DNA vaccines: Future strategies and relevance ... - Wiley Online Library

Immunology and Cell Biology (2001) 79, 537–546

Review Article

DNA vaccines: Future strategies and relevance to intracellular pathogens AK SHARMA and GK KHULLER Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India Summary Increasing awareness of microbial threat has rekindled interest in the great potential of vaccines for controlling infectious diseases. The fact that diseases caused by intracellular pathogens cannot be overcome by chemotherapy alone has increased our interest in the generation of highly efficacious novel vaccines. Vaccines have proven their efficacy, as the immunoprotection they induce appears to be mediated by long-lived humoral immune responses. However, there are no consistently effective vaccines available against diseases such as tuberculosis and HIV, and other infections caused by intracellular pathogens, which are predominantly controlled by T lymphocytes. This review describes the T-cell populations and the type of immunity that should be activated by successful DNA vaccines against intracellular pathogens. It further discusses the parameters that need to be fulfilled by protective T-cell Ag. We then discuss future approaches for DNA vaccination against diseases in which cell-mediated immune responses are essential for providing protection. Key words: cell-mediated immune responses, HIV, T lymphocytes, tuberculosis.

Introduction The first successful human vaccine experiment carried out by Edward Jenner approximately 200 years ago demonstrated that inoculation of a boy with cross-reactive cow-pox virus protected him against two successive infections with small pox virus. Since then, the majority of vaccinologists have focussed on the development of vaccines. Recombinant vectors, subunit vaccines, anti-idiotypic vaccines etc. have all been introduced in the past and some have been valuable in this field. Previous studies in our laboratory also indicated that native and recombinant 30 kDa secretory proteins of Mycobacterium tuberculosis are potential candidates for vaccine development against tuberculosis.1–3 However, due to some disadvantages with these vaccines and their poor efficacy, a new approach to vaccination has begun, that is, DNA vaccination. There is mounting evidence, from numerous studies, that injection of free DNA (naked DNA) stimulates effective and long-lived immune responses to the protein Ags (Ag) encoded by the gene vaccine,4 which are being considered ‘the third generation vaccines’. The advent of DNA-based vaccination strategies not only offers a relatively safe modality capable of inducing both cytotoxic T lymphocytes and antibodies, but also allows engineering of artificial immunogens and coexpression of immunomodulatory proteins.5 The term DNA-based immunization currently refers to the induction of immune responses to a protein Ag expressed in vivo subsequent to the introduction of purified

Correspondence: Prof. GK Khuller, Head, Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh 160 012, India. Email: [email protected] Received 21 March 2001; accepted 14 June 2001.

plasmid DNA encoding the polypeptide sequence. The resulting in situ production of the protein can involve biosynthetic processing and post-translational modifications. If the introduction of plasmid DNA is both efficient and safe, nucleicacid-based immunization may one day be an attractive alternative to classical vaccines. Using a number of reporter genes, it has recently been demonstrated that injection of either purified RNA or DNA can result in the expression of the appropriate enzyme activity within the skeletal muscle. The direct in vivo transfer of genes can be accomplished in various tissues by different means. Because routine vaccination is almost always applied to large populations, direct (in vivo) gene transfer would be the method of choice for prophylactic immunization purposes. Although indirect gene transfer, which involves reimplantation of cells removed from an individual and transfected ex-vivo, might be considered for certain forms of immunotherapy, it is too cumbersome and expensive to consider unless direct gene transfer methods fail. Direct gene transfer may be carried out using either viral vectors or recombinant plasmid DNA carrying cloned genes to be expressed in situ. The use of pure plasmid DNA offers many potential advantages, including quick and easy manufacturing, better quality control and the non-integration of DNA and lack of immunogenicity of the vector itself.6 Also these vaccines are heat stable, easily transportable when stored in lyophilized form and can induce persistent cellmediated immunity (CMI) responses to Ag isolated from a variety of viral, bacterial and parasitic pathogens. These vaccines have also been shown to have protective responses against HIV, influenza, rabies, leishmaniasis, malaria, Herpes simplex virus (HSV) and tuberculosis.7–9 DNA vaccines have the capacity to stimulate CMI without the need for adjuvants and have the potential to encode multiple Ag that may influence the nature of immune responses.

