IL-10 suppresses T cell expansion while promoting tissue-resident memory cell formation during SARS-CoV-2 infection in rhesus macaques.

The regulation of inflammatory responses and pulmonary disease during SARS-CoV-2 infection is incompletely understood. Here we examine the roles of the prototypic pro- and anti-inflammatory cytokines IFNγ and IL-10 using the rhesus macaque model of mild COVID-19. We find that IFNγ drives the development of 18fluorodeoxyglucose (FDG)-avid lesions in the lungs as measured by PET/CT imaging but is not required for suppression of viral replication. In contrast, IL-10 limits the duration of acute pulmonary lesions, serum markers of inflammation and the magnitude of virus-specific T cell expansion but does not impair viral clearance. We also show that IL-10 induces the subsequent differentiation of virus-specific effector T cells into CD69+CD103+ tissue resident memory cells (Trm) in the airways and maintains Trm cells in nasal mucosal surfaces, highlighting an unexpected role for IL-10 in promoting airway memory T cells during SARS-CoV-2 infection of macaques.

Recombinant OC43 SARS-CoV-2 spike replacement virus: An improved BSL-2 proxy virus for SARS-CoV-2 neutralization assays.

We generated a replication-competent OC43 human seasonal coronavirus (CoV) expressing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike in place of the native spike (rOC43-CoV2 S). This virus is highly attenuated relative to OC43 and SARS-CoV-2 in cultured cells and animals and is classified as a biosafety level 2 (BSL-2) agent by the NIH biosafety committee. Neutralization of rOC43-CoV2 S and SARS-CoV-2 by S-specific monoclonal antibodies and human sera is highly correlated, unlike recombinant vesicular stomatitis virus-CoV2 S. Single-dose immunization with rOC43-CoV2 S generates high levels of neutralizing antibodies against SARS-CoV-2 and fully protects human ACE2 transgenic mice from SARS-CoV-2 lethal challenge, despite nondetectable replication in respiratory and nonrespiratory organs. rOC43-CoV2 S induces S-specific serum and airway mucosal immunoglobulin A and IgG responses in rhesus macaques. rOC43-CoV2 S has enormous value as a BSL-2 agent to measure S-specific antibodies in the context of a bona fide CoV and is a candidate live attenuated SARS-CoV-2 mucosal vaccine that preferentially replicates in the upper airway.

Mucosal prime-boost immunization with live murine pneumonia virus-vectored SARS-CoV-2 vaccine is protective in macaques.

Immunization via the respiratory route is predicted to increase the effectiveness of a SARS-CoV-2 vaccine. Here, we evaluate the immunogenicity and protective efficacy of one or two doses of a live-attenuated murine pneumonia virus vector expressing SARS-CoV-2 prefusion-stabilized spike protein (MPV/S-2P), delivered intranasally/intratracheally to male rhesus macaques. A single dose of MPV/S-2P is highly immunogenic, and a second dose increases the magnitude and breadth of the mucosal and systemic anti-S antibody responses and increases levels of dimeric anti-S IgA in the airways. MPV/S-2P also induces S-specific CD4+ and CD8+ T-cells in the airways that differentiate into large populations of tissue-resident memory cells within a month after the boost. One dose induces substantial protection against SARS-CoV-2 challenge, and two doses of MPV/S-2P are fully protective against SARS-CoV-2 challenge virus replication in the airways. A prime/boost immunization with a mucosally-administered live-attenuated MPV vector could thus be highly effective in preventing SARS-CoV-2 infection and replication.

The inflammatory microenvironment of the lung at the time of infection governs innate control of SARS-CoV-2 replication.

