Michael A. McVoy

Cytomegalovirus vaccines fail to induce epithelial entry neutralizing antibodies comparable to natural infection

Antibodies that neutralize cytomegalovirus (CMV) entry into fibroblasts are predominantly directed against epitopes within virion glycoproteins that are required for attachment and entry. However, the mechanism of CMV entry into epithelial and endothelial cells differs from fibroblast entry. Using assays that simultaneously measured neutralizing activities against CMV entry into fibroblasts and epithelial cells, we found that human immune sera and CMV-hyperimmuneglobulins have on on average 48-fold higher neutralizing activities against epithelial cell entry compared to fibroblast entry, suggesting that natural CMV infections elicit neutralizing antibodies that are epithelial entry-specific. This activity could not be adsorbed with recombinant gB. The Towne vaccine and the gB/MF59 subunit vaccine induced epithelial entry-specific neutralizing activities that were on on average 28-fold (Towne) or 15-fold (gB/MF59) lower than those observed following natural infection. These results suggest that CMV vaccine efficacy may be enhanced by the induction of epithelial entry-specific neutralizing antibodies.

Peptides from cytomegalovirus UL130 and UL131 proteins induce high titer antibodies that block viral entry into mucosal epithelial cells.

Cytomegalovirus infections are an important cause of disease for which no licensed vaccine exists. Recent studies have focused on the gH/gL/UL128-131 complex as antibodies to gH/gL/UL128-131 neutralize viral entry into epithelial cells. Prior studies have used cells from the retinal pigment epithelium, while to prevent transmission, vaccine-induced antibodies may need to block viral infection of epithelial cells of the oral or genital mucosa. We found that gH/gL/UL128-131 is necessary for efficient viral entry into epithelial cells derived from oral and genital mucosa, that short peptides from UL130 and UL131 elicit high titer neutralizing antibodies in rabbits, and that such antibodies neutralize viral entry into epithelial cells derived from these relevant tissues. These results suggest that single subunits or peptides may be sufficient to elicit potent epithelial entry neutralizing responses and that secretory antibodies to such neutralizing epitopes have the potential to provide sterilizing immunity by blocking initial mucosal infection.

TRANSFUSION-ASSOCIATED CYTOMEGALOVIRUS INFECTIONS IN SEROPOSITIVE CARDIAC SURGERY PATIENTS

To determine whether reinfection with an exogenous strain of cytomegalovirus (CMV) or reactivation of endogenous CMV is responsible for post-transfusion CMV infections in patients previously infected with the virus, seropositive patients were randomised preoperatively to receive red blood-cells (RBCs) from CMV seronegative donors (group A) or from random donors (group B). Each group B patient received at least one seropositive unit. 5 of 46 group A patients and 7 of 48 group B patients had a four-fold or greater increase of IgG antibody to CMV eight to twelve weeks postoperatively. IgG antibody titre did not increase in a third group (group C) of 57 seropositive patients who received no RBCs. 10 of the 12 patients with increased IgG titres also had CMV-specific IgM antibody after transfusion. These results indicate that reactivation accounts for most post-transfusion CMV infections in seropositive adults.

Analysis of the nucleotide sequence of the guinea pig cytomegalovirus (GPCMV) genome.

In this report we describe the genomic sequence of guinea pig cytomegalovirus (GPCMV) assembled from a tissue culture-derived bacterial artificial chromosome clone, plasmid clones of viral restriction fragments, and direct PCR sequencing of viral DNA. The GPCMV genome is 232,678 bp, excluding the terminal repeats, and has a GC content of 55%. A total of 105 open reading frames (ORFs) of > 100 amino acids with sequence and/or positional homology to other CMV ORFs were annotated. Positional and sequence homologs of human cytomegalovirus open reading frames UL23 through UL122 were identified. Homology with other cytomegaloviruses was most prominent in the central ~60% of the genome, with divergence of sequence and lack of conserved homologs at the respective genomic termini. Of interest, the GPCMV genome was found in many cases to bear stronger phylogenetic similarity to primate CMVs than to rodent CMVs. The sequence of GPCMV should facilitate vaccine and pathogenesis studies in this model of congenital CMV infection.