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Design of a vaccine and plasmid DNA vectors The prerequisite for a DNA vaccine, a plasmid DNA vector, should be in supercoiled form and requires an efficient promoter, such as a viral promoter [e.g. CMV and Rous sarcoma virus (RSV)] for transcription of Ag gene insert. It also requires a polyadenylation region at the 3′ end of the insert for mRNA stability and proper translation. The CMV promoter appears to be the best choice because it provides adequate expression of Ag for eliciting immunity. Thus, the plasmid not only provides coding sequences for the antigenic protein, but also serves as the physical vector carrying the genes as well as an adjuvant in the process of DNA-mediated immunization. For decades, polynucleotides and even high molecular genomic DNA have been shown to be non-specific stimulators of the immune system.10 Certain sequences in the bacterial DNA appear to specifically serve as mitogen for B cells, however, the large amount of DNA after intramuscular injection does not express protein, but is instead being taken up by macrophages and further stimulates the secretion of IL-12. This cytokine could then impinge on NK cells to release IFN-γ, which would help in the development of Th cells, predominantly towards the Th1 pathway, which further leads to secretion of IL-2 and IFN-γ. Thus, this cytokine favours Ag processing by stimulating biochemical catabolism of Agic proteins encoded by the plasmid DNA.

Factors affecting the efficacy of DNA vaccines Plasmid-encoding human GM-CSF may be clinically useful in enhancing the efficacy of DNA vaccines in humans. Recombinant human GM-CSF is used in patients to stimulate cell growth from bone-marrow cells in several clinical settings and its potential toxicities are well defined.11 If safety concerns related to injecting a human with cDNA can be addressed, the potency of DNA vaccines in humans can be enhanced by the addition of either plasmid encoding GMCSF or recombinant GM-CSF cytokine, or by using a coimmunization strategy with vectors expressing cytokines, such as GM-CSF, IFN-γ or IL-12,12,13 or by the use of costimulatory molecules.14,15 It may also be possible to manipulate the DNA backbone to increase its adjuvant effects by the addition of multiple immunostimulatory sequences.16 A combination of DNA and oral vaccines expressing the same candidate Ag may increase the protective response to complex organisms.9,11,17 Thus, a combination of these approaches may be necessary to obtain clinically significant long-lived protective efficacy of DNA vaccines in humans. Therefore, success in achieving long-lived memory responses by DNA vaccination may lie in optimal activation and expansion of most Ag-specific T-cell precursors rather than longterm storage of Ag leading to periodic restimulation. Ag complexed with IFN-γ plasmid may also be trialled and could be beneficial in the treatment of diseases caused by undesired Th2 dominated responses, including allergic diseases and certain parasitic infections. Hence, DNA-mediated immunization may allow rational designs of DNA-expression vectors to induce a particular type of immune response.18 Because changes in Ag composition and coding sequence can be evaluated more rapidly than for recombinant proteins, this approach could also lead to the development of a new

generation of vaccines based on plasmid DNA. Professional APC play a key role in the induction of the immune response evoked by vaccination with plasmid DNA specific epitope. The use of attenuated intracellular bacteria, for example, Shigella fluxneri, Salmonella typhimurium, Listeria monocytogenes, as delivery vehicles has the potential to efficiently target DNA vaccines to professional APC.19 This approach of DNA delivery has the potential to combine the advantages of live vaccine vectors with those of naked DNA vaccines. Thus, a very attractive scenario can be envisaged for DNA immunization whereby favourable conditions are provided for Ag processing/presentation by involving professional APC (e.g. macrophages and dendritic cells). Small amounts of proteins that are generated from introduced plasmids require the most efficient mechanism to stimulate efficient immune responses. Thus, the plasmid DNA for gene vaccination can be divided conceptually into two distinct units: the first is a transcription unit that directs Ag synthesis and the second is an adjuvant/mitogen unit in the plasmid DNA backbone.

