CA2103788A1

CA2103788A1 - Reagents and methods for identification of vaccines

Info

Publication number: CA2103788A1
Application number: CA002103788A
Authority: CA
Inventors: Robert B. Grieve; Frank Glenn; Marcia Mika-Grieve; Janice A. Culpepper
Original assignee: Individual
Current assignee: Colorado State University Research Foundation; Paravax Inc
Priority date: 1991-02-12
Filing date: 1992-01-30
Publication date: 1992-08-13
Also published as: WO1992013560A1; AU1423792A; EP0571536A4; EP0571536A1

Abstract

Cells, serum or fractions thereof from exposed hosts, especially those with demonstrated ability to protect against infection are screening reagents to identify antigens for use in protective vaccines. Biological materials from exposed native hosts can be validated in vivo in an irrelevant host by their ability to destroy or impair the infectious agent. Validation is performed by implanting the infectious agent in a membrane enclosure into an animal host to which the candidate screening reagent has been transferred. The candidate providing sucessful destruction or impairment of the infectious agent can then be used to screen antigens produced by cDNA expression libraries or in extracts of the infectious agents to identify components of effective vaccines. Using this method, candidate heartworm immunogens, in particular one of 39 kd, have been identified.

Description

~VO92/13~60 PC~/US9~/~&~
~10~788 REAGENTS AND METHODS
FOR IDENTIFICATTON OF VACCINES

Technica~ Field The invention is directed to reagents and methods to identify and screen candidates for vaccines.
The identification and screen depends on the capacity of biological materials used as the screening reagents to destroy or impair the infective agent in an ln vivo incubator. More particularly, the invention concerns the u~e of cells, serum or fractions thereof obtained from exposed natural hosts wherein a recoverable implant of infectious agent is used to assess the protective effect when these materials are provided passively to the animal incubator~ The method is illustratively applied to determine useful active agents for an anti-heartworm vaccine.

Background Art The general method provided by the invention below to obtain suitable immunogens for use in vaccines is applied specifically to heartworm infection in canines and other mammals, which is caused by the nematode Dirofilaria immitls. Accordingly, a preliminary discussion of the nature of this infection and the life cycle of the D. immitis parasite may be helpful both in reviewing the background literature and in describing the invention. The adult forms of the parasite are quite large (males are 12-20 cm long and 0.7-Q.~ mm wide;
females are 25-31 cm long and 1.0-1.3 mm wide) and these preferentially inhabit the heart and pulmonary arteries ., : ~
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21~)~7~8 of the dog. The sexually mature adults, after ma~ing, produce as embryos microfilariae which are only 300 ~m long and 7 ~m wide. These traverse capillary beds and circulate in the vascular system of the dog in concentrations of 103-105 microfilariae per ml of blood.
one way of demonstrating infection in the dog is to detect the circulating microfilariae.
If the dog is maintained in an insect-free environment, the life cycle of the parasite cannot progress. However, when microfilariae are ingested by the female mosquito during blcod feeding on an infected dog, subsequent development (but not, of course, increase in numbers) occurs in the mosquito. The microfilariae go through two larval stages (Ll and L2) and finally become lS mature third stage larvae (L3) of about 1.1 mm length, which can then be transmitted back ~o the dog through the bite of the mosquito. It is this L3 stage, therefore, that accounts for the initial infection. As early as three days after infection, the L3 molt to the fourth larval (L4) stage, and subsequently to the fifth stage, or immature adults. The immature adults migrate to the heart and pulmonary arteries, where they mature and reproduce, thus producing the microfilariae in the blood.
"Occult" infection with heartworm in dogs is defined as that wherein no microfilaremia is demonstrable, but the existence of the adult heartworms can be determined through thoracic examination.
Control of heartworm infection in canir.es has largely been chemoprophylactic, and no effective vaccine is available for practical immunization of the dog population against this parasite. Further, there appears no generic method to determine suitable immunogens for use as active ingredients in vaccines directed against infectious diseases caused by parasites in general. The invention provides solutions to both of these problems.

