Open Access

Immunological mechanisms underlying protection mediated by RTS,S: a review of the available data

Malaria Journal20098:312

https://doi.org/10.1186/1475-2875-8-312

Received: 7 October 2009

Accepted: 30 December 2009

Published: 30 December 2009

Abstract

The RTS,S/AS candidate malaria vaccine has demonstrated efficacy against a variety of endpoints in Phase IIa and Phase IIb trials over more than a decade. A multi-country phase III trial of RTS,S/AS01 is now underway with submission as early as 2012, if vaccine safety and efficacy are confirmed. The immunologic basis for how the vaccine protects against both infection and disease remains uncertain. It is, therefore, timely to review the information currently available about the vaccine with regard to how it impacts the human-Plasmodium falciparum host-pathogen relationship. In this article, what is known about mechanisms involved in partial protection against malaria induced by RTS,S is reviewed.

Background

Against a background of variably shifting malaria disease burden and a scale-up in the implementation of artemisinin-based combination therapy, long-lasting insecticidal nets and in some settings, indoor residual spraying, Plasmodium falciparum malaria remains the commonest cause of under-five mortality in several countries[1]. After four decades of malaria vaccine development, a pivotal phase III trial is underway of a vaccine which may be suitable for licensure and assessment for implementation in malaria-endemic countries. This vaccine, RTS,S/AS, is based on the hepatitis B surface antigen virus-like particle (VLP) platform, genetically-engineered to include the carboxy terminus (amino acids 207-395) of the P. falciparum circumsporozoite (CS) antigen[2]. The hybrid malaria-hepatitis B VLP is lyophilized and undergoes point-of-use reconstitution with GlaxoSmithKline's AS01 adjuvant, a mixture of liposomes, MPL and QS21[3]. RTS,S has demonstrated clinical efficacy against both infection and clinical malaria in several well-designed phase II field efficacy trials in both adults and children, replicated at several trial sites [47]. The considerations of generalizability of efficacy in different geographic and transmission settings, duration of efficacy and confirmation of efficacy against severe malaria are all to be addressed in the phase III trial[8]. A large database will also be available to provide information on safety of the novel adjuvant AS01E.

Here, the available evidence is re-assessed from clinical trials of the relationships between parasite biology, vaccine-induced immune responses and efficacy for circumsporozoite (CS) -based malaria vaccines.

Localization and functions of CS protein

What is known about the role of the CS protein in malaria parasite biology and pathogenesis has been reviewed previously[9, 10]. Initially identified as a Plasmodium berghei ortholog antigen Pb44, the CS protein[11] was shown to be the target of protective antibodies to the sporozoite surface in murine models over 25 years ago [1214]. CS covers the entire surface of sporozoites[15], the form of the malaria parasite inoculated into humans by female anopheline mosquitoes, and is found on the plasma membrane of liver-stage parasites, which develop after sporozoite invasion of hepatocytes. CS has been detected in the cytoplasm of infected hepatocytes and a recent report indicated that CS plays a role in suppression of liver-stage inflammatory responses in a P. berghei model[16]. CS is secreted at the apex of sporozoites, becomes an integral component of the plasma membrane and is continuously released in large amounts at the distal tip of the sporozoite during gliding motility[17, 18].

Many observations point to a region of CS as one of the key ligands for adherence to the heparan suphate proteoglycan components of the liver sinusoidal lining prior to hepatocyte invasion[10]. Incubation of live sporozoites in vitro with anti-CS antibodies induces a characteristic morphological change in sporozoite appearance with cessation of motility and shedding of sporozoite surface material. This change, dubbed the circumsporozoite precipitin reaction, was first reported with antibodies raised by irradiated sporozoite immunization[19, 20], and later with antibodies raised through immunization with only the conserved Asparagine-Alanine-Asparagine-Proline (NANP) amino acid repeat sequence which forms the immunodominant B-cell epitope from P. falciparum CS antigen[15]. This sequence is species-specific, but highly conserved for isolates from each species.

Clinical trial immunogenicity and efficacy

CS-based malaria vaccine development has progressed through iterations using clinical challenge model efficacy as a means of guiding improvements to vaccine design [2127]. The story of this iterative development in the late 1980s and 1990s, leading up to selection of RTS,S for field trials, is well documented including several review publications. Interested readers are referred to these reviews[2, 2830]. RTS,S/AS01 induces very high IgG concentrations in vaccinated humans to the NANP CS repeat. In addition, this vaccine induces moderate to high CD4+ Th1 responses against flanking region peptides[31].

Immune correlates of protection are known to exist for some vaccines and these permit licensure of new forms of these vaccines and extension of vaccine indications to new populations based on immunogenicity endpoints without a requirement to demonstrate vaccine efficacy (reviewed in [32]). In the case of malaria vaccines, there is no known link between immunogenicity and protection and, therefore, no accepted in vitro correlates of protection[33]. Moreover, the parasite is complex with multiple antigens that are potential targets of naturally acquired immunity. The CS antigen is not thought to be an important target of naturally acquired immunity by individuals repeatedly exposed to malaria-infected mosquitoes.

Analysis of association between immune responses and clinical efficacy have limited utility where the sample size is small, unless the relationship is simple and generalizable amongst vaccinees. Nevertheless, the available experimental human sporozoite challenge trial data for RTS,S with both AS01 and AS02 adjuvants is consistent with an important role of anti-NANP IgG in protection from infection. Each of the challenge studies were necessarily small, with generally too few protected individuals to usefully explore this relationship, in more than an indicative manner[25]. It should be noted that even life-long exposure to large numbers of malaria-infected mosquitoes rarely induces an anti-CS antibody response in excess of 10 μg/mL[34, 35] using a qualified ELISA where the capture antigen consists of NANP repeats, and in infants and children living under these conditions, the values rarely exceed 0.5 μg/mL[36].

