WO1996030045A1

WO1996030045A1 - Vaccine for the immunoprophylaxis of the infection from feline immunodeficiency virus in the domestic cat

Info

Publication number: WO1996030045A1
Application number: PCT/EP1996/001356
Authority: WO
Inventors: Mauro Bendinelli; Donatella Matteucci; Mauro Pistello; Carlo Garzelli; Gino Maldavi; Alessandro Poli; Franco Tozzini
Original assignee: Istituto Superiore Di Sanita'; Universita' Degli Studi Di Pisa
Priority date: 1995-03-31
Filing date: 1996-03-28
Publication date: 1996-10-03
Also published as: ITRM950209A0; AU5398296A; IT1276509B1; ITRM950209A1

Abstract

It is disclosed an immunizing vaccine for the prophylaxis of the infection from feline immunodeficiency virus and/or the pathological syndrome known as feline AIDS induced by said virus. The vaccine is for veterinary use and finds application in the domestic cat. The vaccine produces a strong protective specific immune response to the virus antigens, when the viruses, utilized for the preparation of such vaccine, are strains of fresh isolation and/or are subtypes prevailing in the geographical area of interest.

Description

Vaccine for the immunoprophylaxis of the infection from feline immunodeficiency virus in the domestic cat

Field of the invention

The present invention provides an immunizing vaccine for the prophylaxis of the infection from feline immunodeficiency virus (FIV) and/or the pathological syndrome known as feline AIDS induced by said virus.

In particular, the present invention relates to vaccine prepared with strains of FIV which are of fresh isolation and/or are subtypes prevalent in the geographical area wherein one intends to use the vaccine.

Prior art

It is known in the state of the art that like the human immunodeficiency virus, or HIV, the feline immunodeficiency virus, or FIV, is a lentivirus, which was described for the first time by Niels

Pedersen et al. in 19&7 (Science, 235. 790-793. 1987). Subsequent research has demonstrated that FIV is spread worldwide and represents a serious problem for domestic cats' health (Felis catus). In fact, the virus is constantly detectable in a significant percentage of animals and, at particular epidemiological conditions, may reach epidemic spread levels.

Analogous viruses are found in several wild feline species. The virus usually penetrates through the skin, following the bite of carrier or ill cats, and is progressively and rapidly amplified in lymphoid organs and in other body districts. Different ways of contagion are also possible. The virus thus becomes easily detectable in peripheral blood and in some other body fluids, e.g. saliva. The host exposed to the virus tries to react to its invasion by an intense antiviral immune response, which, at least in the initial phase, is capable of slowing down virus progression, nevertheless, such immune response never succeeds in eradicating the virus itself, which already too intimately settled in several types of cells.

Within a few months after FIV infection, the infected cats develop a state of immunodepression, easily detectable by analysing the classical immune functionality parameters, i.e. progressive fε.ll in T CD4+ lymphocytes, inverted CD4:CD8 ratio, hypergammaglobulinaemia, reduced antigen-induced antibodypoiesis, reduced cell-mediated responses, reduced lymphokine production, etc., as well as other proteiform immune dysfunctions whose significance is still under evaluation. The decrease of immune response caused by the virus opens the way to the appearance neurological manifestations, to the development of various types of neoplasias and to very frequent and dangerous superinfections sustained by a wide range of infecting agents. The resulting clinical patterns, which are extremely complex and polymorphous, configure a real syndrome, i.e. feline acquired immunodeficiency syndrome (FAIDS) . Further information on FIV and its pathological effects is disclosed in two recent reviews (Pedersen N.C., in "The Retroviruses", vol. 2, J.A. Levy Ed., Plenum Press, New York, 1993; Bendinelli M. et al.. Clinical Microbiology Reviews, 8, 87-112, 1995). Technical problem The possibilities to stop FIV infection by therapeutic means are extremely low or inexistent. In fact, the drugs now available, which are the same as those used to treat human AIDS, have proved to be even less efficacious in FIV-infected cats than in man. This is one of the reasons why it is highly important to provide commercial vaccines administrable as a prophylactic measure, able to prevent the infection and/or the progressive evolution of the disease. Up to now, the attempts to produce vaccines against FIV have run into serious difficulties, which, however, are common to those met when trying to develop vaccines against other lentiviruses, in particular HIV. Among the many problems to be solved, one is the existence of various FIV subtypes (or clades) provided with different surface protective antigens.

