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Mosquitocidal vaccines: a neglected addition to malaria and dengue control strategies Peter F. Billingsley1,2, Brian Foy3 and Jason L. Rasgon4 1

School of Biological Sciences, University of Aberdeen, Tillydrone Avenue, Aberdeen, AB24 2TZ, UK Sanaria Inc., 9800 Medical Center Drive, Rockville, MD 20850, USA 3 Arthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, 1682 Campus Delivery, Fort Collins, CO 80523-1682, USA 4 The Johns Hopkins Malaria Research Institute and The W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA 2

The transmission of vector-borne diseases is dependent upon the ability of the vector to survive for longer than the period of development of the pathogen within the vector. One means of reducing mosquito lifespan, and thereby reducing their capacity to transmit diseases, is to target mosquitoes with vaccines. Here, the principle behind mosquitocidal vaccines is described, their potential impact in malaria and dengue control is modeled and the current research that could make these vaccines a reality is reviewed. Mosquito genome data, combined with modern molecular techniques, can be exploited to overcome the limited advances in this field. Given the large potential benefit to vector-borne disease control, research into the development of mosquitocidal vaccines deserves a high profile. Vaccines against bloodfeeding arthropods The concept of vaccines against bloodfeeding arthropods gained prominence with the successful demonstration of anti-tick immunity in cattle that were immunized with a recombinant protein, Bm86 [1,2]. Boophilus microplus ticks that fed on vaccinated cattle exhibited reduced fecundity and survival. Bm86, which is marketed as TickGARDPLUS, has proven to be robust in the field [3] and maintains effectiveness over several tick generations. Since the immunization of hosts with mosquito antigens in 1948 [4], research into mosquitocidal vaccines has continued intermittently, but successes have been few. Mosquitoes differ greatly from ticks in feeding behavior (mosquitoes do not attach to the host long-term) and digestion (ticks digest their bloodmeal intracellularly). Nevertheless, mosquitoes ingest several times their own weight in host blood [5] and the bloodmeal contains all host immune system components [6]. These immune effectors can remain active in the mosquito for 24 h [7,8] and can kill mosquitoes [9–14]. It is surprising, therefore, that mosquitocidal vaccines continue to be considered as unfeasible and research in the field suffers from a lack of acceptance. Here, the case is made for a more sustained research effort to truly test Corresponding author: Billingsley, P.F. ([email protected]).

the feasibility of mosquitocidal vaccines for the control of diseases such as malaria and dengue. Mosquitocidal vaccines: pros and cons There are strong arguments in favor of immune control of vectors in general and mosquitoes in particular. Vector control is by far the most successful method for reducing the incidences of diseases such as malaria and dengue, but the emergence of widespread insecticide resistance and the potential environmental issues associated with some insecticides (such as DDT) indicate that additional approaches to control the vector are needed. An ‘immune insecticide’ would target biting mosquitoes much more directly than any environmentally applied insecticide and would preferentially kill the oldest females, which tend to drive disease transmission. Mosquitocidal vaccines have already been proven to work in the laboratory [9–14], and it might be possible to target multiple mosquito species with a multivalent vaccine. The most compelling arguments for developing a mosquitocidal vaccine come from modeling. In any epidemiological model for vector-borne diseases, the most influential factor that drives transmission by a competent vector is the daily survival rate (Box 1). Essentially, the vector must survive throughout the extrinsic incubation period (i.e. ingestion into the vector, development and transmission) of the pathogen. Pathogen transmission is exquisitely sensitive to the daily survival rate of mosquitoes [15–23], and changes in survival have an exponential impact upon the transmission and basic reproductive rate (R0) (Box 1), whereas most other parameters in malaria models have outcomes in linear proportion to their efficacy. This has several important implications for mosquito vaccines that add to their attractiveness. Foremost is that high efficacy is not needed; modest reductions in the survival of mosquitoes that feed upon immunized hosts are sufficient to have a major impact upon transmission. In the model presented in Box 1, reducing the survival rate by only 19% (as seen in Ref. [13]) would reduce R0 by >95% for dengue and >99% for malaria (Figure 1a). Compare this to recent vaccine trials with the anti-Plasmodium sporozoite RTS,S vaccine, in which 30% efficacy was noted in the

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Box 1. Modeling mosquiticidal vaccines The basic reproductive number (R0) for a vector-borne pathogen is defined as the number of secondary cases that follow the introduction of a single infected individual into a susceptible population [22]. R0 is a function of both entomological and host parameters and is calculated as: ma2 p n b R0 ¼ rln p

(Eqn I)

