Nucleic Acid-based Vaccines Research Articles

Self-amplifying mRNA vaccines.

This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization.

A Cationic Nanoemulsion for the Delivery of Next-generation RNA Vaccines

Nucleic acid-based vaccines such as viral vectors, plasmid DNA, and mRNA are being developed as a means to address a number of unmet medical needs that current vaccine technologies have been unable to address. Here, we describe a cationic nanoemulsion (CNE) delivery system developed to deliver a self-amplifying mRNA vaccine. This nonviral delivery system is based on Novartis's proprietary adjuvant MF59, which has an established clinical safety profile and is well tolerated in children, adults, and the elderly. We show that nonviral delivery of a 9 kb self-amplifying mRNA elicits potent immune responses in mice, rats, rabbits, and nonhuman primates comparable to a viral delivery technology, and demonstrate that, relatively low doses (75 µg) induce antibody and T-cell responses in primates. We also show the CNE-delivered self-amplifying mRNA enhances the local immune environment through recruitment of immune cells similar to an MF59 adjuvanted subunit vaccine. Lastly, we show that the site of protein expression within the muscle and magnitude of protein expression is similar to a viral vector. Given the demonstration that self-amplifying mRNA delivered using a CNE is well tolerated and immunogenic in a variety of animal models, we are optimistic about the prospects for this technology.

Development of nucleic acid vaccines: use of self-amplifying RNA in lipid nanoparticles.

Self-amplifying RNA or RNA replicon is a form of nucleic acid-based vaccine derived from either positive-strand or negative-strand RNA viruses. The gene sequences encoding structural proteins in these RNA viruses are replaced by mRNA encoding antigens of interest as well as by RNA polymerase for replication and transcription. This kind of vaccine has been successfully assayed with many different antigens as vaccines candidates, and has been shown to be potent in several animal species, including mice, nonhuman primates, and humans. A key challenge to realizing the broad potential of self-amplifying vaccines is the need for safe and effective delivery methods. Ideally, an RNA nanocarrier should provide protection from blood nucleases and extended blood circulation, which ultimately would increase the possibility of reaching the target tissue. The delivery system must then be internalized by the target cell and, upon receptor-mediated endocytosis, must be able to escape from the endosomal compartment into the cell cytoplasm, where the RNA machinery is located, while avoiding degradation by lysosomal enzymes. Further, delivery systems for systemic administration ought to be well tolerated upon administration. They should be safe, enabling the multiadministration treatment modalities required for improved clinical outcomes and, from a developmental point of view, production of large batches with reproducible specifications is also desirable. In this review, the concept of self-amplifying RNA vaccines and the most promising lipid-based delivery systems are discussed.

Enhanced Delivery and Potency of Self-Amplifying mRNA Vaccines by Electroporation in Situ.

Nucleic acid-based vaccines such as viral vectors, plasmid DNA (pDNA), and mRNA are being developed as a means to address limitations of both live-attenuated and subunit vaccines. DNA vaccines have been shown to be potent in a wide variety of animal species and several products are now licensed for commercial veterinary but not human use. Electroporation delivery technologies have been shown to improve the generation of T and B cell responses from synthetic DNA vaccines in many animal species and now in humans. However, parallel RNA approaches have lagged due to potential issues of potency and production. Many of the obstacles to mRNA vaccine development have recently been addressed, resulting in a revival in the use of non-amplifying and self-amplifying mRNA for vaccine and gene therapy applications. In this paper, we explore the utility of EP for the in vivo delivery of large, self-amplifying mRNA, as measured by reporter gene expression and immunogenicity of genes encoding HIV envelope protein. These studies demonstrated that EP delivery of self-amplifying mRNA elicited strong and broad immune responses in mice, which were comparable to those induced by EP delivery of pDNA.