The anti-FIV vaccines described to date have not brought about significant protection levels or have produced modest or controversial protective effects. Paradoxically, in some cases they even enhanced the infection experimentally induced in immunized animals to evaluate the protective action thereof (Pedersen N.C., in "The Retroviruses" , vol. 2. J.A. Levy, ed. , Plenum Press, New York, 1993; Hosie et al., British Veterinary Journal, 150, 25-39, 199*4; Bendinelli M. et al.. Clinical Microbiology Review, 8, 87-112, 1995). The authors of the present invention have found that the low or inexistent protective effectiveness of the anti-FIV vaccines disclosed up to now is, mainly, due to the use of strains of viruses repeatedly propagated in vitro on cell cultures. When passaged more than 20-30 times in vitro, such viruses fail to show the protective epitopes or show them in an inadequate way to induce an effective protective immunity when inoculated into cats. Summary of the invention The purpose of the authors of the present invention was to provide vaccines able to produce a strong immunity against the feline immunodeficiency virus and therefore to protect cats from infection acquisition and/or FAIDS onset.

In particular, the present authors have surprisingly found that when for the preparation of anti-FIV vaccines, strains of freshly isolated virus are used, such virus maintained its immunogenicity and allowed the vaccine to induce a very strong immune protective response. Therefore, a fundamental characteristic of the present invention is an anti-FIV vaccine preparared with freshly isolated virus. Another characteristic of the invention is a vaccine prepared including subtypes of FIV virus prevalent in the geographical area wherein one intends to use them.

A further characteristic of the present invention is a vaccine, including a freshly isolated virus, for use as a therapeutic agent to reinforce the antiviral response in already infected cats.

The present invention also relates to an improved process for the preparation of a vaccine according to the invention.

Detailed description of the invention

As used herein, the expression "vaccine against feline immunodeficiency virus or feline AIDS (FAIDS)" refers to a preparation consisting of (i) one or more FIV subtypes, known as A, B, C and D (e.g., see Bendinelli M, et al., 1995). freshly isolated, according to procedures, on lymphocytes or permissive non-trasformed lymphoid cultures and selected on the basis of the diverse epidemiological situations existing in different geographical areas or (ii) lymphoid cells infected productively with local freshly isolated virus strains or (iii) selected antigens of said freshly isolated viruses. The vaccinal preparations are prepared or treated in such a way as to make them completely harmless to cats , though maintaining their immunogenic properties .

The virus or viruses utilized for the preparation of the anti-FIV vaccine according to the invention must be "strains of fresh isolation" , that is passed in cultures of non-transformed lymphoid cells and for a maximum, of 10-15 times , in order to avoid the loss of the immungenicity properties which allow the virus to induce immune protective responses . That represents an essential prerequisite for the vaccines to be effective .

Our data show that vaccines , prepared with freshly isolated virus and chosing strains of subtypes of the desired geographical areas according to the invention give better results compared to those of the state of the art prepared with virus repeatedly propagated in vitro on cell cultures and without chosing subtypes of the pertinent geographical areas (Tables 1 and 2 ) .

The vaccine preparations according to the invention are treated or prepared in order to render them absolutely innocuous for cats , though maintaining their immunogenic properties . The vaccines of the invention are designated "anti-FIV immunizing vaccines" .

A very active form of said vaccine was developed by growing virus strains , selected in various local epidemiological situations , on cultures of feline cells of lymphoid origin or similar origin optionally in combination with a pharmaceutically acceptable adjuvant . For the present case the selected subtype ( or clade ) was the B subtype , highly prevalent in Italy , and therefore the vaccine according to the invention will contain at least the B subtype, or a mixture comprising it.

