Immunogenicity Of Plasmid DNA Research Articles

Development of neutralizing monoclonal antibodies against the pandemic H1N1 virus (2009) using plasmid DNA immunogen

Reported here is the development and characterization of eighteen mouse monoclonal antibodies (MAbs) against the 2009 pandemic H1N1 influenza virus (A/Mexico/InDRE4487/2009). To our knowledge, this is the first report on pandemic (pH1) H1N1 MAbs developed using plasmid DNA encoding the viral surface glycoprotein, hemagglutinin (HA). All eighteen MAbs were specific for A/Mexico/InDRE4487/2009 HA. Ten MAbs were found to cross-react with A/Swine/Indiana/81 using a dot blot assay. However, there was no cross-reactivity detected against any other strains of influenza A viruses despite screening against all 16 hemagglutinin subtypes. Examination of these MAbs identified individual antibodies suitable for use in several practical applications including ELISA, immunoblot and immunofluorescence assays. Analysis of the kinetics of each MAb revealed significant binding affinities (KD<10−8M) confirming the antibodies are highly specific for A/Mexico/InDRE4487/2009 HA. Functional analysis demonstrated the panel of MAbs included antibodies with HA inhibition and virus neutralization activities. Not all MAbs inhibited hemagglutination or neutralized the virus. Furthermore, the panel of MAbs was not found to be cross-reactive against additional strains tested in hemagglutination inhibition assays. Finally, the MAbs were tested in competitive ELISA (cELISA) using reference serum antibodies developed against different clusters of H1 (pH1, α, β, γ, and δ). The developed MAbs outcompeted serum antibodies of pH1 in 16/18, 15/18 (γ), 3/18 (α), 2/18 (δ1) and 1/18 (β) samples. Overall, this panel of MAbs proved specific and highly sensitive for A/Mexico/InDRE4487/2009 HA and could potentially serve as immunodiagnostic tools for the rapid detection of this specific strain of influenza virus.

Preclinical evaluation of the immunogenicity and safety of plasmid DNA-based prophylactic vaccines for human cytomegalovirus

Human cytomegalovirus (CMV) establishes a lifelong persistent infection characterized by periods of latency and sporadic viral replication and is a major infectious cause of birth defects following congenital infection. Currently, no licensed vaccine is available that would prevent CMV infection. In an effort to develop a prophylactic CMV vaccine, the effects of different formulations, immunization routes and delivery devices on the immunogenicity of plasmid DNA (pDNA)-based vaccines were evaluated in rabbits and mice. Compared with PBS- and poloxamer-based formulations, significantly higher antibody responses were obtained with pDNA formulated with Vaxfectin®, a cationic lipid-based adjuvant. With low vaccine doses, the intradermal (ID) route resulted in higher antibody responses than obtained when the same dose was administered intramuscularly (IM). Since the IM route allowed injection of larger volumes and higher doses than could be administered at a single ID site, better antibody responses were obtained using the IM route. The needle-free injection system Biojector® 2000 and electroporation devices enhanced antibody responses only marginally compared with responses obtained with Vaxfectin®-formulated pDNA injected IM with a needle. A single-vial Vaxfectin® formulation was developed in a dosage form ready for use after thawing at room temperature. Finally, in a GLP-compliant repeat-dose toxicology study conducted in rabbits, single-vial Vaxfectin®-formulated vaccines, containing pDNA and Vaxfectin® up to 4.5 mg and 2 mg/injection, respectively, showed a favorable safety profile and were judged as well-tolerated. The results support further development of a Vaxfectin®-formulated pDNA vaccine to target congenital CMV infection.

