Humoral and Cellular Immunities Elicited by HIV-1 DNA Vaccination

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Humoral and Cellular Immunities Elicited by HIV-1 DNA Vaccination JOHN W. SHIVERX, MARY-ELLEN DAVIES, HELEN C. PERRY, DANIEL C. FREED,

AND

MARGARET A. LIU

Received February 26, 1996, from the Department of Virus and Cell Biology, Merck Research Laboratories, West Point, PA 19486. Final revised manuscript received March 25, 1996. Accepted for publication April 9, 1996X. Abstract 0 Recently it has been shown that immunization with plasmid DNA encoding genes for viral or bacterial antigens can elicit both humoral and cellular immune responses in rodents and nonhuman primates. In this study, mice and nonhuman primates were vaccinated by intramuscular injection with plasmids that express either a secreted form of HIV-1 gp120 or rev proteins. Mice receiving the tPA-gp120 DNA developed antigenspecific antibody responses against recombinant gp120 protein and the V3 peptide neutralization epitope as determined by ELISA. Vaccinated mice also exhibited gp120-specific T cell responses, such as in vitro proliferation of splenocytes and MHC Class I-restricted cytotoxic T lymphocyte (CTL) activities, following antigen restimulation. In addition, supernatants from these lymphocyte cultures showed high levels of γ-interferon production compared with IL-4, suggesting that primarily type 1-like helper T (Th1) lymphocyte responses were induced by both vaccines. Th1-like responses were also obtained for mice vaccinated with rev DNA. Immune responses induced by gp120 or rev vaccines were dose-dependent, boostable, and long-lived (g 6 months). Nonhuman primates vaccinated with tPA-gp120 DNA also showed antigenspecific T lymphocyte proliferative and humoral responses, including moderate levels of neutralizing sera against homologous HIV. These results suggest that plasmid DNA may provide a powerful means for eliciting humoral and cellular immune responses against HIV.

Introduction There is a growing concensus that an effective vaccine for the prevention of HIV infection may need to elicit cellular as well as humoral immunities.1 Among the reasons for this shift are the observations that cytotoxic T lymphocytes (CTL) appear to have a primary role in clearing the initial viremia following infection2,3 and maintenance of disease-free infection,4,5 and that HIV-exposed, uninfected individuals often have anti-HIV T cell responses, including CTL, without antiHIV antibodies.6,7 In addition, an epidemiological study of sex-workers in Gambia showed that prior infection with HIV-2 provided ∼70% protection from subsequent infection with HIV-1, even though the env genes from these viruses encode proteins that are serologically distinct.8 In the simian immunodeficiency virus (SIV) infection model of Rhesus monkeys, which is currently the most stringent test of vaccinemediated protection against primate immunodeficiency viruses, the laboratories of Desrosiers et al.9 and Stott et al.10 have shown that complete protection against SIV may be provided by prior infection with live, attenuated forms of SIV.9,10 Although the correlates of immunity produced by this vaccine are still unknown, protection was achieved at levels of neutralizing serum antibodies substantially lower than levels known to confer protection. Taken together, these results suggest that both T cells and antibody may have the potential to provide protective immunity to HIV infection. Most vaccines for HIV under development employ either recombinant gp120 or killed virus antigens and are formulated in adjuvants that are unable to, or poorly able to, stimulate X

Abstract published in Advance ACS Abstracts, November 1, 1996.

