal portion (VEE amino acids 125 to 275), which is involved in glycoprotein interactions during the virion assembly process (26). X-ray crystallographic structural studies of the SIN capsid also showed that the amino-terminal residues 1 to 113 were disordered, while the carboxy-terminal residues 114 to 264 were folded into a chymotrypsin-like structure (5). The S113V capsid and the VEE envelope glycoprotein genes were then inserted into a SIN-derived defective helper backbone for packaging a SIN RNA replicon within VEE envelope glycoproteins (designated SINrep/VEEenv particles) (Fig. 1).



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  		FIG. 1. SINrep/VEEenv replicon particle chimeras. (A) Schematic illustration of the tripartite RNAs used in generating SINrep/VEEenv particle chimeras: SIN replicon expressing a gene-of-interest (G.O.I.), such as GFP or HIV-1 p55gag, and SIN-derived defective helpers expressing a SIN/VEE hybrid capsid protein and VEE envelope glycoproteins, respectively. SIN-derived sequences (white boxes), VEE-derived sequences (shaded boxes), and the subgenomic promoter (arrows) are indicated. (B) Alignment of capsid protein sequences in the RNA-binding domain. Sequences shown are from two SIN strains, DC+ (15) and HR (51), and two VEE strains, TRD (22) and 6119 (58). Identical residues (bold letters on shaded background), similar residues (residues shown boxed on white background), and the point where the amino terminus of the SIN capsid was fused to the carboxy terminus of VEE capsid (arrow) are indicated.

In the second chimera approach, a replicon vector was modified to contain a heterologous packaging signal derived from a different alphavirus (Fig. 2). A 132-nt sequence containing the well-defined SIN packaging signal (59) was cloned in frame into the nsP3 gene of the pVCR replicon instead of a sequence previously found to be nonessential for VEE (8). Moreover, the 3'UTR from SIN was used in place of the VEE 3'UTR, generating the modified replicon pVCR-Chim2.1. Finally, the SIN capsid and envelope glycoprotein genes were inserted into the VEE-derived defective helper backbone for packaging the modified VEE RNA replicon within SIN envelope glycoproteins (designated VEErep/SINenv particles) (Fig. 2).



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  		FIG. 2. VEErep/SINenv replicon particle chimeras. (A) Construction of the VCR-Chim2.1 replicon. The packaging signal (PS) and 3'UTR (3') from the SIN replicon were used to replace sequences in nsP3 and at the 3' end of the VEE replicon, respectively. (B) VEE-derived defective helpers expressing SIN capsid and envelope glycoproteins. SIN-derived sequences (white boxes), VEE-derived sequences (shaded boxes), and the subgenomic promoter (arrows) are indicated.

RNA replication and packaging efficiency of alphavirus replicon particle chimeras. To investigate possible differences in the RNA replication levels of the SIN and VEE replicons, we examined replication of genomic RNA separately from subgenomic mRNA transcription. The relative levels of minus-strand RNA synthesized by the replicons were measured by semiquantitative RT-PCR on equivalent amounts of total RNA extracted from BHK-21 cells that were electroporated 4, 10, and 22 h earlier with the in vitro-transcribed replicon RNAs. An oligonucleotide complementary to minus-strand RNA was used for cDNA synthesis, and a 420-bp fragment of the heterologous GFP sequence was PCR amplified. Each PCR mixture was divided into multiple aliquots, and one aliquot was analyzed every five amplification cycles. As shown in Fig. 3A, all three replicons exhibited similar levels of minus-strand RNA, indicating no major differences in their replication efficiency. A specific fragment of the housekeeping gene BHKp23 (43) was also synthesized from each sample as an internal standard, and similar amounts of product were obtained in all cases (data not shown). Similar results were obtained using RT-PCR conditions specific for positive-strand replicon RNA and with RNA extracted from BHK cells infected with packaged replicon vector particle preparations (data not shown).



