Unexpected involvement of IL-13 signalling via a STAT6 independent mechanism during murine IgG2a development following viral vaccination.

In this study, recombinant pox viral vaccination was shown to induce highly elevated IgG2a and low IgG1 antibody expression in mice lacking IL-4 or STAT6, whilst IL-13-/- mice exhibited elevated IgG1, but very low IgG2a. These findings revealed that IL-13 and IL-4 differentially regulated antibody development. To understand this further, when STAT6-/- mice were given a vaccine co-expressing IL-13Rα2 that temporarily sequestered IL-13, significantly reduced IgG2a expression, was detected. These findings for the first time demonstrated that IL-13 regulated IgG2a differentiation utilising an alternative IL-13R signalling pathway independent of STAT6 (IL-13Rα2 pathway). This was further corroborated by the (i) elevated IL-13Rα2 expression detected on STAT6-/- lung MHCII+ CD11c+ cells 24 h post IL-13 inhibitor vaccination and ii) significant up-regulation of IL-13Rα2 expression on spleen and lung derived MHCII+ CD11c+ following inhibition of STAT6 signalling in vitro, or vaccination with IL-4R/STAT6 antagonist in vivo. When T follicular helper (Tfh) cells which regulate antibody differentiation were assessed post vaccination, although no difference in IL-4 expression was observed, greatly reduced IFN-γ expression was detected in IL-13-/- and STAT6-/- mice compared to wild-type. These findings support the notion that the balance of IL-13 level at the vaccination site can differentially regulate T and B-cell immune outcomes.

Induction of Genotype Cross-Reactive, Hepatitis C Virus-Specific, Cell-Mediated Immunity in DNA-Vaccinated Mice.

A universal hepatitis C virus (HCV) vaccine should elicit multiantigenic, multigenotypic responses, which are more likely to protect against challenge with the range of genotypes and subtypes circulating in the community. A vaccine cocktail and vaccines encoding consensus HCV sequences are attractive approaches to achieve this goal. Consequently, in a series of mouse vaccination studies, we compared the immunogenicity of a DNA vaccine encoding a consensus HCV nonstructural 5B (NS5B) protein to that of a cocktail of DNA plasmids encoding the genotype 1b (Gt1b) and Gt3a NS5B proteins. To complement this study, we assessed responses to a multiantigenic cocktail regimen by comparing a DNA vaccine cocktail encoding Gt1b and Gt3a NS3, NS4, and NS5B proteins to a single-genotype NS3/4/5B DNA vaccine. To thoroughly evaluate in vivo cytotoxic T lymphocyte (CTL) and T helper (Th) cell responses against Gt1b and Gt3a HCV peptide-pulsed target cells, we exploited a novel fluorescent-target array (FTA). FTA and enzyme-linked immunosorbent spot (ELISpot) analyses collectively indicated that the cocktail regimens elicited higher responses to Gt1b and Gt3a NS5B proteins than those with the consensus vaccine, while the multiantigenic DNA cocktail significantly increased the responses to NS3 and NS5B compared to those elicited by the single-genotype vaccines. Thus, a DNA cocktail vaccination regimen is more effective than a consensus vaccine or a monovalent vaccine at increasing the breadth of multigenotypic T cell responses, which has implications for the development of vaccines for communities where multiple HCV genotypes circulate.IMPORTANCE Despite the development of highly effective direct-acting antivirals (DAA), infections with hepatitis C virus (HCV) continue, particularly in countries where the supply of DAA is limited. Furthermore, patients who eliminate the virus as a result of DAA therapy can still be reinfected. Thus, a vaccine for HCV is urgently required, but the heterogeneity of HCV strains makes the development of a universal vaccine difficult. To address this, we developed a novel cytolytic DNA vaccine which elicits robust cell-mediated immunity (CMI) to the nonstructural (NS) proteins in vaccinated animals. We compared the immune responses against genotypes 1 and 3 that were elicited by a consensus DNA vaccine or a DNA vaccine cocktail and showed that the cocktail induced higher levels of CMI to the NS proteins of both genotypes. This study suggests that a universal HCV vaccine can most readily be achieved by use of a DNA vaccine cocktail.