A plasmid DNA immunogen expressing fifteen protein antigens and complex virus-like particles (VLP+) mimicking naturally occurring HIV

We describe here a single plasmid DNA immunogen representing the broadest antigen repertoire among HIV vaccine candidates. This pDNA was “ANTIGENeered” for the regulated expression of thirteen complete and two non-functional HIV protein antigens. These proteins self assemble into complex virus-like particles (VLP+). Multiple irreversible safety features were introduced by genetic modifications including the complete impairment of integration, reverse transcription, the function of Nef and the 3′LTR. Epitope analysis predicted that the pDNA-derived protein repertoire can potentially present over 3000 T cell epitopes. However, the expressed antigens have different potential to induce HIV-specific CD4+ and CD8+ T cells supporting our hypothesis that HIV vaccines should contain all possible regulatory and structural proteins. This immunogen is the active pharmaceutical ingredient of DermaVir, a therapeutic vaccine product candidate that recently successfully completed Phase II clinical trials and meets the safety, immunogenicity and cost requirements of an HIV vaccine.

Essential Role for TLR9 in Prime but Not Prime-Boost Plasmid DNA Vaccination To Activate Dendritic Cells and Protect from Lethal Viral Infection

One of the requirements for efficient vaccination against infection is to achieve the best combination of an adequate adjuvant with the antigenic information to deliver. Although plasmid DNA is a promising tool bearing the unique potential to activate humoral and cellular immunity, an actual challenge is to increase plasmid immunogenicity in human vaccination protocols in which efficacy has proven rather limited. Previous work showed that the bacterial DNA backbone of the plasmid has potent adjuvant properties because it contains CpG motifs that are particular activating nucleotidic sequences. Among TLRs, which are key sensors of microbial products, TLR9 can detect CpG motifs and confer activation of APCs, such as dendritic cells. However, whether the immunogenic properties of plasmid DNA involve TLR9 signaling has not been clearly established. In the current study, we demonstrate that TLR9 determines the effectiveness of vaccination against lethal lymphocytic choriomeningitis virus infection using plasmid DNA in a prime, but not prime-boost, vaccination regimen. Furthermore, we provide evidence that the presence of TLR9 in dendritic cells is necessary for effective and functional priming of virus-specific CD8+ T cells upon plasmid exposure in vitro or single-dose vaccination in vivo. Therefore, at single or low vaccine doses that are often used in human-vaccination protocols, CpG/TLR9 interactions participate in the immunogenicity of plasmid DNA. These results suggest that the TLR9 signaling pathway is involved in the efficacy of plasmid vaccination; therefore, it should remain a focus in the development or amelioration of vaccines to treat infections in humans.

Localization of CD8+ cells specific for hepatitis B virus surface protein in the liver of immunized mice

DNA plasmids are potent inducers of long-lasting antigen-specific CTL responses. Little is known about the distribution of antigen-specific CD8+ T cells in the lymphoid tissue and the non-lymphoid tissue after DNA immunization. HBsAg-specific CD8+ T cells in peripheral blood mononuclear cells, spleen, lymph nodes, and the liver of Balb/c mice have been quantified after injection with a DNA plasmid expressing the major S protein of hepatitis B virus (HBV). The kinetics of CD8+ T-cell responses in the circulation were measured after priming and boosting, showing that antigen-specific CD8+ T cells undergo first expansion and then decline to a sustainable level in the circulation, although the frequencies of HBsAg-specific CD8+ T cells in the circulation were lower than for the spleen. The greater frequencies of HBsAg-specific CD8+ T cells were found in the liver, whereas the largest numbers of antigen-specific CD8+ T cells were found in the spleen. By day 100 after priming, HBsAg-specific CD8+ T cells were still detected in the circulation, the spleen and the liver. After boosting with the same plasmid DNA immunogen, HBsAg-specific CD8+ T cells proliferated quickly and vigorously. By 150 days after boosting, HBsAg-specific memory CD8+ T cells were sustained at higher levels than those recorded after the first, primary injection, both in the spleen and the liver: anti-HBs antibody-secreting plasma cells persisted in the bone marrow and in the spleen, consistent with the detection of anti-HBs antibodies detected in the blood. These findings indicate that DNA immunization has considerable potential for inducing specific T cell responses in the liver and offers a strategy for the development of post-exposure immunotherapy against persistent hepatitis B infections.

