Protection against Congenital CMV Infection Conferred by MVA-Vectored Subunit Vaccines Extends to a Second Pregnancy after Maternal Challenge with a Heterologous, Novel Strain Variant

Maternal reinfection of immune women with novel human cytomegalovirus (HCMV) strains acquired during pregnancy can result in symptomatic congenital CMV (cCMV) infection. Novel animal model strategies are needed to explore vaccine-mediated protections against maternal reinfection. To investigate this in the guinea pig cytomegalovirus (GPCMV) model, a strictly in vivo-passaged workpool of a novel strain, the CIDMTR strain (dose, 1 × 107 pfu) was used to infect dams that had been challenged in a previous pregnancy with the 22122 strain, following either sham-immunization (vector only) or vaccination with MVA-vectored gB, gH/gL, or pentameric complex (PC) vaccines. Maternal DNAemia cleared by day 21 in the glycoprotein-vaccinated dams, but not in the sham-immunized dams. Mean pup birth weights were 72.85 ± 10.2, 80.0 ± 6.9, 81.4 ± 14.1, and 89.38 ± 8.4 g in sham-immunized, gB, gH/gL, and PC groups, respectively (p < 0.01 for control v. PC). Pup mortality in the sham-immunized group was 6/12 (50%), but reduced to 3/35 (8.6%) in combined vaccine groups (p = 0.0048). Vertical CIDMTR transmission occurred in 6/12 pups (50%) in the sham-vaccinated group, compared to 2/34 pups (6%) in the vaccine groups (p = 0.002). We conclude that guinea pigs immunized with vectored vaccines expressing 22122 strain-specific glycoproteins are protected after a reinfection with a novel, heterologous clinical isolate (CIDMTR) in a second pregnancy.

Inclusion of the Guinea Pig Cytomegalovirus Pentameric Complex in a Live Virus Vaccine Aids Efficacy against Congenital Infection but Is Not Essential for Improving Maternal and Neonatal Outcomes

The development of a vaccine against congenital human cytomegalovirus (HCMV) infection is a major priority. The pentameric complex (PC) of virion envelope proteins gH, gL, UL128, UL130, and UL131A is a key vaccine target. To determine the importance of immunity to the homologous PC encoded by guinea pig cytomegalovirus (GPCMV) in preventing congenital CMV, PC-intact and PC-deficient live-attenuated vaccines were generated and directly compared for immunogenicity and efficacy against vertical transmission in a vertical transmission model. A virulent PC-intact GPCMV (PC/intact) was modified by galK mutagenesis either to abrogate PC expression (PC/null; containing a frame-shift mutation in GP129, homolog of UL128) or to delete genes encoding three MHC Class I homologs and a protein kinase R (PKR) evasin while retaining the PC (3DX/Δ145). Attenuated vaccines were compared to sham immunization in a two-dose preconception subcutaneous inoculation regimen in GPCMV seronegative Hartley guinea pigs. Vaccines induced transient, low-grade viremia in 5/12 PC/intact-, 2/12 PC/null-, and 1/11 3DX/Δ145-vaccinated animals. Upon completion of the two-dose vaccine series, ELISA titers for the PC/intact group (geometic mean titer (GMT) 13,669) were not significantly different from PC/null (GMT 8127) but were significantly higher than for the 3DX/Δ145 group (GMT 6185; p < 0.01). Dams were challenged with salivary gland-adapted GPCMV in the second trimester. All vaccines conferred protection against maternal viremia. Newborn weights were significantly lower in sham-immunized controls (84.5 ± 2.4 g) compared to PC/intact (96 ± 2.3 g), PC/null (97.6 ± 1.9 g), or 3DX/Δ145 (93 ± 1.7) pups (p < 0.01). Pup mortality in sham-immunized controls was 29/40 (73%) and decreased to 1/44 (2.3%), 2/46 (4.3%), or 4/40 (10%) in PC/intact, PC/null, or 3DX/Δ145 groups, respectively (all p < 0.001 compared to control). Congenital GPCMV transmission occurred in 5/44 (11%), 16/46 (35%), or 29/38 (76%) of pups in PC/intact, PC/null, or 3DX/Δ145 groups, versus 36/40 (90%) in controls. For infected pups, viral loads were lower in pups born to vaccinated dams compared to controls. Sequence analysis demonstrated that infected pups in the vaccine groups had salivary gland-adapted GPCMV and not vaccine strain-specific sequences, indicating that congenital transmission was due to the challenge virus and not vaccine virus. We conclude that inclusion of the PC in a live, attenuated preconception vaccine improves immunogenicity and reduces vertical transmission, but PC-null vaccines are equal to PC-intact vaccines in reducing maternal viremia and protecting against GPCMV-related pup mortality.

