Robust and prototypical immune responses toward COVID-19 vaccine in First Nations peoples are impacted by comorbidities.

High-risk groups, including Indigenous people, are at risk of severe COVID-19. Here we found that Australian First Nations peoples elicit effective immune responses to COVID-19 BNT162b2 vaccination, including neutralizing antibodies, receptor-binding domain (RBD) antibodies, SARS-CoV-2 spike-specific B cells, and CD4+ and CD8+ T cells. In First Nations participants, RBD IgG antibody titers were correlated with body mass index and negatively correlated with age. Reduced RBD antibodies, spike-specific B cells and follicular helper T cells were found in vaccinated participants with chronic conditions (diabetes, renal disease) and were strongly associated with altered glycosylation of IgG and increased interleukin-18 levels in the plasma. These immune perturbations were also found in non-Indigenous people with comorbidities, indicating that they were related to comorbidities rather than ethnicity. However, our study is of a great importance to First Nations peoples who have disproportionate rates of chronic comorbidities and provides evidence of robust immune responses after COVID-19 vaccination in Indigenous people.

Parallel use of human stem cell lung and heart models provide insights for SARS-CoV-2 treatment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily infects the respiratory tract, but pulmonary and cardiac complications occur in severe coronavirus disease 2019 (COVID-19). To elucidate molecular mechanisms in the lung and heart, we conducted paired experiments in human stem cell-derived lung alveolar type II (AT2) epithelial cell and cardiac cultures infected with SARS-CoV-2. With CRISPR-Cas9-mediated knockout of ACE2, we demonstrated that angiotensin-converting enzyme 2 (ACE2) was essential for SARS-CoV-2 infection of both cell types but that further processing in lung cells required TMPRSS2, while cardiac cells required the endosomal pathway. Host responses were significantly different; transcriptome profiling and phosphoproteomics responses depended strongly on the cell type. We identified several antiviral compounds with distinct antiviral and toxicity profiles in lung AT2 and cardiac cells, highlighting the importance of using several relevant cell types for evaluation of antiviral drugs. Our data provide new insights into rational drug combinations for effective treatment of a virus that affects multiple organ systems.

Respiratory viruses: New frontiers-a Keystone Symposia report.

Respiratory viruses are a common cause of morbidity and mortality around the world. Viruses like influenza, RSV, and most recently SARS-CoV-2 can rapidly spread through a population, causing acute infection and, in vulnerable populations, severe or chronic disease. Developing effective treatment and prevention strategies often becomes a race against ever-evolving viruses that develop resistance, leaving therapy efficacy either short-lived or relevant for specific viral strains. On June 29 to July 2, 2022, researchers met for the Keystone symposium "Respiratory Viruses: New Frontiers." Researchers presented new insights into viral biology and virus-host interactions to understand the mechanisms of disease and identify novel treatment and prevention approaches that are effective, durable, and broad.

Broad immunity to SARS-CoV-2 variants of concern mediated by a SARS-CoV-2 receptor-binding domain protein vaccine.

BACKGROUND: The SARS-CoV-2 global pandemic has fuelled the generation of vaccines at an unprecedented pace and scale. However, many challenges remain, including: the emergence of vaccine-resistant mutant viruses, vaccine stability during storage and transport, waning vaccine-induced immunity, and concerns about infrequent adverse events associated with existing vaccines. METHODS: We report on a protein subunit vaccine comprising the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 spike protein, dimerised with an immunoglobulin IgG1 Fc domain. These were tested in conjunction with three different adjuvants: a TLR2 agonist R4-Pam2Cys, an NKT cell agonist glycolipid α-Galactosylceramide, or MF59® squalene oil-in-water adjuvant, using mice, rats and hamsters. We also developed an RBD-human IgG1 Fc vaccine with an RBD sequence of the immuno-evasive beta variant (N501Y, E484K, K417N). These vaccines were also tested as a heterologous third dose booster in mice, following priming with whole spike vaccine. FINDINGS: Each formulation of the RBD-Fc vaccines drove strong neutralising antibody (nAb) responses and provided durable and highly protective immunity against lower and upper airway infection in mouse models of COVID-19. The 'beta variant' RBD vaccine, combined with MF59® adjuvant, induced strong protection in mice against the beta strain as well as the ancestral strain. Furthermore, when used as a heterologous third dose booster, the RBD-Fc vaccines combined with MF59® increased titres of nAb against other variants including alpha, delta, delta+, gamma, lambda, mu, and omicron BA.1, BA.2 and BA.5. INTERPRETATION: These results demonstrated that an RBD-Fc protein subunit/MF59® adjuvanted vaccine can induce high levels of broadly reactive nAbs, including when used as a booster following prior immunisation of mice with whole ancestral-strain spike vaccines. This vaccine platform offers a potential approach to augment some of the currently approved vaccines in the face of emerging variants of concern, and it has now entered a phase I clinical trial. FUNDING: This work was supported by grants from the Medical Research Future Fund (MRFF) (2005846), The Jack Ma Foundation, National Health and Medical Research Council of Australia (NHMRC; 1113293) and Singapore National Medical Research Council (MOH-COVID19RF-003). Individual researchers were supported by an NHMRC Senior Principal Research Fellowship (1117766), NHMRC Investigator Awards (2008913 and 1173871), Australian Research Council Discovery Early Career Research Award (ARC DECRA; DE210100705) and philanthropic awards from IFM investors and the A2 Milk Company.