RNA-Based Vaccination: Sending a Strong Message

Nucleic acid–based vaccines (NAVs) for induction of antigen-specific immunity have reemerged as important tools in our vaccine/immune therapeutic arsenal. DNA technology appears to more than hold its own alongside viral vectored systems, particularly when the focus is on driving robust T-cell immunity in the clinic.1 By contrast, studies of vaccines based on RNA in small animals have been disappointing. Instability of the mRNA and manufacturing problems further weakened interest in this platform. However, just as good wine improves with age, cutting-edge technologies can take time to mature, and direct injection of RNA for immunization has recently been shown to be sufficiently improved. Support for RNA delivery as a more effective means of vaccination comes from several very recent reports.2,3,4

HPV and Therapeutic Vaccines: Where are We in 2010?

The discovery of human papillomavirus (HPV) as a necessary etiological factor for cervical cancer has spurred the development of preventive and therapeutic HPV vaccines for the control of HPV-associated malignancies including cervical, vulvar, vaginal, and a subset of head and neck cancers. The commercial preventive HPV vaccines, Gardasil and Cervarix, use HPV virus-like particles to generate neutralizing antibodies against HPV major capsid protein L1. However, they do not exert therapeutic effects on existing lesions and are unlikely to have an immediate impact on the prevalence of cervical cancer due to their cost and limited availability in developing countries, which account for more than 80% of cervical cancers. Thus, there is an urgent need for therapeutic HPV vaccines. Therapeutic HPV vaccines can eliminate preexisting lesions and infections by generating cellular immunity against HPV-infected cells. HPV E6 and E7 oncoproteins represent ideal targets for therapeutic intervention because of their constitutive expression in HPV-associated tumors and their crucial role in the induction and maintenance of HPV-associated disease. This review discusses the current progress of various therapeutic HPV vaccine approaches, including live vector-based, peptide/protein-based, nucleic acid-based and cell-based vaccines targeting E6 and/or E7 antigens, and their future prospects for the control of HPV-associated malignancies.

Innate immune control of nucleic acid-based vaccine immunogenicity

Optimal vaccine efficacy requires not only a protective antigen, but also a strong immune activator as an adjuvant. Most viral vaccines, such as influenza vaccines and nonviral genetic vaccines (e.g., DNA vaccines), contain nucleic acids, which appear to act as essential ‘built-in’ adjuvants. Specific receptors, including Toll-like receptors, retinoic acid-inducible protein-I-like receptors, and nucleotide-binding oligomerization domain-like receptors can detect specific nucleic acid patterns, depending on the immunized tissue, cell type and intracellular localization. The resulting immune activation is uniquely regulated by intra- and intercellular signaling pathways, which are indispensable for the ensuing vaccine immunogenicity, such as antigen-specific T- and B-cell responses. Thus, elucidation and manipulation of immune signaling and interactions by nucleic acid adjuvants are essential for maximizing the immunogenicity and safety of viral and DNA vaccines.

Use of Conventional or Replicating Nucleic Acid-Based Vaccines and Recombinant Semliki Forest Virus-Derived Particles for the Induction of Immune Responses Against Hepatitis C Virus Core and E2 Antigens

Replicating and nonreplicating nucleic acid-based vaccines as well as Semliki Forest-recombinant Viruses (rSFVs) were evaluated for the development of a vaccine against hepatitis C virus (HCV). Replicating SFV-DNA vaccines (pSFV) and rSFVs expressing HCV core or E2 antigens were compared with classical CMV-driven plasmids (pCMV) in single or bimodal vaccine protocols. In vitro experiments indicated that all vaccine vectors produced the HCV antigens but to different levels depending on the antigen expressed. Both replicating and nonreplicating core-expressing plasmids induced, upon injection in mice, specific comparable CTL responses ranging from 10 to 50% lysis (E:T ratio 100:1). Comparison of different injection modes (intramuscular versus intraepidermal) and the use of descalating doses of DNA (1–100 μg) did not show an increased efficacy of the core–SFV plasmid compared with the CMV-driven one. Surprisingly, rSFVs yielded either no detectable anticore CTL or very low anti-E2 antibody titers following either single or bimodal administration together with CMV-expressing counterparts. Prime-boost experiments revealed, in all cases, the superiority of DNA-based only vaccines. The anti-E2 antibody response was evaluated using three different assays which indicated that all generated anti-E2 antibodies were targeted at similar determinants. This study emphasizes the potential of DNA-based vaccines for induction of anti-HCV immune responses and reveals an unexpected and limited benefit of SFV-based vaccinal approaches in the case of HCV core and E2.

