Nelmary Hernandez-Alvarado

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

ABSTRACT 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 Freunds 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.

An attenuated cytomegalovirus vaccine with a deletion of a viral chemokine gene is protective against congenital CMV transmission in a guinea pig model.

Development of a vaccine against congenital cytomegalovirus (CMV) infection is a public health priority, but CMVs encode immune evasion genes that complicate live virus vaccine design. To resolve this problem, this study employed guanosyl phosphoribosyl transferase (gpt) mutagenesis to generate a recombinant guinea pig CMV (GPCMV) with a knockout of a viral chemokine gene, GPCMV MIP (gp1). MIP deletion virus replicated with wild-type kinetics in cell culture but was attenuated in nonpregnant guinea pigs, demonstrating reduced viremia and reduced inflammation and histopathology (compared to a control virus with an intact GPCMV MIP gene) following footpad inoculation. In spite of attenuation, the vaccine was immunogenic, eliciting antibody responses comparable to those observed in natural infection. To assess its protective potential as a vaccine, either recombinant virus or placebo was used to immunize seronegative female guinea pigs. Dams were challenged in the early 3rd trimester with salivary gland-adapted GPCMV. Immunization protected against DNAemia (1/15 in vaccine group versus 12/13 in the control group, P < 0.01). Mean birth weights were significantly higher in pups born to vaccinated dams compared to controls (98.7 g versus 71.2 g, P < 0.01). Vaccination reduced pup mortality, from 35/50 (70%) in controls to 8/52 (15%) in the immunization group. Congenital GPCMV infection was also reduced, from 35/50 (70%) in controls to 9/52 (17%) in the vaccine group (P < 0.0001). We conclude that deletion of an immune modulation gene can attenuate the pathogenicity of GPCMV while resulting in a viral vaccine that retains immunogenicity and demonstrates efficacy against congenital infection and disease.

Development of a novel, guinea pig-specific IFN-γ ELISPOT assay and characterization of guinea pig cytomegalovirus GP83-specific cellular immune responses following immunization with a modified vaccinia virus Ankara (MVA)-vectored GP83 vaccine

The guinea pig (Cavia porcellus) provides a useful animal model for studying the pathogenesis of many infectious diseases, and for preclinical evaluation of vaccines. However, guinea pig models are limited by the lack of immunological reagents required for characterization and quantification of antigen-specific T cell responses. To address this deficiency, an enzyme-linked immunospot (ELISPOT) assay for guinea pig interferon (IFN)-γ was developed to measure antigen/epitope-specific T cell responses to guinea pig cytomegalovirus (GPCMV) vaccines. Using splenocytes harvested from animals vaccinated with a modified vaccinia virus Ankara (MVA) vector encoding the GPCMV GP83 (homolog of human CMV pp65 [gpUL83]) protein, we were able to enumerate and map antigen-specific responses, both in vaccinated as well as GPCMV-infected animals, using a panel of GP83-specific peptides. Several potential immunodominant GP83-specific peptides were identified, including one epitope, LGIVHFFDN, that was noted in all guinea pigs that had a detectable CD8+ response to GP83. Development of a guinea pig IFN-γ ELISPOT should be useful in characterization of additional T cell-specific responses to GPCMV, as well as other pathogens. This information in turn can help focus future experimental evaluation of immunization strategies, both for GPCMV as well as for other vaccine-preventable illnesses studied in the guinea pig model.

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.

Comparison of monovalent glycoprotein B with bivalent gB/pp65 (GP83) vaccine for congenital cytomegalovirus infection in a guinea pig model

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.

Complete Genome Sequence of Pathogenic Guinea Pig Cytomegalovirus from Salivary Gland Homogenates of Infected Animals

ABSTRACT The sequence of guinea pig cytomegalovirus (GPCMV) was determined by direct sequencing of salivary gland homogenates obtained following sustained, serial in vivo passage of pathogenic virus in guinea pigs. The 233,501-nucleotide salivary gland (SG) genome was noted to have 11 differences compared to the tissue culture-passaged virus, although no variations were noted in putative protein coding sequences.