Robust SARS-CoV-2 T cell responses with common TCRαß motifs toward COVID-19 vaccines in patients with hematological malignancy impacting B cells.

Immunocompromised hematology patients are vulnerable to severe COVID-19 and respond poorly to vaccination. Relative deficits in immunity are, however, unclear, especially after 3 vaccine doses. We evaluated immune responses in hematology patients across three COVID-19 vaccination doses. Seropositivity was low after a first dose of BNT162b2 and ChAdOx1 (∼26%), increased to 59%-75% after a second dose, and increased to 85% after a third dose. While prototypical antibody-secreting cells (ASCs) and T follicular helper (Tfh) cell responses were elicited in healthy participants, hematology patients showed prolonged ASCs and skewed Tfh2/17 responses. Importantly, vaccine-induced expansions of spike-specific and peptide-HLA tetramer-specific CD4+/CD8+ T cells, together with their T cell receptor (TCR) repertoires, were robust in hematology patients, irrespective of B cell numbers, and comparable to healthy participants. Vaccinated patients with breakthrough infections developed higher antibody responses, while T cell responses were comparable to healthy groups. COVID-19 vaccination induces robust T cell immunity in hematology patients of varying diseases and treatments irrespective of B cell numbers and antibody response.

Macrophage ACE2 is necessary for SARS-CoV-2 replication and subsequent cytokine responses that restrict continued virion release.

Macrophages are key cellular contributors to the pathogenesis of COVID-19, the disease caused by the virus SARS-CoV-2. The SARS-CoV-2 entry receptor ACE2 is present only on a subset of macrophages at sites of SARS-CoV-2 infection in humans. Here, we investigated whether SARS-CoV-2 can enter macrophages, replicate, and release new viral progeny; whether macrophages need to sense a replicating virus to drive cytokine release; and, if so, whether ACE2 is involved in these mechanisms. We found that SARS-CoV-2 could enter, but did not replicate within, ACE2-deficient human primary macrophages and did not induce proinflammatory cytokine expression. By contrast, ACE2 overexpression in human THP-1-derived macrophages permitted SARS-CoV-2 entry, processing and replication, and virion release. ACE2-overexpressing THP-1 macrophages sensed active viral replication and triggered proinflammatory, antiviral programs mediated by the kinase TBK-1 that limited prolonged viral replication and release. These findings help elucidate the role of ACE2 and its absence in macrophage responses to SARS-CoV-2 infection.

Immune profiling of SARS-CoV-2 infection during pregnancy reveals NK cell and γδ T cell perturbations.

Pregnancy poses a greater risk for severe COVID-19; however, underlying immunological changes associated with SARS-CoV-2 during pregnancy are poorly understood. We defined immune responses to SARS-CoV-2 in unvaccinated pregnant and nonpregnant women with acute and convalescent COVID-19, quantifying 217 immunological parameters. Humoral responses to SARS-CoV-2 were similar in pregnant and nonpregnant women, although our systems serology approach revealed distinct antibody and FcγR profiles between pregnant and nonpregnant women. Cellular analyses demonstrated marked differences in NK cell and unconventional T cell activation dynamics in pregnant women. Healthy pregnant women displayed preactivated NK cells and γδ T cells when compared with healthy nonpregnant women, which remained unchanged during acute and convalescent COVID-19. Conversely, nonpregnant women had prototypical activation of NK and γδ T cells. Activation of CD4+ and CD8+ T cells and T follicular helper cells was similar in SARS-CoV-2-infected pregnant and nonpregnant women, while antibody-secreting B cells were increased in pregnant women during acute COVID-19. Elevated levels of IL-8, IL-10, and IL-18 were found in pregnant women in their healthy state, and these cytokine levels remained elevated during acute and convalescent COVID-19. Collectively, we demonstrate perturbations in NK cell and γδ T cell activation in unvaccinated pregnant women with COVID-19, which may impact disease progression and severity during pregnancy.

