Preclinical efficacy of a prototype DNA vaccine: enhanced protection against antigenic drift in influenza virus.

Vaccination with plasmid DNA expression vectors encoding foreign proteins elicits antibodies and cell-mediated immunity and protects against disease in animal models. We report a comparison of DNA vaccines, using contemporary human strains of virus, and clinically licensed (inactivated virus or subvirion) vaccines in preclinical animal models, to better predict their efficacy in humans. Influenza DNA vaccines elicited antibodies in both non-human primates and ferrets and protected ferrets against challenge with an antigenically distinct epidemic human influenza virus more effectively than the contemporary clinically licensed vaccine. These studies demonstrate that DNA vaccines may be more effective, particularly against different strains of virus, than inactivated virus or subvirion vaccines.

Immunogenicity and protective efficacy of a tuberculosis DNA vaccine.

Tuberculosis is the most widespread and lethal infectious disease affecting humans. Immunization of mice with plasmid DNA constructs encoding one of the secreted components of Mycobacterium tuberculosis, antigen 85 (Ag85), induced substantial humoral and cell-mediated immune responses and conferred significant protection against challenge with live M. tuberculosis and M. bovis bacille Calmette-Guérin (BCG). These results indicate that immunization with DNA encoding a mycobacterial antigen provides an efficient and simple method for generating protective immunity and that this technique may be useful for defining the protective antigens of M. tuberculosis, leading to the development of a more effective vaccine.

Hormone conjugated with antibody to CD3 mediates cytotoxic T cell lysis of human melanoma cells.

Cytotoxic T lymphocytes can be activated by antibodies to their antigen-specific receptor complex (TCR-CD3) to destroy target cells, regardless of the specificity of the cytotoxic T cells. A novel hormone-antibody conjugate, consisting of an analog of melanocyte-stimulating hormone chemically coupled to a monoclonal antibody to CD3, the invariant component of the T cell receptor complex, was used to target human melanoma cells for destruction by human cytotoxic T lymphocytes that bear no specificity for the tumor cells. As targeting components of such anti-CD3 conjugates, hormones or growth factors are expected to prove more effective than antibodies to tumor-associated antigens in focusing the destructive activity of cytotoxic T cells on tumor target cells.

Effect of recombinant soluble CD4 on human peripheral blood lymphocyte responses in vitro.

We have previously demonstrated that recombinant soluble CD4 protein (rsT4) blocks both HIV-1 infection of CD4 bearing lymphocytes and syncytium formation in vitro. (Recombinant soluble CD4 is designated by rsT4). Hence, we suggested the use of rsT4 in therapy for AIDS or the prevention of HIV-1 infection in individuals with a known risk of exposure. However, concerns arose that rsT4 might be immunosuppressive because of its implicated role in the enhancement of certain lymphocyte activation events through its engagement of MHC class II molecules on target cells. We therefore assessed the effect of recombinant soluble CD4 upon a number of functional and activation parameters of lymphocytes, including cellular proliferation, IL-2 secretion, and cytolytic capability, after antigenic or mitogenic stimulation. We report here that rsT4, at 60-fold over the concentration needed to block acute HIV-1 infection in vitro, does not significantly inhibit the activation of human peripheral blood lymphocytes by either PHA, tetanus toxoid or allogeneic cells. These results indicate that rsT4 will potentially exert minimal immunosuppressive effects in vivo, thus supporting the feasibility of clinical trials of rsT4 in the treatment or prevention of AIDS. In addition, the implications of these results for the interactions between CD4 and MHC class II molecules during lymphocyte activation are discussed.

DNA vaccines.

Preclinical DNA vaccine development has continued apace during the past year, with the investigation of several new infectious and non-infectious disease targets as well as advances in our understanding of some of the basic immunologic mechanisms, such as effector cells, responsible for conferring protection. The coming year promises to be at least as exciting, as initial human clinical studies have begun.

Experimental study of environmental tobacco smoke particles under actual indoor environment.

