Title:  Formulations of recombinant papillomavirus vaccines

United States Patent:  6,280,740

Assignee:  Merck & Co., Inc. (Rahway, NJ)

Filed:  January 14, 2000

Vaccine formulations comprising recombinant early (E) and late (L) proteins of papillomavirus and oxidized mannan as well as methods of making and using the formulations are provided.

DETAILED DESCRIPTION OF THE INVENTION

Vaccine formulations comprising recombinant early (E) and late (L) proteins of papillomavirus and oxidized mannan as well as methods of making and using the formulations are provided.

The current invention identifies a new technology that may be useful for eliciting potent cell-mediated and humoral immune responses to a candidate protein antigen for vaccine development.

Aluminum hydroxide generally elicits potent antibody responses to the candidate antigen and little if any cell-mediated immune responses.

Apostolopoulos et al. demonstrated the induction of strong cell-mediated immune responses to mucin 1 antigen (MUC 1) in mice when the animals were immunized with MUC1 antigen conjugated to oxidized mannan (ox-mannan). However, the utility of ox-mannan as an adjuvant or immunomodulator in vaccine development to infectious agents has not been evaluated.

Antigens conjugated to oxidized or reduced mannan are likely to elicit strong cell-mediated and humoral immune responses to the candidate antigen. Alum has been used as an adjuvant that elicits good humoral immune responses and little if any cell-mediated immune responses.

A vaccine against human papillomavirus that may require an adjuvant capable of eliciting both humoral and cell-mediated immune responses to papillomavirus antigens. In this report, we describe the utility of ox-mannan in eliciting protective immune responses to infectious agents in cottontail rabbit papillomavirus model.

In this study we evaluated the utility of oxidized mannan as a carrier for vaccine development and immunotherapy, we conjugated oxidized mannan to, E. coli expressed recombinant CRPV early proteins (E-proteins) antigens and evaluated their efficacy in containing pre-existing infection.

Methods, compositions and processes for the prevention, characterization, detection and treatment of papifiomavirus (PV) infection are provided. The methods are based on the production of recombinant L1 or recombinant L2 or recombinant L1 and L2 proteins in yeast. The recombinant proteins are capable of mimicking the conformational neutralizing epitopes of native PV. The recombinant L1 or L1 and L2 proteins may also be capable of forming virus-like particles (VLP). The compositions of the invention include but are not limited to recombinant DNA molecules encoding the L1 or L2 or L1 and L2 proteins, the recombinant proteins either alone or in combination with other recombinant proteins, VLP comprised of at least one recombinant protein, fragments of the recombinant proteins, pharmaceutical compositions comprising the recombinant proteins, vaccine compositions comprising the recombinant proteins, antibodies to the recombinant proteins or VLP, immunogenic compositions comprising at least one recombinant protein, and diagnostic kits comprising the recombinant DNA molecules or the recombinant proteins. The processes of the present invention include but are not limited to the process of producing a recombinant protein comprising the transformation of an appropriate yeast host cell with a recombinant DNA molecule, cultivating the transformed yeast under conditions that permit the expression of the DNA encoding the recombinant protein, and purifying the recombinant protein. The processes of the present invention also include the administration of the recombinant protein, recombinant protein compositions or VLP to an animal, including but not limited to humans. Appropriate host cells include, but are not limited yeast strains of the genera Saccharomyces, Pichia, ulyermyces, Schizosaccharomyces and Hansenula.

Immunological studies in animals have shown that the production of neutralizing antibodies to papillomavirus capsid proteins prevents infection with the homologous virus. The development of effective papillomavirus vaccines has been slowed by difficulties associated with the cultivation of papillomaviruses in vitro. The development of an effective HPV vaccine has been particularly slowed by the absence of a suitable animal model.

Neutralization of papillomavirus by antibodies appears to be type-specific and dependent upon conformational epitopes on the surface of the virus.

Pharmaceutically useful compositions comprising the proteins or VLP may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the protein or VLP. Such compositions may contain proteins or VLP derived from more than one type of HPV.

Therapeutic or diagnostic compositions of the invention are administered to an individual in amounts sufficient to treat or diagnose PV infections. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration. Generally, the compositions will be administered in dosages ranging from about 1 .mu.g to about 250 .mu.g.

The pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral, mucosal, and intramuscular.

The vaccines of the invention comprise recombinant proteins or VLP that contain the antigenic determinants necessary to induce the formation of neutralizing antibodies in the host. Such vaccines are also safe enough to be administered without danger of clinical infection; do not have toxic side effects; can be administered by an effective route; are stable; and are compatible with vaccine carriers.

The vaccines may be administered by a variety of routes, such as orally, parenterally, subcutaneously, mucosally or intramuscularly. The dosage administered may vary with the condition, sex, weight, and age of the individual; the route of administration; and the type PV of the vaccine. The vaccine may be used in dosage forms such as capsules, suspensions, elixirs, or liquid solutions. The vaccine may be formulated with an immunologically acceptable carrier.

The vaccines are administered in therapeutically effective amounts, that is, in amounts sufficient to generate a immunologically protective response. The therapeutically effective amount may vary according to the type of PV. The vaccine may be administered in single or multiple doses.

The methods of the present invention make possible the formulation of subviral vaccines for preventing PV infection. Using the methods, either monovalent or multivalent PV vaccines may be made. For example, a monovalent HPV type 16 vaccine may be made by formulating recombinant HPV 16 L1 protein or L2 protein or L1 and L2 proteins. Alternatively, a multivalent HPV vaccine may be formulated by mixing L1 or L2 or L1 and L2 proteins or VLP from different HPV types.

The recombinant proteins and VLP of the present invention may be used in the formulation of immunogenic compositions. Such compositions, when introduced into a suitable host, are capable of inducing an immunologic response in the host.

The recombinant proteins and VLP may be used to generate antibodies. The term "antibody" as used herein includes both polyclonal and monoclonal antibodies, as well as fragments thereof, such as, Fv, Fab and F(ab)2 fragments that are capable of binding antigen or hapten.

The recombinant proteins, VLP and antibodies of the present invention may be used to serotype HPV infection and HPV screening. The recombinant proteins, VLP and antibodies lend themselves to the formulation of kits suitable for the detection and serotyping of HPV. Such a kit would comprise a compartmentalized carrier suitable to hold in close confinement at least one container. The carrier would further comprise reagents such as recombinant HPV protein or VLP or anti-HPV antibodies suitable for detecting a variety of HPV types. The carrier may also contain means for detection such as labeled antigen or enzyme substrates or the like.

The recombinant proteins and VLP of the present invention are also useful as molecular weight and molecular size markers.

Claim 1 of 3 Claims

What is claimed:

1. A method of preventing infection of an animal by a papillomavirus comprising administration of a mixture comprising recombinant papillomavirus virus-like particles, recombinant papillomavirus E proteins and oxidized mannan (ox-mannan) to the animal.

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