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As to t~e general approach to a method to obtain vaccine components, the ability of various components of infectious agents to raise antibodies when injected into animal hosts is well understood not to be determinative of the ability of these components to behave as e~fective vaccines. A large number of materials are immunogenic and produce sera which test positive in immunoassays for ability to react with the immunizing antigen, but which fail to protect the hosts against infection. Antibodies which neutralize the infective agent in in vitro assays are much more likely to protect against challenge in v~vo. Accordingly, the use of "immune" serum simply resulting from "immunization" or from infection by the infectious agent to screen for candidate vaccines does not provide sufficient specificity to identify protective immunogens.
on the other hand, serum or other components of blood from immunized animals which is demonstrably protective against infection is assured to contain antibodies, cells, or other fastors reactive with an immunogen or infectious agent which will produce responses that protect against challenge.
In most infectious diseases, particularly those such as parasitic infections that have long and complex development courses, it is difficult to verify the protective effect of serum or T-cells from exposed animals for use as a screening reagent. First, verification of protection against challenge is tedious, since the host animal would first have to be challenged with the infectious agent and shown to be protected before it could be shown that antibody components of serum, for example, could be used as a screen. The definition of "protection" under such a regimen is often complex. Second, even if a protective effect against challenge is shown, it is not clear to what components of -WO92/13560 PC~/US92/~

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~, , the immune system the protection is due. The protective effect could ~e due to antibodies, cells, mediators of the immune system or to combinations thereof. Thus, although this method of obtaining the screening reagent is sometimes used, it is time-consuming and does not permit identification of protective components.
A more useful manner of obtaining blood components and substantiating their protective effects takes advantage of implanted diffusion chambers containing the infectious agents, such as those described by Grieve, R.B., et al. Am J Trop_Med Hyg (1988) 39O373-379, and by Abraham, D., et al., J Parasit (1988) 74:275-282. In the first of these papers, dogs which had been immunized against Dirof1laria immitis infection were supplied diffusicn chambers containing infective larvae.
The larvae in the chambers could then be evaluated for the effect of the previous immunizations. In the second paper referred to, mice were supplied diffusion chambers containing D. immitis third-stage larvae, and the effects on these larvae were used to determine the possible immunity of the mice putatively engendered by previous injections with L3. In the context of these papers, the use of the diffusion chamber containing the infectious agent, therefore, gives a convenient assessment of the effectiveness of certain directly administered active imm~nization protocols, but not of passively transferred protective effects of selected fractions of a ~arget host bloodstream.
The general method of the invention is illustrated specifically below in the context of heartworm infectio~ in dogs, a disease which is identified as one wherein the complexity of the parasitic infection makes the choice of a candidate immunogen for vaccination very difficult. Even naturally conferred immunity cannot be assured to exist, as dogs .. . , , . . , ~ .

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with previous or existing infections with D. immitls can be reinfected (Grieve, R.B., et al. E~idemiolo~ic Reviews (1983) 5:220-246). However, this review also reports that there is some evidence of a naturally occurring protective immune response. This evidence is stated to be the apparent limitation on the population of mature worms in infected dogs.
Furthermore, it has been possible to induce protective immunity artificially. Wong, M.M., et al., Ex~ Parasitol (1974) 35:465-474, reported the immunization of dogs with radiation-attenuated infective larvae. The dogs were protected to varying degrees upon challenge. Blair, L.S., et al., in Fifth International Conaress of Parasitology, Toronto, Canada (August 1982), reported successful immunization by infecting the dogs and terminating the infection at the fourth larval stage by chemotherapy.
Grieve, R.B., Proc Heartworm S~m~ (1989), pp.
187-190, reviewed t~e status of attempts to produce vaccines against heartworm in dogs. This report summarizes the use of infective larvae implanted in an inert diffusion chamber which permits the influx of cells and/or serum from the host and outflow of parasite material from the chamber to assess the effectiveness of inoculation protocols in both dogs and mice. The use of immunization with infective larvae was demonstrated to be partially effective in protection against subsequent challenge.
An alternative approach to finding a heartworm vaccine has been to attempt to identify prominent antigens in the infective stage of D. immitis. Philipp, M., et al., J Immunol (1986) l36:2621_2627, reports a 35 kd major surface antigen of D. immitis third stage larvae which was capable of immunoprecipitation with sera from doss carrying an occult experimental D. immitis infection ~, ' :