Enhancing DNA Immunization

Nucleic acid immunization describes the inoculation of DNA (usually a closed circular plasmid, although linear PCR fragments may be used) or RNA with the intent of inducing an immune response to the protein(s) (usually microbial) encoded therein. It is a relatively recent addition to the vaccine arsenal, and no nucleic acid-based vaccine has yet been approved for clinical or veterinary use. However, experimental studies have demonstrated that plasmid-based DNA vaccines can induce immune responses in humans (Wang et al., 1998), and the approach is currently eing evaluated in several clinical trials. There are good rounds for optimism; the method has many advantages, ot the least of which is its enormous flexibility, and it onfers protective immunity in a multitude of animal models reviewed by Donnelly et al., 1997; Hassett and Whitton, 996). Nevertheless, there remains much room for improveent; nucleic acid vaccines in general appear not to induce s strong responses as do conventional (live attenuated) accines, and consequently many laboratories are attemptng to enhance the immunogenicity of these promising accine candidates. Here we describe some of the aveues which have been explored; since the great majority of tudies have employed plasmid DNA, in this minireview we ocus on DNA vaccines. DNA vaccines may be administered in several ways. erhaps the two most common are, first, intramuscular njection, in which the plasmid DNA is taken up by muscle ells and the genes are expressed therein; and, second, dministration by some form of intradermal inoculation, ometimes using the “gene-gun,” in which tiny gold partiles coated with DNA are impelled into the skin. Following uch inoculation, the DNA is taken up by and expressed in pecialized antigen-presenting cells (APCs) called Langerans’ cells. Space constraints in this minireview prevent us rom providing a thorough comparison of these two popular outes of administration.

Progress on Nucleic Acid-Based Vaccines for the Control of Human and Animal Diseases

Vaccination still remains the most widely used method of controlling infectious diseases in both humans and animals. In view of this, considerable research effort is being devoted to the discovery of new vaccines, and to the refinement of existing ones, with the aim of making them safer, cheaper, and above all, more efficacious. Among the novel developments in vaccine technology is the use of naked DNA encoding the relevant immunogenic component of pathogens, which has been shown to confer protection on animals. Several advantages, such as stability, stimulation of both cellular and humoral immune responses, and antigen expression over prolonged periods, make DNA vaccines an attractive alternative to conventional ones. This paper highlights the progress made so far with the new technology in terms of its immunogenicity, concerns regarding its safety, and future research directions.

Differential Neutralizing Antibody Responses to Varicella-Zoster Virus Glycoproteins B and E Following Naked DNA Immunization

The only available vaccine against varicella-zoster virus (VZV) consists of the VZV-Oka attenuated but persistent virus strain. Development of a safer, subunit vaccine is therefore desirable. In this prospect, nucleic acid vaccines, expressing truncated forms of VZV glycoproteins B (recgB) and E (recgE) from which the anchor and the cytoplasmic domains were deleted, were used to immunize mice. Vaccination with recgB encoding plasmid elicited a strong and specific humoral immune response. Total IgG and neutralizing titres were comparable to those previously obtained by vaccination with purified and adjuvanted native recgB. In contrast, mice immunization with recgE encoding plasmid only induced a very weak immune response whereas we previously showed that vaccination with adjuvanted native or denatured recgE protein led to high neutralizing titres. The weakness of the immune response induced by recgE-encoding plasmid depended neither on the deletion of the anchor domain in the gE gene nor on the animal model. Analysis of antibody isotypes produced by plasmid immunizations revealed a response slightly dominated by IgG2a. Taken together, the data indicate that a VZV subunit vaccine based on adjuvanted recombinant glycoprotein E is more promising than a nucleic acid-based vaccine strategy. As regards recgB, both vaccination approaches might be appropriate.

