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* Department of Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523;
Department of Immunology and Infectious Diseases, Harvard University School of Public Health, Boston, MA 02115; and
Department of Pathology and Center for Tropical Diseases, University of Texas Medical Branch, Galveston, TX 77555
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is now apparent that the saliva of blood-feeding arthropods contains molecules that enhance blood flow (3) and inhibit the immune response of the host (4). While enhanced blood flow insures the feeding success of the arthropod, inhibiting the immune response of the host may prevent the host from becoming sensitized to the bite of the arthropod. However, there is now mounting evidence that the saliva of an arthropod vector can also enhance the infectivity of pathogens that the arthropod transmits (5, 6, 7, 8, 9, 10). Therefore, injecting arthropod-borne pathogens by syringe does not mimic natural transmission.
We originally showed that infection with Leishmania major was dramatically enhanced in mice coinjected with the parasite plus sandfly saliva. Cutaneous lesions caused by the parasite were severalfold larger than lesions on control mice, and parasite burden in those lesions could be as much as several thousand-fold higher (5). Indeed, saliva completely reversed the outcome of infection in L. braziliensis-infected mice (4, 11). We proposed that the protein in saliva responsible for its disease-exacerbating qualities is a vasodilator, and that this vasodilator is related to a mammalian neuropeptide (12). Subsequently, the gene encoding the salivary vasodilator was cloned (13). This salivary vasodilator, termed Lutzomyia longipalpis sandfly maxadilan (MAX),3 appears to be functionally related to the mammalian neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP). Both MAX and PACAP are vasodilators and inhibitors/modulators of an inflammatory and an immune response, and both signal at least through the PACAP type 1 receptor (4, 14, 15).
Taken together, these observations suggest that MAX is indeed responsible for the disease-exacerbating qualities of sandfly saliva, a hypothesis that is tested here. Moreover, when mice are infected with L. major in numbers equivalent to those injected by a naturally infected sandfly 10–100(10–100), the parasite does not survive unless it is coinjected with sandfly saliva (5). This suggests that if mice were vaccinated with MAX, they would be protected against a challenge with L. major plus sandfly saliva. This novel approach for vaccinating against a pathogen is also examined here.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Young adult female CBA/CaH-T6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Stationary phase promastigotes of L. major (LV39 (MRHO/Sv/59/P) were used. Mice were injected s.c. with 105 L. major ± varying doses of salivary gland lysate or MAX in one hind footpad. These mouse experiments were approved by the Institutional Review Board of Colorado State University.
Monitoring lesion development and parasite burden in the lesion
Lesion development was followed by measuring with a caliper the thickness of the infected footpad compared with the control contralateral uninfected footpad. Parasite numbers were determined in infected footpads using a published limiting dilution assay (16).
Sandfly salivary gland lysate and synthetic maxadilan
Salivary glands of Lutzomyia longipalpis (Belo Horizonte, Brazil, Lapinha Cave isolate) were collected and lysed by freezing and thawing as described (5). Synthetic maxadilan was prepared by the Biopolymers Laboratory, Harvard Medical School. The 63-mer amino acid sequence used was based on the predicted sequence of mature, secreted MAX (Ref. 17 ; CDATCQFRKAIEDCRKKAHHSDVLQTSVQTTATFTSMDTSQLPGSGVFKECMKEKAKEFKAGK).
Vaccinating against MAX
Groups of mice (n = 5) were injected s.c. at the
base of the tail with 25 µg of synthetic MAX emulsified in CFA. Ten
days later the mice were injected i.p. with 25 µg of MAX emulsified
in IFA. Two weeks later the mice were boosted i.p. with 25 µg of
soluble MAX. Another group of mice (n = 5) was injected
with adjuvant or vehicle alone. Three days after the soluble boost with
MAX, the titer of anti-MAX Ab in the sera of the MAX-sensitized
mice was determined (see MAX ELISA). If the titer was
sufficient (Table III
), mice were challenged with either L.
major or L. major + 0.5 salivary gland lysate (see
Results).
