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.

Heterologous protection against influenza by injection of DNA encoding a viral protein.

Cytotoxic T lymphocytes (CTLs) specific for conserved viral antigens can respond to different strains of virus, in contrast to antibodies, which are generally strain-specific. The generation of such CTLs in vivo usually requires endogenous expression of the antigen, as occurs in the case of virus infection. To generate a viral antigen for presentation to the immune system without the limitations of direct peptide delivery or viral vectors, plasmid DNA encoding influenza A nucleoprotein was injected into the quadriceps of BALB/c mice. This resulted in the generation of nucleoprotein-specific CTLs and protection from a subsequent challenge with a heterologous strain of influenza A virus, as measured by decreased viral lung titers, inhibition of mass loss, and increased survival.

DNA vaccines against tuberculosis.

DNA plasmids encoding Mycobacterium tuberculosis antigen 85 (Ag85) were tested as vaccines in animal models. Ag85 DNA induced relevant immune responses (i.e. T helper (Th) cells, Th1 cytokines and cytotoxic T lymphocytes) and was protective in mouse and guinea pig models of mycobacterial disease. Therefore, DNA vaccination holds promise as an effective means of preventing tuberculosis in humans. Furthermore, this technique is amenable to identifying the protective antigens of M. tuberculosis.

Immunogenicity of a Haemophilus influenzae polysaccharide-Neisseria meningitidis outer membrane protein complex conjugate vaccine.

Polysaccharide-protein conjugate vaccines made with different carriers vary in their ability to elicit antipolysaccharide IgG antibody responses in young infants and an adult mouse model, suggesting that the carrier proteins used in the conjugate vaccines differ in their ability to act as carriers, or that additional mechanisms of immunogenicity play a role. A conjugate vaccine of Haemophilus influenzae PRP coupled to the outer membrane protein complex (OMPC) of Neisseria meningitidis serogroup B is immunogenic in children as young as 2 mo of age and is immunogenic in infant rhesus monkeys, an animal model for infant humans. In the present study, PRP-OMPC was found to induce efficient IgM to IgG switching of anti-PRP serum antibody in adult mice, whereas PRP conjugated to two other protein carriers did not. Thus the PRP-OMPC conjugate was examined in order to determine why PRP coupled to OMPC was so immunogenic, even more immunogenic than conjugates made with other carrier proteins. The OMPC carrier differs from the other protein carriers in that the proteins are present in a liposomal form containing lipids (including LPS) derived from the outer membrane of N. meningitidis. We studied the OMPC to see whether the different components or the nature of the OMPC carrier could contribute to its enhanced immunogenicity. Specifically we evaluated the OMPC for both classic Th cell carrier activity and adjuvanticity, and the LPS component of OMPC for systemic polyclonal B cell activation. Carrier recognition of the OMPC moiety of PRP-OMPC was demonstrated. In addition the PRP-OMPC conjugate vaccine was observed to have adjuvant properties for both T cell-dependent and T cell-independent Ag in the absence of LPS-induced systemic polyclonal B cell activation. These observations suggest that in addition to functioning as a classic protein carrier whereby the proteins in OMPC provide Th cell epitopes, the OMPC also has adjuvant activity that distinguishes it from other protein carriers and may contribute to the increased immunogenicity of PRP-OMPC conjugates in animal models.

Importance of complement source in measuring meningococcal bactericidal titers.

Complement-mediated bactericidal antibodies in serum confer protection against meningococcal disease. The minimum protective titer is estimated to be between 1:4 and 1:8 when measured by the Goldschneider assay performed with human complement, the assay used in the 1960s to establish the correlation between bactericidal antibodies and protection. A more recently described bactericidal assay standardized by an international consortium uses rabbit complement, which is known to augment bactericidal titers. To define a protective titer measured by the standardized assay, we compared bactericidal titers against serogroup C strains measured by this assay to titers measured by the assay described by Goldschneider et al. A titer of > or =1:128 measured by the standardized assay was needed to predict with > or =80% certainty a positive titer of > or =1:4 as measured by the Goldschneider assay. However, the majority of samples with titers of 1:4 measured by the Goldschneider assay had titers of <1:128 when measured by the standardized assay. Therefore, by the results of the standardized assay such persons would be falsely categorized as being susceptible to disease. In conclusion, high bactericidal titers measured with the standardized assay performed with rabbit complement are predictive of protection, but no threshold titer is both sensitive and specific for predicting a positive titer measured by the Goldschneider assay using human complement. Up to 10% of the U.S. adult population lacks intrinsic bactericidal activity against serogroup C strains in serum and can serve as complement donors. Therefore, use of the Goldschneider assay or an equivalent assay performed with human complement is preferred over assays that use rabbit complement.

Protective immunity by intramuscular injection of low doses of influenza virus DNA vaccines.

