Prevention of maternal-fetal transmission of cytomegalovirus after primary maternal infection in the first trimester by biweekly hyperimmunoglobulin administration.

OBJECTIVE: To examine the efficacy of biweekly hyperimmunoglobulin (HIG) administration to prevent maternal-fetal transmission of cytomegalovirus (CMV) in women with primary first-trimester CMV infection. METHODS: This was a prospective observational study of women with confirmed primary CMV infection in the first trimester who had the first HIG administration at or before 14 weeks' gestation. All women had biweekly HIG treatment until 20 weeks' gestation at a dose of 200 IU/kg of maternal body weight. Each subject underwent amniocentesis at least 6 weeks after first presentation at about 20 weeks. Primary outcome was maternal-fetal transmission at the time of amniocentesis, and secondary outcome was the frequency of congenital CMV infection at birth. The results were compared with a historic cohort of women with first-trimester CMV infection who did not undergo HIG treatment and who had amniocentesis at about 20 weeks. RESULTS: Subjects were 40 pregnant women with a primary CMV infection, with a median gestational age at first presentation of 9.6 (range, 5.1-14.3) weeks. On average, HIG administration started at 11.1 weeks and continued until 16.6 weeks. Within this interval, HIG was administered between two and six times in each patient. While CMV immunoglobulin-G (IgG) monitoring showed periodic fluctuations during biweekly HIG administration cycles, high CMV-IgG avidity indices remained stable over the whole treatment period. Maternal-fetal transmission before amniocentesis occurred in only one of the 40 cases (2.5% (95% CI, 0-13.2%)). At delivery, two additional subjects were found to have had late-gestation transmission. Considering all three cases with maternal-fetal transmission, the transmission rate was 7.5% (95% CI, 1.6-20.4%) in our 40 cases. All infected neonates were asymptomatic at birth. The matched historical control group consisted of 108 pregnancies. Thirty-eight transmissions (35.2% (95% CI, 26.2-45.0%)) occurred in the control group, which was significantly higher (P < 0.0001) than the transmission rate in the HIG treatment group. CONCLUSION: After a primary maternal CMV infection in the first trimester, biweekly HIG administration at a dose of 200 IU/kg prevents maternal-fetal transmission up to 20 weeks' gestation. Copyright © 2018 ISUOG. Published by John Wiley & Sons Ltd.

Human parvovirus B19 infection in infancy associated with acute and chronic lymphocytic myocarditis and high cytokine levels: report of 3 cases and review.

Human parvovirus B19 infection is occasionally associated with acute lymphocytic myocarditis (ALM). Three infants with B19 virus-associated ALM were followed up clinically, histologically, and immunovirologically. Each infant had B19 virus DNA in the blood or B19 virus-specific IgM antibodies. Two infants with postnatal infection recovered after immunosuppressive therapy. The third infant with possible prenatal infection developed chronic persistent myocarditis associated with persistent B19 virus DNA in the blood. All 3 infants had increased levels of interferon-gamma, tumor necrosis factor-alpha, and interleukins -6 and -8. Four newborns with congenital B19 virus infection and 4 infants and children who had postnatally acquired B19 virus infection without myocarditis all had normal levels of these cytokines. These observations suggest that B19 virus infection in infancy causes ALM in some infants and children.

The ends on herpesvirus DNA replicative concatemers contain pac2 cis cleavage/packaging elements and their formation is controlled by terminal cis sequences.

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 and pac2 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 that pac2 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 terminal cis sequences, since ectopic cleavage sites engineered into the murine cytomegalovirus genome mediated formation of pac2 ends on concatemers regardless of the orientation of their insertion. These findings are consistent with a model in which pac2 elements at concatemer ends impart a directionality to concatemer packaging by binding proteins that initiate insertion of concatemer ends into empty capsids.

A canarypox vector expressing cytomegalovirus (CMV) glycoprotein B primes for antibody responses to a live attenuated CMV vaccine (Towne).

