Clinically protective malaria vaccines consistently fail to protect adults and children in endemic settings, and at best only partially protect infants.
We identify and evaluate 1916 immunization studies between 1965-February 2010, and exclude partially or nonprotective results to find 177 completely protective immunization experiments. Detailed reexamination reveals an unexpectedly mundane basis for selective vaccine failure: live malaria parasites in the skin inhibit vaccine function. We next show published molecular and cellular data support a testable, novel model where parasite-host interactions in the skin induce malaria-specific regulatory T cells, and subvert early antigen-specific immunity to parasite-specific immunotolerance. This ensures infection and tolerance to reinfection. Exposure to Plasmodium-infected mosquito bites therefore systematically triggers immunosuppression of endemic vaccine-elicited responses. The extensive vaccine trial data solidly substantiate this model experimentally.
We conclude skinstage-initiated immunosuppression, unassociated with bloodstage parasites, systematically blocks vaccine function in the field. Our model exposes novel molecular and procedural strategies to significantly and quickly increase protective efficacy in both pipeline and currently ineffective malaria vaccines, and forces fundamental reassessment of central precepts determining vaccine development. This has major implications for accelerated local eliminations of malaria, and significantly increases potential for eradication.
Citation: Guilbride DL, Gawlinski P, Guilbride PDL (2010) Why Functional Pre-Erythrocytic and Bloodstage Malaria Vaccines Fail: A Meta-Analysis of Fully Protective Immunizations and Novel Immunological Model. PLoS ONE 5(5): e10685. doi:10.1371/journal.pone.0010685
Editor: Mauricio Martins Rodrigues, Federal University of São Paulo, Brazil
Received: November 9, 2009; Accepted: April 16, 2010; Published: May 19, 2010
Copyright: © 2010 Guilbride et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The Malaria Research Foundation (MRF, P.O. Box 10420, CO 81612 USA) supported this research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The malaria vaccine paradoxes
A solitary subunit vaccine marginally , , , , ,  protects children in endemic areas ,  against malaria, and only partially protects infants , , , similarly to malaria-naïve adults , . Adults in endemic areas remain unprotected , . These data crystallize the paradoxes central to 80 years of malaria vaccine research. Endemic populations display T cell , , , , , , ,  and antibody , , , , ,  responses to all malaria lifecycle stages and rapidly acquire immunity to bloodstage parasites, mitigating adult disease and death , , , , . Immunity to earlier skinstage parasites however does not develop, and endemic populations remain tolerant to continual reinfection , ,  remaining at risk for severe malaria should immunocompetence weaken. Similarly, potentially protective , , , ,  T cell responses elicited by diverse attenuated-parasite , , , , , , , , , , , , ,  and subunit , , , , , , , , , , , , , , , , ,  malaria vaccines, and laboratory infections , , , , , , , , , ,  provide sterile immunity , , , ,  to infection, yet are ineffective in endemic populations , , , , ,  and are effectively blocked by the parasite . Ostensibly, protection is blocked only in endemic areas, implying a conditional difference between laboratory and field infections which systematically triggers an immunological block to vaccine function in the field. This rationale pinpoints an activatable immune mechanism which blocks existing T cell responses. Obvious candidates are the normal immune mechanisms suppressing autoimmunity and allergy, or self- and nonself-tolerance. These mechanisms centre largely around suppressive function of regulatory T cell subsets (Tregs) , . Activated natural (nTreg) and induced (iTreg) Tregs suppress effector T cell ,  and B cell responses , , , ,  and tolerize dendritic cells (DC)  to maintain self-tolerance and regulate inflammatory responses to injury , tissue grafts , , , pathogens and allergens , , .
A major regulatory immune organ, rich in regulatory T cells , , is human skin. This suggests the initial path of malarial infection will profoundly affect systemic host responses to subsequent lifecycle stages. Most experimental infections bypass the skin entirely. Natural malaria infection however, starts in the skin , , , , . Infected mosquitoes inject motile  skinstage (sporozoite) parasites; within minutes, a few migrate to proximal lymphatic vessels and skin-draining lymph nodes (LN) . Another few  invade blood vessels, rapidly , ,  migrating to the liver; the remainder linger in the skin . Liver invading parasites differentiate  and multiply (for days) asymptomatically  until bloodstage-filled vesicles bleb ,  into the blood, causing the systemic inflammatory reaction , immunologically similar to sepsis , underlying initial symptoms of clinical malaria. We compile the immunobiology of Anopheline mosquito bites and human skin, with the molecular and behavioural characteristics of skinstage parasites, to show that natural infection leads inevitably to systemic immunotolerance. We further show, via comprehensive meta-analysis of fully protective vaccine trials, that bypassing or disrupting natural parasite-host immune interactions in the skin profoundly affects host responses to vaccine antigens, and leads to protective immunization against malaria.
Literature Searches for completely protective vaccine trials, for meta-analysis
Searches were performed following MOOSE (Meta-analysis of Observational Studies in Epidemiology) guidelines for conduct and PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analysis) reporting protocol for Systematic Reviews and Meta-analysis . Studies showing complete protection to malaria challenge in mammals were identified by searching the PubMed database (www.ncbi.nlm.nih.gov/pubmed/) with search terms: malaria OR plasmodium & (complete &) protect* OR immuni* OR vaccine OR human/man/chimpanzee/monkey/mouse/rat/rabbit/dog/goat/OR
Snounou/Tartz/Tsuji/Urban/Vanderberg/Vaughan/Weiss/Weidanz/White/Yoshida/Zavala/&/ORyear (1965 – 2009). Searches were performed by adding two or three qualifiers at a time to the basic search string: [“malaria” OR “plasmodium” AND protect*” OR “immuni*”], and rerunning the search each time. We ran searches without, then with, qualifier term “complete” to allow wide retrieval sensitivity. Retrieved records were combined and replicates removed. Compiled single records were then screened by search term and abstract perusal for traveller, bednet, thalassemia, genetic, pharmacokinetic, drug intervention, insecticide, repellent or mosquito physiology content without reinfection follow-up, and excluded; we also contacted authors to verify conditions where necessary. This search strategy, (outlined in Figure 1), provided meticulous coverage, as determined by random spot-checks for coverage. Last complete database searches were carried out 9–13 September 2009; final update searches were run 13 February 2010. We also examined cited reference lists in studies and reviews identified. Late-breaking studies were manually added to this final database.
Selection of studies for meta-analysis data–Inclusion/exclusion criteria
Inclusion criterion: Identified full-text immunization studies were then checked individually by full-text inspection for experimental immunization and challenge conditions, and results of challenge, and sorted with regard to complete protection criteria (defined below). Immunization studies showing complete protection of all immunized subjects and infectivity in all control infections (in any part of the study), were included in a final dataset for analysis (Defined in Figure 1; listed in Table S1). No other inclusion criterion was used. Studies in English, French, Spanish, Portuguese and German were assessed, and sufficient discriminatory data was available in English abstracts, for most studies identified published in further languages. Studies in Oriental languages without abstract (5) were not evaluated. Included studies were further sorted for analysis according to experimental data as described below in Validity and Sorting Criteria. Exclusion criteria: Studies showing incomplete protection in immunized subjects, or incomplete control infectivity, were excluded. No other exclusion criterion was used on the immunization studies identified by the search method described.
Validity Assessment and Sorting Criteria
Complete protection criteria: bloodstage parasites undetectable after challenge in all immunized subjects AND 100% of non-immunized controls became parasitemic. Experiments documenting complete protection were further sorted into 8 categories, (a–h), defined first by route and method of immunization, and then route of challenge. Experimental data publications are listed by category in Supplementary Table S1. The route and conditions defining immunization in each category are represented graphically in Figure 2 (and in written form in Supplementary Table S2) on green background, and explained in the legends to Figure 2 and Supplementary Table S2. The route of challenge, either intravenous or via moquito bite, is indicated graphically on lilac background, (in written form in Table S2) and explained in legends for Figure 2 and Table S2.
A. Exposure to parasites in the skin coincides closely with vaccine failure. Green background- immunization procedures. Lilac background- challenge procedures and percent of total experiments showing complete protection (% total) formed by a subset of studies (category) using a given experimental procedure (categories a-i; supporting data in references listed below). Inclined syringe- administration route is intravenous (i.v.) for live parasites, or, the method does not involve live parasites, but uses dead parasites or purified antigen, antibody, or recombinant DNA (dead/Ag/Ab/subunit) and therefore bypasses parasite interactions with host skin. Multiple mosquitoes- live parasites administered by multiple simultaneous mosquito bites. Single mosquito- live parasites naturally transmitted by 4–15 bites. Mosquito in aromatic ring- live parasites administered by 12–15 bites/session with chloroquine. Vertical syringe- live parasites delivered subcutaneously (s.c) or intradermally (i.d.) or intramuscularly (i.m.); (i), uncontrolled exposure to endemic mosquitos. B. Protective immunization physically bypasses the skin at either immunization or challenge in 90% of cases (a,b,c,e,h). Protective immunization which transits skin during immunization (c,d,e,f,g,h) either: bypasses the skin physically at challenge (c,e,h); or, involves skin immunomodulation during immunization (d,g, 10% of cases). Within A and B: Red lines and numbers- experiment bypasses parasite-skin interactions at stage indicated by red lines; black lines and numbers- parasites interact with skin at stage indicated by black lines. Asterisk (*)- immunization via unmodified skin, limited to less-virulent P. berghei (f). Skin bypass- method physically avoids live parasite interactions in the host skin. Malaria exposure- skin exposure to infected mosquito bite before first immunization; naïve- no pre-exposure; exposed (endemic)- chronic exposure. x- this study shows complete protection of 40 of 41 mice challenged. y: one person in one study  was infected one time via the skin prior to protective immunization and was therefore moderately tolerized. Data pertaining to experimental categories (a–h): a:, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,, , , , , , , , , , , , , , , , , , , , , , ,, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , . b: , , , , , , , , , . c: . d: , , , , , , , , , , , , , , , . e: , , . f: . g: . h: , , , , . Studies containing data for multiple relevant experimental conditions are referenced accordingly in each appropriate category. Multiple experiments contributed by a single study are indicated beneath study reference number (eg. reference 124 X2) in Supplementary Table S1. (Meta-analysis data extended reference list).
Final database searches as described were performed separately twice during a three month period (June-September 2009), and once in February 2010, by the same person, and a further search performed once by a second person. Four separate results databases were combined with longterm manual search archives from over 4 years, and replicates removed. Assessment of compliance with complete protection criteria for the final dataset was reconfirmed in all cases by one person. From this final dataset, by full-text inspection, we retrieved specific experimental data regarding method and route of immunization, and method and route of challenge.
We measured the percentages of our dataset of fully protective vaccination experiments, that involved either absence, or presence, of live parasite interaction within the skin, during immunization, during challenge, or during both.
1. Selection of studies for vaccine trial meta-analysis
Literature search, final selection strategy and data retrieval were designed to return data completely unlinked to and irrespective of any criterion beyond complete protection. Searches targeted any immunization, in any mammal, generating a comprehensive database of immunizations from 1965-February 2010. Random spot-checks confirmed meticulous coverage. Search method, inclusion/exclusion, assessment and sorting criteria are fully described in the Methods section. The probability of missing any important immunization study is therefore finite but exceedingly small. The probability of missing an entire class of vaccines is nil. Literature searches and resulting datasets of factors contributing to protective vaccine function were therefore comprehensive, and completely unfocused and unbiased with regard to parasite life-cycle stage, antigen character, proposed immune mechanism, mode of application or any other criterion beyond complete protection. Data for analysis (specific experimental data regarding method and route of immunization, and method and route of challenge) was included according to a rigorous definition of complete protection.
We identify 1916 immunization studies performed worldwide between 1965 and February 2010 (Fig. 1), and retrieve an unambiguous, comprehensive and unbiased data set of 177 experiments in 161 publications documenting completely protective immunization, versus 1627 nonprotective or partially protective immunization results (Fig. 1).
2. Vaccine trial data implicates the skin in vaccine failure
2-i. Meta-analysis shows complete protection against natural challenge, by immunization via unmodified skin, does not occur.
Completely protective vaccinations (177 experiments, Fig. 2; see also Supplementary Table S2, and Fig. 1) employing diverse vaccines, fall into 8 categories (Fig. 2a–h) according to method, and routes of immunization and challenge. These reveal that, unlike endemic populations (Fig. 2i), all protected subjects (Fig. 2a–h, with verifiable exception of one individual ) are malaria-naïve (unexposed to malaria-infected mosquitoes) prior to first immunization (Fig. 2A). Most protective vaccinations also physically bypass live parasite interactions in the skin at either immunization or challenge (90%, Fig. 2B, a,b,c,e,h). Bypass is either by intravenous (i.v.) injection, or use of dead parasites, purified antibody or protein, or recombinant DNA, at immunization (84%, Fig. 2a,b), or i.v. injection at challenge (84%, Fig. 2a,c,e,h), usually by i.v. injection at both (78.5% Fig. 2a). Skin-based immunization (via infected mosquito bite), completely protective against virulent natural challenge (Fig. 2d,g) in humans , , , , ,  and mice  occurs only under conditions significantly altering the immune context in the skin during immunization. These immunomodulary conditions are illuminating and are examined in detail in Sections 3 and 4, and entirely account for the remaining 10% of completely protective immunization.
2-ii. Partially and non-protective studies strongly suggest parasite skin interactions actively depress host immunity.
Otherwise, in mice, live-parasite immunization “via the skin” (this includes subcutaneous injection (s.c.) which bypasses, but inevitably contaminates, the skin) is usually non-protective , . Where documented, skin-based immunization provides complete  (Fig. 2f) or substantial , ,  immunity only against less infective Plasmodium berghei natural challenge, but not against ~100-fold more infective  P. yoelii natural challenge , . Intravenous immunization with P. yoelii protects completely against intravenous challenge , , but mosquito delivered , , or high-dose injected intradermal  immunization does not. This suggests skin-immunization generates immunity less efficiently , , or increases intrinsic parasite infectivity , , .
However, after identical intravenous infective challenge (bypassing the skin) , diminished immunity with high dose skin immunization (compared to intravenous)  must derive from deficient host responses (activated less efficiently, or actively depressed, or both), not parasite-intrinsic changes.
Therefore, since skin immunization with less infective  P. berghei protects completely against intravenous infection (20,000–50,000 parasites) , , but not against a 20–200X lesser skin challenge (10 bites) , (roughly 250–1000 parasites , ) the data argue strongly for parasite-skin interactions increasing host susceptibility by actively depressing host immunity.
2-iii. Immunity generated via unmodified skin is easily broken.
Intravenous mouse  and primate ,  attenuated-sporozoite immunizations withstand repeated intravenous challenge. Immunity generated by live parasites via skin however, is reversed by small increments (5 additional bites, or 125–500 more parasites) in natural challenge dose , , but withstands heavy intravenous challenge (20,000–100,000 parasites) , , , ,  (see also Fig. 2c,e,h). Likewise, skin-generated immunity in humans ,  (see also Fig. 2d) despite immunizing doses 100-fold greater than challenge, succumbs to increased , , and usually, sequential , , , ,  natural challenge. Immunity generated transiting skin, therefore, is marginal, and reversible.
2-iv. Bloodstage vaccines do not protect against challenge via the skin.
Finally, intravenous immunization with bloodstage parasites , , , , fully protects against intravenous bloodstage challenge in humans  and monkeys  and both bloodstage ,  and sporozoite  intravenous, but not mosquito-bite, challenge  in skinstage-naïve mice. Similarly, major bloodstage-antigen vaccines (eg. MSP-142, AMA-1), show strong antibody-correlated , ,  efficacy against symptomatic malaria after intravenous challenge in monkeys ,  and induce similar antibody responses in people from endemic areas , , . Protective efficacy against infection however, is negligible, despite some evidence of reducing risk of symptom severity and parastaemia density , . No bloodstage antigen in over 16 trials and 10,300 humans vaccinated to date, protects against infection by mosquito bites , , , , , ,  (and Figure 2i).
2-v. Summary: Vaccine trial data implicates the skin in vaccine failure.
Collectively, these data show that in malaria-naïve subjects, (which excludes bloodstage immunosuppressive effects) live-parasite immunization transiting unmodified skin is inefficient. Immunity diminishes after unmodified skin-parasite interactions and is significantly less robust generated via skin than if intravenously. The bulk of experimentation shows immunization avoiding parasite-skin interaction withstands heavy, repeated intravenous challenge, but only limited challenge transiting the epidermis. Importantly, the data imply parasite-skin interactions actively diminish host protective responses. Avoiding parasite/host skin interactions during both immunization and challenge however, associates solidly with immunity.
3. Altered immune context in the skin during immunization protects against natural challenge and suggests a skin-linked immunosuppressive mechanism
3-i. Immunization under pro-inflammatory skin conditions confers protection.
Complete human protection with irradiation-attenuated , , , ,  P. falciparum or P. vivax requires 1000 or more mosquito bites ,  (usually 80–240 bites/session). This causes coalescing skin inflammation , , lasting several hours . Immunity is reversed by relatively small increases ,  in challenge dose, as for mice , . Fewer total immunizing bites (<1000) are not reliably , or (<700) not at all ,  protective when delivered in low density bites/session , , , , or with strongly anti-inflammatory topical cream  and/or heavier parasite irradiation , . However, 440 infected bites, delivered with additional uninfected bites, increasing bite density, is protective . Rather than parasite dose alone , , therefore, protection appears influenced by degree of parasite attenuation (limiting liverstages ), density of simultaneous bites, and pro-inflammatory local context.
3-ii. Immunization via unmodified skin suppresses available protective responses.
Systemic proinflammatory context confers resistance to malaria infection in mice , ,  and correlates strongly with human resistance , , . In uninflamed mouse skin, increasing immunizing dose from 2 mosquito bites (roughly 50–200 parasites , ) to 4 bites, significantly increases parasite-specific (CD8+) T cell responses . More immunizing bites yield no further increases . Yet, 100-fold higher immunizing doses (20 000 parasites) delivered directly (i.v.) to the liver, provide almost twice the protection of 10 000 parasites , proportionally increasing specific T cell responses . This reveals higher protective responses to direct liverstage infection are available, which become unavailable when parasites transit uninflamed skin. An intravenous immunizing dose completely protective against i.v. challenge of 20,000 parasites, but not against 10 bites  (20-80-fold fewer parasites), is further consistent with skin-linked immunosuppression.
These data suggest inflammatory skin context potentiates immunization via skin, implying inflammation relieves a skin-linked suppression of immunity to liverstage malaria unassociated with prior bloodstage infection.
3-iii Skin-immunization under chloroquine (CQ) immunomodulation also confers protection.
CQ, a widely immunomodulatory 4-aminoquinolone, accumulates preferentially in human skin and lymphocytes , to levels 200–20,000 -fold those in the liver, and remains in human skin, (not plasma) for 6 months . Prophylactic in vivo concentrations (100–500 ng/ml liver plasma , ) inhibit antigen presentation in human antigen-presenting dendritic (DC) and B cells in vitro , , .
Immunization via skin with a drastically lower (but normally 100% infective ) dose of unattenuated P. falciparum (15 infected bites/session, x3), if co-administered with prophylactic chloroquine (CQ), instead protects malaria-naïve individuals from challenge (5 infective bites) . CQ provides greater immunity than parasite attenuation: considerably greater immunizing doses of irradiated parasites without CQ do not protect mice  or humans  against natural challenge. CQ treatment either allows greater and/or antigenically broader immune responses consequent to robust expression of liverstage antigens , , , or lowers a barrier to immunity, or both. We examine the evidence for both, below.
4. Protective effects of skin inflammation and CQ immunomodulatory mechanisms implicate early, skin-induced Tregs in systemic immunotolerance
4-i. The liverstage antigen concept for protective immunity.
That protection requires robust expression of liverstage-antigens has significant correlative support. Primaquine (PQ) eliminates all forms of liverstage parasites ,  and concomitantly abrogates protection , ; incremental parasite irradiation increasingly limits parasite liverstage proliferation ,  and corresponds to decreasing antigen synthesis  (implying decreased antigen presentation) and diminished protectivity , , , , . CQ attenuation however, does not affect parasite liver stages , allowing fullscale liver infections, abrogated after emerging from the liver . These data imply robustly expressed mid-lateliverstage parasite antigens provide immunity ,  to subsequent natural challenge.
4-ii. The liverstage antigen concept does not explain all the data.
