Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Reexamination Certificate
2001-09-06
2004-03-30
Housel, James (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
C424S192100, C424S196110, C424S202100, C424S204100, C424S230100, C435S005000
Reexamination Certificate
active
06713070
ABSTRACT:
This application claims priority to German Patent Application No. 19910044.6 filed Mar. 8, 1999.
The present invention relates to viral particles which are released after HCMV infection of mammalian cells, and to their use as vaccine.
Human cytomegalovirus (HCMV), a &bgr;-herpesvirus is a ubiquitously occurring pathogen. In an immunocompetent person, HCMV infection is normally unnoticed, having at the most mild and nonspecific symptoms. By contrast, in certain risk groups, for example in immunosuppressed patients such as AIDS patients or transplant recipients, and after prenatal infection, HCMV infection has serious manifestations.
Chemotherapeutics are available for treating HCMV infections. The success of antiviral chemotherapy of HCMV infection is restricted, however, in particular by the toxicity of the medicaments and the development of resistant variants of the virus if the duration of treatment is prolonged. In addition, the prophylactic or therapeutic use of antiviral hyperimmune sera has proved to be of only limited efficacy.
There has been work on the development of a vaccine against HCMV for many years. Thus, attempts have been made with weakened (attenuated) live vaccines to induce the desired immunity. This vaccine proved to have only limited efficacy, however. The reasons for this may be, inter alia, the restricted viability of such attenuated viruses in humans and strain-specific variations in the antigenicity. Besides the inadequacies in the induction of a permanent immunity, the use of a live vaccine must be regarded critically; lack of knowledge about the pathogenetic mechanisms in HCMV infection and the risk of reactivating the vaccine strain after immunosuppression make the use of a live vaccine appear at least questionable in these clinical situations.
In order to avoid these risks, strategies have recently been preferentially followed to develop subunit vaccines against HCMV which contain proteins from the viral envelope synthesized in various expression systems. Such envelope proteins, especially the glycoproteins gB and gH, are the essential target antigens of neutralizing antibodies against HCMV. Neutralizing antibodies are able to prevent the infection. It was possible both in experimental animals and in clinical studies to induce such neutralizing antibodies with a gB subunit vaccine. However, in humans, the antibody response induced in this way proved to be short-lived and not suitable for preventing the infection in all cases. This is detrimental to the wide use of subunit vaccines based exclusively on the gB of HCMV. The reasons which have been suggested for the limited efficacy of such antigen preparations in turn are the strain-specific variations in the immune response, lack of induction of an adequate cellular immune response, and structural restrictions of the antigen used, whose epitopes are in some cases known to be conformation-dependent.
On the basis of this experience, therefore, the requirements to be met by an effective and widely useful vaccine against HCMV are as follows:
(1) Long-lasting induction of neutralizing antibodies which protect from HCMV infection in a strain-overlapping manner. This requires efficient induction of a so-called “helper cell response” (CD4-positive T lymphocytes) against HCMV to assist the maturation of antibody-secreting B lymphocytes.
(2) Induction of the formation of cytotoxic T cells against HCMV. Lymphocytes of this type are of crucial importance for terminating an HCMV infection which has taken place and limiting the spread of the virus in the body.
(3) Minimizing the side effects by the vaccine. The risk which might derive from an inoculated viable virus which, according to present knowledge, would have the ability to establish latency after immunosuppression cannot be estimated. The aim ought therefore to be to prepare nonviable viral antigen as vaccine.
In order to comply with the conditions mentioned for an HCMV vaccine, the vaccine must contain the relevant antigens for inducing neutralizing antibodies and for stimulating helper cells (T
H
lymphocytes) and cytotoxic T cells (CTL).
Neutralizing antibodies are, according to the present state of knowledge, after HCMV infection formed exclusively against viral envelope proteins, and especially against the glycoproteins gB and gH.
T
H
cells are formed mainly against tegument proteins of the virus, and particularly against the so-called pp65 (ppUL83). In addition, pp65 is an essential antigen for the induction of CTL against HCMV. Presentation of pp65 takes place not only as usual after de novo synthesis by cells in connection with MHC class I molecules; it can also be introduced into the MHC I presentation pathway by so-called “exogenous loading”.
Said antigens are the essential constituents of defective viral particles which, during the infection of primary human fibroblast cultures, are synthesized by HCMV and released into the culture medium. These so-called dense bodies (DB) are structures which are visible under the electron microscope and more than 90% of whose protein mass consists of pp65. They are comparable with virus particles in being provided with a cellular lipid membrane modified by viral glycoproteins and being ejected from the cell. The viral glycoproteins are very probably in the natural conformation in this envelope. Since DB contain no viral DNA and no viral capsid, they are non-infectious. They can be concentrated in large quantity from the cell culture supernatant by established methods.
One object of the invention was to provide an effective and widely usable vaccine against HCMV.
The present invention describes viral particles which are released after infection of mammalian cells by human cytomegalovirus (HCMV). These particles can be employed as preventive or therapeutic vaccine against infections by HCMV.
The particles of the invention are surrounded by a lipid membrane which makes it possible to fuse the particles to certain mammalian cells so that their contents enter the cytoplasm of the cells. The membrane of the particles contains viral glycoproteins which represent the main antigens for virus-neutralizing antibodies. The particles are also characterized in that they contain no viral DNA and no capsid. In addition, they contain the viral T-cell antigen pp65 (ppUL83) which both stimulates the formation of T-helper cells and is an essential antigen for inducing cytotoxic T lymphocytes (CTL) against HCMV.
These properties, especially the combination of antigens able to induce both neutralizing antibodies and an adequate cellular response, make the particles suitable as vaccines against HCMV.
Dense bodies have already been described in the literature (Gibson et al. Birth Defects: Original Article Series 20, 1 (1984) 305-324; Virology 66 (1975) 464-473). In this connection, their possible use as vaccine was suggested. However, no experiments proving that dense bodies in fact show the hoped-for effects were shown. In addition, it has been shown that dense bodies have an antigenic effect (Jahn et al., J. gen. Virol. 68 (1987) 1327-1337). However, these experiments do not characterize the induced immune response, which is why no information about the suitability as vaccine can be inferred.
The fact that the particles of the invention have a high antigenicity and are able to induce the formation of neutralizing antibodies is shown in examples 1 and 2. The induction of virus-neutralizing antibodies is long-lasting (example 3), which is another prerequisite for a successful vaccine. The immune response achieved in examples 1-3 is all the more surprising since the immunization was carried out without using adjuvant. Side effects of vaccines administered with adjuvants thus do not apply. The particles of the invention also activate cytotoxic T lymphocytes (CTL) (examples 4 and 5).
Finally, DB induce, irrespective of the route of administration, T-helper cell responses of the Th1 type (example 6).
These examples demonstrate that the particles of the invention are suitable as vaccine against HCMV.
In a further embodiment, particles w
Foley Shanon
Housel James
Smith Reed
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