SARS-CoV-2 infection leads to vastly divergent clinical outcomes ranging from asymptomatic infection to fatal disease. Co-morbidities, sex, age, host genetics and vaccine status are known to affect disease severity. Yet, how the inflammatory milieu of the lung at the time of SARS-CoV-2 exposure impacts the control of viral replication remains poorly understood. We demonstrate here that immune events in the mouse lung closely preceding SARS-CoV-2 infection significantly impact viral control and we identify key innate immune pathways required to limit viral replication. A diverse set of pulmonary inflammatory stimuli, including resolved antecedent respiratory infections with S. aureus or influenza, ongoing pulmonary M. tuberculosis infection, ovalbumin/alum-induced asthma or airway administration of defined TLR ligands and recombinant cytokines, all establish an antiviral state in the lung that restricts SARS-CoV-2 replication upon infection. In addition to antiviral type I interferons, the broadly inducible inflammatory cytokines TNFα and IL-1 precondition the lung for enhanced viral control. Collectively, our work shows that SARS-CoV-2 may benefit from an immunologically quiescent lung microenvironment and suggests that heterogeneity in pulmonary inflammation that precedes or accompanies SARS-CoV-2 exposure may be a significant factor contributing to the population-wide variability in COVID-19 disease outcomes.

Measuring factors affecting honey bee (Hymenoptera: Apidae) attraction to soybeans using bioacoustics monitoring.

Soybean (Glycine max (L.) Merr.) is an important agricultural crop around the world, and previous studies suggest that honey bees (Apis mellifera Linnaeus) can be a component for optimizing soybean production through pollination. Determining when bees are present in soybean fields is critical for assessing pollination activity and identifying periods when bees are absent so that bee-toxic pesticides may be applied. There are currently several methods for detecting pollinator activity, but these existing methods have substantial limitations, including the bias of pan trappings against large bees and the limited duration of observation possible using manual techniques. This study aimed to develop a new method for detecting honey bees in soybean fields using bioacoustics monitoring. Microphones were placed in soybean fields to record the audible wingbeats of foraging bees. Foraging activity was identified using the wingbeat frequency of honey bees (234 ±â€…14 Hz) through a combination of algorithmic and manual approaches. A total of 243 honey bees were detected over 10 days of recording in 4 soybean fields. Bee activity was significantly greater in blooming fields than in non-blooming fields. Temperature had no significant effect on bee activity, but bee activity differed significantly between soybean varieties, suggesting that soybean attractiveness to honey bees is heavily dependent on varietal characteristics. Refinement of bioacoustics methods, particularly through the incorporation of machine learning, could provide a practical tool for measuring the activity of honey bees and other flying insects in soybeans as well as other crops and ecosystems.

Omicron Spike confers enhanced infectivity and interferon resistance to SARS-CoV-2 in human nasal tissue.

Omicron emerged following COVID-19 vaccination campaigns, displaced previous SARS-CoV-2 variants of concern worldwide, and gave rise to lineages that continue to spread. Here, we show that Omicron exhibits increased infectivity in primary adult upper airway tissue relative to Delta. Using recombinant forms of SARS-CoV-2 and nasal epithelial cells cultured at the liquid-air interface, we show that mutations unique to Omicron Spike enable enhanced entry into nasal tissue. Unlike earlier variants of SARS-CoV-2, our findings suggest that Omicron enters nasal cells independently of serine transmembrane proteases and instead relies upon metalloproteinases to catalyze membrane fusion. Furthermore, we demonstrate that this entry pathway unlocked by Omicron Spike enables evasion from constitutive and interferon-induced antiviral factors that restrict SARS-CoV-2 entry following attachment. Therefore, the increased transmissibility exhibited by Omicron in humans may be attributed not only to its evasion of vaccine-elicited adaptive immunity, but also to its superior invasion of nasal epithelia and resistance to the cell-intrinsic barriers present therein.

Bacterial-induced or passively administered interferon gamma conditions the lung for early control of SARS-CoV-2.

