Highly infectious rubella virus clones and methods of production

Highly infectious rubella virus cDNA clones that are chimeric constructs of an infectious cDNA clone having a low specific infectivity and nucleic acid molecule fragments from a second rubella virus genome, wherein portions of the nucleotide sequence of the infectious cDNA clone having low specific infectivity have been replaced with the corresponding cDNA fragments derived from the second rubella virus genome.

The present invention relates to the field of molecular virology and more 
particularly to construction of highly infectious rubella virus cDNA 
clones. 
BACKGROUND OF THE INVENTION 
Rubella virus is a major human pathogen. Infection with rubella virus can 
cause serious birth defects and chronic disease. There was a mini-epidemic 
of both rubella and congenital rubella syndrome in the United States 
between 1989 and 1991. 
Rubella was first described in the eighteenth century in Germany. The 
symptoms of a rash and mild fever were similar to those of measles, so the 
disease was given the name German measles. The name "rubella" was coined 
in 1814 when physicians realized that the disease was unique and was not 
merely a variant of scarlatina (scarlet fever) or rubeola (measles). 
Rubella is a relatively harmless disease in young children. However, during 
the first trimester of pregnancy, rubella virus infection can cause fetal 
death. If the fetus survives, it may be born deaf or have cataracts, 
cardiac abnormalities, microcephaly, motor deficits or other congenital 
anomalies. The infant may also be born with thrombocytopenic purpura, 
hepatosplenomegaly, icterus, anemia, and low birth weight. The presence of 
one or more of these defects has been termed "congenital rubella syndrome" 
or CRS. 
The rubella virus was isolated in 1962 at the beginning of a worldwide 
rubella epidemic which lasted from 1962 to 1965. This epidemic peaked in 
the United States in 1964, resulting in the birth of approximately 20,000 
infants exhibiting congenital rubella syndrome. 
Scientists began development of an effective vaccine against the rubella 
virus during the rubella epidemic. Effective attenuated vaccines became 
available in the late 1960's and are still used today. These attenuated 
vaccines are live viruses that have been passaged to reduce their 
virulence. Attenuated vaccines produce immunity, but can cause disease. 
Protection is believed to persist for at least 15 years after inoculation 
with the attenuated rubella vaccine. 
Various vaccination schedules have been set up in different parts of the 
world to eliminate rubella infection, especially of the human fetus. The 
rubella immunization program established in Great Britain requires 
vaccination of all girls between the ages of 10 and 14. The United States 
immunization program vaccinates infants at approximately 15 months and 
requires a certificate of vaccination prior to attending school. The 
United States program is designed to eradicate the disease among the 
population that is most responsible for transmission of rubella, whereas 
the program of Great Britain seeks to achieve complete protection for 
those at risk for pregnancy. One disadvantage to the United States program 
is that protection against rubella may dissipate at the very time when 
immunity is most needed, namely, during the child-bearing years. 
Vaccination of women of child-bearing age having undetectable antibody 
titers is recommended in both the United States and Great Britain. 
However, there are several risks to this procedure. First, there is a risk 
that these women may be pregnant and not be aware of their pregnancy, or 
they may become pregnant within a few months following immunization. 
Vaccination against rubella is contraindicated in pregnant women because 
the live virus in the vaccine can cross the placenta and infect the fetus. 
Pregnant women who have not previously been infected with the rubella 
virus or who have not been vaccinated prior to becoming pregnant are 
advised to refrain from becoming vaccinated during their pregnancy. These 
women are therefore at risk for contracting rubella by coming in contact 
with infectious persons, including those recently vaccinated with the 
attenuated vaccine. 
Vaccination of older women has been associated with chronic arthritis and 
neurological symptoms. Scientists believe that these symptoms may be due 
to the persistent nature of the attenuated rubella virus in the currently 
available vaccines. Rubella virus is the sole member of the rubivirus 
genus of the Togavirus family. Compared to other viruses, very little is 
known about the molecular biology of the rubella virus. The rubella virion 
consists of single-stranded RNA encapsidated in an icosahedral 
nucleocapsid surrounded by a lipid envelope. Multiple copies of a viral 
protein, designated the C protein (MW=32,000-38,000 daltons), make up the 
nucleocapsid. Two types of viral glycoprotein, designated E1 and E2 
(MW=53,000-58,000 daltons and 42,000-48,000 daltons, respectively), are 
embedded in the envelope, as reported by Waxham, M. N. and Wolinsky, J. 