Although widely accepted , this concept does not reconcile important data. Genetically attenuated liverstage parasites (p52/36, delivered i.v.), persisting in hepatocytes less than 6 hrs  (minimizing antigen presentation) nonetheless protect against stringent P. yoelii  natural challenge. Similarly, protective responses to liverstage infection (delivered i.v.) are swift , , but do not increase with prolonged antigen presentation . Stable, protective  memory T cell populations to skinstage parasites are induced and maintained by exposure to sporozoite and bloodstage parasites in skinstage immunized, (malaria-naïve) people . Finally, endemic populations remain susceptible to reinfection, despite multiple fullscale liverstage infections, widespread use of CQ in adults, and evidence for unimpaired development and recall of liverstage-cognate memory responses during , ,  and after  bloodstage infection. These discrepancies suggest CQ co-administered with skin-infecting parasites in malaria-naïve people, confers protection in some way beyond allowing a fullscale liverstage antigenic repertoire.
4-iii. CQ inhibits antigen presentation pathways vital to Treg induction in skin.
CQ disrupts all MHC II antigen presentation , ,  to CD4+ T cells. CQ also specifically blocks  the rapid, seconds-to-15-minute  noncanonical MHC I recycling pathway ,  for extracellular antigen cross-presentation to cytotoxic (CD8+) T cells, which in human plasmacytoid-DC (pDC)  enables very rapid cytotoxic responses . CQ does not block classical MHC I antigen cross-presentation , which takes 6-18 hrs  for optimal presentation.
Therefore, only slow, classical MHC I cross-presentation, (efficiently functional in human skin-resident epidermal Langerhans DC (L-DC) , , dermal DC (d-DC)  and other immune and parenchymal cells  such as traversed  skin or malaria-infected liver cells , ), is available to generate protective cytotoxic (CD8+, MHC I) responses to malaria antigens presented at the skinstage , , ,  in humans. In agreement, protection provided by attenuated sporozoites against intravenous challenge in mice, requires MHC I cross-presentation . Also, in humans, one therapeutic dose of CQ strongly inhibits CD4+ T cell responses while strongly inducing CD8+ effector responses in vivo to a particulate viral antigen . Malaria parasites constitute particulate antigen. Evidently, CQ treatment will affect antigen presentation in the skin during the earliest stages of malaria infection: the mosquito bite. Antigen-specific responses dependent on rapid cross-presentation of exogenous MHC I, and all MHC II antigen presentation, including any regulatory T cell induction or activation, will be blocked. CQ therefore, affects the balance of earliest host immune responses triggered to malaria antigens, which are first encountered in the skin.
4-iv. Normal host response to mosquito-bite inflammation triggers tolerizing cascades, facilitating parasite-cognate Treg induction early in infection.
Mosquito bites cause severe local or systemic allergic inflammation , unless rapidly dampened by in situ suppressive activity induced in antigen-cognate T cells (iTregs) . Human skin is rich (~100,000 per cm2) ,  in suppressive , skin-resident , , circulating ,  and LN-homing  nTregs, and local inflammation, which recruits DCs and T cells ,  preferentially increases Treg infiltration , , . Tregs further accumulate at inflammatory sites , ,  via DC (antigen-presenting) dependent and independent  mechanisms.
In skin, sunlight and allergen induced inflammations cause keratinocyte signalling molecules  to interact with densely interwoven epidermal  L-DC. This increases antigen scavenging  in L-DC, and induces L-DC to generate de novo, and proliferate, antigen-specific Tregs . These skin-triggered Tregs repress allergic inflammatory reactions both locally, in skin and draining LNs, and systemically .
Mosquito saliva allergens , in sensitized subjects  such as endemic populations also trigger  almost instantaneous  inflammatory extravasation , , , IgE-mediated  and independent mast-cell TNF-α  and local IFN-γ and inflammatory cytokine secretion , , . This induces chemokines  enabling  local skin inflammations to rapidly recruit leukocytes , , , including circulating immature monocyte-derived DC (mDC) , pDC , , , , , ,  not normally present in skin, and skin-resident dDC and immature L-DC . While immature L-DC are initially tolerogenic , , , secreting high levels of TGF-β , , strong inflammatory stimuli mature L-DC  to preferentially  crosspresent ,  MHC I epitopes. This drives high avidity, antigen-specific cytotoxic T cell responses against epidermally acquired antigens ; human dDC preferentially stimulate antibody responses .
However, in Anopheline mosquito-bites, infiltrating mast cells rapidly degranulate , releasing stored TGF-β1  and bioactivating molecules , activating TGF-β signalling . Exogenous bioactive TGF-β inhibits maturation and endogenous antigen presentation by immature human L-DC after antigen uptake , , and upregulates IL-10 secretion in T cells  and mDC  subsets (this includes L-DC ).
Immature human mDC , , , , ,  and antigen-activated human pDC , , , (which also secrete chemokines that attract circulating T cells ), prime IL-10-secreting, immunosuppressive iTregs from interacting antigen-cognate T cells. Suboptimal , or immature DC antigen presentation , ,  and low antigen dose , ,  preferentially  activate Tregs over effectors, and increasing local TGF-β and IL-10 concentrations further activate Tregs , , , . This tolerogenic cascade will act on recruited T cells and Tregs, potentially including malaria-crossreacting specificities  present in malaria-naïve people , , .
Once activated, nTregs also secrete bioactive  TGF-β1 , , , , and most iTregs produce high levels of IL-10 , , ,  and/or TGF-β , . These cytokines mediate multiple Treg activation , , , , , , , , induction , , , , , , ,  and suppression mechanisms both antigen specific ,  and non-specific . Also, independently of DCs, accumulating human antigen-activated CD4+ Tregs can themselves generate suppressive iTreg via TGF-β-dependent , ,  or IL-10 dependent ,  infectious tolerance. These cascading mechanisms will efficiently expand miniscule numbers of cognate Tregs , ,  initially activated or induced in location, such as an Anopheline bite-site or draining LN.
4-v. Antigen presentation during mosquito bites provides for opportunistic systemic subversion to malaria- specific tolerance.
Critically, Tregs normally home to inflamed tissue  and LNs  according to homing molecules expressed , and in inflamed skin , ,  and LNs , rapidly out-proliferate  and suppress priming ,  and proliferation  of same-specificity effector T cells. Under such Treg-dominated , , or tolerogenic IL-10-rich microenvironments , , mature human mDC will also generate iTregs , , . This further facilitates specific systemic tolerization to skin-encountered antigens. High, tolerogenic IL-10 levels normally prevail within 8 hours in LNs draining uninfected Anopheline mosquito bites , . The normal response to Anopheline bites therefore, provides a tolerogenically predisposed microenvironment, in which parasites, when transmitted, will drive parasite-specific tolerization.
Evidently, antigen presentation in skin during malaria-infected mosquito bites provides major sources of antigen-specific Tregs capable of repressing local , , LN and systemic ,  inflammatory reactions to bite-site antigens. T cell repertoires in malaria-naïve people include malaria-cognate specificities , , . Malaria proteins contain amino acid sequences highly conserved in human housekeeping proteins , implying the pre-existence of malaria-cognate nTregs. These T cell and Treg repertoires, responding to mosquito-allergen inflammation, and the minimal malaria antigen doses transmitted in bites, provide amply for de novo malaria-specific suppressive iTreg generation, and expansion of pre-existing, potentially cognate  nTregs, via Treg induction and expansion mechanisms  documented in the basic literature (see above Section 4-iv). Existing mechanisms for rapidly expanding Treg number , , ,  and specificity spectrum  clearly support rapid induction of antigen-specific Tregs and systemic tolerance . These conditions (part of the normal host response to mosquito bites), occur in malaria infections at earliest skinstage, well before bloodstages emerge. This facilitates opportunistic Treg induction triggered at the skinstage by transmitted parasites. In corroboration, in naïve animals, sand-fly transmitted skinstage Leishmania parasites induce de novo parasite-specific Tregs (previously undetectable in the animal)  which inactivate robust, protective cytotoxic responses  concomitantly elicited by parasite infection. Conversely, and significantly, people in endemic areas deficient in basic functions driving Treg induction (TGF-β production, TGF-β receptors, FOXP3, CTLA-4) resist infection , not just bloodstage disease, specifically implicating the mechanisms of Treg induction and expansion described, in host susceptibility.
4-vi. CQ specifically blocks Treg-inducing antigen presentation: this prevents malaria-specific tolerance induction via bite-site parasite interactions in the skin.
Skin-accumulating CQ disrupts bite-site antigen presentation leading to Treg-induction and activation. Malaria-naïve people treated simultaneously with CQ develop strong immunity via infected bites . A malaria-naïve person therefore, develops protection against natural challenge when rapid cross-presentation of exogenous MHC I, and all MHC II antigen presentation, and consequently also, major sources of rapid antigen-specific activation and de novo induction of tolerogenic CD4+ and CD8+ regulatory T cells in the skin and skin-draining LN, are blocked at the skinstage during immunizing skin infections. Slower, protective CD8+ responses to liverstages, also primed in the peripheral lymph nodes ,  are uninhibited. As seen (Section 4-ii), protection due to expression of a broader liverstage antigen repertoire permitted by CQ, as proposed by others  is inconsistent with lifelong susceptibility in populations from endemic areas, and other, experimental data. Therefore, live parasite immunization via the skin under CQ immunomodulation, results in protection  because CQ coadministered with naturally transmitted parasites, in malaria-naïve people, effects an immunological bypass of rapid, initial skinstage antigen-specific tolerization and suppression, enhancing slower-developing immunity, and memory responses.
4-vii. Enhancing inflammation also counteracts tolerization via the skin.
Multiple-bite immunizations deliver unnaturally high allergen/parasite-antigen doses, provoking strongly pro-inflammatory microenvironments. Pro-inflammatory conditions counteract , , and inhibit ,  DC tolerization and directly disrupt Treg-induction and activation in mucosal skin , and can convert Tregs to effectors . Multiple-bite immunizations therefore, support immediate, predominantly cytotoxic  malaria-specific protective immune responses, effectively disrupting skinstage tolerization, and enhancing protective memory responses.
5. A role for the skin accommodates conflicting data
Consistent with this perspective, genetically attenuated immunizing parasites, (p52/36  or p36 ) administered intravenously, entirely bypass skinstage interactions. Multiple short immunizing liverstage infections, of sufficient dosage , accumulate enough antigen exposure to establish protective responses. Without inhibitory skinstage interactions, intravenous immunization eliciting swift  limited ,  responses to incipient liverstage infection will protect ,  against natural challenge without antigen persistence , , reconciling these (and other , , see Supplementary Text S1) contradictory data.
In strong support, multiple cycles of intravenous infection and subsequent elimination with primaquine (PQ), a parasiticidal drug cure, administered during liverstage development (allowing limited antigen presentation by infected hepatocyte  before PQ-cure) builds robust protection to natural challenge . Immunization via skin, however, with identical PQ-cure cycles, reduces protection  even against intravenous challenge, again implicating the skin in inefficient development of immunity.
Finally, 56%  −66% ,  of infants (3–17 and 2–4 months old, respectively) from seasonal , high transmission areas can be immunized against natural challenge ,  with a skinstage antigen (CSP) vaccine, that negligibly protects, or does not protect adults from endemic areas , .
Adult endemic-region populations are largely pre-exposed , ,  to malaria parasites in the skin: high-transmission areas provide over 2 infected bites per night , , . However, neonates are skinstage malaria-naïve. Babies 0–6 months old, enrolled in medical trials, have significant chances of being skinstage-naïve upon immunization, and remaining so for 3–6 months. Urbanizing environments , increased bednet use (about 80% compliance ) and seasonal transmission relative to birthdate, all reduce exposure to mosquitoes. This allows many intramuscularly immunized infants to pre-establish robust protective responses before encountering skinstage parasites.
6. A critical role for immune mechanisms within the skin in malaria vaccine malfunction
Chronic pre-exposure of skin to live parasites coincides closely with failure of clinically functional vaccines (Figure 2A). Immunization reliably protects against natural challenge only in skinstage-naïve individuals where robust responses are established before infective parasites ever interact with unmodified skin (Figure 2b,d,g). Available epidemiological and vaccine trial data strongly implicate the skin in a block to protective immunity dependent on the presence of skinstage parasites and functional CD4+ and rapid CD8+ exogenous-antigen presentation in the skin, and independent of prior bloodstage infection. T cell memory responses develop normally during , ,  and after  bloodstage infection, and human bloodstage-induced immunosuppression , , ,  is usually limited to acute malaria , , . The low incidence of acute malaria in semi-immune ,  endemic adults cannot, therefore, account for unmitigated susceptibility to reinfection, nor uniform inability of diverse clinically functional vaccines to protect healthy, endemic adults.
We conclude potentially protective liverstage and vaccine-generated T cell responses, which indisputably exist, are disabled by parasites in the skin.
7. Timing, behaviour and molecular characteristics of the malaria parasite skin stage are aggressively tolerogenic
7-i. A role for the bite-site and parasite behaviour in early systemic tolerization.
Malaria-infected mosquito bites of 1 minute deposit around 20  sporozoites in nanolitres  of saliva into epidermal ,  and dermal  skin. Within an hour, in mice, about 10–15%  (normally, 2–3) of deposited sporozoites enter proximal skin-draining lymph nodes (LN) , , , rapidly metamorphosing into bloodstage-like forms , (we propose the term “pseudomorphs”) expressing antigens ,  characteristic of later liver and bloodstages . After 7 hours , most LN parasites are inside  or entwined around CD11c+  (cross-presenting) dendritic cells. Some parasites (~10) remain in the skin , and their exact fate is formally unclear. Antigens draining from these will initially reflect the skinstage. However, host cell environment differentially affects expression profile in malaria parasites , . Given the metamorphic propensity of LN parasites, antigenic representation of further lifecycle stages in skin-lingering parasites cannot be excluded.
These data are crucial: they reveal the very earliest stages of natural infection immediately expose the host immune system, via skin and rapidly developing “pseudomorphs” in skin-draining LN, to very low doses of (minimally) both skinstage and liverstage antigens. These conditions anad location are highly conducive to antigen-specific Treg induction and activation, and particularly rapid induction of systemic tolerance , and therefore, rapid systemic tolerance to parasite antigens arriving in the LN.
7-ii. Normal bite-site responses lead rapidly to systemic tolerance.
Tregs activated in skin bite-sites and LN by migrating parasites and draining antigens will rapidly orchestrate systemic tolerance. Tregs tolerize surrounding microenvironments , , , downregulating mDC antigen presentation  and upregulating mDC TGF-β and IL-10 secretion , , . This favours further Treg activation. Tregs also reverse TLR activation of strong proinflammatory responses in human mDC , , , normally triggered by pathogen ligands . Contact with accumulating Tregs induces immature human L-DC and mDC to remain semi-immature ,  and migrate to draining LNs . LN-migrating mDC can transfer peripheral antigen to LN-resident DC  such as human pDCs, which congregate in densely packed  naïve T cell regions . More immediately, pDC cross-presentation will induce IL-10-secreting Tregs ,  in response to rapidly draining skin-located antigens  (eg. from bite-site) and LN-migrating parasite antigens, potentially within an hour of the mosquito bite. Later arriving  semi-mature L-DC, trafficking antigen  to naive T cell regions of the LN ,  will therefore encounter pre-established, tolerizing microenvironments. Accordingly, skin inflammations , Anopheline mosquito-bites , , and activated TNF-α producing mast cells , all increase immature  L-DC migration and accumulation in the LN , , . Also, for isolated Anopheline mosquito bites leukocyte infiltration  cross-presenting DCs  carrying sporozoite antigen  and IL-10 concentration  rapidly increase in draining LNs.
These data strongly suggest skinstage parasites capitalize on host responses to isolated, uninfected Anopheline bites, (which are normally immunosuppressive ), efficiently misdirecting systemic responses to ensure tolerance to subsequently developing parasites.
7-iii. Skinstage parasite molecules are intrinsically tolerogenic.
Malaria parasites also display aggressive molecular intervention strategies. Critically, gliding sporozoites  transiting skin cells ,  the LN lumen  and invading LN DCs , will shed circumsporozoite protein (CSP) and thrombospondin-related-adhesive protein (TRAP) , . Cytoplasmic CSP, shed by infecting parasites, inhibits host transcription activator NF-Kβ , strongly downregulating multiple pleiotropic pro-inflammatory (anti-parasitic ) activities including IL-6  during plasmodial liver infection. It is well established in non-malarial systems that IL-6 averts CD4+ iTreg formation , , ,  and crucially, suppresses the antigen-specific CD4+ Treg activity , , which inhibits both T cell activation to foreign antigen  and CD4+ and CD8+ T cell memory development , . This directly infers IL-6 blockade by malarial CSP  prevents immediate protective T cell activation and memory responses in the skin, LN and infected hepatocytes, simultaneously stimulating in these locations antigen-specific Treg formation , , , , ,  and expansion , triggered by malaria antigens. This will limit any malaria-specific responses arising. Accordingly, immediate in vivo T cell responses to malaria-infected hepatocytes  are “self-limiting” , , and plasmodial liver infection, (an extensive tissue insult), is non-inflammatory and asymptomatic . Further, high dose antigen-presentation, which counteracts Treg suppression , in the case of malaria-antigens  relieves self-limitation of malaria-specific responses , implicating malaria-specific Tregs in self-limiting immune responses to infected hepatocytes.
Also, P. falciparum TRAP (expressed in both skin and blood stages ), like human thrombospondin, bioactivates latent human TGF-β  via the TSR-1 domain . Thrombospondin, by a TGF-β-dependent  mechanism , , converts cognate CD4+ T cells into suppressive iTregs in situ  conferring localized tissue tolerance in mice . In humans, parasite-driven TGF-β bioactivation ,  precedes and correlates strongly with significantly increased Treg numbers and parasitaemia densities , and suppresses proinflammatory responses in humans  and mice . In unimmunized mice, co-inhibition of TGF-β and IL-10 early  in infection, or depletion of Treg , restores proinflammatory responses and parasite clearance. Correlative, functional data therefore strongly suggest Treg-inducing skinstage function for TRAP molecules.
7-iv. Skinstage parasite behaviour is potently tolerogenic.
Human allergen therapy boosts antigen-specific systemic tolerance by chronic low doses applied via the skin , , and requires Treg induction and activation , . Tolerization is drastically accelerated by low-dose antigen frequently introduced directly into skin-draining LNs .
Similarly, people constantly exposed to infected bites inevitably collect frequent low numbers of CSP/TRAP-shedding, LN-migrating, and skin-lingering “pseudomorphic” parasites. This directly infers parasite instigation of continuous Treg activation and induction in both skin and LN, and inevitably, potent intralymphatic, antigen-specific systemic tolerization to exposed malaria antigens.
8. A model for skinstage-initiated immune subversion incorporates normal immunobiology of Treg induction and mechanism, and the cadence, behaviour and cellular biology of the parasite lifecycle stages
8-i. The model.
Cellular and molecular data indicate isolated malaria-infected mosquito bites drive rapid systemic tolerization to malaria antigens at the skinstage. Since naturally transmitted parasites progress from skin to liver to blood, liver infections inevitably present malaria sporozoite ,  and liverstage antigens to an already efficiently compromised host immune system.
Tregs from human skin are highly proliferative in vivo, develop alongside effector responses at the site of a skin inflammation , and can be induced in vivo and ex vivo from highly differentiated memory T cells, by antigen reencounter , . Tregs in vivo are preferentially ,  induced and activated, and have an in vivo proliferative advantage , ,  over non-Tregs. These properties provide a clear systemic advantage to any suppressive malaria-specific Tregs generated at the skinstage, over simultaneously elicited malaria-specific  protective cytotoxic T cell responses.
Skinstage pre-exposure of later life-cycle antigens  provides an additional temporal advantage: liverstage infections subsequently presenting CSP  and pre-exposed liverstage epitopes (eg TRAP, EXP-1) will enhance pre-established parasite-protective Treg populations, suppressing swift cytotoxic responses initiated against infected hepatocytes. B cells require activated T cell help to initiate IgG antibody responses, and Tregs will also directly, antigen-specifically and non-specifically, inhibit or kill activated B cells , , , . Antibody responses and B cell function will therefore also be vulnerable to repression by skin-induced, malaria-specific Tregs, from the skinstage onwards. Infection, and reinfection, not sterile immunity, will prevail in natural transmission.
