The present invention provides new uses of recombinant adenoviral vectors in vaccination regimens, such as prime/boost set-ups and subsequent vaccinations and applications for gene therapy. Moreover, the invention provides new assays to determine the best regimen for applying the most suitable recombinant viral vector in a vaccination or gene therapy setting.

Description of the Invention

TECHNICAL FIELD

The present invention relates generally to the field of biotechnology, and more particularly to the field of medicine, in particular to the field of vaccination using recombinant adenoviral vectors. The invention specifically relates to the production and controlled use of vaccines based on adenoviruses derived from different serotypes.

BACKGROUND

Many different kinds of vaccines are being employed to prevent pathogenic entities to enter the body or to prevent the pathogenic entities to spread and cause illnesses. Vaccines that are being applied nowadays and/or vaccines that are being tested in different stages of development include whole-inactivated viruses, (live-) attenuated viruses, peptide vaccines, (naked) DNA vaccines, sub-unit vaccines and vaccines that are based on (relatively) harmless viruses that harbor an antigenic determinant from the pathogenic entity towards which the vaccine is directed. Examples of such "vaccine carriers" are influenza virus, alphaviruses such as Semliki Forest Virus or Sindbis virus, and adenoviruses. Wild-type adenoviruses are known to cause relatively mild diseases such as common colds. To date, over 50 different adenovirus serotypes have been identified, subdivided into six subgroups based on their sequence homologies and hemagglutination abilities. Recombinant adenoviruses are being extensively tested in HIV vaccine clinical trials and in vaccines against malaria (WO 01/02607; WO 02/22080; WO 01/21201; Sullivan et al. 2000; Shiver et al. 2002). The results that were obtained in these studies clearly show that adenoviruses provide an excellent tool for delivery of the antigen to the host. One could envision an endless list of other pathogens that could be targeted by using the adenovirus as an antigen carrier providing proper protection. Such pathogens include, but are not limited to, viruses, bacteria, yeasts, fungi, etc.

However, a few important drawbacks exist when the most common and probably the best-studied adenovirus serotype, Adenovirus 5 (Ad5) is used. As has been described extensively elsewhere (PCT International Publication WO 00/70071), it is known that most people across the world have encountered an Ad5 infection at least once in their life. This results in a level of neutralizing antibodies that is relatively high and causes a rapid clearance from the system. Moreover, it is known that almost all Ad5-derived recombinant vectors end up in the liver. This phenomenon presumably prevents the recombinant vector (based on Ad5) from very efficiently entering the antigen-presenting cells such as dendritic cells. The art has recognized that there was a need for alternative adenoviruses that would not home to the liver, but rather would be targeted to the cells involved in the immune system. One way of triggering this was by employing the receptor- or cell-binding moiety of the adenovirus. This moiety was swapped from certain adenoviruses not having a tropism for liver cells to Ad5. An example of such a recombinant adenovirus is Ad5fib16, which is a recombinant adenovirus based on Ad5, but carrying the tropism-determining part of the fiber of adenovirus serotype 16 in its capsid (see. PCT International Publications WO 00/03029 and WO 02/24730).

Nevertheless, significant problems remain to be solved. Many of these are based on the finding that an infection (and injection) with a specific adenovirus elicits a significant immune response in humans and thereby hampers different kinds of vaccinations, using that same specific adenovirus serotype. Thus, if an individual has encountered a specific serotype, it is in general hard to obtain an immune response by using a vaccine based on that particular serotype. This would, therefore, limit the possible use of recombinant adenovirus as an antigen carrier for vaccination purposes.