Type-1 and type-3 interferons (IFNs) are important for control of viral replication; however, less is known about the role of Type-2 IFN (IFNγ) in anti-viral immunity. We previously observed that lung infection with Mycobacterium bovis BCG achieved though intravenous (iv) administration provides strong protection against SARS-CoV-2 in mice yet drives low levels of type-1 IFNs but robust IFNγ. Here we examine the role of ongoing IFNγ responses to pre-established bacterial infection on SARS-CoV-2 disease outcomes in two murine models. We report that IFNγ is required for iv BCG induced reduction in pulmonary viral loads, an outcome dependent on IFNγ receptor expression by non-hematopoietic cells. Importantly, we show that BCG infection prompts pulmonary epithelial cells to upregulate IFN-stimulated genes with reported anti-viral activity in an IFNγ-dependent manner, suggesting a possible mechanism for the observed protection. Finally, we confirm the anti-viral properties of IFNγ by demonstrating that the recombinant cytokine itself provides strong protection against SARS-CoV-2 challenge when administered intranasally. Together, our data show that a pre-established IFNγ response within the lung is protective against SARS-CoV-2 infection, suggesting that concurrent or recent infections that drive IFNγ may limit the pathogenesis of SARS-CoV-2 and supporting possible prophylactic uses of IFNγ in COVID-19 management.

Review: the risks of spray adjuvants to honey bees.

Pesticide applications are often made as tank mixes containing multiple pesticide products and may include spray adjuvants to enhance pesticidal activities. The primary aim of adjuvant products is to increase the spreading and sticking of spray droplets and to increase the penetration of active ingredients through the cuticles of leaves or targeted pests, which can reduce the amount of active ingredient needed for effective pest control. Adjuvants are made up of compounds drawn from the "inert ingredient" list maintained by EPA but are identified as "principal functioning agents" when used in adjuvant products. These inert compounds do not undergo the same testing and risk assessment process that is required of pesticide active ingredients and generally have no mitigation measures that prevent application onto crops during bloom at times of day when bees are foraging. Honey bees (Apis mellifera;Hymenoptera:Apidae) are at an increased risk of exposure to adjuvant tank mixtures while providing agricultural pollination services. Colony losses attributed to pesticide applications thought to have low risk to honey bees have been reported, highlighting the need to better understand the toxicity of adjuvants included in pesticide tank mixtures. This review summarizes current literature on the risks posed to honey bees by agricultural adjuvants and tank mix combinations of adjuvants with pesticides. Based on the current state of knowledge, we make recommendations to pesticide applicators, product manufacturers, regulatory agencies, and researchers regarding adjuvant toxicity to honey bees with the goal of reducing risks that adjuvants pose to honey bees and other beneficial insects.

Quality of Life in Adults with Chronic Cough: A Mixed Methods Study Informing the Development of a Quantitative Patient Preference Study.

OBJECTIVES: This study aimed to describe quality of life for patients with chronic cough (CC) and identify meaningful attributes that affect patient treatment preferences to inform the design of a quantitative preference study. METHODS: Eligible patients (≥ 18 years) with a CC (> 8 weeks) participated in qualitative interviews with two defined steps. Step one: concept elicitation and bidding games were used to collect descriptions of patient experiences with CC and identify important CC-related attributes. Step two: attributes were confirmed using concept elicitation and bidding games and prioritized using structured card sort activities. Purposive sampling ensured diversity of patient experiences. Qualitative content analysis was used to analyze participant narratives, and descriptive statistics were used to summarize card sort results. This study follows a fully mixed concurrent dominant status design, with qualitative (dominant) and quantitative components. RESULTS: A total of 20 participants were interviewed with a mean age of 61.4 years (range 24-79 years). Coughing episodes, described as intense consecutive coughs that made catching breath difficult, were important to most participants (n = 17). Participants emphasized the emotional impact of episodes including feelings of uncertainty, loss of control, self-consciousness, and fear. Severity of CC was most often judged by frequency (n = 11) and intensity (n = 12) of cough. Daily, physical, or social activities were impacted for most participants. Impact on sleep (n = 14) included waking during the night, difficulty falling asleep, and daytime fatigue. Medication-related taste disturbances were an important consideration for what participants were willing to accept in exchange for cough relief. CONCLUSIONS: This study emphasizes the importance of coughing episodes for adults with CC and provides initial evidence that taste alterations are an important component of patient treatment decisions for CC.

