An antigen fragment encompassing the AD2 domains of glycoprotein B from two different strains is sufficient for differentiation of primary vs. recurrent human cytomegalovirus infection by ELISA

Abstract

Primary human cytomegalovirus (HCMV) infection during pregnancy is a frequent cause of fatal damage in populations with low prevalence of HCMV. Differentiation of primary vs. recurrent HCMV infection is an important issue in prenatal counseling. Antibodies specific for viral glycoproteins become detectable only with considerable delay with relation to HCMV infection or IgG seroconversion. Thus, lack of glycoprotein specific (gp-specific) antibodies can serve as a convenient indicator to identify those pregnant women that bear an elevated risk for HCMV transplacental transmission and fetal sequelae. In the opposite case, presence of gp-specific antibodies virtually excludes HCMV primary infection several weeks before sampling. However, no standardized screening assay for HCMV gp-specific antibodies had been available thus far. For this reason, an ELISA based on procaryotically expressed fragments of HCMV glycoprotein B (gB; gpUL55) was developed. Small fragments of gB from two different laboratory strains, encompassing the antigenic domain 2 (AD2) sufficed for sensitive and specific detection of gp-specific antibodies. The gB-ELISA titers correlated with titers of virus neutralizing antibodies in serum samples from primary or recurrent HCMV infections. Seroconversion kinetics of the gB-ELISA in samples from patients with primary HCMV infection closely paralleled the delay in seroconversion of gp-specific antibodies as determined by neutralization assay. Thus this assay provides a diagnostic tool that is easy to perform and can significantly add to available methods for the timely identification of primary HCMV infection during pregnancy. In addition, the gB-ELISA may be helpful in other clinical settings for the differentiation of primary HCMV infection from diseases caused by other pathogens.

Cytomegalovirus transmission to preterm infants during lactation

“…The prevalence of reactivity to a recombinant glycoprotein B (gB AD2) fusion protein immediately after delivery was identical, encompassing about 85% of mothers in each seropositive group (T, NT) indicating recurrent maternal infection. This corresponds well to the sensitivity of this assay in seropositive blood donors (Rothe et al, 2001). Maternal virus shedding into urine or saliva was not observed in either group (T, NT).…”

Association of the Outcome of Renal Transplantation with Antibody Response to Cytomegalovirus Strain--Specific Glycoprotein H Epitopes

“…ELISA using these purified recombinant proteins for the measurement of antibodies against the strain-specific gH epitopes was performed as described previously [20], with slight modification. In brief, microtiter plate wells (ELISA-PLATE 96w; Greiner Bio-One) were coated with 1 mg of purified recombinant gH-GST fusion proteins or of purified GST, and they were then blocked with 3% bovine serum albumin in phosphate buffered saline for 2 h at 37ЊC.…”

Recombinant Modified Vaccinia Virus Ankara Expressing a Soluble Form of Glycoprotein B Causes Durable Immunity and Neutralizing Antibodies against Multiple Strains of Human Cytomegalovirus

“…These include the original design of the Chiron gB vaccine, a molecular fusion protein of 807 aa, that was mutagenized at the protease cleavage site and which contained an internal deletion of the putative membrane-spanning (TM) domain between aa 715 and 772 (48,54,55). This molecule and variant constructs of 680 (gB680) and 692 aa, from which the entire carboxyl terminus was deleted, were shown to be immunogenic in animals and humans and induced virus-neutralizing antibodies (NAb) (7,48,53,54). In fact, a plasmid expressing gB680 induced higher levels of CMV NAb than full-length gB in mice, confirming reports that it is more immunogenic than fulllength gB, making it a suitable candidate for further vaccine development (26,27).…”