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  		FIG. 3. RNA replication and packaging of alphavirus replicon particle chimeras. (A) Minus-strand RNA detection in cultured cells by semiquantitative RT-PCR. BHK-21 cells were electroporated with in vitro-transcribed replicon RNAs and harvested at different times, as indicated to the left of each gel panel. cDNA was synthesized from total RNA with an oligonucleotide primer complementary to minus-strand RNA, and a GFP fragment was amplified by PCR. PCR amplification mixtures were divided into six aliquots, and one aliquot per sample was removed after 0, 10, 15, 20, 25, and 30 amplification cycles as indicated above the gel lanes. (B) In vitro-transcribed replicon and defective helper RNAs were coelectroporated into BHK-21 cells to produce stocks of replicon particles. The titers of the replicon particle preparations were then determined by infecting naive BHK-21 cells with serial dilutions of the different particle preparations expressing GFP reporter and counting GFP-positive cells by flow cytometry at 16 h postinfection. The results are the averages of titers from three independent replicon particle preparations. 1E+0, 100; 2E+7, 2 x 107; 4E+7, 4 x 107.

To evaluate the efficiency of replicon particle production for the two chimera approaches, transient packaging assays were performed by cotransfection of in vitro-transcribed replicon and defective helper RNAs (Table 1). For the hybrid capsid protein strategy, SIN-derived replicon RNA expressing GFP was cotransfected with the SIN-derived defective helper RNAs encoding the hybrid capsid protein and VEE envelope glycoproteins and, for comparison, with defective helper RNAs encoding the SIN capsid and envelope glycoproteins. As shown in Fig. 3B, the SINrep/VEEenv replicon particle chimeras were produced at titers comparable to the titers obtained when packaging the SIN replicon RNA in its native SIN structural proteins. These results demonstrated that the hybrid capsid is functional and that SIN replicons can be packaged with VEE envelope glycoproteins and the hybrid capsid protein.

Similarly, for the heterologous packaging sequence strategy, the VEE chimera replicon RNA expressing GFP was coelectroporated with the VEE-derived defective helper RNAs expressing the SIN capsid protein and SIN envelope glycoproteins. For comparison with existing VEE-based replicon particles (40), the wild-type VEE replicon RNA was cotransfected with defective helper RNAs encoding VEE structural proteins. As shown in Fig. 3B, the VEErep/SINenv replicon particle chimeras were produced at titers comparable to the titers obtained when packaging the VEE replicon RNA in its native VEE structural proteins. These results show that VEE replicons containing a SIN packaging signal can be packaged within SIN structural proteins. Also, the modified VEE replicon with a 3' cis-acting sequence derived from SIN and 47-residue replacement between residues 488 to 522 of nsP3 remains functional.

Finally, these data indicate that both chimera strategies may be useful for the development of other chimeric alphavirus replicon particles.

Comparison of gene expression levels from the replicon particles. Since differences in levels of antigen expression from vectors may affect overall immunogenicity (4), a comparison of expression levels was performed in cultured cells using panels of replicon particles expressing a GFP reporter gene or the vaccine immunogen HIV-1 p55Gag (Fig. 4).



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  		FIG. 4. Analysis of heterologous gene expression in cultured cells. (A) BHK or L929 cells were infected with parental and chimeric replicon particles encoding GFP at an MOI of 0.5 to 1. At the indicated postinfection times, cells were harvested and analyzed by flow cytometry to determine the mean fluorescence intensity (MFI) of the GFP-positive cell population. (B) BHK cells were infected with replicon particles expressing HIV p55gag at an MOI of 0.5 to 1. At the indicated times, supernatants and cell lysates were harvested. Levels of p55gag were determined with the Coulter HIV-1 p24 antigen assay. The percentage of cells infected with the particles was determined with intracellular staining at the 24-h time point.

BHK-21 and mouse L929 fibroblast cells were infected with each of the four different GFP-expressing replicon particle preparations. An MOI of 0.5 to 1 was used to ensure that a large percentage of cells would be infected but likely with only a single infectious particle so that any differences measured between the replicon particle preparations would be based on a large number of events. By using flow cytometry, the levels of GFP expression were measured over a time course. As shown in Fig. 4A, the expression of GFP in both cell lines was higher from those particles containing a VEE-derived replicon RNA vector (i.e., VEE or VEErep/SINenv particles) than from the particles with a SIN-derived replicon RNA (i.e., SIN or SINrep/VEEenv particles), particularly at peak levels. Similar comparisons were made in BHK-21 cells using analogous particles expressing the HIV p55gag antigen (63). For these experiments, expression levels of both intracellular and secreted forms of the gag-derived cleavage product p24 were measured by an enzyme-linked immunosorbent assay. While differences in intracellular levels of Gag could not be discerned, similar expression differences were confirmed for secreted Gag, with the VEE replicons expressing 10- to 15-fold-more p24 than the SIN replicons did (Fig. 4B).