© 1996, American Chemical Society and American Pharmaceutical Association

T lymphocyte responses, particularly for CTL. In addition, there is now recognition that helper T cell effector activity is a function of the types of cytokines these cells secrete in response to stimulating antigen. Mosmann and co-workers11 have shown that type 1-like helper T (Th1) lymphocytes secrete primarily IL-2 and γ-interferon, whereas type 2 T cells (Th2) are associated with IL-4, IL-5, and IL-10 secretion.11 These responding T cells usually have an MHC Class IIrestricted, CD4+ (helper) phenotype, although CD8+ T cells also have the ability to secrete cytokines. Th1 lymphocytes and cytokines promote cellular immunity, including CTL and Type IV (delayed-type) hypersensitivity (DTH) responses, and IgG2a synthesis by B cells, whereas Th2 cells and cytokines promote B cell activation and differentiation primarily for humoral immunity, with predominantly IgG1 production. Differentiation among T cell types on the basis of cytokine profiles was first demonstrated in mice, and there is now a growing body of evidence that suggests that similar types of T cells can be found in humans.12 Vaccination by direct injection of plasmid DNA expression vectors encoding genes derived from microbial pathogens has generated increasing interest recently.13 The initial DNA injection studies were performed with genes-encoding reporter enzymes, such as β-galactosidase, luciferase, and chloramphenicol acetlytransferase.14,15 These experiments demonstrated that skeletal muscles were particularly responsive to plasmid DNA uptake and subsequent gene expression and that the use of “naked” DNA yielded superior results compared with plasmid formulated in cationic liposomes. Liu and coworkers made the initial demonstration that DNA vaccination elicited immune responses to viral proteins and conferred protective immunity against influenza challenge in mice16 and, in a subsequent report, ferrets.17 These studies showed that vaccination with DNA encoding the influenza nucleoprotein (NP) elicited cross-strain reactive, anti-NP cytotoxic T lymphocytes (CTL) that provided protection to lethal heterosubtypic (H1 versus H3) influenza challenge. Vaccination with hemagglutinin (HA)-expressing DNA produced neutralizing antibodies, characterized by measurement of hemagglutination-inhibiting antibodies, which completely protected against homologous influenza challenge.18,19 Robinson and co-workers also reported protection against homologous influenza challenges with HA-encoding plasmids even though pre-challenge titers of neutralizing antibodies were low or undetectable.20,21 The HIV env gene (gp160 or gp120) encodes the protein epitopes that have been shown to be targets of neutralizing antibodies for this virus. There is also substantial documentation in HIV seropositive individuals, as well as in laboratory animal models, that both MHC Class I and II reactive peptide epitopes are contained within env, making it an interesting antigenic target for DNA vaccines.22 Experiments performed by Weiner and co-workers23-25 demonstrated that a gp160 DNA was able to induce detectable antibody and T cell responses in mice and nonhuman primates. In this report, we use plasmid DNA that produces a secreted form of gp120 protein as well as a vector that expresses the HIV regulatory protein (rev) to evaluate the immune responses of mice and two species of nonhuman primates (Rhesus and African green monkeys). We show that these vaccines elicit Th1-like

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Journal of Pharmaceutical Sciences / 1317 Vol. 85, No. 12, December 1996

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responses against each protein and CTLs for gp120, even without concomitant induction of antibodies, although significant levels of gp120-specific antibodies can be induced in each species.

