BCG vaccination stimulates integrated organ immunity by feedback of the adaptive immune response to imprint prolonged innate antiviral resistance.

Bacille Calmette-Guérin (BCG) vaccination can confer nonspecific protection against heterologous pathogens. However, the underlying mechanisms remain mysterious. We show that mice vaccinated intravenously with BCG exhibited reduced weight loss and/or improved viral clearance when challenged with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 B.1.351) or PR8 influenza. Protection was first evident between 14 and 21 d post-vaccination and lasted ∼3 months. Notably, BCG induced a biphasic innate response and robust antigen-specific type 1 helper T cell (TH1 cell) responses in the lungs. MyD88 signaling was essential for innate and TH1 cell responses, and protection against SARS-CoV-2. Depletion of CD4+ T cells or interferon (IFN)-γ activity before infection obliterated innate activation and protection. Single-cell and spatial transcriptomics revealed CD4-dependent expression of IFN-stimulated genes in lung myeloid and epithelial cells. Notably, BCG also induced protection against weight loss after mouse-adapted SARS-CoV-2 BA.5, SARS-CoV and SHC014 coronavirus infections. Thus, BCG elicits integrated organ immunity, where CD4+ T cells feed back on tissue myeloid and epithelial cells to imprint prolonged and broad innate antiviral resistance.

A modified vaccinia Ankara vector-based vaccine protects macaques from SARS-CoV-2 infection, immune pathology, and dysfunction in the lungs.

A combination of vaccination approaches will likely be necessary to fully control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Here, we show that modified vaccinia Ankara (MVA) vectors expressing membrane-anchored pre-fusion stabilized spike (MVA/S) but not secreted S1 induced strong neutralizing antibody responses against SARS-CoV-2 in mice. In macaques, the MVA/S vaccination induced strong neutralizing antibodies and CD8+ T cell responses, and conferred protection from SARS-CoV-2 infection and virus replication in the lungs as early as day 2 following intranasal and intratracheal challenge. Single-cell RNA sequencing analysis of lung cells on day 4 after infection revealed that MVA/S vaccination also protected macaques from infection-induced inflammation and B cell abnormalities and lowered induction of interferon-stimulated genes. These results demonstrate that MVA/S vaccination induces neutralizing antibodies and CD8+ T cells in the blood and lungs and is a potential vaccine candidate for SARS-CoV-2.

SARS-CoV-2 Omicron virus causes attenuated disease in mice and hamsters.

The recent emergence of B.1.1.529, the Omicron variant1,2, has raised concerns of escape from protection by vaccines and therapeutic antibodies. A key test for potential countermeasures against B.1.1.529 is their activity in preclinical rodent models of respiratory tract disease. Here, using the collaborative network of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme of the National Institute of Allergy and Infectious Diseases (NIAID), we evaluated the ability of several B.1.1.529 isolates to cause infection and disease in immunocompetent and human ACE2 (hACE2)-expressing mice and hamsters. Despite modelling data indicating that B.1.1.529 spike can bind more avidly to mouse ACE2 (refs. 3,4), we observed less infection by B.1.1.529 in 129, C57BL/6, BALB/c and K18-hACE2 transgenic mice than by previous SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. In wild-type and hACE2 transgenic hamsters, lung infection, clinical disease and pathology with B.1.1.529 were also milder than with historical isolates or other SARS-CoV-2 variants of concern. Overall, experiments from the SAVE/NIAID network with several B.1.1.529 isolates demonstrate attenuated lung disease in rodents, which parallels preliminary human clinical data.

Safety and Immunogenicity of SARS-CoV-2 mRNA-1273 Vaccine in Older Adults.