It has not proved possible to derive a protective threshold for CS repeat IgG concentration, although, should a threshold exist, it probably lies above 20 μg/mL for most individuals: in 10 challenge trials of CS vaccines conducted between 1986 and 2001, only five of 108 volunteers with CS repeat IgG levels below 20 μg/ml were protected, whereas 14 of 27 volunteers with IgG levels above 20 μg/ml were protected[37]. A phase IIa trial of RTS,S/AS02 in 41 vaccinees reported a statistically significant increase in the seroconversion rate above this 20 μg/ml value for protected vaccinees compared to unprotected volunteers[37]. However, in the same trial there was no trend for protection with increasing IgG levels and receiver operator characteristic analyses did not identify a cut-off level for protection. In an analysis of 19 RTS,S/AS02 vaccinees, in a later phase IIa trial, those protected had higher CS repeat IgG levels than those unprotected[38]. None of the vaccinees with an IgG level below 20 μg/ml were protected; some vaccinees with levels above 20 μg/ml were not protected. In a further phase IIa trial of RTS,S/AS02 with 40 vaccinated volunteers, an analysis was performed dividing the vaccinees into three groups: those completely protected from infection, those with a delay in time to first detection of parasitaemia by microscopy, indicating partial protection, and those not protected[39]. In this analysis, the protected group (n = 16) had a geometric mean antibody concentration of 113.7 μg/ml. The equivalent figures for the partially protected (n = 14) and unprotected (n = 8) groups were 67.5 μg/ml and 29.6 μg/ml. These differences were statistically significant[19], and the absolute peak IgG concentrations induced in immunized protected volunteers are clearly very high. The largest Phase IIa trial of RTS,S confirmed the strong association between anti-CS IgG titre and protection against infection and demonstrated an independent, albeit weaker, association between CS- specific CD4+ T cell responses and protection[31]. This trial has not reported on potential threshold levels to date.

The first Phase IIb field efficacy trial, which involved 306 Gambian adults, reported 34% efficacy against the incidence rate of first blood stage infections over a 15-week period. In this study a linear relationship was found between IgG concentration post dose 3 and protection from blood stage infection, such that the odds ratio for a ten-fold increase in IgG concentration was 0.21 (p = 0.023). After correction for age and pre-vaccination titre the odds ratio was 0.27 (p = 0.07). There is some evidence of naturally acquired immunity to infection as detected by microscopy occurring in adolescence and adulthood. For example, in two adult vaccine efficacy trials with primary infection endpoints, the incidence of infection decreased with increasing age in the 18-45 year age range[4, 40] and a decrease in parasite prevalence over this age range is well documented in several studies from Kilifi, Kenya[41]. Given the very substantially greater data on naturally acquired immunity targeting the blood stages of the parasite compared to the pre-erythrocytic stages, it may be that this naturally acquired immunity to infection is in fact a gradual acquisition of the ability of anti-blood stage immunity to suppress blood stage infection to sub-microscopic parasite densities rather than sterilising pre-erythrocytic immunity. This muddies the water to some extent with regard to the question of whether vaccine or naturally acquired responses account for protection from infection in studies in older children and adults. Nevertheless, the best chance of detecting relationships between immune responses and protection for pre-erythrocytic vaccines is in field trials in endemic populations with similarly low pre-existing antibodies reflecting prior exposure to malaria, primary infection endpoints and, where the entomological inoculation rate is high, pre-treatment of volunteers prior to the efficacy follow-up period. Here the efficacy endpoint is as close as possible to a likely biological target of the immune response.

Questions remained as to whether this relationship between anti-CS IgG and protection against infection would hold in younger children or infants. It also remained to be seen whether a similar relationship might have been seen between IgG concentration and morbidity endpoints. This would introduce a further variable which is difficult to assess because there may not be a direct relationship between infection and disease: not all infections become clinically manifest, it may not be possible to link a specific clinical case to a specific infectious event, and some mild cases of clinical disease may not be detectable. It is likely that all cases of severe morbidity episodes are detected in field trials, but the still poorly understood heterogeneity in risk of malaria introduces major complexities in extrapolating from infection to morbidity at the individual level. Thus, a lack of association between immune responses and anti-morbidity efficacy would not necessarily be surprising. Furthermore exposure may not be uniform and this has been shown to make immunity harder to detect[42].

The largest Phase IIb field efficacy trial of RTS,S/AS02 to date reported data on 2,022 Mozambican children aged 1-4. In a commendable attempt to address the issue of efficacy against both infection and clinical disease, two separate cohorts were utilised. In one cohort (cohort 1), passive case detection only was performed, without pre-treatment, in order to assess efficacy against clinical disease. In cohort 2, children were pre-treated and active detection of infection was performed with regular cross-sectional blood sampling. Clinical malaria efficacy over 18 months after dose 3 in cohort 1 was 35.3%. There was an unexpectedly high rate of severe malaria disease detected during the study, allowing an estimation of vaccine efficacy against severe malaria of 48.6%[5, 6]. A recent paper reports for the first time on the association between anti-NANP IgG and infection efficacy in cohort 2 of this same trial[43, 44]. Again there is a statistically significant association between IgG concentration and efficacy against infection. A similar association was reported in an infant RTS,S Mozambican study [45]. In contrast two paediatric randomized controlled field trials have now reported a lack of association between the anti-NANP IgG concentration and protection against clinical disease; cohort 1 of the Mozambican study in children aged 1-4 and a trial conducted in Kenya and Tanzania in children aged 5-17 months[5, 7].

What can be deduced from the consistent pattern of associations seen for anti-NANP IgG and protection from infection with RTS,S, and the lack of association with morbidity to date? Efficacy against morbidity should be a natural extension of efficacy against infection in that by reducing the incidence of new infections, reducing the multiplicity of infection and a reduction in parasite density of breakthrough infections should logically be expected to interact with the expression of clinical malaria disease and the acquisition of naturally acquired immunity. One hypothesis proposed here is that this naturally acquired blood-stage immunity component which cannot be directly measured confounds analyses seeking to measure an association of immune responses against the sporozoite with morbidity endpoints which involve a completely different stage of the parasite that does not share protective epitopes with the CS protein.