SUMMARY OF THE INVENTION

The present invention discloses methods and means for vaccination purposes using recombinant adenoviral vectors. The invention provides a use of a recombinant adenovirus vector of a first serotype for the preparation of a medicament for the treatment or prevention of a disease in a human or animal treated with a recombinant adenovirus vector of a second serotype, wherein the first serotype is different from the second serotype. The invention also relates to a use of a recombinant adenovirus vector of a first serotype for the preparation of a medicament for the treatment or prevention of a disease in a human or animal having an antibody titer against an adenovirus of a second serotype, wherein the first serotype is different from the second serotype. The invention furthermore provides a kit of parts comprising a priming composition and a boosting composition, both compositions comprising: a recombinant adenovirus vector; a heterologous nucleic acid of interest present in the vector; and a pharmaceutically acceptable carrier, wherein the recombinant adenovirus vector of the priming composition is from a different serotype than the recombinant adenovirus vector of the boosting composition. The invention also provides a method for determining the titer of neutralizing antibodies in a blood sample, wherein the neutralizing antibodies are directed against a virus, comprising the steps of: obtaining a sample; culturing host cells; infecting the host cells with recombinant viral vectors comprising a transgene, in the presence of the sample; and determining the activity of a protein encoded by the transgene. In another embodiment, the invention provides a method for determining the titer of neutralizing antibodies in a blood sample, wherein the neutralizing antibodies are directed against a virus, comprising the steps of: obtaining a sample; culturing host cells; infecting the host cells with recombinant viral vectors in the presence of the sample; and determining the number of viral genomes per cell.

The present invention provides methods and means that solve problems in the field of vaccination. The present invention provides the use of a recombinant adenovirus vector of a first serotype for the preparation of a medicament for the treatment or prevention of a disease in a human or animal treated with a recombinant adenovirus vector of a second serotype, wherein the first serotype is different from the second serotype. The invention also relates to the use of a recombinant adenovirus vector of a first serotype for the preparation of a medicament for the treatment or prevention of a disease in a human or animal having an antibody titer against an adenovirus of a second serotype, wherein the first serotype is different from the second serotype. Preferably, the second serotype is selected from the group consisting of: Ad11, Ad26, Ad34, Ad35, Ad46 and Ad49, and wherein the first serotype is selected from the group consisting of: Ad11, Ad26, Ad34, Ad35, Ad46 and Ad49. Preferred are embodiments in which the first serotype according to the invention is comprised in a vaccine composition (normally a boost composition), while the second serotype is part of a priming composition. It is to be understood that it is part of the invention that if an individual does not have a high titer of neutralizing antibodies against an adenovirus serotype that is known in the art, such as Ad5, Ad2, Ad3, Ad4, Ad7 and Ad12, that the priming composition may comprise a vaccine based on such known adenovirus serotype, preferably Ad5, while the following composition (boost) should comprise another adenovirus serotype for which the individual also does not have significantly high levels of neutralizing antibodies in its serum. Of course, such following compositions may comprise an adenovirus vector selected from the same groups, as long as the first and second serotypes are different. If the human or animal has a significantly high titer to a second adenovirus (obtained through a general infection, or through active vaccination, or through a gene therapy application) the vector of choice for the first adenovirus serotype should be different from the second adenovirus serotype. "Significantly high" in this context means that such titers hamper the immune response elicited by the vector being applied, due to neutralization of the vector, hence, leading to the choice of a serotype that would not encounter titers of neutralizing antibodies that cause the immune response to be so low that a protective effect of the vaccine is not accomplished. Moreover, it is also to be understood that if a vaccine regimen requires more than two shots (prime/boost), but rather extra subsequent shots (prime/boost/boost, etc.), that this is also part of the present invention: the subsequent boost compositions should always (if they comprise an adenovirus vector) comprise an adenovirus vector that is different from the adenovirus vectors that have been used previously, at least as long the titers of neutralizing antibodies hamper the immune response required.

"Based on" or "derived from" as used herein means that a gene delivery vehicle, such as a recombinant adenovirus vector, originates from a certain wild-type adenovirus serotype as they have been recognized in the art. This means in general that certain parts of the genome are deleted to prevent replication (such as a deletion of the E1 region), but it also means that other mutations, deletions, naturally occurring chimeras, additions of nucleic acid, etc., may or may not be present in the recombinant adenoviral vector, as long as the capsid proteins towards which the neutralizing antibodies present in the serum from infected or vaccinated individuals are sufficiently different from one composition to the other. For example, if the backbone of the recombinant vector (this means generally all elements except the immunogenic and tropism-determining parts of the capsid) is identical between prime and boost compositions, this is still considered part of the invention, since the immune response towards such vectors having the same or similar backbone is still different.

In a preferred embodiment, the recombinant adenovirus vector of the first and second serotypes comprise essentially the same heterologous nucleic acid of interest. For vaccination purposes, it is generally required that the same antigen, or the nucleic acid encoding that antigen, is administered several times. "Essentially" as used herein refers to the idea that the antigen might be slightly different, but should still elicit an immune response that would fully (or at least sufficiently) protect the vaccinated individual from the pathogen. Generally, recombinant adenoviruses harbor the nucleic acid encoding the heterologous protein in the E1 region that is normally deleted from the genome.