Overview of congenitally and perinatally acquired cytomegalovirus infections: recent advances in antiviral therapy

Congenital and perinatal infection with human cytomegalovirus (CMV) are commonly encountered in newborns. In recent years there has been increased awareness of the disabilities that result from congenital CMV infection, which in turn has prompted interest in examining the potential efficacy of antiviral agents to prevent or ameliorate neurodevelopmental injury. Currently, there are three licensed systemic antivirals for the treatment of CMV: ganciclovir (Cytovene®, Roche] and its prodrug valganciclovir [Valcyte®, Roche); foscarnet (Foscavir®, AstraZeneca); and cidofovir (Vistide®, Pharmacia). A CMV-specific immunoglobulin is also available. Experience with these agents in the setting of congenital and perinatal CMV infection is very limited, but there are encouraging data from a controlled clinical trial indicating that ganciclovir therapy may be of value in limiting one form of neurodevelopmental injury caused by congenital infection, that of sensorineural hearing loss. Licensed antivirals for the treatment of CMV all share the common mechanism of targeting the viral DNA polymerase, but novel therapies that employ alternative modes of action are in development. Ultimately, the problem of perinatal CMV infection may be best controlled by the development of CMV vaccines, which could be administered to young women of childbearing age to help control this important public health problem.

Requisite H2k Role in NK Cell-Mediated Resistance in Acute Murine Cytomegalovirus-Infected MA/My Mice

Human CMV infections are a major health risk in patients with dysfunctional or compromised immunity, especially in patients with NK cell deficiencies, as these are frequently associated with high morbidity and mortality. In experimental murine CMV (MCMV) infections, Ly49H activation receptors on C57BL/6 (B6) NK cells engage m157 viral ligands on MCMV-infected cells and initiate dominant virus control. In this study, we report that MCMV resistance in MA/My relies on Ly49H-independent NK cell-mediated control of MCMV infection as NK cells in these mice do not bind anti-Ly49H mAb or soluble m157 viral ligands. We genetically compared MA/My resistance with MCMV susceptibility in genealogically and NK gene complex-Ly49 haplotype-related C57L mice. We found that MCMV resistance strongly associated with polymorphic H2k-linked genes, including MHC and non-MHC locations by analysis of backcross and intercross progeny. The H2b haplotype most frequently, but not absolutely, correlated with MCMV susceptibility, thus confirming a role for non-MHC genes in MCMV control. We also demonstrate a definite role for NK cells in H2k-type MCMV resistance because their removal from C57L.M-H2k mice before MCMV infection diminished immunity. NK gene complex-linked polymorphisms, however, did not significantly influence MCMV control. Taken together, effective NK cell-mediated MCMV control in this genetic system required polymorphic H2k genes without need of Ly49H-m157 interactions.

Cloning of the genomes of human cytomegalovirus strains Toledo, TownevarRIT3, and Townelong as BACs and site-directed mutagenesis using a PCR-based technique

The 230-kb human cytomegalovirus genome is among the largest of the known viruses. Experiments to determine the genetic determinants of attenuation, pathogenesis, and tissue tropism are underway; however, a lack of complete sequence data for multiple strains and substantial problems with genetic instability during in vitro propagation create serious complications for such studies. For example, recent findings suggest that common laboratory strains Towne and AD169 passaged in cultured human fibroblasts are missing up to 15 kb of genetic information relative to clinical isolates. To establish standard, genetically stable genomes that can be sequenced, disseminated, and repeatedly reconstituted to produce virus stocks, we have undertaken to clone two variants of Towne, designated Towne(long) and Towne(short) (referred to as TownevarRIT3) (A., Proc. Natl. Acad. Sci. USA 98, 7829-7834), and the pathogenic strain Toledo into bacterial artificial chromosomes (BACs). We further demonstrate the ease with which mutagenesis can be achieved by deleting 13.5 kb from the Toledo genome using a PCR-based technique.

