DNA based vaccines should be able to reduce the problems we face in designing a comprehensive vaccine. DNA plasmid fragments encoding a protein can be expressed in host cells. Such proteins when expressed produce a Th1 cells mediated response, which is aimed at intracellular pathogens. But there is also evidence that these vaccines can produce a Th2 cells initiated humoral response resulting in production of antibodies. (100) In case of complex parasites like plasmodium, A CD8+ cell response against the expressed protein processed by MHC class II can produce immunity. This was proposed as a strategy to develop a DNA vaccine against malaria. (90) In case of intra-hepatocytic and intra-erythrocytic antigens in such a vaccine, the immune response initiated via MHC class I causes the cytotoxic T cell to cause the death and lysis of infected cells, but for the blood stage parasites a strong CD4+ T cell response is required to produce antibodies. And a strong antibody response has been shown to clear the asexual stage parasite from the experimental animals. So far the chimeric or peptide based vaccines have failed to produce a strong antibody response in the human volunteer trials. (91) We know that in order to remove the circulating parasites, antibody dependent immunity must be produced. These antibodies can trigger phagocytosis. Another possible mechanism can be of opsonization by FcγRII and subsequent killing by cytokines released by monocytes. The antibodies implicated in merozoite binding and phagocytosis are IgG3 and IgG1 type.
Whereas the IgG2 type antibodies are thought to be involved in cytotoxic response. (94)
All the MSPs yet discovered have immunogenic structures which can be of use in a multi-epitope vaccine. As opposed to conventional vaccines, DNA vaccine components will need modifications to improve their immune
responses. (99) The A+T rich composition of Plasmodium genome makes it difficult to express in mammalian cells. (102) Additionally, the protein product needs to be folded and presented to the immune system of the host in such a manner that both humoral and cellular arms can be manipulated sufficiently.
Immunization of mice with DNA vaccines encoding the C and N termini of Plasmodium spp. MSP-1 provided partial protection against sporozoite challenge and resulted in boosting of antibody titres after challenge. (80)
Another plasmid DNA vaccine encoding the C-terminal sequence of 42 k-Da fragment of MSP-1 was tested on monkeys. The results showed a high T cell response and when boosted by recombinant MSP-1 19k-Da protein the antibody levels were enhanced. Adding the sequence for an adjuvant in the plasmid also resulted in a better antibody response. (92) The experiments on non-human primates and their results show that DNA vaccine consisting of one or more sequences from antigens coupled with a boost component can be designed for human testing. Taking it step further, multi-antigen vaccines based on DNA sequences were tried with successful CD8+ cell response. (81)
MSP4/5 of P. chabaudi adami which is the murine homologue of P. falciparum MSP-4 and MSP-5 has been tested as a DNA vaccine on laboratory mice.
The data shows that although the antibody production is dependent of the route of administration and the vector used, but the vaccine confers immunity against lethal infection especially if administered in a prime/boost sequence.
(93)
The DNA vaccine technology offers a potentially affordable solution for mass production of malaria vaccines. It is the design of the vaccine and its components which need further research and investigation. At this point in time no single vaccine candidate can be relied upon for complete protection against the parasite and our understanding of the complexities of parasite antigenic structure has indicated that a vaccine has to be based on many epitopes from each stage specific protein. As for the blood stage epitopes are concerned, just one component will not be good enough. Experiments of a multi-epitope DNA vaccine on rhesus monkeys have confirmed that MSP-1 19 k-Da component does not produce protective immunity. Although memory
T-cell response was raised after repeated doses, the vaccine failed to stop the development of parasitemia. (98) This leads us to believe that in addition to MSP-1, more immunogenic segments of MSP class of proteins must be added in DNA vaccines to enhance their effectiveness.
A DNA vaccine consisting of sequences from seven different antigens from different stages of P. falciparum lifecycle was tested on human volunteers in 1998. The DNA construct was vectored in attenuated pox virus and administered to volunteers at different intervals. Just one of the volunteers developed immunity and the others showed immune responses of various degrees. The antibody response was not strong, but there was evidence of cell mediated immune response in all the volunteers. (105) This study has underlined the importance of a proper design for a malaria vaccine. The reasons why it failed to produce desired results are still not fully understood, but we know that a number of strategies can be employed to boost the effectiveness of such preparations.