The cultures infected with a convenient multiplicity were incubated at 36°-39°C (preferably 37^βC) for some days, preferably from 6 to 10, until the number of infected cells expressing surface viral antigens reached adequate levels (4θ-8θ/_, preferably 60 ).

The cells were then collected, cooled (preferably at 4^CC), concentrated by centrifugation (preferably by low-speed centrifugation at 4°C). Then the process could be improved compared to the known methods by treating the cells with 10/. glycerol for 1-5 min (preferably 3 minutes). This last treatment is very important as it causes a greater exposure of protective antigens on the cell surfaces and, therefore, makes them more immvmogenic. However, also a process without using the glycerol step can be performed. After the removal of glycerol, when used in the previous step, the cells were treated with a disinfectant for the time required for viral infectivity inactivation and adapting the time of exposure to the strength and concentration of the inactivating agent as well as to the incubation temperature (e.g. 1-2% paraformaldehyde at 37°C for 24 hours). The cell suspension was then mixed with an equal volume of at least one adjuvant (preferably the Freund's incomplete adjuvant). Each dose of vaccine consisted of about 10 million infected cells. The tests conducted to check the efficacy of said vaccines demonstrated that vaccinated cats develop a strong direct specific immune response to the virus as proved by the appearance of a high level of neutralizing and non-neutralizing antiviral antibodies as well as by the development of cell-mediated antiviral responses. Most importantly, vaccinated cats proved to resist, without being infected, when challenged with highly virulent FIV preparations (as obtained by using virus never passaged in vitro, e.g. plasma of infected cats) inoculated experimentally by a parenteral route.

A comparative example of the better effectiveness of the vaccine prepared according to the present invention than a vaccine prepared according to the state of the art is shown in the combination of Tables 1 and 2.

Table 1 Effectiveness of the anti-FIV immunogenic vaccine prepared according to the invention

Pretreatment of Animals infected/ . the animals (6) animals subjected to challenge

Vaccinated 0/6

Non-vaccinated 6/6

The animals were subjected to challenge four months after the last dose of vaccine. The challenge was carried out via intravenous route using a high virulent homologous FIV. Better results were obtained when the high virulent homologous FIV was never passed in vitro in order not to alter its invasive capacity.

A test with reference to a vaccine according to the state of the art prepared with virus repeatedly propagated in vitro on cell cultures and without chose subtypes of the pertinent geographical areas is reported in Table 2. - 8 -

Table 2

Lack of protection against FIV infection using a vaccine prepared with virus grown in vitro for prolonged periods

Pretreatment of_g Animals infected/ . the animals (6)^*-* animals subjected to challenge

Vaccinated 6/6

Non-vaccinated 6/6

The animals were subjected to challenge four months after the last dose of vaccine, using the same procedure and the same virus stock used in the experiment depicted in the above Table 1. ^•The vaccine was prepared and administered exactly as in the xperiment depicted in Table 1, except that the virus used for its preparation was grown in vitro in ε trasformed lymphoid cell line for 30 passages.

.Anti-FIV vaccines, according to the invention, may alternatively be obtained in different ways, always starting from FIV viruses freshly isolated according to the invention. Said anti-FIV vaccines consist of:

(i) extracellular virus, optionally concentrated, purified by physico- chemical methods and treated so as to destroy its infecting and/or pathogenic power; or ;ii) viral molecules; or (iii) specific selected fragments of said molecules produced by recombinant DNA technology or chemical synthesis. Also said materials may be mixed with immunologic adjuvants of various

RECTIFIED SHEET (RULE 91 )

I ISOAΛ//EPPP types and used as vaccines though generally having lower immunizing and protective efficacy than the aforementioned vaccines based on infected cells .

Conversely , an excellent immunizing effect is obtained wi th FIV modified in such a way as to produce a self -limited infection, which, therefore , does not persist in time .

The vaccine may be used to prevent infection in uninfec ted cats . However , it can possibly have beneficial effects also when used as a therapeutic treatment to enhance the antiviral responses in infected cats ; in this case , it slows down or attenuates the development , in said animals , of the FIV-induced immunodepressive infec tion and pathology .

