The present invention provides vaccine compositions of attenuated human rotavirus. More particularly, the attenuated human rotavirus is produced by cold passage and thus contains attenuating mutations which produce virus having a cold-adapted (ca) and temperature sensitive (ts) phenotype. The attenuated strains are used in methods for stimulating the immune system of an individual to induce protection against human rotavirus by administration of the ca attenuated rotavirus.

SUMMARY OF THE INVENTION

The present invention provides vaccine compositions of attenuated human rotavirus. The attenuated rotavirus is provided in an amount sufficient to induce an immune response in a human host, optionally in conjunction with a physiologically acceptable carrier and an adjuvant to enhance the immune response of the host. In certain embodiments the attenuated rotavirus is a derivative of rotavirus which has been incompletely attenuated by introduction of mutations which produce virus having a temperature sensitive (ts), cold-adapted (ca) or other growth restricting or attenuating mutation. The attenuated virus of the invention belongs to serogroup A or other serogroups, and virus from the serogroup(s) and/or multiple serotypes within any of these serogroup(s) may be combined in vaccine formulations for more comprehensive coverage against prevalent rotavirus infections. A preferred embodiment is attenuated virus of serogroup A having the serotype VP4 1A, VP7 1, such as cold adapted (e.g., 30.degree. C.) strain D or WA. The vaccine will typically be formulated in a dose from about 10.sup.3 to about 10.sup.6 plaque-forming units (PFU) or more for maximal efficacy.

In other embodiments the invention provides methods for stimulating the immune system of an individual to induce protection against human rotavirus. These methods comprise administering to the individual an immunologically sufficient amount of human rotavirus which has been attenuated by introducing mutations that specify the ca phenotype and typically the ts phenotype. In view of the potentially serious consequences of rotavirus infection in infants and young children, and the elderly, these individuals will typically benefit most from immunization according to the present methods. In most instances the attenuated rotavirus is administered to the alimentary tract of the individual, preferably by liquid suspension or other solution.

In yet other embodiments the invention provides pure cultures of attenuated human rotavirus, wherein the virus has been attenuated by the introduction of two or more ca and ts mutations. The attenuated virus is capable of eliciting a protective immune response in an infected human host yet is sufficiently attenuated so as to not cause unacceptable symptoms of severe gastrointestinal disease in the immunized host. The attenuated human rotavirus may be present in a cell culture supernatant, recovered from the culture, or partially or completely purified. The virus may also be lyophilized, and can be combined with a variety of other components for storage or delivery to a host, as desired.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides attenuated rotavirus suitable for vaccine use in humans. The rotavirus described herein is produced by introducing mutations in wild-type or incompletely attenuated strains of human rotaviruses. The mutations are introduced in the strains during virus growth in cell cultures by selection of virus that has acquired the ability to grow efficiently at suboptimal temperature in order to introduce growth mutations.

Thus, the vaccine of the invention comprises the attenuated rotavirus and a physiologically acceptable carrier. The vaccine is administered in an immunogenically sufficient amount to an individual in need of immunological protection against rotavirus, such as, e.g., an infant, child or adult. The vaccine elicits the production of an immune response that is protective against serious rotavirus disease, such as severe diarrhea, when the individual is subsequently exposed to or infected with a wild-type human rotavirus. As the attenuated virus of the vaccine infects the host alimentary tract, some mild disease may occur as a result of the vaccination and possibly following subsequent infection by wild-type virus, but typically the vaccine will not cause clinically relevant disease in the vaccine when the virus is sufficiently attenuated and administered in an amount below that which causes such symptoms. Following vaccination, however, there are detectable levels of host engendered serum and secretory antibodies which are capable of neutralizing homologous (of the same serotypes) wild-type virus in vitro and in vivo. To achieve protection against serotypes which are prevalent epidemiologically, i.e., against serotypes VP7 1, VP7 2, VP7 3, and VP7 4, a cocktail of strains containing the different serotypes can be employed in the vaccine composition, up to and including a quadrivalent vaccine comprising each of the VP7 1 4 serotypes. Additional strains may be added (or omitted) as necessary to provide adequate immunological coverage for the strain prevalent in a particular geographic area.

The attenuated virus which is a component of the vaccine is in an isolated and typically purified form. By isolated is meant to refer to attenuated modified rotavirus which is in other than the native environment of wild-type virus, such as the gastrointestinal tract of an infected individual. More generally, isolated is meant to include the attenuated virus as a heterologous component of a cell culture or other system. For example, attenuated rotavirus of the invention may be produced by an infected cell culture, separated from the cell culture and added to a stabilizer, and may contain other non-naturally occurring rotaviruses to form a multivalent vaccine as described above.