Prime boost vaccine strategy has been tried for malaria vaccines with some very positive results. The problem of inadequate cell mediated immunity in general and humoral immunity in particular has been solved in other pathogens by the use of DNA vaccine in specially designed constructs and vectors. Vectors like fowlpox virus (FPV) and modified vaccinia virus Ankara strain (MVA) are ideal for DNA vaccine delivery. Still the DNA vaccines solely based on viral vectors are not always successful. The prime boost strategy consists of a “priming” dose of DNA vaccine which is boosted by the same or similar DNA construct via FPV or MVA. Results so far have shown excellent humoral and cellular response in case of Influenza virus and HIV in experimental animals. Similar results were obtained by prime boost vaccination in mice against sporozoite challenge. (97)
Until now, the trials conducted on humans have shown that multi-epitope DNA construct vaccines can delay the intrahepatic phase of the parasite by almost 48 hours. A vaccine plasmid containing TRAP (found on sporozoite surface) sequence and other immunogenic epitopes, was administered to human
volunteers followed by a boost by the same sequences through a different vector (MVA). The results obtained showed that hepatic stage to blood stage transition (parsitaemia) was delayed by up to 48 hours. (96) It is postulated that this time can be used to build up immunity against the released merozoites from liver by administering appropriate blood stage vaccines. As naturally acquired immunity in malaria endemic areas is against the blood stage antigens, we know that MSPs and other merozoite proteins will form the essential components of such vaccines. Only further experiments will tell whether this strategy is applicable or not, it may be the right direction for vaccine development for now.
Multi-Stage DNA-based Malaria vaccine Operation (MuStDO)
As the parasite has both intracellular and extracellular phases inside the human host, it is important that the multivalent vaccine induces both cell mediated and humoral immune responses. A collaborative effort called Multi-Stage DNA-based Malaria vaccine Operation (MuStDO) is underway. The MuStDO operation has proposed a multi-stage, multi-antigen malaria vaccine based on DNA Plasmid immunization technology. The proposed vaccine includes the sequences from sporozoite, merozoite and sexual stage antigens. The DNA based vaccine will be able to prime the naturally acquired immunity in populations. Apart from the liver stage components (CSP) the other major components are the MSPs. It is proposed that the 42 k-Da fragment of MSP-1, MSP-2, MSP-3 and MSP-5 should be included in the MuStDO vaccine and tested at phase I and II trials. (82)
The MuStDO programme will consist of two components. The first component of the vaccine is specifically aimed at hepatic (Sporozoite) stage of the plasmodium lifecycle. A multivalent DNA vaccine (MuStDO 5) will be designed to induce CD8+ T cell response against the intracellular liver stage of the lifecycle. There are a number of strong candidate antigens from P. falciparum sporozoites which have been shown to induce a good immune response in DNA vaccine form. (101) It is expected that this component of MuStDO vaccine
programme will successfully inhibit the release of infectious merozoites into the circulation. (82, 83)
A Comprehensive Blood Stage Vaccine and Suggested Improvements
The second component of the MuStDO programme will contain a number of blood stage antigens. The MSP antigens are aimed at producing the immunity to clear the parasitemia which will be much milder after the first phase of the vaccine. Other improvements suggested for MuStDO vaccine programme is addition of Codon optimization, use of adjuvants and Prime/Boost immunization. (82)
Codon optimization means that parasite genes are not transcribed fully because the DNA fragment from P. falciparum is rich in A+T sequences which is very different from the human genome sequence. To solve this problem mammalian codons were inserted in the plasmids containing MSP-1 (42 k-Da). The protein production in mice cell lines increased 10-100 folds and the vaccinated mice needed far less dosage of the vaccine to produce a better antibody response. (102) Similar technique can be employed in the design of human vaccines for better results.
The use of adjuvants in conjunction with DNA vaccine is a proven way of optimizing the immune response. Adjuvants can be chemical or genetic; i.e. a stimulatory gene. Genetic adjuvants are usually cytokine genes, which provide general immune stimulation and can also bias the immune response toward a Th1 or Th2 type. But it is debatable whether adjuvants like cytokine genes play any role in long term immunity. (103) It is suggested that the Cytokine adjuvants should be added to the vaccine to optimize their effect. (82)
A Th1 or Th2 bias can also be directed at different targets according to requirement.
The best enhancement in malaria vaccine protocol can be Prime/Boost immunization strategy. A multi-component plasmid based DNA vaccine containing both pre-erythrocyte and erythrocyte stage antigens was
administered in rhesus monkeys and it was boosted by the same genes inserted in attenuated vaccinia virus. Results showed that the immunized animals resolved their parasitemia while control subjects did not. A strong IFNγ response was measured in the immunized animals. (104) This showed that prime/boost regimen coupled with cytokine adjuvants can be tested on humans.