To develop a vaccine against cytomegalovirus (CMV), a canarypox virus (ALVAC) expressing CMV glycoprotein (gB) was evaluated alone or in combination with a live, attenuated CMV vaccine (Towne). Three doses of 106.5 TCID50 of ALVAC-CMV(gB) induced very low neutralizing or ELISA antibodies in most seronegative adults. However, to determine whether ALVAC-CMV(gB) could prime for antibody responses, 20 seronegative adults randomly received either 106.8 TCID50 of ALVAC-CMV(gB) or 106.8 TCID50 of ALVAC-RG, expressing the rabies glycoprotein, administered at 0 and 1 month, with all subjects receiving a dose of 103.5 pfu of the Towne vaccine at 90 days. For subjects primed with ALVAC-CMV(gB), neutralizing titers and ELISA antibodies to CMV(gB) developed sooner, were much higher, and persisted longer than for subjects primed with ALVAC-RG. All vaccines were well tolerated. These results demonstrate that ALVAC-CMV(gB) primes the immune system and suggest a combined-vaccine strategy to induce potentially protective levels of neutralizing antibodies.

Serologic and virologic evidence for frequent intrauterine transmission of human parvovirus B19 with a primary maternal infection during pregnancy.

OBJECTIVE: To define the intrauterine viral transmission rate during primary maternal parvovirus B19 infection and identify factors that may influence this rate. METHODS: Forty-three pregnant women at two medical centers were identified with a primary B19 infection and followed to delivery. At delivery maternal and infant (umbilical cord) blood was obtained for B19 serologic and virologic PCR testing. RESULTS: All of the women delivered healthy infants at term and none was hydropic. Overall 22 (51%) of the 43 infants had some evidence of a congenital B19 infection. B19-specific IgM was detected in 11 infants at delivery, B19 IgA was detected in 10 and B19 DNA was detectable by PCR in 11 infants. One infant was negative at birth but became positive for IgM, IgA and PCR at 6 weeks of age. No association was found between the likelihood of intrauterine infection and: maternal age; symptomatic maternal infection; method of delivery; maternal IgG titer at delivery; maternal IgG avidity at delivery; or maternal viremia at delivery. Intrauterine infection was associated with maternal IgM positivity at delivery; this association may have been a result of maternal infection occurring later in gestation. CONCLUSION: Although the incidence of intrauterine hydrops and fetal demise after maternal infection is low, there is a high rate of intrauterine viral infection that occurs throughout gestation and yields newborns who, although infected in utero, are asymptomatic at birth.

Safety and immunogenicity of the Towne strain cytomegalovirus vaccine.

BACKGROUND: Women with naturally acquired serum antibodies to cytomegalovirus (CMV) are usually protected against both frequent secondary infection and giving birth to infants severely affected by intrauterine CMV infection. OBJECTIVE: To determine the feasibility of using a live attenuated strain of CMV (Towne) to achieve immunity similar to that provided by wild-type infection, we evaluated a new lot of the Towne strain of CMV in 3 open label trials involving 68 men, 63 women of childbearing age and 13 children, respectively. RESULTS: Mild local reactions occurred among approximately one-third of subjects. There were no systemic reactions. All 45 subjects tested developed lymphoproliferative responses to CMV. CD8+ class I-restricted cytotoxic T cell responses specific for CMV antigens were detected in three of four subjects and persisted for 6 months. Neutralizing titers were maximal at 2 to 4 months postimmunization, were dose-dependent and were comparable to those induced by natural infection. CONCLUSION: These results support further evaluation of the Towne strain of CMV in women at risk for acquiring CMV infection during pregnancy or among children transmitting CMV to pregnant women.

Prospective evaluation of 618 pregnant women exposed to parvovirus B19: risks and symptoms.