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Three days after the soluble boost with MAX, blood was collected from mice and the anti-MAX serum titer was determined by an ELISA. Briefly, ELISA plates were coated with synthetic MAX (10 µg/ml) using standard techniques (18) and developed with alkaline phosphatase-labeled goat anti-mouse IgG (H and L chain) and p-nitrophenyl phosphate (catalog no. 075-1806 and 50-80-01, respectively; Kirkegaard & Perry Laboratories, Gaithersburg, MD).
Cytokine ELISA and Griess reaction
Ten days after mice were injected s.c. at the base of the tail
with 25 µg of synthetic MAX emulsified in CFA, the draining inguinal
and para-aortic lymph nodes were removed and a single-cell suspension
was generated. The cells were placed into culture (5 x
106/ml in 24-well plates, Costar 3524; Corning
Glass, Corning, NY) in DMEM (18) + 0.5% normal mouse
serum. Experimental cultures then received 10 µg/ml synthetic MAX;
control cultures received diluent alone. The supernatants and cells
were harvested after 3 days of culture. Supernatants were analyzed for
their content of IFN-
and NO by published methods (18, 19). The cells were purified over Percoll gradients
(18) and analyzed by flow cytometry.
Flow cytometry
Cells were labeled with the following Abs: FITC anti-mouse CD4 or FITC anti-mouse CD8 (557307 and 553030, respectively; BD PharMingen, San Diego, CA), FITC anti-mouse I-AK MHC class II, private (MM3501; Caltag Laboratories, Burlingame, CA). Appropriate control Abs were purchased from the same suppliers. Labeled cells were then analyzed using methods described elsewhere (20).
Statistical analyses
Data for lesion progression were analyzed using ANOVA for repeated measure and for parasite burdens using unpaired t tests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test whether MAX would substitute for whole saliva, we admixed
varying amounts of synthetic MAX with 105
L. major promastigotes and injected them s.c. into a hind
footpad of CBA mice. Three nanograms of MAX markedly
(p < 0.002 compared with control mice infected
with L. major alone) exacerbated lesion development to
the same degree as the lysate of 0.5 sandfly salivary gland
(p < 0.002, Fig. 1a). One-half of one sandfly
salivary gland is the dose that we reported exacerbates infection with
L. major (5).
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a). However, a higher dose of MAX (10 ng) also
exacerbated infection with L. major to a lesser degree (Fig. 1a). We have reported that doses of <0.5 sandfly salivary
gland exacerbate infection with L. major to a lesser degree
(21), but we had not previously tested doses above 0.5
salivary gland. Therefore, we tested a wide range of doses of sandfly
salivary gland lysate (Fig. 1b). Importantly, the same
phenomenon was observed. Compared with controls (saliva-free),
exacerbation of infection increased with increasing doses of saliva, up
to a maximum of 0.5 and 1.0 gland equivalents. Interestingly, higher
doses (2.0 gland equivalents) had a reduced effect (Fig. 1b). The doses of salivary gland lysate that significantly
enhanced lesion development were 0.5 and 1.0 gland extract;
p < 0.001 and 0.002, respectively (Fig. 1b).
Finally, because 3 ng of MAX and 0.5 salivary gland lysate exacerbated
infection equally, we determined whether similar parasite burdens were
present in the two groups of mice. Both MAX (423-fold increase) and
saliva (193-fold increase) markedly enhanced parasite burden
(p < 0.001, Table I). This degree of enhancement of
infection is similar to what we previously reported for whole saliva
(5).
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Because MAX exacerbated infection with L. major, we
hypothesized that vaccinating against it might neutralize the
disease-enhancing effects of whole saliva and thus protect vaccinated
mice against infection with L. major. CBA mice were injected
with synthetic MAX (25 µg) emulsified in Freund’s adjuvant and were
then boosted with soluble synthetic MAX; other mice received adjuvant
or diluent alone. Mice were then challenged s.c. in one hind footpad
with L. major parasites (105) or
parasites admixed with salivary gland lysate (0.5 gland). Vaccinated
mice were highly resistant to infection. Cutaneous lesions on
vaccinated mice were 3- to 5-fold smaller, and these mice healed their
lesions by day 50 of infection, whereas lesions on mice treated with
adjuvant or diluent alone had not healed their lesions by day 65 of
infection. As a result, lesions on vaccinated mice were significantly
smaller (Fig. 2) than lesions on control
mice. In addition, the parasite burden in lesions on vaccinated mice
was markedly reduced (506.7-fold, p < 0.001, Table II).