In a preferred embodiment of the present invention, the heterologous nucleic acid encodes a viral antigen. More preferably, the viral antigen is an Ebola virus antigen, a measles virus antigen or a West Nile virus antigen. Such antigens can be obtained by sequencing the genomes of the wild-type strains of the different viruses, subcloning the nucleic acids encoding the antigenic determinants from such genomes, and cloning them into the adenoviral genomic sequence.

In another preferred embodiment, the viral antigen according to the invention is an antigen from a retrovirus such as Human Immunodeficiency Virus (HIV) or a Simian Immunodeficiency Virus (SIV). Also preferred are antigens derived from Feline Immunodeficiency Virus (FIV). More preferred are embodiments wherein the HIV, SIV or FIV antigen is gag, env, nef, pol and/or combinations thereof.

In another embodiment of the present invention, the heterologous nucleic acid present in the first and second serotype encodes a malaria antigen, such as the circumsporozoite (CS) or LSA-1 antigen from Plasmodium yoelii or Plasmodium falciparum, or functional equivalents or antigenic determinants/parts or derivatives thereof.

The present invention further provides a kit of parts comprising a priming composition and a boosting composition, both compositions comprising: a recombinant adenovirus vector; a heterologous nucleic acid of interest present in the vector; and a pharmaceutically acceptable carrier, wherein the recombinant adenovirus vector of the priming composition is from a different serotype than the recombinant adenovirus vector of the boosting composition. Preferably, the recombinant adenovirus vector of the priming composition is of a serotype selected from the group consisting of: Ad11, Ad26, Ad34, Ad35, Ad46 and Ad49. Also preferred is a kit of parts according to the invention, wherein the recombinant adenovirus vector of the boosting composition is of a serotype selected from the group consisting of: Ad11, Ad26, Ad34, Ad35, Ad46 and Ad49. It is still to be understood that other adenovirus serotypes may be comprised in the kit of parts according to the invention as long as the individual that is to be treated does not carry neutralizing antibodies to significantly high titers against that particular adenovirus serotype and as long as the second and first serotypes are different.

In a specific aspect of the present invention, the present invention also provides the use of a recombinant adenovirus vector derived from Ad11 for the preparation of a medicament in the treatment of a human or animal suffering from, or at risk of, a disease caused by a virus. Besides Ad35, Ad11 is a highly preferred serotype since most people in the world do not carry neutralizing antibodies against Ad11.

The present invention also provides a method for determining the titer of neutralizing antibodies in a human- or animal-derived blood sample, wherein the neutralizing antibodies are directed against a virus, comprising the steps of: obtaining a sample; culturing host cells; infecting the host cells with recombinant viral vectors comprising a transgene, in the presence of the sample; and determining the activity of a protein encoded by the transgene. Preferably, the determined activity is compared to a standard value. Even more preferred are methods wherein the transgene encodes a protein selected from the group consisting of: luciferase, Green Fluorescent Protein (GFP) and LacZ. The invention also provides a method for determining the titer of neutralizing antibodies in a blood sample, wherein the neutralizing antibodies are directed against a virus, comprising the steps of: obtaining a sample; culturing host cells; infecting the host cells with recombinant viral vectors in the presence of the sample; and determining the number of viral genomes per cell. Preferably, the number of viral genomes is compared to a standard value. Also preferred are methods, wherein the number of viral genomes per cell is determined by Quantitative-PCR (Q-PCR).

In a preferred embodiment of the invention, the methods are applied for determining the titer of neutralizing antibodies that are directed against an adenovirus. These antibodies might have been raised during previous vaccinations, prime and/or boost injections or through natural occurring infections. For determining the titer of neutralizing antibodies against an adenovirus, it is preferred to use a recombinant adenoviral vector in the methods of the present invention. The host cells used in the method of the present invention should be receptive for viral infection, preferably for adenoviral infection. A preferred cell line is the A549 cell line. Since titers may be very high, it is useful to make a curve of serial dilutions of the sample and to compare this with a standard curve.

