Pharmaceutical composition and its use for the prophylactic or therapeutic treatment of retroviral diseases

The invention relates to a pharmaceutical combined composition comprising as active ingredients an effective amount of the retroviral transmembrane envelope protein p15E of a gammaretrovirus and an effective amount of a retroviral surface envelope protein of a gammaretrovirus, whereby said proteins are contained as single molecules, for inducing neutralizing antibodies which are directed against retroviral infections. The invention also relates to a method for inducing an antibody response and a method for passive immunization of a mammal by using neutralizing antibodies which are generated with said pharmaceutical composition.

Immunization is the most effective method to prevent diseases caused by infectious agents. It is known that three types of feline leukemia virus (FeLV) vaccines are currently available: inactivated whole virus preparations, inactivated mixed subunit preparations from FeLV-infected tissue culture filtrate and recombinant FeLV proteins. In detail, three vaccines are composed of inactivated whole virus, two are gp70 subunit vaccines and two are recombinant vaccines. The commercially available vaccines containing inactivated FeLV subunit preparations are Fevaxyn, Leucocine and Leukoceli2. Leucogen (Virbac) is an example of a recombinant vaccine comprising recombinant non-glycosylated surface envelope protein p45. However, FeLV vaccines presently in wide use are generally poor inducers of virus neutralizing antibodies. None of the vaccines regularly induce virus neutralizing antibodies following vaccination: such antibodies are usually detected after recovery from challenge only. Therefore, none of the seven commercial FeLV vaccines currently available in the USA and Europe provide 100% protection against infection, and there is only limited scientific data concerning long-term duration of immunity after vaccination.

It was recently shown that neutralizing antibodies can be induced which are specific for the transmembrane envelope protein p15E of porcine endogenous retrovirus (Fiebig et al. (2003) Virology 307, 406-413) and of FeLV-A in goats and rats (Langhammer et al. (2005), Vaccine 23, 3341-3348). The latter can exhibit a neutralization capacity of up to 99 % at a serum dilution of 1 :5.

EP 0 247 904 A1 discloses the induction of antibodies by a fusion protein containing the complete p15E transmembrane envelope protein and the gp70 surface envelope protein. gp70 is not glycosylated when the viral gp70 genes are expressed in E. coli. The physical form of the recombinant antigen used to immunize cats can be either aggregated or non-aggregated. The composition coufd be effective in producing an active immune reaction to protect the animals against an exposure to FeLV or immunologically related viruses. It is unclear whether neutralizing antibodies have been induced or not. Furthermore, the aggregated form makes the handling difficult.

Therefore, the technical problem forming the basis of the present invention is to provide a pharmaceutical composition which has an improved efficacy in neutralizing retroviruses and is ab!e to protect mammals safely against retroviral infections.

The present invention solves this problem by providing a pharmaceutical combined composition comprising as active ingredients an effective amount of the retroviral transmembrane envelope protein p15E of a gammaretrovirus selected from the group consisting of FeLV (Feline leukemia virus), KoRV (Koala retrovirus) and PERV (Porcine endogeneous retrovirus) and an effective amount of a retroviral surface envelope protein of a gammaretrovirus selected from the group consisting of FeLV, KoRV and PERV, or parts of said proteins, or the DNA encoding said proteins, whereby said proteins are contained as single molecules.

Surprisingly, it has been found that higher titers of neutralizing antibodies are induced by the combined immunization with p15E and a surface envelope protein, such as for instance the non-glycosylated p45, in comparison to responses using either p15E or the surface envelope protein alone. Neutralization relates to a mechanism which prevents or inhibits the infection and propagation of a retrovirus, i.e. the immune system is activated by the inventive pharmaceutical composition. The neutralizing antibodies are especially directed against the viral structures which induced them. Both active ingredients are recognized by distinct antibody fraction. The virus infection is prevented by the direct interaction of the neutralizing antibodies with both the retroviral transmembrane envelope protein and the retroviral surface envelope protein. Although the tota! titer of binding antibodies as well as the single titers of binding antibodies to the respective active substances remain approximately constant, the neutralization capacity is unexpectedly enhanced. The inventive composition generates a retroviral protection of every subject of such species capable of being immunized. In addition, the complete anti-viral protection achieved herewith is of clear advantage over the

partial immunization of a mammal subset if using the components alone. Particularly, the inventive pharmaceutical composition causes high titers of neutralizing antibodies within short periods of immunization along with an effective reduction of the number of retroviruses. The high neutralizing antibody titers are reflected by a high dilution of serum which is obtained after immunization and used in neutralization assays. Simultaneously, adverse effects which could be caused by other serum components are largely reduced due to their diluted presence. The inventive composition is also characterized by advantageous physical/ chemical features of the single molecules, such as high expression rate, solubility, stability and/or size enabling a convenient handling.

The terms "effective amount" or "effective dose" or "dose" are interchangeably used herein and denote an amount of a pharmaceutical compound having a prophylacticaliy or therapeutically relevant effect on a retroviral caused disease or pathological conditions. A prophylactic effect prevents the infection with a retrovirus after the infiltration of single viral representatives such that the subsequent propagation of the virus is strictly diminished, or it is even completely inactivated. A therapeutically relevant effect relieves to some extent one or more symptoms of a retroviral disease or returns to normal either partially or completely one or more physiological or biochemical parameters associated with or causative of the disease or pathological conditions. The determination of an effective amount is a routine exercise in the pharmaceutical arts taking various physical parameters, such as weight, age and the like into account, and is best determined by the attending clinician.

The retroviral transmembrane envelope protein and the surface envelope protein can be combined to the inventive pharmaceutical composition without viral species restriction. It is possible that a specific p15E or a specific surface envelope protein can generally act as enhancer in the meaning of the invention. The principle underlying the present synergistic effect is not known. Preferably, the proteins of the pharmaceutical composition are of the same retroviral origin. It guarantees an antibody induction which is specifically and exclusively directed against the same retrovirus target, thereby minimizing the probability of cross-reactivity.

The family of transmembrane envelope proteins has a homologous primary structure. The transmembrane envelope protein p15E, is associated with the membrane by at least one membrane passage, further comprising at least one fusion domain and at least two alpha-helical structures, NHR and CHR (N-terminal helical region and C-

terminal helical region, respectively). The active ingredient p15E is obtained from any gammaretrovirus comprising a membrane which is associated with p15E. It is more preferably obtained from FeLV, PERV or KoRV, most preferably FeLV.

In an embodiment of the invention, the retroviral transmembrane envelope protein comprises a part thereof. It is preferred that the part comprises the ectodomain of p15E, whereby the ectodomain consists of two alpha-helical structures and a cysteine- cysteine-loop. More preferably, the part is the ectodomain or variants, mutants, parts of the ectodomain or homologous sequences having the same function. A couple of methods are known to the skilled artisan to generate equivalent ectodomains, i.e. proteins which are analog in function to those of the inventive teaching. Therefore, the invention also contains the aforementioned modifications. For example, mutants can be generated by substitution, deletion, insertion, translocation, inversion and/or addition of at least a single amino acid, it is known that certain amino acids exhibit similar physicochemical characteristics making the substitution among each other possible. Variants of the ectodomain can be arise from modifications, such as alkySation, arylation or acetylation of at least a single amino acid, from incorporation of enantiomers and/or from fusion of the ectodomain with a single or multiple amino acids, a peptide or a protein. It is preferred in the meaning of the invention that the ectodomain is fused to a purification tag for affinity chromatography. Parts of the ectodomain relates to a restriction to those regions which are sufficient for the expression of a specific function. All alterations are inevitably limited by the requirement of preserving the function. However, the parts of the ectodomain can be very small due to the characterization of epitope which also induce neutralizing antibodies as peptides, in the meaning of the invention, it is to be clearly distinguished between ectodomain parts of any size and homologous sequences which homology is related to the entire ectodomain. Preferably, the homology between a natural ectodomain and a derivative thereof having the same features amounts to at least 60%, more preferably 75%, most preferably 90%. Simiiariy, the homology is to be considered if the aforementioned part of any size is altered to a variant or mutant. In addition, several techniques are described in prior art to generate non-homologous peptides with the same function. All peptide derivatives which are developed on the basis of the present ingredients by such procedures are covered by the present teaching if solving the problem of the invention.