Predicting the efficacy of variant-modified COVID-19 vaccine boosters.

Booster vaccination for the prevention of Coronavirus Disease 2019 (COVID-19) is required to overcome loss of protection due to waning immunity and the spread of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. Studies have assessed the ability of existing ancestral-based vaccines as well as novel variant-modified vaccine regimens to boost immunity to different variants, and a crucial question is to assess the relative benefits of these different approaches. Here we aggregate data on neutralization titers from 14 reports (three published papers, eight preprints, two press releases and notes of one advisory committee meeting) comparing booster vaccination with the current ancestral-based vaccines or variant-modified vaccines. Using these data, we compare the immunogenicity of different vaccination regimens and predict the relative protection of booster vaccines under different scenarios. We predict that boosting with ancestral vaccines can markedly enhance protection against both symptomatic and severe disease from SARS-CoV-2 variant viruses, although variant-modified vaccines may provide additional protection, even if not matched to the circulating variants. This work provides an evidence-based framework to inform choices on future SARS-CoV-2 vaccine regimens.

Incorporation of SARS-CoV-2 spike NTD to RBD protein vaccine improves immunity against viral variants.

Emerging SARS-CoV-2 variants pose a threat to human health worldwide. SARS-CoV-2 receptor binding domain (RBD)-based vaccines are suitable candidates for booster vaccines, eliciting a focused antibody response enriched for virus neutralizing activity. Although RBD proteins are manufactured easily, and have excellent stability and safety properties, they are poorly immunogenic compared to the full-length spike protein. We have overcome this limitation by engineering a subunit vaccine composed of an RBD tandem dimer fused to the N-terminal domain (NTD) of the spike protein. We found that inclusion of the NTD (1) improved the magnitude and breadth of the T cell and anti-RBD response, and (2) enhanced T follicular helper cell and memory B cell generation, antibody potency, and cross-reactive neutralization activity against multiple SARS-CoV-2 variants, including B.1.1.529 (Omicron BA.1). In summary, our uniquely engineered RBD-NTD-subunit protein vaccine provides a promising booster vaccination strategy capable of protecting against known SARS-CoV-2 variants of concern.

Corrigendum: Fc engineered ACE2-Fc is a potent multifunctional agent targeting SARS-CoV2.

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Detection of influenza in managed quarantine in Australia and the estimated risk of importation.

BACKGROUND: Influenza circulated at historically-low levels during 2020 and 2021 due to COVID-19 pandemic travel restrictions. In Australia, international arrivals to Australia were required to undertake 14 days hotel quarantine to limit new introduction of SARS-CoV-2 virus. METHODS: We used routine testing data for travellers arriving on repatriation flights to Darwin, Australia from 3 January to 11 October 2021 to identify importations of influenza virus into Australia and used this information to estimate the risk of a case exiting quarantine while still infectious. Influenza-positive samples were sequenced and cases were followed-up to identify transmission clusters. Data on the number of cases and total passengers was used to infer the risk of influenza cases existing quarantine while infectious. RESULTS: Despite very low circulation of influenza globally, 42 cases were identified among 15,026 returned travellers, of which 30 were A(H3N2), two were A(H1N1)pdm09 and 10 were B/Victoria. Virus sequencing data identified potential in-flight transmission, as well as independent infections prior to travel. Under the quarantine strategy in place at the time, the probability that these cases could initiate influenza outbreaks in Australia neared 0. However, this probability rose as quarantine requirements relaxed. CONCLUSIONS: Detection of influenza virus infections in repatriated travellers provided a source of influenza viruses otherwise unavailable and enabled development of the A(H3N2) vaccine seed viruses included in the 2022 Southern Hemisphere influenza vaccine. Failing to test quarantined returned travellers for influenza, represents a missed opportunity for enhanced surveillance to better inform public health preparedness.

Correlates of Protection, Thresholds of Protection, and Immunobridging among Persons with SARS-CoV-2 Infection.

Several studies have shown that neutralizing antibody levels correlate with immune protection from COVID-19 and have estimated the relationship between neutralizing antibodies and protection. However, results of these studies vary in terms of estimates of the level of neutralizing antibodies required for protection. By normalizing antibody titers, we found that study results converge on a consistent relationship between antibody levels and protection from COVID-19. This finding can be useful for planning future vaccine use, determining population immunity, and reducing the global effects of the COVID-19 pandemic.

Biparatopic nanobodies targeting the receptor binding domain efficiently neutralize SARS-CoV-2.

