Biology subjects

Crooks, C. M.

Publications and source records attributed to Crooks, C. M..

11 recordsLinked to original sources

bioRxiv2026

B cell response to HCMV gH/UL116 elicits neutralizing and FcγR-activating antibodies

Humoral immune mechanisms that can contribute to limiting HCMV disease include neutralizing antibodies that block viral entry and Fc-mediated effector functions that promote elimination of infected cells. Here, we identify the gH/UL116 envelope complex as a naturally immunogenic target that elicits antibodies capable of mediating both of these immune mechanisms. Using single-cell sorting of antigen-specific memory B cells, we isolated gH/UL116-reactive monoclonal antibodies from HCMV-seropositive donors. Epitope mapping showed that gH binding was required for virus neutralization, whereas UL116 targeting was associated with potent and broad Fc{gamma} receptor (Fc{gamma}R) activation. Furthermore, cryo-electron microscopy provided high-resolution structures revealing how UL116-specific antibodies bind gH/UL116. In mice, gH/UL116 immunization elicited serum IgG responses whose binding titers strongly correlated with both neutralization and mouse Fc{gamma}R activation. Together, these findings provide an epitope-level map linking antibody specificity to neutralization and Fc{gamma}R activation across HCMV gH-associated complexes and position gH/UL116 as a promising HCMV vaccine candidate.

immunology↗

bioRxiv2026

Spondweni virus infection in pregnant rhesus macaques causes placental pathology without apparent fetal harm

The 2015-2016 Zika virus (ZIKV) epidemic revealed the potential of flaviviruses to emerge rapidly, cause severe disease, and affect pregnancy outcomes. In 2016, Spondweni virus (SPOV), the closest known relative of ZIKV, was detected in mosquitoes in Haiti, suggesting it may also have the potential to emerge in the Western Hemisphere. The risks that close relatives of ZIKV pose to pregnant individuals are not well understood. Previously, we showed that SPOV can cause fetal demise, placental pathology, and vertical transmission in a mouse model. Here we report SPOVs pathogenic potential in pregnant rhesus macaques. We inoculated four macaques with SPOV at gestational day 30 (early first trimester) and compared their viral loads and fetal outcomes with those of macaques infected in the first trimester with either African-lineage ZIKV (ZIKV-DAK) or an Asian-lineage ZIKV isolate from Puerto Rico (ZIKV-PR) in previous studies. Plasma viremia persisted 10-31 days in SPOV-inoculated dams, whereas viremia resolved within 10-17 days for ZIKV-DAK and 5-52 days for ZIKV-PR. Cesarean deliveries near term (gestational day 152-157) revealed no demise, premature birth, or gross abnormalities in fetuses of dams inoculated with SPOV or ZIKV-PR. In contrast, under near-identical conditions, all ZIKV-DAK-inoculated dams experienced fetal demise between 12 and 20 days post-inoculation. At cesarean section, we did not detect SPOV RNA above the limit of detection in maternal (e.g., spleen, liver), placental, or fetal tissues, in contrast to previous findings with ZIKV-PR. Histological analysis revealed mononuclear/lymphohistiocytic villitis in all placentas of SPOV-exposed macaques, along with other pathological changes in individual placentas. Our findings suggest that SPOV infection of macaques in early pregnancy may result in placental pathology without overt fetal harm. Our results suggest that flaviviruses in the Spondweni serocomplex, which includes ZIKV and SPOV, may vary in their pathogenic potential during pregnancy. Author SummaryZika virus (ZIKV) can cause fetal harm. Does this risk extend to its closest known relative, Spondweni virus (SPOV)? Should SPOV circulate in humans, what risks would it pose in pregnancy? SPOV can injure fetuses in immunocompromised mice, but the physiology of pregnancy in mice differs greatly from that of humans. We therefore infected pregnant rhesus macaques with SPOV during early gestation and compared maternal viremia, placental pathology, and fetal outcomes with macaques infected with African- or Asian-lineage ZIKVs at the same gestational age. All fetuses survived to near-term pregnancy, fetal tissues were negative for SPOV RNA, and fetal growth tracked within expected ranges. Nonetheless, all SPOV-exposed pregnancies showed placental injury, including mononuclear/lymphohistiocytic villitis and maternal vascular malperfusion. Despite the absence of detectable SPOV RNA in fetal tissues, SPOV RNA persisted at term in maternal-fetal interface tissues in two of four animals. These data indicate placental injury without detectable vertical transmission in this translational model. Our results suggest that SPOV and ZIKV display a wide range of risks to the developing fetus. Identifying viral and host factors that increase the potential for fetal harm will be important for assessing risks posed by emerging viruses in this family.

