CD8+ T-cell responses towards conserved influenza B virus epitopes across anatomical sites and age.

Influenza B viruses (IBVs) cause substantive morbidity and mortality, and yet immunity towards IBVs remains understudied. CD8+ T-cells provide broadly cross-reactive immunity and alleviate disease severity by recognizing conserved epitopes. Despite the IBV burden, only 18 IBV-specific T-cell epitopes restricted by 5 HLAs have been identified currently. A broader array of conserved IBV T-cell epitopes is needed to develop effective cross-reactive T-cell based IBV vaccines. Here we identify 9 highly conserved IBV CD8+ T-cell epitopes restricted to HLA-B*07:02, HLA-B*08:01 and HLA-B*35:01. Memory IBV-specific tetramer+CD8+ T-cells are present within blood and tissues. Frequencies of IBV-specific CD8+ T-cells decline with age, but maintain a central memory phenotype. HLA-B*07:02 and HLA-B*08:01-restricted NP30-38 epitope-specific T-cells have distinct T-cell receptor repertoires. We provide structural basis for the IBV HLA-B*07:02-restricted NS1196-206 (11-mer) and HLA-B*07:02-restricted NP30-38 epitope presentation. Our study increases the number of IBV CD8+ T-cell epitopes, and defines IBV-specific CD8+ T-cells at cellular and molecular levels, across tissues and age.

Prior infection with unrelated neurotropic virus exacerbates influenza disease and impairs lung T cell responses.

Immunity to infectious diseases is predominantly studied by measuring immune responses towards a single pathogen, although co-infections are common. In-depth mechanisms on how co-infections impact anti-viral immunity are lacking, but are highly relevant to treatment and prevention. We established a mouse model of co-infection with unrelated viruses, influenza A (IAV) and Semliki Forest virus (SFV), causing disease in different organ systems. SFV infection eight days before IAV infection results in prolonged IAV replication, elevated cytokine/chemokine levels and exacerbated lung pathology. This is associated with impaired lung IAV-specific CD8+ T cell responses, stemming from suboptimal CD8+ T cell activation and proliferation in draining lymph nodes, and dendritic cell paralysis. Prior SFV infection leads to increased blood brain barrier permeability and presence of IAV RNA in brain, associated with increased trafficking of IAV-specific CD8+ T cells and establishment of long-term tissue-resident memory. Relative to lung IAV-specific CD8+ T cells, brain memory IAV-specific CD8+ T cells have increased TCR repertoire diversity within immunodominant DbNP366+CD8+ and DbPA224+CD8+ responses, featuring suboptimal TCR clonotypes. Overall, our study demonstrates that infection with an unrelated neurotropic virus perturbs IAV-specific immune responses and exacerbates IAV disease. Our work provides key insights into therapy and vaccine regimens directed against unrelated pathogens.

Interim results from a phase I randomized, placebo-controlled trial of novel SARS-CoV-2 beta variant receptor-binding domain recombinant protein and mRNA vaccines as a 4th dose booster.