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To investigate the possible mechanism(s) underlying the protection
against infection seen in MAX-vaccinated mice, we characterized the
anti-MAX response elicited in the mice. First, serum contained a
high titer (1/6,400–1/25,600; Table III)
of anti-MAX Abs. In addition, a cellular response was induced in
vaccinated mice. We isolated inguinal and para-aortic lymph nodes from
mice injected at the base of the tail with MAX emulsified in CFA. These
lymph nodes drain both the base of the tail and the footpad where
L. major parasites were subsequently injected. When these
lymph node cells were stimulated with MAX in vitro, the cells released
substantial quantities of both IFN- and NO (Table III). We also
determined the phenotype of the responding cells because several cell
types can release IFN-. A flow cytometric analysis revealed that the
cells were composed principally of CD4 cells (82%) with some CD8 cells
(10%) and I-A+ cells (4%, Table III). Thus,
both a humoral and cellular anti-MAX response were induced in
treated mice (Table III), and this may explain how immunization not
only negated the exacerbative effect of the saliva, but also reduced
the severity of disease below that seen in animals that were challenged
with L. major but no saliva (compare groups 
and • in
Fig. 2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test this hypothesis, we initially demonstrated that sandfly saliva
dramatically enhanced the infectivity of L. major for mice
(5). Here we show that a single sandfly salivary protein,
MAX, can substitute for whole saliva and exacerbate infection with
L. major to the same degree as whole saliva (Fig. 1, Table I). Different doses of either MAX or saliva had different effects on
infection with L. major. A pair of sandfly salivary glands
contains 
1 µg of total protein (5), and the optimal
effect with whole saliva is achieved with 0.5 gland, or 250 ng of total
protein (5, 21). Because MAX is 1% of the total
protein of the salivary gland (22), one would have
predicted that 2–3 ng of synthetic MAX would have optimal effects, as,
in fact, was the case (Fig. 1). That lower doses of either MAX or
saliva had less of an exacerbative effect on L. major
infection was also expected (Ref. 21 and Fig. 1). That
higher doses of either MAX or saliva were also less effective was not
expected (Fig. 1). However, these observations were quite reproducible,
and a dose of 2 or 3 ng of MAX or 0.5–1.0 salivary glands consistently
yielded maximal effects.
The reason(s) why sandfly saliva and MAX display a biphasic effect on infection with L. major are currently unknown. However, there are many possible explanations for the phenomenon. For example, at different concentrations, MAX may be a homo- or heterodimer (with itself or other salivary proteins) that, as a result, interact with different forms of the PACAP receptor. There are at least eight forms, and each may signal different effects in the target cell (23, 24, 25, 26).
The results presented herein represent the "proof-of-principle"
experiments for our work with arthropod salivary proteins. We show that
a dual-function protein (MAX) is responsible for the effects of sandfly
saliva (Fig. 1, Table I), and that immunizing against MAX can protect
against infection with Leishmania (Fig. 2, Table II).
Moreover, the results suggest that MAX is the major molecule in sandfly
saliva that exacerbates infection with L. major because
vaccinating against this molecule neutralized the effects of whole
saliva. However, other factors may also contribute to exacerbation
because vaccinating against MAX may mask their effects. In addition,
the results confirm and extend the literature regarding the immune
response to Leishmania (27). That is, the
multiple effects that MAX has on the immune system would be predicted
to lead to exacerbation of infection with L. major. For
example, MAX inhibits T cell activation (28), stimulates
the production of cytokines that favor the development of an
exacerbative type 2 response (Refs. 4 and 15 ;
e.g., IL-6), and inhibits the production of molecules that are
important in the destruction of L. major (Refs.
4 and 15 ; e.g., TNF-
,
H2O2, and NO).
Fig. 2 demonstrates that vaccinating against MAX can protect CBA mice
against infection with L. major. We used the CBA model
because infection in this mouse mimics infection with L.
major in humans (i.e., both CBA mice and humans cure an infection
with the parasite). In addition, using this model allowed us to compare
our results with our previous work (e.g., Ref. 5).
