Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1995-07-24
2002-08-13
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S173300, C424S184100, C424S188100, C424S204100, C530S324000, C530S325000
Reexamination Certificate
active
06432675
ABSTRACT:
BACKGROUND OF THE INVENTION
Host defense is a hallmark of vertebrate immune systems. To this end, antibodies perform numerous functions in the defense against pathogens. For instance, antibodies can neutralize a biologically active molecule, induce the complement pathway, stimulate phagocytosis (opsonization), or participate in antibody-dependent cell-mediated cytotoxicity (ADCC).
If the antibody binds to a site critical for the biological function of a molecule, the activity of the molecule can be neutralized. In this way, specific antibodies can block the binding of a virus or a protozoan to the surface of a cell. Similarly, bacterial and other types of toxins can be bound and neutralized by appropriate antibodies. Moreover, regardless of whether a bound antibody neutralizes its target, the resulting antigen-antibody complex can interact with other defense mechanisms, resulting in destruction and/or clearance of the antigen.
Parasites have evolved an array of mechanisms for avoiding an immune response. Antigenic variation is perhaps the most studied of the evasion strategies, in part because such variation makes vaccine development especially difficult. Generally, here are two ways in which antigenic variation can occur: antigenic drift and antigenic shift. Antigenic drift is relatively straightforward. Point mutations arise in genes that encode pathogen antigens, altering some of the epitopes on the antigen such that host immunologic memory to the original antigen is not triggered by the mutant. As immunity to one variant will not necessarily ensure immunity to others, accumulation of such point mutations in a pathogen population can result in multiple infections in the same host. Antigenic drift has been found in most pathogens (including viruses, bacteria, and protozoa), its importance varying among individual species.
Many viruses are capable of great antigenic variation, and large numbers of serologically distinct strains of these viruses have been identified. As a result, a particular strain of a virus becomes insusceptible to immunity generated in the population by previous infection or vaccination. For instance, the progress of HIV-1 vaccine development has been impeded by the amino acid sequence variability among different isolates of HIV-1. This variability is particularly high in the external envelope protein gp120, which is the primary target for antibodies that neutralize virus infectivity (Robey et al. (1986)
PNAS
83:7023; Putney et al. (1986)
Science
234:1392; and Rusche et al. (1987)
PNAS
84:6924, incorporated by reference herein). Studies in humans and mice have revealed a small region of gp120, termed the V3 loop or principal neutralizing determinant (PND), comprising bout 35 residues between two invariant, disulfide-crosslinked cysteines (Cys-303 to Cys-338: HIV-1 nomenclature of Takahashi et al. (1992)
Science
255:333), that evokes the major neutralizing antibodies to the virus (Palker et al. (1988)
PNAS
85:1932; Rusche et al. (1988)
PNAS
85:3198 and Goudsmit et al. (1988)
PNAS
85:4478). While this same region is one of the most variable in sequence among different clonal isolates (Takahashi et al. (1992)
Science
255:333), analysis of the amino acid sequences of this domain revealed conservation to better than 80-percent of the amino acids in 9 out of 14 positions in the central portion of the V3 loop, suggesting that there are constraints on the V3 loop variability (LaRosa et al. (1990)
Science
249:932). However, because of this variability, neutralizing antibodies elicited by the PND from one isolate generally do not neutralize isolates with PND's of different amino acid sequence.
Likewise, attempts to control influenza by vaccination has so far been of limited success and are hindered by continual changes in the major surface antigen of influenza viruses, the hemagglutinin (HA) and neuraminidase (NA), against which neutralizing antibodies are primarily directed (Caton et al.(1982)
Cell
31:417; Cox et al. (1983)
Bulletin W.H.O.
61:143; Eckert, E. A. (1972)
J. Virology
11:183). The influenza viruses have the ability to undergo a high degree of antigenic variation within a short period of time. It is this property of the virus that has made it difficult to control the seasonal outbreaks of influenza throughout the human and animal populations.
Through seralogic and sequencing studies, two types of antigenic variations have been demonstrated in influenza A viruses. Antigenic shift occurs primarily when either HA or NA, or both, are replaced in a new viral strain with a new antigenically novel HA or NA. The occurrence of new subtypes created by antigenic shift usually results in pandemics of infection.
Antigenic drift occurs in influenza viruses of a given subtype. Amino acid and nucleotide sequence analysis suggests that antigenic drift occurs through a series of sequential mutations, resulting in amino acid changes in the polypeptide and differences in the antigenicity of the virus. The accumulation of several mutations via antigenic drift eventually results in a subtype able to evade the immune response of a wide number of subjects previously exposed to a similar subtype. In fact, similar new variants have been selected experimentally by passage of viruses in the presence of small amounts of antibodies in mice or chick embryos. Antigenic drift gives rise to less serious outbreak, or epidemics, of infection. Antigenic drift has also been observed in influenza B viruses.
Purified antigen vaccines directed against the hepatitis B virus currently in clinical trials generally consists of antigens of a single viral subtype. The rational for this decision has been that both S region and pre-S(2) region-specific antibodies, shown to be somewhat effective in neutralizing the hepatitis B virus, are primarily group-specific. However, this logic does not take into consideration the influence of viral subtype on T-cell recognition. It has been demonstrated that murine pre-S(2)-specific T-cell response is highly subtype-specific (Milic H. et al. (1990)
J. Immunol.
144:3535).
The unicellular protozoon
Plasmodium falciparum
is the predominant pathogen causing malaria in humans. The infection starts when sporozoites, present in the salivary glands of Anopheles mosquitos, are inoculated into the blood of susceptible hosts. Sporozoites rapidly penetrate hepatocytes, in which they further develop into liver schizonts. After maturation, infectious merozoites are released into the blood of the host and invade erythrocytes, starting a new schizogonic cycle that is associated with the clinical symptoms of malaria. The number of malaria cases predicted by the World Health Organization is over 100 million worldwide.
Limited success has been reported in the protection of monkeys against infection by certain species of Plasmodium by immunization with purified surface antigens expressed during at least one stage of the life cycle of the parasite. For instance, an attractive candidate for a blood-stage vaccine is the merozoite protein termed p190, or polymorphic schizont antigen (Herra et al. (1992)
Infection and Immunity
60:154-158; Merkli et al. (1984)
Nature
311:379-382; Mackay et al. (1985)
EMBO J.
4:3823-3829). p190 is a large glycoprotein which is synthesized and extensively processed during merozoite formation, the 80 kDa processing product of which is the major coat protein of mature merozoites. Monoclonal antibody probes against P190 and primary sequence analysis reveal that the antigen contains polymorphic sequences giving rise to antigenic variation among species and subspecies of Plasmodium.
SUMMARY OF THE INVENTION
This invention pertains to a set of polypeptide antigens having amino acid sequences derived from amino acid sequences of a population of variants of a protein, or a portion thereof, and to methods of producing the set of polypeptide antigens. In general, the method comprises:
a. selecting a protein, or a portion thereof, which exhibits a population of N variants, represented by the formula
A
1
A
2
A
3
. . . A
n−2
DeConti, Jr. Esq. Giulio A.
DiGiorgio, Esq Jeanne M.
Lahive & Cockfield LLP
Park Hankyel T.
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