Induction of broad-spectrum malaria-specific Tregs in the skin therefore, ensures liverstage infections always develop into transmissible blood stages. At least 281 bloodstage proteins are expressed at the skin stage, including AMA-1, PfEMP and STEVOR proteins . More than one liver- and bloodstage-expressed antigen (eg TRAP, EXP-1) , ,  is definitely exposed at the skinstage , , and therefore to the skin-based tolerizing mechanisms we define. Once Tregs are induced, numerous mechanisms (see Sections 4-iv, 4-v, 7-iii) exist allowing small numbers of Tregs of one specificity to expand, outgrow, convert non-Tregs of different specificities to suppressive function (infectious tolerance), and also non-specifically suppress responses to other antigens (bystander suppression) . Therefore, a Treg response specific and suppressive to one liverstage or bloodstage antigen (eg. TRAP or EXP-1), triggered initially during skinstage, can later re-expand upon specific antigen re-encounter at the liver or bloodstage, and non-specifically suppress T cell (or ablate memory B cell) responses to further parasite antigens co-expressed only at the later stage, eg. responses to MSP-1, specific to late-liver and bloodstages. Pre-induction of bloodstage-specific Tregs provides an obvious protective advantage promoting the establishment of bloodstage parasites emerging from the liver. Re-expansion of pre-induced Tregs will initiate a tolerogenic cascade and immunosuppressive environment conducive to bloodstage expansion, which is further enhanced by the bloodstage parasite themselves. Bloodstage parasites generate Treg activity , , ,  and nonspecific immunosuppressive conditions , , , , , , enhancing systemic tolerance ,  conducive to multiple parasitaemia cycles, thereby increasing probability of gametocyte transmission, and also reinfection , .
8-ii. Model outcomes: experimental versus field infections.
Experimental, intravenous sporozoite infections bypass skin/LN Treg activation (Figure 3B). Consequently, predominant immune responses to liverstage infection, which prime in the peripheral LN ,  are unhindered, rendering the immunodominant CSP molecule and also TRAP, experimentally protective , , .
A: Natural transmission (mosquito bite) allows skinstage parasites (sporozoites, spz, green fragments) to migrate (dotted gray arrows) through skin cells to both lymph nodes (LN) and liver (via blood vessels, bv), and induce malaria-specific regulatory T cells in the skin and LN that suppress (red blocker lines) local and systemic protective immune responses (green arrows), resulting in bloodstage infections (gray fragments below liver), and amplification cycles within red blood cells (rbc). B, C, D: Protective immunity develops where immunization and/or challenge avoids parasite-skin interaction and Treg activation/induction; B: Physical bypass of the skin, (by intravenous (i.v.) attenuated parasites, purified antigen, antibody or intramuscular or intranasal subunit vaccine) at immunization, or i.v. challenge with unattenuated infective parasites, avoids all induction or activation of skin and liverstage-specific Tregs. C: Chloroquine (CQ) accumulates and blocks all MHC II and rapid, (but not classical, slow) MHC I malaria-antigen presentation in the skin, which would otherwise immediately induce/activate antigen-specific Tregs. D: Multiple simultaneous mosquito bites create a strongly pro-inflammatory local skin and/or systemic milieu which inhibit Treg activation and induction processes. LN Inset: Inside LN, metamorphosing skinstage sporozoite parasites (spz) are in close contact with, or invade, host antigen presenting dendritic cells (DC).
Like physical bypass of immune interactions in the skin at immunization or challenge (Fig. 3B), immunological bypass by CQ inhibition of antigen presentation (Fig. 3C), or inflammatory inhibition of tolerizing cellular cascades (Fig. 3D) during immunization, consistently result in protection to natural skinstage challenge for malaria-naïve subjects.
Natural, skin-initiated infections however, pre-establish antigen-specific tolerance to parasite antigens presented in the skin and LN (Fig. 3A). In fundamental opposition to current opinion  and vaccine dogma, this model shows CSP and TRAP are pleiopotently immunosuppressive molecules when deployed within the cadence of natural infection. Other lifecycle-stage antigens pre-exposed by skinstage “pseudomorphs” , such as liver and bloodstage antigens, broaden the scope of immunosuppressive systemic responses to parasite antigens subsequently encountered during liverstage or bloodstage infection (or immunization). Critically, again directly opposing prevailing opinion, further exposure to infected mosquitoes will boost tolerance, and suppression of protective responses, not immunity.
8-iii. Selective vaccine failure: who, where and why.
Adults chronically exposed to infected mosquitoes from birth will accumulate broad repertoires of regulatory T cells, becoming potently pretolerized. Vaccination deploying skinstage-exposed or bloodstage antigens will activate both pre-existing cognate Tregs , ,  as shown for foreign antigen in mouse systems, and malaria-specific T cells, (effector and other subsets) as shown in humans , ; challenge by mosquito bite will preferentially boost pre-existing Treg-based tolerance , , suppressing protective concomitantly elicited and recall responses . Vaccines which substantially protect malaria-naïve adults ,  will negligibly, or not, protect endemic-area adults , , as solidly evidenced by adult vaccine field trials.
Neonates, initially skinstage-malaria-naïve, are easier to protect from mosquito bite. Accumulation of skinstage-induced regulatory T cells specific for malaria will be negligible, and immunization will (potentially unrestrictedly) favour  immunity. Endemic-area infants, like malaria-naïve adults, should be better protected by formulations that negligibly protect endemic-area adults. This profile is precisely corroborated by results for leading CSP-containing RTS,S, vaccine formulations. These protect malaria-naïve adults partially , , endemic-area infants (2–17 month-old) similarly partially , , , older children poorly , ,  and endemic-area adults negligibly , , .
Limitations of the study
Using rigorous systematic literature search, and individual screening with unambiguous criteria on nearly 2000 studies, we have identified the great majority of peer-reviewed, experimental evidence documenting complete protection to malaria infection. Meta-analysis of experimental conditions involved shows only immunizations that avoid live parasite interaction in the skin, or inhibit regulatory T cell induction within the skin during skin-immunization, fully protect against infected mosquito bites. Our main conclusion, that very early, skinstage-induced, antigen-specific regulatory T cells block malaria vaccine function therefore rests empirically but solidly on a straightforward meta-analysis of unbiased experimental data. The data were retrieved from experiments carried out worldwide, by hundreds of groups, using many different protocols over many decades, and drawn from extensive literature reporting skinstage, liverstage and bloodstage vaccine trials. This minimizes both experimental bias and the impact inherent in overlooking any one published study, but does not specify a mechanism. However, detailed molecular/cellular immunological data from experiments carried out independently, in the main by basic research groups unconcerned with malaria (and therefore unbiased), define a mechanism of tolerogenic induction via the skin which strongly supports this conclusion. Combined with the cadence of the parasite lifecycle stages, and the behaviour and detailed cellular and molecular character of skinstage parasites during transmission, we define a novel immuno-epidemiological model for vaccine success/failure. This identifies differential exposure to infected mosquitos and malaria-specific Treg accumulation as the basis for selective vaccine failure in endemic-area populations, and defines a skin-triggered immunological mechanism for early tolerance induction, preceding and independent of later bloodstage immunosuppression. The available epidemiological and experimental vaccine trial data are entirely consistent with the immunological mechanism and model we propose. The model specified by these combined data easily explains myriad long-standing incongruities, paradoxes and contradictions in the malaria literature. In 1916 studies, we could find no experimental immunization or natural infection data which contradict or weaken our conclusions.
Fundamental implications for vaccine efficacy
Circumvention of malaria-specific regulatory T cell activation in the skin fits all identified instances of protective immunization against virulent experimental challenge, and selective protection in endemic skinstage challenge. Fundamental immediate and longterm implications for vaccine development and strategy are highlighted below.
i. Live parasite vaccines.
Unmodified intradermal delivery  will obliterate efficacy in otherwise outstandingly successful attenuated-parasite vaccines, advocating optimization of immune context for dermal delivery.
ii. Antigen selection.
Intrinsically tolerogenic antigens (eg. CSP, TRAP), or those enriching skinstage and/or bloodstage-cognate Tregs (eg. TRAP, EXP-1), (or abundant bloodstream antigens, eg. AMA-1, MSP-1, which will almost inevitably induce homeostatic regulatory T cell activity), are counter-productive in pre-tolerized individuals, although initially effective in malaria-naïve individuals, such as neonates. With age, increasing bite exposure and skin-induced malaria-specific Treg accumulation will repress T cell and B cell responses, eroding ,  skinstage-specific, antibody-correlated , , ,  infant protection elicited by leading vaccines such as RTS,S. Of particular concern, vaccination with intrinsically tolerogenic antigens although initially protective in malaria-naïve infants, will also predispose bite-exposed growing children to severe malaria, increasing individual risk of later childhood death. Late-midliverstage antigens (unexposed at skinstage) however, should initially escape significant Treg induction and memory inhibition in the skinstage, and do show protectivity , , , , ,  into adolescence . This makes late-midliverstage antigens rationally preferred candidates for more universally applicable subunit vaccines with less hindered immune memory.
iii. Infant mortality.
Neonates protected from mosquitoes will not accumulate skinstage-specific Tregs. Immunization under Treg pre-emptive conditions from birth (non-tolerogenic antigen, CQ accumulation in the skin, scrupulous use of bed-nets/insect-repellent), inducing abundant skinstage-specific antibodies should substantially reduce infant infections beyond currently obtainable protection. Additional early-infancy immunization with liverstage  and bloodstage antigens ,  will rapidly , , , ,  provide robust ,  bloodstage immunity. This provides critical protection against increased risk of severe malaria  due to decreased exposure to bloodstage disease. Substantially reduced childhood morbidity and mortality, and shrinking transmission reservoirs, should result.
Discarded vaccines, ineffective in endemic adults but pretested for safety, immunogenicity and tolerability can be rapidly retested (given voluntary patent rescindication ) for protectivity in neonates under Treg pre-emptive conditions. Bednets and CQ are cheap, CQ is usually well-tolerated, including during pregnancy , , and is transmitted transplacentally at therapeutic doses and via breast milk at subtherapeutic dosages . CQ accumulates preferentially in the skin, so significantly reduced, subtherapeutic dosage may suffice. Treg-blocking (immunomodulary) pharmacological effects of CQ are on the host, not parasite, eliminating parasite-resistance obstacles. Current infrastructure (http://www.theglobalfund.org) allows largescale immunization across (primarily African) endemic regions worldwide.
Malaria outside Africa is mostly hypoendemic , and not all Africa is holoendemic . With increasing bednet/insecticide use, transmission drops , , , . Coordinated neonate vaccination generating additional concerted widespread reductions (herd effects) in infectious reservoirs is feasible, even with low efficacy vaccines . Conditions pre-empting Treg induction at early skin stage will amplify trends leading to interrupted transmission, catalyzing  significantly accelerated local elimination, and facilitating worldwide eradication of malaria.
Existing adult transmission reservoirs however, will counteract shrinking infant reservoirs , . Skinstage-induced-Treg evasion and memory enhancing vaccines, preventing adult re-infection, and transmission-blocking vaccines targeting parasite development within the mosquito, and antimalarials blocking bloodstage transmission, are therefore essential to fast-track eradication.
Skinstage activation of parasite-specific Treg-based systemic immunosuppression provides a fundamentally new, experimentally widely substantiated, immunological rationale, and precise focus, for research and vaccination strategy leading to potentially accelerated malaria eradication. The concept, and implications for vaccination, apply to closely related and economically important (Toxoplasma, Theileria, Babesia spp.) pathogens.
Complete protection data (177 experiments) reference list for meta-analysis.
(0.24 MB DOC)
Protective vaccination physically bypasses the skin at immunization or challenge (90%) or involves skin immunomodulation (10%). A. Exposure to parasites in the skin coincides closely with vaccine failure. Green background- immunization procedures. Lilac background- challenge procedures and percent of total experiments showing complete protection (% total) formed by a subset of studies (category) using a given experimental procedure (categories a-i; supporting experimental data for each category is in references listed below; also Supplementary Table S1). Parasite immunization administration routes: (a,b): live parasites given intravenously (i.v.), or the method does not involve live parasites, but uses dead parasites or purified antigen, antibody, or recombinant DNA (subunit) and therefore bypasses parasite interactions with host skin; (c,d,e,f,g,h): live parasites are administered via the skin; (c,d): by multiple simultaneous mosquito bites/session; (e,f): live parasites are naturally transmitted by 4–15 bites; (g): live parasites are administered by 12–15 bites/session with chloroquine; (h): live parasites delivered subcutaneously (s.c) or intradermally (i.d.) or intramuscularly (i.m.); (i): uncontrolled exposure to endemic mosquitos. Challenge route is either i.v. or by mosquito bite, as indicated. B. Protective immunization bypasses the skin at either immunization or challenge in 90% of cases: categories (a,b,c,e,h) shaded blue with red crosses. Protective immunization which transits skin during immunization (c,d,e,f,g,h) either: bypasses the skin physically at challenge (c,e,h) (red cross); or, involves skin immunomodulation during immunization (d,g, 10% of cases). Asterisk (*)- immunization via unmodified skin, limited to P. berghei (f). Skin bypass (red cross)- method physically avoids live parasite interactions in the host skin. Malaria exposure- skin exposure to infected mosquito bite before first immunization; naïve- no pre-exposure; exposed (endemic)- chronic exposure. x- this study shows complete protection of 40 of 41 mice challenged. y: one person in one study  was infected one time via the skin prior to protective immunization and was therefore moderately tolerized. Data pertaining to experimental categories (a–h): a:, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,  , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,, , , , , , , , , , , , , , , , , , , , , , ,, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , . b: , , , , , , , , , . c: . d: , , , , , , , , , , , , , , , . e: , , . f: . g: . h: , , , , . Studies containing data for multiple relevant experimental conditions are referenced accordingly in each appropriate category. Multiple experiments contributed by a single study are indicated beneath study reference number (eg. reference 124 X2) in Supplementary Table S1. (Meta-analysis data extended reference list).
(8.70 MB TIF)
Antigen persistence in liverstages is not required for protection. Directly contradictory data , , ,  is also easily reconciled. Like increasing irradiation , , the drug primaquine (PQ) eliminates liverstages , . PQ however, also disrupts membrane and vesicular trafficking , , temporarily eliminating all antigen presentation. This prevents immunity if used during immunization , creating an apparent correlation between protection and parasite persistence . Used after intravenous immunization , allowing early liverstage antigen presentation, however, multiple PQ-cure cycles provide sufficient cumulative antigen presentation to build immunity, without antigen persistence.
(0.07 MB DOC)
We thank Bernd Bukau (Zentrum für Molekulare Biologie der Universität-Heidelberg, Germany), Uli Göringer (Technical University of Darmstadt, Germany), Theresa Shapiro (Johns Hopkins School of Medicine, USA), Derrick Robinson (CNRS, University of Bordeaux, France) Alvaro Acosta-Serrano (Liverpool School of Tropical Medicine) and Bernie Cohen (University of Glasgow, UK) for critical reading of the manuscript and incisive discussion. D.L.G. thanks Freddy Frischknecht (Universität-Heidelberg, Germany), Kai Matuschewski (MPI-Berlin, Germany) and Volker Schirrmacher (Emeritus; DKFZ, Germany) for supportive discussion over the past five years. We particularly thank the Directors of the Malaria Research Foundation, (MRF) Simon Pinneger, and Carolyne Roehm, and MRF donors for enabling continued support during financial crisis years.
Conceived and designed the experiments: DLG. Performed the experiments: DLG PG. Analyzed the data: DLG PDG. Contributed reagents/materials/analysis tools: DLG PG PDG. Wrote the paper: DLG PDG. Also contributed to figure design and execution: DLG. Contributed significantly to database searches, data acquisition, figure design, figure execution, editing: PG. Contributed substantially to manuscript organization, analysis of data and editing, and expertise on Apicomplexa pathogenic to livestock: PDLG.
- 1. Graves P, Gelband H (2006) Vaccines for preventing malaria (pre-erythrocytic). Cochrane Database Syst Rev: CD006198.
- 2. Matuschewski K, Mueller AK (2007) Vaccines against malaria - an update. FEBS J 274: 4680–4687.
- 3. Doolan DL, Stewart AA (2007) Status of Malaria Vaccine R&D in 2007- Malaria vaccines for the World 2007, September 17 –19th, 2007, London, UK. Expert Rev Vaccines 6: 903–905.
- 4. Wipasa J, Riley EM (2007) The immunological challenges of malaria vaccine development. Expert Opin Biol Ther 7: 1841–1852.
- 5. Greenwood BM, Fidock DA, Kyle DE, Kappe SH, Alonso PL, et al. (2008) Malaria: progress, perils, and prospects for eradication. J Clin Invest 118: 1266–1276.
- 6. Moorthy VS, Diggs C, Ferro S, Good MF, Herrera S, et al. (2009) Report of a consultation on the optimization of clinical challenge trials for evaluation of candidate blood stage malaria vaccines, 18–19 March 2009, Bethesda, MD, USA. Vaccine 27: 5719–5725.
- 7. Alonso PL, Sacarlal J, Aponte JJ, Leach A, Macete E, et al. (2004) Efficacy of the RTS,S/AS02A vaccine against Plasmodium falciparum infection and disease in young African children: randomised controlled trial. Lancet 364: 1411–1420.
- 8. Sacarlal J, Aide P, Aponte JJ, Renom M, Leach A, et al. (2009) Long-term safety and efficacy of the RTS,S/AS02A malaria vaccine in Mozambican children. J Infect Dis 200: 329–336.
- 9. Bejon P, Lusingu J, Olotu A, Leach A, Lievens M, et al. (2008) Efficacy of RTS,S/AS01E vaccine against malaria in children 5 to 17 months of age. N Engl J Med 359: 2521–2532.
- 10. Aponte JJ, Aide P, Renom M, Mandomando I, Bassat Q, et al. (2007) 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 370: 1543–1551.
- 11. Abdulla S, Oberholzer R, Juma O, Kubhoja S, Machera F, et al. (2008) Safety and immunogenicity of RTS,S/AS02D malaria vaccine in infants. N Engl J Med 359: 2533–2544.
- 12. Stoute JA, Slaoui M, Heppner DG, Momin P, Kester KE, et al. (1997) A preliminary evaluation of a recombinant circumsporozoite protein vaccine against Plasmodium falciparum malaria. N Engl J Med 336: 86–91.
- 13. Kester KE, Cummings JF, Ofori-Anyinam O, Ockenhouse CF, Krzych U, et al. (2009) 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 200: 337–346.
- 14. Bojang KA, Milligan PJ, Pinder M, Vigneron L, Alloueche A, et al. (2001) Efficacy of RTS,S/AS02 malaria vaccine against Plasmodium falciparum infection in semi-immune adult men in The Gambia: a randomised trial. Lancet 358: 1927–1934.
- 15. Polhemus ME, Remich SA, Ogutu BR, Waitumbi JN, Otieno L, et al. (2009) Evaluation of RTS,S/AS02A and RTS,S/AS01B in adults in a high malaria transmission area. PLoS One 4: e6465.
- 16. Good MF, Pombo D, Quakyi IA, Riley EM, Houghten RA, et al. (1988) Human T-cell recognition of the circumsporozoite protein of Plasmodium falciparum: Immunodominant T-cell domains map to the polymorphic regions of the molecule. Proc Natl Acad Sci U S A 85: 1199–1203.
- 17. Hill AV, Elvin J, Willis AC, Aidoo M, Allsopp CE, et al. (1992) Molecular analysis of the association of HLA-B53 and resistance to severe malaria. Nature 360: 434–439.
- 18. Doolan DL, Hoffman SL, Southwood S, Wentworth PA, Sidney J, et al. (1997) Degenerate cytotoxic T cell epitopes from P. falciparum restricted by multiple HLA-A and HLA-B supertype alleles. Immunity 7: 97–112.
- 19. Plebanski M, Aidoo M, Whittle HC, Hill AV (1997) Precursor frequency analysis of cytotoxic T lymphocytes to pre-erythrocytic antigens of Plasmodium falciparum in West Africa. J Immunol 158: 2849–2855.
- 20. Aidoo M, Lalvani A, Gilbert SC, Hu JT, Daubersies P, et al. (2000) Cytotoxic T-lymphocyte epitopes for HLA-B53 and other HLA types in the malaria vaccine candidate liver-stage antigen 3. Infect Immun 68: 227–232.
- 21. Lee EAM, Flanagan Katie L, Odhiambo K, Reece WHH, Potter Colin, et al. (2001) Identification of frequently recognized dimorphicepitopes in plasmodium falciparum merozoitesurface protein-1in West and East Africans: lack of correlation of immune recognition and allelic preference. Am J Trop Med Hyg 64: 194–203.