In general, transmembrane envelope proteins comprise an ectodomain, an anchor domain (also termed membrane passage), a fusion domain and a cytoplasmatic

portion, whereby the ectodomain comprises two alpha-heiicai structures and a cysteine-cysteine-loop. It has been found that membrane passage, and cytoplasmatic portion are dispensable for inducing neutralizing antibodies. The ectodomain contains highly conserved regions which do not mutate during virus replication, thereby representing the principal antibody target. These regions termed epitopes E1 and E2 have been identified for several ectodomains. The ectodomain is characterized by the induction of neutralizing antibodies in several species and a better soiubility compared to the total transmembrane envelope protein. Due to its small size, the ectodomain is cost-efficiently produced. In the scope of the invention it is even sufficient that p15E, comprises the essential epitope regions to generate the desired immune reaction of neutralising antibodies.

In a preferred embodiment of the invention the FeLV p15E ectodomain may comprise the amino acid sequences E1a (LETAQFRQL) ₁ E1 b (iQALEESISALEK), E2a (KQRQQLF) and/or E2b (WFEGWFN). Epitope mapping of antibodies induced by simultaneous immunization with a surface envelope protein, such as p45 (Leucogen), and p15E revealed the same pattern of response as described after immunization with p15E alone. The same epitopes are detected, two of them are located at the N- terminus of the ectodomain (designated epitope E1a and E 1b) and two others at the C- terminus of the ectodomain (designated epitope E2a and E2b). The presence of both E1 and E2 seems to be crucial. Furthermore, the general proximity and immunogenicity suggests that the E2a and E2b epitopes represent the functional equivalents of the 2F5 and 4E10 (these are antibodies isolated from HIV infected individuals and neutralising a broad range of HIV) epitopes, respectively. Sera from rats immunized with FeLV p15E recognize a 4E10 equivalent and/or a 2F5 equivalent in the C-terminal end and E1 a and/or E1 b in the N-terminal end. Simultaneous immunization of both antigens increased also the recognition of an epitope in p15E crucial for neutralization (E1 b).

According to the invention the surface envelope protein is originated from a gammaretrovirus, preferably FeLV, PERV, KoRV, more preferably FeLV.

ft is particularly preferred to select both the surface envelope protein and p15E from the same retroviral origin which is FeLV, PERV or KoRV.

Of course, it is possible that variants, mutants, parts of the surface envelope protein or homologous sequences having the same features represent an active ingredient of the present invention. The prior teaching of the present specification concerning the

ectodomain and derivatives thereof is considered as valid and applicable without restrictions to alterations of the surface envelope protein if expedient,

In an embodiment of the invention, the surface envelope protein is gp70 or an unglycosylated form thereof. Either the glycosylated or the non-glycosyfated form increase the neutralizing titer significantly if combined with p15E. The unglycosylated form is preferred, and it can be advantageously produced in prokaryotic expression systems at low costs. In a preferred embodiment of the invention, the surface envelope protein is p45 which corresponds to the unglycosylated form of gp70. Its sequence has always to correlate with the virus against the immunization is directed, preferably gp70 or p45 from FeLV, PERV or KoRV.

The active ingredients of the pharmaceutical combined composition are obtained from natural sources, (t is possible to gather total proteins form the retrovirus, or parts thereof from the retroviral protein, respectively. Preferably, the ingredients are recombinantly expressed and purified. Therefor, one or both ingredients can be fused with a tag for affinity chromatography, such as Strep-tag, His-tag, GST-tag, Arg-tag or the calmodulin binding protein, preferably the calmodulin binding protein (CBP). CBP binds to a calmodulin resin, e.g. to be used as column matrix. The column is loaded with the protein suspension and all components lacking CBP are immediately eluted. After removal of unspecific binders by washing steps, the CBP fused ingredient are removed from the column. The tag does not affect the induction of neutralizing antibodies of the ingredients. Alternatively, the DNA encoding the protein sequences can be obtained, amplified, altered or synthesized with techniques known to the skilled artisan. Subsequently, the DNA can be introduced into a vector and transcribed and translated in cells.

In another embodiment of the present invention the pharmaceutical combined composition comprises as active substances an effective amount of a hybrid protein comprising the backbone of the retroviral transmembrane envelope protein p15E of a gam ma retrovirus selected from the group consisting of FeLV, KoRV and PERV and two domains E1 and E2 derived from gp41 of HIV-1 , and an effective amount of the retroviral surface envelope protein gp120 of HIV-1 or an unglycosylated form thereof, or parts of said proteins, or the DNA encoding said proteins, whereby said proteins are contained as single molecules. That means, in the present hybrid protein the primary structure of the original epitopes E1 and E2 of p15E is partially or completely removed and replaced by the epitopes E1 and E2 of gp41 of HIV-1 , respectively. E1 is located at

the N-terminus of the retroviral transmembrane envelope protein and E2 is located between membrane passage and C-terminal helical region (CHR). Suitable engineering techniques for the preparation of such hybrid protein are known to the skilled artisan.

Object of the present invention is also the use of an effective amount of the retroviral transmembrane envelope protein p15E of a gammaretrovirus selected from FeLV, KoRV or PERV or the described hybrid protein and an effective amount of a retroviral surface envelope protein of the corresponding gammaretrovirus or gp41 of HIV-1 (in the case of the hybrid protein), or parts thereof, or the DNA encoding said proteins, whereby said proteins are used as single molecules, for the manufacture of a pharmaceutical combined composition for the prophylactic or therapeutic treatment and/or monitoring of retroviral diseases, preferably retroviral immune diseases. The ingredients can be either administered to prevent the infection of a mammal with a retrovirus and the outbreak of the disease in advance, or to treat the disease caused by the infectious agent. Particularly, later stages of virus internalization can be prevented. Herein, monitoring is considered as kind of treatment provided that the pharmaceutical combined composition is sequentially administered, e.g. in order to booster the immune response and eradicate the retrovirus and the arisen symptoms completely. Numerous retroviral diseases can be successfully combated by applying the inventive composition, such as leukemia and immunodeficiency caused by FeLV, or lymphoma and immunodeficiency caused by KoRV. Another example is PERV representing a clear threat during xenotransplantation of pig cells or organs because these viruses are present in all pigs and can infect human cells. The prior teaching of the present invention and embodiments thereof is considered as valid and applicable without restrictions to the use of the pharmaceutical combined composition if expedient.

In an embodiment of the invention, the aforementioned active substances are used for the manufacture of a pharmaceutical combined composition for the prophylactic or therapeutic treatment and/or monitoring of a FeLV infection. FeLV is a gammaretrovirus, closely related to PERV, which induces leukaemia, lymphoma and immunosuppression associated with opportunistic infections in infected cats. The cat/FeLV system additionally helps to address the question of whether PERV p15E can induce protective neutralizing antibodies since there is no animal that can be artificially infected with PERV.

The composition of the invention is produced in a known way using the usuai soiid or liquid carriers, diluents and/or additives and the common adjuvants for pharmaceutical engineering and with an appropriate dosage depending on the intended mode of application. These pharmaceutically acceptable excipients comprise salts, buffers, fillers, chelating agents, antioxidants, solvents, bonding agents, lubricants, tablet coatings, flavor additives, flavors, preservatives and suspending agents. In the meaning of the invention, an adjuvant denotes every substance which enables, intensifies or modifies a specific immune response against the inventive composition if administered simultaneously, contemporarily or sequentially. Known adjuvants for vaccines are for example aluminum compositions, such as aluminum hydroxide or aluminum phosphate, saponins, such as QS21 , muramyldipeptide or muramyltripeptide, proteins, such as gamma-interferon or TNF, M59, squalen or polyols. The co-application of egg albumin in complete Freund's adjuvant also increases cell-mediated immunity, thereby supporting the effect of neutralizing antibodies. Preferred adjuvants are Freund's adjuvant, Leucogen adjuvant containing Quil-A and aluminum hydroxide, or Montanide, such as Montanide ISA 720. The amount of excipient material that is combined with the active ingredient(s) to produce a single dosage form varies depending upon the host treated and the particular mode of administration.

Possible formulations of the inventive composition are those forms which are suitable for oral administration, such as tablets, film tablets, lozenges, capsules, pills, powders, solutions, dispersions, suspensions or depot forms thereof, for transdermal administration, such as solutions, suspensions, creams, emulsions or band-aids, for parental administration, such as suppositories, and for intravenous infusion, subcutaneous injection or intramuscular administration, examples for the latter three are solutions and suspensions.

