Host Range and Population Survey of Spodoptera frugiperda Rhabdovirus.

The Sf9 and Sf21 cell lines derived from ovarian tissues of the wide-host-range phytophagous lepidopteran Spodoptera frugiperda are widely used for research and commercial-scale production of recombinant proteins. These cell lines are chronically infected with a rhabdovirus (Sf-RV) that does not cause any overt cytopathic effects. We demonstrate that wild populations of S. frugiperda in the eastern United States and Caribbean are infected with genetically diverse strains of Sf-RV and that this virus is also capable of infecting cells of Spodoptera exigua, Heliothis subflexa, and Bombyx mori Feeding studies demonstrated the ability of S. frugiperda larvae to deposit Sf-RV onto human-consumed vegetables during feeding. Although no evidence for replication in two species of plant cells was detected, subcellular localization studies demonstrated that the Sf-RV nucleocapsid was targeted to plasmodesmata, while two forms of the accessory protein were differentiated on the basis of their ability to localize to nuclei. Collectively, the results from this study suggest that environmental exposure of humans to Sf-RV is likely to be commonplace and frequent, but its inability to replicate in plant or human cells suggests that there is no substantial risk to human health.IMPORTANCE Insect-derived cell lines are widely used commercially for the production of vaccines and protein-based pharmaceuticals. After decades of safe and beneficial use, it was a surprise to the biotechnology industry to discover an endemic rhabdovirus in Sf9 cells. This discovery was made possible only by the substantial advancements in DNA sequencing technologies. Given the public health concerns associated with many rhabdovirus species, several initiatives were undertaken to establish that Spodoptera frugiperda rhabdovirus (Sf-RV) does not pose a threat to humans. Such actions include the generation of cell lines that have been cleared of Sf-RV. Given that Sf9 is derived from a moth whose larvae feed on human-edible foods, we explored the prevalence of Sf-RV in its wild and lab-grown populations, as well as its ability to be deposited on food items during feeding. Collectively, our data suggest that there is no overt risk from exposure to Sf-RV.

Induction of antigen-specific CD8+ T cells, T helper cells, and protective levels of antibody in humans by particle-mediated administration of a hepatitis B virus DNA vaccine.

A DNA vaccine against the hepatitis B virus (HBV) was evaluated for safety and induction of immune responses in 12 healthy, hepatitis-naïve human volunteers using the needle-free PowderJect system to deliver gold particles coated with DNA directly into cells of the skin. Three groups of four volunteers received three administrations of DNA encoding the surface antigen of HBV at one of the three dose levels (1, 2, or 4 microg). The vaccine was safe and well tolerated, causing only transient and mild to moderate responses at the site of administration. HBV-specific antibody and both CD4+ and CD8+ T cell responses were measured before and after each immunization. All the volunteers developed protective antibody responses of at least 10 mIU/ml. In volunteers who were positive for the HLA class I A2 allele, the vaccine also induced antigen-specific CD8+ T cells that bound HLA-A2/HBsAg(335-343) tetramers, secreted IFN-gamma, and lysed target cells presenting a hepatitis B surface antigen (HBsAg) CTL epitope. Enumeration of HBsAg-specific T cells producing cytokine indicated preferential induction of a Type 1 T helper cell response. These results provide the first demonstration of a DNA vaccine inducing protective antibody titers and both humoral and cell-mediated immune responses in humans.

Route and method of delivery of DNA vaccine influence immune responses in mice and non-human primates.