How do DNA vaccines work? Mechanism of DNA vaccination The mechanism of immune stimulation by genetic immunization has been an important issue over the past decade. By simply putting DNA into skin or muscle cells, it was speculated that these cells activate killer T cells. However, before these cells can activate cytotoxic T cells, the killer T cells must be primed, and this requires the interaction of ‘professional’ APC. Recent discoveries of several groups have shown that plasmids do in fact make their way into professional APC or resident dendritic cells, which then display Ag alongside the critical costimulatory molecules and help to prepare T cells for action. Thus, for a rational vaccine design, an understanding of the immunological response of host to infection is very much essential. Intracellularly replicating viruses and some bacteria are processed within the cell and the full-length proteins are chopped off into peptides by an intracellular complex (Proteasome complex). These peptides then move into the endoplasmic reticulum (ER) by a transcription-associated protein (TAP) transporter.20,21 In the ER, the peptides associate with MHC class I molecules and β2-microglobulin. This, tri-molecular complex is then transported to the cell surface for perusal by circulating T cells. Upon encountering the ternary complex (i.e. β2-microglobulin, MHC class I and peptide), Ag-specific CD8+ T cells are activated, which leads to cytotoxic functions. The activated T lymphocytes (CTL) lyse infected cells and thereby restrict the multiplication of the pathogen and contain the spread of the infection. Thus, in DNA immunization, this pathway holds true and has been confirmed by several studies.20 Direct inoculation of plasmid DNA containing open reading frames with appropriate eukaryotic transcription and translation control signals results in the in vivo synthesis of the protein with confirmation and post-translational modification patterns identical in most cases to those that occur during normal infection. The endogenously synthesized protein mimic viral infection allows presentation of foreign Ag by MHC class I, while the

DNA vaccines

uptake of soluble protein by specialized APC allows presentation by MHC class II; thereby inducing both arms of T-cell responses.20–23 However, the mechanism underlying CTL induction remains controversial. The provoked expression of encoded Ag by muscle cells led to suggestions that muscle cells themselves present Ag and directly induce T-cell responses. The T cells can recognize muscle in certain myopathies,21,24–27 but this does not imply that myocytes can induce T-cell responses. Appropriate CTL induction requires two signals: first, the appropriate MHC class I peptide complex, which interacts with T-cell receptors, and second a B7–1 costimulatory molecule, which interacts with CD28 on the T-cell membrane. However, myocytes express only low levels of MHC class I molecules and the level of costimulatory molecules is very low or absent. Thus, the ability of myocytes to induce efficient CTL responses is questionable. Recently, many authors have shown that Ag is released from muscle cells and taken up by APC, thereby inducing CTL as well as Th-cell responses. In contrast, transfected cells in injected muscle do not play a vital role in DNA-induced antibody and CTL responses.21,22,28 After induction of cytotoxic T-lymphocyte responses by DNA vaccines, some activated cells also become memory cells, which are ready to eliminate cells invaded by intracellular pathogens in the future. The high frequency of immunostimulatory CpG motifs in plasmids also strengthens the immune responses evoked by the Ag derived from antigenic genes in plasmids. Plasmid DNA derived from bacteria has a greater frequency of CpG sequences than does the DNA in vertebrates,29 and is nonmethylated. Hence, the vertebrate body interprets a high frequency of non-methylated CG pairs as a danger signal. There is a possibility that DNA vaccination may be involved in the cross-priming method of immune induction. Such a cross-priming process may represent a principle mechanism by which plasmid DNA is delivered to cells such as myocytes. These myocytes effectively shuttle Ag to dendritic cells (DC) or other APC to achieve CTL induction in vivo.30 It is thought that professional APC play a dominant role in the induction of immunity by presenting vaccine peptides on MHC class I molecules after Ag capture and processing within the endocytic pathway. The immunity can be manipulated according to many immunization parameters, including the method of gene vaccine delivery,31 and the presence of genetic adjuvants and vaccine regimens. DNA vaccines were first used in clinical trials 5 years ago and an initial picture of their utility in humans is now emerging. However, further analysis is required to determine the ultimate efficacy and safety of these vaccines in humans. While DNA vaccination resulted in the transfection of only a small proportion of DC, it led to the general activation of all DC, thus providing optimal conditions for effective T-cell activation and the maintenance of memory. These efficacy-limiting mechanisms can be overpowered by coupling the potent Agpresenting capacity of DC with paracrine delivery of potent anti-infective cytokines, such as IL-12, to local immune responses sites.32 The regulatory interactions between CD8 T cells and CD4 T cells are complex and may involve both Ag-specific and non-specific mechanisms. Ag-specific regulation could in principle occur in two ways.33 Both CD4 and CD8 T cells may