Experimental Section Materials and AnimalssFemale BALB/c mice (8 to 12 weeks old) were obtained from Charles River Laboratories (Wilmington, MA). Adult African green (Cercopithecus aethiops) and Rhesus (Macaca mulatta) monkeys were obtained from and maintained within a closed breeding colony. Recombinant gp120 (IIIB, baculovirus-derived) and rev (IIIB, E. coli-derived) were obtained from Repligen (Cambridge, MA). Recombinant gp41(IIIB, E. coli-derived) was obtained from Intracel (Cambridge, MA). V3 peptides corresponding to MN and IIIB viral strains were purchased from American Bio-Technologies, Inc. (Cambridge, MA). Lymphocyte culture media was prepared with RPMI 1640 medium (Gibco Laboratories, Grand Island, NY) supplemented with 10% fetal calf serum containing 4 mM L-glutamine and 100 µg/mL each of penicillin and streptomycin. Vaccination VectorssThe expression vector used for deriving the vaccination constructs, V1Jns, and the bacterial growth conditions and plasmid purification procedures were similar to those described previously.18 This vector utilizes the promoter, enhancer, and intron A from the human cytomegalovirus26 (CMV) and the bovine growth hormone (BGH) termination and polyadenylation sequences27 within a pUC plasmid backbone from which the entire lac operon was removed and the ampicillin resistance gene replaced by one conferring resistance to kanamycin. DNA yields averaged from 15 to 30 mg of purified plasmid per liter of cell culture following purification by alkaline lysis and two CsCl centrifugations. V1Jns-tPA-gp120 vectors (gp120 IIIB or MN) express a secreted form of gp120 from a chimeric gene that substitutes the signal peptide from the human tissuespecific plasminogen activator (tPA) gene for the native gp120 leader and has a termination codon inserted at the end of the gp120 openreading frame similar to that described by Chapman and co-workers26 Constructs were also prepared for both gp120 and gp160 that retained native leader sequences. V1Jns-rev was prepared by PCR amplification of rev from pCV-1 (NIH AIDS Research and Reference Reagent Program Catalogue #303), which was then ligated into V1Jns. Each plasmid was confirmed by dideoxy sequencing of insert junction sites and in vitro expression by Western analysis of transfected human rhabdomyosarcoma cells (American Type Culture Collection, Rockville, MD). Measurement of Anti-gp120 and -rev AntibodiessELISAs were designed to detect antibodies generated against HIV with either specific recombinant protein or synthetic peptides as substrate antigens. Ninety-six-well microtiter plates were coated at 4 °C overnight with recombinant antigen at 2 µg/mL in phosphate buffered saline (PBS) solution using 50 µL/well on a rocking platform. Antigens consisted of either recombinant protein or synthetic V3 peptide. Plates were rinsed four times with wash buffer (PBS/0.05% Tween 20) followed by addition of 200 µL/well of blocking buffer [1% bovine serum albumin (BSA), 0.05% Tween-20 in PBS] and incubation for 2 h at room temperature with agitation. Pre-immune sera and immune sera were diluted in blocking buffer at the desired range of dilutions, and 100 µL was added per well. Plates were incubated for 1 h at room temperature with agitation and then washed four times with wash buffer. Secondary antibodies conjugated with horseradish peroxidase (anti-Rhesus Ig, Southern Biotechnology Associates; antimouse Ig, Jackson Immuno Research) were diluted 1:2000 (monkey) or 1:5000 (mouse) in blocking buffer and then added to each sample at 100 µL/well and incubated 1 h at room temperature with agitation. Plates were washed four times with wash buffer and then developed by addition of 100 µL/well of an o-phenylenediamine (o-PD, Calbiochem) solution at 1 mg/mL in 100 mM citrate buffer and 0.012% hydrogen peroxide at pH 4.5. Replicate samples were read for absorbance at 450 nm both kinetically (first 5 min of reaction) and at a 30 min endpoint with 100 µL of 1 M H2SO4 solution as a stop reagent [absorbance measured at 490 nm with a Thermo-max microplate reader (Molecular Devices Corp.)]. The background reactivities were subtracted for each immune serum using pre-immunization serum controls. Endpoint titers were calculated using SOFTmax (Molecular Devices Corp.) and geometric mean titers (GMT) reported for each animal group.

1318 / Journal of Pharmaceutical Sciences Vol. 85, No. 12, December 1996

HIV-1 Neutralization AssayssHIV-1 (MN) neutralization was detected by measurement of p24 gag antigen following infection of C8166 cells with either 100 or 1000 TCID50 infectious units of virus (Quality Biological, Inc., Gaithersburg, MD). Immune sera was heat inactivated at 56 °C for 30 min and then incubated at various dilutions in the presence of virus for 1 h at 37 °C in culture media prior to addition of C8166 cells. All samples were analyzed in quadruplicate. Viral culture supernatants were tested at 4 and 10 days post-infection for production of p24 gag (Western detection kit, Dupont, Wilmington, DE). For each sample, a matched pre-vaccination bleed was assayed simultaneously. Neutralization activities were calculated as percent of p24 gag expression of a given immune serum relative to a matched dilution of pre-injection serum. Cytotoxicity AssayssPeripheral blood mononuclear cells (PBMC) derived from spleens of vaccinated BALB/c mice were purified by Ficoll-Hypaque centrifugation to separate erythrocytes from white blood cells. CTLs were prepared by culturing splenocytes from vaccinated or control mice with specific peptide antigen using an equal number of irradiated antigen presenting cells. The P18 peptide28 (RIHIGPGRAFYTTKN for HIV MN strain) was used at 10 µM concentration to restimulate CTL in vitro and to sensitize target cells during the cytotoxicity assay at 1-10 µM by incubation at 37 °C for 2 h prior to the assay. The murine mastocytoma cell line P815, which is syngeneic with H-2d MHC haplotype mice, was used as target cells. Antigen-sensitized target cells were loaded with Na51CrO4 by incubation of targets for 1-2 h at 37 °C (0.2 mCi for ∼5 × 106 cells) followed by several washings of the target cells. CTL populations were mixed with target cells at varying ratios of effectors to targets (E/T) pelleted together, and incubated 4 h at 37 °C before harvest of the supernatants. Cytotoxicity was calculated with the following formula:

