Abstract Chikungunya virus (CHIKV) causes periodic outbreaks and is endemic in more than 110 countries. VIMKUNYA, a CHIKV virus-like particle (CHIKV VLP) vaccine, was recently approved by regulators in the United States, European Union, and United Kingdom. Efficacy of VIMKUNYA in endemic settings is difficult to evaluate due to outbreak unpredictability. We used cynomolgus macaques, which model human CHIKV viremia and disease, to assess CHIKV VLP vaccine efficacy. Doses as low as 1.25 μg of CHIKV VLP with aluminum hydroxide adjuvant and passively transferred IgG from vaccinated humans significantly reduced viremia, disease, and joint pathology. Animals that received IgG doses resulting in mean reciprocal 80% neutralization titers of 35, well below the predicted protective threshold of ≥100, exhibited improved clinical outcomes compared with CHIKV-infected control animals, suggesting clinical benefits may occur at lower antibody levels. These findings demonstrate immunogenicity and protective efficacy of CHIKV VLP and relevance of neutralizing antibodies in protection, reinforcing its use in humans to protect against chikungunya disease.

Zika virus (ZIKV) epidemics with infections in pregnant women are associated with severe neurological disease in newborns. Although an arbovirus, ZIKV is also blood transfusion‐transmitted (TT). Greater knowledge of the efficiency of ZIKV TT would aid decisions on testing and pathogen reduction technologies (PRT).

There is enormous variation in the extent to which fetal Zika virus (fZIKV) infection affects the developing brain. Despite the neural consequences of fZIKV infection observed in people and animal models, many open questions about the relationship between infection dynamics and fetal and infant development remain. To further understand how ZIKV affects the developing nervous system and the behavioral consequences of prenatal infection, we adopted a nonhuman primate model of fZIKV infection in which we inoculated pregnant rhesus macaques and their fetuses with ZIKV in the early second trimester of fetal development. We then tracked their health across gestation and characterized infant development across the first month of life. ZIKV-infected pregnant mothers had long periods of viremia and mild changes to their hematological profiles. ZIKV RNA concentrations, an indicator of infection magnitude, were higher in mothers whose fetuses were male, and the magnitude of ZIKV RNA in the mothers' plasma or amniotic fluid predicted infant outcomes. The magnitude of ZIKV RNA was negatively associated with infant growth across the first month of life, affecting males' growth more than females' growth, although for most metrics, both males and females evidenced slower growth rates as compared with control animals whose mothers were not ZIKV inoculated. Compared with control infants, fZIKV infants also spent more time with their mothers during the first month of life, a social behavior difference that may have long-lasting consequences on psychosocial development during childhood.

Zika virus (ZIKV) is a mosquito-borne arbovirus that can cause severe congenital birth defects. The utmost goal of ZIKV vaccines is to prevent both maternal-fetal infection and congenital Zika syndrome. A Zika purified inactivated virus (ZPIV) was previously shown to be protective in non-pregnant mice and rhesus macaques. In this study, we further examined the efficacy of ZPIV against ZIKV infection during pregnancy in immunocompetent C57BL6 mice and common marmoset monkeys (Callithrix jacchus). We showed that, in C57BL/6 mice, ZPIV significantly reduced ZIKV-induced fetal malformations. Protection of fetuses was positively correlated with virus-neutralizing antibody levels. In marmosets, the vaccine prevented vertical transmission of ZIKV and elicited neutralizing antibodies that remained above a previously determined threshold of protection for up to 18 months. These proof-of-concept studies demonstrate ZPIV's protective efficacy is both potent and durable and has the potential to prevent the harmful consequence of ZIKV infection during pregnancy.