In a preferred embodiment of the invention, said composition is a vaccine or vaccine- adjuvant. In accordance with the present invention, the term vaccine composition relates to any composition which can be used as a vaccine. A vaccine means a therapeutic or prophylactic use of the medicament which attacks retroviruses. The vaccination is advantageously performed in such a way that an active protection is provided in the mammal. The initial immunization can be boostered by subsequent inoculations. Furthermore, the inoculation can be administered before or following an infection once or several times acting as therapy. The forms or methods for manufacturing vaccine compositions according to the present invention are not

particularly limited, and a composition in a desired form can be prepared by applying a single method available in the field of the art or methods in an appropriate combination. For the manufacture of a vaccine composition, aqueous media such as distilled water for injection and physiological saline, as well as one or more kinds of pharmaceutical additives available in the field of the art can be used. For example, buffering agents, pH adjusting agents, solubilizing aids, stabilizing agents, soothing agents, antiseptics, and the like can be used, and specific ingredients thereof are well known to those skilled in the art. The vaccine composition can also be prepared as a solid preparation such as a lyophiiized preparation, and then prepared as an injection by adding a solubilizing agent such as distilled water for injection before use. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways as discussed below. The concentration of therapeutically active compound in the formulation may vary from about 0.1 to 100 wt %. The vaccine composition may be administered alone or in combination with other treatments. In a preferred embodiment, the vaccine compositions are in a water-soluble form, such as pharmaceutically acceptable salts, which is meant to include both acid and base addition saits. The vaccine compositions can be prepared in various forms, such as injection solutions, suspensions and the like. Preferably, it is used as an injection solution. The vaccine compositions may also include one or more of the following: carrier proteins, such as serum albumin, buffers, stabilizing agents, coloring agents and the like. Additives are well known in the art, and are used in a variety of formulations.

The pharmaceutical composition can also contain, if desired, other retrovirus-attacking agents. The inventive composition can be used as vaccine adjuvant to enhance the protection afforded by animal or human vaccines that are considered weak, i.e. by providing diminished protection in terms of level, extent, and/or duration. The ingredients as vaccine-adjuvant will normally be administered separately from the vaccine, although it may be administered in combination with the vaccine as well. Administration of the inventive composition as vaccine-adjuvant can be subcutaneous, intravenous, parenteral, intramuscular, or in any other acceptable method. Preferably, the vaccine-adjuvant is administered prior to the administration of the vaccine and at the same site where the vaccine is to be administered. The formulations and pharmaceutical compositions contemplated by the above dosage forms can be prepared with conventional pharmaceutically acceptable excipients, and by using conventional techniques. Other adjuvants may be administered either with the vaccine or together with the inventive pharmaceutical combined composition.

It will be understood that the specific dose level, frequency and period of administration of any particular mammal will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet time of administration, route of administration, rate of excretion, drug combination and the severity of the specific therapy.

The active ingredients of the inventive combined composition can be simultaneously administered as fixed composition, i.e. being a single pharmaceutical formulation which contains both ingredients, it is prepared for example as injection or infusion solution, or lyophilized form thereof, which is filled in ampoules. The fixed composition of the lyophilized form guarantees a simple and secure handling, it is solved in the ampoule by adding an ordinary pharmaceutical injection agent and administered intravenously. The reconstitution solution can be part of the combination package.

it is also possible to provide the transmembrane envelope protein and the surface envelope protein as single pharmaceutical compositions which are either mixed prior administration or sequentially administered. Usually, a single packing unit comprising two boxes is offered, whereby the first one represents a suitable pharmaceutical form for the transmembrane envelope protein (injection or infusion solution, lyophilized form) and the second one represents a suitable pharmaceutical form for the surface envelope protein (injection or infusion solution, lyophilized form). This free combination is of benefit by individually allotting an effective amount of the transmembrane envelope protein and an effective amount of a surface envelope protein to the mammal. Another possibility is the provision of single preparations of the transmembrane envelope protein and the surface envelope protein being independent medicaments. The single preparations are converted to contain the required amounts of ingredient for the inventive combination. Corresponding instructions are given at the package insert concerning the combined administration of the respective medicament. Preferably, the inventive composition is sequentially administered and at different sites of the mammal. Contrary, a decrease of binding antibody titer is recognized while simultaneously administered, whereas the neutralization capacity is not affected.

Another object of the present invention are neutralizing antibodies produced by immunization with the inventive pharmaceutical combined composition. For the production of neutralizing antibodies at least one inventive composition is exposed to a mammal that it is capable to induce antibodies which are directed against the active substances of the composition and obtained by routine procedures known to those

skilled in the art. Any mammal is chosen which produces high antibody titers. The neutralizing antibodies are used for prophylactic or therapeutic treatment and/or monitoring of retroviral diseases, preferably a FeLV infection. The neutralizing effect of the antibodies is demonstrated by inhibiting either the viral infection, the formation of syncytiums or the fusion between virus and target membrane, or everything thereof, or by reducing or stabilizing the propagation rate of a retrovirus in a mammal. The effect of the antibodies is not restricted to the elimination of a retrovirus, but comprises the entire spectrum of advantageous effects in prophylaxis and therapy.

The present invention also relates to a method for treating a retroviral disease, preferably a FeLV infection, wherein as active substances an effective amount of the retroviral transmembrane envelope protein p15E of a gam ma retrovirus selected from FeLV, KoRV or PERV or the described hybrid protein and an effective amount of a retroviral surface envelope protein of the corresponding gammaretrovirus or gp41 of HIV-1 (in the case of the hybrid protein), or parts thereof, or the DNA encoding said proteins, are administered to a mammal in need of such treatment, whereby said proteins are administered as single molecules. However, the transfer of the retrovirus to new hosts is not be excluded due to the viral variability. The prior teaching of the present invention and embodiments thereof is considered as valid and applicable without restrictions to the method of treatment if expedient.

Furthermore, the invention relates to a method for inducing an antibody response, wherein the inventive pharmaceutical combined composition is administered to a mammal, thereby inducing the production of neutralizing antibodies. It has been shown that the best antibody response concerning the neutralization capacity is obtained by the immunization of that mammal being the natural host species for the retrovirus from which the ingredients of the pharmaceutical combined composition are derived. For example, after demonstrating the induction of neutralizing antibodies in rats and goats immunized with the transmembrane envelope protein p15E of FeLV, cats have been immunized with the same antigen. High titers of neutralizing antibodies specific for FeLV are induced and epitope mapping reveals a pattern of recognition similar to that seen following immunization of rats and goats. These epitopes are highly related to epitopes recognized after immunization with PERV p15E and to epitopes recognized by neutralizing antibodies in patients infected with HIV-1. Tolerance against these epitopes is not induced. The skilled artisan can set the concentration and the mode of application by routine experiments as already mentioned in the course of the specification. The administration is performed prophylactically or therapeutically.

Preferably, the composition is injected. It is the intention of a prophylactic inoculation to prevent the infection with a retrovirus after the infiltration of single virai representatives, e.g. into a wound, such that the subsequent propagation of the virus is strictly diminished, or it is even completely inactivated. If an infection of the patient is already given, a therapeutic induction of an immune response is performed in order to inactivate the retrovirus being present in the body or to stop its propagation.

A method for passive immunization of a mammal is still another object of the present invention, wherein the antibodies induced by administering the inventive pharmaceutical combined composition to a mammal are separated and administered to a further mammal. In the meaning of the invention, "further mammal" refers to a mammal which is identical to the species or foreign to the species of that mammal having induced said antibodies, whereas the same mammal is disclaimed. The presence of neutralizing antibodies in cats recovering from natural FeLV infection clearly correfates with resistance to subsequent infection, and passive transfer of antibodies can protect other animals as well. In addition, monoclonal antibodies can be produced and used, such as in humans following humanization. In doing so, it is also possible to obtain antibody-producing cells from inoculated or infected individuals which neutralizing antibodies are directed against the inventive composition and applied as monoclonal antibodies during passive immunization. That mode of immunization does not cause a patient's own immune reaction to certain viruses, but the antibodies are introduced in the body as healing sera. The approach aims at a prompt effect, i.e. to cure the given infection sickness as quickly as possible or to protect against a viral infection immediately. Vaccination schedules are known to those skilled in the art and can be easily adapted to specific retroviruses which are to be attacked by the combined composition of the present invention. Preferably, monoclonal antibodies are used for passive immunization. Their use in a combination therapy is of special benefit.

EXAMPLES

The following examples is provided by way of illustration and not by way of limitation. Within the example, standard reagents and buffers that are free from contaminating activities (whenever practical) are used.

Fig. 1 : Comparison of p15E of different FeLV. (a) Unrooted phylogenetic tree of gp70 and of p15E of the FeLV strains Glasgow- 1 , Rickard, Sarma (FeLV-A) and Gardner-

Arnstein (FeLV-B). (b) Alignment of epitopes designated E1a, E2a, E1 b and E2a identified after immunization of rats with the recombinant ectodomaiπ of p15E of FeLV (indicated in gray) in different FeLV-A strains.