Molecular and Biological Characterization of a New Isolate of Guinea Pig Cytomegalovirus

Development of a vaccine against congenital infection with human cytomegalovirus is complicated by the issue of re-infection, with subsequent vertical transmission, in women with pre-conception immunity to the virus. The study of experimental therapeutic prevention of re-infection would ideally be undertaken in a small animal model, such as the guinea pig cytomegalovirus (GPCMV) model, prior to human clinical trials. However, the ability to model re-infection in the GPCMV model has been limited by availability of only one strain of virus, the 22122 strain, isolated in 1957. In this report, we describe the isolation of a new GPCMV strain, the CIDMTR strain. This strain demonstrated morphological characteristics of a typical Herpesvirinae by electron microscopy. Illumina and PacBio sequencing demonstrated a genome of 232,778 nt. Novel open reading frames ORFs not found in reference strain 22122 included an additional MHC Class I homolog near the right genome terminus. The CIDMTR strain was capable of dissemination in immune compromised guinea pigs, and was found to be capable of congenital transmission in GPCMV-immune dams previously infected with salivary gland‑adapted strain 22122 virus. The availability of a new GPCMV strain should facilitate study of re-infection in this small animal model.

Identification by mass spectrometry and immune response analysis of guinea pig cytomegalovirus (GPCMV) pentameric complex proteins GP129, 131 and 133.

Development of a vaccine against congenital infection with human cytomegalovirus (HCMV) is a major public health priority. A potential vaccine target receiving considerable recent attention is the pentameric complex (PC) of HCMV proteins consisting of gL, gH, UL128, UL130, and UL131, since some antibodies against these target proteins are capable of potently neutralizing virus at epithelial and endothelial cell surfaces. Recently, homologous proteins have been described for guinea pig cytomegalovirus (GPCMV), consisting of gH, gL, and the GPCMV proteins GP129, GP131, and GP133. To investigate these proteins as potential vaccine targets, expression of GP129-GP133 transcripts was confirmed by reverse-transcriptase PCR. Mass spectrometry combined with western blot assays demonstrated the presence of GP129, GP131, and GP133 proteins in virus particles. Recombinant proteins corresponding to these PC proteins were generated in baculovirus, and as GST fusion proteins. Recombinant proteins were noted to be immunoreactive with convalescent sera from infected animals, suggesting that these proteins are recognized in the humoral immune response to GPCMV infection. These analyses support the study of PC-based recombinant vaccines in the GPCMV congenital infection model.

Molecular Detection of Streptococcus pneumoniae on Dried Blood Spots from Febrile Nigerian Children Compared to Culture

Background Nigeria has one of the highest burdens of pneumococcal disease in the world, but accurate surveillance is lacking. Molecular detection of infectious pathogens in dried blood spots (DBS) is an ideal method for surveillance of infections in resource-limited settings because of its low cost, minimal blood volumes involved, and ease of storage at ambient temperature. Our study aim was to evaluate a Streptococcus pneumoniae real-time polymerase chain reaction (rt-PCR) assay on DBS from febrile Nigerian children on Whatman 903 and FTA filter papers, compared to the gold standard of culture. Methods Between September 2011 to May 2015, blood was collected from children 5 years of age or under who presented to six hospital study sites throughout northern and central Nigeria with febrile illness, and inoculated into blood culture bottles or spotted onto Whatman 903 or FTA filter paper. Culture and rt-PCR were performed on all samples. Results A total of 537 DBS specimens from 535 children were included in the study, of which 15 were culture-positive for S. pneumoniae. The rt-PCR assay detected S. pneumoniae in 12 DBS specimens (2.2%). One positive rt-PCR result was identified in a culture-negative specimen from a high-risk subject, and two positive rt-PCR results were negative on repeat testing. Six culture-confirmed cases of S. pneumoniae bacteremia were missed. Compared to culture, the overall sensitivities of Whatman 903 and FTA DBS for detection of S. pneumoniae were 57.1% (95% CI 18.4–90.1%) and 62.5% (95% CI 24.5–91.5%), respectively. Nonspecific amplification was noted in an additional 22 DBS (4.1%). Among these, six were positive for a non-S. pneumoniae pathogen on culture. Conclusions Rt-PCR was able to detect S. pneumoniae from clinical DBS specimens, including from a culture-negative specimen. Our findings show promise of this approach as a surveillance diagnostic, but also raise important cautionary questions. Several DBS specimens were detected as S. pneumoniae by rt-PCR despite growth of a non-S. pneumoniae pathogen on culture. A precise definition of what constitutes a positive result is required to avoid falsely over-identifying specimens.

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

ABSTRACT 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 × 106-fold) viral loads in blood and end organs compared to N13R10. In pregnant guinea pigs, challenge with doses of r129 virus of ≥5 × 106 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.