It should be noted that little is known about the fine specificity or functional activity of anti-NANP IgG and their role in protection. Where it has been analysed, IgG1 and IgG2 account for "nearly all" of the total IgG concentration of anti-NANP antibodies[25], with IgG1 being "the dominant subclass"[37]. Sporozoite-opsonizing activity has been demonstrated in vitro, where monocytes have been shown to internalize and kill live sporozoites exposed to plasma from RTS,S-immunized protected vaccinees[46]. Further characterization of the associations between functional activities of anti-NANP IgG and protection is highly desirable and may inform refinements planned for future CS-based vaccine candidates.

Vaccines may protect either through complete protection of a proportion of vaccinees or through partial protection of all vaccinees (or a combination of the two)[47]. Certain characteristics of clinical efficacy data may point to one or the other mode of vaccine effect. The RTS,S challenge and field trial data to date are consistent with at least partial protection in most or all volunteers. It is possible that complete protection also occurs in some volunteers. It has been established that RTS,S/AS reduces the rate of new blood stage infections[5], reduces the initial inoculum of each blood stage infection[48] and reduces the multiplicity of infection[49] in vaccinees. Taken together these may also foster acquisition of naturally acquired immunity to malaria, whilst reducing the malaria morbidity in vaccinees. This mode of action has been called a "leaky vaccine". If RTS,S is indeed a leaky pre-erythrocytic malaria vaccine, this needs to be taken into account in interpreting associations of immune responses and efficacy, as partial protection from infection would be expected in most individuals. Recent advances in understanding of the skin stage of malaria, help us envisage how such partial protection could occur. When a mosquito probes for a blood meal, sporozoites are deposited intradermally and migrate for up to two hours before entering skin microvasculature or entering lymphatics[50]. However, there is a wide range of skin transit times before sporozoites enter the vasculature[51], with some sporozoites perhaps entering directly into vessels during mosquito probing. Anti-sporozoite antibodies have been shown to reduce the numbers of sporozoites which enter skin blood vessels to begin the journey to the liver[52].

What is the role of cell-mediated immune (CMI) responses in protection afforded by RTS,S? The published literature indicates that there is evidence that CMI has an important role when added to the foundation of robust IgG responses. CMI indicators were used as a down-selection criterion for adjuvant choice in the RTS,S programme[2]. Both CS-specific γ-interferon secreting CD4+ T cell responses (as enumerated by ex vivo ELSISPOT) and multifunctional CS-specific CD4+ T cells (defined as expressing two or more of γ-interferon, TNF, IL-2 and CD40 ligand using an intracellular cytokine staining assay) were greater in protected than in unprotected vaccinees in a recent RTS,S clinical challenge trial[31]. Multifunctional CD4+ T cell responses were reported not to be correlated with anti-NANP IgG responses. There are major limitations in what CMI studies are possible with blood volumes obtainable in paediatric trials, although some data on CMI responses to RTS,S is now available in African children[53]. Most RTS,S studies performing CMI studies have reported an absence of substantial CS-specific CD8+ T cell responses[31, 54]. Weak CS-specific CD8+ T cell responses were reported in 1 study with a highly sensitive ELISPOT assay performed on cultured cells[55].

CS-specific CMI and vaccine efficacy

RTS,S-induced CS-specific CD4+ T cell frequency, as enumerated by both ex vivo ELISPOT and intracellular cytokine staining, is associated with protection against infection[31]. However the available data indicates that IgG plays a more important role in RTS,S-mediated protection than CMI. The potential contribution of CD8+ T cells in killing of intracellular hepatocyte infection is unquestioned. This evidence stems from adoptive transfers and pre-clinical models of whole organism and subunit vaccine immunity [5658] with some indirect evidence from clinical studies[59, 60]. CD8 T cells are thought to be the critical determinant of irradiated sporozoite immunity, at least in mouse models where immune mechanisms can be dissected in detail[58]. There is, therefore, good reason to believe that induction of robust liver-stage specific CD8+ T cell responses in addition to RTS,S-induced IgG and CD4+ T cells, could add to currently achieved levels of clinical protection. There is also evidence for the important role of CD4+ Th1 responses from both pre-clinical adoptive transfer experiments[61] and field trials[62]. Furthermore protective CD8+ responses may be CD4+ T cell dependent[58]. Thus further improving upon currently attained CD4+ T cell magnitude may augment RTS,S-induced protection[63]. It is worth noting that modest CD4+ T cell ex vivo γ-interferon responses alone at an arithmetic mean of 129 spot forming cells/million PBMCs induced by DNA/modified vaccinia virus Ankara prime-boost delivery of P. falciparum CS were not associated with efficacy in a sporozoite challenge trial in the UK[64].

Conclusions

The available evidence about the protective mechanism of RTS,S/AS strongly supports a critical role for IgG against the CS repeat sequence in the protection seen against infection, whether in multiple clinical challenge trials in USA, adult or paediatric field trials in different age groups and across the distinct transmission settings of The Gambia, Kenya, Tanzania and Mozambique. Two conclusions follow from this fact. Firstly, future attempts at improvements of RTS,S-mediated protection should be rooted in at least matching the potent IgG response. Secondly, exploratory studies to shed some light on the fine specificity and protective mechanism of the IgG response are a high priority. These same data do not support identification to date of an absolute correlate of protection in the sense of a threshold level where complete protection is conferred at the individual level against a defined endpoint, but this lack of an absolute correlate does not change the above conclusions. The relationship between a protective immune response and reduction in risk of a defined malaria endpoint, be it infection or clinical disease, could perhaps be described graphically (see Figure 1). This type of representation of correlates of immunity has been performed for some other diseases[32] and it may be beneficial for this sort of analysis to be attempted for RTS,S-induced immune responses and malaria efficacy.
Figure 1

The relationship between a protective immune response and reduction in risk of a defined malaria endpoint. If the risk of a defined malaria endpoint (infection or disease) is inversely related to a vaccine-induced immune response, it may be possible to describe this relationship graphically. Three hypothetical relationships are shown above. The blue dashed line represents a relationship for which a clear threshold could be calculated using efficacy trial data, but in a biological system such a relationship is perhaps less likely than the dotted green and solid red lines.