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recognize Ag–MHC complex on conventional APC. Activated CD8+ T cells would thus suppress/inactivate CD4 cells in the proximity by either secreted products or cell contact, or possibly by killing the APC. Alternatively, CD8+ T cells may recognize activated CD4 T cells directly in a peptide-specific manner. Activated CD8 regulatory T cells would in turn delete or otherwise inactivate ‘inducer’ CD4 T cells.33 When considering the reasons for and against such approaches, it is important to remember that when protection against infection is the target, the type of immunity required is not the same for all diseases. Thus, a vaccine that is effective against systemic infection may not work against localized infection (e.g. mucosal sites). For infections involving mucosal surfaces an attenuated salmonella vector strategy is more suitable for presenting foreign Ag, whereas for systemic infections recombinant vaccinia viruses are likely to be more useful. The combinatorial approach of combining T-cell sites and B-cell sites is also useful for overcoming genetic restriction. Thus, the overall effect of a DNA vaccine depends on several factors. Non-living vaccines are incapable of generating cellular immune responses indispensable for protective immunity against intracellular pathogens. In contrast, liveattenuated vaccines permit efficient MHC class I presentation of Ag, which can stimulate CD8+ T-cell responses. Also, these vaccines have the advantage of antigenic persistence. Attenuated whole-organism vaccines raise issues that may preclude their widespread application for certain diseases such as HIV and malaria. Thus, DNA vaccines have an edge over these vaccine formulations as they induce strong CMI (strong CTL and Th1 responses) and humoral immunity (HMI) responses in mice. However, results have been disappointing in humans when compared with rodent models.34–36 This could be due to different disease conditions in humans and rodents. In order to extrapolate the efficacy of DNA vaccines in animal models to humans, these vaccines need to be tested in animals that closely mimic the course and pathology of the disease as seen in humans. For example, the promising antituberculous DNA vaccine should be tested in a guinea pig model. Enhanced immunogenicity of a heterologous prime-boost immunization strategy, by using DNA priming and boosting37 with a recombinant vector that encodes the same foreign Ag, has been demonstrated in several infectious diseases inducing strong CD8+ CTL responses in mice and primates.37,38 Several pox viruses, including modified vaccinia virus Ankara (MVA) and fowl pox, and replication-defective adenoviruses have the capacity to boost primed CD8+ T-cell responses substantially,39,40 but the questions are: (i) how long are these responses maintained after the MVA boost; and (ii) whether continued boosting will be required for sustaining a sufficient number of cells to confer protection. A comparative analysis of various vaccine formulations known to the authors has been elucidated in Table 1, which shows a clear-cut potential role for DNA vaccines over other vaccines.

DNA vaccination against intracellular pathogens DNA vaccines have been developed against various pathogens, including HIV, rabies, hepatitis virus, HSV, Ebola virus,

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Table 1


Efficacy of various vaccine formulations

Immune responses

Cell-mediated immunity (CMI) CD4+ CD8+ Humoral immunity (HMI) Ag presentation Memory HMI† responses Memory CMI‡ responses Ease of manufacturing and production Cost Transport/storage Safety concerns References

DNA vaccine

Epitopic/multitopic DNA vaccine

Killed vaccine

Live attenuated

Live vector (e.g. Pox virus)

+ + + Strong Th1 ++ ++ MHC 1 & II +++ ++ ++++

++ +++ ++ MHC I & II +++ +++ +++

+– – +++ MHC II only +++ +– ++

+– +++ +++ MHC I & II +++ +++ +

+ +++ ++ MHC I & II ++ ++ +++

+++ +++ ++++ 29,31,34–36,51

+++ +++ ++++ 20,30,41,45

+ +++ ++++ 22,23,29 (reviewed)

+ + ++++ 1,2,29 (reviewed)

++++ + ++ 52,58,59

(+), Live/attenuated vaccines may be precluded for use in the immunocompromised patients and certain infections such as HIV. Memory humoral-mediated immunity. ‡ Memory cell-mediated immunity. †

Mycobacterium spp., Plasmodium yoelii and Schistosoma japonicum. Infecting pathogens are often elusive targets for a host’s defense mechanisms. Rapid multiplication with the possibility of exchange of genes and molecular switches, as well as molecular mimicry, allow these pathogens to invade the immune system of the host. Genetic drift is a common mechanism for a number of pathogens (e.g. influenza virus and HIV) and a variety of innovative approaches are being used to overcome these obstacles.