All the Pups We Cannot See: Cannibalism Masks Perinatal Death in Laboratory Mouse Breeding but Infanticide Is Rare

Perinatal mortality is a major issue in laboratory mouse breeding. We compared a counting method using daily checks (DAILY_CHECK) with a method combining daily checks with detailed video analyses to detect cannibalisms (VIDEO_TRACK) for estimating the number of C57BL/6 pups that were born, that died and that were weaned in 193 litters from trios with (TRIO-OVERLAP) or without (TRIO-NO_OVERLAP) the presence of another litter. Linear mixed models were used at litter level. To understand whether cannibalism was associated with active killing (infanticide), we analysed VIDEO_TRACK recordings of 109 litters from TRIO-OVERLAP, TRIO-NO_OVERLAP or SOLO (single dams). We used Kaplan-Meier method and logistic regression at pup level. For DAILY_CHECK, the mean litter size was 35% smaller than for VIDEO_TRACK (p < 0.0001) and the number of dead pups was twice lower (p < 0.0001). The risk of pup loss was higher for TRIO-OVERLAP than TRIO-NO_OVERLAP (p < 0.0001). A high number of pup losses occurred between birth and the first cage check. Analyses of VIDEO_TRACK data indicated that pups were clearly dead at the start of most of the cannibalism events and infanticide was rare. As most pups die and disappear before the first cage check, many breeding facilities are likely to be unaware of their real rates of mouse pup mortality.

Risk Factors for New Zealand Sea Lion (Phocarctos hookeri) Pup Mortality: Ivermectin Improves Survival for Conservation Management

Septicaemia due to hypervirulent (HV) Klebsiella pneumoniae is the leading cause of neonatal pup mortality in endangered New Zealand sea lions (Phocarctos hookeri) at Enderby Island, in the New Zealand sub-Antarctic. Accounting for approximately 60% of annual pup mortality at this site following an epizootic event in 2001–02, HV K. pneumoniae is also emerging worldwide as a significant community-acquired human pathogen. To facilitate efficient direct mitigation to reduce pup mortality, a case-control study and prospective cohort study were conducted to identify risk factors amenable to active management. Additionally, to investigate impacts of hookworm (Uncinaria spp.), a nested treatment trial with the anthelmintic ivermectin was undertaken concurrently. During two austral summer field seasons (2016–2018), 698 pups were captured for treatment trial recruitment and the collection of morphometric measurements, biological samples and risk factor data. Gastrointestinal carriage of the virulent phenotype of K. pneumoniae was a consistent risk factor, while ivermectin treatment and higher body condition index consistently reduced risk of HV K. pneumoniae mortality. Significantly fewer ivermectin-treated pups were found dead (24.1% control, 11.1% treatment), with a trend towards a higher proportion of HV K. pneumoniae deaths amongst the control group. This study provides evidence to support ivermectin treatment as a pup mortality mitigation strategy in New Zealand sea lions at Enderby Island. If applied to larger colonies where HV K. pneumoniae and hookworm impact pup survival, this intervention could have population-scale benefits for this endangered species. Further work is required to understand how ivermectin prevents HV K. pneumoniae septicaemia, but removal of hookworms before intestinal mucosal damage occurs could limit systemic spread of virulent bacteria from the gastrointestinal tract.

All the Pups We Cannot See: Cannibalism Masks Perinatal Death in Laboratory Mouse Breeding but Infanticide Is Rare

Perinatal mortality is a major issue in laboratory mouse breeding. We compared a counting method using daily checks (DAILY_CHECK) with a method combining daily checks with detailed video analyses to detect cannibalisms (VIDEO_TRACK) for estimating the number of C57BL/6 pups born, died and weaned in 193 litters from trios with (TRIO-OVERLAP) or without (TRIO-NO_OVERLAP) the presence of another litter. Linear mixed models were used at litter level. To understand if cannibalism was associated with active killing (infanticide), we analysed VIDEO_TRACK recordings of 109 litters from TRIO-OVERLAP, TRIO-NO_OVERLAP or SOLO (single dams). We used Kaplan-Meier method and logistic regression at pup level. For DAILY_CHECK, the mean litter size was 35% smaller than for VIDEO_TRACK (P&amp;lt;0.0001) and the number of dead pups was twice lower (P&amp;lt;0.0001). The risk of pup loss was higher for TRIO-OVERLAP than TRIO-NO_OVERLAP (P&amp;lt;0.0001). A high number of pup losses occurred between birth and the first cage checking. Analyses of VIDEO_TRACK data indicated that pups were clearly dead at the start of most of the cannibalism events and infanticide was rare. As most pups die and disappear before the first cage check, many breeding facilities are likely to be unaware of their real rates of mouse pup mortality.