There is supportive evidence, although weaker than that for the role of antibodies, that CS-specific CD4+ T cell responses are independently associated with protection against infection. A parsimonious interpretation is that such CD4+ T cell responses and IgG are additive in their protective effect for RTS,S. In some individuals a protective effect of moderate antibody concentrations may be complemented by a strong T cell response and vice versa. From this it follows that second generation vaccines that are able to match the protective B cell response seen in RTS,S vaccinees, but improve on the CMI aspect, would have a good chance of inducing higher efficacy. A valid hypothesis for vaccine approaches, which induce CMI responses without antibody induction, is whether spectacular CMI responses an order of magnitude higher than has been seen to date with CS-based vaccines could protect in the absence of antibodies. Though an interesting research question, this is a higher risk approach than dual induction of potent IgG and CMI.

Given the recent demonstration of partial efficacy in humans through prime-boost immunization with the ME-TRAP construct in the UK, two approaches appear highly worthy of attention from a technical perspective. These are matching the potent IgG response induced by RTS,S, and improving upon either the malaria-specific CD8+ or the CD4+ T cell responses.

Declarations

Acknowledgements

Authors alone are responsible for the views expressed in this publication and they do not necessarily represent the decisions or the stated policy of the World Health Organization. The authors thank Philip Bejon for his review and critical comments on the manuscript. Financial support to WHO Initiative for Vaccine Research from Fondazione Monte dei Paschi di Siena is acknowledged.

Authors’ Affiliations

(1)
Initiative for Vaccine Research, World Health Organization
(2)
Nuffield Department of Clinical Medicine, John Radcliffe Hospital, University of Oxford
(3)
Infectious Diseases Development, Global Health Division, Bill and Melinda Gates Foundation