Recombinant fowlpox virus vector-based vaccines: expression kinetics, dissemination and safety profile following intranasal delivery.

We have previously established that mucosal uptake of recombinant fowlpox virus (rFPV) vaccines is far superior to other vector-based vaccines. Specifically, intranasal priming with rFPV vaccines can recruit unique antigen-presenting cells, which induce excellent mucosal and systemic HIV-specific CD8+ T-cell immunity. In this study, we have for the first time investigated the in vivo dissemination, safety and expression kinetics of rFPV post intranasal delivery using recombinant viruses expressing green fluorescent protein or mCherry. Both confocal microscopy of tissue sections using green fluorescent protein and in vivo Imaging System (IVIS) spectrum live animal and whole organ imaging studies using mCherry revealed that (i) the peak antigen expression occurs 12 to 24 h post vaccination and no active viral gene expression is detected 96 h post vaccination. (ii) The virus only infects the initial vaccination site (lung and nasal cavity) and does not disseminate to distal sites such as the spleen or gut. (iii) More importantly, rFPV does not cross the olfactory receptor neuron pathway. Collectively, our findings indicate that rFPV vector-based vaccines have all the hallmarks of a safe and effective mucosal delivery vector, suitable for clinical evaluation.

Heparan sulfate as a receptor for poxvirus infections and as a target for antiviral agents.

To establish the importance of virus-heparan sulfate (HS) interactions in virus infectivity, the poxvirus vaccinia virus (VACV) was used, as it binds HS and has both enveloped virus (EV) and non-enveloped mature virus (MV) forms. Initial studies showed that heparin inhibited plaque formation by both MV-rich WR and EV-rich IHD-J strains of VACV, with the EV-rich strain also losing trademark 'comet'-shaped plaques. However, using GFP-tagged EV and MV forms of VACV, based on IC50 values, heparin was 16-fold more effective at inhibiting the infectivity of the EV form compared to the MV form. Furthermore, 6-O and N-sulfation of the glucosamine residues of heparin was essential for inhibition of the infectivity of both VACV forms. Several low-molecular-weight HS mimetics were also shown to have substantial antiviral activity, with glycosidic linkages, chain length and monosaccharide backbone being important contributors towards anti-VACV activity. In fact, the d-mannose-based sulfated oligosaccharide mixture, PI-88 (Muparfostat), was four-fold more active than heparin at inhibiting MV infections. Paradoxically, despite heparin and HS mimetics being potent inhibitors of VACV infections, removal of HS from cell surfaces by enzymatic or genetic means resulted in only a modest reduction in infectivity. It is unlikely that this paradox can be explained by steric hindrance, due to the low molecular weight of the HS mimetics (~1-2.5 kDa), with a more likely explanation being that binding of heparin/HS mimetics to free VACV initiates an abortive viral infection. Based on this explanation, HS mimetics have considerable potential as antivirals against HS-binding viruses.

CCR6 is transiently upregulated on B cells after activation and modulates the germinal center reaction in the mouse.

The CC-chemokine receptor 6 (CCR6) is expressed constitutively at an intermediate level on naïve B cells and is upregulated after activation on pregerminal center (GC) B cells. We hypothesized that it could be involved in the events leading to GC reaction and high-affinity antibody production, and therefore investigated the potential role of CCR6 in B-cell differentiation in vivo. After antigenic challenge of CCR6-/- mice with the T-cell-dependent antigen nitrophenyl-keyhole limpet hemocyanin (NP-KLH), GC B-cell development was found to be accelerated and the number of GC had increased significantly compared with control mice, but the antibodies produced by CCR6-/- B cells were on average of lower affinity. We conclude from these data that the CCR6/CCL20 axis has an important role in regulating the kinetics and efficiency of the GC reaction.

Long-term safety and immunogenicity of an MF59-adjuvanted spike glycoprotein-clamp vaccine for SARS-CoV-2 in adults aged 18-55 years or ≥56 years: 12-month results from a randomised, double-blind, placebo-controlled, phase 1 trial.