The attenuated human rotavirus of the present invention exhibits a substantial diminution of virulence when compared to wild-type virus that is circulating naturally in infected humans. The attenuated human rotavirus is not so attenuated however, that all symptoms of infection will necessarily be absent in all immunized individuals. The attenuated virus is capable of replication, particularly in the gastrointestinal tract of the host. In some instances the attenuated virus may still be capable of dissemination to unvaccinated individuals. However, its virulence is sufficiently abrogated such that severe lower gastrointestinal tract infections or disease in the vaccinated or incidental host do not occur.

The level of attenuation may be determined by, for example, quantifying the amount of virus found in the gastrointestinal tract of an immunized host and comparing the amount to that produced by wild-type rotavirus or other attenuated rotaviruses which have been evaluated as candidate vaccine strains, such as those of rhesus monkey or bovine rotavirus strains. For example, the attenuated virus of the invention will have a greater degree of restriction of replication in the gastrointestinal tract of an infected host, such as an infant, child or adult, compared to the levels of replication of wild-type virus, e.g., 100- to 1000-fold less. The attenuated viruses of the invention must be sufficiently infectious and immunogenic in humans to confer effective protection in vaccinated individuals. Methods for determining levels of rotavirus in the gastrointestinal tract of an immunized host are well known in the literature and are described in the Example section below. For example, specimens obtained by stool sample or swab are suspended in buffer and virus quantified in tissue culture or other by laboratory procedure. See, for example, Kapikian et al., in Lennette and Schmidt (ed.), Diagnostic Procedures for Viral. Rickettsial. and Chlamydial Infections, 5th ed., p. 927 995, Amer. Public Health Assoc., Washington, D.C. (1979), which is incorporated herein by reference.

To produce a satisfactorily attenuated human rotavirus of the present invention mutations are introduced into a parental wild-type viral strain which has been isolated from a clinical sample or other source. For rotavirus of serogroup A, the parental wild-type virus is preferably serotypes VP4 1A or 1B. With regard to VP7 serotype, the parental wild-type virus is preferably serotype VP7 1, VP7 2, VP7 3 or VP7 4, although other parental serotypes may be employed as necessary.

Attenuated human rotavirus mutants can also be produced by biologically cloning wild-type virus in an acceptable cell substrate and developing cold-adapted mutants thereof, and by subjecting the virus to mutagenic pressures, e.g., chemical mutagenesis or even neutralizing monoclonal antibodies, to produce ts mutants or other attenuating mutants. The various selection techniques may also be combined to produce the attenuated mutants of human rotavirus which are useful as described herein.

According to the present invention the ca mutants are produced by subjecting the parental wild-type or even a partially attenuated virus strain to passage in cell culture at progressively lower, suboptimal temperatures. For example, whereas wild-type virus is typically cultivated at about 37 39.degree. C., the partially attenuated mutants are produced by repeated passage in cell cultures (e.g., African green monkey kidney cells, AGMK) at suboptimal temperatures, e.g., from about 30.degree. C. down to about 26.degree. C. These mutants demonstrate evidence of cold adaptation (ca) by increased efficiency of growth at the lower temperature compared to the wild-type (or partially attenuated) parental virus, and, in the process of becoming cold-adapted, typically demonstrate ts mutations, with shutoff temperature or plaque formation in cell cultures ranging from near 36.degree. C. or 37.degree. C., up to about 38.degree. C. or even 39.degree. C. Thus, in one method of the present invention the ca attenuated mutant human rotavirus strains are adapted to efficient growth at a lower temperature by passage in AGMK cells, down to a temperature of about 24 26.degree. C., preferably 26 30.degree. C. This selection of rotavirus mutant during cold-passage substantially eliminates significant virulence in the strains as compared to the virulent wild-type or partially attenuated parent strain. The level of temperature sensitivity of viral replication of the attenuated rotavirus of the invention is determined by comparing its replication at a permissive temperature with that at several restrictive temperatures. The lowest temperature at which the replication of the virus is reduced 100-fold or more in comparison with its replication at the permissive temperature is termed the shutoff temperature. Typically the replication and virulence of rotavirus correlate with the shutoff temperature. Replication of mutants with a shutoff temperature of 39.degree. C. is moderately restricted, whereas mutants with a shutoff of 36 38.degree. C. replicate even less well.

In addition to the criteria of attenuation and immunogenicity, the properties of the attenuated human rotaviruses which are selected must also be as stable as possible so that the desired attributes are maintained. While some genetic instability may be tolerated, virus useful in the vaccines of the present invention must retain the properties of attenuation, restriction of replication in the immunized host, and the ability to effectively elicit the production of an immune response in the vaccinee that is sufficient to confer protection against serious disease caused by subsequent infection by wild-type virus. It may be desirable to use two or more additional mutations or selection criteria, e.g., ca and ts, in one viral strain to more effectively attenuate the virus while retaining the ability to stimulate a protective immune response and in some instances to expand the protection afforded by multiple modifications, e.g., induce protection against different viral strains or subgroups, or protection of a different immunologic basis, e.g., secretory versus serum immunoglobulins, cellular immunity, and the like.