corrected % 51Cr release ) [cpm sample] - [cpm spontaneous]/ [cpm maximal] - [cpm spontaneous] The maximal releasable counts from the target cells was obtained by 0.2% Triton X-100 treatment. T Lymphocyte Proliferation AssayssMice were vaccinated one to three times with varying amounts (1.6-200 µg) of plasmid DNA [gp120 (MN or IIIB) or V1Jns-rev], and their spleens were extracted for in vitro determinations of T lymphocyte proliferation to recombinant antigen. Splenocytes were purified from red blood cells by Ficoll/ Hypaque centrifugation to obtain PBMC and cultured in tissue culture media with recombinant antigen [r-rev or r-gp120 (IIIB)] at 0.06-5 µg/mL of 4 × 105 cells/well (in 200 µL). Basal levels of [3H]thymidine uptake by these cells were obtained by culturing the cells in media alone, and maximum proliferation was induced by ConA stimulation at 2 µg/mL. Because ConA reactivities peak at ∼3 days in culture these samples were pulsed with [3H]thymidine-containing media (100 µL; 1 µCi of radioisotope/well) on day 3, whereas antigen-treated samples were pulsed on day 4. Samples were harvested ∼18 h after radioisotope pulsing. Control samples were prepared for each day of harvest. Stimulation indices (SI) were calculated for each sample by determining the ratio of proliferation of splenocytes in the presence of antigen to that in media alone. For African green and Rhesus monkeys, proliferation assays were performed with PBMC derived from peripheral blood with conditions similar to those just described for murine PBMCs except that 105 cells/ well were used and [3H]thymidine-containing media was added on day 5. Measurement of Murine CytokinessAn ELISA-based assay kit was used as directed by the manufacturer (Endogen, Cambridge, MA) to determine γ-interferon and IL-4 concentrations from supernatants derived from recombinant antigen-treated lymphocyte cultures. Culture supernatants (100 µL) were removed when [3H]thymidine additions were made and stored at -70 °C until analyzed. Supernatants from ConA-treated cultures were used as positive controls for each cytokine. Measurement of Type IV (Delayed-Type) Hypersensitivity (DTH) ResponsessMice were tested for DTH responses 10 ten days following the second of two vaccinations (200 µg/round) with either tPA-gp120 (MN) DNA or control plasmid, V1Jns. Vaccinated and control mice were injected in each hind footpad with 25 µL of either 1.25 µg of rgp120 (IIIB) in PBS or PBS only, and footpad size was measured 24 h later with a caliper-type micrometer in a blinded fashion.

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Table 1sAntibody Responses Elicited by tPA-gp120 DNA Vaccination of Micea Plasmid

Dose (µg)

Anti-gp120b

Anti-revb

tPA-gp120 gp120 gp160 gp160 + rev

200 200 200 100 + 100

5390 <40 <40 <40

n.t.c n.t. n.t. 170

a BALB/c mice (five/group) were vaccinated twice with the indicated plasmid(s) (all derived from HIV-1 MN except rev). b GMT antibody titers determined by ELISA with rgp120 (IIIB) or rev for test antigens. c n.t., not tested.