microbiology↗

bioRxiv2024

A human cytomegalovirus prefusion-like glycoprotein B subunit vaccine elicits similar humoral immunity to that of postfusion gB in mice

Human cytomegalovirus (HCMV) is the leading infectious cause of birth defects. Despite the global disease burden, there is no FDA-approved HCMV vaccine. The most efficacious HCMV vaccine candidates to date have used glycoprotein B (gB), a class III viral fusion protein, in its postfusion form. While some viral fusion proteins have been shown to elicit stronger neutralizing responses in their prefusion conformation, HCMV prefusion-like and postfusion gB were recently shown to elicit antibodies with similar fibroblast neutralization titers in mice. We aimed to define and compare the specificity and functionality of plasma IgG elicited by distinct prefusion-like and postfusion gB constructs. Prefusion-like and postfusion gB elicited comparable IgG responses that predominantly mapped to AD-5 antigenic domain known to elicit neutralizing antibodies. Interestingly, postfusion gB elicited significantly higher plasma IgG binding to cell-associated gB and antibody dependent cellular phagocytosis than that of prefusion-like gB. The vaccines elicited comparable neutralization titers of heterologous HCMV strain AD169r in fibroblasts, yet neither elicited neutralizing titers against the vaccine-matched strain Towne in fibroblasts. Our data indicate that gB in this prefusion-like conformation elicits similar specificity and functional humoral immunity to that of postfusion gB, unlike certain class I viral fusion proteins which have been used as vaccine antigens. These findings deepen our understanding of the immune response elicited by class III fusion proteins and may inform further design and testing of conformationally-dependent herpesvirus glycoprotein vaccine candidates.

immunology↗

bioRxiv2023

The pentameric complex is not required for vertical transmission of cytomegalovirus in seronegative pregnant rhesus macaques

Congenital cytomegalovirus (cCMV) infection is the leading infectious cause of neonatal neurological impairment but essential virological determinants of transplacental CMV transmission remain unclear. The pentameric complex (PC), composed of five subunits, glycoproteins H (gH), gL, UL128, UL130, and UL131A, is essential for efficient entry into non-fibroblast cells in vitro. Based on this role in cell tropism, the PC is considered a possible target for CMV vaccines and immunotherapies to prevent cCMV. To determine the role of the PC in transplacental CMV transmission in a non-human primate model of cCMV, we constructed a PC-deficient rhesus CMV (RhCMV) by deleting the homologues of the HCMV PC subunits UL128 and UL130 and compared congenital transmission to PC-intact RhCMV in CD4+ T cell-depleted or immunocompetent RhCMV-seronegative, pregnant rhesus macaques (RM). Surprisingly, we found that the transplacental transmission rate was similar for PC-intact and PC-deleted RhCMV based on viral genomic DNA detection in amniotic fluid. Moreover, PC-deleted and PC-intact RhCMV acute infection led to similar peak maternal plasma viremia. However, there was less viral shedding in maternal urine and saliva and less viral dissemination in fetal tissues in the PC-deleted group. As expected, dams inoculated with PC-deleted RhCMV demonstrated lower plasma IgG binding to PC-intact RhCMV virions and soluble PC, as well as reduced neutralization of PC-dependent entry of the PC-intact RhCMV isolate UCD52 into epithelial cells. In contrast, binding to gH expressed on the cell surface and neutralization of entry into fibroblasts by the PC-intact RhCMV was higher for dams infected with PC-deleted RhCMV compared to those infected with PC-intact RhCMV. Our data demonstrates that the PC is dispensable for transplacental CMV infection in our non-human primate model. One Sentence SummaryCongenital CMV transmission frequency in seronegative rhesus macaques is not affected by the deletion of the viral pentameric complex.