- 22. Kabilan L, Troye-Blomberg M, Perlmann H, Andersson G, Hogh B, et al. (1988) T-cell epitopes in Pf155/RESA, a major candidate for a Plasmodium falciparum malaria vaccine. Proc Natl Acad Sci U S A 85: 5659–5663.
- 23. Flanagan KL, Lee EA, Gravenor MB, Reece WH, Urban BC, et al. (2001) Unique T cell effector functions elicited by Plasmodium falciparum epitopes in malaria-exposed Africans tested by three T cell assays. J Immunol 167: 4729–4737.
- 24. Nardin EH, Nussenzweig RS, McGregor IA, Bryan JH (1979) Antibodies to sporozoites: their frequent occurrence in individuals living in an area of hyperendemic malaria. Science 206: 597–599.
- 25. Hoffman SL, Oster CN, Plowe CV, Woollett GR, Beier JC, et al. (1987) Naturally acquired antibodies to sporozoites do not prevent malaria: vaccine development implications. Science 237: 639–642.
- 26. Migot-Nabias F, Deloron P, Ringwald P, Dubois B, Mayombo J, et al. (2000) Immune response to Plasmodium falciparum liver stage antigen-1: geographical variations within Central Africa and their relationship with protection from clinical malaria. Trans R Soc Trop Med Hyg 94: 557–562.
- 27. Zhou Z, Xiao L, Branch OH, Kariuki S, Nahlen BL, et al. (2002) Antibody responses to repetitive epitopes of the circumsporozoite protein, liver stage antigen-1, and merozoite surface protein-2 in infants residing in a Plasmodium falciparum-hyperendemic area of western Kenya. XIII. Asembo Bay Cohort Project. Am J Trop Med Hyg 66: 7–12.
- 28. Bongfen SE, Ntsama PM, Offner S, Smith T, Felger I, et al. (2009) The N-terminal domain of Plasmodium falciparum circumsporozoite protein represents a target of protective immunity. Vaccine 27: 328–335.
- 29. Osier FH, Fegan G, Polley SD, Murungi L, Verra F, et al. (2008) Breadth and magnitude of antibody responses to multiple Plasmodium falciparum merozoite antigens are associated with protection from clinical malaria. Infect Immun 76: 2240–2248.
- 30. Roussilhon C, Oeuvray C, Muller-Graf C, Tall A, Rogier C, et al. (2007) Long-term clinical protection from falciparum malaria is strongly associated with IgG3 antibodies to merozoite surface protein 3. PLoS Med 4: e320.
- 31. Michon P, Cole-Tobian JL, Dabod E, Schoepflin S, Igu J, et al. (2007) The risk of malarial infections and disease in Papua New Guinean children. Am J Trop Med Hyg 76: 997–1008.
- 32. Langhorne J, Ndungu FM, Sponaas AM, Marsh K (2008) Immunity to malaria: more questions than answers. Nat Immunol 9: 725–732.
- 33. Doolan DL, Dobano C, Baird JK (2009) Acquired immunity to malaria. Clin Microbiol Rev 22: 13–36.
- 34. Hogh B (1996) Clinical and parasitological studies on immunity to Plasmodium falciparum malaria in children. Scand J Infect Dis: Suppl 1021–53.
- 35. Romero P, Maryanski JL, Corradin G, Nussenzweig RS, Nussenzweig V, et al. (1989) Cloned cytotoxic T cells recognize an epitope in the circumsporozoite protein and protect against malaria. Nature 341: 323–325.
- 36. Wizel B, Houghten R, Church P, Tine JA, Lanar DE, et al. (1995) HLA-A2–restricted cytotoxic T lymphocyte responses to multiple Plasmodium falciparum sporozoite surface protein 2 epitopes in sporozoite-immunized volunteers. J Immunol 155: 766–775.
- 37. Sun P, Schwenk R, White K, Stoute JA, Cohen J, et al. (2003) 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 171: 6961–6967.
- 38. Reece WH, Pinder M, Gothard PK, Milligan P, Bojang K, et al. (2004) 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 10: 406–410.
- 39. Nardin EH, Herrington DA, Davis J, Levine M, Stuber D, et al. (1989) Conserved repetitive epitope recognized by CD4+ clones from a malaria-immunized volunteer. Science 246: 1603–1606.
- 40. Herrington D, Davis J, Nardin E, Beier M, Cortese J, et al. (1991) Successful immunization of humans with irradiated malaria sporozoites: humoral and cellular responses of the protected individuals. Am J Trop Med Hyg 45: 539–547.
- 41. Malik A, Egan JE, Houghten RA, Sadoff JC, Hoffman SL (1991) Human cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein Proc Natl Acad Sci U S A 88: 3300–3304.
- 42. Rodrigues M, Nussenzweig RS, Zavala F (1993) The relative contribution of antibodies, CD4+ and CD8+ T cells to sporozoite-induced protection against malaria. Immunology 80: 1–5.
- 43. Moreno A, Clavijo P, Edelman R, Davis J, Sztein M, et al. (1993) CD4+ T cell clones obtained from Plasmodium falciparum sporozoite-immunized volunteers recognize polymorphic sequences of the circumsporozoite protein. J Immunol 151: 489–499.
- 44. Wizel B, Houghten RA, Parker KC, Coligan JE, Church P, et al. (1995) Irradiated sporozoite vaccine induces HLA-B8-restricted cytotoxic T lymphocyte responses against two overlapping epitopes of the Plasmodium falciparum sporozoite surface protein 2. J Exp Med 182: 1435–1445.
- 45. Krzych U, Lyon JA, Jareed T, Schneider I, Hollingdale MR, et al. (1995) T lymphocytes from volunteers immunized with irradiated Plasmodium falciparum sporozoites recognize liver and blood stage malaria antigens. J Immunol 155: 4072–4077.
- 46. Sano G, Hafalla JC, Morrot A, Abe R, Lafaille JJ, et al. (2001) Swift development of protective effector functions in naive CD8(+) T cells against malaria liver stages. J Exp Med 194: 173–180.
- 47. Belnoue E, Costa FT, Frankenberg T, Vigario AM, Voza T, et al. (2004) Protective T cell immunity against malaria liver stage after vaccination with live sporozoites under chloroquine treatment. J Immunol 172: 2487–2495.
- 48. Mueller AK, Labaied M, Kappe SH, Matuschewski K (2005) Genetically modified Plasmodium parasites as a protective experimental malaria vaccine. Nature 433: 164–167.
- 49. Jobe O, Lumsden J, Mueller AK, Williams J, Silva-Rivera H, et al. (2007) Genetically attenuated Plasmodium berghei liver stages induce sterile protracted protection that is mediated by major histocompatibility complex Class I-dependent interferon-gamma-producing CD8+ T cells. J Infect Dis 196: 599–607.
- 50. Purcell LA, Wong KA, Yanow SK, Lee M, Spithill TW, et al. (2008) Chemically attenuated Plasmodium sporozoites induce specific immune responses, sterile immunity and cross-protection against heterologous challenge. Vaccine 26: 4880–4884.
- 51. Oliveira GA, Kumar KA, Calvo-Calle JM, Othoro C, Altszuler D, et al. (2008) Class II-restricted protective immunity induced by malaria sporozoites. Infect Immun 76: 1200–1206.
- 52. Herrington DA, Clyde DF, Davis JR, Baqar S, Murphy JR, et al. (1990) Human studies with synthetic peptide sporozoite vaccine (NANP)3-TT and immunization with irradiated sporozoites. Bull World Health Organ 68: Suppl33–37.
- 53. Nardin EH, Calvo-Calle JM, Oliveira GA, Nussenzweig RS, Schneider M, et al. (2001) A totally synthetic polyoxime malaria vaccine containing Plasmodium falciparum B cell and universal T cell epitopes elicits immune responses in volunteers of diverse HLA types. J Immunol 166: 481–489.
- 54. Birkett A, Lyons K, Schmidt A, Boyd D, Oliveira GA, et al. (2002) A modified hepatitis B virus core particle containing multiple epitopes of the Plasmodium falciparum circumsporozoite protein provides a highly immunogenic malaria vaccine in preclinical analyses in rodent and primate hosts. Infect Immun 70: 6860–6870.
- 55. Wang R, Doolan DL, Le TP, Hedstrom RC, Coonan KM, et al. (1998) Induction of antigen-specific cytotoxic T lymphocytes in humans by a malaria DNA vaccine. Science 282: 476–480.
- 56. Lalvani A, Moris P, Voss G, Pathan AA, Kester KE, et al. (1999) Potent induction of focused Th1-type cellular and humoral immune responses by RTS,S/SBAS2, a recombinant Plasmodium falciparum malaria vaccine. J Infect Dis 180: 1656–1664.
- 57. Tartz S, Russmann H, Kamanova J, Sebo P, Sturm A, et al. (2008) Complete protection against P. berghei malaria upon heterologous prime/boost immunization against circumsporozoite protein employing Salmonella type III secretion system and Bordetella adenylate cyclase toxoid. Vaccine 26: 5935–5943.
- 58. Perlaza BL, Zapata C, Valencia AZ, Hurtado S, Quintero G, et al. (2003) Immunogenicity and protective efficacy of Plasmodium falciparum liver-stage Ag-3 in Aotus lemurinus griseimembra monkeys. Eur J Immunol 33: 1321–1327.
- 59. McConkey SJ, Reece WH, Moorthy VS, Webster D, Dunachie S, et al. (2003) Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans. Nat Med 9: 729–735.
- 60. Moorthy VS, Imoukhuede EB, Keating S, Pinder M, Webster D, et al. (2004) Phase 1 evaluation of 3 highly immunogenic prime-boost regimens, including a 12-month reboosting vaccination, for malaria vaccination in Gambian men. J Infect Dis 189: 2213–2219.
- 61. Pichyangkul S, Gettayacamin M, Miller RS, Lyon JA, Angov E, et al. (2004) Pre-clinical evaluation of the malaria vaccine candidate P. falciparum MSP142 formulated with novel adjuvants or with alum. Vaccine 22: 3831–3840.
- 62. Calvo-Calle JM, Oliveira GA, Nardin EH (2005) Human CD4+ T cells induced by synthetic peptide malaria vaccine are comparable to cells elicited by attenuated Plasmodium falciparum sporozoites. J Immunol 175: 7575–7585.
- 63. Mettens P, Dubois PM, Demoitie MA, Bayat B, Donner MN, et al. (2008) Improved T cell responses to Plasmodium falciparum circumsporozoite protein in mice and monkeys induced by a novel formulation of RTS,S vaccine antigen. Vaccine 26: 1072–1082.
- 64. Pichyangkul S, Kum-Arb U, Yongvanitchit K, Limsalakpetch A, Gettayacamin M, et al. (2008) Preclinical evaluation of the safety and immunogenicity of a vaccine consisting of Plasmodium falciparum liver-stage antigen 1 with adjuvant AS01B administered alone or concurrently with the RTS,S/AS01B vaccine in rhesus primates. Infect Immun 76: 229–238.
- 65. Schneider J, Langermans JA, Gilbert SC, Blanchard TJ, Twigg S, et al. (2001) A prime-boost immunisation regimen using DNA followed by recombinant modified vaccinia virus Ankara induces strong cellular immune responses against the Plasmodium falciparum TRAP antigen in chimpanzees. Vaccine 19: 4595–4602.
- 66. BenMohamed L, Thomas A, Druilhe P (2004) Long-term multiepitopic cytotoxic-T-lymphocyte responses induced in chimpanzees by combinations of Plasmodium falciparum liver-stage peptides and lipopeptides. Infect Immun 72: 4376–4384.
- 67. Weiss WR, Kumar A, Jiang G, Williams J, Bostick A, et al. (2007) Protection of rhesus monkeys by a DNA prime/poxvirus boost malaria vaccine depends on optimal DNA priming and inclusion of blood stage antigens. PLoS One 2: e1063.
- 68. Daubersies P, Ollomo B, Sauzet JP, Brahimi K, Perlaza BL, et al. (2008) Genetic immunisation by liver stage antigen 3 protects chimpanzees against malaria despite low immune responses. PLoS One 3: e2659.
- 69. Barbosa A, Naniche D, Aponte JJ, Manaca MN, Mandomando I, et al. (2009) P. 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.
- 70. Chen DH, Tigelaar RE, Weinbaum FI (1977) Immunity to sporozoite-induced malaria infeciton in mice. I. The effect of immunization of T and B cell-deficient mice. J Immunol 118: 1322–1327.
- 71. Spitalny GL, Verhave JP, Meuwissen JH, Nussenzweig RS (1977) Plasmodium berghei: T cell dependence of sporozoite-induced immunity in rodents. Exp Parasitol 42: 73–81.
- 72. Schofield L, Villaquiran J, Ferreira A, Schellekens H, Nussenzweig R, et al. (1987) Gamma interferon, CD8+ T cells and antibodies required for immunity to malaria sporozoites. Nature 330: 664–666.
- 73. Weiss WR, Sedegah M, Beaudoin RL, Miller LH, Good MF (1988) CD8+ T cells (cytotoxic/suppressors) are required for protection in mice immunized with malaria sporozoites. Proc Natl Acad Sci U S A 85: 573–576.
- 74. Tsuji M, Romero P, Nussenzweig RS, Zavala F (1990) CD4+ cytolytic T cell clone confers protection against murine malaria. J Exp Med 172: 1353–1357.
- 75. Doolan DL, Hoffman SL (2000) The complexity of protective immunity against liver-stage malaria. J Immunol 165: 1453–1462.
- 76. Belnoue E, Voza T, Costa FT, Gruner AC, Mauduit M, et al. (2008) Vaccination with live Plasmodium yoelii blood stage parasites under chloroquine cover induces cross-stage immunity against malaria liver stage. J Immunol 181: 8552–8558.
- 77. Pombo DJ, Lawrence G, Hirunpetcharat C, Rzepczyk C, Bryden M, et al. (2002) Immunity to malaria after administration of ultra-low doses of red cells infected with Plasmodium falciparum. Lancet 360: 610–617.
- 78. Beaudoin RL, Strome CP, Mitchell F, Tubergen TA (1977) Plasmodium berghei: immunization of mice against the ANKA strain using the unaltered sporozoite as an antigen. Exp Parasitol 42: 1–5.
- 79. Hafalla JC, Sano G, Carvalho LH, Morrot A, Zavala F (2002) Short-term antigen presentation and single clonal burst limit the magnitude of the CD8(+) T cell responses to malaria liver stages. Proc Natl Acad Sci U S A 99: 11819–11824.
- 80. Hafalla JC, Rai U, Bernal-Rubio D, Rodriguez A, Zavala F (2007) Efficient development of plasmodium liver stage-specific memory CD8+ T cells during the course of blood-stage malarial infection. J Infect Dis 196: 1827–1835.
- 81. Moorthy VS, Imoukhuede EB, Milligan P, Bojang K, Keating S, et al. (2004) A randomised, double-blind, controlled vaccine efficacy trial of DNA/MVA ME-TRAP against malaria infection in Gambian adults. PLoS Med 1: e33.
- 82. Bejon P, Mwacharo J, Kai O, Mwangi T, Milligan P, et al. (2006) A phase 2b randomized trial of the candidate malaria vaccines FP9 ME-TRAP and MVA METRAP among children in Kenya. PLoS Clin Trials 1: e29.
- 83. Ogutu BR, Apollo OJ, McKinney D, Okoth W, Siangla J, et al. (2009) Blood stage malaria vaccine eliciting high antigen-specific antibody concentrations confers no protection to young children in Western Kenya. PLoS One 4: e4708.
- 84. Graves P, Gelband H (2006) Vaccines for preventing malaria (SPf66). Cochrane Database Syst Rev CD005966.
- 85. Orjih AU (1985) Acute malaria prolongs susceptibility of mice to Plasmodium berghei sporozoite infection. Clin Exp Immunol 61: 67–71.
- 86. Sakaguchi S, Yamaguchi T, Nomura T, Ono M (2008) Regulatory T Cells and Immune Tolerance. Cell 133: 775–787.
- 87. Horwitz DA, Zheng SG, Gray JD (2008) Natural and TGF-beta-induced Foxp3(+)CD4(+) CD25(+) regulatory T cells are not mirror images of each other. Trends Immunol 29: 429–435.
- 88. Mahnke K, Bedke T, Enk AH (2007) Regulatory conversation between antigen presenting cells and regulatory T cells enhance immune suppression. Cell Immunol 250: 1–13.
- 89. Zhao DM, Thornton AM, DiPaolo RJ, Shevach EM (2006) Activated CD4+CD25+ T cells selectively kill B lymphocytes. Blood 107: 3925–3932.
- 90. Ludwig-Portugall I, Hamilton-Williams EE, Gottschalk C, Kurts C (2008) Cutting edge: CD25+ regulatory T cells prevent expansion and induce apoptosis of B cells specific for tissue autoantigens. J Immunol 181: 4447–4451.
- 91. Curotto de Lafaille MA, Kutchukhidze N, Shen S, Ding Y, Yee H, et al. (2008) Adaptive Foxp3+ regulatory T cell-dependent and -independent control of allergic inflammation. Immunity 29: 114–126.
- 92. Lim HW, Hillsamer P, Banham AH, Kim CH (2005) Cutting edge: direct suppression of B cells by CD4+ CD25+ regulatory T cells. J Immunol 175: 4180–4183.
- 93. Tang Q, Bluestone JA (2008) The Foxp3+ regulatory T cell: a jack of all trades, master of regulation. Nat Immunol 9: 239–244.
- 94. Murphy TJ, Ni Choileain N, Zang Y, Mannick JA, Lederer JA (2005) CD4+CD25+ regulatory T cells control innate immune reactivity after injury. J Immunol 174: 2957–2963.
- 95. Zhang ZX, Yang L, Young KJ, DuTemple B, Zhang L (2000) Identification of a previously unknown antigen-specific regulatory T cell and its mechanism of suppression. Nat Med 6: 782–789.
- 96. Gondek DC, Devries V, Nowak EC, Lu LF, Bennett KA, et al. (2008) Transplantation survival is maintained by granzyme B+ regulatory cells and adaptive regulatory T cells. J Immunol 181: 4752–4760.
- 97. Carvalho-Gaspar M, Jones ND, Luo S, Martin L, Brook MO, et al. (2008) Location and time-dependent control of rejection by regulatory T cells culminates in a failure to generate memory T cells. J Immunol 180: 6640–6648.
- 98. Mills KH (2004) Regulatory T cells: friend or foe in immunity to infection? Nat Rev Immunol 4: 841–855.
- 99. Belkaid Y (2007) Regulatory T cells and infection: a dangerous necessity. Nat Rev Immunol 7: 875–888.
- 100. Mellor AL, Munn DH (2008) Creating immune privilege: active local suppression that benefits friends, but protects foes. Nat Rev Immunol 8: 74–80.
- 101. Clark RA, Chong B, Mirchandani N, Brinster NK, Yamanaka K, et al. (2006) The vast majority of CLA+ T cells are resident in normal skin. J Immunol 176: 4431–4439.
- 102. Clark RA, Kupper TS (2007) IL-15 and dermal fibroblasts induce proliferation of natural regulatory T cells isolated from human skin. Blood 109: 194–202.
- 103. Boyd MF, Kitchen SF (1939) The demonstration of sporozoites in human tissue. Am J Trop Med Hyg 19: 27–31.
- 104. Griffiths RB, Gordon RM (1952) An apparatus which enables the process of feeding by mosquitoes to be observed in the tissues of a live rodent; together with an account of the ejection of saliva and its significance in Malaria. Ann Trop Med Parasitol 46: 311–319.
- 105. Sidjanski S, Vanderberg JP (1997) Delayed migration of Plasmodium sporozoites from the mosquito bite site to the blood. Am J Trop Med Hyg 57: 426–429.
- 106. Vanderberg JP, Frevert U (2004) Intravital microscopy demonstrating antibody-mediated immobilisation of Plasmodium berghei sporozoites injected into skin by mosquitoes. Int J Parasitol 34: 991–996.
- 107. Amino R, Thiberge S, Martin B, Celli S, Shorte S, et al. (2006) Quantitative imaging of Plasmodium transmission from mosquito to mammal. Nat Med 12: 220–224.
- 108. Vanderberg JP (1974) Studies on the motility of Plasmodium sporozoites. J Protozool 21: 527–537.
- 109. Yamauchi LM, Coppi A, Snounou G, Sinnis P (2007) Plasmodium sporozoites trickle out of the injection site. Cell Microbiol 9: 1215–1222.