It is very useful to know what titers of neutralizing antibodies are present in the serum of the individual to be treated. The methods known in the art are not considered accurate and useful for high throughput use. The method provided by the present invention ensures an easy way of determining the presence of neutralizing antibodies against all different adenovirus serotypes known in the art. This can then be followed by a regimen as provided by the present invention in which adenovirus vectors based on different serotypes are used in subsequent vaccine applications, such as prime/boosts. It is to be understood that the method is not limited to the transgenes as described in the present disclosure, or to the materials such as antibodies as described in the provided example. The method can be executed by using a kit of parts comprising a plate, a standard curve of diluted antibodies for possibly all serotypes known and possibly materials such as buffers and antisera for detection.

The present invention relates to methods and means to overcome at least part of the limitations of adenovirus-based vaccines. It has been recognized in the art that a series of vaccine applications would render a better and more potent immune response towards a certain immunogenic antigen. In the HIV vaccine studies (WO 01/02607; WO 02/22080), several regimens were tested, including the use of naked DNA encoding the antigen, as a priming composition, after which a boosting composition comprising a recombinant Ad5 vector was applied. Similar regimens were followed in obtaining a specific response against malaria antigens in other studies (WO 01/21201). It has been suggested in the art by several investigators to use different (low neutralized) serotypes of adenovirus in different rounds of vaccination and gene therapy applications (Parks et al. 1999; Mack et al. 1997; Hsu et al. 1992; Moffat et al. 2000; Kass-Eisler et al. 1996; Mastrangeli et al. 1996; Roy et al. 1998; Lubeck et al. 1997). However, the present invention realizes that such regimens are feasible for subsequent series of vaccinations, applying different antigens directed towards different pathogens, but using the same serotype in one prime/boost setting would still render the boost immune response weaker if the same serotype would have been used in the priming composition. Settings in which different serotypes are used in a prime/boost set-up for the same vaccine have not been suggested, nor have they been used in the art. The art describes either the use of the same serotype (mostly Ad5) in prime/boost set-ups or the use of different kinds of compositions like, for instance, naked DNA encoding the antigen, and a certain serotype (being mostly Ad5) as carrier of the DNA encoding the antigen in prime/boost settings. The inventors of the present invention now show for the first time that pre-existing immunity against a well-known and widely used vector as Ad5 can be overcome by using a recombinant adenoviral vector that is based on an adenovirus serotype that has a low prevalence in humans and that is not neutralized by antibodies in a large percentage of the worldwide population.

The present invention now provides methods and means for repeated vaccination applications, using different serotypes from the same subgroup. Moreover, the present invention discloses that, indeed, subjects that are immunized with Ad5-based vectors do not raise antibodies that are directed against a subsequent adenovirus serotype such as Ad35 or Ad11, while the titer of antibodies directed against the antigen (measles antigen H, or SIV-gag) is higher when an Ad5-Ad35 regimen is applied as compared to an Ad5-Ad5 regimen. This result strongly indicates that subsequent applications of an adenovirus of the same subgroup are not very efficient in vaccination, while subsequent applications of adenoviruses of different serotype are. These results also strongly suggest that an individual that has encountered an Ad5 infection in the past should preferably receive a priming vaccine composition comprising an adenovirus that is at least different from Ad5, while the boosting composition (if applicable) should also comprise yet another serotype that has never infected that particular individual.

The present invention also discloses that cross-neutralization is not an important issue. It was widely believed that a certain extent of cross-neutralization could or may prevent the use of different adenoviruses that are extremely similar. As disclosed herein, sera that harbor neutralizing antibodies against Ad35 do not, in most cases, contain neutralizing antibodies against Ad11 and vice versa. The present invention, therefore, makes it now possible to use prime/boost vaccination applications in which the priming composition comprises one adenovirus serotype, while the boosting composition comprises an adenovirus from another serotype. The present invention discloses which adenovirus serotypes are suitable for such settings. Preferred serotypes that are used in prime/boost applications according to the invention are the subgroup B serotypes Ad11 and Ad35, since these serotypes encounter neutralizing antibodies in only a very limited number of human sera, while humans that have encountered Ad11 in their lifetime most likely do not contain neutralizing antibodies against Ad35, and vice versa. The chance of encountering both serotypes in one lifetime seems to be extremely slim. The use of such adenovirus serotypes, of course, would render a vaccine that needs priming and boosting compositions for a proper immune response more potent than a vaccine that is built up from serotypes that are likely to encounter neutralizing antibodies, such as Ad5.

