WO1995024215A1

WO1995024215A1 - Retroviral vaccines, vaccination and immune-therapy for retroviral infections

Info

Publication number: WO1995024215A1
Application number: PCT/NL1995/000091
Authority: WO
Inventors: Victor Smit; W. Huppes
Original assignee: Victor Smit; Huppes W
Priority date: 1994-03-11
Filing date: 1995-03-09
Publication date: 1995-09-14
Also published as: NL9400384A; AU1825895A

Abstract

The invention is based on living retrovirus that causes a non persistent infection for purpose of vaccination, immune therapy and as a retroviral vaccine against retroviral infections, preferably against HIV. It is preferred that persistence of the retrovirus is prevented by one or more of the following features: (1) it does infect T-cells but not histiocytic cells (macrophage like cells), (2) it is inactivated by a plasma factor, (3) it is inactivated by a natural immune system, (4) it is a virus that kills cells very quickly. In addition an other area of application of the invention is the treatment of forms of cancer, consisting of cells that can be infected with non persisting retroviruses. The hypothesis of this application is that the highly pathogenic virus eliminates the cancer before it is eliminated itself. This description of the invention enables the easy application of the invention to other retroviruses or retrovirus strains by a person skilled in the art. Therefore, these should also be considered to be within the scope of the invention.

Description

Title: RETROVIRAL VACCINES, VACCINATION AND IMMUNE-THERAPY FOR RETROVIRAL INFECTIONS.

Description of the invention:

The invention relates to the subject of retroviruses. A retrovirus is a virus that contains RNA as genetic material which is transcribed into the genome of the host as proviral DNA. Examples of retroviruses are HTLV-I, HTLV-II, STLV-I and viruses of the Lentivirus family, for instance HIV, visna virus, equine infectious anemia virus, feline immunodeficiency virus and bovine immunodeficiency virus. They are described in Science 239: 617 (1988) and its references.

The invention contains products that can be used to immunize both uninfected and infected mammals against retroviral infection. The invention further relates to (1) A living vaccine that causes a non-persisting infection in mammals, (2) an operation procedure for the vaccination, (3) an operation procedure for immune-therapy and (4) The variants and resulting products of one or more of the preceding items.

The vaccine is by preference a living retrovirus that causes a non persisting infection resulting in an effective suppression and eradication of the viral infection, preferably a HIV infection. The vaccine can thus be used against AIDS or AIDS associated diseases. The non persistence of the infection is preferably the result of one or more of the followin *go:-

(1) The retrovirus does infect T-cells but does not infect histiocytic cells (macrophage-like cells). Examples of these 'T-cell tropic viruses are HTLV-IIIB, HTLV-IIIMN, HTLV-IIIRF, HIV-ISF2 (AIDS Research and Reference Reagent program Catalogue, NIH Publication Number 91- 1536, Bethesda MD, U.S.A.)and HIV₃ o.2A 1.2 (AIDS Res. and Human retroviruses 9: 669 (1993)). The last reference also confirms the existence of T-cell tropism and the requirement of infection of histiocytic cells to obtain sustained infection.

(2) The retrovirus is inactivated by the complement system. This form of virus inactivation is described in Immunol. 73: 377 (1991) and its references. (3) The retrovirus is inactivated by natural antibodies. The existence of these usually low affine and broadly neutralizing antibodies is described in Immunol. Invest. 17: 387 (1988), "Advances in Immunology", Eds. Taliaferro W. H. & Humphrey J.H. Academic Press, New York: Vol. 2; 245-265 and "Applications of Immunological Methods in Biomedical Sciences" in "Handbook of Experimental Immunology", Ed. Weir D.M., Blackwell Scientific Publ., Oxford: Vol. 4; 1986, 117.4-118.17 en their references.

(4) An other preferential reason is a high cytopathogenecity of the retrovirus, meaning it causes a rapid killing of the host cells. In general these processes are harmful for the persistence of the virus because the virus producing cells are killed.