Other factors which may increase the efficacy of the vaccine are the route of administration and delivery system, modifications in the vector DNA backbone, modifications in the insert sequence and targeted delivery etc. (118)
The MuStDO vaccine programme will go through the trials and setting any hopes for a quick “miracle” vaccine is unrealistic. We have to bear in mind the fact that DNA vaccine technology is still in its infant stages. We have yet to see a successful gene based vaccine in humans. (99) A lot needs to be learnt about the safety and effectiveness of such preparations. With an anti-GM attitude among the educated public, we have to prove through time consuming testing that a malaria DNA vaccine if effective does not carry any side effects. Whether we can find a preventative vaccine breakthrough or not, we still have the option for working on therapeutic vaccines based on blood stage antigens.
Prospects of therapeutic malaria vaccines and MSPs:
GPI anchor motif is termed as the major malarial toxin involved in dramatic manifestation of the symptoms which include pyrexia, metabolic acidosis, hypoglycaemia, seizures, comma and cerebral oedema etc. (59, 106) The toxic effect of the GPI moiety has been discussed above. We also know that a number of MSPs carry GPI anchors as their integral structural domains. As MSPs are expressed during the blood stage of the parasite life cycle, the GPI domains exhibit their toxicity at this point. The toxicity leads to release of pro-inflammatory cytokines (59), leading to a cascade of events which contribute to the overall clinical picture of malaria. A vaccine targeting the GPI anchor would lead to suppressing of its pathological activity. A vaccine based on synthetic peptide structures derived from the GPI motif was tested on rodent
malaria model. The vaccine substantially reduced malarial acidosis, pulmonary oedema, cerebral syndrome and fatality in the subjects (107). The antibodies produced against the GPI anchor motifs are thought to block the binding of the GPI glycans to cell surface receptors. The receptors, not classified yet, may be the start point for cytokine production events. (106) Data collected from malaria endemic areas confirms that anti-GPI antibodies are present in immune individuals. In addition to this, the asymptomatic individuals show high antibody levels against GPI protein. (113) A GPI anchor vaccine, either DNA or peptide based, can bring us closer to eradicating the disease in near future.
Emergence of more virulent strains: A possible hazard.
A challenge that scientist will have to face is to develop a comprehensive vaccine against the blood stage of the parasite. As such vaccines will be aimed at blocking the toxic effects and growth rate of the parasite, thus providing a selection pressure which will result in evolution of a much more virulent parasite population. As parasites have been evolving under the selection pressure as a result of host immune response, they are capable of maintaining the populations even in the presence of incomplete vaccines. This was proven in a vaccine trial study done in Papua New Guinea in 2002. A combined vaccine consisting of peptides from MSP-1 and MSP-2 was administered to human volunteers. Although the infection by the vaccine allelic forms of parasites was markedly reduced in the vaccinated individuals, the morbidity level was the same owing to the infection by the other allelic form. (70) Keeping in mind the immense nature of antigen diversity and polymorphisms, designing a vaccine of such quality will not be easy. If compared to the projected effect of a vaccine against the pathogen infection, which will not result in increased virulence, we have to be very careful and precise in our approach towards designs, clinical trials and administration of blood stage vaccines. (50) So an ideal blood stage vaccine should include the peptides representative of all the major allelic forms during the entire blood stage of the parasite. (70, 117) But with the available knowledge a safe approach
would be to design a vaccine with both transmission blocking and infection blocking components.
Further research in proteomics and recent advances in the area of recombinant vaccine technology has opened up many options, some of which can be of particular use in developing an anti malarial vaccine. Improved expression of parasite antigens in the cell lines and other expression vectors by use of high quality synthetic techniques (109) means that we are exploring the antigens related to immunity in greater detail. The odds of a malaria vaccine causing increased virulence should be minimum in this post-genomic age.
Conclusion:
The functional studies on MSPs need further improvements. We know that they play an active role in the invasion of erythrocytes. Gradually a vague picture is emerging giving us hints of the structural and functional correlation between MSPs and the molecules of human host cells. New drug targets are being identified which can offer us more effective treatment in coming years.
(116)
The role of merozoite surface proteins in vaccine development is of primary significance. The experiments and trials so far conducted have shown that neither sporozoite antigen based vaccines nor sexual stage antigen based vaccines alone can give complete immunity against malarial infection. The blood stage of the life cycle of parasite is where the parasite can be most vulnerable to an immune attack for a number of reasons; a) owing to the immunogenic structures present on its surface b) The continued exposure of these antigens to the immune cells c) the ability of the host to ward off the challenge in endemic areas due to the immune clearance of the blood stage parasite d) the ability of the natural immune response to boost itself after consecutive infections. Keeping the above factors in mind, we can manipulate the antigen structures and genetic sequences in-vitro to generate more immunogenic vaccine components. The antigens from other stages of the lifecycle can be employed to reinforce the whole vaccine regimen. It seems
very likely that within the next few years we will have transmission (host to vector) blocking (115) and infection (vector to host to blood) blocking vaccines
(96) available. There are a number of other candidate antigens from the merozoite stage which can be useful in a multi-epitope design of a blood stage vaccine. MSPs will be the primary components of such vaccine, but we will have to surmount the challenges of incomplete or inadequate immune responses, parasite evolution and polymorphisms and appropriate vaccine delivery in a huge population of prospective recipients.
