Neutralizing Activity of Saliva against Cytomegalovirus

Saccoccio, Frances M.; Gallagher, Mary K.; Adler, Stuart P.; McVoy, Michael A.

doi:10.1128/cvi.05128-11

Cited by 26 publications

(27 citation statements)

References 28 publications

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“…In strain AD169 repair of the UL131A mutation resulted in efficient epithelial entry and replication (Wang and Shenk, 2005a). Subsequently, a GFP-tagged UL13 1A-repaired AD169 variant, BAD r UL131-Y4 (BADr, Table 1), facilitated development of assays to quantitate epithelial entry neutralizing activities in CMV hyperimmuneglobulin, serum, and saliva (Cui et al, 2008; Saccoccio et al, 2011a, b). To establish similar epithelialtropic variants in the Towne genetic background, two GFP-tagged epithelialtropic variants, TS15-rR and TS15-rN, were derived from TS15 (Cui et al, 2012), a BAC-cloned virus obtained from the Towne vaccine (see Section 2).…”

Section: Resultsmentioning

confidence: 99%

“…GFP-based assays for quantitating CMV neutralizing activity in CMV hyperimmuneglobulin, serum, and saliva have been described previously (Cui et al, 2008; Saccoccio et al, 2011a). To develop a GFP-based assay to measure spread inhibition activity, the experiment described above was modified to include quantitative measurement of GFP levels using a fluorescent plate reader.…”

Section: Resultsmentioning

confidence: 99%

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Antibody inhibition of human cytomegalovirus spread in epithelial cell cultures

Cui¹,

Lee²,

Adler³

et al. 2013

Journal of Virological Methods

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Anti-cytomegalovirus (CMV) antibodies reduce the incidence of CMV transmission and ameliorate the severity of CMV-associated disease. Neutralizing activity, measured as the ability of antibodies to prevent entry of cell-free virus, is an important component of natural immunity. However, in vivo CMV amplification may occur mainly via spread between adjacent cells within tissues. Thus, inhibition of cell-to-cell spread may be important when evaluating therapeutic antibodies or humoral responses to infection or immunization. In vitro CMV cell-to-cell spread is largely resistant to antibodies in fibroblast cultures but sensitive in endothelial cell cultures. In the present study antibodies in CMV hyperimmuneglobulin or seropositive human sera inhibited CMV cell-to-cell spread in epithelial cell cultures. Spread inhibition activity was quantitated with a GFP reporter assay employing GFP-tagged epithelialtropic variants of CMV strains Towne or AD169. Measurement of spread inhibition provides an additional parameter for the evaluation of candidate vaccines or immunotherapeutics and to further characterize the role of antibodies in controlling CMV transmission and disease.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Antibody inhibition of human cytomegalovirus spread in epithelial cell cultures

Cui¹,

Lee²,

Adler³

et al. 2013

Journal of Virological Methods

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“…A potential extension of our assay would be to test the neutralizing capability of antibodies found in saliva and urine. For example, a recent report demonstrated the presence of CMV-neutralizing antibodies in saliva and indicated an important distinction in neutralizing capability between fibroblast and epithelial cells (36).…”

Section: Discussionmentioning

confidence: 99%

Development of a High-Throughput Assay To Measure the Neutralization Capability of Anti-Cytomegalovirus Antibodies