OBJECTIVE: To assess the risk of maternal parvovirus B19 infection from exposure to various sources and the fetal morbidity of those infections. METHODS: We obtained demographic and occupational information about pregnant women exposed to sources of B19 and about the nature and duration of the exposures. We performed serologic testing 10-14 days after exposure using an indirect capture enzyme-linked immunosorbent assay. Women with immunoglobulin (Ig) M were examined with weekly ultrasound until 12 weeks after exposure, and the outcome of the pregnancy was ascertained from interviews with patients and their obstetricians. Logistic regression analysis was used to determine risk factors for maternal immunity and infection by B19. RESULTS: Of 618 pregnant women exposed, 307 (49.7%) were immune to B19, 259 remained susceptible after exposure, and 52 (16.7% of all susceptibles) contracted B19 infection. None of the 52 fetuses of infected women developed nonimmune hydrops, and there were no fetal deaths attributable to B19 in this group. The relative risk of maternal B19 infection was 2.8 if the source was a related child living in the household (95% confidence interval 1.7, 4.6; P < .001). No significant differences were found for maternal B19 infection in eight categories of maternal occupation. Maternal symptoms of polyarthralgia (46%), fever (19%), and nonspecific rash (38%) were significantly more common (P < .001) in IgM-positive patients than in noninfected women (4.1%, 2.8%, and 5.7%, respectively). Only 17 (33%) of the IgM-positive women were entirely asymptomatic. CONCLUSION: The risk of maternal B19 infection in pregnancy could not be predicted by a gravida's occupation, but it was significantly higher when the source of exposure was her own child. The fetal risk of nonimmune hydrops after maternal B19 infection must be very low. As a consequence, exclusion of pregnant women from the workplace during endemic periods with seasonal clusters of cases is not justified. Weekly fetal ultrasound evaluation in these cases carries a low yield.

Prior infection with cytomegalovirus is not a major risk factor for angiographically demonstrated coronary artery atherosclerosis.

To determine if cytomegalovirus (CMV) infection is a risk factor for primary coronary artery disease (CAD), the association between CMV infection and CAD (>50% blockage in any coronary artery) was investigated in nearly 900 successive nontransplant patients undergoing coronary angiography. By use of logistic regression, older age (P <.001), white race (P <.001), gender (P <.001), hypercholesterolemia (P = .04), and other established cardiovascular risk factors (P = .003) were identified as significantly associated with CAD, but CMV seropositivity (P = .462), the level of IgG antibodies to CMV whole cell antigen (P = .98), or the levels of IgG antibodies to CMV glycoprotein B (P = .67) were not. These data suggest that CMV infection is not a major risk factor for the development of primary CAD in adults.

Sequences within the herpesvirus-conserved pac1 and pac2 motifs are required for cleavage and packaging of the murine cytomegalovirus genome.

The DNA sequence motifs pac1 [an A-rich region flanked by poly(C) runs] and pac2 (CGCGGCG near an A-rich region) are conserved near herpesvirus genomic termini and are believed to mediate cleavage of genomes from replicative concatemers. To determine their importance in the cleavage process, we constructed a number of recombinant murine cytomegaloviruses with a second cleavage site inserted at an ectopic location within the viral genome. Cleavage at a wild-type ectopic site occurred as frequently as at the natural cleavage site, whereas mutation of this ectopic site revealed that some of the conserved motifs of pac1 and pac2 were essential for cleavage whereas others were not. Within pac1, the left poly(C) region was very important for cleavage and packaging but the A-rich region was not. Within pac2, the A-rich region and adjacent sequences were essential for cleavage and packaging and the CGCGGCG region contributed to, but was not strictly essential for, efficient cleavage and packaging. A second A-rich region was not important at all. Furthermore, mutations that prevented cleavage also blocked duplication and deletion of the murine cytomegalovirus 30-bp terminal repeat at the ectopic site, suggesting that repeat duplication and deletion are consequences of cleavage. Given that the processes of genome cleavage and packaging appear to be highly conserved among herpesviruses, these findings should be relevant to other members of this family.