Tuberculosis Experimental approaches to develop an improved vaccine against tuberculosis have included the use of attenuated mycobacteria, subunit vaccines and, more recently, DNA vaccines. Recently, in our laboratory, a recombinant 30 kDa (rMtb30) secretory protein of M. tuberculosis H37Rv was found to be highly protective against tuberculosis irrespective of the mouse strain used and the protection was equivalent to bacille Calmette-Guerin (BCG).1–3 However, until recently, none of these vaccines had been shown to be more potent than BCG in any animal model or even to be closely comparable in the highly susceptible and clinically relevant guinea pig model. All these observations led to the construction of a DNA vaccine that can be easily manufactured and can induce better protection than BCG. DNA vaccines against M. tuberculosis have been most extensively explored because of the repeated failure of BCG, which is the only available vaccine against tuberculosis. Thirty-two novel DNA vaccines have been tested, singly and in combination, for their ability to elicit immune responses to specific M. tuberculosis Ag.41,42 The immune responses evoked by DNA vaccines can be altered by expressing the tuberculous Ag as tissue plasminogen activator (tPA), fusion or ubiquitin chimeric proteins.43,44 Tuberculosis DNA vaccines effectively induce IFN-γ, both in splenocytes and lung cells. However, in the lungs the Agspecific responses are usually low and substantially less than the responses elicited by BCG. At least 10 of the vaccines

tested have induced significant protection in the low-dose aerosol mouse challenge model. One combination of single vaccines induced a protective response that was similar to the response elicited by BCG. However, no evidence for CD8+ T-cell priming was observed against the identified 19 kDa and Ahpc peptides after DNA vaccination of mice.45 An intranasal route of vaccination also deserves further attention because a recent study indicated that BCG and Pst1 (DNAencoding-38 kDa protein) impart strong protective immunity, which paves the way towards improving vaccination against tuberculosis.46 Mycobacterial proteins, a 65 kDa heat shock protein (hsp) of Mycobacterium leprae, and Ag85-complex proteins have been evaluated as DNA vaccines in mice.4,47,48 These vaccines have been reported to induce substantial immune responses and protection against experimental tuberculosis, which is equivalent to BCG. Baldwin et al. reported the protective efficacy of a secretory protein-based DNA vaccine.49 Kamath et al. studied the comparative protective efficacy of DNA vaccines encoding three mycobacterial proteins, MPT 64 (23 kDa), Ag85B (30 kDa) and ESAT-6, individually as well as in combination and it has been suggested that multi-subunit vaccination may contribute to future vaccines.42 These authors also reported that codelivery of plasmid expressing GM-CSF in conjuction with DNA vaccines based on mycobacterial secretory proteins, MPT-64 and Ag85B, resulted in significant cellular immune responses. However, there was no improvement in protective efficacy against aerosol challenge with M. tuberculosis H37Rv.42 Previous studies have also shown that plasmid DNA encoding hsp70, 36 kDa and MTB12 mycobacterial proteins to be potent candidates for providing mycobacterial protective immunity.48,50 Reports are available indicating the failure of DNA vaccine based on mycobacterial 19 kDa and Ahpc proteins.45 Until recently, no research has reported DNA vaccines exhibiting higher efficacy than BCG. In addition to the prophylactic potential of DNA vaccines, the immunotherapeutic role of plasmid DNA encoding mycobacterial hsp65 against tuberculosis has also been reported.51

DNA vaccines

Table 2

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Important DNA vaccine candidates for tuberculosis control

Gene

Encoded protein

Immunogenic potential

MPT 64

23 kDa

Ag85B, A,C

30 kDa, 31 kDa & 31.5 kDa

ESAT-6 Phosphate transport gene

6 kDa 38 kDa lipoprotein

Combined DNA-85B, DNA-64, DNA ESAT6 M. bovis 621 bp gene Hsp65 Ag85B + GMCSF Multivalent DNA vaccine (ESAT-6, MPT-64, MPT-63, KATG constructs) Ag85A, 85B, ESAT-6, 38 kDa & 19 kDa constructs

30 kDa + 23 kDa + 6 kDa 22 kDa 65 kDa heat shock protein 30 kDa Combinatorial vaccine containing 6,23,18 and KatG-expressing protein

IFN-γ, IgG1, CD8+, cytotoxic cell induction, good immunogenicity, protective efficacy Increased IFN-γ, increased IgG, immunogenic and very good protective efficacy in murine and guinea pig model Highly immunogenic, less protection Highly protective, comparable to BCG strong CD8+ T cell response Greater efficacy than individual vaccines Immunogenic, but less protective Highly immunogenic and high protective effect Increased Ag specific T-cell responses Protection equivalent to BCG-improved protection than individual vaccines in mice model

Proteins expressed in vaccinia virus

Immune responses and protection in mice

References: 41,42,47–49,93–97.

