Induction of AIDS virus-specific CTL activity in fresh, unstimulated peripheral blood lymphocytes from rhesus macaques vaccinated with a DNA prime/modified vaccinia virus Ankara boost regimen.

The observed role of CTL in the containment of AIDS virus replication suggests that an effective HIV vaccine will be required to generate strong CTL responses. Because epitope-based vaccines offer several potential advantages for inducing strong, multispecific CTL responses, we tested the ability of an epitope-based DNA prime/modified vaccinia virus Ankara (MVA) boost vaccine to induce CTL responses against a single SIVgag CTL epitope. As assessed using both 51Cr release assays and tetramer staining of in vitro stimulated PBMC, DNA vaccinations administered to the skin with the gene gun induced and progressively increased p11C, C-->M (CTPYDINQM)-specific CD8+ T lymphocyte responses in six of six Mamu-A*01+ rhesus macaques. Tetramer staining of fresh, unstimulated PBMC from two of the DNA-vaccinated animals indicated that as much as 0.4% of all CD3+/CD8alpha+ T lymphocytes were specific for the SIVgag CTL epitope. Administration of MVA expressing the SIVgag CTL epitope further boosted these responses, such that 0.8-20.0% of CD3+/CD8alpha+ T lymphocytes in fresh, unstimulated PBMC were now Ag specific. Enzyme-linked immunospot assays confirmed this high frequency of Ag-specific cells, and intracellular IFN-gamma staining demonstrated that the majority of these cells produced IFN-gamma after peptide stimulation. Moreover, direct ex vivo SIV-specific cytotoxic activity could be detected in PBMC from five of the six DNA/MVA-vaccinated animals, indicating that this epitope-based DNA prime/MVA boost regimen represents a potent method for inducing high levels of functionally active, Ag-specific CD8+ T lymphocytes in non-human primates.

Comparison of immunity generated by nucleic acid-, MF59-, and ISCOM-formulated human immunodeficiency virus type 1 vaccines in Rhesus macaques: evidence for viral clearance.

The kinetics of T-helper immune responses generated in 16 mature outbred rhesus monkeys (Macaca mulatta) within a 10-month period by three different human immunodeficiency virus type 1 (HIV-1) vaccine strategies were compared. Immune responses to monomeric recombinant gp120SF2 (rgp120) when the protein was expressed in vivo by DNA immunization or when it was delivered as a subunit protein vaccine formulated either with the MF59 adjuvant or by incorporation into immune-stimulating complexes (ISCOMs) were compared. Virus-neutralizing antibodies (NA) against HIV-1SF2 reached similar titers in the two rgp120SF2 protein-immunized groups, but the responses showed different kinetics, while NA were delayed and their levels were low in the DNA-immunized animals. Antigen-specific gamma interferon (IFN-gamma) T-helper (type 1-like) responses were detected in the DNA-immunized group, but only after the fourth immunization, and the rgp120/MF59 group generated both IFN-gamma and interleukin-4 (IL-4) (type 2-like) responses that appeared after the third immunization. In contrast, rgp120/ISCOM-immunized animals rapidly developed marked IL-2, IFN-gamma (type 1-like), and IL-4 responses that peaked after the second immunization. To determine which type of immune responses correlated with protection from infection, all animals were challenged intravenously with 50 50% infective doses of a rhesus cell-propagated, in vivo-titrated stock of a chimeric simian immunodeficiency virus-HIVSF13 construct. Protection was observed in the two groups receiving the rgp120 subunit vaccines. Half of the animals in the ISCOM group were completely protected from infection. In other subunit vaccinees there was evidence by multiple assays that virus detected at 2 weeks postchallenge was effectively cleared. Early induction of potent type 1- as well as type 2-like T-helper responses induced the most-effective immunity.

Gene gun-based nucleic acid immunization alone or in combination with recombinant vaccinia vectors suppresses virus burden in rhesus macaques challenged with a heterologous SIV.