Results gp120-Specific Antibody Responses in MicesMice were vaccinated in each quadricep with 200 µg of gp120, gp160, or tPA-gp120 (all derived from MN) DNA dissolved in saline solution or an equal mixture (100 µg of each DNA) of gp160 and rev plasmids, boosted after 4 weeks, and tested for antigp120 antibodies 3 weeks later. Only the mice vaccinated with tPA-gp120 DNA developed anti-gp120 antibody responses (see Table 1), and no more experiments were performed with gp120 and gp160 plasmids. All mice receiving 200 µg of tPA-gp120 DNA developed antigp120 antibody responses after two vaccinations, with reciprocal endpoint ELISA titers ranging from 1800 to > 14 500. About 60% of mice responded with antibody titers at the lowest doses tested (1.6 µg). The geometric mean titers (GMT) of these antisera increased with increasing vaccine doses ranging from ∼600 at 1.6 µg up to ∼5400 at the 200-µg dose. All mice seroconverted following a third vaccination, with GMTs of >10 000 achieved at the higher doses. Anti-gp120 reactive mouse sera also bound synthetic V3 peptide corresponding to homologous virus, with endpoint titers that generally ranged from 50 to 200-fold lower than titers obtained against full-length rgp120 (data not shown). Similar results were obtained with tPA-gp120 DNA prepared from an HIV IIIB clone except that endpoint titers were up to 10-fold greater (data not shown). This result was probably because the IIIB gp120 gene was homologous to rgp120 used in the assay but was ∼20% mismatched compared with the MN tPAgp120 DNA. Mouse anti-gp120 responses remained stable for at least 6 months (latest dates tested) following the last inoculation. gp120-Specific Antibody Responses in Nonhuman PrimatessAfrican green monkeys showed detectable ELISA titers to rgp120 following one intramuscular vaccination with tPA-gp120 (MN) DNA, which increased to a GMT of 645 following three rounds (see Figure 1). A peak GMT of 1800 was achieved following four vaccinations. Anti-V3 peptide (MN) ELISA titers were obtained following three vaccinations, reaching a peak GMT of 150 following four rounds. Similar results were obtained for tPA-gp120 (IIIB) DNA with Rhesus monkeys.29,30 These sera also tested positive for gp120 antibodies as determined by a Western immunoblot strip kit (DuPont; data not shown). Sera from the African green monkeys were tested for their ability to neutralize homologous HIV with twofold serial dilutions of immune sera starting at 1:10 dilution. Homologous HIV-1 (MN) virus neutralization was detected following both three and four vaccinations as determined by inhibition of p24 gag production. Neutralization data, using sera obtained at week 20 (see Figure 1), at four weeks after receiving a fourth vaccination are shown in Figure 2. Nearly complete neutralization was observed for two of three antisera in this assay, but the third had detectable (>60%) activity at serum dilutions up to 1:80. However, when this assay was performed with 10-fold higher input levels of HIV (1000 TCID50), little or no detectable neutralization was observed for all three samples (data not

Figure 1sAntibody responses to HIV-1 gp120 antigen in African green monkeys vaccinated with tPA-gp120 (MN) DNA. Three African green monkeys were vaccinated at the times indicated by arrows with 2 mg of DNA, and immune sera was assayed for anti-gp120 (IIIB) (closed circles) ELISA reactivities. Endpoint GMTs were calculated from duplicate samples.

Figure 2sNeutralization of HIV-1 (MN) virus by sera from African green monkeys vaccinated with tPA-gp120 (MN) DNA. Each curve represents sera from an individual monkey that had been vaccinated four times with 2 mg of DNA. Reduction in p24 gag protein production was calculated relative to that for matched pre-bleeds at the indicated dilutions of sera. Data were obtained after 10 days in tissue culture following virus inoculation (using 100 TCID50 per sample).

shown), indicating that these sera have only low or moderate neutralization capabilities. Lymphocyte Responses in tPA-gp120 DNA Vaccinated MicesT cell proliferation assays were performed with spleens obtained from BALB/c mice vaccinated with tPAgp120 (MN) DNA and age-matched, syngeneic control mice. All vaccinees at all vaccine doses tested (down to 1.6 µg) showed rgp120-induced proliferation 6 months (longest timepoints tested) after two vaccinations. There was no apparent correlation between extent of proliferation and anti-gp120 ELISA titer. For example, two of the mice (Figure 3A) were seronegative as determined by ELISA for anti-gp120 antibodies, and the mouse with the highest antibody titer had one of

Journal of Pharmaceutical Sciences / 1319 Vol. 85, No. 12, December 1996

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Table 2sDelayed Hypersensitivity (Type IV) Responses in gp120 DNA-Vaccinated Micea % Difference Mouse No.

Vaccine

R

L

3951 3952 3953 3954 3955 3956 3957 3958 3959 3960

tPA-gp120 (MN) tPA-gp120 (MN) tPA-gp120 (MN) tPA-gp120 (MN) tPA-gp120 (MN) V1Jns V1Jns V1Jns V1Jns V1Jns

12.7 5.5 15.5 9.1 9.2 2.9 −1.4 1.4 1.4 4.5

1.4 1.4 0 4.5 0 −1.4 0 1.4 1.4 -4.3

a Measurements were made 24 h after rgp120 (R) or PBS (L) injections in footpads.