- 110. Jin Y, Kebaier C, Vanderberg J (2007) Direct microscopic quantification of dynamics of Plasmodium berghei sporozoite transmission from mosquitoes to mice. Infect Immun 75: 5532–5539.
- 111. Chakravarty S, Cockburn IA, Kuk S, Overstreet MG, Sacci JB, et al. (2007) CD8+ T lymphocytes protective against malaria liver stages are primed in skin-draining lymph nodes. Nat Med 13: 1035–1041.
- 112. Shortt HE, Bray RS, Cooper W (1954) Further note on the tissue stages of P. cynomolgi. Trans R Soc Trop Med Hyg 48: 122–131.
- 113. Laveran A (1880) Un nouveau parasite trouve dans le sang des malades atteintes de fievre palustre origine parisitaire des accidents de impaludisme. Bulletins et memoires de la Societe Medicale des Hopitaux de Paris 17: 158–164.
- 114. Sturm A, Amino R, van de Sand C, Regen T, Retzlaff S, et al. (2006) Manipulation of host hepatocytes by the malaria parasite for delivery into liver sinusoids. Science 313: 1287–1290.
- 115. Clark IA, Alleva LM, Mills AC, Cowden WB (2004) Pathogenesis of malaria and clinically similar conditions. Clin Microbiol Rev 17: 509–539.
- 116. Liberati A, Altman DG, Tetzlaff J, Mulrow C, Gotzsche PC, et al. (2009) The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration. PLoS Med 6: e1000100.
- 117. Hoffman SL, Goh LM, Luke TC, Schneider I, Le TP, et al. (2002) (Table 1: subject 10) J Infect Dis 185: 1155–1164.
- 118. Clyde DF, Most H, McCarthy VC, Vanderberg JP (1973) Immunization of man against sporozite-induced falciparum malaria. Am J Med Sci 266: 169–177.
- 119. Clyde DF (1975) Immunization of man against falciparum and vivax malaria by use of attenuated sporozoites. Am J Trop Med Hyg 24: 397–401.
- 120. Clyde DF, McCarthy VC, Miller RM, Hornick RB (1973) Specificity of protection of man immunized against sporozoite-induced falciparum malaria. Am J Med Sci 266: 398–403.
- 121. Rieckmann KH, Carson PE, Beaudoin RL, Cassells JS, Sell KW (1974) Sporozoite induced immunity in man against an Ethiopian strain of Plasmodium falciparum. Trans R Soc Trop Med Hyg 68: 258–259.
- 122. Rieckmann KH, Beaudoin RL, Cassells JS, Sell KW (1979) Use of attenuated sporozoites in the immunization of human volunteers against falciparum malaria. Bull World Health Organ 57: Suppl 1261–265.
- 123. Rieckmann KH (1990) Human immunization with attenuated sporozoites. Bull World Health Organ 68: Suppl13–16.
- 124. Vaughan JA, Scheller LF, Wirtz RA, Azad AF (1999) Infectivity of Plasmodium berghei sporozoites delivered by intravenous inoculation versus mosquito bite: implications for sporozoite vaccine trials. Infect Immun 67: 4285–4289.
- 125. Spitalny GL, Nussenzweig RS (1972) Effect of various routes of immunization and methods of parasite attenuation on the development of protection against sporozoite-induced rodent malaria. Proc Helm Soc Wash 39: 506–514.
- 126. Kramer LD, Vanderberg JP (1975) Intramuscular immunization of mice with irradiated Plasmodium berghei sporozoites. Enhancement of protection with albumin. Am J Trop Med Hyg 24: 913–916.
- 127. Sina BJ, do Rosario VE, Woollett G, Sakhuja K, Hollingdale MR (1993) Plasmodium falciparum sporozoite immunization protects against Plasmodium berghei sporozoite infection. Exp Parasitol 77: 129–135.
- 128. Douradinha B, van Dijk MR, Ataide R, van Gemert GJ, Thompson J, et al. (2007) Genetically attenuated P36p-deficient Plasmodium berghei sporozoites confer long-lasting and partial cross-species protection. Int J Parasitol 37: 1511–1519.
- 129. Putrianti ED, Silvie O, Kordes M, Borrmann S, Matuschewski K (2009) Vaccine-like immunity against malaria by repeated causal-prophylactic treatment of liver-stage Plasmodium parasites. J Infect Dis 199: 899–903.
- 130. Sedagah M, Weiss WR, Hoffman SL (2007) Cross-protection between attenuated Plasmodium berghei and P. yoelii sporozoites. Parasite Immunology 29: 559–565.
- 131. Frischknecht F (2007) The skin as interface in the transmission of arthropod-borne pathogens. Cell Microbiol 9: 1630–1640.
- 132. Wong KA, Zhou A, Rodriguez A (2008) Protective immunity induced by daily bites from irradiated mosquitoes infected with Parasite Immunol 30: 482–486.
- 133. Collins WE, Skinner JC, Millet P, Broderson JR, Filipski VK, et al. (1992) Reinforcement of immunity in Saimiri monkeys following immunization with irradiated sporozoites of Plasmodium vivax. Am J Trop Med Hyg 46: 327–334.
- 134. Hoffman SL, Goh LM, Luke TC, Schneider I, Le TP, et al. (2002) Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites. J Infect Dis 185: 1155–1164.
- 135. McCarthy VC, Clyde DF (1977) Plasmodium vivax: correlation of circumsporozoite precipitation (CSP) reaction with sporozoite-induced protective immunity in man. Exp Parasitol 41: 167–171.
- 136. Edelman R, Hoffman SL, Davis JR, Beier M, Sztein MB, et al. (1993) Long-term persistence of sterile immunity in a volunteer immunized with X-irradiated Plasmodium falciparum sporo-zoites. J Infect Dis 168: 1066–1070.
- 137. Fandeur T, Gysin J, Mercereau-Puijalon O (1992) Protection of squirrel monkeys against virulent Plasmodium falciparum infections by use of attenuated parasites. Infect Immun 60: 1390–1396.
- 138. Ting LM, Gissot M, Coppi A, Sinnis P, Kim K (2008) Attenuated Plasmodium yoelii lacking purine nucleoside phosphorylase confer protective immunity. Nat Med 14: 954–958.
- 139. Hirunpetcharat C, Tian JH, Kaslow DC, van Rooijen N, Kumar S, et al. (1997) Complete protective immunity induced in mice by immunization with the 19-kilodalton carboxyl-terminal fragment of the merozoite surface protein-1 (MSP1) of Plasmodium yoelii expressed in Saccharomyces cerevisiae: correlation of protection with antigen-specific antibody titer, but not with effector CD4+ T cells. J Immunol 159: 3400–3411.
- 140. Stowers AW, Kennedy MC, Keegan BP, Saul A, Long CA, et al. (2002) Vaccination of monkeys with recombinant Plasmodium falciparum apical membrane antigen 1 confers protection against blood-stage malaria. Infect Immun 70: 6961–6967.
- 141. Thera MA, Doumbo OK, Coulibaly D, Diallo DA, Sagara I, et al. (2006) Safety and allele-specific immunogenicity of a malaria vaccine in Malian adults: results of a phase I randomized trial. PLoS Clin Trials 1: e34.
- 142. Sagara I, Dicko A, Ellis RD, Fay MP, Diawara SI, et al. (2009) A randomized controlled phase 2 trial of the blood stage AMA1-C1/Alhydrogel malaria vaccine in children in Mali. Vaccine 27: 3090–3098.
- 143. Stanisic DI, Richards JS, McCallum FJ, Michon P, King CL, et al. (2009) Immunoglobulin G subclass-specific responses against Plasmodium falciparum merozoite antigens are associated with control of parasitemia and protection from symptomatic illness. Infect Immun 77: 1165–1174.
- 144. Fowkes F, Richards J, Simpson J, Beeson J (2010) The relationship between anti-merozoite antibodies and incidence of Plasmodium falciparum malaria: A systematic review and meta-analysis. PLoS Med 7: e1000218.
- 145. Graves P, Gelband H (2006) Vaccines for preventing malaria (blood-stage). Cochrane Database Syst Rev: CD006199.
- 146. Thompson FM, Porter DW, Okitsu SL, Westerfeld N, Vogel D, et al. (2008) Evidence of blood stage efficacy with a virosomal malaria vaccine in a phase IIa clinical trial. PLoS One 3: e1493.
- 147. Spring MD, Cummings JF, Ockenhouse CF, Dutta S, Reidler R, et al. (2009) Phase 1/2a study of the malaria vaccine candidate apical membrane antigen-1 (AMA-1) administered in adjuvant system AS01B or AS02A. PLoS One 4: e5254.
- 148. Luke TC, Hoffman SL (2003) Rationale and plans for developing a non-replicating, metabolically active, radiation-attenuated Plasmodium falciparum sporozoite vaccine. J Exp Biol 206: 3803–3808.
- 149. Vanderberg JP, Nussenzweig RS, Most H, Orton CG (1968) Protective immunity produced by the injection of x-irradiated sporozoites of Plasmodium berghei. II. Effects of radiation on sporozoites. J Parasitol 54: 1175–1180.
- 150. Puri SK, Maheshwari RK, Dutta GP, Friedman RM, Dhar MM (1988) Human interferon-gamma protects rhesus monkeys against sporozoite-induced Plasmodium cynomolgi malaria infection. J Interferon Res 8: 201–206.
- 151. Romero JF, Ibrahim GH, Renggli J, Himmelrich H, Graber P, et al. (2007) IL-12p40-independent induction of protective immunity upon multiple Plasmodium berghei irradiated sporozoite immunizations. Parasite Immunol 29: 541–548.
- 152. Donovan MJ, Messmore AS, Scrafford DA, Sacks DL, Kamhawi S, et al. (2007) Uninfected mosquito bites confer protection against infection with malaria parasites. Infect Immun 75: 2523–2530.
- 153. Luty AJ, Lell B, Schmidt-Ott R, Lehman LG, Luckner D, et al. (1999) Interferon-gamma responses are associated with resistance to reinfection with Plasmodium falciparum in young African children. J Infect Dis 179: 980–988.
- 154. Farouk SE, Dolo A, Bereczky S, Kouriba B, Maiga B, et al. (2005) Different antibody- and cytokine-mediated responses to Plasmodium falciparum parasite in two sympatric ethnic tribes living in Mali. Microbes Infect 7: 110–117.
- 155. Torcia MG, Santarlasci V, Cosmi L, Clemente A, Maggi L, et al. (2008) Functional deficit of T regulatory cells in Fulani, an ethnic group with low susceptibility to Plasmodium falciparum malaria. Proc Natl Acad Sci U S A 105: 646–651.
- 156. Bergqvist Y, Domeij-Nyberg B (1983) Distribution of chloroquine and its metabolite desethyl-chloroquine in human blood cells and its implication for the quantitative determination of these compounds in serum and plasma. J Chromatogr 272: 137–148.
- 157. Kalia S, Dutz JP (2007) New concepts in antimalarial use and mode of action in dermatology. Dermatol Ther 20: 160–174.
- 158. Lee SJ, McGready R, Fernandez C, Stepniewska K, Paw MK, et al. (2008) Chloroquine pharmacokinetics in pregnant and nonpregnant women with vivax malaria. Eur J Clin Pharmacol 64: 987–992.
- 159. Reid PA, Watts C (1989) Cycling of cell-surface MHC glycoproteins through primaquine-sensitive intracellular compartments. Nature 346: 655–657.
- 160. Di Pucchio T, Chatterjee B, Smed-Sorensen A, Clayton S, Palazzo A, et al. (2008) Direct proteasome-independent cross-presentation of viral antigen by plasmacytoid dendritic cells on major histocompatibility complex class I. Nat Immunol 9: 551–557.
- 161. Burgdorf S, Scholz C, Kautz A, Tampe R, Kurts C (2008) Spatial and mechanistic separation of crosspresentation and endogenous antigen presentation. Nat Immunol 9: 558–566.
- 162. Verhage DF, Telgt DS, Bousema JT, Hermsen CC, van Gemert GJ, et al. (2005) Clinical outcome of experimental human malaria induced by Plasmodium falciparum-infected mosquitoes. Neth J Med 63: 52–58.
- 163. Roestenberg M, McCall M, Hopman J, Wiersma J, Luty AJ, et al. (2009) Protection against a malaria challenge by sporozoite inoculation. N Engl J Med 361: 468–477.
- 164. Sauerwein RW (2009) Clinical malaria vaccine development. Immunol Lett 122: 115–117.
- 165. Scheller LF, Azad AF (1995) Maintenance of protective immunity against malaria by persistent hepatic parasites derived from irradiated sporozoites. Proc Natl Acad Sci U S A 92: 4066–4068.
- 166. Mueller AK, Deckert M, Heiss K, Goetz K, Matuschewski K, et al. (2007) Genetically attenuated Plasmodium berghei liver stages persist and elicit sterile protection primarily via CD8 T cells. Am J Pathol 171: 107–115.
- 167. Suhrbier A, Winger LA, Castellano E, Sinden RE (1990) Survival and antigenic profile of irradiated malarial sporozoites in infected liver cells. Infect Immun 58: 2834–2839.
- 168. Sigler CI, Leland P, Hollingdale MR (1984) In vitro infectivity of irradiated Plasmodium berghei sporozoites to cultured hepatoma cells. Am J Trop Med Hyg 33: 544–547.
- 169. Mellouk S, Lunel F, Sedegah M, Beaudoin RL, Druilhe P (1990) Protection against malaria induced by irradiated sporozoites. Lancet 335: 721.
- 170. Silvie O, Semblat JP, Franetich JF, Hannoun L, Eling W, et al. (2002) Effects of irradiation on Plasmodium falciparum sporozoite hepatic development: implications for the design of pre-erythrocytic malaria vaccines. Parasite Immunol 24: 221–223.
- 171. Labaied M, Harupa A, Dumpit RF, Coppens I, Mikolajczak SA, et al. (2007) Plasmodium yoelii sporozoites with simultaneous deletion of P52 and P36 are completely attenuated and confer sterile immunity against infection. Infect Immun 75: 3758–3768.
- 172. Palmer DR, Krzych U (2002) Cellular and molecular requirements for the recall of IL-4-producing memory CD4(+)CD45RO(+)CD27(-) T cells during protection induced by attenuated Plasmodium falciparum sporozoites. Eur J Immunol 32: 652–661.
- 173. Orjih AU, Nussenzweig RS (1979) Plasmodium berghei: suppression of antibody response to sporozoite stage by acute blood stage infection. Clin Exp Immunol 38: 1–8.
- 174. Bejon P, Mwacharo J, Kai O, Todryk S, Keating S, et al. (2007) The induction and persistence of T cell IFN-gamma responses after vaccination or natural exposure is suppressed by Plasmodium falciparum. J Immunol 179: 4193–4201.
- 175. Ziegler HK, Unanue ER (1982) Decrease in macrophage antigen catabolism caused by ammonia and chloroquine is associated with inhibition of antigen presentation to T cells. Proc Natl Acad Sci U S A 79: 175–178.
- 176. Nowell J, Quaranta V (1985) Chloroquine affects biosynthesis of ia molecules by inhibiting dissociation of invariant (gamma) chains from alpha-beta dimers in B cells. J Exp Med 162: 1371–1376.
- 177. Stoitzner P, Tripp CH, Eberhart A, Price KM, Jung JY, et al. (2006) Langerhans cells cross-present antigen derived from skin. Proc Natl Acad Sci USA 103: 7783–7788.
- 178. Klechevsky E, Morita R, Liu M, Cao Y, Coquery S, et al. (2008) Functional Specializations of Human Epidermal Langerhans Cells and CD14+ Dermal Dendritic Cells. Immunity 29: 497–510.
- 179. Flacher V, Sparber F, Tripp CH, Romani N, Stoitzner P (2009) Targeting of epidermal Langerhans cells with antigenic proteins: attempts to harness their properties for immunotherapy. Cancer Immunol Immunother 58: 1137–1147.
- 180. Dzierszinski F, Pepper M, Stumhofer JS, LaRosa DF, Wilson EH, et al. (2007) Presentation of Toxoplasma gondii antigens via the endogenous major histocompatibility complex class I pathway in nonprofessional and professional antigen-presenting cells. Infect Immun 75: 5200–5209.
- 181. Bongfen SE, Balam S, Torgler R, Romero JF, Corradin G (2008) Processing of the circumsporozoite protein in infected hepatocytes is not dependent on aspartic proteases. Parasite Immunol 30: 375–378.
- 182. Bongfen SE, Torgler R, Romero JF, Renia L, Corradin G (2007) Plasmodium berghei-infected primary hepatocytes process and present the circumsporozoite protein to specific CD8+ T cells in vitro. J Immunol 178: 7054–7063.
- 183. Doolan DL, Southwood S, Chesnut R, Appella E, Gomez E, et al. (2000) HLA-DR-promiscuous T cell epitopes from Plasmodium falciparum pre-erythrocytic-stage antigens restricted by multiple HLA class II alleles. J Immunol 165: 1123–1137.
- 184. Doolan DL, Southwood S, Freilich DA, Sidney J, Graber NL, et al. (2003) Identification of Plasmodium falciparum antigens by antigenic analysis of genomic and proteomic data. Proc Natl Acad Sci U S A 100: 9952–9957.
- 185. White KL, Snyder HL, Krzych U (1996) MHC class I-dependent presentation of exoerythrocytic antigens to CD8+ T lymphocytes is required for protective immunity against Plasmodium berghei. J Immunol 156: 3374–3381.
- 186. Accapezzato D, Visco V, Francavilla V, Molette C, Donato T, et al. (2005) Chloroquine enhances human CD8+ T cell responses against soluble antigens in vivo. J Exp Med 202: 817–828.
- 187. Peng Z, Simons FE (2004) Mosquito allergy: immune mechanisms and recombinant salivary allergens. Int Arch Allergy Immunol 133: 198–209.
- 188. Iellem A, Colantonio L, D'Ambrosio D (2003) Skin-versus gut-skewed homing receptor expression and intrinsic CCR4 expression on human peripheral blood CD4+CD25+ suppressor T cells. Eur J Immunol 33: 1488–1496.
- 189. Hirahara K, Liu L, Clark RA, Yamanaka K, Fuhlbrigge RC, et al. (2006) The majority of human peripheral blood CD4+CD25highFoxp3+ regulatory T cells bear functional skin-homing receptors oming Receptors. J Immunol 177: 4488–4494.
- 190. Demeure CE, Brahimi K, Hacini F, Marchand F, Peronet R, et al. (2005) Anopheles mosquito bites activate cutaneous mast cells leading to a local inflammatory response and lymph node hyperplasia. J Immunol 174: 3932–3940.
- 191. Iellem A, Mariani M, Lang R, Recalde H, Panina-Bordignon P, et al. (2001) Unique chemotactic response profile and specific expression of chemokine receptors CCR4 and CCR8 by CD4(+)CD25(+) regulatory T cells. J Exp Med 194: 847–853.
- 192. Selvaraj RK, Geiger TL (2008) Mitigation of experimental allergic encephalomyelitis by TGF-beta induced Foxp3+ regulatory T lymphocytes through the induction of anergy and infectious tolerance. J Immunol 180: 2830–2838.
- 193. Huehn J, Hamann A (2005) Homing to suppress: address codes for Treg migration. Trends Immunol 26: 632–636.
- 194. Campanelli AP, Roselino AM, Cavassani KA, Pereira MS, Mortara RA, et al. (2006) CD4+CD25+ T cells in skin lesions of patients with cutaneous leishmaniasis exhibit phenotypic and functional characteristics of natural regulatory T cells. J Infect Dis 193: 1313–1322.
- 195. Loser K, Mehling A, Loeser S, Apelt J, Kuhn A, et al. (2006) Epidermal RANKL controls regulatory T-cell numbers via activation of dendritic cells. Nat Med 12: 1372–1379.
- 196. Romani N, Holzmann S, Tripp CH, Koch F, Stoitzner P (2003) Langerhans cells – dendritic cells of the epidermis. APMIS 111: 725–740.
- 197. Hudson A, Bowman L, Orr CW (1960) Effects of absence of saliva on blood feeding by mosquitoes. Science 131: 1730–1731.
- 198. Peng Z, Yang M, Simons FE (1996) Immunologic mechanisms in mosquito allergy: correlation of skin reactions with specific IgE and IgG antibodies and lymphocyte proliferation response to mosquito antigens. Ann Allergy Asthma Immunol 77: 238–244.
