Amplicon-directed expression of HIV gp120 in 293 cells. 293 cells were infected with either HSV:gp120 amplicon particles or with amplicon particles encoding MIAP (HSV:MIAP) at an MOI of 0.5. At 48 h after infection, cell culture supernatants were collected, concentrated, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analyzed by immunoblot with the gp120-specific monoclonal antibody AD3 (the position of gp120 is indicated by the arrow). The numbers on the left denote the positions of the prestained molecular mass markers (Amersham-Pharmacia) which were included on the gel; sizes are in kilodaltons.

Gamma interferon-producing cells detected in the ELISPOT assay correspond to CD8⁺ T lymphocytes. Splenocytes were collected from immunized animals at 22 days postinfection, and CD8⁺ T lymphocytes were depleted from this population by using anti-CD8a coupled to magnetic particles. Primary splenocytes were isolated and stained with FITC-conjugated anti-CD8 Ly-2 monoclonal antibody either prior to or after depletion with anti-CD8a magnetic beads. Cells were analyzed by using a FACScan cytometer. (A) Percentage of CD8⁺ cells in the splenocyte population (before and after immunodepletion) for each individual mouse. (B) Splenocytes were subsequently tested for gamma interferon release by ELISPOT assay. The results are shown for each individual mouse (A1, A2, and A3) in the presence of the immunodominant V3 peptide from HIV-1 (RGPGRAFVTI). In all cases, three different input levels of splenocytes were analyzed in triplicate (400,000, 200,000 or 100,000 cells as indicated). The results shown represent the mean number of spots ± the standard error of the mean for each datum point. Note that in this experiment, the mean number of spots was calculated as follows: mean number of spots in the presence of the V3 peptide minus mean number of spots detected in the absence of the V3 peptide. This subtraction of nonspecific background reactivity was performed to facilitate comparison between results from the total and CD8-depleted splenocyte populations.

(A) A single inoculation of HSV:gp120 amplicon particles results in durable cellular immunity, as measured by gamma interferon ELISPOT assay. Mice were inoculated with a single dose of 10 6 i.u. of either HSV:gp120 or HSV:lacZ amplicon particles via either the i.d. or the i.m. routes. Mice were sacrificed, and splenocytes were tested by gamma interferon ELISPOT assay 171 days later. The results are shown for each individual mouse (A1, A2, and A3) in the presence (+) or absence (−) of the V3 peptide. As noted in the legend to Fig. 3A, three different input dilutions of splenocytes were used, and results are presented as the mean of triplicate datum points ± the standard error of the mean. (B) A single inoculation of HSV:gp120 amplicon particles results in durable cellular immunity, as measured by cytotoxic (JAM) assay. Splenocytes were harvested as described above, and cells were cultured in the presence of irradiated syngeneic LPS blasts pulsed with the class I-restricted epitope as indicated. The results are shown across a range of effector-to-target (E:T) cell ratios for each individual mouse (A1, A2, and A3) in the presence (+; dark lines) or absence (−; light lines) of the HIV-1 V3 peptide. (C) A single inoculation of HSV:gp120 amplicon particles elicits long-lasting Env-specific humoral immunity. Mice were injected with 10 6 i.u. of HSV:gp120 or HSV:lacZ amplicon particles. At 171 days after inoculation, mice were bled, and endpoint anti-gp160 titers were determined by ELISA.