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21 03'788 -6-or with sera fro~ dogs immunized by irradiated third stage larvae. In addition, this group reported (Davis, T.B., et al., Abstract 404, 37th Annual Meeting, Am. Soc.
Trop. Med. Hyg. (1988)) three major surface proteins of the L4 stage of molecular weights 150 kd, 52 kd, and 25 kd. The 25 kd molecule seemed unique tc the L4 stage.
Ibrahim, M.S., e~ al., Parasitol (19~9) 99:89-97, using D. immi~ls L3s labeled with l25I, showed that a 35 kd and 6 kd component were shed into the culture medium by developing parasites. They further showed that antibodies from immunized rabbits and infected doqs immunoprecipitated the 35 kd, but not the 6 kd, component.
Scott, A.L., et al, Acta Tro~ica (1990) 47:339-353, reported characterization of the surface-associated molecules of L2, L3, and L4 of D. immitis by radiola-beling techniques and SDS-P~GE. They found major labeled components of 35 kd and 6 kd in extracts from iodine-labeled L2 and L3 stages; lactoperoxidase~
catalyzed labeling revealed Components Of apparent molecular weights 66 kd, 48 kd, 25 kd, 16.5 kd, and 12 kd. Iodine labeling of surface-associated molecules of L4 gave molecules of apparent molecular weights of 57 kd, 40 kd, 25 kd, 12 kd, and 10 kd; lactoperoxidase-catalyzed labeling showed additional bands of 45 kd, 43 kd, and 3 kd.- However, these were identified using uncharacterized serum sources or without serum identification.
Other approaches to obtaining vaccines against parasites in general have focused on the production of neutralizing antibodies. For example, bo~h in vitro studies by Tanner, M., et al., Trans Roy Soc Trop Med HYG
(1981) 75:173-174, and by ~im, B.K.L. et al., (ibid.) (1982) 76:362-370, and in vivo by Parab, P.B., et al., Immunol (1988~ 64:169-174, have demonstrated that anti-bodies are effective alone or with other immune - .
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components in killing filarial L3 from Di~etalonema (Acanthocheilonema) viteae or Bruqia malayi.
Furthermore, passive immunity to Schistosoma mansoni has been transferred from immune rats or humans to normal mice (Sher, A. et al., Parasitol (1975) 70:347-357; Jwo, J. et al., Am J Trop Med Hy~ (1989) ~l:553~562). None of these studies involved recovery and evaluation of the infectious agent implanted in an in vivo incubator for the agent treated with candidate protective components.
Disclosure of the Invention The invention provides both a general methodology for identification of suitable immunogens for inclusion in vaccines and, specifically, immunogens identifi~d using this method which are useful in controlling heartworm infections in dogs. The general methodology depends on the verification of the protective qualities of serum or cells used to detect or bind candidate antigens by virtue of the ability of the serum or cells to impair or destroy thè infectious agent in an in vivo incubator animal wherein an irrelevant host contains diffusion chamber implants of the infectious agent.
Accordingly, in one aspect, the invention is directed to a reaqent useful to identify immunogens for inclusion in vaccines against infectious agents. The reagent comprises cells, antiserum or fractions thereof -which has been demonstrated to be protective against the infective agent by its ability to confer, destroy or impair the infectious agent in an in vlvo model wherein an irrelevant host is implanted with a diffusion chamber containing the infectious agent. The method is, of course, limited to infectious agents of sufficient size to be retained by the diffusion chamber.

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, In another aspect, the invention is directed to methods to use these reaqents to screen for candidate vaccines in various potential sources of immunogens.
These sources of immunogens may be extracts or resolved extracts of the infectious agent, or DNA expression libraries obtained from the infectious agents. The invention is also directed to methods to confer passive immunity on hosts by administration of the reagent.
In other aspects, the invention is directed to ; lO the application of the reagents and methods of the invention to heartworm infection and, specifically, to a component of the L3 and L4 larval stage of D. immitis, said component having a molecular weight of 39 kd.
Additional components verified to react exclusively with protective immune serum cells or fractions are also disclosed.
In still another aspect, the invention permits evaluation of the candidate immunogens by providing a short-term test of their protective effect in the target host. Instead of challenging the immunized hosts with infectious agents directly and waiting several months to evaluate the outcome of infection, the host may be bled and the components of the blood or serum tested more directly in the experimental host containing the 2S diffusion chamber. This permits rapid screening of both naturally occurring components of the infectious agent and of synthetic peptides, carbohydrates and glyco-proteins.