- 199. Jawdat DM, Albert EJ, Rowden G, Haidl ID, Marshall JS (2004) IgE-mediated mast cell activation induces Langerhans cell migration in vivo. J Immunol 173: 5275–5282.
- 200. Le Borgne M, Etchart N, Goubier A, Lira SA, Sirard JC, et al. (2006) Dendritic cells rapidly recruited into epithelial tissues via CCR6/CCL20 are responsible for CD8+ T cell crosspriming in vivo. Immunity 24: 191–201.
- 201. Williams IR (2006) CCR6 and CCL20: partners in intestinal immunity and lymphorganogenesis. Ann N Y Acad Sci 1072: 52–61.
- 202. Urosevic M, Dummer R, Conrad C, Beyeler M, Laine E, et al. (2005) Disease-independent skin recruitment and activation of plasmacytoid predendritic cells following imiquimod treatment. J Natl Cancer Inst 97: 1143–1153.
- 203. Ueno H, Klechevsky E, Morita R, Aspord C, Cao T, et al. (2007) Dendritic cell subsets in health and disease. Immunol Rev 219: 118–142.
- 204. Randolph GJ, Inaba K, Robbiani DF, Steinman RM, Muller WA (1999) Differentiation of phagocytic monocytes into lymph node dendritic cells in vivo. Immunity 11: 753–761.
- 205. Wollenberg A, Wagner M, Gunther S, Towarowski A, Tuma E, et al. (2002) Plasmacytoid dendritic cells: a new cutaneous dendritic cell subset with distinct role in inflammatory skin diseases. J Invest Dermatol 119: 1096–1102.
- 206. Vermi W, Riboldi E, Wittamer V, Gentili F, Luini W, et al. (2005) Role of ChemR23 in directing the migration of myeloid and plasmacytoid dendritic cells to lymphoid organs and inflamed skin. J Exp Med 201: 509–515.
- 207. Albanesi C, Scarponi C, Bosisio D, Sozzani S, Girolomoni G (2010) Immune functions and recruitment of plasmacytoid dendritic cells in psoriasis. Autoimmunity.
- 208. Hawiger D, Inaba K, Dorsett Y, Guo M, Mahnke K, et al. (2001) Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo. J Exp Med 194: 769–779.
- 209. Steinman RM, Hawiger D, Nussenzweig MC (2003) Tolerogenic dendritic cells. Annu Rev Immunol 21: 685–711.
- 210. Kaplan DH, Jenison MC, Saeland S, Shlomchik WD, Shlomchik MJ (2005) Epidermal langerhans cell-deficient mice develop enhanced contact hypersensitivity. Immunity 23: 611–620.
- 211. Morelli AE, Rubin JP, Erdos G, Tkacheva OA, Mathers AR, et al. (2005) CD4+ T cell responses elicited by different subsets of human skin migratory dendritic cells. J Immunol 175: 7905–7915.
- 212. Li MO, Wan YY, Sanjabi S, Robertson AK, Flavell RA (2006) Transforming growth factor-beta regulation of immune responses. Annu Rev Immunol 24: 99–146.
- 213. Lindstedt KA, Wang Y, Shiota N, Saarinen J, Hyytiainen M, et al. (2001) Activation of paracrine TGF-beta1 signaling upon stimulation and degranulation of rat serosal mast cells: a novel function for chymase. FASEB J 15: 1377–1388.
- 214. Geissmann F, Revy P, Regnault A, Lepelletier Y, Dy M, et al. (1999) TGF-beta 1 prevents the noncognate maturation of human dendritic Langerhans cells. J Immunol 162: 4567–4575.
- 215. Kitani A, Fuss I, Nakamura K, Kumaki F, Usui T, et al. (2003) Transforming growth factor (TGF)-beta1-producing regulatory T cells induce Smad-mediated interleukin 10 secretion that facilitates coordinated immunoregulatory activity and amelioration of TGF-beta1-mediated fibrosis. J Exp Med 198: 1179–1188.
- 216. Levings MK, Gregori S, Tresoldi E, Cazzaniga S, Bonini C, et al. (2005) Differentiation of Tr1 cells by immature dendritic cells requires IL-10 but not CD25+CD4+ Tr cells. Blood 105: 1162–1169.
- 217. Ginhoux F, Tacke F, Angeli V, Bogunovic M, Loubeau M, et al. (2006) Langerhans cells arise from monocytes in vivo. Nat Immunol 7: 265–273.
- 218. Jonuleit H, Schmitt E, Schuler G, Knop J, Enk AH (2000) Induction of interleukin 10-producing, nonproliferating CD4(+) T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J Exp Med 192: 1213–1222.
- 219. Dhodapkar MV, Steinman RM, Krasovsky J, Munz C, Bhardwaj N (2001) Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells. J Exp Med 193: 233–238.
- 220. Mahnke K, Qian Y, Knop J, Enk AH (2003) Induction of CD4+/CD25+ regulatory T cells by targeting of antigens to immature dendritic cells. Blood 101: 4862–4869.
- 221. Roncarolo MG, Gregori S, Battaglia M, Bacchetta R, Fleischhauer K, et al. (2006) Interleukin-10-secreting type 1 regulatory T cells in rodents and humans. Immunol Rev 212: 28–50.
- 222. Cools N, Van Tendeloo VF, Smits EL, Lenjou M, Nijs G, et al. (2008) Immunosuppression induced by immature dendritic cells is mediated by TGF-beta/IL-10 double-positive CD4+ regulatory T cells. J Cell Mol Med 12: 690–700.
- 223. Gilliet M, Liu YJ (2002) Generation of human CD8+ T regulatory cells by CD40 ligand-activated plasmacytoid dendritic cells. J Exp Med 195: 695–704.
- 224. Ito T, Yang M, Wang YH, Lande R, Gregorio J, et al. (2007) Plasmacytoid dendritic cells prime IL-10-producing T regulatory cells by inducible costimulator ligand. J Exp Med 204: 105–115.
- 225. Kretschmer K, Apostolou I, Hawiger D, Khazaie K, Nussenzweig MC, et al. (2005) Inducing and expanding regulatory T cell populations by foreign antigen. Nat Immunol 6: 1219–1227.
- 226. Roncarolo MG, Levings MK, Traversari C (2001) Differentiation of T regulatory cells by immature dendritic cells. J Exp Med 193: F5–9.
- 227. George TC, Bilsborough J, Viney JL, Norment AM (2003) High antigen dose and activated dendritic cells enable Th cells to escape regulatory T cell mediated suppression in vitro. Eur J Immunol 33: 502–511.
- 228. Sarris M, Andersen KG, Randow F, Mayr L, Betz AG (2008) Neuropilin-1 expression on regulatory T cells enhances their interactions with dendritic cells during antigen recognition. Immunity 28: 402–413.
- 229. Tang Q, Adams JY, Tooley AJ, Bi M, Fife BT, et al. (2006) Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice. Nat Immunol 7: 83–92.
- 230. Dieckmann D, Bruett CH, Ploettner H, Lutz MB, Schuler G (2002) Human CD4(+)CD25(+) regulatory, contact-dependent T cells induce interleukin 10-producing, contact-independent type 1-like regulatory T cells [corrected]. J Exp Med 196: 247–253.
- 231. von Boehmer H (2005) Mechanisms of suppression by suppressor T cells. Nat Immunol 6: 338–344.
- 232. Belkaid Y, Rouse B (2005) Natural regulatory T cells in infectious disease. Nat Immunol 6: 353–360.
- 233. Robson KJ, Hall JR, Jennings MW, Harris TJ, Marsh K, et al. (1988) A highly conserved amino-acid sequence in thrombospondin, properdin and in proteins from sporozoites and blood stages of a human malaria parasite. Nature 335: 79–82.
- 234. Okitsu SL, Silvie O, Westerfeld N, Curcic M, Kammer AR, et al. (2007) A virosomal malaria peptide vaccine elicits a long-lasting sporozoite-inhibitory antibody response in a phase 1a clinical trial. PLoS One 2: e1278.
- 235. Miller A, al-Sabbagh A, Santos LM, Das MP, Weiner HL (1993) Epitopes of myelin basic protein that trigger TGF-beta release after oral tolerization are distinct from encephalitogenic epitopes and mediate epitope-driven bystander suppression. J Immunol 151: 7307–7315.
- 236. Zheng SG, Wang JH, Gray JD, Soucier H, Horwitz DA (2004) Natural and induced CD4+CD25+ cells educate CD4+CD25- cells to develop suppressive activity: the role of IL-2, TGF-beta, and IL-10. J Immunol 172: 5213–5221.
- 237. Li MO, Sanjabi S, Flavell RA (2006) Transforming growth factor-beta controls development, homeostasis, and tolerance of T cells by regulatory T cell-dependent and -independent mechanisms. Immunity 25: 455–471.
- 238. Groux H, O'Garra A, Bigler M, Rouleau M, Antonenko S, et al. (1997) A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389: 737–742.
- 239. Maynard CL, Harrington LE, Janowski KM, Oliver JR, Zindl CL, et al. (2007) Regulatory T cells expressing interleukin 10 develop from Foxp3+ and Foxp3– precursor cells in the absence of interleukin 10. Nat Immunol 8: 931–941.
- 240. Cottrez F, Groux H (2001) Regulation of TGF-beta response during T cell activation is modulated by IL-10. J Immunol 167: 773–778.
- 241. Chen ZM, O'Shaughnessy MJ, Gramaglia I, Panoskaltsis-Mortari A, Murphy WJ, et al. (2003) IL-10 and TGF-beta induce alloreactive CD4+CD25- T cells to acquire regulatory cell function. Blood 101: 5076–5083.
- 242. Chen ML, Pittet MJ, Gorelik L, Flavell RA, Weissleder R, et al. (2005) Regulatory T cells suppress tumor-specific CD8 T cell cytotoxicity through TGF-beta signals in vivo. Proc Natl Acad Sci U S A 102: 419–424.
- 243. Fahlen L, Read S, Gorelik L, Hurst SD, Coffman RL, et al. (2005) T cells that cannot respond to TGF-beta escape control by CD4(+)CD25(+) regulatory T cells. J Exp Med 201: 737–746.
- 244. Chen W, Jin W, Hardegen N, Lei KJ, Li L, et al. (2003) Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med 198: 1875–1886.
- 245. Horwitz DA, Zheng SG, Gray JD (2003) The role of the combination of IL-2 and TGF-beta or IL-10 in the generation and function of CD4+ CD25+ and CD8+ regulatory T cell subsets. J Leukoc Biol 74: 471–478.
- 246. Apostolou I, von Boehmer H (2004) In vivo instruction of suppressor commitment in naive T cells. J Exp Med 199: 1401–1408.
- 247. Andersson J, Tran DQ, Pesu M, Davidson TS, Ramsey H, et al. (2008) CD4+ FoxP3+ regulatory T cells confer infectious tolerance in a TGF-beta-dependent manner. J Exp Med 205: 1975–1981.
- 248. Jonuleit H, Schmitt E, Kakirman H, Stassen M, Knop J, et al. (2002) Infectious tolerance: human CD25(+) regulatory T cells convey suppressor activity to conventional CD4(+) T helper cells. J Exp Med 196: 255–260.
- 249. Filaci G, Fravega M, Negrini S, Procopio F, Fenoglio D, et al. (2004) Nonantigen specific CD8+ T suppressor lymphocytes originate from CD8+CD28- T cells and inhibit both T-cell proliferation and CTL function. Hum Immunol 65: 142–156.
- 250. Lau AW, Biester S, Cornall RJ, Forrester JV (2008) Lipopolysaccharide-activated IL-10-secreting dendritic cells suppress experimental autoimmune uveoretinitis by MHCII-dependent activation of CD62L-expressing regulatory T cells. J Immunol 180: 3889–3899.
- 251. Zhang N, Schroppel B, Lal G, Jakubzick C, Mao X, et al. (2009) Regulatory T cells sequentially migrate from inflamed tissues to draining lymph nodes to suppress the alloimmune response. Immunity 30: 458–469.
- 252. Haribhai D, Lin W, Relland LM, Truong N, Williams CB, et al. (2007) Regulatory T cells dynamically control the primary immune response to foreign antigen. J Immunol 178: 2961–2972.
- 253. Tadokoro CE, Shakhar G, Shen S, Ding Y, Lino AC, et al. (2006) Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo. J Exp Med 203: 505–511.
- 254. Veldhoen M, Moncrieffe H, Hocking RJ, Atkins CJ, Stockinger B (2006) Modulation of dendritic cell function by naive and regulatory CD4+ T cells. J Immunol 176: 6202–6210.
- 255. Steinbrink K, Graulich E, Kubsch S, Knop J, Enk AH (2002) CD4(+) and CD8(+) anergic T cells induced by interleukin-10-treated human dendritic cells display antigen-specific suppressor activity. Blood 99: 2468–2476.
- 256. Bellinghausen I, Konig B, Bottcher I, Knop J, Saloga J (2006) Inhibition of human allergic T-helper type 2 immune responses by induced regulatory T cells requires the combination of interleukin-10-treated dendritic cells and transforming growth factor-beta for their induction. Clin Exp Allergy 36: 1546–1555.
- 257. Depinay N, Hacini F, Beghdadi W, Peronet R, Mecheri S (2006) Mast cell-dependent down-regulation of antigen-specific immune responses by mosquito bites. J Immunol 176: 4141–4146.
- 258. Möbs C, Slotosch C, Loffler H, Pfutzner W, Hertl M (2008) Cellular and humoral mechanisms of immune tolerance in immediate-type allergy induced by specific immunotherapy. Int Arch Allergy Immunol 147: 171–178.
- 259. Suffia IJ, Reckling SK, Piccirillo CA, Goldszmid RS, Belkaid Y (2006) Infected site-restricted Foxp3+ natural regulatory T cells are specific for microbial antigen. J Exp Med 203: 777–788.
- 260. Anderson CF, Mendez S, Sacks DL (2005) Nonhealing infection despite Th1 polarization produced by a strain of Leishmania major in C57BL/6 mice. J Immunol 174: 2934–2941.
- 261. Bowen DG, Zen M, Holz L, Davis T, McCaughan GW, et al. (2004) The site of primary T cell activation is a determinant of the balance between intrahepatic tolerance and immunity. J Clin Invest 114: 701–712.
- 262. Wei J, Duramad O, Perng OA, Reiner SL, Liu YJ, et al. (2007) Antagonistic nature of T helper 1/2 developmental programs in opposing peripheral induction of Foxp3+ regulatory T cells. Proc Natl Acad Sci U S A 104: 18169–18174.
- 263. Kapp JA, Honjo K, Kapp LM, Goldsmith K, Bucy RP (2007) Antigen, in the presence of TGF-beta, induces up-regulation of FoxP3gfp+ in CD4+ TCR transgenic T cells that mediate linked suppression of CD8+ T cell responses. J Immunol 179: 2105–2114.
- 264. Oldenhove G, Bouladoux N, Wohlfert EA, Hall JA, Chou D, et al. (2009) Decrease of Foxp3+ Treg cell number and acquisition of effector cell phenotype during lethal infection. Immunity 31: 772–786.
- 265. Littman DR, Rudensky AY (2010) Th17 and regulatory T cells in mediating and restraining inflammation. Cell 140: 845–858.
- 266. Hafalla JC, Morrot A, Sano G, Milon G, Lafaille JJ, et al. (2003) Early self-regulatory mechanisms control the magnitude of CD8+ T cell responses against liver stages of murine malaria. J Immunol 171: 964–970.
- 267. Tarun AS, Dumpit RF, Camargo N, Labaied M, Liu P, et al. (2007) Protracted sterile protection with Plasmodium yoelii pre-erythrocytic genetically attenuated parasite malaria vaccines is independent of significant liver-stage persistence and is mediated by CD8+ T cells. J Infect Dis 196: 608–616.
- 268. Weiss WR, Mellouk S, Houghten RA, Sedegah M, Kumar S, et al. (1990) Cytotoxic T cells recognize a peptide from the circumsporozoite protein on malaria-infected hepatocytes. J Exp Med 171: 763–773.
- 269. Saute F, Aponte J, Almeda J, Ascaso C, Vaz N, et al. (2003) Malaria in southern Mozambique: incidence of clinical malaria in children living in a rural community in Manhica district. Trans R Soc Trop Med Hyg 97: 655–660.
- 270. Guerra CA, Gikandi PW, Tatem AJ, Noor AM, Smith DL, et al. (2008) The limits and intensity of Plasmodium falciparum transmission: implications for malaria control and elimination worldwide. PLoS Med 5: e38.
- 271. Hay SI, Guerra CA, Tatem AJ, Atkinson PM, Snow RW (2005) Urbanization, malaria transmission and disease burden in Africa. Nat Rev Microbiol 3: 81–90.
- 272. Hay SI, Smith DL, Snow RW (2008) Measuring malaria endemicity from intense to interrupted transmission. Lancet Infect Dis 8: 369–378.
- 273. Beier JC, Killeen GF, Githure JI (1999) Short report: Entomologic inoculation rates and Plasmodium falciparum malaria prevalence in Africa. Am J Trop Med Hyg 61: 109–113.
- 274. Reyburn H, Mbatia R, Drakeley C, Bruce J, Carneiro I, et al. (2005) Association of transmission intensity and age with clinical manifestations and case fatality of severe Plasmodium falciparum malaria. JAMA 293: 1461–1470.
- 275. Wyler DJ, Brown J (1977) Malaria antigen-specific T-cell responsiveness during infection with Plasmodium falciparum. Clin Exp Immunol 29: 401–407.
- 276. Greenwood BM, Bradley-Moore AM, Bryceson AD, Palit A (1972) Immunosuppression in children with malaria. Lancet 1: 169–172.
- 277. Whittle HC, Brown J, Marsh K, Greenwood BM, Seidelin P, et al. (1984) T-cell control of Epstein-Barr virus-infected B cells is lost during P. falciparum malaria. Nature 312: 449–450.
- 278. Cook IF (1985) Herpes zoster in children following malaria. J Trop Med Hyg 88: 261–264.
- 279. Williamson WA, Greenwood BM (1978) Impairment of the immune response to vaccination after acute malaria. Lancet 1: 1328–1329.
- 280. Bradley-Moore AM, Greenwood BM, Bradley AK, Bartlett A, Bidwell DE, et al. (1985) Malaria chemoprophylaxis with chloroquine in young Nigerian children. II. Effect on the immune response to vaccination. Ann Trop Med Parasitol 79: 563–573.
- 281. Riley EM, Andersson G, Otoo LN, Jepsen S, Greenwood BM (1988) Cellular immune responses to Plasmodium falciparum antigens in Gambian children during and after an acute attack of falciparum malaria. Clin Exp Immunol 73: 17–22.
- 282. Sacci JB Jr, Ribeiro JM, Huang F, Alam U, Russell JA, et al. (2005) Transcriptional analysis of in vivo Plasmodium yoelii liver stage gene expression. Mol Biochem Parasitol 142: 177–183.
- 283. Mikolajczak SA, Silva-Rivera H, Peng X, Tarun AS, Camargo N, et al. (2008) Distinct malaria parasite sporozoites reveal transcriptional changes that cause differential tissue infection competence in the mosquito vector and mammalian host. Mol Cell Biol 29: 6196–6207.
- 284. Siau A, Silvie O, Franetich JF, Yalaoui S, Marinach C, et al. (2008) Temperature shift and host cell contact up-regulate sporozoite expression of Plasmodium falciparum genes involved in hepatocyte infection. PLoS Pathog 4: e1000121.
- 285. Senti G, Prinz Vavricka BM, Erdmann I, Diaz MI, Markus R, et al. (2008) Intralymphatic allergen administration renders specific immunotherapy faster and safer: a randomized controlled trial. Proc Natl Acad Sci U S A 105: 17908–17912.
- 286. Houot R, Perrot I, Garcia E, Durand I, Lebecque S (2006) Human CD4+CD25high regulatory T cells modulate myeloid but not plasmacytoid dendritic cells activation. J Immunol 176: 5293–5298.
- 287. Bayry J, Triebel F, Kaveri SV, Tough DF (2007) Human dendritic cells acquire a semimature phenotype and lymph node homing potential through interaction with CD4+CD25+ regulatory T cells. J Immunol 178: 4184–4193.
- 288. Anderson AE, Sayers BL, Haniffa MA, Swan DJ, Diboll J, et al. (2008) Differential regulation of naive and memory CD4+ T cells by alternatively activated dendritic cells. J Leukoc Biol 84: 124–133.