BACKGROUND: In spite of the large number of studies that have evaluated DNA-based immunization, few have directly compared the immune responses generated by different routes of immunization, particularly in non-human primates. Here we examine the ability of a hepatitis B surface antigen (HBsAg)-encoding plasmid to induce immune responses in mice and non-human primates (rhesus monkeys: Macaca mulatta) after delivery by a number of routes. MATERIALS AND METHODS: Eight different injected [intraperitoneal (IP), intradermal (ID), intravenous (IV), intramuscular (IM), intraperineal (IPER), subcutaneous (SC), sublingual (SL), vaginal wall (VW)] and six noninjected [intranasal inhalation (INH), intranasal instillation (INS), intrarectal (IR), intravaginal (IVAG), ocular (Oc), oral feeding (oral)] routes and the gene gun (GG) were used to deliver HBsAg-expressing plasmid DNA to BALB/c mice. Sera were assessed for HBsAg-specific antibodies (anti-HBs, IgG, IgG1, IgG2a) and cytotoxic T lymphocyte (CTL) activity measured. Three of the most commonly used routes (IM, ID, GG) were compared in rhesus monkeys, also using HBsAg-expressing vectors. Monkeys were immunized with short (0-, 4- and 8-week) or long (0-, 12- and 24-week) intervals between boosts, and in the case of GG, also with different doses, and their sera were assessed for anti-HBs. RESULTS: In one study, anti-HBs were detected in plasma of mice treated by five of eight of the injected and none of the six noninjected routes. The highest levels of anti-HBs were induced by IM and IV injections, although significant titers were also obtained with SL and ID. Each of these routes also induced CTL, as did IPER and VW and one noninjected route (INH) that failed to induce antibodies. In a second study, GG (1.6 microg) was compared to ID and IM (100 microg) delivery. Significant titers were obtained by all routes after only one boost, with the highest levels detected by IM. Delivery to the skin by GG induced exclusively IgG1 antibodies (Th2-like) at 4 weeks and only very low IgG2a levels at later times; ID-immunized mice had predominantly IgG1 at 4 weeks and this changed to mixed IgG1/IgG2a over time. Responses with IM injection (in the leg or tongue) were predominantly IgG2a (Th1-like) at all times. IV injection gave mixed IgG1/IgG2a responses. In monkeys, in the first experiment, 1 mg DNA IM or ID at 0, 4, and 8 weeks gave equivalent anti-HB titers and 0.4 microg at the same times by GG induced lower titers. In the second experiment, 1 mg DNA IM or ID, or 3.2 microg by GG, at 0, 12, and 24 weeks, gave anti-HB values in the hierarchy of GG > IM > ID. Furthermore, high titers were retained after a single immunization in mice but fell off over time in the monkeys, even after boost. CONCLUSIONS: Route of administration of plasmid DNA vaccines influences the strength and nature of immune responses in mice and non-human primates. However, the results in mice were not always predictive of those in monkeys and this is likely true for humans as well. Optimal dose and immunization schedule will most likely vary between species. It is not clear whether results in non-human primates will be predictive of results in humans, thus additional studies are required. http://link.springer-ny.com/link/service/journals/00020/bibs /5n5p287. html

Genetic vaccines.

In a few short years, genetic vaccine technology has moved rapidly from a novel concept to an important strategy for the development of human and veterinary vaccines, for numerous indications. This article discusses current areas in which further refinements in technology will influence a variety of infectious disease treatments, including intramuscular and intradermal inoculation, gene gun inoculation, the mechanism of antigen presentation, and the use of genetic adjuvants.

Immunization of dogs and cats with a DNA vaccine against rabies virus.

The applicability of DNA immunization technology for vaccine development in companion animals was investigated by immunizing dogs and cats by the intramuscular (i.m.) and intradermal (i.d.) routes with a plasmid DNA vector encoding the rabies virus glycoprotein G. In dogs, administration of 100 microg DNA doses by the i.m. route resulted in stronger and more durable rabies virus neutralizing antibody (RVNA) titers than those obtained by i.d. inoculation. In contrast, i.m. vaccination of cats with a similar dose was less effective in terms of mean titer and seroconversion frequency. However, efficacy was improved by increasing the dosage to 300 microg of DNA per immunization. Interestingly, i.d. inoculation of cats appeared to be a superior route of delivery in this species, resulting in higher seroconversion frequency than i.m. administration. In addition, geometric mean RVNA titers in i.d. inoculated cats increased over four-fold during a seven month period following a second and final immunization. These results demonstrate that non-facilitated, naked DNA vaccines can elicit strong, antigen-specific immune responses in dogs and cats, and DNA immunization may be a useful tool for future development of novel vaccines for these species.