BACKGROUND: Testing of vaccine candidates to prevent infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in an older population is important, since increased incidences of illness and death from coronavirus disease 2019 (Covid-19) have been associated with an older age. METHODS: We conducted a phase 1, dose-escalation, open-label trial of a messenger RNA vaccine, mRNA-1273, which encodes the stabilized prefusion SARS-CoV-2 spike protein (S-2P) in healthy adults. The trial was expanded to include 40 older adults, who were stratified according to age (56 to 70 years or ≥71 years). All the participants were assigned sequentially to receive two doses of either 25 µg or 100 µg of vaccine administered 28 days apart. RESULTS: Solicited adverse events were predominantly mild or moderate in severity and most frequently included fatigue, chills, headache, myalgia, and pain at the injection site. Such adverse events were dose-dependent and were more common after the second immunization. Binding-antibody responses increased rapidly after the first immunization. By day 57, among the participants who received the 25-µg dose, the anti-S-2P geometric mean titer (GMT) was 323,945 among those between the ages of 56 and 70 years and 1,128,391 among those who were 71 years of age or older; among the participants who received the 100-µg dose, the GMT in the two age subgroups was 1,183,066 and 3,638,522, respectively. After the second immunization, serum neutralizing activity was detected in all the participants by multiple methods. Binding- and neutralizing-antibody responses appeared to be similar to those previously reported among vaccine recipients between the ages of 18 and 55 years and were above the median of a panel of controls who had donated convalescent serum. The vaccine elicited a strong CD4 cytokine response involving type 1 helper T cells. CONCLUSIONS: In this small study involving older adults, adverse events associated with the mRNA-1273 vaccine were mainly mild or moderate. The 100-µg dose induced higher binding- and neutralizing-antibody titers than the 25-µg dose, which supports the use of the 100-µg dose in a phase 3 vaccine trial. (Funded by the National Institute of Allergy and Infectious Diseases and others; mRNA-1273 Study ClinicalTrials.gov number, NCT04283461.).

The ciliary phosphatidylinositol phosphatase Inpp5e plays positive and negative regulatory roles in Shh signaling.

Sonic hedgehog (Shh) signal transduction specifies ventral cell fates in the neural tube and is mediated by the Gli transcription factors that play both activator (GliA) and repressor (GliR) roles. Cilia are essential for Shh signal transduction and the ciliary phosphatidylinositol phosphatase Inpp5e is linked to Shh regulation. In the course of a forward genetic screen for recessive mouse mutants, we identified a functional null allele of inositol polyphosphate-5-phosphatase E (Inpp5e), ridge top (rdg), with expanded ventral neural cell fates at E10.5. By E12.5, Inpp5erdg/rdg embryos displayed normal neural patterning and this correction over time required Gli3, the predominant repressor in neural patterning. Inpp5erdg function largely depended on the presence of cilia and on smoothened, the obligate transducer of Shh signaling, indicating that Inpp5e functions within the cilium to regulate the pathway. These data indicate that Inpp5e plays a more complicated role in Shh signaling than previously appreciated. We propose that Inpp5e attenuates Shh signaling in the neural tube through regulation of the relative timing of GliA and GliR production, which is important in understanding how the duration of Shh signaling regulates neural tube patterning.

Evaluation of Cellular and Serological Responses to Acute SARS-CoV-2 Infection Demonstrates the Functional Importance of the Receptor-Binding Domain.

The factors that control the development of an effective immune response to the recently emerged SARS-CoV-2 virus are poorly understood. In this study, we provide a cross-sectional analysis of the dynamics of B cell responses to SARS-CoV-2 infection in hospitalized COVID-19 patients. We observe changes in B cell subsets consistent with a robust humoral immune response, including significant expansion of plasmablasts and activated receptor-binding domain (RBD)-specific memory B cell populations. We observe elevated titers of Abs to SARS-CoV-2 RBD, full-length Spike, and nucleoprotein over the course of infection, with higher levels of RBD-specific IgG correlating with increased serum neutralization. Depletion of RBD-specific Abs from serum removed a major portion of neutralizing activity in most individuals. Some donors did retain significant residual neutralization activity, suggesting a potential Ab subset targeting non-RBD epitopes. Taken together, these findings are instructive for future vaccine design and mAb strategies.

CD4+ and CD8+ T cells are required to prevent SARS-CoV-2 persistence in the nasal compartment.