Fig. 2: ELISA reactivity of rat antisera induced by immunization with 500μg p15E alone (group 50), with 100μg Leucogen alone (group 51 ) and with a combination of both antigens (group 52), As antigen in the ELISA FeLV-A p15E (a) or Leucogen (b) were used.

Fig. 3: ELISA reactivity (a, b, d, e) and neutralizing activity (c, f) of rat antisera induced by immunization with 500μg p15E alone (group 55), with 100μg p15E alone (group 57) and with 100μg Leucogen- alone (group 56). As antigen in the ELISA FeLV-A p15E (a, d) or Leucogen (b, e) were used.

Fig. 4: ELISA reactivity (a, b, d, e) and neutralizing activity (c, T) of rat antisera induced by immunization with 500μg p15E in combination with 100μg Leucogen in a single injection site (group 54) or in two different injection sites (group 53) and with Leucogen alone (group 56). As antigen in the ELISA FeLV-A p15E (a, d) or Leucogen (b, e) were used.

Fig. 5: ELISA reactivity (a, b, d, e) and neutraiizing activity (c, f) of rat antisera induced by immunization with 500μg p15E in combination with 100μg Leucogen in a single injection site (group 54), with 100μg p15E in combination with 100μg Leucogen in a single injection site (group 60), with 500μg p15E alone (group 55) and with 100μg p15E alone (group 57). As antigen in the ELISA FeLV-A p15E (a, d) or Leucogen (b, e) were used.

Fig. 6: Epitope mapping of the neutralizing rat antiserum 54.2 obtained after immunization with recombinant p15E of FeLV-A. (a) Result of the dot blot ECL method using overlapping peptides and in (b) sequence aiignment of the recognized peptides. (c) Entire sequence of p15E of FeLV-A is given, the sequence of the recombinant protein used for immunization is printed in bold, and the epitopes are framed.

Fig. 7: Summary of the specific epitope mapping of all antisera obtained after immunization with p15E, Leucogen or both, p15E and Leucogen. The sequence of the FeLV-A p15E ectodomain and the animal number and the antigen(s) used for immunization are given, epitopes are framed.

Fig. 8: (a) Western blot analysis of cat antisera following immunization with FeLV-A p15E (14, 34 and 44) and of sera from FeLV-infected cats (54748-6452). The same recombinant p15E used for immunization was used as antigen. Lane 1 shows the preimmune serum of goat 27, lane 2 the corresponding immune serum. Only one preimmune cat serum is shown (#14, lane 3) (b) ELiSA reactivity of cat sera induced by immunization with 500μg p15E in comparison with the corresponding preimmune sera. Recombinant p15E was used as antigen.

Fig. 9: Neutralizing activity of cat antisera after the second immunization with 500μg p15E. Infection was measured as provirus integration by real-time PCR. Percent of provirus integration was obtained by comparing antisera with the corresponding preimmune sera.

Fig. 10: Epitope mapping using sera from immunized and FeLV-infected cats, (a) As an example, binding of serum from FeLV-infected cat #55409 to a pepspot membrane carrying overlapping peptides is shown and epitopes are identified, (b) Summary of the epitopes identified recognized by each serum. Sequences corresponding to the recombinant p15E of FeLV-A, strain Glasgow 1 , and the corresponding sequence of an endogenous p15E are given at the top. Cats 14, 34 and 44 were immunized with p15E while the others are representative of FeLV-infected cats. Strong epitopes are marked in black, weak epitopes in gray. Common groups of epitopes are framed (E1 a, E1b, E2a, E2b). In addition, epitopes recognized by the serum from goat 27, immunized with p15E, and consensus epitopes recognized by 8 rats immunized with p15E are shown (hatched). For comparison, the C-terminal part of the HIV-1 transmembrane envelope protein gp41 and the localization of epitopes recognized by two monoclonal antibodies (mAb) broadly neutralizing HIV-1 , (2F5 and 4E10) are shown (framed).

Fig. 11 : Indirect immunofluorescence, visualized by confocal laser microscopy, using antiserum from cat 44 (immunized with p15E) and FITC-conjugated anti-cat IgG. FITC staining was measured at 488nm and unspecific fluorescence measured at 543nm was subtracted, (a) Uninfected FEA cells, (b) FeLV-A producing FEA cells.

Fig. 12: Schematic representation of the transmembrane protein of retroviruses at a defined stage of infection. After introduction of the fusion peptide (FP) the N-terminal helical region (NHR) and the C-terminal helical region (CHR), which are connected by

a cysteine-cysteine loop (Cys-Cys-!oop), interact, bringing the epitope regions E1 and E2 into close proximity. TM indicates the transmembrane domain of the protein.

Fig. 13: Titres of binding antibodies against p15E (A) and p45 (B) determined in ELISA with a four fold serial dilution of antisera compared to corresponding preimmmune sera. (C) Neutralising activity in percentage obtained for sera from immunisation with p15E, Leucogen or with a combination of both as shown by indicated brackets. The neutralising activity was calculated in relative to corresponding preimmmune sera.

Fig. 14: Changes in the neutralising activity of sera from animals immunised with p15E, Leucogen or with a combination of both. Sera were taken 4 weeks and 20 weeks after booster immunisation (the last corresponds to three days before virus challenge).

Fig. 15: Response of cats to challenge with FeLV: (A) Summary of presence (+) or absence (-) of p27 antigen from blood samples. (B) Kinetics of FeLV-A p27 antigen detection in blood cells (in % relative to FeLV-A sera from persistently infected control cats) in non-immunised control animals (red), in animals immunised with p15E (blue), with Leucogen alone (green) or in combination with p15E (yellow). FeLV-A p27 antigen measurement was performed in an ELISA and values below a predefined cut off were considered as FeLV negative. (C) Kinetics of FeLV provirus integration in copies/μl blood measured of the animals described in A. The FeLV-A provirus integration was determined using a virus-specific real time PCR.

Fig. 16: Kinetics of provirus integration in copies/μl blood, prevalence of p27 antigen in percentage and neutralising activity (measured at a serum dilution of 1 :100 in % relative to preimmune serum) in cats.

Fig. 17: Epitope mapping using neutralising sera from cats immunised with p15E of FeLV. (A) As an example, binding of serum cat 74 to overlapping peptides on a pepspot membrane is shown and epitopes were framed. (B) Summary of the epitopes recognised by each serum. Cats 14, 34, 44, 53, 74, and 53.3 were immunised with p15E. Cats 50.3 and 51.3 were immunised with p15E and Leucogen. Strong epitopes are marked in black, weak epitopes in grey. Common groups of epitopes are framed (E1a, E1 b, E2a, E2b). In addition, epitopes (hatched) recognised by the serum from goat 27, immunised with p15E, and consensus epitopes recognised by sera from 8 rats immunised with p15E are shown. For comparison, the localisation of epitopes recognised by two monoclonal antibodies broadly neutralising HIV-1 , 2F5 and 4E10, on

the C-terminal part of the HIV-1 transmembrane envelope protein gp41 are shown (framed). (C) Localisation of the epitopes recognised by sera from cats immunised with p15E and neutralising FeLV and localisation of the epitopes recognised by the broadly HIV neutralising antibodies 2F5 and 4E10 in the folded transmembrane envelope proteins p15E and gp41 , respectively. The E1 domain in gp41 of HIV-1 corresponds to a peptide enhancing the binding of 2F5 and 4E10 to their epitopes.

Example 1

Affinity-purified recombinant fusion protein p15E of FeLV-A, strain Glasgow, was produced and characterized as described by Langhammer et al. (2005), Vaccine 23, 3341-3348. Briefly, the ectodomaϊn (aa 476-583) of p15E of FeLV-A was cloned into the pCal-n vector (Stratagene, Europe, Amsterdam, Netherlands), expressed in E. coli BL21 DE3 cells, and the fusion protein containing p15E N-terminal!y fused to a 4kDa calmodulin binding protein (CBP) was purified by calmodulin resin affinity chromatography (Stratagene). Protein to be used for immunization was extensively dialyzed against phosphate-buffered saline (PBS).

Two immunization experiments were performed using Wistar rats. In the first experiment, 9 rats were immunized twice intramuscularly (i.m.) and subcutaneously (s.c.) (at weeks 0 and 3). In the first experiment Leucogen, containing 0.1 mg p45 plus Quil-A and aluminum hydroxide as adjuvant (Virbac, lot number 80986902143521 ) was given alone or mixed with p15E. Freund's adjuvant was used when p15E was injected alone. In the second experiment, 18 rats were immunized i.m. and s.c. at week 0 and 3 (Table 1). Immunization with p15E alone was performed at a dilution 7:3 in Montanide ISA 720 (Seppic, France, lot number 143521).