Effects of Type I Interferons on the Adjuvant Properties of Plasmid Granulocyte-Macrophage Colony-Stimulating Factor In Vivo

While administration of granulocyte-macrophage colony-stimulating factor (GM-CSF) can induce the local recruitment of activated antigen-presenting cells at the site of vaccine inoculation, this cellular recruitment is associated with a paradoxical decrease in local vaccine antigen expression and vaccine-elicited CD8+ T-cell responses. To clarify why this cytokine administration does not potentiate immunization, we examined the recruited cells and expressed inflammatory mediators in muscles following intramuscular administration of plasmid GM-CSF in mice. While large numbers of dendritic cells and macrophages were attracted to the site of plasmid GM-CSF inoculation, high concentrations of type I interferons were also detected in the muscles. As type I interferons have been reported to damp foreign gene expression in vivo, we examined the possibility that these local innate mediators might decrease plasmid DNA expression and therefore the immunogenicity of plasmid DNA vaccines. In fact, we found that coadministration of an anti-beta interferon monoclonal antibody with the plasmid DNA immunogen and plasmid GM-CSF restored both the local antigen expression and the CD8+ T-cell immunogenicity of the vaccine. These data demonstrate that local innate immune responses can change the ability of vaccines to generate robust adaptive immunity.

Plasmid DNA Vaccine-Elicited Cellular Immune Responses Limit In Vivo Vaccine Antigen Expression through Fas-Mediated Apoptosis

Particularly potent cellular or humoral immune responses are needed to confer protection in animal models against such pathogens as HIV/SIV, Mycobacterium tuberculosis, and malarial parasites. Persistent, high-level vaccine Ag expression may be required for eliciting such potent and durable immune responses. Although plasmid DNA immunogens are being explored as potential vaccines for protection against these pathogens, little is known about host factors that restrict long-term plasmid DNA vaccine Ag expression in vivo. We observed rapid damping of transgene expression from a plasmid DNA immunogen in wild-type, but not in T cell-deficient mice. This damping of Ag expression was temporally associated with the emergence of Ag-specific cellular immune responses. A requirement for Fas and the appearance of apoptotic nuclei at the site of vaccine inoculation suggest that T cells induce Fas-mediated apoptosis of plasmid DNA vaccine Ag-expressing cells. These studies demonstrate that high levels of in vivo Ag expression are associated with high-frequency cellular immune responses that in turn rapidly down-regulate vaccine Ag expression in vivo. These findings argue that it may not be possible to maintain persistent, high-level production of vaccine Ag in vivo to drive persistent immune responses as long as vaccine Ag production can be limited by host immune responses.

Characterization of immune response in mice to plasmid DNA encoding dog zona pellucida glycoprotein-3

To investigate the immunogenicity of plasmid DNA encoding dog zona pellucida glycoprotein-3 (dZP3), the cDNA corresponding to dZP3, was cloned in mammalian expression vector VR1020 downstream of tissue plasminogen activator signal sequence under cytomegalovirus promoter (VRdZP3). In vitro transfection of COS-1 mammalian cells with VRdZP3 plasmid DNA led to its cytosolic expression. The expressed dZP3 has an apparent molecular weight of 45 kDa as compared to calculated molecular weight of 38.4 kDa, suggesting possible glycosylation. Immunization of male BALB/cJ mice with VRdZP3 plasmid DNA in saline, by electroporation or adsorbed onto gold microcarriers (delivered by gene gun) generated antibody response against Escherichia coli expressed recombinant dZP3 (r-dZP3). Administration of r-dZP3 in saline following immunization with plasmid DNA led to boosting of the antibody response. Although mice immunized with gene gun exhibited highest antibody titres, the differences in the antibody titres seen by the three modes of plasmid DNA delivery were not statistically significant ( P>0.05). Interestingly, female mice immunized with VRdZP3 plasmid DNA using gene gun also generated antibodies against r-dZP3. A dominant IgG1 isotype response was observed in mice immunized with VRdZP3 plasmid DNA using gene gun as compared to a mixed IgG1–IgG2a isotype response when delivered in saline or by electroporation. Immunization with VRdZP3 plasmid DNA also generated cell mediated immune response. The antibodies generated by VRdZP3 plasmid DNA recognized dog native zona pellucida. These studies for the first time, demonstrate the feasibility of generating an immune response to ZP3 by DNA vaccine and that the antibodies thus generated recognize native zona pellucida.