Zika virus (ZIKV) is unique among mosquito-borne flaviviruses in that it is also vertically and sexually transmitted by humans. The male reproductive tract is thought to be a ZIKV reservoir; however, the reported magnitude and duration of viral persistence in male genital tissues vary widely in humans and non-human primate models. ZIKV tissue and cellular tropism and potential effects on male fertility also remain unclear. The objective of this study was to resolve these questions by analyzing archived genital tissues from 51 ZIKV-inoculated male macaques and correlating data on plasma viral kinetics, tissue tropism, and ZIKV-induced pathological changes in the reproductive tract. We hypothesized that ZIKV would persist in the male macaque genital tract for longer than there was detectable viremia, where it would localize to germ and epithelial cells and associate with lesions. We detected ZIKV RNA and infectious virus in testis, epididymis, seminal vesicle, and prostate gland. In contrast to prepubertal males, sexually mature macaques were significantly more likely to harbor persistent ZIKV RNA or infectious virus somewhere in the genital tract, with detection as late as 60 days post-inoculation. ZIKV RNA localized primarily to testicular stem cells/sperm precursors and epithelial cells, including Sertoli cells, epididymal duct epithelium, and glandular epithelia of the seminal vesicle and prostate gland. ZIKV infection was associated with microscopic evidence of inflammation in the epididymis and prostate gland of sexually mature males, pathologies that were absent in uninfected controls, which could have significant effects on male fertility. The findings from this study increase our understanding of persistent ZIKV infection which can inform risk of sexual transmission during assisted reproductive therapies as well as potential impacts on male fertility.

Human infection by Zika virus (ZIKV) during pregnancy can lead to vertical transmission and fetal aberrations, including microcephaly. Prophylactic administration of antibodies can diminish or prevent ZIKV infection in animal models, but whether passive immunization can protect nonhuman primates and their fetuses during pregnancy has not been determined. Z004 and Z021 are neutralizing monoclonal antibodies to domain III of the envelope (EDIII) of ZIKV. Together the two antibodies protect nonpregnant macaques against infection even after Fc modifications to prevent antibody-dependent enhancement (ADE) in vitro and extend their half-lives. Here we report on prophylactic coadministration of the Fc-modified antibodies to pregnant rhesus macaques challenged three times with ZIKV during first and second trimester. The two antibodies did not entirely eliminate maternal viremia but limited vertical transmission, protecting the fetus from neurologic damage. Thus, maternal passive immunization with two antibodies to EDIII can shield primate fetuses from the harmful effects of ZIKV.

Although fetal death is now understood to be a severe outcome of congenital Zika syndrome, the role of viral genetics is still unclear. We sequenced Zika virus (ZIKV) from a rhesus macaque fetus that died after inoculation and identified a single intrahost substitution, M1404I, in the ZIKV polyprotein, located in nonstructural protein 2B (NS2B). Targeted sequencing flanking position 1404 in 9 additional macaque mothers and their fetuses identified M1404I at a subconsensus frequency in the majority (5 of 9, 56%) of animals and some of their fetuses. Despite its repeated presence in pregnant macaques, M1404I has occurred rarely in humans since 2015. Since the primary ZIKV transmission cycle is human-mosquito-human, mutations in one host must be retained in the alternate host to be perpetuated. We hypothesized that ZIKV I1404 increases viral fitness in nonpregnant macaques and pregnant mice but is less efficiently transmitted by vectors, explaining its low frequency in humans during outbreaks. By examining competitive fitness relative to that of ZIKV M1404, we observed that ZIKV I1404 produced lower viremias in nonpregnant macaques and was a weaker competitor in tissues. In pregnant wild-type mice, ZIKV I1404 increased the magnitude and rate of placental infection and conferred fetal infection, in contrast to ZIKV M1404, which was not detected in fetuses. Although infection and dissemination rates were not different, Aedes aegypti mosquitoes transmitted ZIKV I1404 more poorly than ZIKV M1404. Our data highlight the complexity of arbovirus mutation-fitness dynamics and suggest that intrahost ZIKV mutations capable of augmenting fitness in pregnant vertebrates may not necessarily spread efficiently via mosquitoes during epidemics.IMPORTANCE Although Zika virus infection of pregnant women can result in congenital Zika syndrome, the factors that cause the syndrome in some but not all infected mothers are still unclear. We identified a mutation that was present in some ZIKV genomes in experimentally inoculated pregnant rhesus macaques and their fetuses. Although we did not find an association between the presence of the mutation and fetal death, we performed additional studies with ZIKV with the mutation in nonpregnant macaques, pregnant mice, and mosquitoes. We observed that the mutation increased the ability of the virus to infect mouse fetuses but decreased its capacity to produce high levels of virus in the blood of nonpregnant macaques and to be transmitted by mosquitoes. This study shows that mutations in mosquito-borne viruses like ZIKV that increase fitness in pregnant vertebrates may not spread in outbreaks when they compromise transmission via mosquitoes and fitness in nonpregnant hosts.