Gene gun-based DNA immunization alone or in combination with recombinant vaccinia vectors was evaluated for the ability to elicit protective immune responses in rhesus macaques challenged with a pathogenic, heterologous simian immunodeficiency virus (SIV). Six monkeys primed with seven consecutive doses of DNA encoding SIVmac239 gp120 and gp160 (DNA + DNA) were divided into two groups. Three of these animals received another DNA booster immunization and the remaining three received a booster immunization containing a homologous, live recombinant vaccinia virus expressing SIVmac251 gp160 (DNA + VAC). In addition, a group of 15 animals primed with recombinant vaccinia vectors were divided into two groups. One group of six monkeys received another immunization of vaccinia (VAC + VAC) and the other nine animals received a DNA (mac239) booster immunization (VAC + DNA). Geometric mean end-point IgG titres in the DNA + VAC and VAC + DNA groups were substantially higher than the responses seen in the VAC + VAC and DNA + DNA groups, demonstrating a synergistic relationship between DNA-based vaccines and recombinant vaccinia virus-based vaccines. All vaccinates and five naive controls were challenged 19 weeks after the final booster immunization with 10 animal infectious doses of SIVDelta/B670. The vaccines did not prevent infection. However, all vaccine groups showed significant virus load reductions from seven to 56 days post challenge when compared to controls. Although the DNA + DNA group developed the lowest prechallenge antibody responses, the most significant reduction (200-fold) in virus load was associated with this group. In addition, a significant delay in CD4+ T cell loss relative to controls was observed in the DNA + DNA group. These results demonstrate that a gene gun-based DNA vaccine provided some attenuation of infection and CD4+ T cell loss after a heterologous challenge.

Vaccination with HIV-1 gp120 DNA induces immune responses that are boosted by a recombinant gp120 protein subunit.

Small animals were immunized with plasmid DNA encoding HIV-1 envelope gp120 either intramuscularly by needle injection (mice and guinea pigs) or epidermally with the Accell gene gun (guinea pits). Subsequently, the animals were boosted with a recombinant gp120 protein subunit vaccine in an oil-in-water based adjuvant, MF59. Antibodies and cytotoxic T-lymphocyte (CTL) immune responses to the HIV envelope glycoprotein were observed in animals immunized with gp120 DNA derived from the HIV-1SF2 laboratory strain or from HIV-1 field isolates. Titers of ELISA antibodies and serum neutralizing antibodies against the HIV-1SF2 laboratory isolate were substantially increased in DNA-immunized animals following a single boost with recombinant gp120 protein subunit. This DNA prime/protein subunit boost immunization approach may be important for vaccination against infectious agents such as HIV for which it is difficult to raise strong antiviral humoral responses with DNA vaccination alone.

Enhancement of immunodeficiency virus-specific immune responses in DNA-immunized rhesus macaques.

In contrast to results obtained with plasmid DNA vectors encoding antigens from viruses such as influenza and hepatitis B, plasmids coding for antigens from primate immunodeficiency viruses have elicited relatively weak antibody responses following gene gun-mediated DNA immunization of rhesus monkeys. In an effort to augment these responses, the importance of the immunization schedule was investigated, as well as the possible synergy that might result from boosting gene gun-primed animals with other routes of immunization. Here we demonstrate that endpoint gp120-specific antibody titers can be enhanced as much as tenfold by reducing the number of immunizations and lengthening the resting period between immunizations. In addition, boosting gene gun-primed animals with either recombinant subunits or gp120-expressing vaccinia recombinants resulted in synergistic responses.

Induction of immunodeficiency virus-specific immune responses in rhesus monkeys following gene gun-mediated DNA vaccination.

The Accell gene delivery system (gene gun) was used to deliver gold particles coated with HIV-1LAI and SIVmac239 expression constructs into the epidermis of rhesus macaques, resulting in the elicitation of env- and gag-specific humoral responses. One microgram of vector DNA per dose was sufficient to induce immune responses in monkeys using SIVmac239 gp160 and gp120 vectors driven by the CMV-intron A promoter. Several parameters, including the identity of the vector, the length of the rest period between immunizations, the number of immunizations, and the amount of DNA per immunization, are all important in designing an optimal DNA immunization regimen. In addition, gene gun-based DNA immunization using low efficiency expression vectors is an effective means of priming for the induction of vigorous antibody responses in macaques following boosting with recombinant subunits.

