Title:  Replication defective HIV vaccine

United States Patent:  6,500,623

Issued:  December 31, 2002

Inventors:  Tung; Frank Yao Tsung (Lawrenceville, GA)

Assignee:  Genecure LLP (Atlanta, GA)

Appl. No.:  700304

Filed:  November 9, 2000

PCT Filed:  November 18, 1999

PCT NO:  PCT/US99/10523

371 Date:  May 12, 1999

A replication-defective HIV particle pseudotyped with vesicular stomatitis virus G protein (VSV-G). The pol gene of the HIV genome in the particle is modified to inactivate the pol reverse transcriptase and protcase activity. This pseudotyped HIV particle can infect many cell types, including human and simian cells, and only undergoes one round of replication. Furthermore, a virus-specific immune response can be detected in mice immunized with the VSV-G pseudotyped replication-defective HIV.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is a method for producing a replication-defective HIV virus particle, a virus particle prepared according to the method and to a vaccine including the virus particles. The virus particles exhibit increased ability to produce antigen, expanded cell tropism and the ability to elicit strong humoral and cellular immunity. As a result of the expanded tropism, the virus particles can infect rhesus monkeys in a single cycle of replication. Thus, the protective immunity can be further evaluated in rhesus monkeys by challenge with an SIV/HIV chimera virus (SHIV).

The method of the present invention is a method for preparing a replication-defective retroviral particle including the steps of 1) preparing a DNA molecule which includes a complete retroviral genome; 2) modifying a portion of the pol gene including the protease and reverse transcriptase coding regions, to the extent that the remaining pol gene cannot produce a protease and reverse transcriptase capable of functioning in replication of the genome (the pol^- construct); 3) transferring the pol^- construct into a suitable host cell prior to, along with or after the step of transferring a pseudotyping construct and a packaging construct into the host cell or a predecessor cell or a progeny cell thereof; 4) growing the host cell under conditions suitable for expression of the constructs and for production of the replication-defective retrovirus particle; and 5) collecting the virus particles.

The DNA molecule can be prepared from any DNA containing a complete retroviral genome. In theory, a genome suitable for preparing the pol^- construct is any substantially complete retroviral genome which is intact, and encodes and is capable of expressing all viral proteins other than those inactivated by the described modification of the pol gene, whether or not one or more of the viral proteins are modified or unmodified as compared to the wild-type retrovirus. It later may be found that expression of certain viral proteins (non-pol proteins) are unnecessary to elicit protective immunity. A genome having these viral proteins modified or deleted could be a suitable genome for modification according to the methods of the present invention.

According to the present invention, the pol gene of the retroviral genome is modified to cause the protease and the reverse transcriptase functions of the pol gene to lose their viral replication-associated functionality. This loss of function can be induced by a variety of methods, including: by insertions of amino acids, by replacement of amino acids and by deletions of amino acids. Preferably, a significant portion of the pol gene is deleted, as in the example, below.

The packaging construct can be any construct which provides in trans all functions missing from the pol^- construct to allow packaging of the pol^- genome into a replication-defective viral particle which, when transduced, can achieve one round of viral replication. The packaging construct will at least encode a complete and functional reverse transcriptase for incorporation into the replication-defective virus particles and to copy the pol^- genome into DNA once the replication-defective virus particle infects a target cell.

The pseudotyping construct is, preferably, a recombinant construct for expressing the VSV-G protein. Expression of the protein is under transcriptional control of a suitable promoter. In the example below, the common constitutive CMV promoter is used. Other promoters, whether inducible or constitutive, are suitable so long as the construct encodes the VSV-G protein. Alternative pseudotyping proteins may be encoded by and expressed by the pseudotyping construct. An example of an alternative tropism-modifying protein is the Murine Leukemia Virus (MLV) envelope protein. The appropriate pseudotyping protein can be selected according to the application of the vaccine. The VSV-G protein is an appropriate pseudotyping protein for human or simian applications. Different pseudotyping proteins are appropriate for specific veterinary applications, such as if the virus particle is to serve as a Feline Leukemia Virus vaccine.

The host cell for production of replication-defective retrovirus particles can be virtually any mammalian cell so long as the cell is capable of producing infectious virus particles according to the methods described herein. In the examples described below, the preferred, and widely available, 293 cells (human transformed embryonal kidney cells; ATCC CRL-1573, American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md., U.S.A. 20852) are used as host cells to produce the HIV pol^- particles. This cell line is particularly useful due to its ease of growth, its rapid growth characteristics and its ease of transformation by standard protocols. Cells with similar characteristics are particularly suited as host cells for preparation of the replication-defective retrovirus particles.

The example provided below utilizes a three component transfection to produce virus particles. In the example, a first plasmid contains a DNA fragment corresponding to the pol^- HIV genome ("the HIVpol^- construct"). A second plasmid provides suitable functions in trans which are necessary for production of complete retroviral virus particles (the packaging construct). A third plasmid provides the genetic material for expression of the VSV-G protein (a VSV G-encoding or pseudotyping construct). Transfection of the host cell can be accomplished by a variety of methods, including, without limitation: calcium phosphate, liposome and electroporation methods. Virtually any transfection method is suitable, so long as the functionality of each construct is maintained. Further, transfer of the constructs into the host cell can be accomplished simultaneously, or in any desired order.

