In vitro and in vivo functions of SARS-CoV-2 infection-enhancing and neutralizing antibodies

SARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-g (FcgR)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated FcgR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Three of 46 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can rarely occur in SARS-CoV-2 antibody-infused macaques

Aedes aegypti D7 long salivary proteins modulate blood feeding and parasite infection

Mosquito saliva facilitates blood meal acquisition through pharmacologically active compounds that prevent host hemostasis and immune responses. Here, we generated two knockout (KO) mosquito lines by CRISPR/Cas9 to functionally characterize D7L1 and D7L2, two abundantly expressed salivary proteins from the yellow fever mosquito vector Aedes aegypti. The D7s bind and scavenge biogenic amines and eicosanoids involved in hemostasis at the bite site. The absence of D7 proteins in the salivary glands of KO mosquitoes was confirmed by mass spectrometry, enzyme-linked immunosorbent assay, and fluorescence microscopy of the salivary glands with specific antibodies. D7-KO mosquitoes had longer probing times than parental wildtypes. The differences in probing time were abolished when mutant mice resistant to inflammatory insults were used. These results confirmed the role of D7 proteins as leukotriene scavengers in vivo. We also investigated the role of D7 salivary proteins in Plasmodium gallinaceum infection and transmission. Both KO lines had significantly fewer oocysts per midgut. We hypothesize that the absence of D7 proteins in the midgut of KO mosquitoes might be responsible for creating a harsh environment for the parasite. The information generated by this work highlights the biological functionality of salivary gene products in blood feeding and pathogen infection. IMPORTANCE: During blood feeding, mosquitoes inject saliva into the host skin, preventing hemostasis and inflammatory responses. D7 proteins are among the most abundant components of the saliva of blood-feeding arthropods. Aedes aegypti, the vector of yellow fever and dengue, expresses two D7 long-form salivary proteins: D7L1 and D7L2. These proteins bind and counteract hemostatic agonists such as biogenic amines and leukotrienes. D7L1 and D7L2 knockout mosquitoes showed prolonged probing times and carried significantly less Plasmodium gallinaceum oocysts per midgut than wild-type mosquitoes. We hypothesize that reingested D7s play a vital role in the midgut microenvironment with important consequences for pathogen infection and transmission.This research was supported by the Division of Intramural Research Program of the NIH/NIAID (AI001246, to E.C.) and by a subcontract from grant 1R01AI099483 (to Z.N.A.)S

Prolonging culture of primary human keratinocytes isolated from suction blisters with the Rho kinase inhibitor Y-27632.

Keratinocytes are the most abundant cell type in the epidermis. They prevent desiccation and provide immunological and barrier defense against potential pathogens such as Staphylococcus aureus and Candida albicans. The study of this first line of immune defense may be hindered by invasive isolation methods and/or improper culture conditions to support stem cell maintenance and other potential mechanisms contributing to long-term subcultivation in vitro. Primary keratinocytes have been successfully isolated from blister roofs induced by negative pressure, which separates the epidermis from the dermis in vivo in human subjects. This method allows collection of pure epidermal cells without dermal contamination in a minimally invasive manner. However, the isolated keratinocytes differentiate and senesce when cultured in vitro beyond five passages. Here, we present evidence that the Rho kinase (ROCK) inhibitor Y-27632 can be used to effectively increase the proliferative capabilities of keratinocytes isolated using the suction blister method, similar to what has been previously reported for primary keratinocytes isolated using alternative methods. We show that the increase in passage number is directly correlated to delayed differentiation, and that cells passaged long term with the inhibitor retain their ability to stratify in organotypic raft cultures and respond to cytokine treatment; additionally, the late passage cells have a heterogeneous mix of differentiated and non-differentiated cells which may be predicted by a ratio of select differentiation markers. The described method presents a minimally invasive procedure for keratinocyte isolation and prolonged culture that allows analysis of keratinocyte function in both healthy volunteers and patients with dermatologic diseases

Enhanced inflammation in New Zealand white rabbits when MERS-CoV reinfection occurs in the absence of neutralizing antibody

<div><p>The Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic betacoronavirus that was first detected in humans in 2012 as a cause of severe acute respiratory disease. As of July 28, 2017, there have been 2,040 confirmed cases with 712 reported deaths. While many infections have been fatal, there have also been a large number of mild or asymptomatic cases discovered through monitoring and contact tracing. New Zealand white rabbits are a possible model for asymptomatic infection with MERS-CoV. In order to discover more about non-lethal infections and to learn whether a single infection with MERS-CoV would protect against reinfection, we inoculated rabbits with MERS-CoV and monitored the antibody and inflammatory response. Following intranasal infection, rabbits developed a transient dose-dependent pulmonary infection with moderately high levels of viral RNA, viral antigen, and perivascular inflammation in multiple lung lobes that was not associated with clinical signs. The rabbits developed antibodies against viral proteins that lacked neutralizing activity and the animals were not protected from reinfection. In fact, reinfection resulted in enhanced pulmonary inflammation, without an associated increase in viral RNA titers. Interestingly, passive transfer of serum from previously infected rabbits to naïve rabbits was associated with enhanced inflammation upon infection. We further found this inflammation was accompanied by increased recruitment of complement proteins compared to primary infection. However, reinfection elicited neutralizing antibodies that protected rabbits from subsequent viral challenge. Our data from the rabbit model suggests that people exposed to MERS-CoV who fail to develop a neutralizing antibody response, or persons whose neutralizing antibody titers have waned, may be at risk for severe lung disease on re-exposure to MERS-CoV.</p></div

Pathogenesis and outcome of VA1 astrovirus infection in the human brain are defined by disruption of neural functions and imbalanced host immune responses.