BACKGROUND: SARS-CoV-2 booster vaccination should ideally enhance protection against variants and minimise immune imprinting. This Phase I trial evaluated two vaccines targeting SARS-CoV-2 beta-variant receptor-binding domain (RBD): a recombinant dimeric RBD-human IgG1 Fc-fusion protein, and an mRNA encoding a membrane-anchored RBD. METHODS: 76 healthy adults aged 18-64 y, previously triple vaccinated with licensed SARS-CoV-2 vaccines, were randomised to receive a 4th dose of either an adjuvanted (MF59®, CSL Seqirus) protein vaccine (5, 15 or 45 µg, N = 32), mRNA vaccine (10, 20, or 50 µg, N = 32), or placebo (saline, N = 12) at least 90 days after a 3rd boost vaccination or SARS-CoV-2 infection. Bleeds occurred on days 1 (prior to vaccination), 8, and 29. CLINICALTRIALS: govNCT05272605. FINDINGS: No vaccine-related serious or medically-attended adverse events occurred. The protein vaccine reactogenicity was mild, whereas the mRNA vaccine was moderately reactogenic at higher dose levels. Best anti-RBD antibody responses resulted from the higher doses of each vaccine. A similar pattern was seen with live virus neutralisation and surrogate, and pseudovirus neutralisation assays. Breadth of immune response was demonstrated against BA.5 and more recent omicron subvariants (XBB, XBB.1.5 and BQ.1.1). Binding antibody titres for both vaccines were comparable to those of a licensed bivalent mRNA vaccine. Both vaccines enhanced CD4+ and CD8+ T cell activation. INTERPRETATION: There were no safety concerns and the reactogenicity profile was mild and similar to licensed SARS-CoV-2 vaccines. Both vaccines showed strong immune boosting against beta, ancestral and omicron strains. FUNDING: Australian Government Medical Research Future Fund, and philanthropies Jack Ma Foundation and IFM investors.

Newborn and child-like molecular signatures in older adults stem from TCR shifts across human lifespan.

CD8+ T cells provide robust antiviral immunity, but how epitope-specific T cells evolve across the human lifespan is unclear. Here we defined CD8+ T cell immunity directed at the prominent influenza epitope HLA-A*02:01-M158-66 (A2/M158) across four age groups at phenotypic, transcriptomic, clonal and functional levels. We identify a linear differentiation trajectory from newborns to children then adults, followed by divergence and a clonal reset in older adults. Gene profiles in older adults closely resemble those of newborns and children, despite being clonally distinct. Only child-derived and adult-derived A2/M158+CD8+ T cells had the potential to differentiate into highly cytotoxic epitope-specific CD8+ T cells, which was linked to highly functional public T cell receptor (TCR)αß signatures. Suboptimal TCRαß signatures in older adults led to less proliferation, polyfunctionality, avidity and recognition of peptide mutants, although displayed no signs of exhaustion. These data suggest that priming T cells at different stages of life might greatly affect CD8+ T cell responses toward viral infections.

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.

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.

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.

In Semliki Forest virus encephalitis, suppressor of cytokine signaling 4 (SOCS4) is an essential modulator of immune responses that mediates the balance between immunopathology and virus clearance.

Central nervous system virus infections are a major cause of morbidity and mortality worldwide and a significant global public health concern. As in many tissues, inflammation and immune responses in the brain, despite their protective roles, can also be harmful. Control of brain inflammation is important in many neurological diseases from encephalitis to multiple sclerosis and neurogenerative disease. The suppressors of cytokine signaling (SOCS) proteins are a key mechanism controlling inflammatory and immune responses across all tissues including the brain. Using a mouse model system, we demonstrate that lack of SOCS4 results in changes in the pathogenesis and clinical outcome of a neurotropic virus infection. Relative to wild-type mice, SOCS4-deficient mice showed accelerated clearance of virus from the brain, lower levels of persisting viral RNA in the brain, increased neuroinflammation and more severe neuropathology. We conclude that, in the mouse brain, SOCS4 is a vital regulator of antiviral immunity that mediates the critical balance between immunopathology and virus persistence.

Immunization with inactivated whole virus particle influenza virus vaccines improves the humoral response landscape in cynomolgus macaques.

