The leishmaniases comprise a group of largely neglected tropical diseases, transmitted during the blood meal of the phlebotomine sandfly (Figure 1). The disease outcome ranges from the mild cutaneous, more severe mucocutaneous to the almost fatal visceral leishmaniasis (followed by PKDL in a small proportion of VL patients) depending upon the transmitted species of Leishmania parasite. With more than 90% of the VL patients concentrated in south-east Asia and Africa, the statistics indicate that almost 200 million people are at risk worldwide, which is only a rough estimate, as a major population remains asymptomatic and hence unrecognized (1). VL ranks fourth in morbidity among all tropical diseases with an annual incidence of 2.5/1000 persons (2) and is second only to malaria in terms of mortality (3).

Despite abundant research in recent years, the available treatment options are far from satisfactory. The drugs are associated with toxicity, high cost, and/or resistance. In this context, multi-drug combinatorial therapies have shown some promise (4). Prevention by vaccination is favored by the fact that healing from leishmaniasis is almost always associated with lifelong resistance to infection. A desirable vaccine would provide long term immunity; elicit a T-cell immune response that would be a balance of Th1 mediated immune activation against the pathogen and Th2 mediated suppression to avoid excess tissue damage, produce a strong memory and effector response upon subsequent challenge, be persistent, and highly immunogenic (3). However, the vaccine should not elicit an auto-immune response and be safe even in immune-compromised SCID mice and HIV patients (5).

Based on the general nature of the formulation, there are three types of anti-leishmanial vaccines (6). The first generation of vaccines is comprised of live, virulent parasites injected at hidden body parts so as to avoid lesion visibility (leishmanization) or of inactivated parasites achieved by heat, radiation, antibiotics, chemical mutagenesis, and selection for temperature sensitivity or long passages in-culture (7). The second generation includes crude whole cell lysates, purified fractions, or subunit vaccines composed of single or multiple recombinant or native antigens. The only approved vaccine for human trial is Leish111f, a multivalent vaccine, composed of a thiol-specific antioxidant, Leishmania major stress inducible protein 1, and L. major elongation initiation factor (8). The third generation of vaccines consists largely of DNA in the form of mammalian expression plasmids or viral vectors encoding virulence factors (9). Unfortunately, the efficacy of available DNA and protein subunit vaccine candidates are limited (10). Recent concepts introduce the use of sandfly salivary antigens, T-cell epitope based peptides, antigen pulsed DC’s, and genetically modified live attenuated parasites (11). In contrast, vaccination using live attenuated parasites mimics natural infection and overcomes most of these limitations (12). Additionally, their persistence and display of parasites entire antigenic repertoire alleviates the need for an adjuvant. The recent success of live attenuated vaccination (LAV) in malaria, the clear genetic profile, and safety from reversion of complete knock-outs further encourages this endeavor.

Due to advances in axenic parasite culture, transfection efficiency, availability of genetic manipulation vectors (for expression, recombination, or integration), and the plethora of sequence based information available (from databases, like GeneDB, LeishCyc, LeishBase, KEGG, TriTrypDB, and TDR Targets), the ease and scope of creating live attenuated parasites has increased tremendously (13). Such parasites can be used to elucidate novel drug targets as well as vaccine candidates based on whether the gene under study is essential for both the promastigote and amastigote stages of the parasite or, only the amastigote stage. In addition, genetically modified organisms can also be used in metabolic pathways studies, structure-function relationship investigates (14), screening of new drugs (15), host–parasite interaction, and post-infection analysis among others, to enhance our understanding of these lower eukaryotes. Considering the success of LAV strategies against many viral, bacterial, and protozoan diseases (although to different extents), these are now considered the gold standard for protection against intra-cellular pathogens (12).

Foreign or self genes can be introduced in either episomal or integrated form, for expression of particular proteins to study their effects on various aspects of the parasites life cycle. In the episomal form, the gene’s expression is under the control of the vector specific promoter, which can be inducible or not (for stage specific expression analysis). For integration, the genes are generally targeted downstream of the ribosomal RNA locus to study the effects of constitutive expression at all stages of the life cycle. In either case, the genes can be fused to fluorescent reporter genes for ease of monitoring their expression (15). In addition, there are methods to selectively knock-out particular regions of interest heterologously or homologously using gene specific targeting constructs (16–18). During deletion, the targeted region is replaced by an antibiotic selection marker. Its expression makes the modified cells resistant to that antibiotic, thereby facilitating selection. Multiple genes can be targeted simultaneously. This exchange is generally brought about by the double strand break repair model of homologous recombination (19) whose major role has been the maintenance of its multi-gene families, conferring a selective advantage to parasites stressed by antifolate drugs (by upregulation of resistance genes) (14, 16). Alternatively, the transcripts of the genes can also be simply knocked down by anti-sense RNA interference technique, thereby blocking translation. However, with a few exceptions most leishmanial species lack the RNAi machinery (20).