Manipulation of HIV-1 gp120-specific immune responses elicited via gene gun-based DNA immunization.

Gene gun-based DNA immunization using vectors encoding HIV-1 gp120 or influenza virus nucleoprotein result in Th2-like immune responses following successive immunizations. The codelivery of vectors encoding IL-2, IL-7, or IL-12 blocked this effect by markedly enhancing gp120-specific interferon gamma production, and suppressing IL-4 and IgG1 responses. An unbiased augmentation of all immune responses was observed by increasing the resting period between immunizations. In this case, IFN-gamma production following in vitro stimulation increased by over 1000-fold, while IL-4, IgG1, and IgG2a responses were elevated as well. Interestingly, cytokine gene codelivery, in the context of the longer resting period, provided no additional stimulation of Th1-like responses such as IFN-gamma and IgG2a production, although there was still some suppression of IL-4 production. These data demonstrate that the quality and magnitude of responses elicited following epidermal administration of DNA vaccines can be manipulated by multiple means.

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.

Cross-protection among lethal H5N2 influenza viruses induced by DNA vaccine to the hemagglutinin.

Inoculation of mice with hemagglutinin (HA)-expressing DNA affords reliable protection against lethal influenza virus infection, while in chickens the same strategy has yielded variable results. Here we show that gene gun delivery of DNA encoding an H5 HA protein confers complete immune protection to chickens challenged with lethal H5 viruses. In tests of the influence of promoter selection on vaccine efficacy, close correlations were obtained between immune responses and the dose of DNA administered, whether a cytomegalovirus (CMV) immediate-early promoter or a chicken beta-actin promoter was used. Perhaps most important, the HA-DNA vaccine conferred 95% cross-protection against challenge with lethal antigenic variants that differed from the primary antigen by 11 to 13% (HA1 amino acid sequence homology). Overall, the high levels of protection seen with gene gun delivery of HA-DNA were as good as, if not better than, those achieved with a conventional whole-virus vaccine, with fewer instances of morbidity and death. The absence of detectable antibody titers after primary immunization, together with the rapid appearance of high titers immediately after challenge, implicates efficient B-cell priming as the principal mechanism of DNA-mediated immune protection. Our results suggest that the efficacy of HA-DNA influenza virus vaccine in mice extends to chickens and probably to other avian species as well. Indeed, the H5 preparation we describe offers an attractive means to protect the domestic poultry industry in the United States from lethal H5N2 viruses, which continue to circulate in Mexico.

Influenza virus nucleoprotein-specific immunoglobulin G subclass and cytokine responses elicited by DNA vaccination are dependent on the route of vector DNA delivery.

Endpoint immunoglobulin G (IgG) titers and cytotoxic T-lymphocyte (CTL) activities were identical between mice immunized via the intramuscular and epidermal (gene gun) routes with 100 and 1 micrograms, respectively, of an influenza virus nucleoprotein (NP) expression vector. However, examination of the relative levels of two IgG subclasses demonstrated that muscle inoculation resulted in predominantly IgG2a responses, whereas gene gun immunization yielded a preponderance of IgG1 antibodies. Inasmuch as these data suggested that muscle inoculation and gene gun delivery elicited Th1-like and Th2-like responses, respectively, gamma interferon release profiles from antigen-stimulated splenocytes were remarkably similar between these groups. Interleukin-4 (IL-4) production assays, on the other hand, revealed qualitative differences that could be correlated with the divergent IgG subclass data. Waning gamma interferon production in gene gun-immunized animals was countered by a marked increase in IL-4 production following the third immunization, as was the case in control animals immunized with inactivated influenza virus formulated with Freund's adjuvant. In contrast, significant levels of IL-4 production were not observed in the intramuscular DNA inoculation group, despite similar decreases in gamma interferon production with increasing immunizations. These data show that intramuscular inoculation leads to Th1-like responses due to elevated IgG2a levels, production of gamma interferon, CTL activity, and lack of IL-4. However, gene gun responses are more difficult to categorize because of the presence of significant gamma interferon and CTL activity on the one hand and elevated IgG1 antibodies and increasing IL-4 production with successive immunizations on the other. In addition, there was a lack of correlation between IgG isotype ratios and cytokine production in all of the NP DNA-immunized animals, in that IgG subclass ratios remained fixed while cytokine production patterns fluctuated with successive immunizations. These data are consistent with the idea that the types of responses elicited following DNA immunization. are dependent on both the identity of the antigen and the route of DNA administration.