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g Brief Description of _he Dr~
Figure l shows Western blots of D. immitis proteins immunoreacted with canine sera derived from immune and nonimmune dogs.
5Figure 2 shows Western blots of D immitis proteins immunoreacted with canine sera at various time points (days) after immunization.
Figure 3 shows the results of SDS-PAGE on proteins labeled with S-35 methionine extracted from D.
immitis L4 larvae and reacted with control and immune sera at various time points after immunization.
Figure 4 shows the results of proteins analyzed as set forth in Figure 3, but wherein the larval surface proteins are labeled with I-125.
Figure 5 shows the results of proteins analyzed as in Figure 3, but wherein the larval surface proteins are labeled using biotin.
Figure 6 shows the results of analysis of ; proteins present in the~excretory/secretory material which characterizes the transition from L3 to L4 and maintenance of L4's for 3-4 days thereafter. s Modes oP Carrvinq Out the Invention The method of the invention takes advantage of a model system which has been used to evaluate immunization protocols using the active response of the model host, but not to ascertain or validate the protective capacity of specific immune reagents generated in the natural target host and passively transferred into the experimental animal system. Thus, the method has not been used to identify suitable reagents which themselves are useful to _dentify and evaluate vaccine candidates.
The active immunization model as applied to heartworm is described in the 1988 papers of Grieve and Abraham cited above. In this model, the infectious a~ent, third-stage .' ~ , .
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larvae, were first obtained as follows. D. immitis was maintained in a dog infected with parasites which had been passed once from an infection obtained through the U.S.-Japan Cooperative Medical Sciences Program, National Institutes of Health. Aedes aegypti mosquitos Liverpool tblackeye strain) were infected with the parasite by feeding on microfilaremic blood using the artificial feeding apparatus described by Rutledge, L.L., et al., Mosq News (1964) 24:407-419. Fifteen days after infection, the mosquitoes were cold-anesthetized and surface sterilized by immersion in 95% ethanol followed by a 3 min wash in 1% benzalkonium chloride in 0.01 M
phosphate-buffered saline, pH 7.2. The mosquitoes were washed three times in PBS and incubated over a 60 mesh screen inside a funnel filled with medium (the medium was described in Abraham, D. et al., J Parasitol (1987) `~ 73:377-383) and the larvae were collected 90 min after incubation.
The recovered L3 larvae are then placed in diffusion chambers and implanted subcutaneously. For implantation in dogs, chambers are composed of two 14 mm Lucite rings sealed with S.0 ~m hydrophilic Durapore membranes (Millipore, Bedford, MA). The larvae are inserted through a hole in one of the Lucite rings which ; 25 is subsequently sealed with nylon thread. The dogs are anaesthetized and a subcutaneous pocket is formed in the dorsal skin of the neck; the chamber is implanted in the pocket and the wound closed with suture.
For implantation in mice, similar chambers are used and implanted into a subcutaneous pocket formed laterally to the lumbar spine. The chambers can be removed at the desired time for evaluation of the contained larvae.
A variety of chamber desiqns can be used, and 3S the porosity of the diffusion membrane chosen according .

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to the nature of the infectious agent and ~o desired llmitation on the nature of the inward diffusion.
In the use of the model to prepare screening reagents or to evaluate samples from target animal hosts, the diffusion chambers are allowed to remain in vivo in an irrelevant host which has not been actively immunized and a portion of serum or other component believed to be capable of conferring protective immunity is administered to the irrelevant host. A portion is retained for use in the invention method to screen immunogens if destruction or impairment of infectious agent is shown. For use in dogs as a model, for example, about 0.5 ml of serum, for example, is administered by placing the serum in the pocket along with the diffusion chamber. For mice, similar amounts but fewer chambers are used. The chamber containing the infectious agent is allowed to remain in place for a time sufficient to evaluate the protective effects of the serum or other component. This time is determined by the nature of the infection and the protective capacity of the test sample.
After the required experimental time has lapsed, the chamber is removed and the infectious agents are retrieved. The protective capacity of the passively transferred components from the natural target host is evaluated by any deleterious effects seen in the infectious agents. These effects may include, but are not limited to, such parameters as killing, stunting, alterations in normal morphology, alterations in measurable metabolism, failure to mature in in vitro culture, and failure to infect conventional target hosts.
A fraction of the sample which has been retained during the evaluation is then used to screen candidate immunogens. Any technique which results in the complexation of the validated component with the candidate immunogen can be used. For example, an extract :~' ~::

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WO92/13560 PCr/US92/00~8 210378~ -12-of the infectious agent is subjected to resolution using a variety of chromatographic techniques, including size separation using gel permeation chromatography, electrophoresis on polyacrylamide gels, ion exchange chromatography, affinity chromatography, and the like.
The whole extract or resolved extract is then tested for reactive effect with the protective component. If serum is the protective component, a complex will be formed.
If the component is a cell subfraction with receptor for antigen, the antigen will be bound. The complex is recovered, and the immunogen recovered from the complex.
In applying the method to crude extracts, the protective serum or cells can be used as an affinity ligand in chromatographic techniques to isolate immunoreactive components. Alternatively, as set forth above, the extract can first be resolved and the appropriate fractions identified by complexation with the protective cells or serum.
In an alternative approach, the protective cells or serum can be used as a screening reagent for a cDNA library prepared from the infectious stage or later stage of the infective agen~ which is constructed in expression vectors. A commonly used and convenient such library is the Agtll library described by Young, R.A., and Davis, R.W., Proc Natl Acad sci !USA) (1983) 80:1194-1198. The expression library is platecl and screened with the protective cells or serum to identify colonies which produce immunoreactive components. The positive colonies are then purified, and the cDNA inserts in the expression vectors recovered and sequenced to identify the encoded proteins.
The cDNA inserts identified as expressing immunogens using the reagent of the invention can then be ligated into alternative conventional expression systems for production of the proteins useful as vaccines.