Several nonspecific methods for immune-therapy against virus-mediated diseases have been described. For instance, such is described in the patent "Method for treating viral diseases with attenuated virus " U.S.A. 5215745 (June 1, 1993) of Csatary et al.. The purpose was to enhance the number of CD4 positive cells by an aspecific infection using avian paramyxovirus and/or avian rotavirus. This elevation of the level of CD4 positive cells served the purpose to at least partially restore the disrupted immune system. It was mentioned that this technique could be used in AIDS-therapy of HIV-infected subjects. However, an aspecific immune response of the host can at best lead to a delay of the disease because newly formed CD4 positive cells will be infected soon after they are generated. In contrary, we describe a vaccine that causes a specific immune response against the corresponding retrovirus. Therefore, an efficient neutralization of the retrovirus can be expected.

Until now, specific methods of immune-therapy are limited to the so called peptide vaccines, virus subunit vaccines or inactivated virus vaccines. However, presently none of these methods are effective against a retrovirus infection because the structure and function of the virus is not sufficiently mimicked. As an example with more simple viruses like the measles virus, vaccination with non living virus still only results in moderate protection. Therefore, the short term- and midterm-expectations for such a vaccine are very low. Also immune-therapy with such peptide vaccines, virus subunit vaccines or inactivated virus vaccines is not expected onshort term- and midterm because the natural immunity of mammals can be very strong and yet remain insufficiently effective. This is demonstrated in the asymptomatic phase of a HIV infection. In this stage the high levels of in vitro neutralizing antibodies and cellular immunity against HIV are not able to stop the progressing infection. One explanation could be the protection of the HIV virus against the cellular immunity by the hosts own antibodies. The difficulty to obtain an effective immunity is also demonstrated in Patent number U.S.A. 5081226 (January 14, 1992) from Berzofsky et al. In this case antibodies were generated against small segments of the glycoprotein 160/120 complex of HIV. These generated antibodies were not able to protect against HIV infection.

One possible explanation is that only antibodies against only a small number of natural virus-epitopes are obtained. The chance that these vaccines are able to protect against a progressing infection is very small as demonstrated by the fact that there has not been one effective peptide vaccine against AIDS until now. In addition, when only such a limited number of epitopes are used, chances for an effective cellular immunity are minimal if present at all and no success has been achieved as yet. In contrast, in present invention there is an optimal expectation for diversity of natural epitopes and induction of cellular immunity.

The hypothesis that antibodies against the HIV envelop even protect the HIV retrovirus virus and also even promote successful integration in histiocytic cells has been incorporated in patent " Retroviral antigens" U.S.A. 5256767 (October 26, 1993) by Salk et al. Their invention is based on the generation of antibodies against HIV particles without the envelope. The basic hypothesis of this patent is the fact that these internal segments of the HIV virus will still be expressed on the outside of HIV containing host cells, thereby allowing the efficient elimination of virus producing cells. An important first objection of this method is that the inner parts of the virus can be expected to express poorly on the outside of the host cell membrane because of their nature. This severely limits the chance on effective elimination of virus producing cells. In addition, and seconly, one can expect an inferior presentation of these epitopes to the immune system, as compared the living virus. These two arguments together form a plausible reason why this invention of Salk et. al did not function. In contrast, in our invention the natural situation is optimally mimmicked, resulting in the expectation of an effective cellular immune-respons. Either one or more of these problems is also valid in the use of inactivated viruses. In contrast, the vaccination against smallpox did achieve success because of a living vaccine. Also for polio, there is a successful living vaccine: the Sabin vaccine. There are three possible reasons for this success^ 1) Temporarily, a high dose of viral antigens can be achieved. (2) A potent cellular response is induced. (3)

An immune response against a broad range of different viral antigens can be induced.