Exposed ðe MÞ  ðe Nx;t Þ ¼ e Nx;tþ0:5 0 0 0  0 B e p0 0  0 B e B p1    0 ¼ B0 B .. .. . @. .    .. 0



e

1 0e N0;tþ0:5 C B e N1;tþ0:5 C C Be C C B N2;tþ0:5 C C¼B C C B .. C A @. A e e Nv;t Nv;tþ0:5

1 0e N0;t 0 Be 0C C B e N1;t B 0C C  B N2;t .. C B .. .A @. pv1

0

1

(Eqn II) e

Nx;tþ1 ¼ e Nx;tþ0:5 þ ðK  n Nx;tþ0:5 Þ

(Eqn III)

Naı¨ve ðn MÞ  ðn Nx;t Þ ¼ n Nx;tþ0:5 0 B0 B1    B v1 B n p0 0  0 B n B p1    0 ¼ B0 B .. .. . @. .    .. 0

0

0

n

pv1

Nx;tþ1 ¼ ðn Nx;tþ0:5 Þ  ð1  K Þ

0n

N0;t Bv Bn 0 C C B n N1;t B 0 C C  B N2;t .. C B .. A @. . 0

1

0n

1

N0;tþ0:5 C B n N1;tþ0:5 C C Bn C C B N2;tþ0:5 C C¼B C C B .. C A @. A n n Nv;t Nv;tþ0:5 (Eqn V)

To examine changes in R0 that were due to a mosquitocidal vaccine campaign, we used the age-structured extension of Eqn I [20]. Total R0 for a pathogen with an extrinsic incubation period (the time between when a vector acquires a pathogen from an infected vertebrate host and when it is able to transmit the pathogen to a susceptible vertebrate host) of n days (nRT) was calculated (for each age class) as R0 of the vaccine-exposed fraction of the population

2

RN ¼

v X

n

x¼1

RNx ¼

xY v  þ1 X ma2 b x¼1

r

N

  p j ðN exþn ÞðVx Þð1  G x Þ

j¼x

(Eqn VII)

cumulative reduction in time to infection and all vaccinees would eventually become infected [24]. Low efficacy has other advantages – for example, discovering antigens that only moderately induce a mosquitocidal immune response should be much easier than discovering antigens with a strong mosquitocidal response, and the former would have the added benefit of acting with a lower selection pressure on mosquito populations, which would extend the life of the vaccine. Indeed, such a vaccine could preferentially select for zoophilic-feeding vectors [25,26]. In nature, adult mosquito distributions are extremely aggregated [27–29], which results in highly skewed contributions of individual hosts to transmission that are based upon the scale of their interaction with mosquitoes [30]. Thus, one important potential advantage is that a much smaller proportion of the population could be targeted with a mosquito vaccine to have a significant outcome [22]. In the model in Box 1, vaccinating only 20% of hosts upon which 80% of mosquitoes feed [30] is only negligibly different from vaccinating 100% of hosts. The cases against mosquitocidal vaccines are often based on biological criticisms that m not be accurate. It is argued that because mosquitoes take small bloodmeals, they will not ingest enough immune components to have an effect – but mosquitoes ingest several times their own body mass at a single meal, anthropophilic mosquitoes will often take a bloodmeal every other day [27,29,31–33], the serum components are in intimate contact with the midgut cells immediately after feeding and these components are active for up to 24 h [34,35]. It is also argued that constantly high antibody titres will be needed in vaccinated persons, but evidence with Bm86 indicates otherwise, and long-lasting cellular immunity might also contribute to the mosquitocidal effects of a vaccine. There is also sometimes concern Table I. Model parameter definitions

1

(Eqn IV) n

and n

where parameters are defined in Table I. Traditional vaccines that affect the parasite (reducing 1/r) or transmission-blocking vaccines that affect the ability of the mosquito to transmit pathogens (reducing b) affect R0 in a linear manner and must have strong effects to result in a significant reduction in pathogen transmission. By contrast, mosquitocidal vaccines reduce p, the probability that a mosquito will survive to the next day. Mosquito survival is the most sensitive component of R0. Disease control strategies that decrease p are highly efficient in reducing pathogen transmission because small changes in daily survival can result in large changes in the number of new vertebrate host infections. Previous modeling efforts have documented the increased efficacy of mosquitocidal vaccines compared with traditional or transmission-blocking vaccines when examined over several bloodfeeding and oviposition cycles, but these previous modeling efforts did not include the continuous recruitment of naı¨ve mosquitoes into the population or the age-structure dynamics of the mosquito population [19,22]. We modified previous age-structured modeling efforts [20,21] to examine the potential efficacy of a mosquitocidal vaccine in a more realistic age-structured mosquito population with continuous recruitment and overlapping generations. Life-table parameters were based on estimated values for Anopheles gambiae and were taken from previous simulations [21]. The model is in the form of a Leslie matrix, where e,nM = the life-table matrix for exposed or naı¨ve mosquitoes and e,nNx,t = the age class vector at generation t for exposed or naı¨ve mosquitoes. K represents the matrix describing host–vector contact (age-specific biting). For these analyses, we assume that mosquitoes start feeding on Day 3 of adult life and feed every three days thereafter. The oldest mosquito age class (v) was set at 30 days.