Circularization and cleavage of guinea pig cytomegalovirus genomes.

The mechanisms by which herpesvirus genome ends are fused to form circles after infection and are re-formed by cleavage from concatemeric DNA are unknown. We used the simple structure of guinea pig cytomegalovirus genomes, which have either one repeated DNA sequence at each end or one repeat at one end and no repeat at the other, to study these mechanisms. In circular DNA, two restriction fragments contained fused terminal sequences and had sizes consistent with the presence of single or double terminal repeats. This result implies a simple ligation of genomic ends and shows that circularization does not occur by annealing of single-stranded terminal repeats formed by exonuclease digestion. Cleavage to form the two genome types occurred at two sites, and homologies between these sites identified two potential cis elements that may be necessary for cleavage. One element coincided with the A-rich region of a pac2 sequence and had 9 of 11 bases identical between the two sites. The second element had six bases identical at both sites, in each case 7 bp from the termini. To confirm the presence of cis cleavage elements, a recombinant virus in which foreign sequences displaced the 6- and 11-bp elements 1 kb from the cleavage point was constructed. Cleavage at the disrupted site did not occur. In a second recombinant virus, restoration of 64 bases containing the 6- and 11-bp elements to the disrupted cleavage site restored cleavage. Therefore, cis cleavage elements exist within this 64-base region, and sequence conservation suggests that they are the 6- and 11-bp elements.

Salivary antibodies to cytomegalovirus (CMV) glycoprotein B accurately predict CMV infections among preschool children.

Among preschool children, we found an association between cytomegalovirus (CMV) infection and salivary immunoglobulin G antibodies to CMV glycoprotein B. All of the 20 infected children had immunoglobulin G to CMV glycoprotein B in their saliva, whereas 38 of 38 uninfected children lacked these antibodies. Testing saliva provides a sensitive and specific alternative to obtaining serum.

Mucosal antibodies to human cytomegalovirus glycoprotein B occur following both natural infection and immunization with human cytomegalovirus vaccines.

Because antibodies against human cytomegalovirus (HCMV) glycoprotein B (gB) neutralize, levels of IgG, secretory IgA (sIgA), and mucosal IgA1 antibodies to HCMV were measured in saliva and nasal washes. Ten seronegative adults lacked these antibodies, but of 10 naturally seropositive adults, 10 had IgG to gB, 5 had sIgA, and 0 had mucosal IgA. Among 12 recipients of a live HCMV vaccine, 8 had IgG to gB, 4 had sIgA, and 2 had mucosal IgA in samples collected 10-20 months after immunization; of 10 recipients of a gB vaccine, 8 had IgG to gB, 7 had sIgA, and 7 had mucosal IgA in samples collected just before or 1 month after a booster. IgG to gB and neutralizing titers in serum correlated with IgG to gB in mucosal samples. IgG to gB was in the saliva of 25 of 26 subjects with serum neutralizing titers > 1:64. Serum neutralizing titers > 1:64, whether induced by vaccine or wild type virus, are associated with mucosal IgG to HCMV.

Prevention of child-to-mother transmission of cytomegalovirus by changing behaviors: a randomized controlled trial.