BACKGROUND: We previously demonstrated the safety and immunogenicity of an MF59-adjuvanted COVID-19 vaccine based on the SARS-CoV-2 spike glycoprotein stabilised in a pre-fusion conformation by a molecular clamp using HIV-1 glycoprotein 41 sequences. Here, we describe 12-month results in adults aged 18-55 years and ≥56 years. METHODS: Phase 1, double-blind, placebo-controlled trial conducted in Australia (July 2020-December 2021; ClinicalTrials.govNCT04495933; active, not recruiting). Healthy adults (Part 1: 18-55 years; Part 2: ≥56 years) received two doses of placebo, 5 µg, 15 µg, or 45 µg vaccine, or one 45 µg dose of vaccine followed by placebo (Part 1 only), 28 days apart (n = 216; 24 per group). Safety, humoral immunogenicity (including against virus variants), and cellular immunogenicity were assessed to day 394 (12 months after second dose). Effects of subsequent COVID-19 vaccination on humoral responses were examined. FINDINGS: All two-dose vaccine regimens were well tolerated and elicited strong antigen-specific and neutralising humoral responses, and CD4+ T-cell responses, by day 43 in younger and older adults, although cellular responses were lower in older adults. Humoral responses waned by day 209 but were boosted in those receiving authorised vaccines. Neutralising activity against Delta and Omicron variants was present but lower than against the Wuhan strain. Cross-reactivity in HIV diagnostic tests declined over time but remained detectable in most participants. INTERPRETATION: The SARS-CoV-2 molecular clamp vaccine is well tolerated and evokes robust immune responses in adults of all ages. Although the HIV glycoprotein 41-based molecular clamp is not being progressed, the clamp concept represents a viable platform for vaccine development. FUNDING: This study was funded by the Coalition for Epidemic Preparedness Innovations, the National Health and Medical Research Council of Australia, and the Queensland Government.

Fluorescent target array killing assay: a multiplex cytotoxic T-cell assay to measure detailed T-cell antigen specificity and avidity in vivo.

Here we describe a multiplex, fluorescence-based, in vivo cytotoxic T-cell assay using the three vital dyes carboxyfluorescein diacetate succinimidyl ester, cell trace violet, and cell proliferation dye efluor 670. When used to label cells in combination, these dyes can discriminate >200 different target cell populations in the one animal due to each target population having a unique fluorescence signature based on fluorescence intensity and the different emission wavelengths of the dyes. This allows the simultaneous measurement of the in vivo killing of target cells pulsed with numerous peptides at different concentrations and the inclusion of many replicates. This fluorescent target array killing assay can be used to measure the fine antigen specificity and avidity of polyclonal cytotoxic T-cell responses in vivo, immunological parameters that were previously impossible to monitor.

Evaluation of Innate Lymphoid Cells and Dendritic Cells Following Viral Vector Vaccination.

Recently, we have shown that fate of a vaccine is determined by the cytokine milieu in the innate immune compartment at the early stage of vaccination. Specifically, 24 h post-delivery, level of innate lymphoid cell type 2 (ILC2)-derived IL-13/IL-13Rα2 are the master regulators of DC and also different ILC subsets responsible for modulating the downstream immune outcomes. Here, we provide step-by-step details how to assess different ILC and DC subsets in lung and muscle following intranasal and intramuscular viral vector vaccination, respectively, using multi-color flow cytometry and confocal microscopy.

Human immunodeficiency virus-1 vaccine design: where do we go now?

Numerous human immunodeficiency virus (HIV)-1 vaccines have been developed over the last three decades, but to date an effective HIV-1 vaccine that can be used for prophylactic or therapeutic purposes in humans has not been identified. The failures and limited successes of HIV-1 vaccines have highlighted the gaps in our knowledge with regard to fundamental immunity against HIV-1 and have provided insights for vaccine strategies that may be implemented for designing more effective HIV-1 vaccines in the future. Recent studies have shown that robust mucosal immunity, high avidity and polyfunctional T cells, and broadly neutralizing antibodies are important factors governing the induction of protective immunity against HIV-1. Furthermore, optimization of vaccine delivery methods for DNA or live viral vector-based vaccines, elucidating the immune responses of individuals who remain resistant to HIV-1 infections and also understanding the core immune responses mediating protection against simian immunodeficiency viruses (SIV) and HIV-1 in animal models following vaccination, are key aspects to be regarded for designing more effective HIV-1 vaccines in the future.