microbiology↗

bioRxiv2022

Primary infection with Zika virus provides one-way heterologous protection against Spondweni virus infection in rhesus macaques

Spondweni virus (SPONV) is the closest known relative of Zika virus (ZIKV). SPONV pathogenesis resembles that of ZIKV in pregnant mice, and both viruses are transmitted by Aedes aegypti mosquitoes. We aimed to develop a translational model to further understand SPONV transmission and pathogenesis. We found that cynomolgus macaques (Macaca fascicularis) inoculated with ZIKV or SPONV were susceptible to ZIKV, but resistant to SPONV infection. In contrast, rhesus macaques (Macaca mulatta) supported productive infection with both ZIKV and SPONV and developed robust neutralizing antibody responses. Crossover serial challenge in rhesus macaques revealed that SPONV immunity did not protect against ZIKV infection, whereas ZIKV immunity was fully protective against SPONV infection. These findings establish a viable model for future investigation into SPONV pathogenesis, and suggest the risk of SPONV emergence is low in areas with high ZIKV seroprevalence due to one-way cross-protection between ZIKV and SPONV. TeaserIdentification of asymmetric immune interactions between Zika and Spondweni viruses in macaque monkeys.

microbiology↗

bioRxiv2022

Infection of the maternal-fetal interface and vertical transmission following low-dose inoculation of pregnant rhesus macaques (Macaca mulatta) with an African-lineage Zika virus

BackgroundCongenital Zika virus (ZIKV) infection can result in birth defects, including malformations in the fetal brain and visual system. There are two distinct genetic lineages of ZIKV: African and Asian. Asian lineage ZIKVs have been associated with adverse pregnancy outcomes in humans; however, recent evidence from experimental models suggests that African-lineage viruses can also be vertically transmitted and cause fetal harm. Methodology/Principal FindingsTo evaluate the potential for vertical transmission of African-lineage ZIKV, we inoculated nine pregnant rhesus macaques (Macaca mulatta) subcutaneously with 44 plaque- forming units of a ZIKV strain from Senegal, (ZIKV-DAK). Dams were inoculated either at gestational day 30 or 45. Following maternal inoculation, pregnancies were surgically terminated seven or 14 days later and fetal and maternal-fetal interface tissues were collected and evaluated. Infection in the dams was evaluated via plasma viremia and neutralizing antibody titers pre- and post- ZIKV inoculation. All dams became productively infected and developed strong neutralizing antibody responses. ZIKV RNA was detected in maternal-fetal interface tissues (placenta, decidua, and fetal membranes) by RT-qPCR and in situ hybridization. In situ hybridization detected ZIKV predominantly in the decidua and revealed that the fetal membranes may play a role in ZIKV vertical transmission. Infectious ZIKV was detected in the amniotic fluid of three pregnancies and one fetus had ZIKV RNA detected in multiple tissues. No significant pathology was observed in any fetus; however, we did find an increase in the occurrence of decidual vasculitis and necrosis in ZIKV-exposed pregnancies compared to gestational-age-matched controls. Conclusions/SignificanceThis study demonstrates that African-lineage ZIKV, like Asian-lineage ZIKV, can be vertically transmitted to the macaque fetus during pregnancy. The low inoculating dose used in this study suggests a low minimal infectious dose for rhesus macaques. Vertical transmission with a low dose in macaques further supports the high epidemic potential of African ZIKV strains. Author SummaryZika virus infection during pregnancy can result in adverse pregnancy outcomes including birth defects and miscarriage. There are two distinct genetic backgrounds of Zika virus: Asian-lineage and African-lineage. Currently, only Asian-lineage Zika virus is causally associated with adverse pregnancy outcomes in people. However, experimental studies have shown that African-lineage Zika virus can infect the fetus during pregnancy and cause adverse outcomes. Adverse pregnancy outcomes may not be associated with infection in people until there is an outbreak in a naive population. Thus, as African-lineage Zika virus continues to spread globally, the risk that it may pose to pregnant people remains a public health concern. In this study, we demonstrate that African-lineage Zika virus can be transmitted from the mother to the fetus during pregnancy. This study is significant because we used rhesus macaques, an animal that shares many key elements of Zika virus infection in pregnant people. This study is also significant because we inoculated pregnant macaques with a very small amount of virus, suggesting that fetal infection reported in previously published macaque studies is not limited to high-dose inoculation.