Congenital Zika syndrome (CZS) is associated with microcephaly and various neurological, musculoskeletal, and ocular abnormalities, but the long-term pathogenesis and postnatal progression of ocular defects in infants are not well characterized. Rhesus macaques are superior to rodents as models of CZS because they are natural hosts of the virus and share similar immune and ocular characteristics, including blood-retinal barrier characteristics and the unique presence of a macula. Using a previously described model of CZS, we infected pregnant rhesus macaques with Zika virus (ZIKV) during the late first trimester and characterized postnatal ocular development and evolution of ocular defects in 2 infant macaques over 2 years. We found that one of them exhibited colobomatous chorioretinal atrophic lesions with macular and vascular dragging as well as retinal thinning caused by loss of retinal ganglion neuron and photoreceptor layers. Despite these congenital ocular malformations, axial elongation and retinal development in these infants progressed at normal rates compared with healthy animals. The ZIKV-exposed infants displayed a rapid loss of ZIKV-specific antibodies, suggesting the absence of viral replication after birth, and did not show any behavioral or neurological defects postnatally. Our findings suggest that ZIKV infection during early pregnancy can impact fetal retinal development and cause congenital ocular anomalies but does not appear to affect postnatal ocular growth.

Zika virus (ZIKV) infection of pregnant women is associated with congenital Zika syndrome (CZS) and no vaccine is available, although several are being tested in clinical trials. We tested the efficacy of ZIKV DNA vaccine VRC5283 in a rhesus macaque model of congenital ZIKV infection. Most animal vaccine experiments have a set pathogen exposure several weeks or months after vaccination. In the real world, people encounter pathogens years or decades after vaccination, or may be repeatedly exposed if the virus is endemic. To more accurately mimic how this vaccine would be used, we immunized macaques before conception and then exposed them repeatedly to ZIKV during early and mid-gestation. In comparison to unimmunized animals, vaccinated animals had a significant reduction in peak magnitude and duration of maternal viremia, early fetal loss, fetal infection, and placental and fetal brain pathology. Vaccine-induced neutralizing antibody titers on the day of first ZIKV exposure were negatively associated with the magnitude of maternal viremia, and the absence of prolonged viremia was associated with better fetal outcomes. These data support further clinical development of ZIKV vaccine strategies to protect against negative fetal outcomes.

Recent data in a nonhuman primate model showed that infants postnatally infected with Zika virus (ZIKV) were acutely susceptible to high viremia and neurological damage, suggesting the window of vulnerability extends beyond gestation. In this pilot study, we addressed the susceptibility of two infant rhesus macaques born healthy to dams infected with Zika virus during pregnancy. Passively acquired neutralizing antibody titers dropped below detection limits between 2 and 3 months of age, while binding antibodies remained detectable until viral infection at 5 months. Acute serum viremia was comparatively lower than adults infected with the same Brazilian isolate of ZIKV (n = 11 pregnant females, 4 males, and 4 non-pregnant females). Virus was never detected in cerebrospinal fluid nor in neural tissues at necropsy two weeks after infection. However, viral RNA was detected in lymph nodes, confirming some tissue dissemination. Though protection was not absolute and our study lacks an important comparison with postnatally infected infants born to naïve dams, our data suggest infants born healthy to infected mothers may harbor a modest but important level of protection from postnatally acquired ZIKV for several months after birth, an encouraging result given the potentially severe infection outcomes of this population.