Environmental tobacco smoke (ETS) is a major source of human exposure to airborne particles. In order to provide more information necessary for human exposure investigations, the aim of the work presented here is to investigate experimentally the variation of the ETS particle concentration and size distribution under an actual indoor environment, in a room of 30 m3, using human smokers. The effect of number of cigarettes and brands of cigarettes, the effect of sampling location and the effect of ventilation rates were investigated. The results indicated little difference in the geometric mean diameter (GMD) of the ETS particles from those in background air. Under a ventilation rate of 0.03 m3/s, the concentration of the ETS particles reached a peak value at the sampling point shortly after completing the smoking process. The GMD first increased due to coagulation and diffusion deposition, and finalize decreased due to the effect of ventilation. Smoking two cigarettes at the same time would increase the initial concentration and led to an increase in GMD of the ETS particles. Two different brands of cigarette with different tar contents released ETS particles of different GMDs but similar particle concentrations. Spatial variation in particle concentration was obvious only in the first 600 s of the tests and tended to fade out subsequently. Stronger ventilation would reduce the concentration and GMD of the particles.

DNA vaccines.

In just a few years, injection of plasmid DNA to elicit immune responses in vivo has developed from an interesting observation to a viable vaccine strategy. DNA vaccines have been shown to elicit both cellular and humoral immune responses and to be effective in a variety of preclinical bacterial, viral, and parasitic animal models. This review will discuss the current knowledge of vector design, methods of plasmid delivery, immune responses elicited by various DNA vaccines, safety issues, and production and release of plasmid as a vaccine product. The potential of this new vaccine strategy and its future prospects is summarized.

Toward the development of DNA vaccines.

DNA vaccination has proved to be a generally applicable technology in various preclinical animal models of infectious and noninfectious disease and several DNA vaccines have now entered phase I human clinical trials. It is too early to predict the effectiveness of DNA vaccines in humans and whether improved formulations of DNA vaccines will be required but several lines of investigation have suggested ways in which DNA vaccines may be improved, such as increases in expression, facilitation of DNA targeting or uptake, and enhancement of immune responses.

Protein expression in vivo by injection of polynucleotides.

Over the past few years, intramuscular injection of non-replicating DNA expression vectors has been demonstrated to be generally applicable as an effective method of producing functional proteins in vivo. This technique has been useful in the study of growth factors, regulation of protein expression, transplantation rejection, gene therapy, immune regulation and the production of monoclonal antibodies. The most successful application of DNA injection has, however, been the generation of immune responses in animal models, with the ultimate goal of developing vaccines for humans. Therefore, this approach has the potential to be a new vaccine technology, in addition to its utility in other areas of research.

Heterologous and homologous protection against influenza A by DNA vaccination: optimization of DNA vectors.

We have recently shown that direct injection of DNA can be an effective vaccine strategy eliciting both humoral and cell-mediated immune responses. Vectors were designed specifically for vaccination by direct DNA injection and refined to improve plasmid production in Escherichia coli. The vectors consist of a pUC-19 backbone with the cytomegalovirus (CMV) IE1 enhancer, promoter, and intron A transcription regulatory elements and the BGH polyadenylation sequences driving the expression of the reporter gene CAT or influenza A nucleoprotein (NP) or hemagglutinin (HA). The respective vectors expressed high levels of chloramphenicol acetyltransferase (CAT) and NP in tissue culture, and yielded 14-15 mg of purified plasmid per liter of Escherichia coli culture. Immunization of mice with the NP and HA expression vectors resulted in protection from subsequent lethal challenges of influenza using either heterologous or homologous strains, respectively.

Cytotoxic T lymphocyte and helper T cell responses following HIV polynucleotide vaccination.

Expression vectors encoding either HIV-1 gp160/rev, gp120, or rev alone were used for direct vaccination of mice and nonhuman primates. Each vaccine elicited long-lived (> 7 months) helper T cell responses in mice and monkeys as measured by in vitro proliferation of splenocytes following recombinant antigen treatment. Cytokine assays of the cell supernatants showed that approximately 100-fold more gamma-interferon than IL-4 was secreted during culture indicating that these vaccines elicited TH1-like responses. CD8+ CTL activities were also observed both in mice and rhesus. The gp120 and gp160/rev vaccines elicited antigen-specific antibodies, although these responses were more variable and lower magnitude for gp160/rev, and gp120 DNA-vaccinated African green monkeys had moderate levels of neutralizing antibodies. No antibodies were found against rev (an intracellular protein) with either rev vaccine. Similar antibody titers were obtained for gp120 by either intramuscular or intradermal injection although T cell responses were generally lower by intradermal route. These results indicate that DNA vaccines may provide a powerful means to elicit cellular and humoral immune responses against HIV.