0

plus R0 of the uninfected fraction of the population (nRT = nRE + nRN) and summed over all age classes (x), such that xþn   v v  Y X X ma2 n E n RE ¼ REx ¼ p j ðE exþn ÞðVx ÞðG x Þ (Eqn VI) r x¼1 x¼1 j¼x

Parameter m a p n b r V Bx e,n px E,N

ex+n

Vx Gx

Definition Mosquito density Mosquito daily biting rate Daily probability of mosquito survival Pathogen extrinsic incubation period Mosquito vector competence Host recovery rate Proportion of human hosts vaccinated Daily fecundity of mosquitoes in age class x Probability of survival for vaccine-exposed or naı¨ve mosquitoes at age class x + n Expectation of infective life for vaccine-exposed or naı¨ve mosquitoes at age class x + n Proportion of adult mosquitoes at age class x Proportion of adult mosquitoes at age class x exposed to vaccinated host

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ever, because of the heterogeneity of homogenates that cause variable host immunity, these approaches have probably been pursued to the limits of their usefulness (for reviews, see Refs [6,19,44]). Refined antigens have included GlcNAc-rich midgut glycoproteins [45], chondroitin glycosaminoglycans [46,47], fractions enriched for microvilli from the midgut epithelial cells [48] and monoclonal antibodies (mABs) against immunizing fractions [13]. When fed to mosquitoes, some of these antigens resulted in significant reductions in survival or fecundity in Anopheles stephensi and Anopheles gambiae, or they blocked transmission of malaria parasites to the mosquitoes. Unfortunately, many of these studies highlighted the problems of variable host immunity and inconsistent effects. High variation in controls and experimental replicates, even within a single study, has also been a major problem; for example, in a study by Almeida and Billingsley [12], mosquito survival ranged from 0.3 to 0.6, 13 days after feeding.

Figure 1. The relative reduction in R0 that results from the use of a mosquitocidal vaccine (a 5%, 10% or 20% reduction in p) for pathogens with extrinsic incubation periods (EIPs) that range from 3 to 20 days. Relative R0 is calculated after the host population has been vaccinated and the system has come to equilibrium, and is calculated as nRR = (nRT/n R0), where nR0 = prevaccination R0 when the population is at the stable age distribution [20]. (a) 100% vaccination coverage of host population. (b) Targeted vaccination of 20% of the human hosts, which were responsible for infecting 80% of the mosquito population. If targeting is accurate, limited vaccination can give comparable results to complete vaccination coverage. The EIP range for malaria (9 to >20 days, depending on temperature and parasite species) is shown in pink. The EIP range for the dengue virus (10–14 days, depending on temperature) is shown in green.

that a mosquitocidal vaccine would not prevent sickness, but used in combination with anti-pathogen vaccines or drugs it would effectively prevent breakthrough parasites from propagating and, thus, inhibit the development of drug or vaccine resistance. A brief history of mosquitocidal vaccines Most attempts at developing mosquitocidal vaccines have taken the approach of repeatedly immunizing vertebrate hosts with homogenates of various tissues, which leads to reductions in survival, fecundity and pathogen transmission in numerous mosquito species [9–12,36–43]. How-

Current state-of-the-art Antigen discovery With the deciphering of the An. gambiae and Aedes aegypti genomes, more targeted pathways to antigen discovery are now possible. Ideal antigens would be crucial to the vector, exposed to immune factors from the bloodmeal, susceptible to immune attack and of low abundance. The mosquito midgut remains the best tissue to target because it stores, diureses, digests and absorbs the bloodmeal; it is an essential part of the immune and neuroendocrine systems of the insect [49]; and it is in intimate association with the immune components of the blood at measurable titres. Antigens from other tissues might also be considered as targets because antibody can pass into the mosquito hemolymph and bind to antigens of other tissues [8,50,51]. Mining catalogued libraries of mosquito expressed sequence tags (ESTs) for midgut-specific sequences that contain secretion signal and/or signal anchor peptide signatures (e.g. using SignalP) identifies extracellular proteins that are accessible to ingested immune factors. Selected EST sequences can be cloned for protein expression or DNA vaccination. Efforts can be focused upon groups of midgut-associated genes that are likely to be involved in essential physiological processes, the disruption of which would reduce mosquito survival (e.g. proteins involved in diuresis or bloodmeal detoxification). Microarray studies of transcripts that are differentially regulated after bloodfeeding might usefully pinpoint target molecules, but constitutively expressed antigens might serve equally important functions in mosquito homeostasis. Target efficacy Once the gene target is cloned, there are several ways to ‘attack’ it in the mosquito. Ultimately, the idea is to prove that the target is crucial for mosquito homeostasis, and the final test for this is increased mosquito mortality. Silencing the transcript in the mosquito by using RNA interference (RNAi) has the advantage that only a portion of the gene needs cloning and no immunizations are required. The verification of target silencing via quantitative reversetranscription PCR, northern blots, ELISAs or western 3