The development of therapeutics to prevent or treat COVID-19 remains an area of intense focus. Protein biologics, including monoclonal antibodies and nanobodies that neutralize virus, have potential for the treatment of active disease. Here, we have used yeast display of a synthetic nanobody library to isolate nanobodies that bind the receptor-binding domain (RBD) of SARS-CoV-2 and neutralize the virus. We show that combining two clones with distinct binding epitopes within the RBD into a single protein construct to generate biparatopic reagents dramatically enhances their neutralizing capacity. Furthermore, the biparatopic nanobodies exhibit enhanced control over clinically relevant RBD variants that escaped recognition by the individual nanobodies. Structural analysis of biparatopic binding to spike (S) protein revealed a unique binding mode whereby the two nanobody paratopes bridge RBDs encoded by distinct S trimers. Accordingly, biparatopic nanobodies offer a way to rapidly generate powerful viral neutralizers with enhanced ability to control viral escape mutants.

Heparin Inhibits SARS-CoV-2 Replication in Human Nasal Epithelial Cells.

SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Vaccination, supported by social and public health measures, has proven efficacious for reducing disease severity and virus spread. However, the emergence of highly transmissible viral variants that escape prior immunity highlights the need for additional mitigation approaches. Heparin binds the SARS-CoV-2 spike protein and can inhibit virus entry and replication in susceptible human cell lines and bronchial epithelial cells. Primary infection predominantly occurs via the nasal epithelium, but the nasal cell biology of SARS-CoV-2 is not well studied. We hypothesized that prophylactic intranasal administration of heparin may provide strain-agnostic protection for household contacts or those in high-risk settings against SARS-CoV-2 infection. Therefore, we investigated the ability of heparin to inhibit SARS-CoV-2 infection and replication in differentiated human nasal epithelial cells and showed that prolonged exposure to heparin inhibits virus infection. Furthermore, we establish a method for PCR detection of SARS-CoV-2 viral genomes in heparin-treated samples that can be adapted for the detection of viruses in clinical studies.

Fc engineered ACE2-Fc is a potent multifunctional agent targeting SARS-CoV2.

Joining a function-enhanced Fc-portion of human IgG to the SARS-CoV-2 entry receptor ACE2 produces an antiviral decoy with strain transcending virus neutralizing activity. SARS-CoV-2 neutralization and Fc-effector functions of ACE2-Fc decoy proteins, formatted with or without the ACE2 collectrin domain, were optimized by Fc-modification. The different Fc-modifications resulted in distinct effects on neutralization and effector functions. H429Y, a point mutation outside the binding sites for FcγRs or complement caused non-covalent oligomerization of the ACE2-Fc decoy proteins, abrogated FcγR interaction and enhanced SARS-CoV-2 neutralization. Another Fc mutation, H429F did not improve virus neutralization but resulted in increased C5b-C9 fixation and transformed ACE2-Fc to a potent mediator of complement-dependent cytotoxicity (CDC) against SARS-CoV-2 spike (S) expressing cells. Furthermore, modification of the Fc-glycan enhanced cell activation via FcγRIIIa. These different immune profiles demonstrate the capacity of Fc-based agents to be engineered to optimize different mechanisms of protection for SARS-CoV-2 and potentially other viral pathogens.

Enhanced metanephric specification to functional proximal tubule enables toxicity screening and infectious disease modelling in kidney organoids.

While pluripotent stem cell-derived kidney organoids are now being used to model renal disease, the proximal nephron remains immature with limited evidence for key functional solute channels. This may reflect early mispatterning of the nephrogenic mesenchyme and/or insufficient maturation. Here we show that enhanced specification to metanephric nephron progenitors results in elongated and radially aligned proximalised nephrons with distinct S1 - S3 proximal tubule cell types. Such PT-enhanced organoids possess improved albumin and organic cation uptake, appropriate KIM-1 upregulation in response to cisplatin, and improved expression of SARS-CoV-2 entry factors resulting in increased viral replication. The striking proximo-distal orientation of nephrons resulted from localized WNT antagonism originating from the organoid stromal core. PT-enhanced organoids represent an improved model to study inherited and acquired proximal tubular disease as well as drug and viral responses.

SARS-CoV-2 Omicron BA.1 Challenge after Ancestral or Delta Infection in Mice.

We assessed cross-reactivity to BA.1, BA.2, and BA.5 of neutralizing antibodies elicited by ancestral, Delta, and Omicron BA.1 SARS-CoV-2 infection in mice. Primary infection elicited homologous antibodies with poor cross-reactivity to Omicron strains. This pattern remained after BA.1 challenge, although ancestral- and Delta-infected mice were protected from BA.1 infection.