In these cases of polio- and smallpox vaccine it concerns weak and for healthy people non pathogenic living viruses that do not persist. In contrary, retroviruses do have the capability to persist. Therefore, they are much more capable to distort the immune system and cause disease. In addition, retroviruses in general have the capability to mutate. The combination of persistence and mutation-capability allows them to circumvent the immune system.

The feature of this invention is the discovery of a living retrovirus that causes a non persisting infection. Therefore, it can be expected that the virus will not be able to disrupt the immune sytem. In addition it can be expected that the virus is not able to mutate, and circumvent the immune system in this way.

The same above mentioned problems are valid for vaccines based on weakened forms of retroviruses. Even more harmful however, is the possible generation of dangerous forms because these forms would cause both disruption of the immune system and disease after all. In our invention we are able to exclude the generation of these dangerous forms because we used a virus infection that does not persist. Therefore, there is no opportunity for the virus to mutate. The proposed vaccine against the HIV infection is even the very pathogenic strain HIV- 1 SF2 (AIDS Research and Reference Reagent Program Catalog, NIH Publication No. 91-1536, Bethesda, MD, U.S.A.). The result is that the virus kills its own host cells very quickly. Therefore, even at short term it can be expected that successful vaccination and immune therapy for HIV infected subjectscan be achieved.

The description of the invention as above enables the application of the invention to other retroviruses by a person skilled in the art. Therefore, other retrovirus vaccines based on principles that are either identical or comparable to one or more of the above mentioned principles should be considered to be within the scope of the invention. In general, only non infectious virus is used for vaccination in order to prevent infection with transferable genetic material such as infectious retrovirus, that integrates in the host genome as provirus. More specifically, the transfer of this genetic material to gonad cells is not acceptable because this would induce genetic changes in the progeny. However when an individual is infected by a retrovirus the retrovirus attaches to a unique and specific cell receptor that is not present at all cells. For instance HIV only attaches to a CD4-receptor, present only on specific cells. Cells with such a receptor will remove the free virus particles . Therefore, the concentration of unbound virus particles remains low. Since gonad cells do not have receptors for any of the known retroviruses they will not be infected by them.

In contrast, a persisting infection with a retrovirus does have the capability to cause both a neutralization and saturation of cells that , carry the retroviral receptor. This phenomenon is for instance observed in the final stage of AIDS. It is even probable that transfer of genetic materials to gonad cells is occurring at a large scale during retroviral epidemics. In view of the severity of for instance the HIV epidemic it is justified to introduce a living retrovirus vaccine to the market. Therefore, the preference of our invention is the prevention and treatment of AIDS and AIDS associated diseases.

Examples:

Example 1

The HIV-lsF2 vaccine can be eliminated by a plasma factor.

Materials and methods:

The test system consisted of human to mouse chimerical "severe combined immunodeficient " (SCID) mice at the age of 3 weeks. The chimerical state was induced by conditioning with total body irradiation (TBI)and transplantation of human CB 15 cells into the peritoneal cavity (ip). The dose of the CB15 cells was 1.3 million cells /gram of recipient. These monoclonal CB15 cells are described in (Proc. Natl. Acad. Sci. U.S.A. 89: 3116(1992). The TBI was 2.5 Gy gamma. Comparable transplantation procedures are described in Eur. J. Immunol. 22: 197 (1992).

Daily, the mice were injected ip with 10.000 I.U. human Interleukin-2 (Eurocetus, Amstelveen, Benelux). The infection of the CB15 cells with HIV- 1SF2 or HIV- IQΠ (AIDS Res. and Human Retroviruses 9: 699 (1993) was performed with a dose of 10 times the minimal infectious amount of culture supernatant in an "infectious center test" (ICT) of HIV-1SF2 °^r HIV-1QI7 either in vitro for 1 hour at 37 °C or in vivo by ip injection 1 hour after transplantation of the CB15 cells. The ICT was determined twice with SUPT1 (AIDS Research and reference Reagent Program Catalog, NIH Publication Nr. 91-1536, Bethesda,MD, U.S.A.). Alternatively, CB15 cells were used in an amount of 10000 cells per well of a 96 well culture plate, followed by 5-7 days of cell culture and subsequent ELISA analysis on HIV-p24 presence (Organon Technica Oss, Holland).