Herein, we will discuss LAV strategies in various mosquito borne, viral, protozoal, and bacterial diseases. Malaria, which exerts significant mortality, morbidity, and economic burden, is spread by intra-cellular parasitic apicomplexans of the genus Plasmodium. Like Leishmania, Plasmodium has multiple hosts and forms and rapid amplification is key to its survival and spread. Their pathogenic liver and transmission stages have been the most often chosen targets for attenuation because compared to the blood stages, they are low in numbers and exhibit limited antigenic variation, making it less probable that a vaccine will fail against heterologous parasite strains. The search for a live attenuated malaria vaccine provided some invaluable insights that can be applied to leishmanial as well as other infectious diseases. The failure of the inactivated sporozoites, and success of γ-irradiated ones, demonstrated the requirement of live and host cell invasive parasites to confer protection (21–23). The ability of the UIS3^−/− sporozoites to confer protection against sporozoite re-infection but not blood stage transfusion, demonstrates stage specific immunity, herein, liver stage. Hence, not all stages of a parasites cycle may be equally useful for LAV approaches (24). The deletion of liver stage specific fatty acid synthesis pathway genes, however, had no effect on replication and gametogenesis, indicating that only essential metabolic pathways should be targeted for attenuation. Furthermore, multiple deletions sometimes may be more effective, as combined p26/p52 knock-out provided better protection than either of the single knock-outs in both chimeric mouse harboring human hepatocytes as well as both low/high dose human trials (22, 25, 26). These mutants exhibited complete growth arrest during the liver stages. However, their pre-erythrocytic stages were unhampered, thereby not hindering the possibility of large-scale production. Similarly, for leishmania, an unaffected promastigote growth stage would be desirable for a strain to be used for vaccination.

Another virus that largely affects the cloven hoofed animals worldwide is the foot and mouth disease virus (FMDV). Control by limiting animal movements and herd destruction has been mostly practiced due to insufficient protection by the available inactivated vaccine against all three FMDV variants. Recently, however, a reverse genetics approach has yielded a novel vaccine candidate by substitutions in a few amino-acids showing remarkable protection. These mutants too had normal growth properties as desirable for large-scale vaccine production (27).

One of the most successful and oldest examples of live attenuated vaccines is the 17D strain of yellow fever virus. It has also served as a model for vaccination strategies against dengue, a viral disease caused by transmission of one of its four serotypes 1–4 by the Aedes mosquito. Sanofi Pasteur’s ChimeriVax Dengue tetravalent vaccine (CVD1–4) is the most advanced product so far and a chimera in the truest sense utilizing the licensed YFV 17D vaccine as backbone, each expressing the prM and E genes of one of the four DENV serotypes. An effective dengue vaccine should consist of a tetravalent formulation, with components representing each serotype (28). A “stem-loop” genomic region implicated in its pathogenicity has been deleted to create the rDEN(1,2,4)Δ30 strains that impart adequate protection. However, the rDEN3Δ30 was not protective, indicating differences among strains. Hence, a novel chimerization led to a creation of rDEN3/4Δ30(ME) – a recombinant virus backbone of serotype 4 with Δ30 deletion, containing the ME region of a naturally attenuated serotype 3 strain, having manifold lower replication and transmission. This is a perfect example of successful extrapolation from sabin polio virus whose second component was also a naturally attenuated polio strain (29).

The MMR vaccine against measles, mumps, and rubella given to expecting mothers is another successful example of a multivalent vaccine that reduces the number of doses and avoids unnecessary delays and problems of spacing live attenuated vaccines (30). With pandemic capacity (31), the influenza vaccine, has been a huge challenge with its constantly varying epitopes resulting in antigenically drifted strains (32). In such cases, focusing on the most constant regions is the best strategy. However, till a strain specific vaccine is available, reasonable protection can be offered by a recombinant adenoviral vector expressing antigens from H5, H7, and H9 avian influenza virus strains (33). The success of multivalent, dengue, influenza, and MMR vaccines offers the idea for such a vaccine against CL, ML, and VL too.