References

  1. Greenwood BM, Fidock DA, Kyle DE, Kappe SH, Alonso PL, Collins FH, Duffy PE: Malaria: progress, perils, and prospects for eradication. J Clin Invest. 2008, 118: 1266-1276. 10.1172/JCI33996.PubMed CentralView ArticlePubMedGoogle Scholar
  2. Garcon N, Heppner DG, Cohen J: Development of RTS,S/AS02: a purified subunit-based malaria vaccine candidate formulated with a novel adjuvant. Expert Rev Vaccines. 2003, 2: 231-238. 10.1586/14760584.2.2.231.View ArticlePubMedGoogle Scholar
  3. Reed SG, Bertholet S, Coler RN, Friede M: New horizons in adjuvants for vaccine development. Trends Immunol. 2009, 30: 23-32. 10.1016/j.it.2008.09.006.View ArticlePubMedGoogle Scholar
  4. Bojang KA, Milligan PJ, Pinder M, Vigneron L, Alloueche A, Kester KE, Ballou WR, Conway DJ, Reece WH, Gothard P, Yamuah L, Delchambre M, Voss G, Greenwood BM, Hill A, McAdam KP, Tornieporth N, Cohen JD, Doherty T, RTS,S Malaria Vaccine Trial Team: Efficacy of RTS,S/AS02 malaria vaccine against Plasmodium falciparum infection in semi-immune adult men in The Gambia: a randomised trial. Lancet. 2001, 358: 1927-1934. 10.1016/S0140-6736(01)06957-4.View ArticlePubMedGoogle Scholar
  5. Alonso PL, Sacarlal J, Aponte JJ, Leach A, Macete E, Milman J, Mandomando I, Spiessens B, Guinovart C, Espasa M, Bassat Q, Aide P, Ofori-Anyinam O, Navia MM, Corachan S, Ceuppens M, Dubois MC, Demoitié MA, Dubovsky F, Menéndez C, Tornieporth N, Ballou WR, Thompson R, Cohen J: Efficacy of the RTS,S/AS02A vaccine against Plasmodium falciparum infection and disease in young African children: randomised controlled trial. Lancet. 2004, 364: 1411-1420. 10.1016/S0140-6736(04)17223-1.View ArticlePubMedGoogle Scholar
  6. Alonso PL, Sacarlal J, Aponte JJ, Leach A, Macete E, Aide P, Sigauque B, Milman J, Mandomando I, Bassat Q: Duration of protection with RTS,S/AS02A malaria vaccine in prevention of Plasmodium falciparum disease in Mozambican children: single-blind extended follow-up of a randomised controlled trial. Lancet. 2005, 366: 2012-2018. 10.1016/S0140-6736(05)67669-6.View ArticlePubMedGoogle Scholar
  7. Bejon P, Lusingu J, Olutu A: Efficacy of the RTS,S/AS01E vaccine against clinical episodes of falciparum malaria in 5 to 17 month old children in Kenya and Tanzania. NEJM. 2008, 359: 2521-2532. 10.1056/NEJMoa0807381.PubMed CentralView ArticlePubMedGoogle Scholar
  8. Moorthy V, Reed Z, Smith PG: Measurement of malaria vaccine efficacy in phase III trials: report of a WHO consultation. Vaccine. 2007, 25: 5115-5123. 10.1016/j.vaccine.2007.01.085.View ArticlePubMedGoogle Scholar
  9. Kappe SH, Buscaglia CA, Nussenzweig V: Plasmodium sporozoite molecular cell biology. Annu Rev Cell Dev Biol. 2004, 20: 29-59. 10.1146/annurev.cellbio.20.011603.150935.View ArticlePubMedGoogle Scholar
  10. Sinnis P, Nardin E: Sporozoite antigens: biology and immunology of the circumsporozoite protein and thrombospondin-related anonymous protein. Chem Immunol. 2002, 80: 70-96. full_text.View ArticlePubMedGoogle Scholar
  11. Nardin EH, Nussenzweig V, Nussenzweig RS, Collins WE, Harinasuta KT, Tapchaisri P, Chomcharn Y: Circumsporozoite proteins of human malaria parasites Plasmodium falciparum and Plasmodium vivax. J Exp Med. 1982, 156: 20-30. 10.1084/jem.156.1.20.View ArticlePubMedGoogle Scholar
  12. Aikawa M, Yoshida N, Nussenzweig RS, Nussenzweig V: The protective antigen of malarial sporozoites (Plasmodium berghei) is a differentiation antigen. J Immunol. 1981, 126: 2494-2495.PubMedGoogle Scholar
  13. Yoshida N, Nussenzweig RS, Potocnjak P, Nussenzweig V, Aikawa M: Hybridoma produces protective antibodies directed against the sporozoite stage of malaria parasite. Science. 1980, 207: 71-73. 10.1126/science.6985745.View ArticlePubMedGoogle Scholar
  14. Potocnjak P, Yoshida N, Nussenzweig RS, Nussenzweig V: Monovalent fragments (Fab) of monoclonal antibodies to a sporozoite surface antigen (Pb44) protect mice against malarial infection. J Exp Med. 1980, 151: 1504-1513. 10.1084/jem.151.6.1504.View ArticlePubMedGoogle Scholar
  15. Nussenzweig RS, Nussenzweig V: Antisporozoite vaccine for malaria: experimental basis and current status. Rev Infect Dis. 1989, 11 (Suppl 3): S579-585.View ArticlePubMedGoogle Scholar
  16. Singh AP, Buscaglia CA, Wang Q, Levay A, Nussenzweig DR, Walker JR, Winzeler EA, Fujii H, Fontoura BM, Nussenzweig V: Plasmodium circumsporozoite protein promotes the development of the liver stages of the parasite. Cell. 2007, 131: 492-504. 10.1016/j.cell.2007.09.013.View ArticlePubMedGoogle Scholar
  17. Stewart MJ, Vanderberg JP: Malaria sporozoites leave behind trails of circumsporozoite protein during gliding motility. J Protozool. 1988, 35: 389-393.View ArticlePubMedGoogle Scholar
  18. Stewart MJ, Vanderberg JP: Malaria sporozoites release circumsporozoite protein from their apical end and translocate it along their surface. J Protozool. 1991, 38: 411-421.View ArticlePubMedGoogle Scholar
  19. Gysin J, Barnwell J, Schlesinger DH, Nussenzweig V, Nussenzweig RS: Neutralization of the infectivity of sporozoites of Plasmodium knowlesi by antibodies to a synthetic peptide. J Exp Med. 1984, 160: 935-940. 10.1084/jem.160.3.935.View ArticlePubMedGoogle Scholar
  20. Stewart MJ, Nawrot RJ, Schulman S, Vanderberg JP: Plasmodium berghei sporozoite invasion is blocked in vitro by sporozoite-immobilizing antibodies. Infect Immun. 1986, 51: 859-864.PubMed CentralPubMedGoogle Scholar