SARS-CoV-2 is the causative agent of COVID-19 and continues to pose a significant public health threat throughout the world. Following SARS-CoV-2 infection, virus-specific CD4+ and CD8+ T cells are rapidly generated to form effector and memory cells and persist in the blood for several months. However, the contribution of T cells in controlling SARS-CoV-2 infection within the respiratory tract are not well understood. Using C57BL/6 mice infected with a naturally occurring SARS-CoV-2 variant (B.1.351), we evaluated the role of T cells in the upper and lower respiratory tract. Following infection, SARS-CoV-2-specific CD4+ and CD8+ T cells are recruited to the respiratory tract and a vast proportion secrete the cytotoxic molecule Granzyme B. Using antibodies to deplete T cells prior to infection, we found that CD4+ and CD8+ T cells play distinct roles in the upper and lower respiratory tract. In the lungs, T cells play a minimal role in viral control with viral clearance occurring in the absence of both CD4+ and CD8+ T cells through 28 days post-infection. In the nasal compartment, depletion of both CD4+ and CD8+ T cells, but not individually, results in persistent and culturable virus replicating in the nasal compartment through 28 days post-infection. Using in situ hybridization, we found that SARS-CoV-2 infection persisted in the nasal epithelial layer of tandem CD4+ and CD8+ T cell-depleted mice. Sequence analysis of virus isolates from persistently infected mice revealed mutations spanning across the genome, including a deletion in ORF6. Overall, our findings highlight the importance of T cells in controlling virus replication within the respiratory tract during SARS-CoV-2 infection.

Eosinophils protect against SARS-CoV-2 following a vaccine breakthrough infection.

Waning immunity and the emergence of immune evasive SARS-CoV-2 variants jeopardize vaccine efficacy leading to breakthrough infections. We have previously shown that innate immune cells play a critical role in controlling SARS-CoV-2. To investigate the innate immune response during breakthrough infections, we modeled breakthrough infections by challenging low-dose vaccinated mice with a vaccine-mismatched SARS-CoV-2 Beta variant. We found that low-dose vaccinated infected mice had a 2-log reduction in lung viral burden, but increased immune cell infiltration in the lung parenchyma, characterized by monocytes, monocyte-derived macrophages, and eosinophils. Single cell RNA-seq revealed viral RNA was highly associated with eosinophils that corresponded to a unique IFN-γ biased signature. Antibody-mediated depletion of eosinophils in vaccinated mice resulted in increased virus replication and dissemination in the lungs, demonstrating that eosinophils in the lungs are protective during SARS-CoV-2 breakthrough infections. These results highlight the critical role for the innate immune response in vaccine mediated protection against SARS-CoV-2.

CD4+ and CD8+ T cells are required to prevent SARS-CoV-2 persistence in the nasal compartment.

SARS-CoV-2 infection induces the generation of virus-specific CD4+ and CD8+ effector and memory T cells. However, the contribution of T cells in controlling SARS-CoV-2 during infection is not well understood. Following infection of C57BL/6 mice, SARS-CoV-2-specific CD4+ and CD8+ T cells are recruited to the respiratory tract, and a vast proportion secrete the cytotoxic molecule granzyme B. Using depleting antibodies, we found that T cells within the lungs play a minimal role in viral control, and viral clearance occurs in the absence of both CD4+ and CD8+ T cells through 28 days postinfection. In the nasal compartment, depletion of both CD4+ and CD8+ T cells, but not individually, results in persistent, culturable virus replicating in the nasal epithelial layer through 28 days postinfection. Viral sequencing analysis revealed adapted mutations across the SARS-CoV-2 genome, including a large deletion in ORF6. Overall, our findings highlight the importance of T cells in controlling virus replication within the respiratory tract during SARS-CoV-2 infection.

Atovaquone and Pibrentasvir Inhibit the SARS-CoV-2 Endoribonuclease and Restrict Infection In Vitro but Not In Vivo.

The emergence of SARS-CoV-1 in 2003 followed by MERS-CoV and now SARS-CoV-2 has proven the latent threat these viruses pose to humanity. While the SARS-CoV-2 pandemic has shifted to a stage of endemicity, the threat of new coronaviruses emerging from animal reservoirs remains. To address this issue, the global community must develop small molecule drugs targeting highly conserved structures in the coronavirus proteome. Here, we characterized existing drugs for their ability to inhibit the endoribonuclease activity of the SARS-CoV-2 non-structural protein 15 (nsp15) via in silico, in vitro, and in vivo techniques. We have identified nsp15 inhibition by the drugs pibrentasvir and atovaquone which effectively inhibit SARS-CoV-2 and HCoV-OC43 at low micromolar concentrations in cell cultures. Furthermore, atovaquone, but not pibrentasvir, is observed to modulate HCoV-OC43 dsRNA and infection in a manner consistent with nsp15 inhibition. Although neither pibrentasvir nor atovaquone translate to clinical efficacy in a murine prophylaxis model of SARS-CoV-2 infection, atovaquone may serve as a basis for the design of future nsp15 inhibitors.