DNA vaccines.

Immunization with plasmid DNA encoding antigenic proteins elicits both antibody and cell-mediated immune responses. This method of producing the protein antigens of interest directly in host cells can provide appropriate tertiary structure for the induction of conformationally specific antibodies, and also facilitates the induction of cellular immune responses. DNA immunization has provided effective protective immunity in various animal models. The immune responses induced by DNA vaccines may in some instances be preferable to those produced by immunization using conventional methods. DNA vaccination appears to be applicable to a variety of pathogens and is a useful method of raising immune responses. Thus this approach to vaccination has the potential to be a successful method of rapidly screening for antigens capable of inducing protective immunity, and of inducing protective immunity against pathogens of clinical importance.

Humoral and cellular immunities elicited by HIV-1 vaccination.

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 antigen-specific antibody responses against recombinant gp120 protein and the V2 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 gamma-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 (> or = 6 months). Nonhuman primates vaccinated with tPA-gp120 DNA also showed antigen-specific 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.

DNA vaccines.

The use of DNA and mRNA as vectors for immunization is a relatively recent development in the field of vaccines. The first paper demonstrating the efficacy of a DNA vaccine in an animal model of viral disease was published in 1993 (1). The rationale for using nucleic acids as vaccines came from the Initial observations that mtramuscular (im) injection of nonrephcating plasmid DNA expression vectors or mFWA-encoding reporter genes could result in the in vivo expression of proteins in mouse muscle cells (2). This ability to express proteins in vivo offers the opportunity to generate immune responses against foreign antigens encoded by the nucleic acid. In addition, both humoral and cell-mediated immune (CMI) responses, such as cytotoxic T-lymphocytes (CTL), can be induced. In general, CTL responses require endogenous expression of the antigen, such as during immunization with live viruses or replicating vectors, whereas subunit protein, polysaccharide conjugate, or inactivated virus vaccines generate humoral immune responses, but not CTL. Therefore, the technique of DNA injection has potential advantages over certain other vaccine technologies.

Alphavirus DNA and particle replicons for vaccines and gene therapy.

Alphaviruses have several features that make them attractive as gene delivery platforms, and vectors derived principally from Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEE), are currently being developed as prophylactic and therapeutic vaccines for infectious diseases and cancer. Alphavirus vectors, termed "replicons", retain the nonstructural protein genes encoding the viral replicase, that in turn programme high level cytoplasmic amplification of the vector RNA. We have developed plasmid DNA and recombinant vector particle delivery systems derived from the prototype alphavirus, SIN. Each system uses RNA polymerase II-based expression of alphavirus genome components and both vector formats are highly efficacious towards inducing robust antigen-specific immune responses in vaccinated animals. To increase the potency of SIN vector particles, which are not known to be lymphotropic, the tropism was re-directed for efficient infection of dendritic cells, both in vitro and in vivo.

Consistency of response and image recognition, pulmonary nodules.

OBJECTIVE: To investigate the effect of recognition of a previously encountered radiograph on consistency of response in localized pulmonary nodules. METHODS: 13 radiologists interpreted 40 radiographs each to locate pulmonary nodules. A few days later, they again interpreted 40 radiographs. Half of the images in the second set were new. We asked the radiologists whether each image had been in the first set. We used Fisher's exact test and Kruskal-Wallis test to evaluate the correlation between recognition of an image and consistency in its interpretation. We evaluated the data using all possible recognition levels-definitely, probably or possibly included vs definitely, probably or possibly not included by collapsing the recognition levels into two and by eliminating the "possibly included" and "possibly not included" scores. RESULTS: With all but one of six methods of looking at the data, there was no significant correlation between consistency in interpretation and recognition of the image. When the possibly included and possibly not included scores were eliminated, there was a borderline statistical significance (p = 0.04) with slightly greater consistency in interpretation of recognized than that of non-recognized images. CONCLUSION: We found no convincing evidence that radiologists' recognition of images in an observer performance study affects their interpretation on a second encounter. ADVANCES IN KNOWLEDGE: Conscious recognition of chest radiographs did not result in a greater degree of consistency in the tested interpretation than that in the interpretation of images that were not recognized.