Immunogenicity of alphavirus replicon particles expressing HIV p55gag. Since both SIN- and VEE-derived vectors are being developed for vaccine applications (38, 42, 45), we compared the immunogenicities of replicon particles expressing sequence-modified HIV-1 p55Gag (63) in animal models. Similar to the GFP-expressing particles, SIN, VEE, SINrep/VEEenv, and VEErep/SINenv replicon particles expressing p55gag were efficiently produced, chromatographically purified, and titrated on BHK-21 cells with final titers ranging between 1 x 108 and 9 x 108 IU/ml. These data clearly indicate the absence of any packaging limitations when moving from the GFP reporter to the HIV Gag antigen.

Immunogenicity was evaluated in two different mouse strains, BALB/c and CB6F1, either alone or as a booster dose, following initial immunization with a plasmid DNA expressing the same HIV p55gag antigen.

Initially, BALB/c mice were immunized two times intramuscularly with either 106 or 107 IU of replicon particles (Fig. 5A). Two weeks following the final immunization, splenocytes were obtained and tested directly for the percentage of HIV Gag-specific CD8+ T cells by flow cytometry (see Materials and Methods). As shown in Fig. 5A, immunization of the BALB/c mice with each of the replicon particle preparations resulted in HIV Gag-specific CD8+ T-cell responses that increased in a dose-dependent manner. No background responses were observed in the nonimmunized control group (not shown). Striking differences in potency were observed among the replicon particles expressing Gag, and the potency correlated with the origin of the RNA replicon, irrespective of the envelope glycoproteins used to package the particles. Both the VEE and VEErep/SINenv replicon particles stimulated a significantly greater number of Gag-specific CD8+ T cells than the SIN and SINrep/VEEenv replicon particles. Similar differences in potency were also observed in CB6F1 mice after two intramuscular immunizations with 106 IU of replicon particles (Fig. 5B).



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  		FIG. 5. Induction of HIV p55Gag-specific CD8+ T cells in mice by alphavirus replicon particles. (A) Groups of four BALB/c mice were immunized twice with either 106 (1E6) or 107 (1E7) IU of HIV p55gag-encoding replicon particles, or (B) groups of four BALB/c mice and four CB6F1 mice were immunized twice with 106 IU of HIV p55gag-encoding replicon particles. As a control, a DNA plasmid expressing HIV p55Gag was used. Two weeks after the final immunization, pooled spleen cell suspensions were prepared, and samples were analyzed for IFN-{gamma}-secreting CD8+ T cells following stimulation with Gag peptides. (C) Groups of four BALB/c or CB6F1 mice primed with 10 µg of DNA plasmid expressing HIV p55gag were given a single booster immunization of 106 IU of replicon particles. Five days later, pooled spleen cell suspensions were prepared, and samples were analyzed for IFN-{gamma}-secreting CD8+ T cells following stimulation with Gag peptides. The upper limit of the 95% confidence interval for the measured frequency is indicated by the error bar. Each panel represents the results of one of the two experiments done. In each experiment, the nonimmunized control group gave no background responses.

In the second immunization regimen, mice were immunized with 10 µg of plasmid DNA expressing Gag and then given a single intramuscular booster immunization of replicon particles at a dose of 106 IU (Fig. 5C). Five days following the replicon particle immunization, HIV Gag-specific CD8+ T-cell responses were determined. As shown in Fig. 5C, each of the replicon particle preparations significantly increased the number of Gag-specific CD8+ T cells over the plasmid DNA alone. Again, no background responses were observed in the nonimmunized control group (not shown). Interestingly, although the potencies of the different replicon particles paralleled the single modality data (Fig. 5A and B), the differences were not as dramatic. Both the SIN and SINrep/VEEenv replicon particles showed a potent increase in Gag-specific CD8+ T cells although not as potent as VEE and VEErep/SINenv replicon particles. These data were also confirmed by Gag peptide-H-2Kd tetramer analysis of splenocyte samples (data not shown).