Evaluating antitumor activity of Escherichia coli purine nucleoside phosphorylase against head and neck patient-derived xenografts.

BACKGROUND: Purine nucleoside phosphorylase (PNP) gene transfer represents a promising approach to treatment of head and neck malignancies. We tested recombinant adenovirus already in phase I/II clinical testing and leading-edge patient-derived xenografts (PDX) as a means to optimize this therapeutic strategy. METHODS: Our experiments investigated purine base cytotoxicity, PNP enzyme activity following treatment of malignant tissue, tumor mass regression, viral receptor studies, and transduction by tropism-modified adenovirus. RESULTS: Replication deficient vector efficiently transduced PDX cells and mediated significant anticancer effect following treatment with fludarabine phosphate in vivo. Either 6-methylpurine or 2-fluoroadenine (toxic molecules generated by the PNP approach) ablated head and neck cancer cell proliferation. High levels of adenovirus-3 specific receptors were detected in human tumor models, and vector was evaluated that utilizes this pathway. CONCLUSIONS: Our studies provide the scientific foundation necessary to improve PNP prodrug cleavage and advance a new treatment for head and neck cancer.

A wild boar cathelicidin peptide derivative inhibits severe acute respiratory syndrome coronavirus-2 and its drifted variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a clear threat to humanity. It has infected over 200 million and killed 4 million people worldwide, and infections continue with no end in sight. To control the pandemic, multiple effective vaccines have been developed, and global vaccinations are in progress. However, the virus continues to mutate. Even when full vaccine coverage is achieved, vaccine-resistant mutants will likely emerge, thus requiring new annual vaccines against drifted variants analogous to influenza. A complimentary solution to this problem could be developing antiviral drugs that inhibit SARS CoV-2 and its drifted variants. Host defense peptides represent a potential source for such an antiviral as they possess broad antimicrobial activity and significant diversity across species. We screened the cathelicidin family of peptides from 16 different species for antiviral activity and identified a wild boar peptide derivative that inhibits SARS CoV-2. This peptide, which we named Yongshi and means warrior in Mandarin, acts as a viral entry inhibitor. Following the binding of SARS-CoV-2 to its receptor, the spike protein is cleaved, and heptad repeats 1 and 2 multimerize to form the fusion complex that enables the virion to enter the cell. A deep learning-based protein sequence comparison algorithm and molecular modeling suggest that Yongshi acts as a mimetic to the heptad repeats of the virus, thereby disrupting the fusion process. Experimental data confirm the binding of Yongshi to the heptad repeat 1 with a fourfold higher affinity than heptad repeat 2 of SARS-CoV-2. Yongshi also binds to the heptad repeat 1 of SARS-CoV-1 and MERS-CoV. Interestingly, it inhibits all drifted variants of SARS CoV-2 that we tested, including the alpha, beta, gamma, delta, kappa and omicron variants.

Ablation of Adar1 in myeloid cells imprints a global antiviral state in the lung and heightens early immunity against SARS-CoV-2.

Under normal homeostatic conditions, self-double-stranded RNA (self-dsRNA) is modified by adenosine deaminase acting on RNA 1 (ADAR1) to prevent the induction of a type I interferon-mediated inflammatory cascade. Antigen-presenting cells (APCs) sense pathogen-associated molecular patterns, such as dsRNA, to activate the immune response. The impact of ADAR1 on the function of APCs and the consequences to immunity are poorly understood. Here, we show that ADAR1 deletion in CD11c+ APCs leads to (1) a skewed myeloid cell compartment enriched in inflammatory cDC2-like cells, (2) enhanced numbers of activated tissue resident memory T cells in the lung, and (3) the imprinting of a broad antiviral transcriptional signature across both immune and non-immune cells. The resulting changes can be partially reversed by blocking IFNAR1 signaling and promote early resistance against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Our study provides insight into the consequences of self-dsRNA sensing in APCs on the immune system.

A modified vaccinia Ankara vaccine expressing spike and nucleocapsid protects rhesus macaques against SARS-CoV-2 Delta infection.