Evaluating vaccinia virus cytokine co-expression in TLR GKO mice.

Using Toll-like receptor (TLR) and MyD88 gene knock-out (GKO) mice the effect of TLRs and MyD88 on virus replication, interferon (IFN)-ß production, natural killer (NK) cell and CD8T cell responses were assessed following ectromelia virus (ECTV) and recombinant vaccinia virus (rVV) infection. The capacity for rVVs encoding cytokines to restore immune function in MyD88(-/-) mice was clearly demonstrated. Results showed that TLR2(-/-), TLR4(-/-)and TLR7(-/-) mice survived ECTV infection whereas MyD88(-/-) and TLR9(-/-)mice, in contrast, were highly susceptible. Next, following infection with rVV, MyD88(-/-) mice elicited reduced serum IFN-ß, NK cell and CD8T cell responses compared with wild-type mice, whereas TLR9(-/-) mice showed elevated CD8T cell responses. When MyD88(-/-)mice were infected with rVV co-expressing IFN-ß these mice were able to restore IFN-ß levels and CD8T cell responses but not NK cell activation. Interestingly, even though rVV co-expressing interleukin (IL)-2 enhanced NK cell activation in MyD88(-/-) mice, this was not associated with an antiviral effect, as observed in normal mice. Surprisingly, co-infection with rVV IL-2/rVV IL-12, but not rVV IL-2/rVV IFN-ß, restored the attenuated phenotype of rVV IL-2 in MyD88(-/-) mice indicating that the IL-2/IL-12 combination promotes antiviral responses. Our results clearly show that the CD8T cell defect observed in MyD88(-/-) mice to vaccinia virus infection can be restored by rVV-encoding IFN-ß demonstrating the critical role of this cytokine in T cell mediated immunity and illustrates that the model can provide an effective platform for the elucidation of cytokine immunobiology.

IL-13Rα2 Regulates the IL-13/IFN-γ Balance during Innate Lymphoid Cell and Dendritic Cell Responses to Pox Viral Vector-Based Vaccination.

We have shown that manipulation of IL-13 and STAT6 signaling at the vaccination site can lead to different innate lymphoid cell (ILC)/dendritic cell (DC) recruitment, resulting in high avidity/poly-functional T cells and effective antibody differentiation. Here we show that permanent versus transient blockage of IL-13 and STAT6 at the vaccination site can lead to unique ILC-derived IL-13 and IFN-γ profiles, and differential IL-13Rα2, type I and II IL-4 receptor regulation on ILC. Specifically, STAT6-/- BALB/c mice given fowl pox virus (FPV) expressing HIV antigens induced elevated ST2/IL-33R+ ILC2-derived IL-13 and reduced NKp46+/- ILC1/ILC3-derived IFN-γ expression, whilst the opposite (reduced IL-13 and elevated IFN-γ expression) was observed during transient inhibition of STAT6 signaling in wild type BALB/c mice given FPV-HIV-IL-4R antagonist vaccination. Interestingly, disruption/inhibition of STAT6 signaling considerably impacted IL-13Rα2 expression by ST2/IL-33R+ ILC2 and NKp46- ILC1/ILC3, unlike direct IL-13 inhibition. Consistently with our previous findings, this further indicated that inhibition of STAT6 most likely promoted IL-13 regulation via IL-13Rα2. Moreover, the elevated ST2/IL-33R+ IL-13Rα2+ lung ILC2, 24 h post FPV-HIV-IL-4R antagonist vaccination was also suggestive of an autocrine regulation of ILC2-derived IL-13 and IL-13Rα2, under certain conditions. Knowing that IL-13 can modulate IFN-γ expression, the elevated expression of IFN-γR on lung ST2/IL-33R+ ILC2 provoked the notion that there could also be inter-regulation of lung ILC2-derived IL-13 and NKp46- ILC1/ILC3-derived IFN-γ via their respective receptors (IFN-γR and IL-13Rα2) at the lung mucosae early stages of vaccination. Intriguingly, under different IL-13 conditions differential regulation of IL-13/IL-13Rα2 on lung DC was also observed. Collectively these findings further substantiated that IL-13 is the master regulator of, not only DC, but also different ILC subsets at early stages of viral vector vaccination, and responsible for shaping the downstream adaptive immune outcomes. Thus, thoughtful selection of vaccine strategies/adjuvants that can manipulate IL-13Rα2, and STAT6 signaling at the ILC/DC level may prove useful in designing more efficacious vaccines against different/chronic pathogens.