Use of DNAs expressing HIV-1 Env and noninfectious HIV-1 particles to raise antibody responses in mice.

Two DNA constructs encoding portions of the human immunodeficiency virus type-1 (HIV-1) genome have been used to raise antibody responses in BABL/c mice. One DNA (pNL4-3.env) expresses the natural form of the envelope glycoprotein (Env) of HIV-1-NL4-3 (NL4-3). The second (pNL4-3.dpol) produces noninfectious NL4-3 particles. These two DNAs (alone or in combination) raised only transient titers of anti-Env IgG. In the same group in which pNL4-3.dpol DNA raised only transient antibody responses to Env, this DNA raised persistent antibody responses to the p24 virion capsid protein (CA). Antibody responses to Env and CA also showed different abilities to be boosted. The final boosts of pNL4-3.dpol DNA increased titers of anti-CA antibody, but failed to boost the falling titers of anti-Env antibody. At peak titers of anti-Env activity, sera with relatively low ELISA titers of anti-Env IgG (end points of 1:6250) had good titers of neutralizing antibody (approximately 1:3800 for 50 TCID50 of NL4-3). At the end of the experiment (a time when anti-Env antibodies had fallen to near background levels), in vitro-restimulated splenocytes from both pNL4-3.env and pNL4-3.dpol DNA vaccine groups exhibited similar cytotoxic activity.

DNA vaccines: a novel approach to immunization.

Direct DNA inoculations are being developed as a method of subunit vaccination. Plasmid DNAs encoding influenza virus hemagglutinin glycoproteins have been tested for the ability to provide protection against lethal influenza challenges. In immunization trials using inoculations of purified DNA in saline, 67-95% of test mice and 25-63% of test chickens were protected against the lethal challenge. Good protection was achieved by intramuscular, intravenous and intradermal injections. In mice, 95% protection was achieved by gene gun delivery of 250-2500 times less DNA than the saline inoculations. Successful DNA vaccination by multiple routes of inoculation and the high efficiency of gene-gun delivery highlight the potential of this promising new approach to immunization.

Gene gun-based nucleic acid immunization: elicitation of humoral and cytotoxic T lymphocyte responses following epidermal delivery of nanogram quantities of DNA.

Particle-mediated (gene gun) DNA transfer to the epidermis was evaluated for its ability to elicit humoral and cytotoxic T lymphocyte responses using decreasing quantities of plasmid DNA-based antigen expression vectors. Using plasmids encoding human growth hormone, human alpha-1-antitrypsin, and influenza virus nucleoprotein, strong immune responses were observed in mice following immunization with as little as 16 ng of DNA using an electric discharge gene delivery system. Significant antibody titers were observed against these antigens following a primary immunization, with responses rising dramatically following a boost. Increasing the DNA dose above 16 ng per immunization had little beneficial effect. In contrast to particle-mediated DNA delivery, intramuscular or intradermal inoculation required greater than 5000-fold more DNA to achieve comparable results. Data are also presented demonstrating that a simple, hand-held version of the Accell DNA delivery system, employing compressed helium as the particle motive force, achieves immune responses comparable to the traditional electric discharge device.

A qualitative progression in HIV type 1 glycoprotein 120-specific cytotoxic cellular and humoral immune responses in mice receiving a DNA-based glycoprotein 120 vaccine.