Intranasal murine pneumonia virus-vectored SARS-CoV-2 vaccine induces mucosal and serum antibodies in macaques.

Next-generation SARS-CoV-2 vaccines are needed that induce systemic and mucosal immunity. Murine pneumonia virus (MPV), a murine homolog of respiratory syncytial virus, is attenuated by host-range restriction in nonhuman primates and has a tropism for the respiratory tract. We generated MPV vectors expressing the wild-type SARS-CoV-2 spike protein (MPV/S) or its prefusion-stabilized form (MPV/S-2P). Both vectors replicated similarly in cell culture and stably expressed S. However, only S-2P was associated with MPV particles. After intranasal/intratracheal immunization of rhesus macaques, MPV/S and MPV/S-2P replicated to low levels in the airways. Despite its low-level replication, MPV/S-2P induced high levels of mucosal and serum IgG and IgA to SARS-CoV-2 S or its receptor-binding domain. Serum antibodies from MPV/S-2P-immunized animals efficiently inhibited ACE2 receptor binding to S proteins of variants of concern. Based on its attenuation and immunogenicity in macaques, MPV/S-2P will be further evaluated as a live-attenuated vaccine for intranasal immunization against SARS-CoV-2.

Mucosal prime-boost immunization with live murine pneumonia virus-vectored SARS-CoV-2 vaccine is protective in macaques.

Immunization via the respiratory route is predicted to increase the effectiveness of a SARS-CoV-2 vaccine. We evaluated the immunogenicity and protective efficacy of one or two doses of a live-attenuated murine pneumonia virus vector expressing SARS-CoV-2 prefusion-stabilized spike protein (MPV/S-2P), delivered intranasally/intratracheally to rhesus macaques. A single dose of MPV/S-2P was highly immunogenic, and a second dose increased the magnitude and breadth of the mucosal and systemic anti-S antibody responses and increased levels of dimeric anti-S IgA in the airways. MPV/S-2P also induced S-specific CD4+ and CD8+ T-cells in the airways that differentiated into large populations of tissue-resident memory cells within a month after the boost. One dose induced substantial protection against SARS-CoV-2 challenge, and two doses of MPV/S-2P were fully protective against SARS-CoV-2 challenge virus replication in the airways. A prime/boost immunization with a mucosally-administered live-attenuated MPV vector could thus be highly effective in preventing SARS-CoV-2 infection and replication.

Advances and Challenges in Molecular Imaging of Viral Infections.

Molecular imaging of viral infection, using a variety of advanced imaging techniques such as optical and nuclear imaging, can and has been used for direct visualization of the virus as well as assessment of virus-host interactions. Unlike imaging of other pathogens such as bacteria and fungi, challenging aspects of imaging viral infections include the small size of viruses, the complexity of viral infection animal models (eg, species dependence), and the high-level containment needs for many high-consequence pathogens, among others. In this review, using representative viral infections, we discuss how molecular imaging can reveal real-time infection dynamics, improve our understanding of disease pathogenesis, and guide optimization of treatment and prevention strategies. Key findings from human and animal studies are highlighted.

SARS-CoV-2 infection of human lung epithelial cells induces TMPRSS-mediated acute fibrin deposition.