Dose-response relationships were investigated between dose of influenza virus haemagglutinin (HA) or nucleoprotein (NP) DNA vaccines, and immunogenicity and protective efficacy based on humoral and cellular immunity. In mice, intramuscular (i.m.) injection of HA or NP DNA, at doses of 100 ng to 1 microgram, was found to generate haemagglutination inhibiting (HI) antibodies and cytotoxic T-lymphocytes, respectively, and provide protection in influenza virus challenge models. A direct correlation between the amount of DNA injected and the level of HI antibody was observed. In non-human primates, high-titre neutralizing antibodies were induced in animals vaccinated with as little as 10 micrograms of HA DNA. These results indicate that low doses of DNA administered by i.m. injection provide protective efficacy against influenza.

Characterization of humoral immune responses induced by an influenza hemagglutinin DNA vaccine.

We have examined in detail the characteristics of the humoral immune response and protective efficacy induced by an influenza hemagglutinin (HA) DNA vaccine. In mice injected intramuscularly with HA DNA, the magnitude of the immune responses generated, as measured by ELISA and hemagglutination inhibiting (HI) antibodies, was directly related to the amount of DNA injected and the number of doses administered. The level of anti-HA antibodies in DNA-vaccinated mice was higher than that in convalescent immune mice and was maintained for at least 1.5 years. The immunoglobulin isotype profile of the antibodies was predominantly IgG2a, similar to that induced by live virus infection but in contrast to the relative abundance of IgG1 antibodies observed after inoculation with formalin-inactivated whole virus. The presence of pre-challenge HI antibodies was found to be a good correlate of protection, in that every animal with a detectable HI titer was protected from a lethal challenge. Complete protection from a lethal dose of influenza virus (A/PR/34), as judged by 100% survival and no weight loss, was conferred by as little as 1 microgram of DNA (given twice). Furthermore, mice injected with 10 to 100 micrograms doses, when subsequently challenged with virus, showed no increase in HI titer and no production of antibodies directed against the challenge virus, suggesting a substantial inhibition of virus replication after challenge.

Generation of MHC class I-restricted cytotoxic T lymphocytes by expression of a viral protein in muscle cells: antigen presentation by non-muscle cells.

Expression of reporter genes in muscle cells has been achieved by intramuscular (i.m.) injection of plasmid DNA expression vectors. We previously demonstrated that this technique is an effective means of immunization to elicit both antibodies capable of conferring homologous protection and cell-mediated immunity leading to cross-strain protection against influenza virus challenge in mice. These results suggested that expression of viral proteins by muscle cells can result in the generation of cellular immune responses, including cytotoxic T lymphocytes (CTL). However, because DNA has the potential to be internalized and expressed by other cell types, we sought to determine whether or not induction of CTL required synthesis of antigen in non-muscle cells and if not whether transfer of antigen to antigen-presenting cells from muscle cells may be involved. In the present study we demonstrate that transplantation of nucleoprotein (NP)-transfected myoblasts into syngeneic mice led to the generation of NP-specific antibodies and CTL and cross-strain protective immunity against a lethal challenge with influenza virus. Furthermore transplantation of NP-expressing myoblasts (H-2k) intraperitoneally into F1 hybrid mice (H-2d x H-2k) elicited NPCTL restricted by the MHC haplotype of both parental strains. These results indicate that NP expression by muscle cells after transplantation was sufficient to generate protective cell-mediated immunity and that induction of the CTL response was mediated at least in part, by transfer of antigen from the transplanted muscle cells to a host cell.

Expression and immunogenicity of Mycobacterium tuberculosis antigen 85 by DNA vaccination.

Plasmid DNA expression vectors encoding Mycobacterium tuberculosis antigen 85 (Ag85) were tested as vaccines in preclinical animal models. Expression of secreted and nonsecreted forms of Ag85 was observed after transient transfection of cells in vitro. In mice, both types of Ag85 DNA constructs induced strong humoral and cell-mediated immune responses, as measured by ELISA of sera and recall responses of spleen cells restimulated in vitro, respectively, Therefore, DNA vaccination is an effective means of expressing mycobacterial proteins in eukaryotic cells leading to the induction of potent immune responses.

Expression of a viral protein by muscle cells in vivo induces protective cell-mediated immunity.

Intramuscular injection of plasmid DNA expression vectors results in transfection of myocytes in situ. To determine whether expression of antigen by myocytes is sufficient to induce protective cell-mediated immunity, stably transfected myoblasts expressing influenza nucleoprotein (NP) were transplanted into mice. These animals produced high-titer anti-NP antibodies and MHC class I-restricted cytotoxic T lymphocytes, and were protected from a cross-strain lethal challenge with influenza A virus. Therefore, antigen expression by muscle cells in vivo is sufficient to confer protective cell-mediated immunity.

Induction of humoral and cellular immune responses by vaccination with M. tuberculosis antigen 85 DNA.

DNA vaccines have been demonstrated to be effective in inducing protective cell-mediated immune responses in animal models of infectious disease. In order to investigate this approach for potential use as a vaccine for tuberculosis, DNA constructs encoding Mycobacterium tuberculosis antigen 85A (Ag85A) were prepared. Expression of Ag85A in mammalian cells was demonstrated by transient transfection of cells in vitro. Intramuscular injection of Ag85A DNA vaccines resulted in the generation of anti-Ag85A antibodies and robust cell-mediated immune responses, as measured by lymphoproliferation of spleen cells in vitro upon specific antigen restimulation, leading to protection in animal challenge models. Therefore, the technique of DNA vaccination is effective in inducing relevant immune responses for protection against tuberculosis and may be used to identify the protective antigens of M. tuberculosis.