Sensitivity of replicon particles to IFN. In addition to expression levels, another possible mechanism to explain potency differences is sensitivity to the IFN response. Thus, we examined the sensitivities of the alphavirus replicon vector particles to the antiviral effects of IFN-{alpha}/ß. For these studies, we chose two mouse fibroblast cell lines, L929 and SV-BALB, which are both capable of responding to viral infection with induction of IFN-{alpha}/ß. First, we determined the ability of the vector particles to infect these cell lines in comparison to BHK-21 cells. As shown in Fig. 6, the infectivity for all replicon particles was significantly reduced in L929 cells compared with BHK-21 cells. Similar results were obtained for SV-BALB cells (data not shown). However, particles containing the SIN replicon RNA exhibited a greater impact than particles containing the VEE replicon RNA. Such differences do not seem related to induction of endogenous IFN-{alpha}/ß in these cells, since the replicon particle titers were unaffected by the addition of neutralizing antibodies against IFN-{alpha}/ß (data not shown).



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  		FIG. 6. Replicon particle infection of different cell lines. Cells were infected with serial dilutions of the different replicon particle preparations expressing a GFP reporter. At 16 h postinfection, GFP-positive cells were counted by flow cytometry. Infectivity of the murine fibroblast L929 cell line was compared to the infectivity of BHK-21 cells as a ratio of the titer obtained in the BHK-21 to the titer obtained in the L929 cells. 1E+0, 100; 1E+01, 101; 1E+02, 102.

To determine the sensitivities of the replicons to IFN-{alpha}/ß, L929 cells were pretreated with increasing concentrations of IFN-{alpha}/ß for 24 h prior to infection. The cells were then infected with each of the vector particles at an MOI of 0.5, based on the titers determined in L929 cells. In addition, the cells were infected in either the presence or absence of IFN-{alpha}/ß-specific neutralizing antibodies to evaluate the effect of endogenous IFN-{alpha}/ß induced by the replicon particle infection. At 24 h postinfection, inhibition of vector replication was evaluated as the ratio of the number of cells expressing GFP in the presence of IFN-{alpha}/ß to the number of cells expressing GFP in the absence of IFN-{alpha}/ß. As shown in Fig. 7, pretreatment with the IFN-{alpha}/ß reduced the number of GFP-positive cells compared with the untreated controls for all replicon particles. However, the amount of IFN-{alpha}/ß required to inhibit the replicon expression varied, with the particles containing a VEE replicon RNA being approximately four- to fivefold-less sensitive to IFN-{alpha}/ß than particles containing a SIN replicon RNA. Similar results were obtained with the SV-BALB mouse fibroblast cells (data not shown). Infections done in the presence of IFN-{alpha}/ß neutralizing antibodies doubled the amount of IFN-{alpha}/ß units necessary to achieve 50% inhibition on VEE-derived replicons and had no effect on the SIN-derived replicons (data not shown).



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  		FIG. 7. IFN-{alpha}/ß sensitivity assay. Monolayers of cells were grown for 24 h in the presence of twofold dilutions of mouse-derived IFN-{alpha}/ß, with concentrations ranging from 0 to 500 U/ml. Cells were then infected at an MOI of 0.5 to 1 (based on titers obtained on L929 cells). At 24 h postinfection, GFP-positive cells were counted by flow cytometry. Fifty percent inhibition (dotted lines) is indicated and represents the amount of IFN-{alpha}/ß necessary to halve the number of cells expressing GFP [GFP(+)] compared to the samples that were not treated with IFN-{alpha}/ß [(-)IFN{alpha}/ß].