BACKGROUND: To determine whether a behavioral prevention approach reduces child-to-parent transmission of cytomegalovirus. METHODS: Subjects were seronegative mothers whose child was less than 36 months of age and was shedding cytomegalovirus. Nonpregnant women were randomly assigned to three groups. Mothers in the education group (E) were given instructions about protective behaviors (frequent hand washing, wearing latex gloves) and risky behaviors to avoid (intimate contact with the child). Disposable diapers, liquid soap and latex gloves were provided. During biweekly home visits glove and soap use were monitored for an indirect objective measure of adherence to the protective behaviors. Throughout the study mothers self-reported the frequency they engaged in protective and risky behaviors. In addition to the procedures for Group E the adherence and education group (A) also received social reinforcement for adherence and problem solving for any perceived problems with the behavioral recommendations. The control group (C) received no intervention. A fourth group of pregnant women received an intervention equivalent to that of the education group. RESULTS: Eight of 17 women in Group C and 4 of 11 women in Group E seroconverted. For both E and Group C the average time from enrollment to infection was 4 months (range, 2 to 7 months). Two of 8 women in Group A seroconverted (1 at 3 months and 1 at 8 months). None of 14 pregnant women observed for an average of 8.4 months during pregnancy seroconverted. CONCLUSIONS: These results suggest that intervention for pregnant women is effective because pregnant women will perceive a higher risk and be more motivated to adhere to recommendations than nonpregnant women.

Current prospects for immunization against cytomegaloviral disease.

Cytomegaloviral (CMV) seronegative women who acquire a primary CMV infection during pregnancy are at the greatest risk for delivering infants who may be deaf or intellectually handicapped because of congenital CMV infection. Maternal immunization before pregnancy may protect newborns from congenital disease because mothers who are naturally seropositive are protected against secondary infection and because newborns who acquire CMV either via transfusion or transplacentally are protected if their mothers had antibodies to CMV prior to pregnancy. Further evidence for the feasibility of immunization for CMV comes from studies of patients immunocompromised following solid organ transplantation protection. These patients are protected against severe cytomegaloviral disease by immunity acquired either by wild-type infection prior to transplantation or by passive or active immunization. In three randomized placebo-controlled studies, live attenuated CMV Towne vaccine has successfully protected seronegative recipients of seropositive kidneys from severe CMV disease by inducing humoral and cellular immunity. Subunit vaccines comprised of glycoprotein gB, the viral component containing the majority of viral neutralizing epitopes, are in the early phases of study, as are studies with highly immunogenic preparations of Towne vaccine. Given all of these facts, safe and effective CMV immunoprophylaxis against CMV disease is possible.

Guinea pig and human cytomegaloviruses do not share cross-reactive neutralizing epitopes.

Human (HCMV) and guinea pig (gpCMV) cytomegaloviruses share biological similarities and DNA sequence homologies. Therefore, human and guinea pig sera were tested for cross-reactive antibodies. In eight human sere (four HCMV-seropositive and four HCMV-seronegative), low levels of neutralizing activity against gpCMV were not associated with antibodies to HCMV. The convalescent sera of one of three humans infected with either HCMV Towne vaccine or wild-type HCMV developed a fourfold increase in neutralizing titers to gpCMV after infection, but none of seven guinea pigs immunized with either HCMV Towne or purified HCMV gB developed neutralizing activity against gpCMV. Guinea pigs immunized with gpCMV did not develop antibodies to HCMV or human gB. Neither gpCMV, HCMV Towne stain or purified HCMV gB induced cross-reactive antibodies against the heterologous virus as detected by enzyme immunoassay. Our results indicate that gpCMV and HCMV share a very limited number, if any, of cross-reactive neutralizing epitopes.

Immunity induced by primary human cytomegalovirus infection protects against secondary infection among women of childbearing age.

To determine if immunity to human cytomegalovirus (HCMV) protects women from acquiring HCMV from their children, a blinded, randomized protocol was used to monitor seronegative women who received placebo or Towne vaccine (approximately 500 pfu) and seropositive women. Each group was similar for mean maternal (33 years) and child age (18 months) and duration of viral shedding by the child (15 months). Among 19 placebo recipients, 9 developed primary infection; 8 of 19 vaccines but only 3 of 42 naturally seropositive subjects had evidence of acquiring HCMV from their child. Wild type infection and Towne vaccine induced similar mean lymphoproliferative responses to HCMV antigens, but one dose of Towne vaccine produced mean neutralizing titers 10- to 20-fold lower than those after wild type infection. Thus, a vaccine that induces immune responses equal to those induced by wild type virus may protect healthy women from acquiring HCMV from their children.