Although antibody-inducing split virus vaccines (SV) are currently the most effective way to combat seasonal influenza, their efficacy can be modest, especially in immunologically-naïve individuals. We investigated immune responses towards inactivated whole influenza virus particle vaccine (WPV) formulations, predicated to be more immunogenic, in a non-human primate model, as an important step towards clinical testing in humans. Comprehensive analyses were used to capture 46 immune parameters to profile how WPV-induced responses differed to those elicited by antigenically-similar SV formulations. Naïve cynomolgus macaques vaccinated with either monovalent or quadrivalent WPV consistently induced stronger antibody responses and hemagglutination inhibition (HI) antibody titres against vaccine-matched viruses compared to SV formulations, while acute reactogenic effects were similar. Responses in WPV-primed animals were further increased by boosting with the same formulation, conversely to modest responses after priming and boosting with SV. 28-parameter multiplex bead array defined key antibody features and showed that while both WPV and SV induced elevated IgG responses against A/H1N1 nucleoprotein, only WPV increased IgG responses against A/H1N1 hemagglutinin (HA) and HA-Stem, and higher IgA responses to A/H1N1-HA after each vaccine dose. Antibodies to A/H1N1-HA and HA-Stem that could engage FcγR2a and FcγR3a were also present at higher levels after one dose of WPV compared to SV and remained elevated after the second dose. Furthermore, WPV-enhanced antibody responses were associated with higher frequencies of HA-specific B-cells and IFN-γ-producing CD4+ T-cell responses. Our data additionally demonstrate stronger boosting of HI titres by WPV following prior infection and support WPV administered as a priming dose irrespective of the follow up vaccine for the second dose. Our findings thus show that compared to SV vaccination, WPV-induced humoral responses are significantly increased in scope and magnitude, advocating WPV vaccination regimens for priming immunologically-naïve individuals and also in the event of a pandemic outbreak.

Immature Brain Cortical Neurons Have Low Transcriptional Competence to Activate Antiviral Defences and Control RNA Virus Infections.

Virus infections of the central nervous system (CNS) cause important diseases of humans and animals. As in other tissues, innate antiviral responses mediated by type I interferons (IFNs) are crucially important in controlling CNS virus infections. The maturity of neuronal populations is an established critical factor determining the outcome of CNS virus infection. Using primary cultures of mouse cortical neurons, we investigated the relationships between neuronal maturation, type I IFN responses, and the outcome of Semliki Forest virus infection. The virus replicated better, infected more cells, and produced higher titres of infectious viruses in immature neurons. Complete transcriptome analysis demonstrated that resting immature neurons have low transcriptional competence to mount antiviral responses. They had no detectable transcription of the genes Ddx58 and Ifih1, which encode key RNA virus cytoplasmic sensors RIG-I and MDA5, and very low expression of genes encoding key regulators of associated signalling pathways. Upon infection, immature neurons failed to mount an antiviral response as evidenced by their failure to produce chemokines, IFNs, and other cytokines. Treatment of immature neurons with exogenous IFNß prior to infection resulted in antiviral responses and lower levels of virus replication and infectious virus production. In contrast, resting mature neurons generated a robust antiviral response. This was augmented by pretreatment with IFNß. Infection of mature neurons derived from IFNAR-/- mice did not make an antiviral response and replicated virus to high levels.

SARS-CoV-2-specific T cell memory with common TCRαß motifs is established in unvaccinated children who seroconvert after infection.

As the establishment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell memory in children remains largely unexplored, we recruited convalescent COVID-19 children and adults to define their circulating memory SARS-CoV-2-specific CD4+ and CD8+ T cells prior to vaccination. We analyzed epitope-specific T cells directly ex vivo using seven HLA class I and class II tetramers presenting SARS-CoV-2 epitopes, together with Spike-specific B cells. Unvaccinated children who seroconverted had comparable Spike-specific but lower ORF1a- and N-specific memory T cell responses compared with adults. This agreed with our TCR sequencing data showing reduced clonal expansion in children. A strong stem cell memory phenotype and common T cell receptor motifs were detected within tetramer-specific T cells in seroconverted children. Conversely, children who did not seroconvert had tetramer-specific T cells of predominantly naive phenotypes and diverse TCRαß repertoires. Our study demonstrates the generation of SARS-CoV-2-specific T cell memory with common TCRαß motifs in unvaccinated seroconverted children after their first virus encounter.

TLR2-mediated activation of innate responses in the upper airways confers antiviral protection of the lungs.