Figure 3sAntigen-specific T cell responses by tPA-gp120 DNA-vaccinated mice. Splenic T cells from mice vaccinated twice at a 4 week interval with 1.6 µg of tPA-gp120 DNA 4 months prior to sacrifice were cultured in vitro with recombinant gp120 protein at 5 µg/mL. Control mice were age-matched, unvaccinated mice. (A) Proliferation to rgp120 is represented by a stimulation index (SI). Anti-gp120 ELISA titers are indicated above each sample. (B) Cell culture supernatants from the samples shown in panel A were assayed for IL-4 (solid bar) and γ-interferon (cross-hatched bar) secretion 4 days following treatment with rgp120. Samples producing undetectable amounts of cytokine are shown as the minimum detection limit (15 and 47 pg/mL for IL-4 and γ-interferon, respectively). Supernatants from concanavalin A-treated splenocytes were used as positive controls for each cytokine.

the lowest SIs. Proliferative responses ranged from 6- to 35fold over media controls, whereas naive mice gave SI values of 1 to 3 (Figure 3A). Mice receiving further vaccination with tPA-gp120 DNA (three to four times) often showed very vigorous proliferative responses, with SIs ranging up to ∼100; again, without a simple correlation between antibody and proliferation responses to gp120 induced by this vaccine. T cells responded at antigen concentrations ranging from 5 µg/ mL to 60 ng/mL. No responses were detected for rgp41 treatment of these cells indicating that proliferation was antigen-specific. These experiments demonstrate that tPAgp120 DNA efficiently activated T cells as well as elicited 1320 / Journal of Pharmaceutical Sciences Vol. 85, No. 12, December 1996

strong antibody responses. In addition, each of these immune responses was determined with antigen that was heterologous (∼20%) compared with that encoded by the inoculating DNA (IIIB versus MN), indicating that T cells responded to conserved regions within gp120. Similar results were found in mice vaccinated with tPA-gp120 (IIIB) DNA (data not shown). Supernatants from the cell cultures just described were tested for cytokine secretion following rgp120 treatment by quantitative ELISA-based kits specific for either γ-interferon or IL-4. Naive mouse splenocytes cultured with rgp120 (the same samples shown in Figure 3A) showed no IL-4, whereas 20-75 pg/mL of IL-4 was detected in cultures of splenocytes from vaccinees (detection sensitivity ) 15 pg/mL for IL-4; Figure 3B). Cultures of splenocytes from naive samples produced 27-37 pg/mL of γ-interferon, whereas 430-16 000 pg/mL of γ-interferon was detected in vaccinees (detection sensitivity ) 47 pg/mL for γ-interferon). Taken together, these results suggest that the tPA-gp120 DNA elicits primarily Th1-like responses in mice. DTH responses to subcutaneous injections of antigen are indicative of antigen-specific cellular immunity that is attributed to Th1-like lymphocyte responses. Mice were tested for DTH responses by footpad injection of rgp120 solution or saline 10 days following the second of two vaccinations (200 µg/round) with either tPA-gp120 (MN) or control plasmid, V1Jns. Four of five mice receiving tPA-gp120 DNA showed swelling only in the footpad receiving rgp120, and no control vector mice showed swelling in either foot (see Table 2). These experiments confirm that plasmid DNA can induce Th1 cellular immunities and provide an in vivo correlate to our proliferation/cytokine experiments. tPA-gp120 (MN) DNA-vaccinated mice also were tested for CTL activities with an MHC Class I-restricted P18 peptide corresponding to a region of the V3 peptide loop of gp120 for both in vitro restimulation of splenocytes from vaccinees as well as for antigen sensitization of P815 target cells. Splenocytes from immunized mice showed significant lysis of P18 peptide-sensitized P815 target cells (∼60% lysis at E/T ) 30), with no cytotoxicities observed in the absence of P18 or with effector cells from unimmunized mice (see Figure 4). In an experiment performed simultaneously, splenocytes from vaccinated mice also showed gp120-specific proliferation with SI values of 36.7 and 5.2 (data not shown). Lymphocyte Responses Induced by tPA-gp120 DNA in Nonhuman PrimatessBlood-derived PBMCs from Rhesus and African green monkeys vaccinated with tPA-gp120 DNA (IIIB or MN, respectively; 2 mg/round) were tested for in vitro proliferative responses to rgp120. Vaccinated Rhesus monkey PBMCs showed proliferation when tested 1 week after an

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Figure 4sAnti-gp120 CTL responses in tPA-gp120 DNA-vaccinated mice. Two vaccinated mice (filled circle and square) and an unvaccinated control mouse (filled triangle) were tested for cytotoxicities against P815 cells in the presence (solid line) or absence (broken line, open symbols) of gp120 P18 peptide.