Expression of M. tuberculosis secreted proteins, Ag85A, Ag85B, Ag85C and ESAT-6, in vaccinia virus as tPA signal sequence fusion proteins further enhanced the expression of M. tuberculosis Ag.52 Some of the potential mycobacterial genes encoding proteins of immunological relevance have been illustrated in Table 2. Although several tuberculosis DNA vaccines that elicited cell-mediated and protective immune responses have been identified, further refinements and gene manipulations in the DNA immunization technology will be needed to generate tuberculosis DNA vaccines that are better than BCG.

AIDS When HIV infection first occurs the immune system suppresses the virus very well, but with time this ability is lost. It is questionable how the loss of immune control takes place and to maintain immune control a balance must be established between antiretrovirals versus immune-based therapies. An enumerable number of studies report the efficient use of antiretroviral therapy against this disease,53–55 but for one report that contradicts these observations.56 Two years ago, it was hoped that if the virus were continuously suppressed to undetectable levels with antiretrovirals, the infection would die out by itself within a few years. But, some cells can live for a long time as there is evidence of ongoing viral replication/activity despite very good control of viral load with drugs.53 Among immune-based therapies, recombinant pox viruses have been shown to be efficient inducers of in vivo expansion of primed CD8+ T cells.57 Mature DC infected with canary pox virus elicited strong antihuman immune deficiency virus CD8+ and CD4+ cell responses from a chronically infected individual.58 Robinson et al. suggested the containment of the HIV infection by initial DNA priming followed by recombinant pox-virus

booster immunizations and this approach was totally antibody independent.59 This type of combinatorial approach has been speculated to be more efficient in containing an immunodeficiency virus.39,59–61 Strong CTL responses as well as antibody response(s) have been shown against HIV in mice and non-human primates.28,61–64 Further, the efficacy of the DNA vaccine showed improvement by replacement and proper usage of codons.65 Begarazzie et al. reported the safer use of these vaccines in various chimpazee models inducing both CMI and humoral responses.66 Co-delivery of proinflammatory cytokines (IL-1α and TNF-α), Th1 cytokines (IL-2, IL-12, IL15 and IL18) with an HIV immunogen also yielded better results with enhancement in antibody and cellular responses.65,67 Ishii et al. obtained strong HIV-1 specific cytotoxic T-lymphocyte responses on immunization with DNA and liposomes.68 Sasaki et al. also observed mucosal immune responses to HIV-1 using QS-21 as an adjuvant, which resulted in the induction of Th1 subsets.69 The hope for an effective CTL-epitope-based AIDS vaccine increases with the growing evidence for a central protective role of CTL during HIV infection.70,71 Such a vaccine reduces the amount of protein or genetic material that needs to be delivered during vaccination and facilitates construction of combined multitopic vaccines. It also enables focusing of the immune responses towards important or conserved protein regions and reduces the chance of incorporating undesired proteins, for example, immunopathogenic or immunosuppressive proteins. Hanke et al. also emphasized the fact that particular sequences of DNA and vaccine vehicles are critical for an effective elicitation of CTL.40 Ishii et al. reported strong delayed-type hypersensitivity reaction (DTH) responses induced by HIV-1 DNA vaccine among different mouse strains, suggesting its potential to be used as a DNA vaccine against HIV-1.68 DNA vaccine candidates tried against AIDS have been illustrated in Table 3.

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Table 3


DNA vaccine candidates tested against AIDS

Vaccine objective

Cells involved

Proteins encoded by vaccine genes

Immune responses

HIV prevention

T, B cells

HIV therapy

T cells

Envelope and regulatory proteins or core proteins and enzymes involved in HIV replication (all genes tested in one vaccine) Tat, nef and regulatory proteins or envelope and regulatory proteins

HIV therapy

T cells

HIV prevention HIV prevention HIV prevention

T, B cells T cells T cells

HIV prevention

T, B cells

Strong cellular responses (phase I trials) Humoral and cellular responses immune analysis in progress (phase I trials) Vaccine combined with aggressive drug therapy (HAART) Immune analysis in progress Protection in chimpanzees Increased antibody response, improved protection Strong mucosal and cell-mediated immune responses against HIV Ag; strong CTL responses

Envelope, regulatory core proteins and enzymes involved in HIV replication Regulatory proteins and HIV replication enzymes Combination vaccine (envelope DNA + protein boosting) Combinatorial vaccine approach containing IL-12 and GM-CSF-expressing plasmids in liposomes DNA expressing gp160 protein along with rev and gag/pol construct

References: 12,13,29,64,66,68,82,98.