Severe COVID-associated lung injury is a major confounding factor of hospitalizations and death with no effective treatments. Here, we describe a non-classical fibrin clotting mechanism mediated by SARS-CoV-2 infected primary lung but not other susceptible epithelial cells. This infection-induced fibrin formation is observed in all variants of SARS-CoV-2 infections, and requires thrombin but is independent of tissue factor and other classical plasma coagulation factors. While prothrombin and fibrinogen levels are elevated in acute COVID BALF samples, fibrin clotting occurs only with the presence of viral infected but not uninfected lung epithelial cells. We suggest a viral-induced coagulation mechanism, in which prothrombin is activated by infection-induced transmembrane serine proteases, such as ST14 and TMPRSS11D, on NHBE cells. Our finding reveals the inefficiency of current plasma targeted anticoagulation therapy and suggests the need to develop a viral-induced ARDS animal model for treating respiratory airways with thrombin inhibitors.

Toxicity of spray adjuvants and tank mix combinations used in almond orchards to adult honey bees (Apis mellifera).

Commercial beekeepers transporting honey bees across the United States to provide almond pollination services have reported honey bee deaths, possibly due to pesticide applications made during crop bloom. Pesticides are often applied as "tank mixes", or mixtures of fungicides and insecticides combined into a single application. Spray adjuvants are often added to tank mixes to improve the application characteristics of a pesticide and include spreaders, stickers, or surfactants. The goal of this research was to determine toxicity of adjuvants to adult worker honey bees, both when applied alone and in adjuvant-pesticide tank mixtures. Field-relevant combinations of formulated products were applied to 3-day-old adult worker honey bees using a Potter Spray Tower, and mortality was assessed 48 h following exposure. Adjuvants tested included Activator-90, Attach, Choice Weather Master, Cohere, Dyne-Amic, Induce, Kinetic, LI 700, Liberate, Nu-Film P, PHT Latron B-1956, and Surf-90; fungicides tested include Luna Sensation (Fluopyram and Trifloxystrobin), Pristine (Pyraclostrobin and Boscalid), Tilt (Propiconazole), and Vangard (Cyprodinil), and insecticides tested include Altacor (Chlorantraniliprole), Intrepid 2F (Methoxyfenozide), and a positive control Mustang Maxx (Zeta-cypermethrin). Results demonstrated that exposure to some adjuvants causes acute honey bee mortality at near-field application rates, both when applied alone and in combination with pesticides. Some adjuvant-pesticide combinations demonstrated increased toxicity compared with the adjuvant alone, while others demonstrated decreased toxicity. A better understanding of adjuvant and adjuvant-pesticide tank mixture toxicity to honey bees will play a key role in informing "Best Management Practices" for pesticide applicators using spray adjuvants during bloom when honey bee exposure is likely.

Effects of pesticide-adjuvant combinations used in almond orchards on olfactory responses to social signals in honey bees (Apis mellifera).

Exposure to agrochemical sprays containing pesticides and tank-mix adjuvants has been implicated in post-bloom mortality, particularly of brood, in honey bee colonies brought into California almond orchards for pollination. Although adjuvants are generally considered to be biologically inert, some adjuvants have exhibited toxicity and sublethal effects, including decreasing survival rates of next-generation queens. Honey bees have a highly developed olfactory system to detect and discriminate among social signals. To investigate the impact of pesticide-adjuvant combinations on honey bee signal perception, we performed electroantennography assays to assess alterations in their olfactory responsiveness to the brood ester pheromone (BEP), the volatile larval pheromone ß-ocimene, and the alarm pheromone 2-heptanone. These assays aimed to uncover potential mechanisms underlying changes in social behaviors and reduced brood survival after pesticide exposure. We found that combining the adjuvant Dyne-Amic with the fungicide Tilt (propiconazole) and the insecticide Altacor (chlorantraniliprole) synergistically enhanced olfactory responses to three concentrations of BEP and as well exerted dampening and compensatory effects on responses to 2-heptanone and ß-ocimene, respectively. In contrast, exposure to adjuvant alone or the combination of fungicide and insecticide had no effect on olfactory responses to BEP at most concentrations but altered responses to ß-ocimene and 2-heptanone. Exposure to Dyne-Amic, Altacor, and Tilt increased BEP signal amplitude, indicating potential changes in olfactory receptor sensitivity or sensilla permeability to odorants. Given that, in a previous study, next-generation queens raised by nurses exposed to the same treated pollen experienced reduced survival, these new findings highlight the potential disruption of social signaling in honey bees and its implications for colony reproductive success.

