One major group that has parasites of medical and veterinary importance is the Subclass Hematozoa of the phylum Apicomplexa. This subclass has 2 orders, one of which has the infamous intraerythrocytic genera - Plasmodium, Leucocytozoon and Hemoproteus, and the other has the Piroplasm genera Theileria and Babesia. (see my previous post to see the relationship between these)
Parasites and vectors - Two worlds collide
While Theileria is the more famous of the parasites in the Family Theileriidae, a new cousin parasite was identified in 1976 in domestic cats in the southern US. Cytauxzoon felis, whose definitive host is the Lynx rufus (Bobcat), causes an acute, lethal disease in Felis domesticus . Few survive. Fantastic reviews on the clinical manifestation are found here and here. Cytauxzoon is vector borne and carried by the lone star tick Amblyomma americanum. Transmission is indirect and prevention has relied on disrupting the life cycle at the tick stage. There have been no traditional vaccines so far, because the piroplasms cannot be grown in culture. It has been found previously that of the three life cycle stages of the parasite, viz., sporozoites, schizonts and merozoites, protective immunity is seen with the schizont stage. Cats that had survived the schizogenous phase of the disease were the rare ones that survived a challenge with the organism again. Merozoites do not protect against infection.
(Source : CDC ; http://www.cdc.gov/stari/disease/)
If we gotta start somewhere, I say here
In a recent article published in PLoS One in Oct 2013, Tarigo J. et al. have used the molecular genomics approach to expedite the identification of a new protein antigen to serve as a vaccine candidate. They started with blood from a cat that had died of C.felis. They isolated the parasitic merozoites from the erythrocytes (RBCs) and sequenced the 9.1 megabase genome using Roche's 454 pyrosequencing.
Next Generation Sequencing is all the rage these days. Sanger's first generation method is near-obsolete.But, you can still get a short product sequenced (using the method) for as low as $5. Roche's 454 pyrosequencing, Illumina's reversible terminator sequencing, ABI's SOLiD sequencing by ligation and Helicos' single molecule sequencing , which are/were the second gen, are being supplanted slowly even as I write by third generation technologies, viz., PacBio Real Time single molecule sequencing, Complete Genomics' combined proanchor hybridization and ligation, and Life Tech's Ion Torrent sequencing. (Hui, 2014).
Now, if you have never been a part of a genome sequencing project, you could be under the incorrect impression that the 454 sequencer spits out perfectly ordered nucleotide data that can be uploaded to GenBank and analysed at leisure. The reality is, however, disturbingly far from such an assumption. You typically get about a hundred thousand (or more) short 'reads' as output. Imagine someone cutting up the front page of a newspaper into individual alphabets and then asking you to reassemble the little pieces into coherent words, sentences, paragraphs and sections. You would think that the person was crazy because the task is near impossible manually. What if you were given a software that could do it for you? With reasonable error, the software could probably do it. Such software that handle genomic data are called assemblers. They put nucleotide data together into cogent contigs de novo, i.e. they form sentences in a new language that they do not know. Some of the most popular of these (for 454 and other platforms) are Newbler, Edena, EULER- SR, Gapcloser, Oases, Ray, SOAPdenovo, Vcake, velvet and wgs. For C.felis genome assembly, the authors used Newbler (Tarigo, 2013; Kumar, 2010)
To study genes that code proteins, transcribed messenger RNA is usually utilized to construct a cDNA library. Traditional cDNA library construction involves a polyT primer sequence and two PCR runs to make double stranded cDNA, followed by cloning into vectors. Improved methods have now appeared on the scene including Clontech's SMARTer PCR cDNA synthesis kit, which is a single step reverse transcription reaction using a 'Switching Mechanism At the 5' end of the RNA Template'. SMART, indeed! The cDNA library was then sequenced to generate ESTs or Expressed Sequence Tags using the 454 platform and Newbler. ESTs, in the newspaper analogy, would be the equivalent of the first and the last few words of a sentence. Since the complete genome was available, ESTs were used to map protein-coding regions back onto the full length genome using other software. In this paper, GeneMark-ES 2.5 was used. Other software that can do the same are Apollo, Augustus, CEGMA, ESTScan, Eval, FrameDP, Genethreader and PASA. These are basic annotation software that model gene structures onto genome data. After the first proteome was computed, a second predicted proteome was created using GlimmerHMM. The data was then used to run BLAST searches on NCBI, focusing specifically on the already deposited transcriptomes of Theileria parva, Babesia bovis and Plasmodium falciparum.