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DISCUSSION
 
A promising gene-based vaccine strategy, moving rapidly toward clinical evaluation for HIV and other infections, involves the use of replication-defective viral vector particles derived from alphaviruses (38, 42, 45). While an expanding body of literature indicates the tremendous potential of SIN, Semliki Forest virus, and VEE replicon particle-based vaccines in a variety of animal models, little is known about the potencies or uniquely beneficial features of the different alphavirus systems relative to each other. To address these questions, we generated replicon particles derived from two very different alphaviruses, VEE and SIN. In addition, we evaluated the potential advantages of combining features from each parent alphavirus into novel chimeric replicon particles. To this end, using two distinct approaches, we constructed replicon particle chimeras in which the RNA replicon component of VEE was packaged within the envelope glycoproteins of SIN and the converse, in which SIN replicon RNA was packaged in VEE envelope glycoproteins. The new replicon particle chimeras were then compared to their parental counterparts with respect to a number of parameters.

To compare the immunogenicities of these different replicon particle vectors, HIV-1 p55Gag protein was chosen as an antigen. The HIV Gag antigen is a primary target for CD8+ T cells in HIV-infected long-term nonprogressors (32, 39), and Gag-specific CD8+ cytotoxic T lymphocytes have been shown to be important in controlling virus load in both acute and asymptomatic phases of infection (1, 23, 24, 32). In fact, alphavirus vectors expressing Gag, as well as a number of other HIV antigens, are very promising candidates for HIV vaccine development (6, 33, 55, 56). Therefore, evaluation of the abilities of the different vectors to induce HIV Gag-specific CD8+ T cells is important. Immunization of mice with the various replicon particles revealed that each configuration was able to induce Gag-specific CD8+ T-cell responses in a dose-dependent manner, albeit with different levels of potency.

The potencies of the four different replicon particle preparations correlated with the origin of the RNA vector replicon itself and appeared to be independent of the origin of the envelope glycoproteins. These results were somewhat surprising, since much of the focus on enhancing alphavirus replicon particle-based vaccine potency has targeted the envelope glycoproteins, which are known to play a major role in cell tropism, and single amino acid mutations have been shown to dramatically alter the in vivo phenotype (3, 7, 30, 31, 37). Our data with the replicon particle chimeras suggest that while there may be differences in the cell repertoire infected based on the presence of VEE or SIN envelope glycoproteins, ultimately any such potential difference with these specific envelope glycoproteins does not impact immunogenicity.

Several factors may contribute to the difference in potency observed between the SIN and VEE replicon RNAs expressing HIV p55gag, including vector replication and/or antigen expression levels. It has been suggested that increased antigen expression might enhance plasmid DNA-based genetic vaccines (4). Indeed, among the alphavirus replicon particles, we observed higher expression levels of both a reporter protein and HIV Gag antigen from the VEE replicon RNAs than from SIN replicon RNA. A priori, these expression differences might be expected to result from variations in the level of RNA replication or transcription from the subgenomic promoter. However, semiquantitative RT-PCR did not indicate any significant variations in either genomic or subgenomic RNA levels. It is possible that the expression differences observed are related to the translation efficiencies of VEE versus SIN subgenomic mRNAs. It is known that SIN utilizes a translation enhancer element embedded in the subgenomic mRNA 5'UTR and capsid gene open reading frame (13). This element is typically not present in the SIN replicon, but its addition to the open reading frame of a reporter gene was shown to increase expression of the gene from a SIN replicon (13). Unfortunately, its inclusion in a SIN-derived vector for vaccine applications might not be useful, as it would produce a fusion protein with the alphavirus capsid rather than the native antigen. In contrast, VEE does not appear to have or require a similar element for efficient translation from the subgenomic mRNA (40). We have shown the VEE subgenomic 5'UTR to be compatible when substituted into the SIN defective helpers (tDH-VUTR), and it would be interesting to evaluate whether incorporation into a SIN replicon would enhance expression levels.