Interleukin-13 as a target to alleviate severe coronavirus disease 2019 and restore lung homeostasis.

The ongoing coronavirus disease (COVID-19) pandemic urgently requires the availability of interventions that improve outcomes for those with severe disease. Since severe acute respiratory syndrome coronavirus 2 infection is characterized by dysregulated lung mucosae, and that mucosal homeostasis is heavily influenced by interleukin (IL)-13 activity, we explore recent findings indicating that IL-13 production is proportional to disease severity. We propose that excessive IL-13 contributes to the progression of severe/fatal COVID-19 by (1) promoting the recruitment of immune cells that express inflammatory cytokines, causing a cytokine storm that results in widespread destruction of lung tissue, (2) directly facilitating tissue-remodeling that causes airway hyperinflammation and obstruction, and (3) diverting the immune system away from developing high-quality cytotoxic T cells that confer effective anti-viral immunity. These factors may cumulatively result in significant lung distress, multi-organ failure, and death. Here, we suggest repurposing existing IL-13-inhibiting interventions, including antibody therapies routinely used for allergic lung hyperinflammation, as well as viral vector-based approaches, to alleviate disease. Since many of these strategies have previously been shown to be both safe and effective, this could prove to be a highly cost-effective solution. Relevance for Patients: There remains a desperate need to establish medical interventions that reliably improves outcomes for patients suffering from COVID-19. We explore the role of IL-13 in maintaining homeostasis at the lung mucosae and propose that its dysregulation during viral infection may propagate the hallmarks of severe disease - further exploration may provide a platform for invaluable therapeutics.

STAT3 determines IL-4 signalling outcomes in naïve T cells.

IL-4 production is associated with low-avidity, poorly cytotoxic T cell induction that contributes to viral immune evasion and the failure of T cell-based vaccines. Yet, the precise mechanisms that regulate IL-4 signalling in T cells remain elusive. Mounting evidence indicates that cells can dynamically alter their IL-4/IL-13 receptor signature to modulate downstream immune outcomes upon pathogen encounter. Here, we describe how naïve (CD62L+CD44lo-mid) CD4 and CD8 T cells distinctly engage both STAT6 and STAT3 in response to IL-4. We further show that IL-4R⍺ expression is both time- and IL-4 concentration-dependent. Remarkably, our findings reveal that STAT3 inhibition can ablate IL-4R⍺ and affect transcriptional expression of other Stat and Jak family members. By extension, the loss of STAT3 lead to aberrant STAT6 phosphorylation, revealing an inter-regulatory relationship between the two transcription factors. Moreover, IL-4 stimulation down-regulated TGF-ß1 and IFN-γR1 expression on naïve T cells, possibly signifying the broad regulatory implications of IL-4 in conditioning lineage commitment decisions during early infection. Surprisingly, naïve T cells were unresponsive to IL-13 stimulation, unlike dendritic cells. Collectively, these findings could be exploited to inform more efficacious vaccines, as well as design treatments against IL-4/IL-13-associated disease conditions.

Adjuvant Selection for Influenza and RSV Prefusion Subunit Vaccines.