Co-infection of mice with SARS-CoV-2 and Mycobacterium tuberculosis limits early viral replication but does not affect mycobacterial loads.

Viral co-infections have been implicated in worsening tuberculosis (TB) and during the COVID-19 pandemic, the global rate of TB-related deaths has increased for the first time in over a decade. We and others have previously shown that a resolved prior or concurrent influenza A virus infection in Mycobacterium tuberculosis (Mtb)-infected mice resulted in increased pulmonary bacterial burden, partly through type I interferon (IFN-I)-dependent mechanisms. Here we investigated whether SARS-CoV-2 (SCV2) co-infection could also negatively affect bacterial control of Mtb. Importantly, we found that K18-hACE2 transgenic mice infected with SCV2 one month before, or months after aerosol Mtb exposure did not display exacerbated Mtb infection-associated pathology, weight loss, nor did they have increased pulmonary bacterial loads. However, pre-existing Mtb infection at the time of exposure to the ancestral SCV2 strain in infected K18-hACE2 transgenic mice or the beta variant (B.1.351) in WT C57Bl/6 mice significantly limited early SCV2 replication in the lung. Mtb-driven protection against SCV2 increased with higher bacterial doses and did not require IFN-I, TLR2 or TLR9 signaling. These data suggest that SCV2 co-infection does not exacerbate Mtb infection in mice, but rather the inflammatory response generated by Mtb infection in the lungs at the time of SCV2 exposure restricts viral replication.

Endogenous and Therapeutic 25-Hydroxycholesterols May Worsen Early SARS-CoV-2 Pathogenesis in Mice.

Oxysterols (i.e., oxidized cholesterol species) have complex roles in biology. 25-Hydroxycholesterol (25HC), a product of the activity of cholesterol-25-hydroxylase (CH25H) on cholesterol, has recently been shown to be broadly antiviral, suggesting therapeutic potential against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, 25HC can also amplify inflammation and be converted by CYP7B1 (cytochrome P450 family 7 subfamily B member 1) to 7α,25-dihydroxycholesterol, a lipid with chemoattractant activity, via the G protein-coupled receptor EBI2 (Epstein-Barr virus-induced gene 2)/GPR183 (G protein-coupled receptor 183). Here, using in vitro studies and two different murine models of SARS-CoV-2 infection, we investigate the effects of these two oxysterols on SARS-CoV-2 pneumonia. We show that although 25HC and enantiomeric-25HC are antiviral in vitro against human endemic coronavirus-229E, they did not inhibit SARS-CoV-2; nor did supplemental 25HC reduce pulmonary SARS-CoV-2 titers in the K18-human ACE2 (angiotensin-converting enzyme 2) mouse model in vivo. Treatment with 25HC also did not alter immune cell influx into the airway, airspace cytokines, lung pathology, weight loss, symptoms, or survival but was associated with increased airspace albumin, an indicator of microvascular injury, and increased plasma proinflammatory cytokines. Conversely, mice treated with the EBI2/GPR183 inhibitor NIBR189 displayed a modest increase in lung viral load only at late time points but no change in weight loss. Consistent with these findings, although Ch25h and 25HC were upregulated in the lungs of SARS-CoV-2-infected wild-type mice, lung viral titers and weight loss in Ch25h-/- and Gpr183-/- mice infected with the ß variant were similar to those in control animals. Taken together, endogenous 25HCs do not significantly regulate early SARS-CoV-2 replication or pathogenesis, and supplemental 25HC may have proinjury rather than therapeutic effects in SARS-CoV-2 pneumonia.

