Method to detect CMV by hybridization with a gp55 specific DNA probe

#The invention relates to recombinant human cytomegalovirus (CMV) proteins, and is directed to the production of neutralizing forms of gB protein and truncated forms thereof, their vaccine potential, and diagnostic DNA fragments thereof.

#Human cytomegalovirus (CMV) is a ubiquitous agent in human populations. Infections are generally asymptomatic, but there can be serious medical manifestations of the disease in immunocompromised individuals (transplant recipients and AIDS patients) and in congenitally infected newborns. In immunodeficient patients, primary CMV infection and reactivation of latent virus is associated with serious disease including retinitis and pneumonia. CMV infection also predisposes the patient to fungal and bacterial infections. Congenital CMV infection of the fetus occurs in about 1% (36,000) of infants born in the U.S. per year. Of these infants 10-20% will have symptomatic infection at birth or within two years of birth with a mortality rate of 10-15%. Among the survivors, many will have mild to severe neurologic complications including hearing loss, learning disabilities and mental retardation.

#Vaccines that prevent or reduce CMV-associated disease are clearly needed. The CMV (Towne) strain has been tested as a vaccine candidate in normal individuals and renal transplant patients (Quinnan, Jr., G. V. et al. (1984) Am Intern Med 101:478-483); (Plotkin, S. A. 1985, CMV Vaccines, In: The Herpes Viruses vol. 4, ed., Roizman and Lopez, Plenum Press, N.Y., p. 297-312). While this vaccine appeared to have no deleterious effects and did reduce symptoms of CMV disease in transplant recipients, there are many objections to the use of experimental live attenuated virus vaccines, including the possibility of immune impairment resulting from virus infection and reports of possible association between CMV and oncogenesis.

#FIG. 1 is a HindIII restriction map of the CMV (Towne) genome displayed in the parental orientation. Unique sequences are denoted by a thin line, and inverted repeats of the Long (L), and Short (S) components are denoted by boxes, ab-b′,a′, and a′c′-ca. The a sequence, distinguished as a white box, is a terminal direct repeat with an inverted copy (a′) at the L/S junction.

#The lower restriction map illustrates the ˜4.96 kb BamHI E/R to HindIII D/A fragment encoding the gB gene. The Towne DNA fragment cloned into pXgB1 is shown above the line and the largely colinear AD169 fragment is shown below the line. Restriction enzyme abbreviations are B, BamHI; Bg, BglII; C, ClaI; E, EcoRI; Eg, EagI; H, HindIII; Hc, HincII; K, KpnI; P, PstI; S, SacII; Sa, SalI; X, XhoI.

#FIG. 2 illustrates the nucleotide and deduced amino acid sequences for the gB envelope protein of CMV strains Towne and AD169. The gp55 cleavage site between amino acids 460 and 461 is indicated by the arrow. The N-terminal sequence analysis of gp55, which revealed this cleavage site, is shown in Table 2.

#Definitions

#As used herein, a polynucleotide “derived from” a designated sequence, for example, the DNA from the CMV gB gene, refers to a polynucleotide sequence which is comprised of a sequence of at least 6-20 nucleotides, more preferably at least 15 to 20 nucleotides corresponding, i.e., identical to or complementary to, a region of the designated nucleotide sequence. The correspondence to the nucleic acid sequence will be approximately 70% or greater, will preferably be at least 80%, and even more preferably will be at least 90%.

#The correspondence or non-correspondence of the derived sequence to other sequence can be determined by hybridization under the appropriate stringency conditions, using standard DNA hybridization technologies in liquid phases or on solid supports. Hybridization techniques for determining the complementarity of nucleic acid sequences are known in the art (see, for example, Maniatis et al. (1982)), and are discussed infra. In addition, mismatches of duplex polynucleotides formed by hybridization can be determined by known techniques, including digestion with a nuclease such as S1 that specifically digests single-stranded areas in duplex polynucleotides. Regions from which typical DNA sequences may be “derived” include but are not limited to, regions encoding specific epitopes.