Search

The global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated coronavirus disease (COVID-19) has led to a pandemic of unprecedented scale. An intriguing feature of the infection is the minimal disease in most children, a demographic at higher risk for other respiratory viral diseases. To investigate age-dependent effects of SARS-CoV-2 pathogenesis, we inoculated two rhesus macaque monkey dam–infant pairs with SARS-CoV-2 and conducted virological and transcriptomic analyses of the respiratory tract and evaluated systemic cytokine and Ab responses. Viral RNA levels in all sampled mucosal secretions were comparable across dam–infant pairs in the respiratory tract. Despite comparable viral loads, adult macaques showed higher IL-6 in serum at day 1 postinfection whereas CXCL10 was induced in all animals. Both groups mounted neutralizing Ab responses, with infants showing a more rapid induction at day 7. Transcriptome analysis of tracheal airway cells isolated at day 14 postinfection revealed significant upregulation of multiple IFN-stimulated genes in infants compared with adults. In contrast, a profibrotic transcriptomic signature with genes associated with cilia structure and function, extracellular matrix composition and metabolism, coagulation, angiogenesis, and hypoxia was induced in adults compared with infants. Our study in rhesus macaque monkey dam–infant pairs suggests age-dependent differential airway responses to SARS-CoV-2 infection and describes a model that can be used to investigate SARS-CoV-2 pathogenesis between infants and adults.

The global spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its associated coronavirus disease (COVID-19) has led to a pandemic of unprecedented scale. An intriguing feature of the infection is the minimal disease in most children, a demographic at higher risk for respiratory viral diseases. To elucidate age-dependent effects of SARS-CoV-2 pathogenesis, we inoculated two rhesus macaque monkey dam-infant pairs with SARS-CoV-2 and conducted virological and transcriptomic analysis of the respiratory tract and evaluated systemic cytokine and antibody responses. Viral RNA levels in all sampled mucosal secretions were comparable across dam-infant pairs in the respiratory tract. Despite comparable viral loads, adult macaques showed higher IL-6 in serum while CXCL10 was induced in all animals. Both groups mounted neutralizing antibody (nAb) responses, with infants showing a more rapid induction at day 7. Transcriptome analysis of tracheal tissue isolated at day 14 post-infection revealed significant upregulation of multiple interferon-stimulated genes in infants compared to adults. In contrast, a profibrotic transcriptomic signature with genes associated with cilia structure and function, extracellular matrix (ECM) composition and metabolism, coagulation, angiogenesis, and hypoxia was induced in adults compared to infants. Our observations suggest age-dependent differential airway responses to SARS-CoV-2 infection that could explain the distinction in pathogenesis between infants and adults.

Human clinical studies investigating use of convalescent plasma (CP) for treatment of coronavirus disease 2019 (COVID-19) have produced conflicting results. Outcomes in these studies may vary at least partly due to different timing of CP administration relative to symptom onset. The mechanisms of action of CP include neutralizing antibodies but may extend beyond virus neutralization to include normalization of blood clotting and dampening of inflammation. Unresolved questions include the minimum therapeutic titer in the CP units or CP recipient as well as the optimal timing of administration. Here, we show that treatment of macaques with CP within 24 h of infection does not reduce viral shedding in nasal or lung secretions compared to controls and does not detectably improve any clinical endpoint. We also demonstrate that CP administration does not impact viral sequence diversity in vivo, although the selection of a viral sequence variant in both macaques receiving normal human plasma was suggestive of immune pressure. Our results suggest that CP, administered to medium titers, has limited efficacy, even when given very early after infection. Our findings also contribute information important for the continued development of the nonhuman primate model of COVID-19. These results should inform interpretation of clinical studies of CP in addition to providing insights useful for developing other passive immunotherapies and vaccine strategies. IMPORTANCE Antiviral treatment options for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain very limited. One treatment that was explored beginning early in the pandemic (and that is likely to be tested early in future pandemics) is plasma collected from people who have recovered from coronavirus disease 2019 (COVID-19), known as convalescent plasma (CP). We tested if CP reduces viral shedding or disease in a nonhuman primate model. Our results demonstrate that administration of CP 1 day after SARS-CoV-2 infection had no significant impact on viral loads, clinical disease, or sequence diversity, although treatment with normal human plasma resulted in selection of a specific viral variant. Our results demonstrate that passive immunization with CP, even during early infection, provided no significant benefit in a nonhuman primate model of SARS-CoV-2 infection.