FeLV-A p15E and Leucogen p45-specific antibody titers were determined by ELISA. Briefly, ELISA plates (Nunc) were coated for 1h at 37 ⁰ C with affinity-purified recombinant p15E protein diluted in PBS or for 18h at 37°C with Leucogen p45 vaccine component diluted in PBS (100ng/weH). Then, ELISA plates were washed one time with PBS containing 0.1% Tween 20 and blocked for 1 h at room temperature with PBS containing 0.1 % Tween 20 and 5% BSA. Serum samples diluted with PBS containing 2.5% BSA and 0.1 % Tween-20 were added to the ELISA plate at a starting dilution of 1 :1000, diluted further (four-fold dilution series) and incubated for 1 h at 37°C. Then, ELISA plates were washed three times with PBS containing 0.1 % Tween 20. A horseradish peroxidase conjugated secondary antibody specific for rat IgG (Bethyl,

USA) diluted 1 :3,500 with PBS containing 2.5% BSA and 0.1% Tween-20 was used to detect antigen-specific immunoglobulin. Incubation for 1 h at 37°C was followed by washing five times with PBS containing 0.1 % Tween 20. Finaily, ELISA plates were developed by addition of OPD (alpha-phenyienediamine dihydrochioride) diluted in PBS (50μg/we!l) plus 0.1% H ₂ O ₂ followed by inactivation with 30μl H ₂ SO ₄ (5N) after 10 minutes. Protein-specific antibody endpoint titers are reported as the dilution giving an OD _492/ 6 ₂ o _nm reading above that of preimmune sera.

When, in the first experiment, rats were immunized twice with 500μg p15E (group 50), 100μg Leucogen (group 51 ) or a mixture of Leucogen and p15E (group 52), all sera showed strong ELISA reactivity specific for the corresponding antigen used for immunization (Fig. 2a, b). Interestingly, the titer of binding antibodies specific for p45 was lower when Leucogen and p15E were injected simultaneously (group 52) in comparison to immunization with Leucogen alone (group 51) (Fig. 2b). When this experiment was repeated immunizing with 500μg p15E (group 55), 100μg p45 (group 56) and a mixture of both (group 54), ELISA titers of up to 4x10 ⁶ were observed against the corresponding antigen used for immunization (Fig. 3a, b, d, e; Fig. 4a, b). Again, mixing of p45 and p15E decreased the antibody response to p45 in two cases, animals 54.1 and 54.2 when compared with animals that received only p45 (animal group 56) (Fig. 4b). Firstly, the addition of p15E with its known immunosuppressive properties may have reduced the production of antibodies specific for p45. Alternatively, the addition of 500μg p15E to only 100μg p45 may have led to an antigenic dominance of p15E, or the addition of p15E to p45 may have led to interactions between domains of these two proteins, hiding epitopes of p45 from the immune system. In vivo, three transmembrane envelope proteins and three surface envelope proteins interact when building up the so-called knobs on the virus surface.

To investigate this further, Leucogen and p15E were injected at different sites (group 53) and the results were compared with the injection of a mixture at one site (group 54) (Fig. 4d, e) as had been performed in the first experiment (group 52) (Fig. 4b). Since in two cases (animals 54.1 and 54.2, Fig. 4e) the titer of the binding antibodies specific for p45 was reduced in comparison to all sera of group 53, it seems likely that the simultaneous injection of both antigens when injected into a single site was responsible for the decrease in antibody response.

To study the influence of the amount of p15E antigen on the induction of antibodies specific for p15E, injection of 500μg p15E (group 55) was compared with injection of

100μg p15E (group 57) (Fig. 5d). There was no obvious difference in the titers of binding antibodies as measured by ELISA. To investigate whether higher amounts of p15E in the antigen mixture has any influence on the antibody induction specific for p45, Leucogen vaccines containing 100μg p45 were injected together with 500μg p15E (group 54) or together with 100μg p15E (group 60, Fig. 5b). There was no obvious effect of the increased p15E amounts on the production of binding antibodies specific for p45 or p15E.

The virus stock for the neutralization assay was prepared as cell-free supernatant from feline embryonic fibroblast (FEA) cells infected with the FeLV-A Glasgow strain. The stock was titrated on uninfected FEA cells and was shown to have a titer of 10 ^{4 76} TCID ₅₀ /ml. Neutralization assays were performed as follows: One day before the assay, uninfected FEA cells were seeded at 6000 cells per well into 96-well microtiter plates. Preimmune and immune sera were heat-inactivated at 56°C for 30 min. 50μl of stock virus were incubated with four-fold serial dilutions of serum for 30 min at 37°C and than transferred to the cells. After 3 days incubation, cells were freeze-thawed three times and a lysis buffer containing 20 mg/ml of proteinase K in PCR buffer (50 mM KCI, 1.5 mM MgCI ₂ , 10 mM Tris-HCL, pH 8.4) was added. The cells were incubated for 3h at 56°C followed by 10 min at 95 ⁰ C to inhibit proteinase K activity. Proviral DNA was measured by real time PCR as described below.

An internal probe FAM-5'-TTAAGCACCTGGGCCCCGGC-3'-DQ (Eurogentec) was used together with FeLV-specific primers. The sense primer 5'- TCAAGTATGTTCCCATGAGATACAA-3 ^' and antisense primer 5 ^' - GAAG GTCG AACTCTGGTCAACT-3 ^' were used to amplify and to quantify a 185bp product from the exogenous U3 sequence in the LTR region of the FeLV-A provirus genome. The 25μl reaction mixture consisted of 1x PCR buffer with 1 mM IvIgCI ₂ , 0.5μM each of dATP, dCTP, dGTP, dTTP, 5pmol of each primer, 5pmol of probe, 1.25 U Amplitaq Gold polymerase and 2μl lysis mixture. The thermal cycling conditions used were 12 minutes at 95°C followed by 50 cycles of 1 minute at 95 ⁰ C, 1 minute at 59°C and 30 seconds at 72 ⁰ C in a Stratagene MX4000 machine.

When tested for neutralizing activity, 14 out of 18 antisera induced in the second experiment were able to inhibit the infection of FEA cells by FeLV-A with varying efficacy, whereas all preimmune sera had no such neutralizing activity (Fig. 3c, f; Fig. 4c, f; Fig. 5c, f). Sera from animals 55.1 , 55.3, 57.2, and 57.3 did not show neutralizing

activity. To analyze the neutralization efficacy, serial dilutions (1 :4, 1 :16 and 1 :64) of the antisera were tested.

Antisera generated by immunizing with 500μg p15E (group 55) showed neutralization of FeLV-A ranging from nearly 0% (55.1 and 55.3) to 80% (55.2) at a 1 :4 dilution (Fig. 3c). None of these sera had the ability to neutralize the virus at the final serum dilution of 1 :64. This confirmed previous data showing the induction of neutralizing antibodies after immunizing rats with 500μg p15E. Whereas in the previous report Freund's adjuvant was used, Montanide was employed as adjuvant in the present investigation. However, due to the smail number of animais the influence of the adjuvant cannot be analyzed. When the amount of p15E antigen used for immunization was reduced to 100μg (group 57), no reduction of the titer of neutralizing antibodies was observed (Fig. 3f). One antiserum (57.1 ) neutralized 90% of virus at a serum dilution of 1 :4, but did not neutrafize at 1 :64. Two other antisera from animals of this group, 57.2 and 57.3, did not neutralize at any serum dilution.

Antisera obtained after immunization with 100μg Leucogen alone (group 56) neutralized at a range from 80% to 100% at a dilution of 1 :4 and did not neutralize at 1 :64. These data show that the neutralizing capacity in-vitro is much higher after immunization with Leucogen when compared with immunization with 500μg (Fig. 3c, group 56 versus 55) or 100μg p15E (Fig. 3f, group 56 versus 55).

When the neutralizing capacity of the sera obtained after simultaneous immunization with Leucogen and p15E (group 54) was compared with the neutralizing capacity of sera obtained with Leucogen alone (group 56), better neutralization was observed when both antigens were injected (Fig. 4c). Interestingly, the increase in neutralizing activity was associated with a decrease in the titer of binding antibodies specific for p45 (Fig. 4b).