Table 4

DNA vaccine candidates tested against some diseases

Malaria prevention Malaria prevention

T, B cells T, B cells

Leishmaniasis prevention Mycoplasma infection

T cells T, B cells

Circumsporozoite protein (CSP) Sporozoite protein in a common vector; Salmonella typhimurium; Plasodium yoelii CSP protein and DNA-encoding CSP and Py Hep17, a hepatocyte erythrocyte protein Major surface glycoproteins gp63 of L. major All Ag (expression library)

Hepatitis B prevention Influenza prevention

T, B cells B cells

Hepatitis B surface Ag (HBSAg) Haemaglutinin (HA)

Cellular responses Strong CD8+ T-cell responses. High level protective immunity in animals

Strong protection in murine model Strong immunity (CMI and humoral immunity in mice) Humoral and cellular responses Immune analysis in progress (trials ended). Strong humoral responses in mice

References: 11,29,72,73,79,80,81.

DNA vaccines against other intracellular pathogens Efficient induction of antibodies as a result of plasmid DNA administration encoding haemagglutinin (HA) protein72 led researchers to the potential of this vaccine both in homologous and heterologous strains. DNA encoding the nuclear protein (NP) was observed to be highly protective against two influenza strains73 mediated by effective CTL responses and antibodies. The combinatorial DNA vaccine encoding for HA and NP has been found to be more effective than DNA encoding NP alone against flu in animal models.9 CD4+ T cells appear to mediate protection against HSV infection when immunized with plasmid DNA encoding HSV glycoprotein B against viral challenge.74 The main aim of vaccinologists was to develop approaches beneficial when used postinfection as well as for prophylaxis.75–77 Bourne et al. have investigated the immunogenicity of two proteins of HSV-1, a regulatory protein ICP27 and a major glycoprotein gB.78 Efforts are in progress to improve the efficacy of DNA vaccines by co-expressing GM-CSF with HSV DNAenhancing Ab induction, cytokine production, CTL responses and, thereby, inducing resistance to challenge.74 DNA vaccines have also been found to play a pivotal role in providing protective immunity against other intracellular

agents such as leishmania,79 plasmodium,11,29,80 mycoplasma,81 hepatitis82 and influenza infections.72,73 DNAencoding rabies virus glycoproteins have been shown to generate strong humoral and T-cell mediated immunity, leading to protection against virulent challenge in mice and monkeys.83,84 A combinatorial recombinant vaccinia virus approach along with plasmid DNA has been trialled recently against malaria and significant protection has been achieved in two strains of mice.37 Multigene strategy trialled with four Plasmodium falciparum proteins also yielded encouraging results.85 These DNA vaccines also hold promise against autoimmune diseases, allergies and cancer diseases. Various DNA vaccines tested against some diseases caused by intracellular pathogens have been illustrated in Table 4.

Future strategies for improving the DNA vaccine 1. Currently vaccine research requires optimization of DNA vaccine immunogenicity with immunomodulators (e.g. IL-12, GM-CSF, IL-2, IL-15, TCA3, B7.2, CD40 ligand and macrophage inflammatory protein (MIP)-1α) and chemical adjuvants, such as monophosphoryl lipid A (MPL), QS21, Ubenimex, liposomes, carboxyl methyl cellulose, calcium phosphate and aluminium hydroxide.