Anti-viral monoclonal antibody (mAb) treatments may provide immediate but short-term immunity from coronavirus disease 2019 (COVID-19) in high-risk populations, such as people with diabetes and the elderly; however, data on their efficacy in these populations are limited. We demonstrate that prophylactic mAb treatment blocks viral replication in both the upper and lower respiratory tracts in aged, type 2 diabetic rhesus macaques. mAb infusion dramatically curtails severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-mediated stimulation of interferon-induced chemokines and T cell activation, significantly reducing development of interstitial pneumonia. Furthermore, mAb infusion significantly dampens the greater than 3-fold increase in SARS-CoV-2-induced effector CD4 T cell influx into the cerebrospinal fluid. Our data show that neutralizing mAbs administered preventatively to high-risk populations may mitigate the adverse inflammatory consequences of SARS-CoV-2 exposure., Graphical abstract, Verma et al. observe that prophylactic mAbs limit SARS-CoV-2 replication and immune activation. In aged diabetic rhesus macaques, these protective mechanisms took place in the areas of the body most highly targeted by the virus and the respiratory, nervous, and circulatory systems.

Early in the SARS-CoV-2 pandemic, there was a high level of optimism based on observational studies and small controlled trials that treating hospitalized patients with convalescent plasma from COVID-19 survivors (CCP) would be an important immunotherapy. However, as more data from controlled trials became available, the results became disappointing, with at best moderate evidence of efficacy when CCP with high titers of neutralizing antibodies was used early in infection. To better understand the potential therapeutic efficacy of CCP, and to further validate SARS-CoV-2 infection of macaques as a reliable animal model for testing such strategies, we inoculated 12 adult rhesus macaques with SARS-CoV-2 by intratracheal and intranasal routes. One day later, 8 animals were infused with pooled human CCP with a high titer of neutralizing antibodies (RVPN NT50 value of 3,003), while 4 control animals received normal human plasma. Animals were monitored for 7 days. Animals treated with CCP had detectable levels of antiviral antibodies after infusion. In comparison to the control animals, they had similar levels of virus replication in the upper and lower respiratory tract, but had significantly reduced interstitial pneumonia, as measured by comprehensive lung histology. By highlighting strengths and weaknesses, data of this study can help to further optimize nonhuman primate models to provide proof-of-concept of intervention strategies, and guide the future use of convalescent plasma against SARS-CoV-2 and potentially other newly emerging respiratory viruses., Author summary The results of treating SARS-CoV-2 infected hospitalized patients with COVID-19 convalescent plasma (CCP), collected from survivors of natural infection, have been disappointing. The available data from various studies indicate at best moderate clinical benefits only when CCP with high titer of neutralizing antibodies was infused early in infection. The macaque model of SARS-CoV-2 infection can be useful to gain further insights in the value of CCP therapy. In this study, animals were infected with SARS-CoV-2 and the next day, were infused with pooled human convalescent plasma, selected to have a very high titer of neutralizing antibodies. While administration of CCP did not result in a detectable reduction in virus replication in the respiratory tract, it significantly reduced lung inflammation. These data, combined with the results of monoclonal antibody studies, emphasize the need to use products with high titers of neutralizing antibodies, and guide the future development of CCP-based therapies.

There is an urgent need for effective therapeutic interventions against SARS-CoV-2, including new variants that continue to arise. Neutralizing monoclonal antibodies have shown promise in clinical studies. We investigated the therapeutic efficacy of a combination of two potent monoclonal antibodies, C135-LS and C144-LS that carry half-life extension mutations, in the rhesus macaque model of COVID-19. Twelve young adult macaques (three groups of four animals) were inoculated intranasally and intra-tracheally with a high dose of SARS-CoV-2 and 24 hours later, treated intravenously with a high (40 mg/kg) or low (12 mg/kg) dose of the C135-LS and C144-LS antibody combination, or a control monoclonal antibody. Animals were monitored for 7 days. Compared to the control animals, animals treated with either dose of the anti-SARS-CoV-2 antibodies showed similarly improved clinical scores, lower levels of virus replication in upper and lower respiratory tract, and significantly reduced interstitial pneumonia, as measured by comprehensive lung histology. In conclusion, this study provides proof-of-concept in support of further clinical development of these monoclonal antibodies against COVID-19 during early infection., Author summary Monoclonal antibodies that neutralize SARS-CoV-2 have shown promise in treating recently infected individuals who are at high risk of progression to severe COVID-19 disease. Although several monoclonal antibodies are currently being used in the clinic, there is an ongoing need to develop additional antibodies. The ideal monoclonal antibodies, or combinations, should be potent and durable, and maintain activity against emerging viral variants. In this study, we tested a combination of two potent monoclonal antibodies, C135-LS and C-144-LS, engineered to have long half-lives, in the macaque model of SARS-CoV-2 infection. Animals treated early after infection fared better than placebo-treated controls, manifesting fewer clinical signs, less virus replication in the respiratory tract, and reduced lung inflammation. These promising data support clinical testing of these monoclonal antibodies in humans and further development of similar antibody-based prophylactic and therapeutic strategies.