DETAILED DESCRIPTION OF THE INVENTION

Since it was found that many individuals in different populations carry neutralizing antibodies to many different serotypes, the serotype that was best suited to serve as an antigen carrier in vaccine applications or as a therapeutic/heterologous nucleic acid carrier for gene therapy applications was investigated. Only a few adenovirus serotypes encountered neutralizing antibodies in relatively few sera. The sera used in these studies were obtained from a large number of individuals from across the world, as described herein (see also, PCT International Publications WO 00/70071 and WO 02/40665 and in U.S. Pat. No. 6,492,169). Two adenoviruses of particular interest that encountered antibodies in only a few sera were Ad11 and Ad35, which are both serotypes from the B-subgroup. Generally, B-group adenovirus serotypes have a low tropism for liver cells and are capable of efficiently infecting dendritic cells in vitro. In vivo studies are hampered by the fact that mice do not seem to be a proper host for subgroup B adenoviruses. Nevertheless, Ad35 has been studied in great detail and several recombinant derivatives based on this particular adenovirus were generated (PCT International Publication WO 00/70071 and in U.S. Pat. No. 6,492,169). Since Ad5-complementing cell lines were not able to support the growth of high titers of recombinant Ad35- and Ad11-complementing cell lines, constructs and methods were also generated to provide all necessary elements to generate batches of recombinant adenoviruses based on B-subgroup adenoviruses such as Ad35 and Ad11 (U.S. Pat. No. 6,492,169). The sequences of the Ad11 and Ad35 genomes were obtained in full (WO 00/70071; WO 02/53759).

Clearly, if one wants to apply a certain adenovirus serotype in a vaccine composition, one should be certain that no or a low titer of neutralizing antibodies are present in the subject that is being treated. It is known in the art that different levels of anti-adenovirus antibodies circulate in human individuals (D'Ambrosio et al. 1982) that determine the level of therapeutic preparation that should be applied. To be able to determine in vitro the anti-adenovirus antibody titers in human sera, a validated adenovirus neutralization assay is required. Such a neutralization assay is also extremely useful to monitor vaccination efficiency in experimental and clinical settings and allows standardization. Thus, one determines the titer of neutralizing antibodies against the adenovirus serotype of interest. For this, the present invention also provides a method for determining such titers, allowing the proper adjustment of vaccine regimens suggested by the present invention. In situations that such determinations are not feasible or easily accessible, for instance in mass-vaccination programs in developing countries with poor medical infrastructure or in emergency situations, the chance of success is highest by using the serotypes disclosed herein since those serotypes are unlikely to encounter neutralizing activity in most humans.

Non-limiting examples are measles, rabies virus, Ebola virus, malaria, human Metapneumovirus, etc. Antigens that could be applied are, for instance, nucleic acids encoding measles F and H, SIV-gag, Circumsporozoite (CS) protein or LSA-1 from Plasmodium Yoelii and Plasmodium falciparum, HIV-gag/pol/nef/env, and HA and NA from Influenza virus.

It is to be understood that differences in the capsid of the adenoviral vector would enable one to use the same backbone virus for subsequent vaccinations and prime/boost set ups, provided that the capsid is modified by proteins that would normally be recognized by neutralizing antibodies. For instance, an Ad5 backbone carrying a fiber and/or hexon and/or a penton protein from Ad11 could be followed by a viral vector based on Ad5 (thus, another Ad5 backbone), wherein the capsid comprises a fiber and/or hexon and/or a penton protein from Ad35 and vice versa. Such recombinant vectors are also encompassed by the present invention. As long as the priming composition does not elicit an immune response that significantly hampers the infectivity of the boosting composition (as far as the adenoviral capsid proteins are concerned), then such prime and boost compositions are part of the invention.
 

Claim 1 of 5 Claims

1. An improvement in a method of delivering a nucleic acid sequence of interest to a subject using an adenoviral delivery vehicle, the method comprising: administering to the subject a recombinant adenovirus vector of Ad35 serotype having a nucleic acid sequence encoding a malaria antigen; and administering to the subject, subsequent to administering the recombinant adenovirus vector of the Ad35 serotype, a recombinant adenovirus vector of Ad5 serotype having a nucleic acid sequence encoding essentially the same malaria antigen.

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