In vitro-synthesized infectious RNA as an attenuated live vaccine in a flavivirus model.

Live virus vaccines have in many cases proven to be an extremely effective tool for the prevention of viral diseases. However, the production of conventional live vaccines in eukaryotic cell cultures has many disadvantages, including the potential for contamination with adventitious agents and genetic alterations during propagation, making it necessary to do extensive testing before distribution. Based on results obtained with a flavivirus (tick-borne encephalitis virus) in an experimental animal system, we propose a novel live attenuated virus vaccination strategy consisting of the application of in vitro-synthesized infectious RNA instead of the live virus itself. When administered using the GeneGun, less than 1 ng of RNA was required to initiate replication of virus that was attenuated by a specifically engineered deletion and this induced a protective immunity in laboratory mice. Because this approach uses RNA, it does not have the potential drawbacks of DNA vaccines and thus combines the advantages of conventional live virus vaccines (for example, mimicking natural infection and inducing long-lasting immunity) with those of nucleic acid-based vaccines (for example, ease of production without a requirement for eukaryotic cell culture, stability and purity).

Nucleic acid-based vaccines as an approach to immunization against human immunodeficiency virus type-1.

The spread of HIV-1 has resulted in significant morbidity and mortality worldwide. Traditional therapeutic approaches have included chemotherapy directed at several different viral components either alone or in combination. Studies evaluating combinations of two or three different anti-retroviral agents have recently generated optimism regarding the ability to control viral replication in vivo. It is too early to tell whether this approach is capable of controlling established infection without additional measures including immunotherapy. In any event, the majority of the worldwide at-risk population will not have easy access to these expensive therapeutic regimens. If achievable, protective vaccination represents the best solution to the worldwide problem of infection with HIV-1 and any strategy designed to control the epidemic is likely to rely heavily on mass immunization campaigns (Musgrove 1993). Unfortunately, the effort to design effective vaccines for HIV-1 has encountered a number of significant obstacles. In a 1993 poll by the journal Science (Cohen 1993) 150 top AIDS researchers agreed that the most significant obstacle has been the lack of consistent correlates of protection from natural or experimental infection. It is therefore not surprising that a wide variety of therapeutic modalities have been proposed in pursuit of effective therapy for HIV-1 (Table 1).

Nucleic acid vaccines.

The use of nucleic acid-based vaccines is a novel approach to immunization that elicits immune responses similar to those induced by live, attenuated vaccines. Administration of nucleic acid vaccines results in the endogenous generation of viral proteins with native conformation, glycosylation profiles, and other posttranslational modifications that mimic antigen produced during natural viral infection. Nucleic acid vaccines have been shown to elicit both antibody and cytotoxic T-lymphocyte responses to diverse protein antigens. Advantages of nucleic acid-based vaccines include the simplicity of the vector, the ease of delivery, the duration of expression, and, to date, the lack of evidence of integration. Further studies are needed to assess the feasibility, safety, and efficacy of this new and promising technology.

Nucleic acid vaccines.

The use of nucleic acid-based vaccines is a novel approach to immunization that elicits immune responses similar to those induced by live, attenuated vaccines. Administration of nucleic acid vaccines results in the endogenous generation of viral proteins with native conformation, glycosylation profiles, and other posttranslational modifications that mimic antigen produced during natural viral infection. Nucleic acid vaccines have been shown to elicit both antibody and cytotoxic T-lymphocyte responses to diverse protein antigens. Advantages of nucleic acid-based vaccines include the simplicity of the vector, the ease of delivery, the duration of expression, and, to date, the lack of evidence of integration. Further studies are needed to assess the feasibility, safety, and efficacy of this new and promising technology.