However, pilot studies with BALB/c mice are showing that vaccinating
these mice also protects against L. major infection.
The results presented here suggest that arthropod vectors of disease are not simply "flying/crawling syringes." Rather, they play a dynamic role in the host/vector/pathogen relationship, and vaccinating against components of arthropod vector saliva holds promise as a novel approach toward vaccinating against vector-borne disease. Because arthropods transmit many pathogens, a single vector-based vaccine may help control the transmission of multiple diseases. Indeed, earlier work by others indicated that a vector-based vaccine might be effective. For example, vector saliva can modify the course of infection with bacteria (29, 30), viruses (8, 10, 31), and parasites (32, 33). Finally, recent work by Kamhawi et al. (34) showed that the bite of uninfected sandflies (this work used Phlebotomus papatasi sandflies; MAX is a protein found in L. longipalpis salivary glands) conferred resistance to a subsequent infection with L. major. Although the component in P. papatasi saliva that was responsible for the protection was not identified, these results suggested that a sandfly vector saliva-based vaccine for leishmaniasis might be feasible, and the results presented here demonstrate that this is the case.
For the experiments presented here we used the L.
longipalpis-L. major experimental combination so that
we could compare our results with those of previous publications and
thus interpret the findings within the context of this previous work.
We have not yet examined the effect that vaccination against MAX would
have on infection with L. chagasi, a parasite which is
vectored by L. longipalpis (35). However,
because L. longipalpis saliva enhances infection with
L. chagasi (36) and because vaccination against
MAX elicits a Th1 type response (Table III), it is likely that
MAX-vaccinated mice would be protected against challenge against any
species of Leishmania, so long as the parasite was
coinjected with MAX.
We elected to vaccinate against MAX here because it is an immunomodulatory protein in sandfly salivary glands. However, there may be other proteins in salivary glands that are more immunogenic and thus more suitable for vaccine formulations. This is particularly important because the level of MAX expression differs between different geographical isolates of the fly (17), and MAX is not present in the salivary glands of the Old World fly, P. papatasi. Rather, the saliva contains large amounts of adenosine and AMP (37).
It is perhaps not surprising that it has proven so difficult to develop effective vaccines against vector-borne pathogens/parasites. These organisms often have very complex life cycles. Moreover, a parasite by definition is an organism that lives in or on another organism and often this parasitic existence lasts for the lifetime of both organisms. Therefore, it may be very difficult to develop pathogen-based vaccines that are long-lived and that induce sterile immunity to parasites. However, vaccines that target more than one facet of the life cycle of a parasite (e.g., the pathogen itself, vector salivary factors, vector-pathogen interactions) may prove to be effective.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Richard G. Titus, Department of Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523. E-mail address: Richard.Titus{at}colostate.edu
3 Abbreviations used in this paper: MAX, Lutzomyia longipalpis sandfly maxadilan; PACAP, pituitary adenylate cyclase-activating polypeptide.