(A) Inoculation of mice with wild-type HSV-1 Patton elicits a strong humoral response against HSV-1. Mice were infected with 4 × 10⁷ PFU of HSV-1 Patton via the i.p. route, and 5 weeks later, sera were collected and analyzed by anti-HSV-1 IgG ELISA; as a control, HSV-1-naive mice were also analyzed. Results are expressed in terms of OD readings at 405 nm for each individual mouse; the results from naive animals are shown in the left panel, while the results from the Patton strain-infected animals are shown in the right panel. All animals are identified with respect to the experimental group to which they were assigned (for example, mice which subsequently received HSV:gp120 amplicon particles via the i.d. route are denoted as “gp120 ID,” whereas mice which subsequently received HSV:lacZ amplicon particles are denoted as “LacZ ID”). (B) Mice preinfected with wild-type HSV still mount a strong Env-specific cellular response after inoculation with HSV:gp120 amplicon particles, as determined by the ELISPOT assay. Mice were either left untreated (naive) or preinfected with wild-type HSV-1, as outlined above. All mice were then inoculated with either HSV:gp120 or HSV:lacZ amplicon particles (10 6 i.u. via the i.d. route). Splenocytes were harvested at 21 days after inoculation with amplicon particles and were analyzed for gamma interferon release by ELISPOT assay. The results are shown for each individual mouse (each bar reflects one mouse), in the presence (+ peptide) or absence of the HIV-1 V3 peptide. Three different input levels of splenocytes were analyzed in triplicate (400,000, 200,000, or 100,000 cells), and the results shown represent the mean ± the standard error of the mean for each datum point. Data denoted with an asterisk correspond to mean of only two values, with the corresponding standard deviation; one outlier datum point was discarded. The four panels represent data from naive mice treated with HSV:gp120 or HSV:lacZ (top panels, left and right, respectively) and from HSV-preinfected mice treated with HSV:gp120 or HSV:lacZ (lower panels, left and right, respectively). (C) Mice preinfected with wild-type HSV still mount a strong Env-specific cellular response after inoculation with HSV:gp120 amplicon particles, as determined by functional cytolytic (JAM) assay. Mice were either left untreated (naive) or preinfected with wild-type HSV-1; they were then inoculated with either HSV:gp120 or HSV:lacZ amplicon particles, as outlined above. Splenocytes were harvested at 21 days after immunization with amplicon and were analyzed for functional cytotoxicity by the JAM assay after a 5-day period of in vitro restimulation. The results are shown across a range of effector-to-target (E:T) cell ratios for each individual mouse (A1, A2, and A3) in the presence (+; dark lines) or absence (−; light lines) of the HIV-1 V3 peptide. The four panels represent data from naive mice treated with HSV:gp120 or HSV:lacZ (top panels, left and right, respectively) and from HSV-preinfected mice treated with HSV:gp120 or HSV:lacZ (lower panels, left and right, respectively). (D) Mice preinfected with wild-type HSV mount a weak or undetectable Env-specific humoral response after immunization with HSV:gp120 amplicon particles. Endpoint antibody titers were calculated by a gp160 IgG ELISA. Note that the data shown in this panel are representative of three independent experiments that yielded similar results; this particular data set derives from a different experiment than the datasets shown in panels A to C, as reflected by the use of a different negative control amplicon (HSV:MIAP).

HSV-1 amplicon vector system. The amplicon plasmid contains the HSV-1 origin of DNA replication, the viral cleavage and packaging site, and a mammalian expression cassette encoding the protein of interest (in this case, gp120). This plasmid is cotransfected into BHK cells, along with pBSvhs (which encodes the HSV-1 virion host shutoff protein), and the defective packaging construct (BAC-HSV), which lacks all viral cleavage and packaging elements. Immediately after transfection, the amplicon plasmid undergoes DNA replication (driven by proteins produced from the helper virus genome); this results in the generation of head-to-tail concatemers of the plasmid. These concatemers are cleaved into genome unit-length molecules and then packaged into virus particles, which can be harvested and concentrated and are ready for use.

A single inoculation of HSV:gp120 amplicon particles results in potent and cellular immune responses to gp120. (A) Analysis of gamma interferon release by ELISPOT assay. Mice were inoculated with either 10 6 infectious units of HSV:gp120 or HSV:lacZ amplicon particles or 10 6 PFU of MVA.H. Splenocytes were analyzed 22 days postinfection for gamma interferon release by ELISPOT assay. Results are shown for each individual mouse (A1, A2, and A3) in the presence (+) or absence (−) of the immunodominant V3 peptide from HIV-1 (RGPGRAFVTI). In all cases, three different input levels of splenocytes were analyzed in triplicate (400,000, 200,000, or 100,000 cells as indicated). The results shown represent the mean the standard error of the mean for each datum point. This experiment is representative of two experiments that were performed, both of which yielded similar results. In all cases, relatively few gamma interferon-producing cells were detected in the absence of the specific HIV-1 peptide or in animals immunized with an irrelevant vector (HSV:lacZ). (B) Vaccine-elicited T cells possess potent cytolytic activity. Splenocytes were harvested from immunized mice at 22 days postinfection. The cells were then cultured for 5 days in the presence of irradiated syngeneic LPS blasts pulsed with the class I-restricted epitope. Cytolytic function was determined by incubating various amounts of syngeneic P815 target cells with effector cells, generating different effector-to-target (E:T) ratios. The results are shown for each individual mouse (A1, A2, and A3) in the presence (+; solid lines) or absence (−; shaded lines) of the immunodominant V3 peptide from HIV-1 (RGPGRAFVTI). The results represent the mean values from triplicate samples. This experiment is representative of two experiments that were performed, both of which yielded similar results. (C) Inoculation of mice with a single dose of HSV:gp120 amplicon particles results in a substantial Env-specific humoral response. Estimated endpoint titers are shown; this experiment is representative of two experiments that were performed, both of which yielded similar results.