Priming of cytotoxic T lymphocytes by DNA vaccines: requirement for professional antigen presenting cells and evidence for antigen transfer from myocytes.

BACKGROUND: MHC class I molecule-restricted cytotoxic T-lymphocyte (CTL) responses are induced following either intramuscular (i.m.) injection of a DNA plasmid encoding influenza virus nucleoprotein (NP) or transplantation of myoblasts stably transfected with the NP gene, the latter indicating that synthesis of NP by myocytes in vivo is sufficient to induce CTL. The present study was designed to investigate the role of muscle cells and involvement of professional antigen-presenting cells (APCs) in priming CTL responses following DNA vaccination. MATERIALS AND METHODS: Parent-->F1 bone marrow (BM) chimeric mice were generated whose somatic cells include muscle cells bearing both parental MHC haplotypes, while their professional APCs express only the donor MHC haplotypes. RESULTS AND CONCLUSIONS: Upon injection of NP DNA, or after infection with influenza virus, CTL responses generated in the chimeras were restricted to the donor MHC haplotype. Thus cells of BM lineage were definitively shown to be responsible for priming such CTL responses after infection or DNA immunization. Moreover, expression of antigen by muscle cells in BM chimeric mice after myoblast transplantation is sufficient to induce CTL restricted only by the MHC haplotype of the donor BM. This indicates that transfer of antigen from myocytes to professional APCs can occur, thus obviating a requirement for direct transfection of BM-derived cells.

A vaccine carrier derived from Neisseria meningitidis with mitogenic activity for lymphocytes.

Protein carriers vary in their ability to increase the immunogenicity of poorly immunogenic or T-lymphocyte-independent antigens. We examined one such carrier, the outer membrane protein complex derived from Neisseria meningitidis serogroup B strain B11, in an attempt to determine why this outer membrane protein complex was more immunogenic in young infants and in relevant animal models than two other carriers used in conjugates made with Haemophilus influenzae type b polysaccharide, a T-cell-independent antigen. A single protein of the outer membrane protein complex, the class 2 porin protein, was purified and shown to function as a T-helper lymphocyte carrier protein. Unexpectedly, it was also found to have mitogenic activity for lymphocytes that was not due to lipopolysaccharide. This mitogenic activity appears to date to be unique to this carrier protein of the carrier proteins tested and may contribute to the ability of the H. influenzae type b conjugate vaccine made with the outer membrane protein complex to generate IgG anti-polysaccharide antibody responses in mice and infant monkeys and protective immune responses in infants less than 6 months of age.

Induction of immunity by DNA vaccination: application to influenza and tuberculosis.

DNA vaccination is an effective means of inducing both humoral and cell-mediated immunity in animal models of infectious disease. Presented here are data generated in two distinct disease models; one viral (influenza) and one bacterial (tuberculosis). Specifically, plasmid DNA encoding an influenza virus antigen (nucleoprotein; NP) and a Mycobacterium tuberculosis antigen (antigen 85; Ag85) were prepared and tested as DNA vaccines in mice. In both cases, high titer antibody responses and robust cell-mediated immune responses were induced against the respective antigens. With respect to the latter, lymphocyte proliferation, Th1-type cytokine secretion, and cytotoxic T lymphocyte responses were observed upon restimulation with antigen in vitro. Furthermore, protective efficacy in animal challenge models was demonstrated in both systems. The data support the hypothesis that DNA vaccination will prove to be a broadly applicable technique for inducing immunity against various infectious diseases.

Protective CD4+ and CD8+ T cells against influenza virus induced by vaccination with nucleoprotein DNA.

DNA vaccination is an effective means of eliciting both humoral and cellular immunity, including cytotoxic T lymphocytes (CTL). Using an influenza virus model, we previously demonstrated that injection of DNA encoding influenza virus nucleoprotein (NP) induced major histocompatibility complex class I-restricted CTL and cross-strain protection from lethal virus challenge in mice (J. B. Ulmer et al., Science 259:1745-1749, 1993). In the present study, we have characterized in more detail the cellular immune responses induced by NP DNA, which included robust lymphoproliferation and Th1-type cytokine secretion (high levels of gamma interferon and interleukin-2 [IL-2], with little IL-4 or IL-10) in response to antigen-specific restimulation of splenocytes in vitro. These responses were mediated by CD4+ T cells, as shown by in vitro depletion of T-cell subsets. Taken together, these results indicate that immunization with NP DNA primes both cytolytic CD8+ T cells and cytokine-secreting CD4+ T cells. Further, we demonstrate by adoptive transfer and in vivo depletion of T-cell subsets that both of these types of T cells act as effectors in protective immunity against influenza virus challenge conferred by NP DNA.