Another variable that might contribute to the observed differences in vaccine immunogenicity attributable to the replicon RNA is sensitivity to IFN-{alpha}/ß. The IFN-{alpha}/ß system is the first line of defense against viral infections in animals (for recent reviews, see references 21 and 48) and plays an important role in determining the virulence of alphaviruses (17, 44, 54). Various degrees of resistance to IFN-{alpha}/ß have been shown for VEE, and the resistance phenotype appears to be associated with more-virulent strains (50, 60). In contrast, SIN is generally quite sensitive to the antiviral effects of IFN-{alpha}/ß in vitro (9), while in vivo induction of IFN-{alpha}/ß seems to correlate with high viral replication (49, 53, 57). Interestingly, our experimental results suggest that while replicon particles containing SIN-derived RNA are more sensitive than particles containing VEE-derived RNA, the origin of envelope glycoproteins does not significantly affect the sensitivity of these vectors. The differences in sensitivity might be linked to the corresponding 5'UTR elements of SIN and VEE (50, 60). Alternatively, differences between the nsP2 protein of SIN and VEE may play a role. Recent studies suggest that SIN nsP2 can affect the production of IFN-{alpha}/ß in infected cells and determine the outcome of SIN infection (14). It will be interesting to further analyze the roles of all these elements, as the differences in IFN-{alpha}/ß sensitivity are likely to impact the in vivo potency of these vectors.

In the immunogenicity studies, we evaluated a panel of alphavirus replicon particles for induction of HIV Gag-specific CD8+ T cells using two different immunization regimens. In one regimen, naive mice were immunized twice with replicon particles; while in the other, mice were first primed with a plasmid DNA expressing the same antigen and then given a single booster dose of the replicon particles. In recent years, vaccine strategies of DNA priming followed by a viral vector booster dose have been used to induce potent T-cell responses (reviewed in reference 41). It is believed that the high efficiency of these prime-boost strategies might reflect the ability of DNA vaccines to focus the immune responses on the antigen, followed by the viral vector further stimulating the antigen-specific response, as well as other proinflammatory responses that enhance immunity. The use of viral vectors alone is also quite attractive, given the lower complexity of manufacturing and immunizing for a single-component vaccine. Our studies indicate significant potency of the alphavirus replicon particles by either approach, as neither required any in vitro proliferation or differentiation to detect these antigen-specific CD8+ T-cell responses. Interestingly, while replicon particles containing the VEE replicon RNA (VEE and VEErep/SINenv) induced significantly higher Gag-specific CD8+ T-cell responses than SIN replicon RNA (SIN and SINrep/VEEenv) as a stand-alone vaccine modality, this difference in potency was not as dramatic following a prior DNA priming regimen, as all replicon particle configurations gave a strong boost effect. Also, both particles containing the VEE replicon were more potent than plasmid DNA alone delivered to mice in optimal amounts.

The safety of any replicon vector particle-based platform is essential for vaccine delivery to humans. While the use of two "split" defective helpers was designed as a means toward the elimination of RNA recombination that could lead to contaminating replication-competent virus (11, 12, 40), the perception of risk would be further reduced if any potential replication-competent virus were nonpathogenic to humans. Chimeras between distant alphaviruses such as SIN and Ross River virus or the highly pathogenic western and eastern equine encephalitis viruses have been generated and shown to be viable but also attenuated in animals (25, 47). Similarly, a virus chimera of SIN and VEE also might be attenuated, although further studies would be needed to demonstrate such attenuation.

Finally, the design and use of chimeric replicon particles, which utilize components from both SIN and VEE, represent a novel approach toward rationally designed alphavirus vaccine vectors with selected features that emphasize in vivo potency, ease of production, and overall safety. Certainly our initial immunogenicity evaluations with an HIV p55Gag antigen in the VEErep/SINenv replicon particle chimera indicate potent induction of cellular immune responses. Coupled with the previously developed SIN packaging cell line technology (36), these systems should provide a basis for advancement toward human clinical vaccine studies.


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ACKNOWLEDGMENTS
 
This project has been funded in part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, under contract no. N01-AI-05396.

We thank Nelle Cronen for assistance in preparing the illustrations.


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FOOTNOTES
 
* Corresponding author. Mailing address: Chiron Corporation, 4560 Horton St., MS 4.3, Emeryville, CA 94608. Phone: (510) 923-8144. Fax: (510) 923-2586. E-mail: silvia_perri{at}chiron.com. Back


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Journal of Virology, October 2003, p. 10394-10403, Vol. 77, No. 19
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.19.10394-10403.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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