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21~37~8 Alternatively, the inserts may be ligated into expression systems which are live recombinant carriers such as Sindbis virus, vaccinia virus or other pox viruses, Herpes viruses, Adenoviruses, Salmonella or Mycrobacteria. These infectious agents can then be used directly to immunize the target hosts by generation of immunogen in situ.
The vaccines of the invention are administered in a manner consistent with the nature of the vaccine and the nature of the disease and subject. If the recombinantly produced or native immunogens are administered as proteins, they are formulated with conventional excipients for injection or other systemic administration to the host. In addition to injection, lS formulations may be prepared for other administration methods which include transmucosal or transdermal delivery into the bloodstream. If the immunogens are administered in the form of recombinant DNA expression systems in infectious agents, administration is typically by injection or other mode of conventionally administering the infective agent.
The foregoing approach is applicable to the discovery of suitable immunogens for any disease wherein the infectious agents can conveniently be used in the animal model. In general, such diseases are those caused by organisms of sufficient size to be retained in a diffusion chamber. Thus, in general, the method is applicable to a variety of parasitic diseases including those caused by other filarial nematodes, such as Di~etalonema ~erstans, DiPetalonema stre~tocerca, Wuchereria bancrofti, B. malavi, Mansonella o7zardi, Loa loa~ and o. volvulus.
Other disease-causing oryanisms to which the method is applicable include Stronqyloides spp., Stronaylus spp., Haemonchus spp., Trichostronqvlus spp., - . ..

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2 1~37 8~ -14-ostertaqia spp., Coo~eria spp., Dictyocaulus spp., Nematodirus spp., Cyathostominae (small strongyles of horses), Oesophaqostomum spp., Chabertia ovina, Ancylostoma spp., Uncinaria spp., Bunostomum spp., Filaroldes spp., Aelurostronqylus abstrusus, those nematodes of the Order Ascaridida (Ascarids), Trichinella spiralis, Trichuris spp., Angiostrongylus spp., Enterobius vermicularis. In applying the method, the infectious stage of the agent is determined, if applicable, for identification of the form to be enclosed in the chamber. Protocols are adjusted to take account of the time required for exertion of the prctective ef~ect of the target animal-derived component, such as cells or serum.
Using the invention reagent, several immunogens associated with D. immitis were obtained. One class of such immunogens have molecular weights of 66 kd, 65 kd, 59 kd, 39 kd, 33 kd, 23l24 kd, 22/20.5 ~d and 14 kd. The foregoing proteins are produced by L3 and/or L4 larvae.
DNAs encoding these proteins can be recovered from cDNA
expression libraries prepared from the mRNA of these larval stages by screening for expression with the reagent of the invention. The appropriate cDNA inserts can then be used to produce recombinant immunogen in a suitable expression system which yields practical amounts of the protein or can be ligated into recombinant systems which generate the immunogen in sltu, such as the vaccinia virus system.
The following examples are intended to illus-trate, but not to limit the invention.

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21a~7~s Example 1 Production_of_Sera for Passlve Transfer Four dogs were immunized with chemically-abbreviated infections, and two dogs served as chemically-treated controls. The dogs were housed in indoor mosquito-free individual cages at a temperature of 22C and 40-65% humidity. On day 532, post initial immunization, each dog was challenged with 100 L3 D. immitis larvae contained in 5 diffusion chambers, described above. Concomitant with chamber implantation, the dogs were injected subcutaneously with 50 L3 and the infection was allowed to proceed beyond the anticipated pre-patent period. Challenge in~ections were repeated on day 588 with 100 larvae within diffusion chambers and 30 L3 inoculated subcutaneously. Serum was collected at numerous time points from the immunized dogs, including 554, 588, 602 and 642 days after initial immunization whi~h corresponded to days 22, 56, 77 and 117 after initial challenge. The isolation of serum provides a source of immunoglobulins and soluble factors, but not of cells. Antibody levels were measured to L3 and L4 surf~ce antigens using an indirect fluorescent antibody assay and to L3 and L4 soluble antigens and an excretory-secretory antigen fraction by an indirect ELISA, as dPscribed by Grieve et al., ~19~8) (supra). The sera were pooled and validated as a significant factor in the protective effect in the mouse incubator as described in Example 2.