Subunit vaccines exhibit favorable safety and immunogenicity profiles and can be designed to mimic native antigen structures. However, pairing with an appropriate adjuvant is imperative in order to elicit effective humoral and cellular immune responses. In this study, we aimed to determine an optimal adjuvant pairing with the prefusion form of influenza haemagglutinin (HA) or respiratory syncytial virus (RSV) fusion (F) subunit vaccines in BALB/c mice in order to inform future subunit vaccine adjuvant selection. We tested a panel of adjuvants, including aluminum hydroxide (alhydrogel), QS21, Addavax, Addavax with QS21 (AdQS21), and Army Liposome Formulation 55 with monophosphoryl lipid A and QS21 (ALF55). We found that all adjuvants elicited robust humoral responses in comparison to placebo, with the induction of potent neutralizing antibodies observed in all adjuvanted groups against influenza and in AdQS21, alhydrogel, and ALF55 against RSV. Upon HA vaccination, we observed that none of the adjuvants were able to significantly increase the frequency of CD4+ and CD8+ IFN-γ+ cells when compared to unadjuvanted antigen. The varying responses to antigens with each adjuvant highlights that those adjuvants most suited for pairing purposes can vary depending on the antigen used and/or the desired immune response. We therefore suggest that an adjuvant trial for different subunit vaccines in development would likely be necessary in preclinical studies.

Preclinical development of a molecular clamp-stabilised subunit vaccine for severe acute respiratory syndrome coronavirus 2.

OBJECTIVES: Efforts to develop and deploy effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue at pace. Here, we describe rational antigen design through to manufacturability and vaccine efficacy of a prefusion-stabilised spike (S) protein, Sclamp, in combination with the licensed adjuvant MF59 'MF59C.1' (Seqirus, Parkville, Australia). METHODS: A panel recombinant Sclamp proteins were produced in Chinese hamster ovary and screened in vitro to select a lead vaccine candidate. The structure of this antigen was determined by cryo-electron microscopy and assessed in mouse immunogenicity studies, hamster challenge studies and safety and toxicology studies in rat. RESULTS: In mice, the Sclamp vaccine elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S-specific CD4+ and cytotoxic CD8+ T cells in vivo. In the Syrian hamster challenge model (n = 70), vaccination results in reduced viral load within the lung, protection from pulmonary disease and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level. CONCLUSION: The SARS-CoV-2 Sclamp vaccine candidate is compatible with large-scale commercial manufacture, stable at 2-8°C. When formulated with MF59 adjuvant, it elicits neutralising antibodies and T-cell responses and provides protection in animal challenge models.

Safety and immunogenicity of an MF59-adjuvanted spike glycoprotein-clamp vaccine for SARS-CoV-2: a randomised, double-blind, placebo-controlled, phase 1 trial.