The impact of respiratory virus infections on global health is felt not just during a pandemic, but endemic seasonal infections pose an equal and ongoing risk of severe disease. Moreover, vaccines and antiviral drugs are not always effective or available for many respiratory viruses. We investigated how induction of effective and appropriate antigen-independent innate immunity in the upper airways can prevent the spread of respiratory virus infection to the vulnerable lower airways. Activation of TLR2, when restricted to the nasal turbinates, resulted in prompt induction of innate immune-driven antiviral responses through action of cytokines, chemokines, and cellular activity in the upper but not the lower airways. We have defined how nasal epithelial cells and recruitment of macrophages work in concert and play pivotal roles to limit progression of influenza virus to the lungs and sustain protection for up to 7 days. These results reveal underlying mechanisms of how control of viral infection in the upper airways can occur and support the implementation of strategies that can activate TLR2 in nasal passages to provide rapid protection, especially for at-risk populations, against severe respiratory infection when vaccines and antiviral drugs are not always effective or available.

Geometry of a TLR2-Agonist-Based Adjuvant Can Affect the Resulting Antigen-Specific Immune Response.

Targeted delivery of otherwise nonimmunogenic antigens to Toll-like receptors (TLRs) expressed on dendritic cells (DCs) has proven to be an effective means of improving immunogenicity. For this purpose, we have used a branched cationic lipopeptide, R4Pam2Cys, which is an agonist for TLR2 and enables electrostatic association with antigen for this purpose. Here, we compare the immunological properties of ovalbumin formulated with different geometrical configurations of R4Pam2Cys. Our results demonstrate that notwithstanding the presence of the same adjuvant, branched forms of R4Pam2Cys are more effective at inducing immune responses than are linear geometries. CD8+ T-cell-mediated responses are particularly improved, resulting in significantly higher levels of antigen-specific cytokine secretion and cytolysis of antigen-bearing target cells in vivo. The results correlate with the ability of branched R4Pam2Cys conformations to encourage higher levels of DC maturation and facilitate superior antigen uptake, leading to increased production of proinflammatory cytokines. These differences are not attributable to particle size because both branched and linear lipopeptides associate with antigen-forming complexes of similar size, but rather the ability of branched lipopeptides to induce more efficient TLR2-mediated cell signaling. Branched lipopeptides are also more resistant to trypsin-mediated proteolysis, suggesting greater stability than their linear counterparts. The branched lipopeptide facilitates presentation of antigen more efficiently to CD8+ T cells, resulting in rapid cell division and upregulation of early cell surface activation markers. These results as well as cognate recognition of Pam2Cys by TLR2 indicate that the adjuvant's efficiency is also dependent on its geometry.

CD4+ T help promotes influenza virus-specific CD8+ T cell memory by limiting metabolic dysfunction.

There is continued interest in developing novel vaccine strategies that induce establish optimal CD8+ cytotoxic T lymphocyte (CTL) memory for pathogens like the influenza A viruses (IAVs), where the recall of IAV-specific T cell immunity is able to protect against serologically distinct IAV infection. While it is well established that CD4+ T cell help is required for optimal CTL responses and the establishment of memory, when and how CD4+ T cell help contributes to determining the ideal memory phenotype remains unclear. We assessed the quality of IAV-specific CD8+ T cell memory established in the presence or absence of a concurrent CD4+ T cell response. We demonstrate that CD4+ T cell help appears to be required at the initial priming phase of infection for the maintenance of IAV-specific CTL memory, with "unhelped" memory CTL exhibiting intrinsic dysfunction. High-throughput RNA-sequencing established that distinct transcriptional signatures characterize the helped vs. unhelped IAV-specific memory CTL phenotype, with the unhelped set showing a more "exhausted T cell" transcriptional profile. Moreover, we identify that unhelped memory CTLs exhibit defects in a variety of energetic pathways, leading to diminished spare respiratory capacity and diminished capacity to engage glycolysis upon reactivation. Hence, CD4+ T help at the time of initial priming promotes molecular pathways that limit exhaustion by channeling metabolic processes essential for the rapid recall of memory CD8+ T cells.