Chikungunya virus (CHIKV) is a reemerging global health threat that produces debilitating arthritis in people. Like other RNA viruses with high mutation rates, CHIKV produces populations of genetically diverse genomes within a host. While several known CHIKV mutations influence disease severity in vertebrates and transmission by mosquitoes, the role of intrahost diversity in chikungunya arthritic disease has not been studied. In this study, high-and low-fidelity CHIKV variants, previously characterized by altered in vitro population mutation frequencies, were used to evaluate how intrahost diversity influences clinical disease, CHIKV replication, and antibody neutralization in immunocompetent adult mice inoculated in the rear footpads. Both high-and low-fidelity mutations were hypothesized to attenuate CHIKV arthritic disease, replication, and neutralizing antibody levels compared to wild-type (WT) CHIKV. Unexpectedly, high-fidelity mutants elicited more severe arthritic disease than the WT despite comparable CHIKV replication, whereas a low-fidelity mutant produced attenuated disease and replication. Serum antibody developed against both high-and low-fidelity CHIKV exhibited reduced neutralization of WT CHIKV. Using next-generation sequencing (NGS), the high-fidelity mutations were demonstrated to be genetically stable but produced more genetically diverse populations than WT CHIKV in mice. This enhanced diversification was subsequently reproduced after serial in vitro passage. The NGS results contrast with previously reported population diversities for fidelity variants, which focused mainly on part of the E1 gene, and highlight the need for direct measurements of mutation rates to clarify CHIKV fidelity phenotypes. IMPORTANCE CHIKV is a reemerging global health threat that elicits debilitating arthritis in humans. There are currently no commercially available CHIKV vaccines. Like other RNA viruses, CHIKV has a high mutation rate and is capable of rapid intrahost diversification during an infection. In other RNA viruses, virus population diversity associates with disease progression; however, potential impacts of intrahost viral diversity on CHIKV arthritic disease have not been studied. Using previously characterized CHIKV fidelity variants, we addressed whether CHIKV population diversity influences the severity of arthritis and host antibody response in an arthritic mouse model. Our findings show that CHIKV populations with greater genetic diversity can cause more severe disease and stimulate antibody responses with reduced neutralization of low-diversity virus populations in vitro. The discordant high-fidelity phenotypes in this study highlight the complexity of inferring replication fidelity indirectly from population diversity.

Zika virus (ZIKV) will remain a public health threat until effective vaccines and therapeutics are made available in the hardest hit areas of the world. Recent data in a nonhuman primate model showed that infants postnatally infected with ZIKV were acutely susceptible to high viremia and neurological damage, suggesting the window of vulnerability extends beyond gestation. We addressed the susceptibility of two infant rhesus macaques born healthy to dams infected with Zika virus during pregnancy. Passively acquired neutralizing antibody titers dropped below detection limits between 2 and 3 months of age, while binding, possibly non-neutralizing antibodies remained detectable until viral infection at 5 months of age. Post-infection acute serum viremia was substantially reduced relative to adults infected with the same dose of the same stock of a Brazilian isolate of ZIKV (n=11 pregnant females) and another stock of the same isolate (n=4 males and 4 non-pregnant females). Virus was never detected in cerebrospinal fluid nor in neural tissues at necropsy two weeks after infection, suggesting reduced viral burden relative to adults and published data from infants. However, viral RNA was detected in lymph nodes, confirming some tissue dissemination. Though protection was not absolute, our data suggest infants born healthy to infected mothers may harbor a modest but important level of protection from postnatally acquired ZIKV for several months after birth, an encouraging result given the potentially severe infection outcomes of this population.