Effect of the route of delivery on immune responses to HSV:gp120 amplicon particles. (A) i.d. inoculation results in higher frequency of gamma interferon-secreting cells. A single inoculation of 10 6 i.u. was introduced in mice via i.d., i.m., or i.p. routes. At 21 days postinoculation, mice were sacrificed, and splenocytes were tested for gamma interferon release by ELISPOT assay. The results are shown for each individual mouse (A1, A2, and A3) in the presence (+) or absence (−) of the V3 peptide. As noted in the legend to Fig. 3A, three different input dilutions of splenocytes were used, and results are presented as the mean of triplicate datum points ± the standard error of the mean. The i.d. and i.m. routes of amplicon delivery resulted in higher frequencies of gamma interferon-secreting cells compared to the i.p. route, with the i.d. route resulting in the highest frequency. (B) i.p. inoculation yields the highest level of anti-gp160 antibodies. Of the three routes of inoculation tested, the i.p. route resulted in a stronger anti-gp160 humoral response compared to either the i.m. or i.d. routes.

The immune response to HSV:gp120 amplicon particles is dose dependent, but strong responses are detected even at very low amplicon doses. (A) Dose dependence of the cellular response to HSV:gp120 amplicon particles, as determined by the gamma interferon ELISPOT assay. Decreasing 0.5-log increments of HSV:gp120 amplicon particles, ranging from 10⁶ to 10⁴ i.u. (as indicated), were inoculated into BALB/c mice via the i.d. route; as a negative control, an additional group of animals received an irrelevant amplicon construct (HSV:MIAP) at the high-dose level (10⁶ i.u.) via the i.d. route. At 21 days postinfection, mice were sacrificed and splenocytes were tested for gamma interferon release by ELISPOT assay. Results are shown for each individual mouse (each bar reflects one mouse), in the presence (+; solid bars) or absence (hatched bars) of the HIV-1 V3 peptide. Three different input levels of splenocytes were analyzed in triplicate (400,000, 200,000, or 100,000 cells as denoted in the three panels). The results shown represent the mean ± the standard error of the mean for each datum point; the experiment shown is representative of two experiments that yielded similar results. (B) Comparison of data from the gamma interferon ELISPOT assay and the tetramer assay. Freshly isolated splenocytes from immunized mice (see above) were also evaluated by staining with an H-2D^d tetramer folded with the V3 peptide (RGPGRAFVTI; see Materials and Methods). The results of tetramer staining (solid bars) and gamma interferon ELISPOT analysis (hatched bars) are expressed as percentages of positive splenocytes; the data show represent mean values from the three mice that were analyzed in each group ± the standard error of the mean. (C) Dose dependence of the cellular response to HSV:gp120 amplicon particles, as determined by functional cytolytic (JAM) assay. Splenocytes were isolated from immunized animals, as described in panel A, and then analyzed for cytolytic activity using the JAM assay (see Materials and Methods) after a 5-day period of in vitro expansion. The results are shown across a range of effector-to-target (E:T) cell ratios for each individual mouse (A1, A2, and A3) in the presence (+; solid lines) or absence (−; shaded lines) of the HIV-1 V3 peptide. (D) Dose dependence of the humoral response to HSV:gp120 amplicon particles. Endpoint antibody titers were calculated by the gp160 IgG ELISA assay.