However, when Leucogen and p15E were injected at different injection sites (group 53), the titers of binding antibodies specific for p45 was not reduced (Fig. 4e). This means that the titers of binding antibodies specific for p45 were comparable with titers in the sera from animals which received Leucogen alone (group 56, Fig. 4b) and the neutralizing capacity was as high as in the sera from animals which received simultaneously Leucogen and p15E at a single injection site (group 54, Fig. 4f). In the case that 500μg p15E and 100μg p45 were separately injected at different injection sites (group 53), neutraiization efficacy ranging from 100% at a serum dilution of 1 :4 to

about 75% or more at a serum dilution of 1 :64 were observed (Fig. 4f). The antisera obtained after immunization using a single injection site for 500μg p15E and 100μg p45 (group 54) showed neutralization efficacy ranging from 100% at a serum dilution of 1 :4 to about 80% or more at a serum dilution of 1 :64 (Fig. 4c, 4f). This indicates that the titer of neutralizing antibodies is not affected by the addition of p15E to p45 despite the finding that the titer of binding antibodies specific for p45 was reduced in two cases (Fig. 4e).

To evaluate the influence of the amount of p15E on the antibody response against p45, animals were immunized with Leucogen either together with 500μg (group 54) or together with only 100μg p15E (group 60) (Fig. 5). The binding antibody response specific for p15E was slightly lower in the group that received only 100μg p15E (Fig. 5a). As already mentioned above, the binding antibody response specific for p45 was identical in both groups (Fig. 5b). In contrast, the neutralizing capacity was significantly higher in sera from animals that received 500μg p15E (Fig. 5c), indicating that higher amounts of p15E induced more neutralizing antibodies in connection with Leucogen. When 500μg (group 55) or 100μg p15E (group 57) were applied without Leucogen, no differences in the binding antibody response specific for p15E (Fig. 5d) and in the neutralizing capacity (Fig. 5f) were observed.

An epitope mapping of the induced sera was performed using a cellulose-adsorbed peptide spot library of linear 15-mer peptides overlapping by 13 amino acids. The peptides corresponding to the entire p15E of FeLV-A Glasgow strain were covalentiy bound to the cellulose membrane by the C-terminus (Fig. 6). A standard protocol was applied for synthesis (Jerini Biotools). Sera diluted 1 :1000 were incubated with the membrane for 3h, washed three times for 15 min with Tris-buffered saline, pH 7,5 containing 0.05% Tween 20 (Sigma) and incubated for 2 h with a peroxidase- conjugated secondary antibody diluted 1:10,000. Binding was detected using a chemiluminescence detection solution (ECL, Amersham Pharmacia Biotech).

When sera from rats immunized with recombinant p15E alone (groups 55, 57) were analyzed, four main epitope regions were found, two at the N-terminus and two at the C-terminus of the ectodomain (Fig. 7). These findings confirmed previous studies, in which the same epitope regions were identified and were designated E1a (LETAQFRQL) and E1b (IQALEESISALEK) as well as E2a (KQRQQLF) and E2b (FDSQQGWFEGWFN). In contrast to the previous study, the E2a epitope was better defined (KQRQQLF instead of MAKLRERLKQRQQL). These four epitopes were

identified, regardless of whether 500μg (group 55) or 100μg (group 57) of p15E were applied (Fig. 7). Interestingly, none of the non-neutralizing sera recognized the E1 epitopes, suggesting that this epitope is essential for neutralization.

The same epitopes were identified when rats were immunized with p15E and Leucogen (animal groups 53, 54 and 60), indicating that Leucogen did not change the recognition of the epitopes by the immune system (Fig. 7). It was also shown that the adjuvant does not influence the recognition of the epitopes: regardless of whether Freund's adjuvant, Montanide (groups 55 and 57) or the adjuvant contained in the Leucogen preparation (groups 53, 54 and 60) were used, the same epitopes were recognized.

As expected, no p15E specific epitopes were detected when sera were tested from rats immunized with Leucogen p45 alone (group 56), indicating that the Leucogen vaccine does not contain parts of the transmembrane envelope protein.

Sera induced with 100μg or 500μg p15E (groups 55 and 57) always detected the E2b epitope, while the E1 b epitope was recognized only by one antiserum of each group (55.2 and 57.1). However, only these two antisera efficiently neutralized FeLV (Fig. 5f), indicating a critical role of the E1 b epitope in virus neutralization. The E2 epitopes also seem to be crucial for neutralization since the serum from animal 60.1 did not recognize E2 and had only a weak neutralization activity when compared with the sera from two other animats of the same group that recognize E2 (Fig. 5c). As mentioned above, only two of the sera from animals immunized with p15E recognized E1 b and only these sera were neutralizing. On the other hand, sera from all animals immunized with p15E and Leucogen recognized E1 b (Fig. 7), indicating that simultaneous immunization of both antigens increased the recognition of the crucial epitope E1 b.

Example 2

For immunization recombinant p15E of FeLV-A was prepared as described above.

6-10 month old cats, obtained from the University of Dϋsseldorf and housed in groups of 3, were immunized intramuscularly (Lm.) twice (at weeks 0 and 3) with p15E (Table 2). Montanide ISA 720 (Seppic, France, lot number 143521) was used as adjuvant mixed with p15E at a ratio of 3:7.

SDS-PAGE and Western blotting were performed as described by Tacke et at. (2001), Xenotransplantation 8, 125-135, using 1 μg of the affinity purified recombinant FeLV p15E per lane. In Western blot analyses, antisera from all three cats immunized with 500μg p15E (#14, #34, and #44) specifically detected the recombinant p15E protein at a size of 15kDa, while the preimmune sera did not react (Fig. 8a). When sera from FeLV-infected housecats were tested in the same assay, 44 of 75 sera (58.6%) also specifically detected p15E, four of which are shown in Fig. 8a. Sera were obtained from household cats in Germany at the time of first diagnosis of infection using a commercial p27 Gag antigen detection assay (Feline leukemia virus antigen test kit, Symbiotics, USA). These data indicate that immunized and infected animals are able to produce antibodies specific for p15E.

Pre- and post-immunization cat sera were titrated in ELISA as performed above. The same affinity-purified recombinant p15E used for immunization as in example 1 was applied as antigen herein. All three immune sera, but not the preimmune sera, strongly reacted in ELlSA using recombinant p15E as antigen (Fig. 8b). After the boost immunization the titer of binding antibodies increased markedly (Table 2). Sera from cat 44 showed the highest titers of binding antibodies in this group (2.56 x10 ⁵ after the first immunization and 1x10 ⁶ after the boost). The sera from cats 14 and 34 showed titers of 6.4x10 ⁴ that increased to 2.56 x10 ⁶ after the boost immunization. In contrast, sera obtained from FeLV-infected housecats had only titers between 1x10 ³ and 4x10 ³ (Table 2).

The virus neutralization assay and real time PCR were performed as already described with the following exception: 75μl of stock virus were incubated with four-fold serial dilutions of serum (previously heat-inactivated at 56 ⁰ C for 45 min) for 45 min at 37 ⁰ C and than transferred to the cells. Neutralization of FeLV-A strain Glasgow infection of feline embryonic fibroblast cells was measured using four-fold serial dilutions (from 1 : 16 to 1 :16384) of the sera. All sera taken after the first immunization had titers of 1 :256 (Fig. 9) whereas no preimmune sera showed neutraiizing activity. Similar to the titers of binding antibodies, the titers of neutralizing antibodies increased after the booster immunization in two animals (cats 14 and 34) up to 1 :1024 (Table 2) although the titer of neutralizing antibodies in the serum of cat 44 did not increase.

To identify the epitopes recognized by the immune sera, epitope mapping was performed according to example 1 (Fig 10a). Four major epitopes were identified (Fig. 10b) using sera from the cats immunized with p15E (#14, #34, and #44). The first

epitope, KALLETAQF, is nearly identical to an epitope identified by immunizing a goat with FeLV p15E and to a consensus epitope (LETAQFRQL) recognized by sera from 8 rats immunized with the same antigen. This epitope group was designated E1a. The second epitope (ALEESISALEK, E1b) was also recognized by all 8 rat sera but not by the goat serum. The third epitope is located in the immunosuppressive domain, LQNRRGLDILFLQEGGL, which is highly conserved amongst all retroviruses. Synthetic peptides corresponding to this domain inhibit lymphocyte proliferation and modulate cytokine production. This epitope in the immunosuppressive domain was also recognized by the goat serum, but not by any of the rat sera. The fourth epitope, MAKLRERLKQRQQLF, corresponds to an epitope E2a, recognized both by the goat serum and by 7 of 8 rat sera. Similar to the goat serum the cat sera did not recognize an epitope recognized by all rat sera and designated E2b (FDSQQGWFEGWFN). The cat sera bind to main epitopes described following immunization of rats and goats. These data support the existence of main target epitopes after immunization with p15E and minor species-specific differences.