Cloning the complete guinea pig cytomegalovirus genome as an infectious bacterial artificial chromosome with excisable origin of replication

Congenital human cytomegalovirus infections are the major infectious cause of birth defects in the United States. How this virus crosses the placenta and causes fetal disease is poorly understood. Guinea pig cytomegalovirus (GPCMV) is a related virus that provides an important model for studying cytomegaloviral congenital transmission and pathogenesis. In order to facilitate genetic analysis of GPCMV, the 232kb GPCMV genome was cloned as an infectious bacterial artificial chromosome (BAC). The BAC vector sequences were flanked by LoxP sites to allow efficient excision using Cre recombinase. All initial clones contained spontaneous deletions of viral sequences and reconstituted mutant viruses with impaired growth kinetics in vitro. The deletions in one BAC were repaired using Escherichia coli genetics. The resulting repaired BAC reconstituted a virus with in vitro replication kinetics identical to the wild type parental virus; moreover, its genome was indistinguishable from that of the wild type parental virus by restriction pattern analysis using multiple restriction enzymes. These results suggest that the repaired BAC is an authentic representation of the complete GPCMV genome. It should provide a valuable tool for evaluating the impact of genetic modifications on the safety and efficacy of live attenuated vaccines and for identifying genes important for congenital transmission and fetal disease.

Molecular epidemiology of cytomegalovirus in a nursery: Lack of evidence for nosocomial transmission

During a 3-year period, cytomegalovirus (CMV) was recovered from the urine of 35 hospitalized newborn infants (15 with congenital and 20 with acquired infections). Two of the infants with acquired infections lacked maternal antibody to CMV (seronegative) and received transfusions from multiple seropositive blood donors. After seronegative blood products were used exclusively for seronegative low birth weight (less than 1300 gm) infants, none of 154 seronegative infants acquired CMV. CMV was recovered from one seronegative nurse who became infected during the study period. EcoRl digestion of the DNA of the nurses isolate and of 34 of the 35 infant isolates revealed that no two were identical. LBW seropositive infants were randomized to receive either seronegative blood products or blood products from random donors; there was no significant difference in the number of acquired CMV infections. There were no deaths among 18 infants with acquired CMV infection. Hepatosplenomegaly and worsening bronchopulmonary dysplasia developed in one LBW infant. These results prove that nosocomial transmission of CMV did not occur frequently during the 3-year period.

The Ends on Herpesvirus DNA Replicative Concatemers Contain pac2 cis Cleavage/Packaging Elements and Their Formation Is Controlled by Terminal cis Sequences

ABSTRACT Herpesviruses have large double-stranded linear DNA genomes that are formed by site-specific cleavage from complex concatemeric intermediates. In this process, only one of the two genomic ends are formed on the concatemer. Although the mechanism underlying this asymmetry is not known, one explanation is that single genomes are cleaved off of concatemer ends in a preferred direction. This implies that cis elements control the direction of packaging. Two highly conserved cis elements named pac1 andpac2 lie near opposite ends of herpesvirus genomes and are important for cleavage and packaging. By comparison of published reports and by analysis of two additional herpesviruses, we found thatpac2 elements lie near the ends formed on replicative concatemers of four herpesviruses: herpes simplex virus type 1, equine herpesvirus 1, guinea pig cytomegalovirus, and murine cytomegalovirus. Formation of pac2 ends on concatemers depended on terminalcis sequences, since ectopic cleavage sites engineered into the murine cytomegalovirus genome mediated formation ofpac2 ends on concatemers regardless of the orientation of their insertion. These findings are consistent with a model in whichpac2 elements at concatemer ends impart a directionality to concatemer packaging by binding proteins that initiate insertion of concatemer ends into empty capsids.