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Opinion blots is essential and must be performed in conjunction with phenotypic assays. RNAi studies come with at least two caveats. (i) It is possible that the gene product is not regulated at the transcriptional level and that there is sufficient protein in the mosquito, even after doublestranded RNA injection, to maintain homeostasis. (ii) There might also be some disconnect between mosquito death owing to the silencing of the gene target and mosquito death owing to immune attack against the gene target, the latter being the ultimate goal. By expressing the cloned target as a protein, one can generate antibodies or single-chain, phage-displayed antibody fragments (scFvs) that can bind to and inactivate the target gene product. mAbs can either be used to immunize mice passively for mosquito challenge during bloodfeeding or be fed directly to mosquitoes using a membrane feeder [13]. Attempts to generate scFvs that target mosquito proteins have been met with some limited success. Target-binding scFvs can be constructed de novo from mice immunized with the target protein [52] or selected (by phage panning) from a large and diverse scFv library [53]. The scFv library has been used successfully to select mosquito protein-binding scFv. However, a bottleneck during phage panning might select for only a few scFvs [54,55], possibly because of excessive carbohydrates on the microvillar surface [45,56] that block binding to effective target epitopes. The immunization of host animals and subsequent mosquito challenge by bloodfeeding is the most direct method to prove that the target is effective. If protein is used as the antigen, the cost and time involved can quickly become prohibitive to mass screening because each cloned gene must be separately expressed and purified before immunization. Alternative procedures rely on DNA immunization, in which the cloned gene in an expression plasmid is directly immunized into the animal. An adaptation of this method is expression-library immunization, in which libraries of cloned genes and fractions thereof are immunized, which has proven successful in identifying crucial tick antigens [57] and initially successful for mosquitoes [14]. Understanding the mosquitocidal immune response An immune response can vary greatly depending upon the antigen, its presentation, the immunization regimen and the host. These factors have been insufficiently characterized in previous mosquitocidal vaccine discovery studies, primarily because of a lack of defined targets to study. However, it is becoming clear that understanding the nature of a mosquitocidal immune response is crucial for this field to progress. It is likely that many targets are not identified in the first place because the correct type of immune response is never stimulated. Immunization with one or a few pure antigens is important to prevent nonspecific immunity or the immunodominance of one or more antigens. Previous studies have also fostered the assumption that antibodies are the sole key to a mosquitocidal immune response. Immunization of mice with an expression plasmid that contains the An. gambiae mucin 1 (AgMuc1) gene provides contrasting evidence [14]; results indicated that both cell-mediated activity and antibodies, not antibodies alone, were responsible for mosquito killing. 4

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Together, these data indicate that there are multiple host immune pathways towards killing mosquitoes, but each pathway is probably defined by the target. For example, if the target has a function in the mosquito gut, then antibodies that block the target’s active site are likely to work to kill the mosquito. Conversely, there might be targets that serve as general anchors for antibody- and cell-mediated attack of the insect cells. Key future directions for mosquitocidal vaccine research are to define these types of targets and determine the relative contributions of the immune response elements for mosquitocidal activity. Concluding remarks Current data strongly indicate that targeting mosquitoes with vaccine-type approaches can work. The commercial success of Bm86 as an anti-tick vaccine, along with new molecular techniques, offers encouragement to continue with the approach, but there are important obstacles to success. Not least of these is gaining acceptance from vector and pathogen research communities that the approach has proven merit, and fostering the field alongside more tried-and-tested areas of vector-borne disease control. The models indicate that even a modest breakthrough in the field could have a major impact on disease transmission. Acknowledgements P.F.B. was funded by the European Commission during the work leading up to this article; the views presented here are those of the author, not of Sanaria Inc. B.D.F. acknowledges support on this paper from National Institutes of Health (NIH) contract N01 AI25489, and J.L.R. was funded by the Johns Hopkins Malaria Research Institute and the NIH.

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