S., Virology 126:194-203 (1983). The E2 glycoprotein has been further 
subdivided into two subgroups, designated E2a and E2b, by their ability to 
migrate differently when resolved by polyacrylamide gel electrophoresis, 
as described by Oker-Blom, C., et al., J. Virol. 46:964-973 (1983). E1- is 
the viral hemagglutinin. Neutralizing epitopes have been found on both E1 
and E2 by Waxham, M. N. and Wolinsky, J. S., Virology 143:153-165 (1985) 
and Green, K. Y., and Dorsett, P. H., J. Virol., 57:893-898 (1986). 
The rubella virus genomic RNA is of positive polarity and is capped and 
polyadenylated. In infected cells, a second positive polarity RNA strand 
is synthesized to serve as messenger RNA for translation of structural 
proteins. This second strand is the first 3327 nucleotides beginning from 
the 3' end of the genomic RNA. The structural proteins are proteolytically 
processed from a polyprotein precursor during translation. The order of 
these proteins in the polyprotein is NH.sub.2 --C--E2--E1--COOH, as 
reported by Oker-Blom, C., et al. (1983); Oker-Blom, C., J. Virol. 
51:354-358 (1984). 
Recombinant vaccines are based on live microorganisms which have been 
genetically manipulated so that they are not pathogenic, but result in 
immunity against the virulent organism. Recombinant vaccines can only 
cause disease if a rare genetic mutation or recombinant event occurs which 
allows the microorganism to revert to wild type. A recombinant vaccine is 
generally safer and more effective than an attenuated vaccine because the 
engineered mutations remove or inactivate only specific portions of the 
genome, whereas attenuated vaccines contain random mutations. In order to 
develop a recombinant vaccine, one must first have the nucleic acid 
sequence of the entire viral genome, including both the information 
required for infection and at least limited replication of the virus, and 
for antigenicity. Once the entire sequence has been determined, a cDNA 
clone can be produced that is infectious and can be modified to be 
non-virulent. 
An infectious cDNA clone is a complete DNA copy of an RNA virus genome 
contained in a vector, such as a plasmid, from which RNA transcripts of 
the genome can be synthesized in vitro. In the case of positive-polarity 
RNA viruses such as rubella, such transcripts are infectious when 
transfected into cells. The development of an infectious clone is a 
landmark event in the molecular biology of any RNA virus. Although an 
infectious clone for rubella virus has been described (Wang, et al., J. 
Virol. 68:3550-3557 (1994)), this cDNA clone displayed low infectivity 
(approximately 5 plaques/10 .mu.g of transcripts). Increasing the 
infectivity of this clone would increase the efficiency of a recombinant 
attenuated rubella vaccine derived from the clone and would provide an 
improved molecular biology tool for studying rubella virus replication. 
However, successful generation of highly infectious cDNA clones has often 
been problematic due to the presence of mutations in the virus RNA 
template population caused by the inherent mutability of RNA viruses, the 
relatively low fidelity of the DNA polymerases used in cDNA synthesis, 
instability and toxicity of viral sequences in bacterial hosts, and the 
infidelity of the RNA polymerases used for in vitro transcriptions. 
Therefore, it is clear that there remains a strong need for an infectious 
cDNA clone of the rubella virus genome having a higher infectivity than 
currently available rubella virus clones. The isolation of a highly 
infectious cDNA clone will be useful for the development of a rubella 
vaccine that can be safely administered to pregnant and older women 
without risk of birth defects, auto immune disease, or neurologic 
symptoms. 
SUMMARY OF THE INVENTION 
Highly infectious cDNA clones of the rubella virus are provided herein. The 
clones are chimeric constructs containing portions of both a cDNA clone 
having a low specific infectivity and a second rubella virus genome. The 
infectious rubella virus clones are useful as molecular biology tools for 
studying rubella virus and can be useful for developing recombinant 
vaccines against rubella. 
The highly infectious cDNA clones have a specific infectivity greater than 
0.5 plaques/.mu.g of transcript. In several preferred embodiments of the 
invention, the specific infectivities of viral transcripts were 
approximately 10.sup.4 plaques/.mu.g of transcript. 
In the preferred embodiments, the cDNA clones are prepared by replacing one 
or more large fragments of a w-Therien-derived infectious cDNA clone with 
corresponding fragments from an f-Therien rubella virus strain. 
It is therefore an object of the present invention to provide a highly 
infectious cDNA clone of the rubella virus genomic RNA. 
It is a further object of the present invention to provide a molecular 
biology tool for studying rubella, particularly rubella virus replication. 
It is a further object of the present invention to provide cDNA clones for 
the development of a recombinant rubella virus vaccine.