SARS-CoV-2 vaccines should induce broadly cross-reactive humoral and T cell responses to protect against emerging variants of concern (VOCs). Here, we inactivated the furin cleavage site (FCS) of spike expressed by a modified vaccinia Ankara (MVA) virus vaccine (MVA/SdFCS) and found that FCS inactivation markedly increased spike binding to human ACE2. After vaccination of mice, the MVA/SdFCS vaccine induced eightfold higher neutralizing antibodies compared with MVA/S, which expressed spike without FCS inactivation, and protected against the Beta variant. We next added nucleocapsid to the MVA/SdFCS vaccine (MVA/SdFCS-N) and tested its immunogenicity and efficacy via intramuscular (IM), buccal (BU), or sublingual (SL) routes in rhesus macaques. IM vaccination induced spike-specific IgG in serum and mucosae (nose, throat, lung, and rectum) that neutralized the homologous (WA-1/2020) and heterologous VOCs, including Delta, with minimal loss (<2-fold) of activity. IM vaccination also induced both spike- and nucleocapsid-specific CD4 and CD8 T cell responses in the blood. In contrast, the SL and BU vaccinations induced less spike-specific IgG in secretions and lower levels of polyfunctional IgG in serum compared with IM vaccination. After challenge with the SARS-CoV-2 Delta variant, the IM route induced robust protection, the BU route induced moderate protection, and the SL route induced no protection. Vaccine-induced neutralizing and non-neutralizing antibody effector functions positively correlated with protection, but only the effector functions correlated with early protection. Thus, IM vaccination with MVA/SdFCS-N vaccine elicited cross-reactive antibody and T cell responses, protecting against heterologous SARS-CoV-2 VOC more effectively than other routes of vaccination.

Adjuvanting a subunit SARS-CoV-2 vaccine with clinically relevant adjuvants induces durable protection in mice.

Adjuvants enhance the magnitude and the durability of the immune response to vaccines. However, there is a paucity of comparative studies on the nature of the immune responses stimulated by leading adjuvant candidates. In this study, we compared five clinically relevant adjuvants in mice-alum, AS03 (a squalene-based adjuvant supplemented with α-tocopherol), AS37 (a TLR7 ligand emulsified in alum), CpG1018 (a TLR9 ligand emulsified in alum), O/W 1849101 (a squalene-based adjuvant)-for their capacity to stimulate immune responses when combined with a subunit vaccine under clinical development. We found that all four of the adjuvant candidates surpassed alum with respect to their capacity to induce enhanced and durable antigen-specific antibody responses. The TLR-agonist-based adjuvants CpG1018 (TLR9) and AS37 (TLR7) induced Th1-skewed CD4+ T cell responses, while alum, O/W, and AS03 induced a balanced Th1/Th2 response. Consistent with this, adjuvants induced distinct patterns of early innate responses. Finally, vaccines adjuvanted with AS03, AS37, and CpG1018/alum-induced durable neutralizing-antibody responses and significant protection against the B.1.351 variant 7 months following immunization. These results, together with our recent results from an identical study in non-human primates (NHPs), provide a comparative benchmarking of five clinically relevant vaccine adjuvants for their capacity to stimulate immunity to a subunit vaccine, demonstrating the capacity of adjuvanted SARS-CoV-2 subunit vaccines to provide durable protection against the B.1.351 variant. Furthermore, these results reveal differences between the widely-used C57BL/6 mouse strain and NHP animal models, highlighting the importance of species selection for future vaccine and adjuvant studies.

mRNA-1273 and BNT162b2 mRNA vaccines have reduced neutralizing activity against the SARS-CoV-2 omicron variant.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) omicron variant emerged in November 2021 and consists of several mutations within the spike. We use serum from mRNA-vaccinated individuals to measure neutralization activity against omicron in a live-virus assay. At 2-4 weeks after a primary series of vaccinations, we observe a 30-fold reduction in neutralizing activity against omicron. Six months after the initial two-vaccine doses, sera from naive vaccinated subjects show no neutralizing activity against omicron. In contrast, COVID-19-recovered individuals 6 months after receiving the primary series of vaccinations show a 22-fold reduction, with the majority of the subjects retaining neutralizing antibody responses. In naive individuals following a booster shot (third dose), we observe a 14-fold reduction in neutralizing activity against omicron, and over 90% of subjects show neutralizing activity. These findings show that a third dose is required to provide robust neutralizing antibody responses against the omicron variant.