Basic Local Alignment Search Tool for proteins (BLASTp) was then used to predict shared genes. The authors accepted 60% similarities across at least 30% of the query sequence as matches. They identified the cf67 gene on the genome of C.felis, which is syntenic to p67/SPAG-1/BOV57 on Theileria parva. Going back to the newspaper analogy, syntenous genes are equivalent to words that occur at the same place in a sentence. For example, cf67 and p67 are like the fourth word, on a headline spanning ten words, from two different newspapers - they are located at the same spot but are not the same. In the case of cf67 and p67, the three genes/ words preceding them were conserved/the same.The gene was then directionally cloned into a plasmid vector, and the product was used to perform in vitro transcription and translation reactions. The His tagged translation products were purified using Ni-NTA magnetic agarose beads and their quality assessed using western blot analysis with anti-His antibodies. Another western blot was carried out with serum from cats that had been infected but had survived the infection. There was a high degree of immunoreactivity observed with the sera against the C terminal and the whole ORF clones of cf67. This makes cf67 a great candidate antigen for immunological studies.
The authors also found that cf67 is conserved in samples of C.felis from eight states in the southern US, with very little variation at the C terminal region. The cf67 was expressed in the schizogony stage. This was determined with insitu hybridization using a negative sense riboprobe directed against cytoplasmic mRNA in the schizont stage.Thus, the authors were able to identify a highly conserved sequence that was transcribed into an antigenic protein at the lifestage that could be targeted by the immune system to provide protective immunity. The heavy reliance on bioinformatics and in silico genomics is remarkable.
In a related review, Sirskyj et al., labelled such new innovative uses of bioinformatics for vaccine design as 'vaccinomics'. Conventional methods often miss proteins that are expressed at certain life cycle stages, but not at others, and so are potentially missed and overlooked as vaccine candidates. Software are available to predict immunodominant T and B cell epitopes. But despite the best predictions, field and clinical trials have not always been successful (Sirskyj,2011).
There is hope in the distance
Translational medicine techniques, to make the jump form epitope prediction to commercially viable vaccine, are still lacking across the veterinary and human medical and research fields. Although safety and efficacy are big concerns with conventional cell derived vaccines, they are not so with recombinant protein vaccines. Immune responses are slightly more controlled with the latter. The biggest concerns however arise from the degradation of the vaccine (either the insert or the vector) inside the body, lack of an effective strong immunity and delivery routes. These issues are more easily overcome in the veterinary field than in the human field because of sanctions on testing of vaccine candidates on animals. So far, the licensed recombinant vaccines available in the US for animals include the gI and gX deleted DIVA vaccine for pseudorabies in pigs and 11 poultry vaccines including fowlpox vectored vaccines for avian influenza, ND, Avian encephalomyelitis, ILT and Mycoplasma gallisepticum; Marek's disease virus vectored vaccine for ND, ILT deletion mutants of Salmonella typhimurium and deletion mutant of ILTV expressing HA from avian influenza.Vaccines for companion animals include canarypox vectored canines vaccines against Lyme disease, CDV and rabies; canary pox vectored feline vaccines against FeLV and rabies. Vaccines for horses include canary pox vectored vaccines against west nile virus and equine influenza. The newly identified vaccine candidate antigen for Cytauxzoon felis needs a lot of dedicated labwork ex silico before it can be made into a commercial vaccine for your pet. But in silico predictions are a great way to start.
References :
Tarigo JL, Scholl EH, McK Bird D, Brown CC, Cohn LA, Dean GA, Levy MG, Doolan DL, Trieu A, Nordone SK, Felgner PL, Vigil A, Birkenheuer AJ. A novel candidate vaccine for cytauxzoonosis inferred from comparative apicomplexan genomics. PLoS One. 2013 Aug 20;8(8):e71233
Kumar S, Blaxter ML. Comparing de novo assemblers for 454 transcriptome data. BMC Genomics. 2010 Oct 16;11:571.
Hui P. Next generation sequencing: chemistry, technology and applications. Top Curr Chem. 2014;336:1-18.
Sirskyj D, Diaz-Mitoma F, Golshani A, Kumar A, Azizi A. Innovative bioinformatic approaches for developing peptide-based vaccines against hypervariable viruses. Immunol Cell Biol. 2011 Jan;89(1):81-9.
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