References:
1) WHO annual report. 2002. http://www.who.int/infectious-disease-report/2002/introduction.html
2) Langreth, S G; Jensen, J B; Reese, R T; Trager, W. 1978. Fine structure of human malaria in vitro. The Journal Of Protozoology 25(4): 443-452
3) L. H. Bannister, J. M. Hopkins, R. E. Fowler, S. Krishna and G. H.
Mitchell. 2003. A Brief Illustrated Guide to the Ultrastructure of Plasmodium falciparum Asexual Blood Stages. The Journal of Experimental Biology 206:
3789-3802
4) A.A. Holder. 1994. Proteins on the surface of malaria parasites and cell invasion. Parasitology 108: S5–S18
5) V.M. Marshall, A. Silva, M. Foley et al. 1997 A second merozoite surface protein (MSP-4) of Plasmodium falciparum that contains an epidermal growth factor-like domain. Infect. Immun. 65(11): 4460–4467
6) Anders RF. Brown GV. Coppel RL. Stahl HD. Bianco AE. Favaloro JM.
Crewther PE. Culvenor JG. Kemp DJ. 1985. Potential vaccine antigens of the asexual blood-stages of Plasmodium falciparum. Developments in Biological Standardization. 62:81-9
7) Ballou WR. Rothbard J. Wirtz RA. Gordon DM. Williams JS. Gore RW.
Schneider I. Hollingdale MR. Beaudoin RL. Maloy WL. Et al. 1985.
Immunogenicity of synthetic peptides from circumsporozoite protein of Plasmodium falciparum. Science. 228(4702):996-9.
8) Moelans II. Klaassen CH. Kaslow DC. Konings RN. Schoenmakers JG.
1991. Minimal variation in Pfs16, a novel protein located in the membrane of
gametes and sporozoites of Plasmodium falciparum. Molecular & Biochemical Parasitology. 46(2):311-3
9) Gruner AC. Snounou G. Brahimi K. Letourneur F. Renia L. Druilhe P.
2003. Pre-erythrocytic antigens of Plasmodium falciparum: from rags to riches? Trends in Parasitology. 19(2):74-8
10) Vanderberg, J.P. and Frevert, U. 2004. Intravital microscopy demonstrating antibody-mediated immobilization of Plasmodium berghei sporozoites injected into skin by mosquitoes. International Journal for Parasitology, 34(9): 991-996
11) G. Pradel and U. Frevert. 2001. Plasmodium sporozoites actively enter and passage through Kupffer cells prior to hepatocyte invasion. Hepatology 33:
1154–1165
12) Ute Frevert. 2004. Sneaking in through the back entrance: the biology of malaria liver stages. Trends in Parasitology. 20(9): 417-424
13) Pasvol G. 2003. How many pathways for invasion of the red blood cell by the malaria parasite? Trends in Parasitology. 19(10):430-2. located in an electron lucent compartment in the neck of the rhoptries. J.
Eukaryot. Microbiol. 42: 224–231
16) Peter Preiser, Mallika Kaviratne, Shahid Khan, Lawrence Bannister and William Jarra. 2000. The apical organelles of malaria merozoites: host cell selection, invasion, host immunity and immune evasion.
Microbes and Infection. 2 (12): 1461-1477
17) M.J. Gardner, N. Hall, E. Fung, O. White, M. Berriman, R.W. Hyman, J.M.
Carlton, A. Pain, K.E. Nelson and S. Bowman et al. 2002. Genome sequence of the human malaria parasite Plasmodium falciparum, Nature 419:
498–511
18) Z. Bozdech, M. Llinas, B.L. Pulliam, E.D. Wong, J. Zhu and J.L. DeRisi.
2003. The Transcriptome of the Intraerythrocytic Developmental Cycle of Plasmodium falciparum. PLoS Biol 1: E5
19) Holder AA, Freeman RR. 1984. The three major antigens on the surface of Plasmodium falciparum merozoites are derived from a single high molecular weight precursor. J Exp Med. 160(2):624-9
19) Holder AA, Freeman RR. 1984. The three major antigens on the surface of Plasmodium falciparum merozoites are derived from a single high molecular weight precursor. J Exp Med. 160(2):624-9