  21. Ballou WR, Hoffman SL, Sherwood JA, Hollingdale MR, Neva FA, Hockmeyer WT, Gordon DM, Schneider I, Wirtz RA, Young JF: Safety and efficacy of a recombinant DNA Plasmodium falciparum sporozoite vaccine. Lancet. 1987, 1: 1277-1281.View ArticlePubMedGoogle Scholar
  22. Herrington DA, Clyde DF, Losonsky G, Cortesia M, Murphy JR, Davis J, Baqar S, Felix AM, Heimer EP, Gillessen D, Nardin E, Nussenzweig RS, Nussenzweig V, Hollingdale MR, Levine MM: Safety and immunogenicity in man of a synthetic peptide malaria vaccine against Plasmodium falciparum s porozoites. Nature. 1987, 328: 257-259. 10.1038/328257a0.View ArticlePubMedGoogle Scholar
  23. Hoffman SL, Edelman R, Bryan JP, Schneider I, Davis J, Sedegah M, Gordon D, Church P, Gross M, Silverman C, Hollingdale M, Clyde D, Sztein M, Losonsky G, aparello S, Jones TR: Safety, immunogenicity, and efficacy of a malaria sporozoite vaccine administered with monophosphoryl lipid A, cell wall skeleton of mycobacteria, and squalane as adjuvant. Am J Trop Med Hyg. 1994, 51: 603-612.PubMedGoogle Scholar
  24. Stoute JA, Kester KE, Krzych U, Wellde BT, Hall T, White K, Glenn G, Ockenhouse CF, Garcon N, Schwenk R, Lanar DE, Sun P, Momin P, Wirtz RA, Golenda C, Slaoui M, Wortmann G, Holland C, Dowler M, Cohen J, Ballou WR: Long-term efficacy and immune responses following immunization with the RTS,S malaria vaccine. J Infect Dis. 1998, 178: 1139-1144. 10.1086/515657.View ArticlePubMedGoogle Scholar
  25. Stoute JA, Slaoui M, Heppner DG, Momin P, Kester KE, Desmons P, Wellde BT, Garcon N, Krzych U, Marchand M: A preliminary evaluation of a recombinant circumsporozoite protein vaccine against Plasmodium falciparum malaria. RTS,S Malaria Vaccine Evaluation Group. NEJM. 1997, 336: 86-91. 10.1056/NEJM199701093360202.View ArticlePubMedGoogle Scholar
  26. Gordon DM, McGovern TW, Krzych U, Cohen JC, Schneider I, LaChance R, Heppner DG, Yuan G, Hollingdale M, Slaoui M, Hauser P, Voet P, Sadoff JC, Ballou WR: Safety, immunogenicity, and efficacy of a recombinantly produced Plasmodium falciparum circumsporozoite protein-hepatitis B surface antigen subunit vaccine. J Infect Dis. 1995, 171: 1576-1585.View ArticlePubMedGoogle Scholar
  27. Vreden SG, Verhave JP, Oettinger T, Sauerwein RW, Meuwissen JH: Phase I clinical trial of a recombinant malaria vaccine consisting of the circumsporozoite repeat region of Plasmodium falciparum coupled to hepatitis B surface antigen. Am J Trop Med Hyg. 1991, 45: 533-538.PubMedGoogle Scholar
  28. Heppner DG, Kester KE, Ockenhouse CF, Tornieporth N, Ofori O, Lyon JA, Stewart VA, Dubois P, Lanar DE, Krzych U, Moris P, Angov E, Cummings JF, Leach A, Hall BT, Dutta S, Schwenk R, Hillier C, Barbosa A, Ware LA, Nair L, Darko CA, Withers MR, Ogutu B, Polhemus ME, Fukuda M, Pichyangkul S, Gettyacamin M, Diggs C, Soisson L, Milman J, Dubois MC, Garçon N, Tucker K, Wittes J, Plowe CV, Thera MA, Duombo OK, Pau MG, Goudsmit J, Ballou WR, Cohen J: Towards an RTS,S-based, multi-stage, multi-antigen vaccine against falciparum malaria: progress at the Walter Reed Army Institute of Research. Vaccine. 2005, 23: 2243-2250. 10.1016/j.vaccine.2005.01.142.View ArticlePubMedGoogle Scholar
  29. Ballou WR, Cahill CP: Two decades of commitment to malaria vaccine development: GlaxoSmithKline Biologicals. Am J Trop Med Hyg. 2007, 77 (6 Suppl): 289-295.PubMedGoogle Scholar
  30. Ballou WR: The development of the RTS,S malaria vaccine candidate: challenges and lessons. Parasite Immunol. 2009, 31: 492-500. 10.1111/j.1365-3024.2009.01143.x.View ArticlePubMedGoogle Scholar
  31. Kester KE, Cummings JF, Ofori-Anyinam O, Ockenhouse CF, Krzych U, Moris P, Schwenk R, Nielsen RA, Debebe Z, Pinelis E, Juompan L, Williams J, Dowler M, Stewart VA, Wirtz RA, Dubois MC, Lievens M, Cohen J, Ballou WR, Heppner DG, RTS,S Vaccine Evaluation Group: Randomized, double-blind, phase 2a trial of falciparum malaria vaccines RTS,S/AS01B and RTS,S/AS02A in malaria-naive adults: safety, efficacy, and immunologic associates of protection. J Infect Dis. 2009, 200: 337-346. 10.1086/600120.View ArticlePubMedGoogle Scholar
  32. Plotkin SA: Vaccines: correlates of vaccine-induced immunity. Clin Infect Dis. 2008, 47: 401-409. 10.1086/589862.View ArticlePubMedGoogle Scholar
  33. Langhorne J, Ndungu FM, Sponaas AM, Marsh K: Immunity to malaria: more questions than answers. Nat Immunol. 2008, 9: 725-732. 10.1038/ni.f.205.View ArticlePubMedGoogle Scholar
  34. Polhemus ME, Remich SA, Ogutu BR, Waitumbi JN, Otieno L, Apollo S, Cummings JF, Kester KE, Ockenhouse CF, Stewart A, Ofori-Anyinam O, Ramboer I, Cahill CP, Lievens M, Dubois MC, Demoitie MA, Leach A, Cohen J, Ballou RW, Heppner GD: Evaluation of RTS,S/AS02A and RTS,S/AS01B in adults in a high malaria transmission area. PLoS One. 2009, 4: e6465-10.1371/journal.pone.0006465.PubMed CentralView ArticlePubMedGoogle Scholar
  35. Bojang K, Milligan P, Pinder M, Doherty T, Leach A, Ofori-Anyinam O, Lievens M, Kester K, Schaecher K, Ballou WR, Cohen J: Five-year safety and immunogenicity of GlaxoSmithKline's candidate malaria vaccine RTS,S/AS02 following administration to semi-immune adult men living in a malaria-endemic region of The Gambia. Hum Vaccin. 2009, 5: 242-247. 10.4161/hv.5.4.7050.PubMed CentralView ArticlePubMedGoogle Scholar