Exam~le 2 Validation in Mouse A subcutaneous pocket was formed in male Balb/C
BYJ mice approximately 10 weeks old and 20 L3 inoculated into diffusion chambers as described above were implanted into the pocket, along with 0.5 ml of the demonstrated .
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21037~8 -16-protective serum to be tPsted. Serum samples were retained for future use. The diffuslon chambers werP re covered two or three weeks later. Living larvae in the .
chambers were counted and placed into glacial ace~ic acid followed by 70% ethanol con~aining 5% glycerin. The ethanol was allowed to evaporate leaving the larvae in glycerin; the larvae were measured using projected images in the Macmeasure image analysis system on a Macintosh computer.
Three groups were used: experiment 1 used equal portions of serum from individual dogs a~ each of the three collection points described in Example 1 (days 56, 77 and 117). In experiments 2 and 3 only sera from immune dogs 117 days after initial challenge were used.
Control sera were used in all cases; in experiment 2 this was a pool from 12 naive dogs; in experiment 3 from an individual dog. These groups also contained controls which received no serum.
In experiment 1, chambers were recovered two weeks post-inoculation. The survival rate of larvae in chambers from mice receiving serum from immune dogs were lower than those from mice receiving normal dog serum, but the difference was not statistically significant.
Also, no difference was seen between the length of larvae in each case.
. In experiments 2 and 3, the chambers were recovered three weeks after infection. There were significant differences în the larval recoveries between those receiving serum from naive dogs and those from immune dogs--about 34-33%. The lengths of the larvae were also significantly shorter in those chambers receiving sera from immune dogs.

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Example~3 Identification of Antiqens Crude extracts of L3 and L4 larvae were prepared as follows:
All procedures are performed at 4C or on ice.
The worms were collected and washed twice with wash buffer (PBS/0.1% Triton X-100) and then with extraction buffer (0.05 M Tris/HCl~ pH 6.8; 2% CHAPSO; 1 mM PMSF; 1 mM EDTA; 1 mg/l leupeptin; 1 mg/l pepstatin). (Other detergents may be used in place of CHAPSO, includiny O.5%
Triton X-100, 0.5% CTAB, 2% DOC, or 2% SDS/5% 2-ME/8M
urea.) The worms are then homogenized 5x for 1 minute each, with l minute rest periods, using 250 to 500 ~l for 10,000-20,000 worms (- 500 ~g). This volume i5 transferred to an additional tube, and the homogenizer washed with a clean 100-250 ~l of extraction buffer and the wash pooled with the homogenate. The tube is rocked 4 hours-overnight and centrifuged at 12,000g for 10 minutes. The supernate is harvested and the pellet is washed once with extraction buffer and saved for additional extractions if desired. The combined total volume o~ extract is less than 1 ml and about 20 ng of protein is solubilized per L4 larva used.
The procedure for L3 is identical, except that the.wash buffer is PBS without detergent.
The extracts were subjected to polyacrylamide gel electrophoresis and tested with portions of the serum shown to be protective in the murine model. When pooled canine sera which had been shown to stunt larval growth as described in Example 2 were used as the immuno-reactant in the Western blots, the results were as shown in Figure 1. The 39 kd band shown in Figure l is separated from a 45 kd band when a second dimension is added to the electrophoresis. This 45 kd protein is not . . . .
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w092/l3560 PCT/US92/00&~