Successful prenatal therapy of anti-CD36-mediated severe FNAIT by deglycosylated antibodies in a novel murine model

Recent studies have demonstrated that maternal anti-CD36 antibodies represent a frequent cause of fetal/neonatal alloimmune thrombocytopenia (FNAIT) in Asian and African populations. However, little is known about the pathomechanism and antenatal treatment of anti-CD36-mediated FNAIT. Here, we established a novel animal model to examine the clinical features of pups from immunized Cd36-/- female mice after breeding with wild-type male mice. Mild thrombocytopenia was observed, but high pup mortality was also documented (40.26%). IVIG (1 g/kg) administration on days 7, 12, and 17 to immunized Cd36-/- mothers after breeding reduced fetal death (12.70%). However, delaying the IVIG administration series on days 10, 15, and 20 did not reduce fetal death (40.00%). In contrast, injection of deglycosylated anti-CD36 (deg-anti-CD36) polyclonal antibodies (5 mg/kg) on days 10, 15, and 20 significantly reduced fetal death (5.26%). Subsequently, monoclonal antibodies (mAbs) against mouse CD36 were developed, and one clone producing high-affinity anti-CD36 (termed 32-106) effectively inhibited maternal antibody binding and was therefore selected. Using the same approach of deg-anti-CD36, the administration of deg-32-106 significantly reduced fetal death (2.17%). Furthermore, immunized Cd36-/- mothers showed placenta deficiency. Accordingly, maternal anti-CD36 antibodies inhibited angiogenesis of placenta endothelial cells, which could be restored by deg-32-106. In summary, maternal anti-CD36 antibodies caused a high frequency of fetal death in our animal model, associated with placental dysfunction. This deleterious effect could be diminished by the antenatal administration of IVIG and deg-mAb 32-106. Interestingly, treatment with deg-32-106 appears more beneficial considering the lower dose, later start of treatment, and therapy success.

Evidence for an Allee effect in a declining fur seal population

Allee effects play an important role in the dynamics of many populations and can increase the risk of local extinction. However, some authors have questioned the weight of evidence for Allee effects in wild populations. We therefore exploited a natural experiment provided by two adjacent breeding colonies of contrasting density to investigate the potential for Allee effects in an Antarctic fur seal ( Arctocephalus gazella ) population that is declining in response to climate change-induced reductions in food availability. Biometric time-series data were collected from 25 pups per colony during two consecutive breeding seasons, the first of which was among the worst on record in terms of breeding female numbers, pup birth weights and foraging trip durations. In previous decades when population densities were higher, pup mortality was consistently negatively density dependent, with rates of trauma and starvation scaling positively with density. However, we found the opposite, with higher pup mortality at low density and the majority of deaths attributable to predation. In parallel, body condition was depressed at low density, particularly in the poor-quality season. Our findings shed light on Allee effects in wild populations and highlight a potential emerging role of predators in the ongoing decline of a pinniped species.

Pre-reproductive stress in adolescent female rats alters oocyte microRNA expression and offspring phenotypes: pharmacological interventions and putative mechanisms

Pre-reproductive stress (PRS) to adolescent female rats alters anxiogenic behavior in first (F1)- and second-generation (F2) offspring and increases mRNA expression of corticotropin-releasing factor receptor type 1 (Crhr1) in oocytes and in neonate offspring brain. Here, we ask whether the expression of Crhr1 and Crhr1-targeting microRNA is altered in brain, blood, and oocytes of exposed females and in the brain of their neonate and adult F1 and F2 offspring. In addition, we inquire whether maternal post-stress drug treatment reverses PRS-induced abnormalities in offspring. We find that PRS induces a selective increase in Crhr1-targeting mir-34a and mir-34c in blood and oocytes, while non-Crhr1 microRNA molecules remain unaltered. PRS induces similar microRNA changes in prefrontal cortex of F1 and F2 neonates. In adult animals, cortical Crhr1, but not mir-34, expression is affected by both maternal and direct stress exposure. Post-PRS fluoxetine (FLX) treatment increases pup mortality, and both FLX and the Crhr1 antagonist NBI 27914 reverse some of the effects of PRS and also have independent effects on F1 behavior and gene expression. PRS also alters behavior as well as gene and miRNA expression patterns in paternally derived F2 offspring, producing effects that are different from those previously found in maternally derived F2 offspring. These findings extend current knowledge on inter- and trans-generational transfer of stress effects, point to microRNA changes in stress-exposed oocytes as a potential mechanism, and highlight the consequences of post-stress pharmacological interventions in adolescence.

Evidence for an Allee effect in a declining fur seal population

Allee effects play an important role in the dynamics of many populations and can increase the risk of local extinction. However, some authors have questioned the weight of evidence for Allee effects in wild populations. We therefore exploited a natural experiment provided by two adjacent breeding colonies of contrasting density to investigate the potential for Allee effects in an Antarctic fur seal (Arctocephalus gazella) population that is declining in response to climate-change induced reductions in food availability. Biometric time-series data were collected from 25 pups per colony during two consecutive breeding seasons, the first of which was amongst the worst on record in terms of breeding female numbers, pup birth weights and foraging trip durations. In previous decades when population densities were higher, pup mortality was consistently negatively density-dependent, with rates of trauma and starvation scaling positively with density. However, we found the opposite, with higher pup mortality at low density and the majority of deaths attributable to predation. In parallel, body condition was also depressed at low density, particularly in the poor-quality season. Our findings shed light on Allee effects in wild populations and highlight a potential emerging role of predators in the ongoing decline of a pinniped species.