CD4 T follicular helper (Tfh) cells are important for the generation of durable and specific humoral protection against viral infections. The degree to which SARS-CoV-2 infection generates Tfh cells and stimulates the germinal center (GC) response is an important question as we investigate vaccine induced immunity against COVID-19. Here, we report that SARS-CoV-2 infection in rhesus macaques, either infused with convalescent plasma, normal plasma, or receiving no infusion, resulted in transient accumulation of pro-inflammatory monocytes and proliferating Tfh cells with a Th1 profile in peripheral blood. CD4 helper cell responses skewed predominantly toward a Th1 response in blood, lung, and lymph nodes. SARS-CoV-2 Infection induced GC Tfh cells specific for the SARS-CoV-2 spike and nucleocapsid proteins, and a corresponding early appearance of antiviral serum IgG antibodies. Collectively, the data show induction of GC responses in a rhesus model of mild COVID-19., Induction of CD4 T follicular helper (Tfh) cells is important for antibody responses to viral infections. Here, the authors show in a rhesus macaque model of mild COVID-19 that SARS-CoV-2 infection results in transient accumulation of proliferating Tfh cells with a Th1 profile in peripheral blood and generation of germinal center Tfh cells specific for viral proteins.

Anti-viral monoclonal antibody (mAb) treatments may provide immediate but short-term immunity from COVID-19 in high-risk populations such as aged and diabetic individuals, however, data on their efficacy in these populations is limited. We demonstrate that prophylactic mAb treatment prevented viral replication specifically in the upper respiratory tract in aged, type-2-diabetic rhesus macaques. While activation of innate inflammatory pathways was observed, mAb infusion dramatically curtailed SARS-CoV-2-mediated stimulation of interferon-induced chemokines and T cell activation, significantly reducing the development of interstitial pneumonia. Effector T cell differentiation was reduced in the draining mediastinal lymph nodes, resulting in significantly lower representation of activated cells in the spleen and blood. Consequently, mAb infusion significantly dampened the greater than three-fold increase in SARS-CoV-2-induced effector CD4 T cell influx into the cerebrospinal fluid. Our data indicate that neutralizing mAbs administered preventatively to high-risk populations may mitigate the adverse inflammatory consequences of SARS-CoV-2 exposure. Funding Information: This study was supported by grants R21 AI143454-02S1 (SSI), FAST GRANT- George Mason 358 University (SSI), 3RF1AG061001-01S1 (SSI/JHM) and the CNPRC base grant P51OD011107 Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: All study procedures were approved by the Institutional Animal Care and Use Committee at UC Davis.

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.

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., 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.

CD4 T follicular helper (Tfh) cells are important for the generation of durable and specific humoral protection against viral infections. The degree to which SARS-CoV-2 infection generates Tfh cells and stimulates the germinal center response is an important question as we investigate vaccine options for the current pandemic. Here we report that SARS-CoV-2 infection resulted in transient accumulation of pro-inflammatory monocytes and proliferating Tfh cells with a Th1 profile in peripheral blood. CD4 helper cell responses were skewed predominantly toward a Th1 response in blood, lung, and lymph nodes. We observed the generation of germinal center Tfh cells specific for the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins, and a corresponding early appearance of antiviral serum IgG antibodies. Our data suggest that a vaccine promoting Th1-type Tfh responses that target the S protein may lead to protective immunity.

Significance Zika virus (ZIKV) infection during pregnancy can cause fetal abnormalities. Vaccines against ZIKV are under development, but because of potential safety concerns due to disease-enhancing antibodies, and the time required by active immunization to induce protective antibodies, there is a need to explore alternative strategies. Recombinant monoclonal antibodies can be modified to prevent enhancement of infection, and thus could be an efficacious and safe alternative to vaccines to confer rapid protection. We show that prophylactic administration of two engineered antibodies, Z004 and Z021, to pregnant macaques partially protects against fetal neurologic damage and limits vertical transmission of ZIKV., 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.

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 prior to 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.