ObjectivesOur aims were to examine the prevalence and genetic predictors of aspirin and clopidogrel high on-treatment platelet reactivity (HoTPR), and associated adverse cardiovascular outcomes in patients with peripheral arterial disease (PAD). BackgroundThe association of aspirin and clopidogrel HoTPR with outcomes in PAD remains unclear. MethodsThis is a prospective cohort study of patients with angiographically documented PAD involving carotid and lower extremity arteries. Aspirin and clopidogrel HoTPR (using the VerifyNow Assay) and associated genetic predictors were compared to clinical outcomes. The primary end-point was a composite of major adverse cardiovascular events: all-cause mortality, myocardial infarction, stroke, target vessel revascularization (TVR) and limb-loss in patients who underwent extremity intervention. ResultsThe study was stopped prematurely due to slow patient enrolment. Of 195 patients enrolled, the primary analysis was performed in 154 patients taking both drugs. Aspirin HoTPR was present in 31 (20%) and clopidogrel HoTPR in 76 (49%) patients. There was a trend toward more primary composite outcome events with PRU235 (52% freedom-from-event rate vs. 70% for PRU<235; P=0.09). TVR was higher in those with PRU235 (20 vs. 6%, unadjusted P=0.02). There was no association between aspirin HoTPR and combined outcomes. Single nucleotide polymorphisms in serum paraoxonase/arylesterase 1 (PON1) gene was associated with aspirin HoTPR (P=0.005) while SNP in phospholipase A2, group III (PLA2G3) gene was associated with clopidogrel HoTPR (P=0.002). ConclusionClopidogrel HoTPR was significantly associated with TVR, while aspirin HoTPR was not associated with adverse clinical outcomes in patients with PAD.

Zika virus (ZIKV) causes severe neurologic complications and fetal aberrations. Vaccine development is hindered by potential safety concerns due to antibody cross-reactivity with dengue virus and the possibility of disease enhancement. In contrast, passive administration of anti-ZIKV antibodies engineered to prevent enhancement may be safe and effective. Here, we report on human monoclonal antibody Z021, a potent neutralizer that recognizes an epitope on the lateral ridge of the envelope domain III (EDIII) of ZIKV and is protective against ZIKV in mice. When administered to macaques undergoing a high-dose ZIKV challenge, a single anti-EDIII antibody selected for resistant variants. Co-administration of two antibodies, Z004 and Z021, which target distinct sites on EDIII, was associated with a delay and a 3-to 4-log decrease in peak viremia. Moreover, the combination of these antibodies engineered to avoid enhancement prevented viral escape due to mutation in macaques, a natural host for ZIKV.

Scented sugar baits deployed in California deserts detected early West Nile virus (WNV) transmission by mosquitoes, representing a potential improvement to conventional arbovirus surveillance that relies heavily on infection rates in mosquito pools. In this study, we expanded deployment of scented sugar baits into suburban Sacramento and Yolo (2015, 2016) and Riverside Counties (2016), California. The goal of the study was to determine whether scented sugar baits detect WNV and St. Louis encephalitis virus (SLEV) concurrent with mosquito infections in trapped pools in areas of high human density. Between 8 and 10% of sugar baits were WNV RNA positive in both study years across the three counties. In Riverside County, where SLEV re-emerged in 2015, 1% of sugar baits were SLEV positive in 2016. Rates of sugar bait positives were at least 100 times higher than infection rates in trapped mosquitoes in the same districts. The prevalence of sugar bait positives varied temporally and did not coincide with infections in mosquitoes collected at the same sites each week. WNV RNA positive sugar baits were detected up to 2 wk before and after concurrent surveillance detected infection in mosquito pools at the same sites. Sugar baits also detected WNV in Riverside County at locations where no WNV activity was detected in mosquito pools. Sugar baits generated between 0.8 and 1.2 WNV positives per $1,000 and can be more economical than carbon dioxide baited traps that produce 0.8 positives per $1,000. These results indicate that the sugar bait approach enhances conventional arbovirus surveillance in mosquitoes in suburban California.

Fluconazole-induced alopecia is a significant problem for patients receiving long-term therapy. We evaluated the hair cycle changes of fluconazole in a rat model and investigated potential molecular mechanisms. Plasma and tissue levels of retinoic acid were not found to be causal. Human patients with alopecia attributed to fluconazole also underwent detailed assessment and in both our murine model and human cohort fluconazole induced telogen effluvium. Future work further examining the mechanism of fluconazole-induced alopecia should be undertaken.