Received for publication June 22, 2001. Accepted for publication August 23, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and induces IL-6 by mouse macrophages through interaction with the PACAP receptor. J. Immunol. 160:1811.This article has been cited by other articles:
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  W. H. Wheat, K. E. Pauken, R. V. Morris, and R. G. Titus Lutzomyia longipalpis Salivary Peptide Maxadilan Alters Murine Dendritic Cell Expression of CD80/86, CCR7, and Cytokine Secretion and Reprograms Dendritic Cell-Mediated Cytokine Release from Cultures Containing Allogeneic T Cells J. Immunol., June 15, 2008; 180(12): 8286 - 8298. [Abstract] [Full Text] [PDF]
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 R. Gomes, C. Teixeira, M. J. Teixeira, F. Oliveira, M. J. Menezes, C. Silva, C. I. de Oliveira, J. C. Miranda, D.-E. Elnaiem, S. Kamhawi, et al. Immunity to a salivary protein of a sand fly vector protects against the fatal outcome of visceral leishmaniasis in a hamster model PNAS, June 3, 2008; 105(22): 7845 - 7850. [Abstract] [Full Text] [PDF]
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 T. M. Brodie, M. C. Smith, R. V. Morris, and R. G. Titus Immunomodulatory Effects of the Lutzomyia longipalpis Salivary Gland Protein Maxadilan on Mouse Macrophages Infect. Immun., May 1, 2007; 75(5): 2359 - 2365. [Abstract] [Full Text] [PDF]
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G. Caljon, J. Van Den Abbeele, B. Stijlemans, M. Coosemans, P. De Baetselier, and S. Magez Tsetse Fly Saliva Accelerates the Onset of Trypanosoma brucei Infection in a Mouse Model Associated with a Reduced Host Inflammatory Response Infect. Immun., November 1, 2006; 74(11): 6324 - 6330. [Abstract] [Full Text] [PDF]
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 J. V. BISHOP, J. S. MEJIA, A. A. P. DE LEON, W. J. TABACHNICK, and R. G. TITUS SALIVARY GLAND EXTRACTS OF CULICOIDES SONORENSIS INHIBIT MURINE LYMPHOCYTE PROLIFERATION AND NO PRODUCTION BY MACROPHAGES. Am J Trop Med Hyg, September 1, 2006; 75(3): 532 - 536. [Abstract] [Full Text] [PDF]
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 V. B. Reddy, A. O. Iuga, K. Kounga, and E. A. Lerner Functional Analysis of Recombinant Mutants of Maxadilan with a PAC1 Receptor-expressing Melanophore Cell Line J. Biol. Chem., June 16, 2006; 281(24): 16197 - 16201. [Abstract] [Full Text] [PDF]
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C. E. Demeure, K. Brahimi, F. Hacini, F. Marchand, R. Peronet, M. Huerre, P. St.-Mezard, J.-F. Nicolas, P. Brey, G. Delespesse, et al. Anopheles Mosquito Bites Activate Cutaneous Mast Cells Leading to a Local Inflammatory Response and Lymph Node Hyperplasia J. Immunol., April 1, 2005; 174(7): 3932 - 3940. [Abstract] [Full Text] [PDF]
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K. A. ROGERS and R. G. TITUS CHARACTERIZATION OF THE EARLY CELLULAR IMMUNE RESPONSE TO LEISHMANIA MAJOR USING PERIPHERAL BLOOD MONONUCLEAR CELLS FROM LEISHMANIA-NAIVE HUMANS Am J Trop Med Hyg, November 1, 2004; 71(5): 568 - 576. [Abstract] [Full Text] [PDF]
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N. B. Norsworthy, J. Sun, D. Elnaiem, G. Lanzaro, and L. Soong Sand Fly Saliva Enhances Leishmania amazonensis Infection by Modulating Interleukin-10 Production Infect. Immun., March 1, 2004; 72(3): 1240 - 1247. [Abstract] [Full Text] [PDF]
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D. J. Costa, C. Favali, J. Clarencio, L. Afonso, V. Conceicao, J. C. Miranda, R. G. Titus, J. Valenzuela, M. Barral-Netto, A. Barral, et al. Lutzomyia longipalpis Salivary Gland Homogenate Impairs Cytokine Production and Costimulatory Molecule Expression on Human Monocytes and Dendritic Cells Infect. Immun., March 1, 2004; 72(3): 1298 - 1305. [Abstract] [Full Text] [PDF]
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R. S. MILLERON, J.-P. MUTEBI, S. VALLE, A. MONTOYA, H. YIN, L. SOONG, and G. C. LANZARO ANTIGENIC DIVERSITY IN MAXADILAN, A SALIVARY PROTEIN FROM THE SAND FLY VECTOR OF AMERICAN VISCERAL LEISHMANIASIS Am J Trop Med Hyg, March 1, 2004; 70(3): 286 - 293. [Abstract] [Full Text] [PDF]
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S. Ahmed, M. Colmenares, L. Soong, K. Goldsmith-Pestana, L. Munstermann, R. Molina, and D. McMahon-Pratt Intradermal Infection Model for Pathogenesis and Vaccine Studies of Murine Visceral Leishmaniasis Infect. Immun., January 1, 2003; 71(1): 401 - 410. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, 1 ng of
MAX; 
, 3 ng of MAX; 
, 10 ng of MAX; 