Among bacteria, Streptococcus suis, that causes swine flu is a global health hazard to the swine industry, associated with septic shock, pneumonia, meningitis, and arthritis. The current vaccine against it is a Sly gene deletion attenuated strain undergoing some refinement by association with other surface antigens and adjuvants (34). The Bacillus Calmette Guerin vaccine for tuberculosis is created by long in vitro passaging of the intra-cellular bacteria Mycobacterium tuberculosis. The gradual loss of the RD loci has been reported as the major cause for this attenuation. Hence, attempts at manually creating these deletions are on. Recombinant BCG vaccines co-expressing other antigens from pathogens are also in clinical trials (35, 36). For cholera too, many endogenously produced live attenuated vaccines (Peru15 and Bengal15) are available as a traveler’s vaccine in different countries (37–39).

In contrast to leishmanial species causing CL, research on genetic modification in VL has been limited. However, recent years have seen a significant improvement in this scenario (Table 1). Though mostly focused at elucidating metabolic pathways, cellular processes, and host–parasite interactions; it has simultaneously led to the discovery of novel drug targets and vaccine candidates. The major pathways targeted were those that are unique to the parasite’s life cycle or metabolism, components sufficiently different from the homolog in hosts. Today, bio-informatic databases, proteomic screens (40), and reverse vaccinology, aid in the identification of novel vaccine candidates based on their expression stage, abundance, sub-cellular localization, sequence conservation in leishmanial species, non-homology to their human counterparts, trans-membrane helix predictions, and T-cell epitopic regions (12). Using the same genetically modified strain, research collaborations between labs working on different aspects of leishmaniasis can greatly speed up and enhance this search. Some of the most important pathways and their components, that have surfaced, are briefly discussed below.

Although a large proportion of currently licensed vaccines are based on inactivated or whole live attenuated organisms, the scope of LAV gets largely restricted due to safety issues. Foremost, is the risk of reversion to wild type or expression of compensatory genes. The Leishmania genome being highly plastic, this has a high probability. Additionally, critical consideration of the position of knock-outs, their effects on upstream and downstream genes, the restriction to manipulate only amastigote stage specific and single copy genes and availability of few selectable markers limits the potential targets and simultaneous multi-gene targeting, respectively (12). Furthermore, the retention of antibiotic resistance genes (20) and generation of cross resistance to anti-leishmanial drugs as in the case of neomycin to paromomycin is undesirable (83). Moreover, prior to human clinical trials, the cultivation of parasites in serum free media, their large-scale production, storage, validation of the best challenge methods-syringe or sandfly mediated, and many months of post challenge follow-up impose practical and as yet unresolved issues (84). In contrast, subunit and DNA vaccines are relatively safe and without these limitations. However, the low predictive power of available pre-clinical models to determine the human outcome of vaccination and the lack of knowledge of convincing markers to monitor their safety or efficacy remain common to all vaccination strategies (2).

The following road map may be considered a basic guideline while working with live attenuated vaccines. Preliminary phenotypic and genotypic screening of the parasites after each recombination event should be followed by vigorous in vitro studies on human cell lines. The parasites compartmentalization, proliferation, cellular responses, and activation markers should be closely monitored (85). After successful in vitro screening, the in vivo experiments in Golden Syrian hamsters and BALB/c mice models should be supported by those on chimeric humanized mice (25, 86). Continuous monitoring assays to test for reversion or attenuation retention by sensitive molecular biology techniques like PCR, microarrays should be done (87). Timely splenic biopsies for parasite load and multiparametric FACS analysis and ELISA for monitoring cytokine responses would help in elucidating the immune correlates of protection or disease development (6). Additionally, the comparison of these results among different groups, namely asymptomatic carriers, non-endemic healthy, endemic healthy, infected-cured, and infected individuals would greatly enhance our knowledge of disease pathogenesis. With the advent of modern imaging techniques, bioluminescent parasites can provide unsurpassable insight at each level of disease progression in real time (beginning from host cell–parasite interaction to dissemination and homing to various organs) also requiring lower number of animals to obtain statistically significant data (88). Lastly, human trials to provide proof of concept studies would strengthen our hypothesis derived from pre-clinical studies.

Parasite gene deletion mutants have helped in numerous pathway studies and elucidation of novel drug targets and vaccine candidates (Table 1). They also offer the possibility of co-administration with adjuvants or drugs to improve disease outcome. Moreover, vectored formulations in recombinant vaccinia (89), Lactobacillus (90), adenovirus, or Salmonella (91) carriers offer non-pathogenic and genetically modifiable alternatives for safe mucosal delivery, the major entry portal of pathogens. The concept of the flying vaccinator, genetically engineered blood-feeding insects to deliver vaccines to replace mosquito populations is a novel attempt tried in antimalarial programs and can be applied for sandfly eradication (92) too. Lastly, well-defined clinical trials with attenuated parasites will enhance the number of potential therapeutic targets, which are urgently needed to combat leishmaniasis.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.