  36. Macete EV, Sacarlal J, Aponte JJ, Leach A, Navia MM, Milman J, Guinovart C, Mandomando I, Lopez-Pua Y, Lievens M, Owusu-Ofori A, Dubois M-C, Cahill CP, Koutsoukos M, Sillman M, Thompson R, Dubovsky F, Ballou WR, Cohen J, Alonso PL: Evaluation of two formulations of adjuvanted RTS,S malaria vaccine in children aged 3 to 5 years living in a malaria-endemic region of Mozambique: a Phase I/IIb randomized double-blind bridging trial. Trials. 2007, 8: 11-10.1186/1745-6215-8-11.PubMed CentralView ArticlePubMedGoogle Scholar
  37. Kester KE, McKinney DA, Tornieporth N, Ockenhouse CF, Heppner DG, Hall T, Krzych U, Delchambre M, Voss G, Dowler MG, Palensky J, Wittes J, Cohen J, Ballou WR, RTS,S Malaria Vaccine Evaluation Group: Efficacy of recombinant circumsporozoite protein vaccine regimens against experimental Plasmodium falciparum malaria. J Infect Dis. 2001, 183: 640-647. 10.1086/318534.View ArticlePubMedGoogle Scholar
  38. Kester KE, McKinney DA, Tornieporth N, Ockenhouse CF, Heppner DG, Hall T, Wellde BT, White K, Sun P, Schwenk R, Krzych U, Delchambre M, Voss G, Dubois MC, Gasser RA, Dowler MG, O'Brien M, Wittes J, Wirtz R, Cohen J, Ballou WR, RTS,S Malaria Vaccine Evaluation Group: A phase I/IIa safety, immunogenicity, and efficacy bridging randomized study of a two-dose regimen of liquid and lyophilized formulations of the candidate malaria vaccine RTS,S/AS02A in malaria-naive adults. Vaccine. 2007, 25: 5359-5366. 10.1016/j.vaccine.2007.05.005.View ArticlePubMedGoogle Scholar
  39. Kester KE, Cummings JF, Ockenhouse CF, Nielsen R, Hall BT, Gordon DM, Schwenk RJ, Krzych U, Holland CA, Richmond G, Dowler MG, Williams J, Wirtz RA, Tornieporth N, Vigneron L, Delchambre M, Demoitie MA, Ballou WR, Cohen J, Heppner DG, RTS,S Malaria Vaccine Evaluation Group: Phase 2a trial of 0, 1, and 3 month and 0, 7, and 28 day immunization schedules of malaria vaccine RTS,S/AS02 in malaria-naive adults at the Walter Reed Army Institute of Research. Vaccine. 2008, 26: 2191-2202. 10.1016/j.vaccine.2008.02.048.View ArticlePubMedGoogle Scholar
  40. Moorthy VS, Imoukhuede EB, Milligan P, Bojang K, Keating S, Kaye P, Pinder M, Gilbert SC, Walraven G, Greenwood BM, Hill AS: A randomised, double-blind, controlled vaccine efficacy trial of DNA/MVA ME-TRAP against malaria infection in Gambian adults. PLoS Med. 2004, 1: e33-10.1371/journal.pmed.0010033.PubMed CentralView ArticlePubMedGoogle Scholar
  41. Marsh K, Kinyanjui S: Immune effector mechanisms in malaria. Parasite Immunol. 2006, 28: 51-60. 10.1111/j.1365-3024.2006.00808.x.View ArticlePubMedGoogle Scholar
  42. Bejon P, Warimwe G, Mackintosh CL, Mackinnon MJ, Kinyanjui SM, Musyoki JN, Bull PC, Marsh K: Analysis of immunity to febrile malaria in children that distinguishes immunity from lack of exposure. Infect Immun. 2009, 77: 1917-1923. 10.1128/IAI.01358-08.PubMed CentralView ArticlePubMedGoogle Scholar
  43. Guinovart C, Aponte JJ, Sacarlal J, Aide P, Leach A, Bassat Q, Macete E, Dobano C, Lievens M, Loucq C, Ballou WR, Cohen J, Alonso PL: Insights into long-lasting protection induced by RTS,S/AS02A malaria vaccine: further results from a phase IIb trial in Mozambican children. PLoS One. 2009, 4: e5165-10.1371/journal.pone.0005165.PubMed CentralView ArticlePubMedGoogle Scholar
  44. Sacarlal J, Aide P, Aponte JJ, Renom M, Leach A, Mandomando I, Lievens M, Bassat Q, Lafuente S, Macete E, Vekemans J, Guinovart C, Sigaúque B, Sillman M, Milman J, Dubois MC, Demoitié MA, Thonnard J, Menéndez C, Ballou WR, Cohen J, Alonso PL: Long-term safety and efficacy of the RTS,S/AS02A malaria vaccine in Mozambican children. J Infect Dis. 2009, 200: 329-336. 10.1086/600119.View ArticlePubMedGoogle Scholar
  45. Aponte JJ, Aide P, Renom M, Mandomando I, Bassat Q, Sacarlal J, Manaca MN, Lafuente S, Barbosa A, Leach A, Lievens M, Vekemans J, Sigauque B, Dubois MC, Demoitié MA, Sillman M, Savarese B, McNeil JG, Macete E, Ballou WR, Cohen J, Alonso PL: Safety of the RTS,S/AS02D candidate malaria vaccine in infants living in a highly endemic area of Mozambique: a double blind randomised controlled phase I/IIb trial. Lancet. 2007, 370: 1543-1551. 10.1016/S0140-6736(07)61542-6.View ArticlePubMedGoogle Scholar
  46. Schwenk R, Asher LV, Chalom I, Lanar D, Sun P, White K, Keil D, Kester KE, Stoute J, Heppner DG, Krzych U: Opsonization by antigen-specific antibodies as a mechanism of protective immunity induced by Plasmodium falciparum circumsporozoite protein-based vaccine. Parasite Immunol. 2003, 25: 17-25. 10.1046/j.1365-3024.2003.00495.x.View ArticlePubMedGoogle Scholar
  47. Smith PG, Rodrigues LC, Fine PE: Assessment of the protective efficacy of vaccines against common diseases using case-control and cohort studies. Int J Epidemiol. 1984, 13: 87-93. 10.1093/ije/13.1.87.View ArticlePubMedGoogle Scholar
  48. Bejon P, Andrews L, Andersen RF, Dunachie S, Webster D, Walther M, Gilbert SC, Peto T, Hill AV: Calculation of liver-to-blood inocula, parasite growth rates, and preerythrocytic vaccine efficacy, from serial quantitative polymerase chain reaction studies of volunteers challenged with malaria sporozoites. J Infect Dis. 2005, 191: 619-626. 10.1086/427243.View ArticlePubMedGoogle Scholar