210378~ -18-immunoreactive. As seen, the serum is specifically immunoreactive with a 39 kd protein present in the L4 larval stage. This protein has a pI of about 5. Control serum shows no immunoreactivity with this protein.
Reactivity to the 39 kd molecule is present in immune dogs, but not in control dogs. Sera from dogs with microfilaremic infection or amicrofilaremic infection do not recognize this molecule.
In addition, bands were present at 66 kd, 24/23 kd, and 14 kd, as shown in Figure 2.
The proteins associated with the larval stages were also metabolically labeled using S-35 methionine; or the surfaces were labeledt prior to extraction, with I-125 or with biotin. For labeling with S-35 methionine, the radiolabeled amino acid was added to the parasites after 48 hrs in culture according to the method of Abraham, D., et al., J P _asitol (1987) 73:377-383. For labeling with I-125, the method of Mok, M., et al., Molec Biochem Parasitol (1988) 31:173-182, was used. For biotinylation, a modification of the method of Alvarez, R.~., et al., Molec Biochem Parasitol (1989) 33:183-190, was employed. In the modified procedure, NHS-longchain biotin was substituted for biotin per se.
Thus, additional identification could be had using these prelabeled proteins which immunoprecipitated with.the successfully validated immune serum. These results are shown in Figures 3, 4 and 5. As shown in Figure 3, additional candidates are found at 59 kd and 16 kd, as indicated by the arrows. The radioactive iodine-labeled material shows a candidate at about 33 kd with ahigher moiecular weight smear at 35.8-34.5 kd. This was present beginning at day 345 and persisting until day 642 in some, but not all, immune dogs. An additional band was present at 14.5 kd. This is indicated in Figure 4.

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2~03788 Figure 5 shows the results when the proteins were labeled by biotinylation in an enhanced chemi-luminescence assay. A transient band represented by 65.3 kd was recognized by 3 of 4 immune dogs.
In addition, passive transfer of the earliest immune dog serum which showed uniform responses to the 39 kd protein (i.e., the day-142 immune serum shown in Figure 3) was able to effect killing of the entrapped larvae; recoveries of intact larvae were only S8.3~ in the case of immune serum compared to 65.8~ for controls.
To summarize, the following antigen candidates were obtained:
A 39 kd protei~ which reacted with sera from all immune dogs but not with sera from naive cohorts.
The protein is shown to be present in Western blots obtained from L4 soluble antigen and solubilized L4 larval pellets and is shown to be present, although apparently to a lesser degre~, in L3. This protein appears to be absent from~adult D. immitis and the microfilariae. It is clearly a distinct protein from the p35 protein described bv Scott, A.L., et al., Acta Trop~ (1990) (supra), and is relatively acidic, having a pI of approximately 5.
A 14 kd immunogen is detected with immune dog seru~ using Western blots and immunoprecipitation employing S-35 and iodine-labeled components. The protein is detected with immune dog serum, but not by serum from controls.
Additional proteins detected are of 66 kd and 30 23/24 kd.
Another potential source of protective antigens in parasitic diseases are excretory/secretory products which are associated with various stages of the parasite.
The transition between L3 and L4 involves excretionl 3S secretion of a number of proteins which are harvested as ' . :

W092/l3560 PCT/US92/00~

follows: Larvae are cultured at 250-400/ml, washed at 48 hr and cultured an additional 4 days. The worms are then settled out and the supernate collected. This is filtered through a 0.45 ~m filter and protease inhibitors added as in L4 solubilization. The ES is then concen-trated and buffer exchanged by ultrafiltration over a 10 kd membrane (Amicon Centriprep-10 and/or Centricon-10). The final buffer is 0.05 M Tris/HCl pH 6.8 with protease inhibitors. ~ields may be app. 5 ng/larvae.
10 Final volume frequently 150-250 ~1. This extract, referred to as DILEX, was prepared using larvae which were metabolically labeled with S-35 methionine and tasted with respect td immune and control sera from dogs.
The immune serum was that obtained on day 554 post immunization as set forth in Example 3.
Immunoprecipitation with respect to the immune serum was - obtained at 22/20.5 kd and 14.3 kd, as shown in Figure 6.
In Figure 6, lane 1 shows molecular weight standards;
lane 2, the immunoprecipitates from immune dog; lane 3, - 20 from control dog; lane 4, bead control; and lane 5, DILEX
~ itself.
, Example 4 Isolation of cDNA's and Genes Encodina Potential D. immitis Protective ImmunQ~ens . Genomic and cDNA expression libraries in AZapII
(Short, J.M., et al., Nucleic Aclds Res (1988) 16:7583_ 7600), a derivative of ~gtll, were prepared from total genomic DNA, or L4 or L3 larval stage mRNA's, respectively, using standard procedures (Short Protocols in Molecular Biolo~y (1989) Ausubel, M.F., et al., eds.) Screening of these libraries with pooled immune dog sera permits identification of clones which contain candidate antigens. The clones identified as immunoreactive with the immune serum provide a source of DNA encoding desired WO92~135~ PCT~US92/~0 proteins which can conveniently be produced as fusion proteins in E. CQli.