It should be noted that, although the examples herein utilize plasmid DNA to provide the three constructs, other equivalent vector systems may be utilized to transfer the constructs to the host cell, including, for example and without limitation: linear DNA fragments, plasmids and other recombinant DNA vectors and recombinant virus genomes or particles. In the case of recombinant virus particles, the method for transferring the constructs to the host cell is by transduction with the virus particles. For instance, virus particles including the transfer vectors described in Naldini et al., Science 272 (1996): 263-267 would be suitable for delivery of one or both of the packaging construct and the VSV-G-encoding construct. Further, the replication-defective HIV virus particles of the present invention can be used as a suitable source of the particles to transduce the HIV pol^- construct.

Further, a host cell may be established having one or both of the packaging construct and the pseudotyping construct integrated permanently into the host cell DNA. Packaging cell lines for retroviruses are well known in the art. (Delwart, E. L. et al., Aids Research and Human Retroviruses 8 (1992): 1669-1677). The integrated packaging construct and pseudotyping construct may be under the control of a constitutive promoter such as the cytomegalovirus (CMV) promoter, as described in the examples, or under the control of an inducible promoter, allowing exact control of virus production and preventing interference with host cell growth during passage of the packaging cell line. Transfection inefficiencies are lessened by the integration of one or more of the constructs into the host cell genome.

Virus particles prepared according to the above-described method include all components of a wild-type virus particle with the exception that the particles include, at least, the above-described modified (pol^- retroviral) genome, as opposed to the wild-type RNA genome. Further, the virus particles preferably include the VSV-G (pseudotyping) protein in order to alter the tropism of the virus particles. Virus particles are prepared according to the above-described methods and are, then, collected by standard methodology. In the example provided herein, the virus particles are purified by sucrose gradient ultracentrifugation. Other methods for purification can be employed, such as affinity chromatography, so long as the method enables concentrate of the titer of the infectious replication-defective virus particles.

In use, the replication-defective HIV virus particles are formulated as a vaccine. The only limitation is that the vaccine includes, in addition to the virus particles a pharmaceutically or veterinarilly acceptable excipient, which can include, without limitation: virus stabilizing compounds (i.e., sugars such as sucrose or other saccharides or polysaccharides); buffer systems (i.e., TNE or its equivalents); antibiotic compounds (i.e., bacteriocides, bacteriostatic agents and fungicides); viscosity enhancing agents (i.e., polyethylene glycol); polymeric materials (i.e., polymers, copolymers, block polymers and cross-linked polymers) for use as viscosity enhancing agents, as tackifying agents or as a matrix or reservoir for harboring the particles; tackifying agents which promote adhesion of the vaccine mixture to the skin or mucosa; viral transduction-enhancing agents; penetration enhancing agents; flavoring agents; coloring agents; adsorption enhancing agents and adjuvants. For example, as shown below, mice were immunized with virus particles in tissue culture media, which served as a suitable excipient.

The vaccine can be prepared in many forms (and with an excipient suitable for each form), such as, without limitation: an oral liquid for either swallowing or for application to a surface of the mouth, including sublingual or buccal application; an oral capsule or tablet; a liquid for parenteral injection; a cream or ointment for topical application; a lyophilized product for reconstitution or for vaccination by scarification; a transdermal preparation; and a liquid or a suppository for rectal or intravaginal application. A "lyophilized product" is understood to include virus particles freeze-dried, lyophilized or vitrified in the presence of a suitable buffer system, such as TNE, and a suitable carrier, such as a sugar (i.e., trehalose or sucrose), a polysaccharide, or other compounds suitable for stabilizing labile biological compounds.

The vaccine can also contain two or more pol^- variants of the same retrovirus. Therefore, a subject can be immunized once or multiple times with two or more variants of the virus which reflect common antigenic variations of HIV. Production of a variant virus would be straight-forward once the nucleotide sequence of the variant HIV is known. Appropriate modification of DNA encoding the replication-defective genome would be accomplished through well known cloning and mutagenesis methods.

Claim 1 of 23 Claims

I claim:

1. A method for producing replication-defective retrovirus particles, comprising the steps of:

a. providing a DNA molecule which includes a complete retroviral genome;

b. modifying a portion of the pol gene of the DNA molecule including the protease and reverse transcriptase activity coding regions to the extent that the remaining pol gene cannot produce a protease and reverse transcriptase functioning in replication of the genome, thereby forming a pol^- construct;

c. transferring the pol^- construct into a suitable host cell;

d. prior to, during or after the step of transferring the pol^- construct into the host cell, transferring into the host cell or a predecessor cell or a progeny cell thereof, a pseudotyping construct and a packaging construct;

e. growing the host cell under conditions suitable for expression of the constructs and for production of the replication-defective retrovirus particles; and

f. collecting the replication-defective virus particles.
 

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