In the last case , therefore , the invention provides a vaccine comprising freshly isolated virus ( es ) with or without a pharmaceutically acceptable excipient that can also be used as drug to reinforce the antiviral response in already infected cats . The present invention further provides a method for determining whether a vaccine is indeed protec tive consis ting in the administration of a FIV derived directly from the tissues of infected cat as a challenge .

The vaccine according to the invention may be prepared from FIV , from viral molecules in general or one or more fragments thereof . Such a viral molecule or fragments may be produced by recombinant DNA technology or chemical synthesis . The invention will be now more properly illustrated with reference to a preferred embodiment . Example

Process for the preparation of an anti-FIV vaccine

Feline cells of lymphoid source were infected at high multiplicity with freshly isolated B subtype FIV, prevalent in Italy, and incubated at 37°C for 6 days, until the number of infected cells expressing surface viral antigens reached the level of 60/..

The cells were then collected, cooled at 4°C, concentrated by low- speed centrifugation at 4^βC, and treated with 10% glycerol for 3 min. Once glycerol had been removed, the cells were treated with 1 % paraformaldehyde at 37°C for 2k hours for viral infectivity inactivation.

The cell suspension was then mixed with an identical volume of Freund's incomplete adjuvant. Each dose of vaccine consisted of about 10 million infected cells.

Claims

Claims : 1. Vaccine for the immunoprophylaxis of the infection from feline immunodeficiency virus and/or^' against FAIDS in the domestic cat comprising freshly isolated FIV virus ( es ) with or without a pharmaceutically acceptable excipient . 2. A vaccine according to claim 1 , wherein said FIV-virus comprises at least one subtype of FIV virus prevalent in the geographical area wherein one intends to use it . 3 - A vaccine according to claim 2 , wherein said at least one subtype is of B-subtype. 4. A vaccine according to claim 1 , wherein said vaccine consists of extracellular freshly isolated virus , optionally concentrated , modified by known physico-chemical methods and treated in such a way as to destroy its infecting and/or pathogenic power. 5 • A vaccine according to claims 1 and 4 , wherein the said vaccine comprises viral molecules or fragment thereof . 6. A vaccine according to claims 1 and 4 , wherein the said vaccine comprises specific selected fragments of said viral molecules produced by recombinant DNA technology or chemical synthesis . 7 - Use of vaccine according to claims 1-6, as therapeutic agent to reinforce the antiviral response in already infected cats . 8. Process for the preparation of a vaccine for the immunoprophylaxis of the infection from feline immunodeficiency virus , consisting of : < ) one or more FIV subtypes , reshly isolated and grown in primary lymphocytes or permissive lymphoid cultures and selected on the basis of the various epidemiological si tuations exis ting in di fferent geographical areas ; or (ii) lymphoid cells infected productively with local virus strains freshly isolated; or (iϋ) selected antigens of said local virus strains; said preparations being prepared or treated in such a way as to make them completely harmless to cats, though maintaining their immunogenic properties. 9• A process for the preparation of a vaccine according to claim 1, wherein the virus strains used, selected in various local epidemiological situations, are grown on feline lymphoid cell cultures or on cellular substrates of similar origin, incubated at 36°-39°C for 6-10 days until 40-80% of infected cells exhibit surface viral antigens, said cells being cooled, concentrated, centrifuged, subjected to a treatment making them more immunogenic, by amplifying the cell surface viral antigenic stimulus and to a subsequent treatment allowing viral infectivity inactivation, and therefore becoming harmless to the feline, the preparation of said vaccine being completed by the addition of at least one adjuvant; each dose of vaccine consisting of approx. 10 million infected cells. 10. Process according to claim 9. wherein the strains selected comprise at least the B subtype. 11. Process according to claims 8-10, wherein the treatment making the cells more immunogenic consists of subjecting said cells treatment with 10% glycerol for 1-5 minute. 12. Method, for determining whether a vaccine, according to claims 1- 4. is indeed protective, consisting in the administration of a FIV derived directly from the tissues of infected cat as a challenge.