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

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.

Repair of a Mutation Disrupting the Guinea Pig Cytomegalovirus Pentameric Complex Acquired during Fibroblast Passage Restores Pathogenesis in Immune-Suppressed Guinea Pigs and in the Context of Congenital Infection.

UNLABELLED: Guinea pig cytomegalovirus (GPCMV) provides a valuable model for congenital cytomegalovirus transmission. Salivary gland (SG)-passaged stocks of GPCMV are pathogenic, while tissue culture (TC) passage in fibroblasts results in attenuation. Nonpathogenic TC-derived virus N13R10 (cloned as a bacterial artificial chromosome [BAC]) has a 4-bp deletion that disrupts GP129, which encodes a subunit of the GPCMV pentameric complex (PC) believed to govern viral entry into select cell types, and GP130, an overlapping open reading frame (ORF) of unknown function. To determine if this deletion contributes to attenuation of N13R10, markerless gene transfer in Escherichia coli was used to construct virus r129, a variant of N13R10 in which the 4-bp deletion is repaired. Virions from r129 were found to contain GP129 as well as two other PC subunit proteins, GP131 and GP133, whereas these three PC subunits were absent from N13R10 virions. Replication of r129 in fibroblasts appeared unaltered compared to that of N13R10. However, following experimental challenge of immunocompromised guinea pigs, r129 induced significant weight loss, longer duration of viremia, and dramatically higher (up to 1.5 × 10(6)-fold) viral loads in blood and end organs compared to N13R10. In pregnant guinea pigs, challenge with doses of r129 virus of ≥5 × 10(6) PFU resulted in levels of maternal viremia, congenital transmission, pup viral loads, intrauterine growth restriction, and pup mortality comparable to that induced by pathogenic SG virus, although higher doses of r129 were required. These results suggest that the GP129-GP130 mutation is a significant contributor to attenuation of N13R10, likely by abrogating expression of a functional PC. IMPORTANCE: Tissue culture adaptation of cytomegaloviruses rapidly selects for mutations, deletions, and rearrangements in the genome, particularly for viruses passaged in fibroblast cells. Some of these mutations are focused in the region of the genome encoding components of the pentameric complex (PC), in particular homologs of human cytomegalovirus (HCMV) proteins UL128, UL130, and UL131A. These mutations can attenuate the course of infection when the virus is reintroduced into animals for vaccine and pathogenesis studies. This study demonstrates that a deletion that arose during the process of tissue culture passage can be repaired, with subsequent restoration of pathogenicity, using BAC-based mutagenesis. Restoration of pathogenicity by repair of a frameshift mutation in GPCMV gene GP129 using this approach provides a valuable genetic platform for future studies using the guinea pig model of congenital CMV infection.

Comparison of monovalent glycoprotein B with bivalent gB/pp65 (GP83) vaccine for congenital cytomegalovirus infection in a guinea pig model: Inclusion of GP83 reduces gB antibody response but both vaccine approaches provide equivalent protection against pup mortality.

Cytomegalovirus (CMV) subunit vaccine candidates include glycoprotein B (gB), and phosphoprotein ppUL83 (pp65). Using a guinea pig cytomegalovirus (GPCMV) model, this study compared immunogenicity, pregnancy outcome, and congenital viral infection following pre-pregnancy immunization with a three-dose series of modified vaccinia virus Ankara (MVA)-vectored vaccines consisting either of gB administered alone, or simultaneously with a pp65 homolog (GP83)-expressing vaccine. Vaccinated and control dams were challenged at midgestation with salivary gland-adapted GPCMV. Comparisons included ELISA and neutralizing antibody responses, maternal viral load, pup mortality, and congenital infection rates. Strikingly, ELISA and neutralization titers were significantly lower in the gB/GP83 combined vaccine group than in the gB group. However, both vaccines protected against pup mortality (63.2% in controls vs. 11.4% and 13.9% in gB and gB/GP83 combination groups, respectively; p<0.0001). Reductions in pup viral load were noted for both vaccine groups compared to control, but preconception vaccination resulted in a significant reduction in GPCMV transmission only in the monovalent gB group (26/44, 59% v. 27/34, 79% in controls; p<0.05). We conclude that, using the MVA platform, the addition of GP83 to a gB subunit vaccine interferes with antibody responses and diminishes protection against congenital GPCMV infection, but does not decrease protection against pup mortality.

Gas exchange and lung inflammation using nasal intermittent positive-pressure ventilation versus synchronized intermittent mandatory ventilation in piglets with saline lavage-induced lung injury: an observational study.

OBJECTIVE: Physiologic and pathologic comparison of two modes of assisted ventilation, nasal intermittent positive-pressure ventilation (NIPPV) and synchronized intermittent mandatory ventilation (SIMV), in spontaneously breathing term newborn piglets with saline lavage-induced lung injury. DESIGN: After inducing acute lung injury via repetitive saline lavage, piglets were randomized to NIPPV (n = 12) or SIMV (n = 11) and treated for 6 hrs. SETTING: Clinical laboratory. SUBJECTS: Spontaneously breathing term newborn piglets. INTERVENTIONS: Invasive (SIMV) or noninvasive (NIPPV) assisted ventilation for 6 hrs. MEASUREMENTS: Physiologic parameters and arterial blood gases were continuously monitored. At the conclusion of the study, lung tissue was obtained to analyze for evidence of inflammation, including myeloperoxidase, interleukin-8, and hydrogen peroxide levels, as well as for evidence of pathologic injury. MAIN RESULTS: Piglets treated with NIPPV demonstrated higher arterial blood gas pH (p < .001), lower PaCO2 (p < .05), and a lower set respiratory rate (p < .0001) as compared with the SIMV-treated piglets. The piglets in the SIMV group had higher PaO2/PaO2 ratio than those in the NIPPV group (p = .001). There was significantly more interstitial inflammation (p = .04) in the SIMV-treated piglets compared with the NIPPV-treated piglets. Total respiratory rate, heart rate, blood pressure, oxygen saturation, and biochemical markers of lung inflammation were not different between the groups. CONCLUSION: In surfactant-deficient term newborn piglets, NIPPV offers an effective and noninvasive ventilatory strategy with the potential for less pathologic lung inflammation.