Antibodies generated in response to plasmid DNA encoding zona pellucida glycoprotein-B inhibit in vitro human sperm-egg binding.

To investigate the immunogenicity of plasmid DNA encoding bonnet monkey (Macaca radiata) zona pellucida (ZP) glycoprotein-B (bmZPB), the cDNA corresponding to bmZPB, excluding the N-terminal signal sequence and C-terminus transmembrane-like domain, was cloned in mammalian expression vector VR1020 downstream of tissue plasminogen activator signal sequence under cytomegalovirus promoter (VRbmZPB). In vitro transfection of COS-1, COS-7, CHO, HEK-293, and UM-449 mammalian cells with VRbmZPB plasmid DNA led to the expression of bmZPB. Expression of bmZPB in transfected cells was cytosolic. Flow cytometry analysis of COS-1 cells transfected with VRbmZPB revealed that approximately 15% cells expressed bmZPB. The expressed bmZPB has an apparent molecular weight of 57 kDa. Immunization of male BALB/cJ mice with VRbmZPB plasmid DNA in saline as compared to VR1020 immunized group, elicited significant antibodies against E. coli expressed recombinant bmZPB as evaluated in ELISA. The antibodies generated by VRbmZPB plasmid DNA recognized bonnet monkey as well as human ZP. The immune sera obtained from mice immunized with VRbmZPB plasmid DNA also inhibited, in vitro, the binding of spermatozoa to the ZP in the hemizona assay. These studies, for the first time, demonstrate the feasibility of DNA vaccine to generate antibodies against ZP that recognize native protein and inhibit human sperm-oocyte binding.

Enhanced CTL responses mediated by plasmid DNA immunogens encoding costimulatory molecules and cytokines.

In the course of examining epitope-specific CTL responses to intramuscular plasmid DNA immunization with influenza nucleoprotein (NP)-expressing vectors, a nonimmunogenic mutant NP (NP(o)) was identified. The coding region of NP(o) differed from the wild-type A/PR/8/34 NP sequence (designated NP(v)) by three amino acid alterations in the carboxyl-terminal portion of the molecule remote from the H-2K(d) epitope (147-155) being monitored. Correction of these mutations restored the immunogenicity of the native sequence, indicating that sequence alterations remote from the CTL epitope in question can profoundly influence its immunogenicity. In an effort to identify general, nonstructural means of enhancing the CTL response to weak plasmid DNA immunogens, vectors were constructed expressing NP(o) in tandem with the costimulatory molecules B7-1 or B7-2. Co-linear expression of NP(o) with B7-2, but not B7-1, significantly increased the NP epitope-specific CTL response. In addition, coinjection of these NP(o) plasmids with granulocyte-macrophage CSF- and/or IL-12-expressing vectors also restored near native NP-specific CTL responses. Thus, the coexpression of certain costimulatory molecules and/or cytokines, in concert with a non-self gene delivered as an intramuscular plasmid DNA immunogen, can significantly enhance Ag-specific CTL responses.

Immunostimulatory DNA sequences necessary for effective intradermal gene immunization.

Vaccination with naked DNA elicits cellular and humoral immune responses that have a T helper cell type 1 bias. However, plasmid vectors expressing large amounts of gene product do not necessarily induce immune responses to the encoded antigens. Instead, the immunogenicity of plasmid DNA (pDNA) requires short immunostimulatory DNA sequences (ISS) that contain a CpG dinucleotide in a particular base context. Human monocytes transfected with pDNA or double-stranded oligonucleotides containing the ISS, but not those transfected with ISS-deficient pDNA or oligonucleotides, transcribed large amounts of interferon-alpha, interferon-beta, and interleukin-12. Although ISS are necessary for gene vaccination, they down-regulate gene expression and thus may interfere with gene replacement therapy by inducing proinflammatory cytokines.