On a predetermined day the mice were sacrificed and the cells were rinsed from the peritoneal cavity with medium containing heparin (Organon Technica, Oss, Holland). Subsequently, 5 million of the obtained cells were added to the same number of SUPT1 or CB15 cells and were cultured in presence of 100 I.U. human Interleukin-2 per ml of culture medium. After 5-7 days, a HIV-p24-ELISA was performed on the culture supernatant.

Results:

The results (Table 1) show that the HIV- 1SF2 is effectively neutralized after in vivo infection of the human cells in the SCID mice. However HIV-IQ₁₇ was not effectively neutralized under these circumstances. Since irradiated SCID mice- that are transplanted with CB15 do not have any form of immunity, there must be a plasma factor that neutralizes the free HIV-1 SF2- Possibly this factor is complement. In addition it is demonstrated that the HIV- 1 SF2 infection can not be neutralized in vivo if the CB 15 cells have been infected in vitro. Because free HIV- 1 SF2 is neutralized by plasma, the in vivo HIV- 1 SF2 transmission has to be mediated by cell-cell contact of the CB 15 cells.

Example 2

HIV-1SF2 can be inactivated by human peripheral blood lymphocytes.

Materials and methods:

The test system consisted of human chimerical "X-linked immunodeficient" (CBA/N) mice at the age of 4 weeks and SCID mice as described in example 1. The chimerical state was induced by conditioning with total body irradiation and transplantation of human blood cells, so called peripheral blood leukocytes (PBL) in the peritoneal cavity (ip). The TBI of the SCID mice was 2.5 Gy gamma and of the CBA/N mice 9 Gy gamma. The latter animals also obtained a blood supporting treatment in the form of 0.5 million autologous bone-marrow cells intravenously. The dose of the PBL tranplant was 4 million blood lymphocytes per gram of recipient. The CBA/N mice also received intravenous treatment with

In some of the SCID mice CB 15 cells were transplanted as described in example 1 and in some CBA/N of the mice CB15 cells were transplanted as described above. Similar irradiation's, preparations of human blood and transplantation procedures are described in Eur. J. Immunol. 22: 197 (1992). The mice received daily ip injections with 10000 I.U. of human Interleukin-2. The infection of the human cells with HIV was performed as described in example 1.

On a predetermined day the mice were sacrificed and the cells were rinsed from the peritoneal cavity as described in example 1. Subsequently, 5 million of the obtained cells were added to the same amounts of SUPT1 or CB 15 cells and were cultureddays in presence of 100 I.U. human Interleukin-2 per ml of culture medium. After 5-7 days a HIV-p24-ELISA was performed on the culture supernatant. Results:

The results (Table 2) show that this experimental set up results in an efficient HIV infection of both PBL and CB15 cells. After the in vitro infection of PBL with HIV- 1SF2 the virus is eliminated within 2-4 days after transplantation. Since this elimination does not occur with the CB15 transplants, the elimination of the virus and/or virus containing cells is performed by the human PBL. As the generation of a newly induced humoral or cellular immunity takes at least 6 days, the prompt response of these human PBL against HIV- 1SF2 ^must already have been present in the transplant when the transplantation was performed.

Finally, in table 2 it is also demonstrated that there is no difference in infection and/or elimination of HIV between CBA N mice and SCID mice. The above used CBA/N mice have an almost normal antibody repertoire, except for the absence of natural antibodies. Since this antibody repertoire of the CBA/N mice does not cause this prompt elimination of HIV- 1SF2. the cause of the elimination of HIV-1SF2 is therefore the already present resistance of the human PBL. The most probable explanation for the elimination of the HIV- 1 SF2 i the neutralization by natural antibodies produced by the human PBL transplant. However, other systems cannot be excluded. For instance human natural killer cells or in the mouse by the PBL transplant produced other human antibodies are alternative explanations.