  49. Enosse S, Dobano C, Quelhas D, Aponte JJ, Lievens M, Leach A, Sacarlal J, Greenwood B, Milman J, Dubovsky F, Cohen J, Thompson R, Ballou WR, Alonso PL, Conway DJ, Sutherland CJ: RTS,S/AS02A malaria vaccine does not induce parasite CSP T cell epitope selection and reduces multiplicity of infection. PLoS Clin Trials. 2006, 1: e5-10.1371/journal.pctr.0010005.PubMed CentralView ArticlePubMedGoogle Scholar
  50. Amino R, Thiberge S, Martin B, Celli S, Shorte S, Frischknecht F, Menard R: Quantitative imaging of Plasmodium transmission from mosquito to mammal. Nat Med. 2006, 12: 220-224. 10.1038/nm1350.View ArticlePubMedGoogle Scholar
  51. Vanderberg JP, Frevert U: Intravital microscopy demonstrating antibody-mediated immobilisation of Plasmodium berghei sporozoites injected into skin by mosquitoes. Int J Parasitol. 2004, 34: 991-996. 10.1016/j.ijpara.2004.05.005.View ArticlePubMedGoogle Scholar
  52. Kebaier C, Voza T, Vanderberg J: Kinetics of mosquito-injected Plasmodium sporozoites in mice: fewer sporozoites are injected into sporozoite-immunized mice. PLoS Pathog. 2009, 5: e1000399-10.1371/journal.ppat.1000399.PubMed CentralView ArticlePubMedGoogle Scholar
  53. Barbosa A, Naniche D, Aponte JJ, Manaca MN, Mandomando I, Aide P, Sacarlal J, Renom M, Lafuente S, Ballou WR, Alonso PL: Plasmodium falciparum specific cellular immune responses after immunization with the RTS,S/AS02D candidate malaria vaccine in infants living in a highly endemic area of Mozambique. Infect Immun. 2009, 77: 4502-450. 10.1128/IAI.00442-09.PubMed CentralView ArticlePubMedGoogle Scholar
  54. Lalvani A, Moris P, Voss G, Pathan AA, Kester KE, Brookes R, Lee E, Koutsoukos M, Plebanski M, Delchambre M, Flanagan KL, Carton C, Slaoui M, Van Hoecke C, Ballou WR, Hill AV, Cohen J: Potent induction of focused Th1-type cellular and humoral immune responses by RTS,S/SBAS2, a recombinant Plasmodium falciparum malaria vaccine. J Infect Dis. 1999, 180: 1656-1664. 10.1086/315074.View ArticlePubMedGoogle Scholar
  55. Sun P, Schwenk R, White K, Stoute JA, Cohen J, Ballou WR, Voss G, Kester KE, Heppner DG, Krzych U: Protective immunity induced with malaria vaccine, RTS,S, is linked to Plasmodium falciparum circumsporozoite protein-specific CD4+ and CD8+ T cells producing IFN-gamma. J Immunol. 2003, 171: 6961-6967.View ArticlePubMedGoogle Scholar
  56. Romero P, Maryanski JL, Corradin G, Nussenzweig RS, Nussenzweig V, Zavala F: Cloned cytotoxic T cells recognize an epitope in the circumsporozoite protein and protect against malaria. Nature. 1989, 341 (6240): 323-326. 10.1038/341323a0.View ArticlePubMedGoogle Scholar
  57. Doolan DL, Hoffman SL: The complexity of protective immunity against liver-stage malaria. J Immunol. 2000, 165: 1453-1462.View ArticlePubMedGoogle Scholar
  58. Overstreet MG, Cockburn IA, Chen YC, Zavala F: Protective CD8 T cells against Plasmodium liver stages: immunobiology of an 'unnatural' immune response. Immunol Rev. 2008, 225: 272-283. 10.1111/j.1600-065X.2008.00671.x.PubMed CentralView ArticlePubMedGoogle Scholar
  59. Lalvani A, Hill AV: Cytotoxic T-lymphocytes against malaria and tuberculosis: from natural immunity to vaccine design. Clin Sci (Colch). 1998, 95: 531-538. 10.1042/CS19980201.View ArticleGoogle Scholar
  60. Hill AV, Allsopp CE, Kwiatkowski D, Anstey NM, Twumasi P, Rowe PA, Bennett S, Brewster D, McMichael AJ, Greenwood BM: Common west African HLA antigens are associated with protection from severe malaria. Nature. 1991, 352: 595-600. 10.1038/352595a0.View ArticlePubMedGoogle Scholar
  61. Tsuji M, Romero P, Nussenzweig RS, Zavala F: CD4+ cytolytic T cell clone confers protection against murine malaria. J Exp Med. 1990, 172 (5): 1353-1357. 10.1084/jem.172.5.1353.View ArticlePubMedGoogle Scholar
  62. Reece WH, Pinder M, Gothard PK, Milligan P, Bojang K, Doherty T, Plebanski M, Akinwunmi P, Everaere S, Watkins KR, Voss G, Tornieporth N, Alloueche A, Greenwood BM, Kester KE, McAdam KP, Cohen J, Hill AV: A CD4(+) T-cell immune response to a conserved epitope in the circumsporozoite protein correlates with protection from natural Plasmodium falciparum infection and disease. Nat Med. 2004, 10: 406-410. 10.1038/nm1009.View ArticlePubMedGoogle Scholar
  63. Stewart VA, McGrath SM, Dubois PM, Pau MG, Mettens P, Shott J, Cobb M, Burge JR, Larson D, Ware LA, Demoitie MA, Weverling GJ, Bayat B, Custers JH, Dubois MC, Cohen J, Goudsmit J, Heppner DG: Priming with an adenovirus 35-circumsporozoite protein (CS) vaccine followed by RTS,S/AS01B boosting significantly improves immunogenicity to Plasmodium falciparum CS compared to that with either malaria vaccine alone. Infect Immun. 2007, 75: 2283-2290. 10.1128/IAI.01879-06.PubMed CentralView ArticlePubMedGoogle Scholar
  64. Dunachie SJ, Walther M, Epstein JE, Keating S, Berthoud T, Andrews L, Andersen RF, Bejon P, Goonetilleke N, Poulton I, Webster DP, Butcher G, Watkins K, Sinden RE, Levine GL, Richie TL, Schneider J, Kaslow D, Gilbert SC, Carucci DJ, Hill AV: A DNA prime-modified vaccinia virus ankara boost vaccine encoding thrombospondin-related adhesion protein but not circumsporozoite protein partially protects healthy malaria-naive adults against Plasmodium falciparum sporozoite challenge. Infect Immun. 2006, 74: 5933-5942. 10.1128/IAI.00590-06.PubMed CentralView ArticlePubMedGoogle Scholar

Copyright

© Moorthy and Ballou; licensee BioMed Central Ltd. 2009

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.