Figure 5sProliferation of peripheral blood T cells from tPA-gp120 DNA-vaccinated Rhesus monkeys. PBMCs from monkeys were tested for in vitro proliferation induced by rgp120 (5 µg/mL) 10 days following the second of two vaccinations (4 week interval) with 2 mg of tPA-gp120 (IIIB) DNA. A control Rhesus had an SI of 3.6 (PBMCs from seven different control monkeys had an average SI of 1.4 under similar conditions).

initial vaccination with tPA-gp120 (IIIB) DNA (data not shown). Of the three Rhesus monkeys tested 10 days after a second vaccination, only one monkey had clearly developed anti-gp120 antibodies (ELISA endpoint titers ) 56, 1556, and <20), but PBMCs from all three monkeys proliferated following rgp120 treatment (Figure 5). In a separate experiment with tPA-gp120 (MN) DNA, PBMCs from two of three African green monkeys (anti-gp120 ELISA GMT ) 1778 with individual serum end-point titers of 1179, 2830, and 1696; see Figure 1) showed specific proliferation (SI ) 4 and 9.2 compared with <2 in controls) when tested 4 weeks following four vaccination rounds (earlier dates not tested). Similar results were found 4 and 8 weeks following a fifth vaccination. Taken together, these data demonstrate that tPA-gp120 DNA can elicit T cell responses in nonhuman primates whether or not detectable levels of antibodies against gp120 were found.

Antibody and Lymphocyte Responses Induced by rev DNA in Mice and African Green MonkeyssMice were vaccinated either 3X or 1X with 200 µg of rev DNA at 4-week intervals. Low or undetectable levels of antibodies (endpoint titers up to ∼500) to r-rev were detected in vaccinee sera, although the rev ELISA assay easily detected anti-rev antibodies when testing sera obtained from an HIV-1 seropositive patient (data not shown). Splenocytes from vaccinated mice were also tested for in vitro proliferation and cytokine secretion following in vitro culture with r-rev protein (see Figure 6A and B). Splenocytes from mice vaccinated three times had SIs of 9-12, whereas splenocytes from mice receiving one vaccination were the same as background (SIs ) 2-3). None of sera tested from these mice had detectable levels of anti-rev antibodies. Splenic T cells from all rev vaccinees, but not control mice, secreted γ-interferon in response to r-rev antigen (2.4-2.8 ng/mL, 3X; 0.4-0.7 ng/mL, 1X), but no IL-4 was detected in culture supernatants above the background, indicating that Th1-like helper T cell responses were induced as found following tPAgp120 DNA vaccination. Based on this ability to detect cytokine secretion in the absence of cell proliferation, cytokine secretion may be a more sensitive assay than proliferation to specific antigen for determining T cell memory responses. Similar proliferation and cytokine results were found for mice tested at least 6 months post-vaccination (data not shown). No anti-rev antibodies were detected in three African green monkeys vaccinated up to four times with 2 mg of rev DNA. However, all three monkeys showed strong in vitro T cell proliferation (SIs ) 9-30) to r-rev following two vaccinations with V1Jns-rev (see Figure 7). These results corroborate the aforementioned mouse/rev experiments and confirm that strong T cell responses can be induced by plasmid DNA without concomitant induction of antibody responses.

Discussion We have used direct injection of plasmid DNA to elicit immune responses against the HIV-1 gp120 and rev proteins in mice and nonhuman primates. Because this type of vaccination allows in vivo production and, most likely, ap-

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Figure 6sAntigen-specific T cell responses of rev DNA-vaccinated mice. Splenic T cells from mice vaccinated three times (solid bars) or one time (cross-hatched bars) with 200 µg of rev DNA were cultured in vitro with r-rev protein at 5 or 1 µg/mL. A control mouse (open bar) did not receive any vaccination. (A) Proliferation to rgp120 is represented by a stimulation index (SI). (B) Cell culture supernatants from the samples shown in panel A were assayed for IL-4 (solid bar) and γ-interferon (cross-hatched bar) secretion 4 days following treatment with rgp120. Samples producing undetectable amounts of cytokine are shown as the minimum detection limit (15 and 47 pg/mL for IL-4 and γ-interferon, respectively). Supernatants from concanavalin A-treated splenocytes were used as positive controls for each cytokine.