Zika virus (ZIKV) infection of pregnant women can cause fetal microcephaly and other neurologic defects. We describe the development of a non-human primate model to better understand fetal pathogenesis. To reliably induce fetal infection at defined times, four pregnant rhesus macaques are inoculated intravenously and intraamniotically with ZIKV at gestational day (GD) 41, 50, 64, or 90, corresponding to first and second trimester of gestation. The GD41-inoculated animal, experiencing fetal death 7 days later, has high virus levels in fetal and placental tissues, implicating ZIKV as cause of death. The other three fetuses are carried to near term and euthanized; while none display gross microcephaly, all show ZIKV RNA in many tissues, especially in the brain, which exhibits calcifications and reduced neural precursor cells. Given that this model consistently recapitulates neurologic defects of human congenital Zika syndrome, it is highly relevant to unravel determinants of fetal neuropathogenesis and to explore interventions., Zika virus infection of pregnant women can cause congenital brain defects. Here, Coffey et al. establish a pregnant rhesus macaque model, using intravenous and intraamniotic route of infection, that reliably reproduces fetal neurologic defects of congenital Zika syndrome in humans.

We tested the hypothesis that oxidant-injured cells upregulate thioredoxin, whereas oxidant-stressed, but not injured, cells upregulate interleukin (IL)-8 after injury. We exposed primary human tracheobronchial epithelial cells and transformed human bronchial epithelial cells (BEAS-2B S.6) to 0, 200, 400, or 600 microM H(2)O(2) for 1 h followed by an additional 7 h of incubation. Subsequently, the cells were double-labeled with markers of injury (either Ethidium Homodimer-1 for cellular injury or MitoTracker dye for functional mitochondria) or oxidant stress (5-[and 6]-chloromethyl-2',7'-dicholorodihydrofluorescein diacetate) and antibodies specific for the chemoattractants IL-8 or thioredoxin. We found significant inverse relationships between numbers and stained chemoattractant volumes of IL-8 and thioredoxin-positive cells with increasing H(2)O(2) dose. Cells with mitochondrial injury produced thioredoxin but not IL-8, and oxidant-stressed cells were more likely to produce thioredoxin than IL-8. Isolated human neutrophils were more likely to colocalize with thioredoxin-positive BEAS-2B S.6 cells than thioredoxin-negative cells. The H(2)O(2) injury did not induce significant apoptosis in the BEAS-2B S.6 cells as measured by caspase 3 activation. We conclude that oxidant-injured and stressed airway epithelial cells upregulate thioredoxin, but produce little IL-8, which may be important in airway epithelial cell-mediated multistep navigation of neutrophils to sites of oxidant injury.

Leukocyte recruitment from the circulation into the airways is a multi-step process, involving both chemotactic and adhesive mechanisms. Using an in vitro model of leukocyte transepithelial trafficking, we show that movement of human peripheral blood neutrophils (PMN) across airway epithelium in the optimal basolateral-to-apical surface direction is partially blocked by pertussis toxin, an inhibitor of Gαi-protein-linked receptors. A neutralizing monoclonal antibody against interleukin-8 (IL-8; constitutively expressed by airway epithelium) did not inhibit PMN transepithelial migration, suggesting that alternative pertussis toxin-sensitive signaling mechanisms are involved in this process. However, a neutralizing antibody against thioredoxin, a redox enzyme with pertussis toxin-insensitive chemoattractant activity, did reduce PMN migration across airway epithelium. We conclude that trafficking of PMN across airway epithelium is mediated by both thioredoxin- and pertussis toxin-sensitive signaling mechanisms that are independent of IL-8.

Changes in the density of bronchial vessels have been proposed as a part of airway remodeling that occurs in chronic asthma.Using an established nonhuman primate model of chronic allergic asthma, we evaluated changes in vascular density as well as the contribution of bronchial epithelium to produce vascular endothelial growth factor (VEGF).Eight juvenile rhesus macaques were divided into two groups of four. One group was exposed to 11 cycles of aerosolized house dust mite allergen (HDMA), whereas the other was exposed to filtered air. Bronchial wall vasculature was identified using an immunohistochemical approach, and vascular density was quantified stereologically. A semiquantitative polymerase chain reaction approach was used to estimate VEGF splice variant gene expression at discrete airway generations. Cell culture of primary tracheal epithelial cells with varying concentrations of HDMA was used to quantify the direct contribution of the epithelium to VEGF production.Bronchial vascular density was increased at mid- to lower airway generations, which was independent of changes in the interstitial compartment. The VEGF121 splice variant was significantly increased at lower airway generations. VEGF protein increased in a dose-dependant fashion in vitro primarily by an increase in VEGF121 gene expression.This study highlights that increased vascular density in an animal model of chronic allergic asthma is airway generation specific and associated with a unique increase of VEGF splice variant gene expression. Airway epithelium is the likely source for increased VEGF.