pathology↗

bioRxiv2022

Avian H7N9 influenza viruses are evolutionarily constrained by stochastic processes during replication and transmission in mammals

H7N9 avian influenza viruses (AIV) have caused over 1,500 documented human infections since emerging in 2013. Although wild type H7N9 AIV can transmit by respiratory droplets in ferrets, they have not yet caused widespread outbreaks in humans. Previous studies have revealed molecular determinants of H7N9 AIV virus host-switching, but little is known about potential evolutionary constraints on this process. Here we compare patterns of sequence evolution for H7N9 AIV and mammalian H1N1 viruses during replication and transmission in ferrets. We show that three main factors - purifying selection, stochasticity, and very narrow transmission bottlenecks - combine to severely constrain the ability of H7N9 AIV to effectively adapt to mammalian hosts in isolated, acute spillover events. We find rare evidence of natural selection favoring new or mammalian-adapting mutations within ferrets, but no evidence of natural selection acting during transmission. We conclude that human-adapted H7N9 viruses are unlikely to emerge during typical spillover infections. Our findings are instead consistent with a model in which the emergence of a human-transmissible virus would be a rare and unpredictable, though highly consequential, "jackpot" event. Strategies to limit the total number of spillover infections will limit opportunities for the virus to win this evolutionary lottery.

microbiology↗

bioRxiv2022

Fetal loss in pregnant rhesus macaques infected with high-dose African-lineage Zika virus

Countermeasures against Zika virus (ZIKV), including vaccines, are frequently tested in nonhuman primates (NHP). Macaque models are important for understanding how ZIKV infections impact human pregnancy due to similarities in placental development. The lack of consistent adverse pregnancy outcomes in ZIKV-affected pregnancies poses a challenge in macaque studies where group sizes are often small (4-8 animals). Studies in small animal models suggest that African-lineage Zika viruses can cause more frequent and severe fetal outcomes. No adverse outcomes were observed in macaques inoculated with a low dose of African-lineage ZIKV at gestational day (GD) 45. Here, we inoculate eight pregnant rhesus macaques with a higher dose of African-lineage ZIKV at GD 45 to test the hypothesis that adverse pregnancy outcomes are dose-dependent. Three of eight pregnancies ended prematurely with fetal death. ZIKV was detected in both fetal and placental tissues from all cases of early fetal loss. Further refinements of this challenge system (e.g., varying the dose and timing of infection) could lead to an even more consistent, unambiguous fetal loss phenotype for assessing ZIKV countermeasures in pregnancy. These data demonstrate that high-dose inoculation with African-lineage ZIKV causes pregnancy loss in macaques and also suggest that ZIKV-induced first trimester pregnancy could be strain-specific. Author summaryAlthough pregnant rhesus macaques are susceptible to infection with Zika virus (ZIKV), fetal phenotypes can be subtle and variable. Most macaque studies of ZIKV have involved infection with Asian-lineage viruses because these viruses caused Western Hemisphere outbreaks beginning in 2015. African-lineage ZIKV yields more severe adverse fetal outcomes in small animal models. Here, we provide evidence that pregnant macaques infected late in the first trimester using a high dose of African-lineage ZIKV have frequent ZIKV-associated pregnancy loss. This severe phenotype establishes a new model for evaluating countermeasures and reinforces the idea that African-lineage ZIKV infection may be a frequent cause of pregnancy loss in areas where it is endemic depending on the amount of transmitted virus during infection.

pathology↗

bioRxiv2021

Prior dengue immunity enhances Zika virus infection of the maternal-fetal interface in rhesus macaques