Sequences homologous to the epitopes are also present in endogenous retroviruses. When the sequence of the FeLV-A p15E used for immunization was compared with that of the endogenous feline retrovirus CFE-6 (NCBI accession no. gi:74706), sequence homologies were identified in the epitope domains (Fig. 10b). Despite such sequences being present in the genomes of the immunized cats, binding and neutralizing antibodies specific for these domains were induced.

To elucidate the possible mechanisms of neutralization, the localization on the cell surface of the epitopes recognized by the p15E-specific sera were analyzed by immunofluorescence using non-permeabilized FeLV-infected FEA feline embryonic fibroblast cells. FeLV-A producing FEA cells were grown on chamber slides, washed three times with PBS and fixed with 3.5% formaldehyde. Unspecific binding sites were blocked with 5% BSA in PBS for 20 minutes followed by washing with PBS. Cat sera were applied in 2.5% BSA/PBS at a dilution of 1 :1000 and incubated at 37°C for 1 h. After five washes with PBS the cells were incubated with FITC-labeled goat anti-cat IgG (Bethyl, USA). Finally, the ceils were embedded in Prolong antifade reagent (Molecular Probes) and the surface fluorescence was analyzed by confocal microscopy (Zeiss, LSM510). Unspecific cell fluorescence at 543nm was subtracted from the specific signal at 488nm. Uninfected ceils were not recognized by cat sera #14, #34, and #44 were used (Fig. 1 1a). However, all three bound to the cell surface (Fig. 1 1a) whereas the corresponding preimmune sera did not. To increase picture quality,

unspecific ceil fluorescence at 543nm was subtracted from FITC-specific signal at 488nm. This binding of immune sera to the cell surface indicates that the epitopes identified are accessible to FeLV on the surface of infected cells.

To compare neutralizing antibody responses in FeLV-infected cats with those of p15E- immunised animals, sera from infected housecats were analyzed. As described above, 44 of 75 sera investigated showed antibodies specific for p15E by Western blot analysis (Fig. 8a), and the titers of p15E-specific antibodies in ELISA ranged from ≤1 x10 ³ to 4 x10 ³ (Table 2). Neutralizing titers of these sera were found to be between 0 and 1 :256 (Table 2), although it must be kept in mind that in the infected cat neutralizing antibodies might also be directed against other viral proteins such as gp70. Epitope mapping using overlapping peptides spanning the entire p15E (Fig. 10) was carried out. Serum from cat 9425 only recognized the epitope E2a, while serum from cat 6452 recognized an epitope located outside E1a as well as E1 b and E2a, and serum from cat 27047 recognized the epitopes E1a and E1 b weakly, but E2a more strongly. Cat 27047 had initially been immunized with Leucogen containing the non- glycosylated surface envelope protein p45, but became infected despite this immunization. Sera from cats 54748 and 55409 (Fig. 10a) weakly detected epitopes E2a and E2b, but none of these epitopes were recognized by serum from cat 55284, despite this cat being infected and having low titer neutralizing antibodies.

Example 3

When the antibody response of cats which had been immunised twice with the ectodomain of p15E, with Leucogen or with a combination of both, and of non- immunised control cats, was investigated four weeks after the last boost and 16 weeks later, three days before challenge. Four weeks after immunisation with p15E alone or in combination with Leucogen, binding antibodies specific for p15E were detected in all animals. In an ELISA using the ectodomain of p15E as antigen, titres of binding antibodies ranging from 2.56x10 ⁵ to 4x10 ⁶ were found (Fig. 13A). Cat 53.3 showed the highest titre. As expected, no antibodies specific for p15E were found in the sera from cats immunised with Leucogen alone. The sera from cats immunised with Leucogen alone reacted with p45 and showed ELISA titres ranging from 6.4x10 ⁴ to 2.56x10 ⁵ (Fig. 1 B). As expected, none of the sera from cats immunised with p15E showed antibodies specific for p45 (Fig. 13B). After immunisation with a combination of p15E and Leucogen (animals 50.3 and 51.3) ELISA titres of 1x10 ^e for p15E-specific antibodies and of 2.56x10 ⁵ of p45-specific antibodies was found (Fig. 13A, B). The titres of binding

antibodies to each antigen, p15E or p45, were in the same range as after immunisation with each antigen alone, indicating that each does not influence the response to the other.

The neutralising capacity of the sera from immunised cats was determined in a neutralisation assay using FeLV-A, measuring provirus integration by real time PCR. The neutralising capacity was expressed as percent neutralisation in comparison with the corresponding preimmune serum (see experimental procedures). The sera were used at a dilution 1 :100. Four weeks after the boost immunisation, all but one serum (51.3) showed neutralising activity (Fig. 13C) and the neutralising activity of sera after immunisation with p15E was comparable with that of sera after immunisation with Leucogen. Sera from three animals immunised with p15E (cats 34, 44 and 53.3) showed high neutralising activity (up to 92%), while the other three had low or medium activity. Sera obtained after immunisation with Leucogen alone showed a neutralising activity between 47% (cat 54) and 91% (cat 32.3). The serum from cat 50.3, immunised with a combination of p15E and Leucogen showed a neutralising activity of 90%, while serum from cat 51.3 as an exception did not show any neutralising activity (Fig. 13C). To study how long these neutralising antibody titers persisted, sera were tested again after 4 months, which is 3 days before challenge. It was interesting to see a nearly parallel decline of the neutralising titers of sera in this short time (with the exception of the serum from cat 51.3, which showed an increased activity) (Fig. 14). Nevertheless, three sera from cats immunised with p15E alone (cat 14, 34) or in combination with Leucogen (cat 50) stiff showed significant neutralising activity, although it was reduced about 2-fold in comparison to the activity 4 months earlier. It is important to note that all antisera still showing neutralising activity at that time were derived from animals immunised with p15E, either alone or in combination with Leucogen.

Animals were challenged with infectious FeLV, the outcome of the challenge is summarised in Fig. 15. None of the cats showed p27 antigen in the blood when tested 3 days before and 10 days after challenge (Fig. 15A). However, 30 days after the challenge, p27 antigen was detected in the sera of all non-immunised control cats (16.4, 22.4, 35.3), with activities ranging from 47% to 92% of the control (Fig. 15B). Therefore all of these animals were considered to be FeLV positive. The non- immunised animais also showed the highest p27 antigen levels (values between 82% and 125%), 80 days after the challenge. Thereafter the level decreased to values between 18% and 46%. Analysing the animals immunised with p15E alone, two cats (14 and 74) were p27 positive and four cats (44, 34, 53, 53.3) were negative 30 days

after challenge. The p27 antigen detected in the peripheral biood of cat 14 was in the same range (98%) as in the blood of non-immunised animals of the control group, whereas cat 74 showed a significantly lower p27 antigen level (11 %). Sixty days after challenge, four cats were found to be positive. In addition to cats 14 and 74 that were already positive at day 30, cat 44 with 18% and cat 53 with 94% p27 antigen were also found positive. The level of p27 antigen in the serum from cat 44 was significantly lower than those from all other cats that were positive.

At day 80 and day 100 after challenge cats 14, 53 and 74 were FeLV positive while cats 34, 44 and 53.3 remained negative. It appears that cat 44 was transiently viraemic while cat 53 became viraemic later; this interpretation is consistent with the development of virus neutralising antibodies in cat 44 and a decline in antibody titer in cat 53 by day 100. In the cats immunised with Leucogen alone or in combination with p15E (Leucogen: 54, 64, 32.3; combination: 50.3, 51.3) p27 antigen was never detected in the blood. In addition, p27 antigen was never detected in cats 34, 44 (except day 60) and 53.3 immunised with p15E. Therefore, according to the commercial ELISA used, all 5 animals immunised with Leucogen, either alone or together with p15E and three of the 6 animals immunised with p15E alone were protected.

To characterise the level of protection in more detail, the provirus load was analyzed in peripheral blood cells. For this, a real time PCR specific for FeLV was developed and the provirus integration was measured as copies/μl blood. No cat showed provirus integration in the blood cells tested before challenge, confirming that uninfected animals were used and that the primers used in the PCR did not detect feline endogenous retroviruses. In the blood cells of the non-immunised control animals (16.4, 22.4, 35.3) provirus integration was observed starting on day 10 after challenge. At that time, between 1.08x10 ² and 1.26x10 ² copies/μl blood were observed, increasing up to 5.82x10 ⁴ and 2.66x10 ⁵ copies/μl blood at day 30 after challenge. This level decreased to below 1x10 ⁴ copies/μl blood at day 100 after challenge.