- 289. Krishnegowda G, Hajjar AM, Zhu J, Douglass EJ, Uematsu S, et al. (2005) Induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols of Plasmodium falciparum: cell signaling receptors, glycosylphosphatidylinositol (GPI) structural requirement, and regulation of GPI activity. J Biol Chem 280: 8606–8616.
- 290. Allan RS, Waithman J, Bedoui S, Jones CM, Villadangos JA, et al. (2006) Migratory dendritic cells transfer antigen to a lymph node-resident dendritic cell population for efficient CTL priming. Immunity 25: 153–162.
- 291. Lammermann T, Sixt M (2008) The microanatomy of T-cell responses. Immunol Rev 221: 26–43.
- 292. Rothenfusser S, Tuma E, Endres S, Hartmann G (2002) Plasmacytoid dendritic cells: the key to CpG. Hum Immunol 63: 1111–1119.
- 293. Kissenpfennig A, Henri S, Dubois B, Laplace-Builhe C, Perrin P, et al. (2005) Dynamics and function of Langerhans cells in vivo: Dermal dendritic cells colonize lymph node areas distinct from slower migrating Langerhans cells. Immunity 22: 643–654.
- 294. Macatonia SE, Knight SC, Edwards AJ, Griffiths SF, Fryer P (1987) Localization of antigen on lymph node dendritic cells after exposure to the contact sensitizer fluorescein isothiocyanate. Functional and morphological studies. J Exp Med 166: 1654–1667.
- 295. Randolph GJ, Ochando J, Partida-Sanchez S (2008) Migration of dendritic cell subsets and their precursors. Annu Rev Immunol 26: 293–316.
- 296. Geissmann F, Dieu-Nosjean MC, Dezutter C, Valladeau J, Kayal S, et al. (2002) Accumulation of immature Langerhans cells in human lymph nodes draining chronically inflamed skin. J Exp Med 196: 417–430.
- 297. Stewart MJ, Vanderberg JP (1988) Malaria sporozoites leave behind trails of circumsporozoite protein during gliding. J Protozool 35: 389–393.
- 298. Mota MM, Pradel G, Vanderberg JP, Hafalla JC, Frevert U, et al. (2001) Migration of Plasmodium sporozoites through cells before infection. Science 291: 141–144.
- 299. Amino R, Giovanni D, Thiberge S, e al (2008) Host cell traversal is important for progression of the malaria parasite through the dermis to the liver. Cell Host Microbe 3: 88–96.
- 300. Stewart MJ, Vanderberg JP (1992) Electron microscopic analysis of circumsporozoite protein trail formation by gliding malaria sporozoites. J Protozool 39: 663–671.
- 301. Singh AP, Buscaglia CA, Wang Q, Levay A, Nussenzweig DR, et al. (2008) Plasmodium circumsporozoite protein promotes the development of the liver stages of the parasite. Cell 131: 492–504.
- 302. Doolan DL, Sedegah M, Hedstrom RC, Hobart P, Charoenvit Y, et al. (1996) Circumventing genetic restriction of protection against malaria with multigene DNA immunization: CD8+ cell-, interferon gamma-, and nitric oxide-dependent immunity. J Exp Med 183: 1739–1746.
- 303. Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B (2006) TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 24: 179–189.
- 304. Bettelli E, Korn T, Oukka M, Kuchroo VK (2008) Induction and effector functions of T(H)17 cells. Nature 453: 1051–1057.
- 305. Chen X, Das R, Komorowski R, Beres A, Hessner MJ, et al. (2009) Blockade of interleukin-6 signaling augments regulatory T-cell reconstitution and attenuates the severity of graft-versus-host disease. Blood 114: 891–900.
- 306. Piconese S, Gri G, Tripodo C, Musio S, Gorzanelli A, et al. (2009) Mast cells counteract regulatory T cell suppression through interleukin-6 and OX40/OX40L axis toward Th17 cell differentiation. Blood 114: 2639–2648.
- 307. Pasare C, Medzhitov R (2003) Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science 299: 1033–1036.
- 308. Longhi MP, Wright K, Lauder SN, Nowell MA, Jones GW, et al. (2008) Interleukin-6 is crucial for recall of influenza-specific memory CD4 T cells. PLoS Pathog 4: e1000006.
- 309. Heit A, Gebhardt F, Lahl K, Neuenhahn M, Schmitz F, et al. (2008) Circumvention of regulatory CD4+ T cell activity during cross-priming strongly enhances T cell-mediated immunity. Eur J Immunol 38: 1585–1597.
- 310. Tan PH, Sagoo P, Chan C, Yates JB, Campbell J, et al. (2005) Inhibition of NF-kappa B and oxidative pathways in human dendritic cells by antioxidative vitamins generates regulatory T cells. J Immunol 174: 7633–7644.
- 311. Cong Y, Konrad A, Iqbal N, Hatton RD, Weaver CT, et al. (2005) Generation of antigen-specific, Foxp3-expressing CD4+ regulatory T cells by inhibition of APC proteosome function. J Immunol 174: 2787–2795.
- 312. Cockburn IA, Chakravarty S, Overstreet MG, Garcia-Sastre A, Zavala F (2008) Memory CD8+ T cell responses expand when antigen presentation overcomes T cell self-regulation. J Immunol 180: 64–71.
- 313. Cowan G, Krishna S, Crisanti A, Robson KJ (1992) Expression of thrombospondin-related anonymous protein in Plasmodium falciparum sporozoites. Lancet 339: 1412–1413.
- 314. Omer FM, de Souza JB, Corran PH, Sultan AA, Riley EM (2003) Activation of transforming growth factor β by malaria parasite-derived metalloproteinases and a thrombospondin-like molecule. J Exp Med 198: 1817–1827.
- 315. Lawler J (2000) The functions of thrombospondin-1 and-2. Curr Opin Cell Biol 12: 634–640.
- 316. Futagami Y, Sugita S, Vega J, Ishida K, Takase H, et al. (2007) Role of thrombospondin-1 in T cell response to ocular pigment epithelial cells. J Immunol 178: 6994–7005.
- 317. Walther M, Tongren JE, Andrews L, Korbel D, King E, et al. (2005) Upregulation of TGF-beta, FOXP3, and CD4+CD25+ regulatory T cells correlates with more rapid parasite growth in human malaria infection. Immunity 23: 287–296.
- 318. Omer FM, de Souza JB, Riley EM (2003) Differential induction of TGF-beta regulates proinflammatory cytokine production and determines the outcome of lethal and nonlethal Plasmodium yoelii infections. J Immunol 171: 5430–5436.
- 319. Hisaeda H, Maekawa Y, Iwakawa D, Okada H, Himeno K, et al. (2004) Escape of malaria parasites from host immunity requires CD4+ CD25+ regulatory T cells. Nat Med 10: 29–30.
- 320. Birch KE, Vukmanovic-Stejic M, Reed JR, Akbar AN, Rustin MH (2005) The immunomodulatory effects of regulatory T cells: implications for immune regulation in the skin. Br J Dermatol 152: 409–417.
- 321. Vukmanovic-Stejic M, Agius E, Booth N, Dunne PJ, Lacy KE, et al. (2008) The kinetics of CD4+Foxp3+ T cell accumulation during a human cutaneous antigen-specific memory response in vivo. J Clin Invest 118: 3639–3650.
- 322. Akbar AN, Vukmanovic-Stejic M, Taams LS, Macallan DC (2007) The dynamic co-evolution of memory and regulatory CD4+ T cells in the periphery. Nat Rev Immunol 7: 231–237.
- 323. Vukmanovic-Stejic M, Zhang Y, Cook JE, Fletcher JM, McQuaid A, et al. (2006) Human CD4+ CD25hi Foxp3+ regulatory T cells are derived by rapid turnover of memory populations in vivo. J Clin Invest 116: 2423–2433.
- 324. Janssens W, Carlier V, Wu B, VanderElst L, Jacquemin MG, et al. (2003) CD4+CD25+ T cells lyse antigen-presenting B cells by Fas-Fas ligand interaction in an epitope-specific manner. J Immunol 171: 4604–4612.
- 325. Florens L, Washburn MP, Raine JD, Anthony RM, Grainger M, et al. (2002) A proteomic view of the Plasmodium falciparum life cycle. Nature 419: 520–526.
- 326. Hope IA, Hall R, Simmons DL, Hyde JE, Scaife JG (1984) Evidence for immunological cross-reaction between sporozoites and blood stages of a human malaria parasite. Nature 308: 191–194.
- 327. Meraldi V, Nebie I, Tiono AB, Diallo D, Sanogo E, et al. (2004) Natural antibody response to Plasmodium falciparum Exp-1, MSP-3 and GLURP long synthetic peptides and association with protection. Parasite Immunol 26: 265–272.
- 328. Couper KN, Blount DG, Wilson MS, Hafalla JC, Belkaid Y, et al. (2008) IL-10 from CD4+ CD25− Foxp3− CD127− adaptive regulatory T cells modulates parasite clearance and pathology during malaria infection. PLoS Pathog 4: e1000004.
- 329. Scholzen A, Mittag D, Rogerson SJ, Cooke BM, Plebanski M (2009) Plasmodium falciparum-mediated induction of human CD25hi Foxp3hi CD4 T cells is independent of direct TCR stimulation and requires IL-2, IL-10 and TGFβ. PLoS Pathog 5: e1000543.
- 330. Urban BC, Ferguson DJP, Pain A, Willcox N, Plebanski M, et al. (1999) Plasmodium falciparum infected erythrocytes modulate the maturation of dendritic cells. Nature 400: 73–77.
- 331. Ocaña-Morgner C, Mota MM, Rodriguez A (2003) Malaria blood stage suppression of liver stage immunity by dendritic cells. J Exp Med 197: 143–151.
- 332. Wilson NS, Behrens GM, Lundie RJ, Smith CM, Waithman J, et al. (2006) Systemic activation of dendritic cells by Toll-like receptor ligands or malaria infection impairs cross-presentation and antiviral immunity. Nat Immunol 7: 165–172.
- 333. Belkaid Y, Blank RB, Suffia I (2006) Natural regulatory T cells and parasites: a common quest for host homeostasis. Immunol Rev 212: 287–300.
- 334. Todryk SM, Bejon P, Mwangi T, Plebanski M, Urban B, et al. (2008) Correlation of memory T cell responses against TRAP with protection from clinical malaria, and CD4 CD25 high T cells with susceptibility in Kenyans. PLoS One 3: e2027.
- 335. Kumar KA, Sano G, Boscardin S, Nussenzweig RS, Nussenzweig MC, et al. (2006) The circumsporozoite protein is an immunodominant protective antigen in irradiated sporozoites. Nature 444: 937–940.
- 336. Khusmith S, Charoenvit Y, Kumar S, Sedegah M, Beaudoin RL, et al. (1991) Protection against malaria by vaccination with sporozoite surface protein 2 plus CS protein. Science 252: 715–718.
- 337. Kumar KA, Baxter P, Tarun AS, Kappe SH, Nussenzweig V (2009) Conserved protective mechanisms in radiation and genetically attenuated uis3(-) and uis4(-) Plasmodium sporozoites. PLoS One 4: e4480.
- 338. Overstreet MG, Cockburn IA, Chen YC, Zavala F (2008) Protective CD8 T cells against Plasmodium liver stages: immunobiology of an ‘unnatural’ immune response. Immunol Rev 225: 272–283.
- 339. Klein L, Khazaie K, von Boehmer H (2003) In vivo dynamics of antigen specific regulatory T cells not predicted from behavior in vitro. Proc Natl Acad Sci U S A 100: 8886–8891.
- 340. Moorthy VS, Pinder M, Reece WH, Watkins K, Atabani S, et al. (2003) Safety and immunogenicity of DNA/modified vaccinia virus ankara malaria vaccination in African adults. J Infect Dis 188: 1239–1244.
- 341. Hill AV, Reece W, Gothard P, Moorthy V, Roberts M, et al. (2000) DNA-based vaccines for malaria: a heterologous prime-boost immunisation strategy. Dev Biol (Basel) 104: 171–179.
- 342. Macete EV, Sacarlal J, Aponte JJ, Leach A, Navia MM, et al. (2007) 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 8: 11.
- 343. Kester KE, Cummings JF, Ockenhouse CF, Nielsen R, Hall BT, et al. (2008) 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 26: 2191–2202.
- 344. Kester KE, McKinney DA, Tornieporth N, Ockenhouse CF, Heppner DG, et al. (2001) Efficacy of recombinant circumsporozoite protein vaccine regimens against experimental Plasmodium falciparum malaria. J Infect Dis 183: 640–647.
- 345. Kester KE, McKinney D, Tornieporth N, Ockenhouse CF, Heppner DG, et al. (2007) 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 25: 5359–5366.
- 346. Daubersies P, Thomas AW, Millet P, Brahimi K, Langermans JA, et al. (2000) Protection against Plasmodium falciparum malaria in chimpanzees by immunization with the conserved preerythrocytic liver-stage antigen 3. Nat Med 6: 1258–1263.
- 347. Toure-Balde A, Perlaza BL, Sauzet JP, Ndiaye M, Aribot G, et al. (2009) Evidence for multiple B- and T-cell epitopes in Plasmodium falciparum liver-stage antigen 3. Infect Immun 77: 1189–1196.
- 348. McCallum FJ, Persson KE, Mugyenyi CK, Fowkes FJ, Simpson JA, et al. (2008) Acquisition of growth-inhibitory antibodies against blood-stage Plasmodium falciparum. PLoS One 3: e3571.
- 349. Guinovart C, Aponte JJ, Sacarlal J, Aide P, Leach A, et al. (2009) Insights into long-lasting protection induced by RTS,S/AS02A malaria vaccine: further results from a phase IIb trial in Mozambican children. PLoS One 4: e5165.
- 350. Whalen J (2009) Patent rescindication/release (Glaxo-Smith and Kline). Wall Street Journal.
- 351. Law I, Ilett KF, Hackett LP, Page-Sharp M, Baiwog F, et al. (2008) Transfer of chloroquine and desethylchloroquine across the placenta and into milk in Melanesian mothers. Br J Clin Pharmacol 65: 674–679.
- 352. Okiro EA, Hay SI, Gikandi PW, Sharif SK, Noor AM, et al. (2007) The decline in paediatric malaria admissions on the coast of Kenya. Malar J 6: 151.
- 353. O'Meara WP, Bejon P, Mwangi TW, Okiro EA, Peshu N, et al. (2008) Effect of a fall in malaria transmission on morbidity and mortality in Kilifi, Kenya. Lancet 372: 1555–1562.
- 354. Penny MA, Maire N, Studer A, Schapira A, Smith TA (2008) What should vaccine developers ask? Simulation of the effectiveness of malaria vaccines. PLoS One 3: e3193.
- 355. Martinez PA, Yandar N, Lesmes LP, Forero M, Perez-Leal O, et al. (2009) Passive transfer of Plasmodium falciparum MSP-2 pseudopeptide-induced antibodies efficiently controlled parasitemia in Plasmodium berghei-infected mice. Peptides 30: 330–342.
- 356. Amante FH, Good MF (1997) Prolonged Th1-like response generated by a Plasmodium yoelii-specific T cell clone allows complete clearance of infection in reconstituted mice. Parasite Immunol 19: 111–126.
- 357. Brahimi K, Badell E, Sauzet JP, BenMohamed L, Daubersies P, et al. (2001) Human antibodies against Plasmodium falciparum liver-stage antigen 3 cross-react with Plasmodium yoelii preerythrocytic-stage epitopes and inhibit sporozoite invasion in vitro and in vivo. Infect Immun 69: 3845–3852.
- 358. Bruna-Romero O, Gonzalez-Aseguinolaza G, Hafalla JC, Tsuji M, Nussenzweig RS (2001) Complete, long-lasting protection against malaria of mice primed and boosted with two distinct viral vectors expressing the same plasmodial antigen. Proc Natl Acad Sci U S A 98: 11491–11496.
- 359. Burns JM Jr, Flaherty PR, Nanavati P, Weidanz WP (2004) Protection against Plasmodium chabaudi malaria induced by immunization with apical membrane antigen 1 and merozoite surface protein 1 in the absence of gamma interferon or interleukin-4. Infect Immun 72: 5605–5612.
- 360. Cabrera EJ, Barr ML, Silverman PH (1977) Long-term studies on rhesus monkeys (Macaca mulatta) immunized against Plasmodium knowlesi. Infect Immun 15: 461–465.
- 361. Cao Y, Zhang D, Pan W (2009) Construction of transgenic Plasmodium berghei as a model for evaluation of blood-stage vaccine candidate of Plasmodium falciparum chimeric protein 2.9. PLoS One 4: e6894.
- 362. Charoenvit Y, Brice GT, Bacon D, Majam V, Williams J, et al. (2004) A small peptide (CEL-1000) derived from the beta-chain of the human major histocompatibility complex class II molecule induces complete protection against malaria in an antigen-independent manner. Antimicrob Agents Chemother 48: 2455–2463.
- 363. Charoenvit Y, Collins WE, Jones TR, Millet P, Yuan L, et al. (1991) Inability of malaria vaccine to induce antibodies to a protective epitope within its sequence. Science 251: 668–671.
- 364. Charoenvit Y, Majam VF, Corradin G, Sacci JB Jr, Wang R, et al. (1999) CD4(+) T-cell- and gamma interferon-dependent protection against murine malaria by immunization with linear synthetic peptides from a Plasmodium yoelii 17-kilodalton hepatocyte erythrocyte protein. Infect Immun 67: 5604–5614.
- 365. Charoenvit Y, Sedegah M, Yuan LF, Gross M, Cole C, et al. (1990) Active and passive immunization against Plasmodium yoelii sporozoites. Bull World Health Organ 68: Suppl26–32.
- 366. Chatterjee S, Druilhe P, Wery M (1999) Irradiated sporozoites prime mice to produce high antibody titres upon viable Plasmodium berghei sporozoite challenge, which act upon liver-stage development. Parasitology 118: 219–225.
- 367. Chatterjee S, Francois G, Druilhe P, Timperman G, Wery M (1996) Immunity to Plasmodium berghei exoerythrocytic forms derived from irradiated sporozoites. Parasitol Res 82: 297–303.
- 368. Chatterjee S, Ngonseu E, Van Overmeir C, Correwyn A, Druilhe P, et al. (2001) Rodent malaria in the natural host–irradiated sporozoites of Plasmodium berghei induce liver-stage specific immune responses in the natural host Grammomys surdaster and protect immunized Grammomys against P. berghei sporozoite challenge. Afr J Med Med Sci. 30. pp. 25–33.
- 369. Chattopadhyay R, Conteh S, Li M, James ER, Epstein JE, et al. (2009) The Effects of radiation on the safety and protective efficacy of an attenuated Plasmodium yoelii sporozoite malaria vaccine. Vaccine 27: 3675–3680.
- 370. Clark IA, Allison AC, Cox FE (1976) Protection of mice against Babesia and Plasmodium with BCG. Nature 259: 309–311.
- 371. Clark IA, Cox FE, Allison AC (1977) Protection of mice against Babesia spp. and Plasmodium spp. with killed Corynebacterium parvum. Parasitology 74: 9–18.
- 372. Daly TM, Long CA (1995) Humoral response to a carboxyl-terminal region of the merozoite surface protein-1 plays a predominant role in controlling blood-stage infection in rodent malaria. J Immunol 155: 236–243.
- 373. Degano P, Schneider J, Hannan CM, Gilbert SC, Hill AV (1999) Gene gun intradermal DNA immunization followed by boosting with modified vaccinia virus Ankara: enhanced CD8+ T cell immunogenicity and protective efficacy in the influenza and malaria models. Vaccine 18: 623–632.
- 374. Doolan DL, Hoffman SL (1999) IL-12 and NK cells are required for antigen-specific adaptive immunity against malaria initiated by CD8+ T cells in the Plasmodium yoelii model. J Immunol 163: 884–892.
- 375. Egan JE, Weber JL, Ballou WR, Hollingdale MR, Majarian WR, et al. (1987) Efficacy of murine malaria sporozoite vaccines: implications for human vaccine development. Science 236: 453–456.
- 376. Gilbert SC, Schneider J, Plebanski M, Hannan CM, Blanchard TJ, et al. (1999) Ty virus-like particles, DNA vaccines and Modified Vaccinia Virus Ankara; comparisons and combinations. Biol Chem 380: 299–303.
- 377. Gramzinski RA, Doolan DL, Sedegah M, Davis HL, Krieg AM, et al. (2001) Interleukin-12- and gamma interferon-dependent protection against malaria conferred by CpG oligodeoxynucleotide in mice. Infect Immun 69: 1643–1649.