The potential for eliciting humoral and cytotoxic T lymphocyte (CTL) responses to HIV-1 gp120 by gene gun-based DNA immunization in mice was examined. We speculated that the induction of de novo antigen production in the epidermis of BALB/c mice following particle bombardment-based gene delivery would result in both MHC class I- and class II-mediated antigen presentation for the elicitation of CTL and antibody responses, respectively. Following epidermal delivery of microgram quantities of an expression plasmid, gp120 production resulted in the appearance of MHC class I-restricted, CD8+ CTL responses. gp120-specific CTL responses peaked following a booster immunization, then declined with the appearance of gp120-specific IgG responses when additional booster immunizations were administered. This qualitative progression in the nature of gp120-specific immune responses with subsequent immunizations was paralleled by a simultaneous shift in the interferon-gamma and interleukin 4 release profiles following antigen stimulation of splenocytes in vitro. The simultaneous shifts in immune responses and cytokine release profiles indicate that the progression of antigen-specific CTL and IgG responses in gp120 DNA-immunized mice may be mediated through changes in the in vivo production of cytokines, such as those associated with the Th1 and Th2 subsets of CD4+ cells.

Accell particle-mediated DNA immunization elicits humoral, cytotoxic, and protective immune responses.

Accell particle-mediated gene delivery technology was employed for the intracellular delivery of antigen-encoding expression vectors in epidermal tissues in laboratory animals. Delivery of plasmid DNA-coated gold microparticles using the Accell gene delivery system resulted in de novo antigen expression in epidermal cells that stimulated the induction of antigen-specific humoral and cytotoxic cellular immune responses. Optimal DNA delivery conditions favoring maximal humoral responses required the delivery of 5 x 10(7) micron-sized gold particles containing 300 plasmid copies per particle (80 ng of vector total) into a 4-cm2 area of abdominal skin. Comparison of immune responses between animals that received intramuscular injections of relatively large quantities of vector DNA (100 micrograms) and those that received intracellular deliveries of submicrogram quantities of the same DNA to the epidermis demonstrated that the latter approach was considerably more effective at eliciting strong humoral responses. In addition, cytotoxic cellular immune responses were elicited to HIV-1 gp120 following epidermal delivery of HIV-1 gp160 or gp120 expression constructs. A qualitative shift from predominantly cytotoxic cellular to predominantly humoral immune responses with continued immunization indicated the potential for optimizing delivery conditions to favor specifically one type of response over the other.

DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations.

Plasmid DNAs expressing influenza virus hemagglutinin glycoproteins have been tested for their ability to raise protective immunity against lethal influenza challenges of the same subtype. In trials using two inoculations of from 50 to 300 micrograms of purified DNA in saline, 67-95% of test mice and 25-63% of test chickens have been protected against a lethal influenza challenge. Parenteral routes of inoculation that achieved good protection included intramuscular and intravenous injections. Successful mucosal routes of vaccination included DNA drops administered to the nares or trachea. By far the most efficient DNA immunizations were achieved by using a gene gun to deliver DNA-coated gold beads to the epidermis. In mice, 95% protection was achieved by two immunizations with beads loaded with as little as 0.4 micrograms of DNA. The breadth of routes supporting successful DNA immunizations, coupled with the very small amounts of DNA required for gene-gun immunizations, highlight the potential of this remarkably simple technique for the development of subunit vaccines.

Examination of parameters affecting the elicitation of humoral immune responses by particle bombardment-mediated genetic immunization.

A human growth hormone expression construct was delivered intracellularly into the abdominal skin of mice by particle bombardment-mediated gene transfer. Using this technology, the in vivo delivery of antigen-encoding expression vectors affixed to gold microprojectiles results in de novo antigen production in target skin and development of specific antibody responses. In this study, we examined the contribution of various delivery parameters to the resultant protein expression and related antibody responses. The highest levels of both protein expression and antibody production were correlated with particle delivery to the epidermis while deliveries extending into the dermis resulted in decreased protein and antibody production. Optimal immune responses were also shown to be dependent upon the delivery of a sufficient number of DNA-coated gold particles, indicating that a dose-response relationship exists between the number of particles delivered and the resultant protein expression and antibody production. Further, maximal protein expression and associated antibody titers were elicited with surprisingly small amounts of DNA. The practicality of targeting skin and the use of three to four orders of magnitude less DNA than is typically required in direct DNA inoculation studies demonstrates the potential utility of this emerging technology for the rapid production of antibodies in laboratory animals, and in the development of a new class of human clinical vaccines based upon direct, intracellular DNA delivery.