Concerns have arisen that pre-existing immunity to dengue virus (DENV) could enhance Zika virus (ZIKV) disease, due to the homology between ZIKV and DENV and the observation of antibody-dependent enhancement (ADE) among DENV serotypes. To date, no study has examined the impact of pre-existing DENV immunity on ZIKV pathogenesis during pregnancy in a translational non-human primate model. Here we show that prior DENV-2 exposure enhanced ZIKV infection of maternal-fetal interface tissues in macaques. However, pre-existing DENV immunity had no detectable impact on ZIKV replication kinetics in maternal plasma, and all pregnancies progressed to term without adverse outcomes or gross fetal abnormalities detectable at delivery. Understanding the risks of ADE to pregnant women worldwide is critical as vaccines against DENV and ZIKV are developed and licensed and as DENV and ZIKV continue to circulate.

microbiology↗

bioRxiv2020

African-lineage Zika virus replication dynamics and maternal-fetal interface infection in pregnant rhesus macaques

Following the Zika virus (ZIKV) outbreak in the Americas, ZIKV was causally associated with microcephaly and a range of neurological and developmental symptoms, termed congenital Zika syndrome (CZS). The isolates responsible for this outbreak belonged to the Asian lineage of ZIKV. However, in-vitro and in-vivo studies assessing the pathogenesis of African-lineage ZIKV demonstrated that African-lineage isolates often replicated to high titer and caused more severe pathology than Asian-lineage isolates. To date, the pathogenesis of African-lineage ZIKV in a translational model, particularly during pregnancy, has not been rigorously characterized. Here we infected four pregnant rhesus macaques with a low-passage strain of African-lineage ZIKV and compared its pathogenesis to a cohort of four pregnant rhesus macaques infected with an Asian-lineage isolate and a cohort of mock-infected controls. Viral replication kinetics were not significantly different between the two experimental groups and both groups developed robust neutralizing antibody titers above levels considered to be protective. There was no evidence of significant fetal head growth restriction or gross fetal harm at delivery in either group. However, a significantly higher burden of ZIKV vRNA was found in maternal-fetal interface tissues in the macaques exposed to an African-lineage isolate. Our findings suggest that ZIKV isolates of any genetic lineage pose a threat to women and their infants. IMPORTANCEZIKV was first identified over 70 years ago in Africa, but most of our knowledge of ZIKV is based on studies of the distinct Asian genetic lineage, which caused the outbreak in the Americas in 2015-16. In its most recent update, the WHO stated that improved understanding of African-lineage pathogenesis during pregnancy must be a priority. Recent detection of African-lineage isolates in Brazil underscores the need to understand the impact of these viruses. Here we provide the first comprehensive assessment of African-lineage ZIKV infection during pregnancy in a translational non-human primate model. We show African-lineage isolates replicate with similar kinetics to Asian-lineage isolates and are capable of infecting the placenta. However, there was no evidence of more severe outcomes with African-lineage isolates. Our results highlight both the threat that African-lineage ZIKV poses to women and their infants and the need for future epidemiological and translational in-vivo studies with African-lineage ZIKV.

microbiology↗

bioRxiv2020

Rapid evolution of enhanced Zika virus virulence during direct vertebrate transmission chains

Zika virus (ZIKV) has the unusual capacity to circumvent natural alternating mosquito-human transmission and be directly transmitted human-to-human via sexual and vertical routes. The impact of direct transmission on ZIKV evolution and adaptation to vertebrate hosts is unknown. Here we show that molecularly barcoded ZIKV rapidly adapted to a mammalian host during direct transmission chains in mice, coincident with the emergence of an amino acid substitution previously shown to enhance virulence. In contrast, little to no adaptation of ZIKV to mice was observed following chains of direct transmission in mosquitoes or alternating host transmission. Detailed genetic analyses revealed that ZIKV evolution in mice was generally more convergent and subjected to more relaxed purifying selection than in mosquitoes or alternate passages. These findings suggest that prevention of direct human transmission chains may be paramount to resist gains in ZIKV virulence.

microbiology↗