- 378. Gruner AC, Mauduit M, Tewari R, Romero JF, Depinay N, et al. (2007) Sterile protection against malaria is independent of immune responses to the circumsporozoite protein. PLoS One 2: e1371.
- 379. Guebre-Xabier M, Schwenk R, Krzych U (1999) Memory phenotype CD8(+) T cells persist in livers of mice protected against malaria by immunization with attenuated Plasmodium berghei sporozoites. Eur J Immunol 29: 3978–3986.
- 380. Hirunpetcharat C, Vukovic P, Liu XQ, Kaslow DC, Miller LH, et al. (1999) Absolute requirement for an active immune response involving B cells and Th cells in immunity to Plasmodium yoelii passively acquired with antibodies to the 19-kDa carboxyl-terminal fragment of merozoite surface protein-1. J Immunol 162: 7309–7314.
- 381. Hirunpetcharat C, Wipasa J, Sakkhachornphop S, Nitkumhan T, Zheng YZ, et al. (2003) CpG oligodeoxynucleotide enhances immunity against blood-stage malaria infection in mice parenterally immunized with a yeast-expressed 19 kDa carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (MSP1(19)) formulated in oil-based Montanides. Vaccine 21: 2923–2932.
- 382. Hoffman SL, Berzofsky JA, Isenbarger D, Zeltser E, Majarian WR, et al. (1989) Immune response gene regulation of immunity to Plasmodium berghei sporozoites and circumsporozoite protein vaccines. Overcoming genetic restriction with whole organism and subunit vaccines. J Immunol 142: 3581–3584.
- 383. Hoffman SL, Crutcher JM, Puri SK, Ansari AA, Villinger F, et al. (1997) Sterile protection of monkeys against malaria after administration of interleukin-12. Nat Med 3: 80–83.
- 384. Hunter RL, Kidd MR, Olsen MR, Patterson PS, Lal AA (1995) Induction of long-lasting immunity to Plasmodium yoelii malaria with whole blood-stage antigens and copolymer adjuvants. J Immunol 154: 1762–1769.
- 385. Imai T, Shen J, Chou B, Duan X, Tu L, et al. (2010) Involvement of CD8(+) T cells in protective immunity against murine blood-stage infection with Plasmodium yoelii 17XL strain. Eur J Immunol.
- 386. Jaffe RI, Lowell GH, Gordon DM (1990) Differences in susceptibility among mouse strains to infection with Plasmodium berghei (ANKA clone) sporozoites and its relationship to protection by gamma-irradiated sporozoites. Am J Trop Med Hyg 42: 309–313.
- 387. Aly AS, Mikolajczak SA, Rivera HS, Camargo N, Jacobs-Lorena V, et al. (2008) Targeted deletion of SAP1 abolishes the expression of infectivity factors necessary for successful malaria parasite liver infection. Mol Microbiol 69: 152–163.
- 388. Aly AS, Downie MJ, Mamoun CB, Kappe SH (2010) Subpatent infection with Nucleoside Transporter 1-deficient Plasmodium blood stage parasites confers sterile protection against lethal malaria in mice. Cell Microbiol.
- 389. Draper SJ, Goodman AL, Biswas S, Forbes EK, Moore AC, et al. (2009) Recombinant viral vaccines expressing merozoite surface protein-1 induce antibody- and T cell-mediated multistage protection against malaria. Cell Host Microbe 5: 95–105.
- 390. Jobe O, Donofrio G, Sun G, Liepinsh D, Schwenk R, et al. (2009) Immunization with radiation-attenuated Plasmodium berghei sporozoites induces liver cCD8alpha+DC that activate CD8+T cells against liver-stage malaria. PLoS One 4: e5075.
- 391. Jones TR, Obaldia Nr, Gramzinski RA, Hoffman SL (2000) Repeated infection of Aotus monkeys with Plasmodium falciparum induces protection against subsequent challenge with homologous and heterologous strains of parasite. Am J Trop Med Hyg 62: 675–680.
- 392. Kaba SA, Brando C, Guo Q, Mittelholzer C, Raman S, et al. (2009) A nonadjuvanted polypeptide nanoparticle vaccine confers long-lasting protection against rodent malaria. J Immunol 183: 7268–7277.
- 393. Kaur A, Kinhikar AG, Singh PP (2004) Bioimmunotherapy of rodent malaria: co-treatment with recombinant mouse granulocyte-macrophage colony-stimulating factor and an enkephalin fragment peptide Tyr-Gly-Gly. Acta Trop 91: 27–41.
- 394. Khan ZM, Vanderberg JP (1992) Specific inflammatory cell infiltration of hepatic schizonts in BALB/c mice immunized with attenuated Plasmodium yoelii sporozoites. Int Immunol 4: 711–718.
- 395. Khullar N, Sehgal S (1990) Use of adjuvants in modulating the behaviour of Plasmodium berghei. Indian J Exp Biol 28: 1112–1117.
- 396. Khusmith S, Sedegah M, Hoffman SL (1994) Complete protection against Plasmodium yoelii by adoptive transfer of a CD8+ cytotoxic T-cell clone recognizing sporozoite surface protein 2. Infect Immun 62: 2979–2983.
- 397. Kumar S, Good MF, Dontfraid F, Vinetz JM, Miller LH (1989) Interdependence of CD4+ T cells and malarial spleen in immunity to Plasmodium vinckei vinckei. Relevance to vaccine development. J Immunol 143: 2017–2023.
- 398. Lanar DE, Tine JA, de Taisne C, Seguin MC, Cox WI, et al. (1996) Attenuated vaccinia virus-circumsporozoite protein recombinants confer protection against rodent malaria. Infect Immun 64: 1666–1671.
- 399. Li S, Rodrigues M, Rodriguez D, Rodriguez JR, Esteban M, et al. (1993) Priming with recombinant influenza virus followed by administration of recombinant vaccinia virus induces CD8+ T-cell-mediated protective immunity against malaria. Proc Natl Acad Sci U S A 90: 5214–5218.
- 400. Ling IT, Ogun SA, Holder AA (1994) Immunization against malaria with a recombinant protein. Parasite Immunol 16: 63–67.
- 401. Marussig M, Renia L, Motard A, Miltgen F, Petour P, et al. (1997) Linear and multiple antigen peptides containing defined T and B epitopes of the Plasmodium yoelii circumsporozoite protein: antibody-mediated protection and boosting by sporozoite infection. Int Immunol 9: 1817–1824.
- 402. Mauduit M, Gruner AC, Tewari R, Depinay N, Kayibanda M, et al. (2009) A role for immune responses against non-CS components in the cross-species protection induced by immunization with irradiated malaria sporozoites. PLoS One 4: e7717.
- 403. McColm AA, Bomford R, Dalton L (1982) A comparison of saponin with other adjuvants for the potentiation of protective immunity by a killed Plasmodium yoelii vaccine in the mouse. Parasite Immunol 4: 337–347.
- 404. McColm AA, Dalton L (1983) Heterologous immunity in rodent malaria: comparison of the degree of cross-immunity generated by vaccination with that produced by exposure to live infection. Ann Trop Med Parasitol 77: 355–377.
- 405. Mueller AK, Camargo N, Kaiser K, Andorfer C, Frevert U, et al. (2005) Plasmodium liver stage developmental arrest by depletion of a protein at the parasite– host interface. Proc Natl Acad Sci U S A 102: 3022–3027.
- 406. Orjih AU, Nussenzweig RS (1980) Immunization against rodent malaria with cryopreserved irradiated sporozoites of Plasmodium berghei. Am J Trop Med Hyg 29: 343–347.
- 407. Patterson PS, Bosshardt SC, Udhayukumar V, Xiao L, Kidd M, et al. (1999) Prolonged expression of IFNgamma induced by protective blood-stage immunization against Plasmodium yoelii malaria. Vaccine 18: 173–180.
- 408. Perlaza BL, Valencia AZ, Zapata C, Castellanos A, Sauzet JP, et al. (2008) Protection against Plasmodium falciparum challenge induced in Aotus monkeys by liver-stage antigen-3-derived long synthetic peptides. Eur J Immunol 38: 2610–2615.
- 409. Roberts DW, Rank RG, Weidanz WP, Finerty JF (1977) Prevention of recrudescent malaria in nude mice by thymic grafting or by treatment with hyperimmune serum. Infect Immun 16: 821–826.
- 410. Playfair JH, De Souza JB (1979) Antibody responses in mice protected against malaria by vaccination. Parasite Immunol 1: 197–208.
- 411. Playfair JH, De Souza JB, Cottrell BJ (1977) Protection of mice against malaria by a killed vaccine: differences in effectiveness against P. yoelii and P. berghei. Immunology 33: 507–515.
- 412. Potocnjak P, Yoshida N, Nussenzweig RS, Nussenzweig V (1980) Monovalent fragments (Fab) of monoclonal antibodies to a sporozoite surface antigen (Pb44) protect mice against malarial infection. J Exp Med 151: 1504–1513.
- 413. Puri SK, Dutta GP, Levy HB, Maheshwari RK (1996) Poly ICLC inhibits Plasmodium cynomolgi B malaria infection in rhesus monkeys. J Interferon Cytokine Res 16: 49–52.
- 414. Pye D, O'Brien CM, Franchina P, Monger C, Anders RF (1994) Plasmodium falciparum infection of splenectomized and intact Guyanan Saimiri monkeys. J Parasitol 80: 558–562.
- 415. Reed RC, Louis-Wileman V, Wells RL, Verheul AF, Hunter RL, et al. (1996) Re-investigation of the circumsporozoite protein-based induction of sterile immunity against Plasmodium berghei infection. Vaccine 14: 828–836.
- 416. Reed RC, Louis-Wileman V, Cosmai EV, Fang S, Jue DL, et al. (1997) Multiple antigen constructs (MACs): induction of sterile immunity against sporozoite stage of rodent malaria parasites, Plasmodium berghei and Plasmodium yoelii. Vaccine 15: 482–488.
- 417. Renia L, Grillot D, Marussig M, Corradin G, Miltgen F, et al. (1993) Effector functions of circumsporozoite peptide-primed CD4+ T cell clones against Plasmodium yoelii liver stages. J Immunol 150: 1471–1478.
- 418. Reyes-Sandoval A, Berthoud T, Alder N, Siani L, Gilbert SC, et al. (2010) Prime-boost immunization with adenoviral and modified vaccinia virus Ankara vectors enhances the durability and polyfunctionality of protective malaria CD8+ T-cell responses. Infect Immun 78: 145–153.
- 419. Rodrigues EG, Claassen J, Lee S, Wilson JM, Nussenzweig RS, et al. (2000) Interferon-gamma-independent CD8+ T cell-mediated protective anti-malaria immunity elicited by recombinant adenovirus. Parasite Immunol 22: 157–160.
- 420. Rodrigues MM, Cordey AS, Arreaza G, Corradin G, Romero P, et al. (1991) CD8+ cytolytic T cell clones derived against the Plasmodium yoelii circumsporozoite protein protect against malaria. Int Immunol 3: 579–585.
- 421. Romero JF, Ciabattini A, Guillaume P, Frank G, Ruggiero P, et al. (2009) Intranasal administration of the synthetic polypeptide from the C-terminus of the circumsporozoite protein of Plasmodium berghei with the modified heat-labile toxin of Escherichia coli (LTK63) induces a complete protection against malaria challenge. Vaccine 27: 1266–1271.
- 422. Romero JF, Eberl G, MacDonald HR, Corradin G (2001) CD1d-restricted NK T cells are dispensable for specific antibody responses and protective immunity against liver stage malaria infection in mice. Parasite Immunol 23: 267–269.
- 423. Sadoff JC, Ballou WR, Baron LS, Majarian WR, Brey RN, et al. (1988) Oral Salmonella typhimurium vaccine expressing circumsporozoite protein protects against malaria. Science 240: 336–338.
- 424. Schmidt LH, Rossan RN, Fradkin R, Sullivan R, Schulemann W, et al. (1985) Antimalarial activities and subacute toxicity of RC-12, a 4-amino-substituted pyrocatechol. Antimicrob Agents Chemother 28: 612–625.
- 425. Schmidt NW, Podyminogin RL, Butler NS, Badovinac VP, Tucker BJ, et al. (2008) Memory CD8 T cell responses exceeding a large but definable threshold provide long-term immunity to malaria. Proc Natl Acad Sci U S A 105: 14017–14022.
- 426. Schneider J, Gilbert SC, Blanchard TJ, Hanke T, Robson KJ, et al. (1998) Enhanced immunogenicity for CD8+ T cell induction and complete protective efficacy of malaria DNA vaccination by boosting with modified vaccinia virus Ankara. Nat Med 4: 397–402.
- 427. Sedegah M, Brice GT, Rogers WO, Doolan DL, Charoenvit Y, et al. (2002) Persistence of protective immunity to malaria induced by DNA priming and poxvirus boosting: characterization of effector and memory CD8(+)-T-cell populations. Infect Immun 70: 3493–3499.
- 428. Sedegah M, Finkelman F, Hoffman SL (1994) Interleukin 12 induction of interferon gamma-dependent protection against malaria. Proc Natl Acad Sci U S A 91: 10700–10702.
- 429. Siddiqui WA, Tam LQ, Kan SC, Kramer KJ, Case SE, et al. (1986) Induction of protective immunity to monoclonal-antibody-defined Plasmodium falciparum antigens requires strong adjuvant in Aotus monkeys. Infect Immun 52: 314–318.
- 430. Spitalny GL, Nussenzweig RS (1973) Plasmodium berghei: relationship between protective immunity and anti-sporozoite (CSP) antibody in mice. Exp Parasitol 33: 168–178.
- 431. Taylor-Robinson AW, Phillips RS (1994) Th1 and Th2 CD4+ T cell clones specific for Plasmodium chabaudi but not for an unrelated antigen protect against blood stage P. chabaudi infection. Eur J Immunol 24: 158–164.
- 432. Taylor-Robinson AW, Phillips RS, Severn A, Moncada S, Liew FY (1993) The role of TH1 and TH2 cells in a rodent malaria infection. Science 260: 1931–1934.
- 433. Tsuji M, Miyahira Y, Nussenzweig RS, Aguet M, Reichel M, et al. (1995) Development of antimalaria immunity in mice lacking IFN-gamma receptor. J Immunol 154: 5338–5344.
- 434. Trimnell A, Takagi A, Gupta M, Richie TL, Kappe SH, et al. (2009) Genetically attenuated parasite vaccines induce contact-dependent CD8+ T cell killing of Plasmodium yoelii liver stage-infected hepatocytes. J Immunol 183: 5870–5878.
- 435. van Dijk MR, Douradinha B, Franke-Fayard B, Heussler V, van Dooren MW, et al. (2005) Genetically attenuated, P36p-deficient malarial sporozoites induce protective immunity and apoptosis of infected liver cells. Proc Natl Acad Sci U S A 102: 12194–12199.
- 436. Vinetz JM, Kumar S, Good MF, Fowlkes BJ, Berzofsky JA, et al. (1990) Adoptive transfer of CD8+ T cells from immune animals does not transfer immunity to blood stage Plasmodium yoelii malaria. J Immunol 144: 1069–1074.
- 437. Waki S, Takagi T, Suzuki M (1989) Acquirement of protective immunity in mice through infection with an attenuated isolate and its failure in parent virulent Plasmodium berghei. Parasitol Res 75: 614–618.
- 438. Wang R, Charoenvit Y, Corradin G, De La Vega P, Franke ED, et al. (1996) Protection against malaria by Plasmodium yoelii sporozoite surface protein 2 linear peptide induction of CD4+ T-cell and IFN-dependent elimination of infected hepatocytes. J Immunol 157: 4061–4067.
- 439. Wang R, Charoenvit Y, Corradin G, Porrozzi R, Hunter RL, et al. (1995) Induction of protective polyclonal antibodies by immunization with a Plasmodium yoelii circumsporozoite protein multiple antigen peptide vaccine. J Immunol 154: 2784–2793.
- 440. Weiss WR, Berzofsky JA, Houghten RA, Sedegah M, Hollindale M, et al. (1992) A T cell clone directed at the circumsporozoite protein which protects mice against both Plasmodium yoelii and Plasmodium berghei. J Immunol 149: 2103–2109.
- 441. White KL, Jarboe DL, Krzych U (1994) Immunization with irradiated Plasmodium berghei sporozoites induces IL-2 and IFN gamma but not IL-4. Parasite Immunol 16: 479–491.
- 442. Wykes MN, Zhou YH, Liu XQ, Good MF (2005) Plasmodium yoelii can ablate vaccine-induced long-term protection in mice. J Immunol 175: 2510–2516.
- 443. Pacheco ND, McConnell E, Beaudoin RL (1979) Duration of immunity following a single vaccination with irradiated sporozoites of Plasmodium berghei. Bull World Health Organ 57: Suppl 1159–163.
- 444. Nussenzweig RS, Vanderberg JP, Most H, Orton C (1969) Specificity of protective immunity produced by x-irradiated Plasmodium berghei sporozoites. Nature 222: 488–489.
- 445. Nussenzweig R, Vanderberg J, Most H (1969) Protective immunity produced by the injection of x-irradiated sporozoites of Plasmodium berghei. IV. Dose response, specificity and humoral immunity. Mil Med 134: 1176–1182.
- 446. Orjih AU, Cochrane AH, Nussenzweig RS (1982) Comparative studies on the immunogenicity of infective and attenuated sporozoites of Plasmodium berghei. Trans R Soc Trop Med Hyg 76: 57–61.
- 447. Renia L, Rodrigues MM, Nussenzweig V (1994) Intrasplenic immunization with infected hepatocytes: a mouse model for studying protective immunity against malaria pre-erythrocytic stage. Immunology 82: 164–168.
- 448. Favila-Castillo L, Monroy-Ostria A, Kobayashi E, Hirunpetcharat C, Kamada N, et al. (1996) Protection of rats against malaria by a transplanted immune spleen. Parasite Immunol 18: 325–331.
- 449. Butler NS, Schmidt NW, Harty JT (2010) Differential Effector Pathways Regulate Memory CD8 T Cell Immunity against Plasmodium berghei versus P. yoelii Sporozoites. J Immunol 184: 2528–2538.
- 450. Yoshida S, Araki H, Yokomine T (2010) Baculovirus-based nasal drop vaccine confers complete protection against malaria by natural boosting of vaccine-induced antibodies in mice. Infect Immun 78: 595–602.
- 451. Falae A, Combe A, Amaladoss A, Carvalho T, Menard R, et al. (2010) Role of Plasmodium berghei cGMP-dependent protein kinase in late liver stage development. J Biol Chem 285: 3282–3288.
- 452. Sina BJ, Wright C, Atkinson CT, Ballou R, Aikawa M, et al. (1995) Characterization of a sporozoite antigen common to Plasmodium falciparum and Plasmodium berghei. Mol Biochem Parasitol 69: 239–246.
- 453. Sina BJ, Wright C, Ballou R, Hollingdale M (1992) A protective monoclonal antibody with dual specificity for Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins. Exp Parasitol 74: 431–440.
- 454. Clyde DF (1990) Immunity to falciparum and vivax malaria induced by irradiated sporozoites: a review of the University of Maryland studies, 1971-75. Bull World Health Organ 68: Suppl9–12.
- 455. Egan JE, Hoffman SL, Haynes JD, Sadoff JC, Schneider I, et al. (1993) Humoral immune responses in volunteers immunized with irradiated Plasmodium falciparum sporozoites. Am J Trop Med Hyg 49: 166–173.
- 456. Herrington DA, Nardin EH, Losonsky G, Bathurst IC, Barr PJ, et al. (1991) Safety and immunogenicity of a recombinant sporozoite malaria vaccine against Plasmodium vivax. Am J Trop Med Hyg 45: 695–701.
- 457. Schmidt NW, Butler NS, Harty JT (2009) CD8 T cell immunity to Plasmodium permits generation of protective antibodies after repeated sporozoite challenge. Vaccine 27: 6103–6106.
- 458. Butcher GA, Mitchell GH, Cohen S (1978) Antibody mediated mechanisms of immunity to malaria induced by vaccination with Plasmodium knowlesi merozoites. Immunology 34: 77–86.
- 459. Hommel M, David PH, Guillotte M, Pereira da Silva L (1982) Protection against Plasmodium chabaudi malaria. I.–Vaccination of mice with merozoites and Freund's adjuvants. Ann Immunol (Paris) 133C: 57–67.