DNA vaccines

2. DNA priming and recombinant vaccinia virus/pox-virus boosting may lead to stronger protective immunity against intracellular pathogens especially in the containment of HIV infections.59,60 3. Route of administration of the vaccine has an important role to play because, depending on the pathogenic invasion, both mucosal and systemic immune responses may need to be taken care of. Both mucosal and systemic immune responses are equally important in containing pathogen attack as compared to a single type of immunity.12,86,87 Because HIV-1 invades through the rectovaginal mucosa, both mucosal and systemic immunities are indispensable and must be elicited to prevent the spread of the pathogen. As systemic vaccination is not fully protective despite its strong Th1 bias (as is clearly evident from numerous studies); we have to explore oral/mucosal vaccination as well. In a recent study mucosal immunity has been found to overcome an organ-specific failure of the lungs to benefit from otherwise potent systemic immunity against tuberculosis. In addition, secretory IgA may act against intracellular infectious pathogens. Thus, we have to take both systemic and mucosal immunity into account while administering a DNA vaccine.69,88 4. DNA vaccines must surmount major safety concerns, including a theoretical potential for integration into host genome and insertional mutagenesis and induction of autoimmunity, immunological tolerance or a prolonged allergic reaction to an encoded protein in which synthesis is not readily terminated. 5. Dual intervention is essential using vaccinations to boost immunity and drugs to kill the bacteria/virus, for example, antiretroviral therapy along with immunotherapy against HIV infection.53 6. The expression of Ag from DNA vaccines as fusion proteins with a destabilizing ubiquitin molecule (which enhances proteasome-dependent degradation of the endogenously synthesized Ag) results in strong CMI and humoral response(s) to some extent. This is required to achieve a boost in genetic immunization vaccine technology. Vaccination with plasmids expressing multiple epitopes, either as a combination of different plasmids or in the minigene form,89 may be needed to further improve the protective immunity evoked. Morris et al. showed that DNA vaccines encoding immunoreactive proteins cotranslated with tissue plasminogen activator (tPA) modulate the immune responses.41 Other constructs should also be trialled. Despite the long-term potential of Ub-conjugated vaccines, the protective effect of tPA-fusion vaccines and Ub-conjugated vaccines is more or less similar.43,44,90 7. There is a need to understand to a great extent the distribution, cellular uptake and expression of DNA vaccines to know the limitations to transfection in situ. Strategies to increase DNA uptake by muscle cells or to facilitate DNA entry into the nucleus of APC are likely to increase the potency of DNA vaccines. In one study, Dupuis et al. showed that the only cells transfected after i.m. injection were muscle cells and established a direct relationship between muscle transfection and DNA vaccine potency.91 Therefore, the above approach may yield useful results if trialled successfully. 8. It is important to determine for each new vaccine the optimal immunization regime, in terms of vaccine doses, timing of boosters, delivery routes and vaccine vehicles.

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Previous studies have shown that a newly developed multiCTL epitopic vaccine(s) efficiently induced HIV-specific CTL responses when delivered by skin bombardment and these responses can be enhanced by using a particular sequence of intramuscular DNA injection and genegun-mediated DNA immunization. 9. The gene gun vaccination combined with skin excision/ grafting provides a novel method for analysing the effect of Ag-dose and the duration of the induction of primary and memory responses as T- and B-cell memory is triggered by cells that migrate from the epidermis 5–12 h post-DNA immunization.92 Several routes of immunization have been shown to be efficacious, but on the basis of the potential use in humans and the economy of dose of DNA, the gene-gun approach was favoured.22 10. Finally, promising DNA vaccines should be tested in animal models that closely mimic the human disease and efforts should be made to reduce the cost of DNA vaccination to make it commercially viable for use in higher animals (e.g. humans).

Concluding remarks Studies are now aimed at characterizing T-cell epitopes, for example, for the development of antitumour vaccines using mainly CD8+ epitopes along with CD4+ epitope peptides derived from tumour associated Ag. Several studies have demonstrated the presence of T-cell epitopes on intracellular parasites, viruses and fungi, which can be envisaged for the preparation of safe DNA vaccines that are able to induce protective CMI. Considering vital factors, including the optimization of DNA vaccine immunogenicity with immunomodulators, mucosal site-directed immunity, DNA priming and protein boosting delivery by intracellular bacteria, as well as the easy manipulability of DNA should make DNA vaccine an appropriate choice for examining the body’s complex immune responses to different disease-causing agents. With such information in hand, vaccine manufacturers in future should be able to design effective vaccines that will target selected pathways and could induce both systemic and mucosal immune responses. In the future, such rationally designed DNA vaccines are likely to provide new immunotherapy for cancer, AIDS, tuberculosis, etc. and also provide powerful ways to prevent or minimize infections that elude human control today. Therefore, exciting prospects lie ahead for the control of major infectious diseases.

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