Astroviruses (AstVs) can cause of severe infection of the central nervous system (CNS) in immunocompromised individuals. Here, we identified a human AstV of the VA1 genotype, HAstV-NIH, as the cause of fatal encephalitis in an immunocompromised adult. We investigated the cells targeted by AstV, neurophysiological changes, and host responses by analyzing gene expression, protein expression, and cellular morphology in brain tissue from three cases of AstV neurologic disease (AstV-ND). We demonstrate that neurons are the principal cells targeted by AstV in the brain and that the cerebellum and brainstem have the highest burden of infection. Detection of VA1 AstV in interconnected brain structures such as thalamus, deep cerebellar nuclei, Purkinje cells, and pontine nuclei indicates that AstV may spread between connected neurons transsynaptically. We found transcriptional dysregulation of neural functions and disruption of both excitatory and inhibitory synaptic innervation of infected neurons. Importantly, transcriptional dysregulation of neural functions occurred in fatal cases, but not in a patient that survived AstV-ND. We show that the innate, but not adaptive immune response was transcriptionally driving host defense in the brain of immunocompromised patients with AstV-ND. Both transcriptome and molecular pathology studies showed that most of the cellular changes were associated with CNS-intrinsic cells involved in phagocytosis and injury repair (microglia, perivascular/parenchymal border macrophages, and astrocytes), but not CNS-extrinsic cells (T and B cells), suggesting an imbalance of innate and adaptive immune responses to AstV infection in the brain as a result of the underlying immunodeficiencies. These results show that VA1 AstV infection of the brain in immunocompromised humans is associated with imbalanced host defense responses, disruption of neuronal somatodendritic compartments and synapses and increased phagocytic cellular activity. Improved understanding of the response to viral infections of the human CNS may provide clues for how to manipulate these processes to improve outcomes

Antibody-dependent enhancement (ADE) assay using rabbit sera throughout the infection series.

<p>Sera from naïve rabbits (week 0), following primary infection (week 8), and following secondary infection (week 13) were collected from both the 10³ and 10⁵ infection schedules. Week 8 serum had no neutralizing activity while week 13 serum had neutralizing activity. Sera were tested in Vero81 cells (A), Raji cells (B) and THP-1 cells (C). None = virus only control. p values **<0.01, ***<0.001.</p

Serum ELISA and neutralizing antibody titers in rabbits following primary and secondary infection.

<p>Serum ELISA and neutralizing antibody titers in rabbits following primary and secondary infection.</p

Viral RNA titers and histopathology in the lungs following MERS-CoV infection in rabbits that received passive transfer (PT) of serum from infected rabbits.

<p>Viral RNA titers in the lungs upon infection with 10⁵ TCID₅₀ of MERS-CoV in rabbits either previously infected with a low dose of MERS-CoV (10³ TCID₅₀) four weeks prior or naïve rabbits following PT of post-infection sera at either a full dose or 1:10 dilution (A). Images show the H&E staining (left) and IHC with an antibody against the MERS-CoV N protein (right) following infection for the 10^3//10⁵ TCID₅₀ (reinfection) group (B,E), the group that received passive transfer of undiluted post-infection serum (C, F), and the group that received passive transfer of post-infection serum at 1:10 dilution (D,G). n = 3 rabbits per group. Images from day 3 post-infection at 10x, bar equivalent to 100μm.</p

Viral RNA titers and histopathology in the lungs of rabbits following reinfection with MERS-CoV.

<p>Viral RNA titers in the lungs of rabbits following reinfection with EMC/2012 (A). Images show H&E (left) and IHC for the MERS-CoV N protein (right) following reinfection for the 10^3//10⁵ TCID₅₀ reinfection group (B,E) and 10^5//10⁵ TCID₅₀ reinfection group (C,F). The 10⁵ TCID₅₀^//media control group was included to demonstrate that the observed inflammation was not residual from the primary infection (D,G). n = 3 rabbits per group. All images at 10x, (bar equivalent to 100μm) with 40x inset (bar equivalent to 20μm). Images from day 3 post-infection.</p

Viral RNA titers in the respiratory tract following primary infection with MERS-CoV.

<p>Viral RNA titers in the nasal turbinates (A) and lungs (B) of rabbits following infection with either 10³ or 10⁵ TCID₅₀ of EMC/2012 strain of MERS-CoV through day 5 after infection. In a separate experiment, viral RNA titers were determined in the nasal turbinates (C) and lungs (D) following infection with 10^6.5 TCID₅₀. n = 3 rabbits per group. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test. p values *<0.05, ***<0.001.</p