Field of the invention

From the examples it can be concluded that HIV-1SF2 is a safe vaccine against retroviral infections. In addition it might be applicable to immune therapy. Although only two examples with one vaccine are given they are to be considered as an illustration and not a limitation of the invention. Since someone skilled in the art can easily apply this to other retrovirus strains and or retroviruses these applications should also be considered to be within the scope of the invention.

Moreover, an other field of applications of the invention can be found in the treatment of forms of cancer, consisting of cells that can be infected with non persisting retroviruses. The examples of this invention lead to a possible application of the HIV- 1SF2 against a CD4 positive cancer like some non Hodgkin lymphoma's. The idea of this application is that the highly pathogenic virus eliminates the cancer before it is eliminated itself. Since someone skilled in the art can easily apply this to other retrovirus strains and or retroviruses these applications should also be considered to be within the scope of the invention.

Table 1:

HIV- 1SF2 can be eliminated by a plasma factor.*

Number of SCID mice Way of infection Virus strain HIV p24 -ELISA

3 in vivo SF2 negative

5 in vivo Q17 positive

6 in vitro SF2 positive

In all tests the mice were sacrificed 8 days after the transplantation with CB 15 cells.

Table 2:

HIV- 1SF2 can be inactivated by human peripheral blood cells.

Mouse Number Way of Virus- Trans- Day of HIV p24- of mice infection strain plant sacrifice* ELISA

SCID 6 in vitro SF2 CB 15 8 positive

CBA/N 3 in vitro SF2 CB 15 8 positive

SCID 3 in vitro SF2 PBL 2 positive

CBA/N 6 in vitro SF2 PBL 2 positive

SCID 3 in vitro SF2 PBL 4 negative

CBA/N 6 in vitro SF2 PBL 4 negative

SCID 3 in vitro SF2 PBL 8 negative

CBA/N 3 in vitro SF2 PBL 8 negative

SCID 5 in vivo Q17 PBL 8 positive

CBA/N 5 in vivo Q17 PBL 8 positive

The number of days after the transplantation procedure.

Claims

What is claimed is:

1- A vaccine to counteract retroviral infections in mammals, preferably human subjects, with the feature that it contains a retrovirus, a derived virus thereof or a derived product thereof which causes a non persisting infection in the host.

A vaccine according to claim 1, with the feature that the counteraction of the retroviral infection is a vaccination to prevent retroviral infection.

A vaccine according to one or more of the preceding claims, with the feature that the counteraction of the retroviral infection is an immune-therapy for the retroviral infection.

4- A vaccine according to one or more of the preceding claims, with the feature that it is effective against HIV-infection and/or AIDS and / or AIDS associated diseases.

A vaccine according to one or more of the preceding claims, with the feature that it causes an elevated number of CD4 positive cells.

6- A vaccine according to one or more of the preceding claims, with the feature that it is a T-cell tropic retrovirus.

A vaccine according to one or more of the preceding claims, with the feature that it is not a histiotropic, preferably not a macrophage-tropic virus.

8- A vaccine according to one or more of the preceding claims, with the feature that it contains either one or more of the following: HTLV-IIIB, HTLV-III N_. HTLV- IIIRF, HIV-ISF2- - A procedure to counteract retroviral infections with a vaccine with one or more features as described in one or more of the preceding claims.

0- A vaccine according to one or more of the preceding claims, with the feature that it can is used in mammals , preferably human subjects, to counteract malignancies that can be infected by the vaccine.

1- A procedure to counteract malignancies by using a vaccine as described in one or more of the preceding calims.