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.

Simian immunodeficiency virus DNA vaccine trial in macaques.

An experimental vaccine consisting of five DNA plasmids expressing different combinations and forms of simian immunodeficiency virus-macaque (SIVmac) proteins has been evaluated for the ability to protect against a highly pathogenic uncloned SIVmac251 challenge. One vaccine plasmid encoded nonreplicating SIVmac239 virus particles. The other four plasmids encoded secreted forms of the envelope glycoproteins of two T-cell-tropic relatives (SIVmac239 and SIVmac251) and one monocyte/macrophage-tropic relative (SIVmac316) of the uncloned challenge virus. Rhesus macaques were inoculated with DNA at 1 and 3, 11 and 13, and 21 and 23 weeks. Four macaques were inoculated intravenously, intramuscularly, and by gene gun inoculations. Three received only gene gun inoculations. Two control monkeys were inoculated with control plasmids by all three routes of inoculation. Neutralizing antibody titers of 1:216 to 1:768 were present in all of the vaccinated monkeys after the second cluster of inoculations. These titers were transient, were not boosted by the third cluster of inoculations, and had fallen to 1:24 to 1:72 by the time of challenge. Cytotoxic T-cell activity for Env was also raised in all of the vaccinated animals. The temporal appearance of cytotoxic T cells was similar to that of antibody. However, while antibody responses fell with time, cytotoxic T-cell responses persisted. The SIVmac251 challenge was administered intravenously at 2 weeks following the last immunization. The DNA immunizations did not prevent infection or protect against CD4+ cell loss. Long-term chronic levels of infection were similar in the vaccinated and control animals, with 1 in 10,000 to 1 in 100,000 peripheral blood cells carrying infectious virus. However, viral loads were reduced to the chronic level over a shorter period of time in the vaccinated groups (6 weeks) than in the control group (12 weeks). Thus, the DNA vaccine raised both neutralizing antibody and cytotoxic T-lymphocyte responses and provided some attenuation of the acute phase of infection, but it did not prevent the loss of CD4+ cells.

Particle-mediated nucleic acid immunization.

Nucleic acid immunization involves the direct in vivo administration of antigen-encoding plasmid DNA molecules that results in the de novo production of correctly folded microbial antigens at the site of DNA delivery. While this process can lead to the development of neutralizing antibody responses recognizing authentic protein conformations, in vivo antigen production also results in epitope presentation via the MHC class I antigen processing pathway, leading to the elicitation of cytotoxic cellular immune responses. Recent efforts in the authors' laboratories have focused on use of the Accell gene delivery system (gene gun) to achieve the direct, intracellular delivery of small quantities of DNA into cells of the epidermis. The gene gun approach to nucleic acid vaccination capitalizes on the synergistic combination of an effective DNA delivery system and a target tissue that serves as a major immunological inductive site. Experimental gene gun-based nucleic acid vaccines can achieve potent humoral and cytotoxic cellular immune responses in rodent models following immunization with as little as 16 ng of DNA. Equally strong responses have also been elicited in larger animals, such as pigs and monkeys, following epidermal immunization with as little as 2 to 4 micrograms of DNA.

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.