The attenuated virus of the invention may be propagated in a number of cell lines which permit rotavirus growth. Preferred cell lines for propagation of attenuated human rotavirus for vaccine use include AGMK and Vero cells. Cells are typically inoculated with virus at a multiplicity of infection ranging from about 0.1 to 1.0 or more, and are cultivated under conditions permissive to replication of the virus, e.g., at about 28.degree. 35.degree. C. and for about 3 5 days, or as long as necessary for virus to reach an adequate titer. Virus is removed from cell culture and separated from cellular components, typically by well known clarification procedures, e.g., centrifugation, and may be further purified as desired using procedures well known to those in the art.

Human rotavirus which has been attenuated as described herein can be tested in in vitro and in vivo models to confirm adequate attenuation and immunogenicity for vaccine use. In in vitro assays the modified virus is tested for the ability to (i) replicate at a suboptimal temperature when compared to the parental strain, and (ii) exhibit temperature sensitivity of plaque formation when compared to the parental strain. Mutant human rotaviruses are further tested in animal models of rotavirus infection. Animal models such as the gnotobiotic piglet are described in the Example section hereinbelow.

For vaccine use, the attenuated virus of the invention can be used directly in vaccine formulations, or lyophilized, as desired, using lyophilization protocols well known to the artisan, and then hydrated immediately prior to use. Lyophilized virus will typically be maintained at about 4.degree. C. When ready for use the lyophilized virus is reconstituted in a stabilizing solution, e.g., citrate buffer, saline or the like, with or without adjuvant, as further described below.

Thus, rotavirus vaccines of the invention contain as an active ingredient an immunogenically effective amount of an attenuated cold adapted human rotavirus as described herein. The attenuated virus may be introduced into a host, particularly humans, with a physiologically acceptable carrier and/or adjuvant. Useful carriers are well known in the art, and include, e.g., citrate-bicarbonate buffer, buffered water, 0.4% saline, and the like. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration, as mentioned above. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.

Upon immunization with a attenuated rotavirus composition as described herein, via oral or parenteral route, the immune system of the host responds to the vaccine by producing antibodies, both secretory and serum, specific for rotavirus proteins. As a result of the vaccination the host becomes at least partially or completely immune to rotavirus infection, or if infection occurs the host is resistant to developing moderate or severe rotaviral disease, particularly of the lower gastrointestinal tract.

The vaccine compositions containing the attenuated human rotavirus of the invention are administered to a person susceptible to or otherwise at risk of rotavirus disease to enhance the individual's own immune response capabilities. Such an amount is defined to be a "immunogenically effective dose." In this use, the precise amounts again depend on the patient's age, state of health and weight, the mode of administration, the nature of the formulation, etc., but generally range from about 10.sup.3 to about 10.sup.6 plaque forming units (PFU) or more of virus per patient, more commonly from about 10.sup.4 to 10.sup.5 PFU virus per patient. In any event, the vaccine formulations should provide a quantity of attenuated rotavirus of the invention sufficient to effectively protect the patient against serious or life-threatening rotavirus infection.

The attenuated rotavirus of the invention of one particular rotavirus serotype can be combined with attenuated viruses of the other serotypes to achieve protection against multiple serotypes of human rotaviruses. Typically the different modified viruses will be in admixture and thus administered simultaneously, but in some instances they may be administered separately.

In some instances it may be desirable to combine the attenuated rotavirus vaccines of the invention with vaccines which induce protective responses to other agents, particularly other gastrointestinal viruses. For example, the attenuated virus vaccine of the present invention can be administered simultaneously (but typically separately) or sequentially with a Norwalk virus vaccine.

Single or multiple administrations of the vaccine compositions of the invention can be carried out. In neonates and infants, multiple administration may be required to elicit sufficient levels of immunity. Administration should begin within the first 2 4 months of life, and continue at intervals such as at one to two months or more after the initial immunization, as necessary to induce and maintain sufficient levels of protection against native (wild-type) rotavirus infection. Similarly, adults who are particularly susceptible to repeated or serious rotavirus infection, such as, for example, health care workers, day care workers, family members of young children, the elderly, etc. may require multiple immunizations to establish and/or maintain protective immune responses. Levels of induced immunity can be monitored by measuring amounts of neutralizing antibodies in serum and secretions, and dosages adjusted or vaccinations repeated as necessary to maintain desired levels of protection.
 

Claim 1 of 49 Claims

1. A human rotavirus composition which comprises a cloned, genetically stable, live, cold adapted, temperature sensitive, attenuated human rotavirus of serogroup A wherein the attenuated rotavirus displays increased replication efficiency as compared to wild-type rotavirus when cultured in vitro at a temperature between 26 to 30.degree. C. and the attenuated rotavirus displays a decreased replication efficiency as compared to wild-type rotavirus when cultured in vitro at a temperature between 36 to 39.degree. C., while retaining the ability to induce an immune response in a human host without causing a severe lower gastrointestinal infection.

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