propriate post-translational modifications and trafficking of vaccine-encoded gene products within the cell, these particular proteins provided an opportunity to compare immune responses generated against proteins such as gp120, which is secreted from cells, or rev, which is retained within cells. DNA vaccines also may present antigen in native structural conformation, including the formation of membrane protein oligomers. The generation of conformational epitopes may provide at least part of the reason that we have observed substantial neutralizing antibody responses to trimer-forming influenza HA proteins following vaccination with HA-encoding DNAs.17-19 For HIV the ability to generate native conformation may have great importance because of the observation that a conformational epitope, such as the CD4 binding site 1322 / Journal of Pharmaceutical Sciences Vol. 85, No. 12, December 1996

of gp120, is a major determinant of cross-strain neutralizing antibodies.31 More recent data suggest that further advantage may be gained for obtaining neutralizing antibodies by use of gp160 oligomers, rather than monomeric gp120 as the immunogen, because gp160 oligomers appear to contain additional linear32 as well as oligomer-dependent epitopes.33,34 In the current study we obtained significant antibody titers in all animal species vaccinated with tPA-gp120 DNA. By contrast, little or no antibody was obtained for animals vaccinated with gp120 and gp160 constructs containing genes with the native leader sequences or rev DNAs. For the gp120 and gp160 DNA, this result may be due to lower levels of protein expression produced by these constructs than that obtained with tPA-gp120 as determined by in vitro cell

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of plasmid DNA to elicit CTL responses is not limited to rodents. Antisera from tPA-gp120 DNA-vaccinated African green monkeys showed only low to moderate levels of virus neutralization activities against homologous virus based upon the inability of these sera to neutralize higher input levels of virus, as already noted, despite extensive vaccination (up to 5). It will be of interest to determine which antigenic form, gp120 versus gp160, provides better neutralization capability. Future experiments will include comparison of different forms of HIV env DNA for relative abilities to generate neutralizing antibody responses as well as the use of vaccines containing additional components of HIV to expand T cell reactivities.

References and Notes

Figure 7sProliferation of peripheral blood T cells from rev DNA-vaccinated African green monkeys. T cells from monkeys were tested for antigen-specific in vitro proliferation using r-rev protein (5 or 1 µg/mL) 10 days following a second vaccination with 2 mg of rev (IIIB) DNA. Control African green monkeys from five different monkeys had an average SI of 1.5 under similar conditions.

transfection studies (Perry and Shiver, unpublished results, and ref 26). Although the V1Jns-rev construct produced large quantities of rev in transfected cells, this protein has intracellular locations and may not be readily available for presentation to B cells for antibody generation. Interestingly, although antibody responses were not always obtained for the rev or tPA-gp120 (especially at low dose) DNA, all vaccinated animals exhibited T cell reactivities to either recall antigen. Although this result may appear paradoxical, in light of the already described observation that some HIV-exposed but seronegative individuals exhibit T cell memory responses to HIV-derived antigens,6,7 it is possible that a lower amount of antigen is required to stimulate T cell responses compared with antibodies. Alternatively, T cell proliferation/cytokine secretion assays may be more sensitive indicators of immunity than our ELISAs for gp120 and rev antibodies. Nevertheless, we have shown that small quantities of DNA elicit long-lived T cell memory that appears to be Th1-like by cytokine profile as well as by measurement of DTH responses. It is interesting to note that Haynes and coworkers35 recently reported that DNA vaccination of mice by particle bombardment with a gene gun initially elicited Th1like responses, which over time converted to Th2-like cytokine profiles, unlike the results presented here. Gene guns deliver DNA to the epidermis and dermis and, although the identity of antigen presenting cells is unknown for these vaccinations as well as intramuscular injection, it may be that the different inoculation routes lead to functionally different T cell responses. In addition to antibody and T cell proliferative responses, we also found that tPA-gp120 DNA elicited anti-gp120 CTL responses in mice with an MHC Class I-restricted peptide as antigen for both in vitro restimulation and sensitization of target cells for cytotoxicity assays. This result is similar to the previous finding in our laboratory that an influenza NP DNA was able to elicit a CTL response to the immunodominant epitope within NP and adds further evidence that plasmid DNA provides a potent means for generating CTLs against a variety of antigens. We have also recently found that tPA-gp120 DNA vaccination elicited potent CTL responses in Rhesus monkeys,36 thus confirming that the ability

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Acknowledgments We thank the veterinary associates who assisted with monkey vaccinations and bleedings, and W. McClement for his help in preparing the tPA-gp120 (MN) construct.

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