Immunoprophylaxis against cytomegalovirus disease.

Patients with either deficient or immature immune systems need protection against cytomegalovirus (CMV) disease. That maternal immunization prior to pregnancy will protect newborns from congenital disease is suggested by the fact that newborns who acquire CMV either via transfusion or transplacentally are relatively protected if their mothers had antibodies to CMV prior to pregnancy. For patients becoming partially immunocompromised following solid organ transplantation, protection against severe CMV disease is afforded by immunity acquired either by wild-type infection prior to transplantation or passive or active immunization. In three randomized placebo-controlled studies, live attenuated CMV vaccine has successfully protected seronegative recipients of kidneys from seropositive donors from severe CMV disease by efficiently inducing humoral and cellular immunity. Subunit vaccines comprised of glycoprotein gB, the viral component containing the majority of viral neutralizing epitopes, are in the early phases of study, as are strategies to provide patients with CD8+ deficiency immunoprophylaxis via adoptive transfer of cytotoxic T-cells expanded in vitro against CMV structural proteins. Given all of these facts, safe and effective CMV immunoprophylaxis against CMV disease is possible.

Human cytomegalovirus DNA replicates after early circularization by concatemer formation, and inversion occurs within the concatemer.

To determine the replicative mechanism for human cytomegalovirus (HCMV) DNA, field inversion gel electrophoresis was used to separate HCMV replicative DNAs during lytic infection. Unit-length circular HCMV genomes lacking terminal restriction fragments were detected starting 4 h after infection even when cells were treated with aphidicolin, phosphonoacetic acid, or cycloheximide. Viral DNA synthesis began 24 h after infection and produced large amounts of high-molecular-weight replicative DNA that was a precursor of progeny genomes. Replicative DNA contained rare terminal restriction fragments, and long-arm termini were much less frequent than short-arm termini. Replicative DNA was not composed of unit-length circles because low-dose gamma irradiation of replicative DNA generated numerous random high-molecular-weight fragments rather than unit-length molecules. PacI digestion of replicative DNA from a recombinant HCMV with two closely spaced PacI sites revealed that replicative DNA is concatemeric and genome segment inversion occurs after concatemer synthesis. These results show that after circularization of the parental genome, DNA synthesis produces concatemers and genomic inversion occurs within concatemeric DNA. The results further suggest that concatemers acquire genomic termini during the cleavage/packaging process which preferentially inserts short-arm termini into empty capsids, causing a predominance of short-arm termini on the concatemer.

Antibodies induced by a primary cytomegalovirus infection react with human herpesvirus 6 proteins.

After a primary human cytomegalovirus (HCMV) infection, antibody titer to human herpes-virus 6 (HHV-6) rises. To determine if this occurs because of simultaneous infection with both viruses, serologic responses to these viruses were investigated among healthy women who received a live HCMV vaccine or acquired HCMV from an infected child. Both vaccination and natural infection caused four-fold or greater titer rises to HHV-6. Analysis of sera from 5 children with primary HHV-6 showed no serologic response to HCMV. Absorption of 7 sera with HCMV antigens reduced titers to HHV-6 by two- to fourfold. Immunoblot analysis of sera obtained from 10 persons before and after a primary HCMV infection revealed that after HCMV infection, 2 persons developed IgG reactivity to the 116-kDa protein (gp116) of HHV-6 and to two cleavage products of this protein. Reactivity to HHV-6 gp116 appeared after HCMV sero-conversion and was removed by absorption of sera with HCMV glycoprotein gB (gB), indicating that antibodies to HCMV gB cross-react with HHV-6 gp116, the likely gB homologue of HHV-6. The serologic diagnosis of HHV-6 infection requires excluding a primary HCMV infection.