Gardner

Bolovan-Fritts

Teng

et al. 2013

Clin Vaccine Immunol

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e Infection by human cytomegalovirus (CMV) elicits a strong humoral immune response and robust anti-CMV antibody production. Diagnosis of virus infection can be carried out by using a variety of serological assays; however, quantification of serum antibodies against CMV may not present an accurate measure of a patient's ability to control a virus infection. CMV strains that express green fluorescent protein (GFP) fusion proteins can be used as screening tools for evaluating characteristics of CMV infection in vitro. In this study, we employed a CMV virus strain, AD169, that ectopically expresses a yellow fluorescent protein (YFP) fused to the immediate-early 2 (IE2) protein product (AD169 IE2-YFP ) to quantify a CMV infection in human cells. We created a high-throughput cell-based assay that requires minimal amounts of material and provides a platform for rapid analysis of the initial phase of virus infection, including virus attachment, fusion, and immediate-early viral gene expression. The AD169 IE2-YFP cell infection system was utilized to develop a neutralization assay with a monoclonal antibody against the viral surface glycoprotein gH. The high-throughput assay was extended to measure the neutralization capacity of serum from CMV-positive subjects. These findings describe a sensitive and specific assay for the quantification of a key immunological response that plays a role in limiting CMV dissemination and transmission. Collectively, we have demonstrated that a robust high-throughput infection assay can analyze the early steps of the CMV life cycle and quantify the potency of biological reagents to attenuate a virus infection.T he coevolution of human herpesviruses with their hosts over the past millions of years has led to the development of complex strategies of immune evasion that allow persistent viral infection despite the presence of an active immune response (1). A comprehensive understanding of cytomegalovirus (CMV) infection is essential to delineate the molecular and cellular interactions necessary for priming a targeted humoral immune response and how a pathogen may coopt these processes to establish a persistent and lifelong infection (2). Furthermore, a more complete comprehension of CMV entry, replication, and immune evasion is paramount in developing strategies to diagnose and alleviate CMV disease in immunocompromised patients such as transplant recipients, AIDS patients, and neonates.Analysis of viral protein expression during CMV infection can be useful in studying viral entry, cellular manipulation, and egress. The replication cycle of CMV is temporally controlled and regulated by different segments of the viral genome. The replicative cycle is divided into immediate-early (IE), early (E), and late (L) phases of replication. CMV IE proteins are produced first and appear within 6 h postinfection (hpi). IE proteins are potent transactivators that stimulate the transcription of E genes (3, 4). IE1 and IE2 are the best-characterized IE gene products and are essential for viral replication a...

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“…Virus neutralization assays were performed at Virginia Commonwealth University in the laboratory of M. McVoy (38)(39)(40)(41) (Fig. 3A) and by the authors by applying an analogous method (Fig.…”

Section: Methodsmentioning

confidence: 99%

Additive Protection against Congenital Cytomegalovirus Conferred by Combined Glycoprotein B/pp65 Vaccination Using a Lymphocytic Choriomeningitis Virus Vector

Schleiss

Berka²,

Watson³

et al. 2017

Clin Vaccine Immunol

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Subunit vaccines for prevention of congenital cytomegalovirus (CMV) infection based on glycoprotein B (gB) and pp65 are in clinical trials, but it is unclear whether simultaneous vaccination with both antigens enhances protection. We undertook evaluation of a novel bivalent vaccine based on nonreplicating lymphocytic choriomeningitis virus (rLCMV) vectors expressing a cytoplasmic tail-deleted gB [gB(dCt)] and full-length pp65 from human CMV in mice. Immunization with the gB(dCt) vector alone elicited a comparable gB-binding antibody response and a superior neutralizing response to that elicited by adjuvanted subunit gB. Immunization with the pp65 vector alone elicited robust T cell responses. Comparable immunogenicity of the combined gB(dCt) and pp65 vectors with the individual monovalent formulations was demonstrated. To demonstrate proof of principle for a bivalent rLCMV-based HCMV vaccine, the congenital guinea pig cytomegalovirus (GPCMV) infection model was used to compare rLCMV vectors encoding homologs of pp65 (GP83) and gB(dCt), alone and in combination versus Freund's adjuvanted recombinant gB. Both vectors elicited significant immune responses, and no loss of gB immunogenicity was noted with the bivalent formulation. Combined vaccination with rLCMV-vectored GPCMV gB(dCt) and pp65 (GP83) conferred better protection against maternal viremia than subunit or either monovalent rLCMV vaccine. The bivalent vaccine also was significantly more effective in reducing pup mortality than the monovalent vaccines. In summary, bivalent vaccines with rLCMV vectors expressing gB and pp65 elicited potent humoral and cellular responses and conferred protection in the GPCMV model. Further clinical trials of LCMV-vectored HCMV vaccines are warranted.

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Neutralizing Activity of Saliva against Cytomegalovirus

Cited by 26 publications

References 28 publications

Antibody inhibition of human cytomegalovirus spread in epithelial cell cultures

Antibody inhibition of human cytomegalovirus spread in epithelial cell cultures

Development of a High-Throughput Assay To Measure the Neutralization Capability of Anti-Cytomegalovirus Antibodies

Additive Protection against Congenital Cytomegalovirus Conferred by Combined Glycoprotein B/pp65 Vaccination Using a Lymphocytic Choriomeningitis Virus Vector

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