Example 5 Construction of GST ~usion Proteins The DNA inserts are recovered from the AZapII
phaqemid by digesting with EcoRI and purified using agarose gel electrophoresis. The purified DNA is ligated into the expression vector pGEX-3X such that when the plasmid is expressed in E. coli the protein enooded by the DNA insert produces a fusion protein with glutathione-S-transferase. This procedure is described in detail by Smith, D.B., et al., Gene (19g8) 67:31-40.
Plasmids containing the DNA inserts are transformed into E. coli and successful transformants are grown in the presence of IPTG. The induced fusion protein is purified from the lysate by affinity chromatography with glutathione-beads as described by Smith et al. (supra).

Example 6 Immunization of Doas Recombinant peptides derived from any of a variety of expression systems are used to immunize dogs for the purpose of obtaining specifically reactive blood components. Recombinant antigens are administered to dogs with or without adjuvant by the subcutaneous, intramuscular, intradermal or intravenous routes.
Following single or multiple immunization, blood is collected from dogs by routine venipuncture. Serum is collected from coagulated blood and used directly or stored frozen prior to use. Leukocytes are collected from anticoagulant-treated blood by density gradient centrifugation and used directly or stored by freezing at -1C/minute with storage in liquid nitrogen.

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Claims

1. A reagent useful to screen candidate components for vaccines against infectious diseases, which reagent comprises cells, serum or at least one fraction thereof, obtained from an exposed native host, and which cells, serum or fraction has been demonstrated to destroy or impair the infectious agent by providing said cells, serum or fraction to an animal which has been implanted with the infectious agent encapsulated in a diffusion membrane, and observing said destroying or impairing of said infectious agent.

2. The reagent of claim 1 wherein the animal is a mouse.

3. The reagent of claim 1 wherein said infectious agent is a nematode.

4. The reagent of claim 3 wherein said nematode is Dirofilaria immitis.

5. The reagent of claim 4 wherein said D.
immitis is the L3 or L4 larval stage.

6. A method to screen for immunogens useful as vaccines to protect against infection, which method comprises contacting a source of candidate immunogens with the reagent of claim 1 under conditions wherein complexation occurs between said immunogens and said reagent, and recovering the complexed immunogens.

7. The method of claim 6 which further includes recovering said immunogen from the complex.

8. The method of claim 6 wherein said source of candidate immunogens comprises an extract from the infectious agent.

9. The method of claim 8 wherein said extract has been resolved into fractions.

10. The method of claim 6 wherein the source of candidate immunogens is an expression library obtained from the infectious agent.

11. The method of claim 10 wherein said expression library is cloned into .lambda.gtll or a derivative thereof.

12. The method of claim 6 wherein said source of candidate immunogen comprises a synthetic peptide or glycopeptide.

13. The method of claim 6 wherein said infectious agent is a nematode.

14. The method of claim 13 wherein said nematode is D. immitis.

15. The method of claim 14 wherein said infectious agent is the L3 or L4 larval stage.

16. A method to identify a reagent useful to screen candidate components for vaccines against infectious diseases, which method comprises providing cells, serum or at least one fraction thereof, obtained from an exposed native host, to an animal which has been implanted with the infectious agent encapsulated in a diffusion membrane, and observing said destroying or impairing of said infectious agent.

17. The method of claim 16 wherein said infectious agent is a nematode.

18. The method of claim 17 wherein said nematode is D. immitis.

19. The method of claim 18 wherein said infectious agent is the L3 or L4 larval stage.

20. A composition of matter useful to protect against infection identified by the method of claim 16.

21. A vaccine useful to protect against infection containing as active ingredient an immunogen identified by the method of claim 6.

22. The vaccine of claim 20 wherein said immunogen has a molecular weight selected from the group consisting of 66 kd, 65 kd, 59 kd, 39 kd, 33 kd, 23/24 kd, 22/20.5 kd and 14 kd.

23. A method to confer passive immunity on an animal host, which method comprises administering to said host the reagent of claim 1 in an amount effective to confer immunity.

24. An immunogen effective to protect a host against heartworm infection, said immunogen in purified and isolated form, and having a molecular weight of 39 kd, a pI of about 5 and obtainable from extracts of the L3 or L4 stage of D. immitis.

25. A pharmaceutical composition for immunization of dogs against heartworm, which composition comprises a component obtainable from L3 or L4 larvae of D. immitis having a molecular weight of 39 kd, and a pI
of about 5.

26. A method to immunize dogs against heartworm, which method comprises administering to a canine subject in need of such treatment an amount of the composition of claim 21 effective to protect said subject against heartworm infection.

27. A pharmaceutical composition for immunization of dogs against heartworm, which composition comprises an immunogen identified from L3 or L4 larvae of D. immitis by the method of claim 6.