Exposure to lung-migrating helminth protects against murine SARS-CoV-2 infection through macrophage-dependent T cell activation.

Helminth endemic regions report lower COVID-19 morbidity and mortality. Here, we show that lung remodeling from a prior infection with a lung-migrating helminth, Nippostrongylus brasiliensis, enhances viral clearance and survival of human-ACE2 transgenic mice challenged with SARS-CoV-2 (SCV2). This protection is associated with a lymphocytic infiltrate, including increased accumulation of pulmonary SCV2-specific CD8+ T cells, and anti-CD8 antibody depletion abrogated the N. brasiliensis-mediated reduction in viral loads. Pulmonary macrophages with a type 2 transcriptional and epigenetic signature persist in the lungs of N. brasiliensis-exposed mice after clearance of the parasite and establish a primed environment for increased CD8+ T cell recruitment and activation. Accordingly, depletion of macrophages ablated the augmented viral clearance and accumulation of CD8+ T cells driven by prior N. brasiliensis infection. Together, these findings support the concept that lung-migrating helminths can limit disease severity during SCV2 infection through macrophage-dependent enhancement of antiviral CD8+ T cell responses.

Omicron Spike confers enhanced infectivity and interferon resistance to SARS-CoV-2 in human nasal tissue.

Omicron emerged following COVID-19 vaccination campaigns, displaced previous SARS-CoV-2 variants of concern worldwide, and gave rise to lineages that continue to spread. Here, we show that Omicron exhibits increased infectivity in primary adult upper airway tissue relative to Delta. Using recombinant forms of SARS-CoV-2 and nasal epithelial cells cultured at the liquid-air interface, enhanced infectivity maps to the step of cellular entry and evolved recently through mutations unique to Omicron Spike. Unlike earlier variants of SARS-CoV-2, Omicron enters nasal cells independently of serine transmembrane proteases and instead relies upon metalloproteinases to catalyze membrane fusion. This entry pathway unlocked by Omicron Spike enables evasion of constitutive and interferon-induced antiviral factors that restrict SARS-CoV-2 entry following attachment. Therefore, the increased transmissibility exhibited by Omicron in humans may be attributed not only to its evasion of vaccine-elicited adaptive immunity, but also to its superior invasion of nasal epithelia and resistance to the cell-intrinsic barriers present therein.

Live-attenuated pediatric parainfluenza vaccine expressing 6P-stabilized SARS-CoV-2 spike protein is protective against SARS-CoV-2 variants in hamsters.

The pediatric live-attenuated bovine/human parainfluenza virus type 3 (B/HPIV3)-vectored vaccine expressing the prefusion-stabilized SARS-CoV-2 spike (S) protein (B/HPIV3/S-2P) was previously evaluated in vitro and in hamsters. To improve its immunogenicity, we generated B/HPIV3/S-6P, expressing S further stabilized with 6 proline mutations (S-6P). Intranasal immunization of hamsters with B/HPIV3/S-6P reproducibly elicited significantly higher serum anti-S IgA/IgG titers than B/HPIV3/S-2P; hamster sera efficiently neutralized variants of concern (VoCs), including Omicron variants. B/HPIV3/S-2P and B/HPIV3/S-6P immunization protected hamsters against weight loss and lung inflammation following SARS-CoV-2 challenge with the vaccine-matched strain WA1/2020 or VoCs B.1.1.7/Alpha or B.1.351/Beta and induced near-sterilizing immunity. Three weeks post-challenge, B/HPIV3/S-2P- and B/HPIV3/S-6P-immunized hamsters exhibited a robust anamnestic serum antibody response with increased neutralizing potency to VoCs, including Omicron sublineages. B/HPIV3/S-6P primed for stronger anamnestic antibody responses after challenge with WA1/2020 than B/HPIV3/S-2P. B/HPIV3/S-6P will be evaluated as an intranasal vaccine to protect infants against both HPIV3 and SARS-CoV-2.