!n all six animals immunised with p15E alone (cats 14, 44, 34, 53, 74, 53.3), a similar cell associated virus ioad was found as in the non-immunised control group. Only cat 53.3 (the animal with the highest titer of binding and neutralising antibodies) showed significantly lower provirus load starting at day 60 after challenge until the end of study. At day 60 and at day 100 cat 53.3 showed a provirus load below 1x10 ³ copies/μl blood. In addition, cats 14 and 44 also showed a reduced level of provirus integration in

comparison to the celS-associated virus load in non-immunised control animals 100 days after the challenge (Fig. 15B),

In contrast to the animals of the non-immunised control group and to the animate immunised with p15E alone, low levels of provirus integration were observed in the blood of animals immunised with Leucogen alone or in combination with p15E (Leucogen: 54, 64, 32.3; combination: 50.3, 51.3). Starting with day 10 after challenge the level of provirus integration ranged between 1.32x10 ¹ and 1.57x10 ² copies/μl blood decreasing to 7.05 copies/μl blood (animal 50.3) or to a complete clearance of provirus at day 100 after the challenge. There was no detectable difference in the provirus load between animals immunised with Leucogen alone or with the combination. In both groups provirus load was about 100 fold lower when compared with the non-immunised control animals or in the animals immunised with p15E alone beginning with day 30 after challenge (Fig. 15B).

To analyse whether neutralising antibodies represented a correlate of protection, the relationship between provirus load in copies/μl blood and the p27 antigen load in percentage on the one hand, and the neutralising capacity of the serum on the other, was investigated. Sera from all cats, including the non-immunised animals, had neutralising antibodies beginning on day 10 (except animal 44 immunised with p15E) (Fig. 16). Some of the immunised cats, however, already had immunisation-induced neutralising antibodies at the day of challenge. Such antibodies were found in two of six animals immunised with p15E alone and in both animals immunised with p15E and Leucogen, but in none of the animals immunised with Leucogen alone. The kinetics of the titers of neutralising antibodies showed two maxima in most animals. In the case of the non-immunised animals (where the neutralising antibodies were induced solely by infection) the titer decreased finally to zero at day 100. In all other animals the neutralising activity increased up to 100%. However, an inverse relationship between p27 antigen and neutralising activity was observed: animals with high p27 antigen load had neutralising activities below 100%, whereas in all animals where p27 antigen was not detected, the neutralising activity always reached 100% at day 100 (except cat 32.3).

When comparing the animals immunised with Leucogen alone with the animals immunised with the combination of Leucogen and p15E, one important difference was observed: in both animals immunised with p15E, neutralising antibodies existed

already at the day of challenge (obviously induced by immunisation with p15E), whereas animais immunised with Leucogen did not have neutralising antibodies.

Taken together, these data show that at the day of challenge no neutralising antibodies were detected in animals immunised with Leucogen, although immediately after immunisation these antibodies could be detected. Most important, we clearly show that there is a strong inverse correlation between the neutralising activity and the p27 antigen load. This may indicate that the neutraiising antibodies suppress the virus load or that the developing persistent viraemia inhibits the production of FeLV-specific antibodies.

In order to localize the epitopes of the neutraiising antibodies induced by immunisation with p15E of FeLV, epitope mapping was performed using overlapping peptides corresponding to the entire p15E (Fig. 17). Four major epitopes were identified when the sera from three cats immunised with p15E (cats 14, 34, and 44) were analysed immediately after immunisation. The first, KALLETAQF (designated E1 a), and the second epitope (ALEESISALEK, E1 b) were located at the N-terminal part of the ectodomain. In the membrane proximal region the epitope MAKLRERLKQRQQLF (E2a) was identified. This epitope was also recognised by sera from a goat and from 7 of 8 rats, ail immunised with the same p15E sera. Interestingly, the cat sera also recognised an epitope (WFEGWFNKS) that had been recognised by rat sera after immunisation with the same p15E of FeLV (FDSQQGWFEGWFN, E2b). This epitope shares three identical amino acids with the epitope recognised by the broadly HJV neutralising antibody 4E10 (NWFN(D)IT, identical amino acids in bold). Sera 14, 34 and 44 also recognised a third epitope that is located in the immunosuppressive domain, LQNRRGLDILFLQEGGL. This domain is highly conserved among all retroviruses; and synthetic peptides corresponding to this domain inhibit lymphocyte proliferation and modulate cytokine production.

Whereas animals 34 and 44 were protected from antigenaemia, the neutralising capacity of sera 14 and 74 was rather low and the animals had a high level of p27 antigen (Fig. 17). While sera 14, 34 and 44 all recognised the same epitopes E 1a, E1b and E2a, the titer of neutralising antibodies was lowest in serum 14 (Fig. 13C).

When the antibody response was analysed 150 days after challenge, a clear difference between the animals immunized with Leucogen alone and Leucogen and p15E was detected: Whereas in the sera from the first group the titres of neutralizing antibodies

decreased, in the sera from the last group the titres of neutralizing antibodies remained high. Surprisingly, the amount of antibodies binding p45 is much higher in the sera from animal immunized with Leucogen and p15E compared to sera immunized with Leucogen alone.

Table 1 Titers of binding and neutralizing antibodies after immunization with Leucogen, pl5E or both antigens

Group No. of Antigen(μg) Application site for Adjuvant"' Titers of Titers of Neutralization Titer Number of sera Number of sera animals combined antibodies antibodies with neutralizing with neutralizing immunizations specific foτ specific for activity at activity at dilution pl5E Leucogen p 15E (ELlSA) p45 (ELISA) dilution 1 :4 1 :64

53.1 LA and IxIO ⁶ 6.4x10 ⁴ 1 :64

53 53.2 500 100 Two separate Montanide Ix IO ⁶ 6.4xlO ⁴ 1:64 3 3 53.3 IxIO ⁶ 6.4xlO ⁴ 1:64

54.1 IxIO ⁶ 1.6xlO ⁴ 1:64

54 54.2 500 100 Single LA 2.56 xlO ⁵ 1.6x10 ⁴ 1 :64 3 3 54.3 1x10" 6.4xlO ⁴ 1:64 o 55.1 Montanide 1x10* 0

55 55.2 500 - - 4x10 ^s 1: 16 1 0 55.3 4x10 ⁶ _ 0

56.1 6.4x10 ⁴ 1: 16

56 56.2 - 100 - LA 6.4xlO ⁴ 1: 16 3 0 56.3 _ 6.4x10 ⁴ 1: 16

57.1 Montanide 4x10" 1:16

57 57.2 100 - - IxIO ⁶ - 0 1 0 57.3 4x10 ⁶ 0

60.1 2.56 xlO ⁵ 1.6x10* 1 : 16

60 60.2 100 100 Single LA 2.56 xlO ⁵ 1.6x10 ⁴ 1:64 3 2 60.3 2.56 xlO ⁵ 6.4x10 ⁴ 1:64

LA - Leucogen adjuvant containing Quil-A and aluminum hydroxide, Montanide- Montanide ISA 720

Table 2 Characterization of sera from immunized and FeLV- infected cats

Serum Immunization FeLV Western blotting ELISA titre ¹ Neutralization titer Epitope mapping diagnosis pI5E

EIa EIb E2a E2b

14 pl5E + (6.4x10 ⁴ ) 2.56 xlO ⁵ (1 :256) 1 : 1024 ++ ++

34 p !5E + (6.4xlO ⁴ ) 2.56 xlO ⁵ (1 :256) 1 :1024 ++ ++

44 pl5E + (2.56 xl0 ⁵ ) l xl0 ⁶ (1:256) 1 :256 ++ ++

9425 none <i xlO ³ 1:16 -/+

w 6452 none + 4 xlO ³ 1 :256 -!-+

27470 various 4 xlO ³ 1 :256 -/+ vaccines

54748 none 4 xlO ³ 0 +

55409 none -/+ 4 xlO ³ 1 :64 +

55284 none <1 xlO ³ 1:64

1 : ELISA using recombinant pi 5 E , titers after the booster immunization (titers obtained after first immunization are shown in brackets)

2: Inhibition of provirus integration measured by real time PCR in comparison to preimmune sera, titers after the booster immunization (titers obtained after first immunization are shown in brackets)

3: ++ strong detection, + weak detection, - no detection