BACKGROUND: Given the scale of the ongoing COVID-19 pandemic, the development of vaccines based on different platforms is essential, particularly in light of emerging viral variants, the absence of information on vaccine-induced immune durability, and potential paediatric use. We aimed to assess the safety and immunogenicity of an MF59-adjuvanted subunit vaccine for COVID-19 based on recombinant SARS-CoV-2 spike glycoprotein stabilised in a pre-fusion conformation by a novel molecular clamp (spike glycoprotein-clamp [sclamp]). METHODS: We did a phase 1, double-blind, placebo-controlled, block-randomised trial of the sclamp subunit vaccine in a single clinical trial site in Brisbane, QLD, Australia. Healthy adults (aged ≥18 to ≤55 years) who had tested negative for SARS-CoV-2, reported no close contact with anyone with active or previous SARS-CoV-2 infection, and tested negative for pre-existing SARS-CoV-2 immunity were included. Participants were randomly assigned to one of five treatment groups and received two doses via intramuscular injection 28 days apart of either placebo, sclamp vaccine at 5 µg, 15 µg, or 45 µg, or one dose of sclamp vaccine at 45 µg followed by placebo. Participants and study personnel, except the dose administration personnel, were masked to treatment. The primary safety endpoints included solicited local and systemic adverse events in the 7 days after each dose and unsolicited adverse events up to 12 months after dosing. Here, data are reported up until day 57. Primary immunogenicity endpoints were antigen-specific IgG ELISA and SARS-CoV-2 microneutralisation assays assessed at 28 days after each dose. The study is ongoing and registered with ClinicalTrials.gov, NCT04495933. FINDINGS: Between June 23, 2020, and Aug 17, 2020, of 314 healthy volunteers screened, 120 were randomly assigned (n=24 per group), and 114 (95%) completed the study up to day 57 (mean age 32·5 years [SD 10·4], 65 [54%] male, 55 [46%] female). Severe solicited reactions were infrequent and occurred at similar rates in participants receiving placebo (two [8%] of 24) and the SARS-CoV-2 sclamp vaccine at any dose (three [3%] of 96). Both solicited reactions and unsolicited adverse events occurred at a similar frequency in participants receiving placebo and the SARS-CoV-2 sclamp vaccine. Solicited reactions occurred in 19 (79%) of 24 participants receiving placebo and 86 (90%) of 96 receiving the SARS-CoV-2 sclamp vaccine at any dose. Unsolicited adverse events occurred in seven (29%) of 24 participants receiving placebo and 35 (36%) of 96 participants receiving the SARS-CoV-2 sclamp vaccine at any dose. Vaccination with SARS-CoV-2 sclamp elicited a similar antigen-specific response irrespective of dose: 4 weeks after the initial dose (day 29) with 5 µg dose (geometric mean titre [GMT] 6400, 95% CI 3683-11 122), with 15 µg dose (7492, 4959-11 319), and the two 45 µg dose cohorts (8770, 5526-13 920 in the two-dose 45 µg cohort; 8793, 5570-13 881 in the single-dose 45 µg cohort); 4 weeks after the second dose (day 57) with two 5 µg doses (102 400, 64 857-161 676), with two 15 µg doses (74 725, 51 300-108 847), with two 45 µg doses (79 586, 55 430-114 268), only a single 45 µg dose (4795, 2858-8043). At day 57, 67 (99%) of 68 participants who received two doses of sclamp vaccine at any concentration produced a neutralising immune response, compared with six (25%) of 24 who received a single 45 µg dose and none of 22 who received placebo. Participants receiving two doses of sclamp vaccine elicited similar neutralisation titres, irrespective of dose: two 5 µg doses (GMT 228, 95% CI 146-356), two 15 µg doses (230, 170-312), and two 45 µg doses (239, 187-307). INTERPRETATION: This first-in-human trial shows that a subunit vaccine comprising mammalian cell culture-derived, MF59-adjuvanted, molecular clamp-stabilised recombinant spike protein elicits strong immune responses with a promising safety profile. However, the glycoprotein 41 peptide present in the clamp created HIV diagnostic assay interference, a possible barrier to widespread use highlighting the criticality of potential non-spike directed immunogenicity during vaccine development. Studies are ongoing with alternative molecular clamp trimerisation domains to ameliorate this response. FUNDING: Coalition for Epidemic Preparedness Innovations, National Health and Medical Research Council, Queensland Government, and further philanthropic sources listed in the acknowledgments.

Immunisation route-dependent expression of IL-4/IL-13 can modulate HIV-specific CD8(+) CTL avidity.

All HIV-1 'systemic vaccine trials' in humans have yielded poor outcomes. Thus, it is important to understand whether the route of delivery influences the quality of protective CTL immunity. Using heterologous poxvirus immunisation we have shown that systemically (i.m./i.m.) immunised CD8(+) T cells generated higher levels of IL-4/IL-13 compared to mucosal delivery and expression also correlated with i.m./i.m. immunised mice eliciting CTL of lower avidity. Studies using IL-4(-/-) and IL-13(-/-) KO mice have shown that the capacity to express IFN-gamma, IL-4 and/or IL-13 by K(d)Gag(197-205)-specific CTL differed between these groups and was inversely correlated with CTL avidity (IL-13(-/-)>IL-4(-/-)>BALB/c), although no significant differences in the magnitude of CTL responses were observed between IL-13(-/-) and wild type mice. When IL-13 was reconstituted in IL-13(-/-) splenocytes in vitro, their ability to bind tetramers also decreased significantly. Our data reveal that total absence of IL-13 can greatly enhance CTL avidity. In contrast, extracellular IL-4 appears to be important in maintaining long-term Th1/Th2 balance in CTL, even though expression of IL-4 by CTL markedly reduced avidity. STAT6(-/-) mice also showed memory CTL of higher avidity. Furthermore, CCL5 expression in K(d)Gag(197-205)-specific CTL was also regulated by IL-4/IL-13.

Unique IL-13Rα2/STAT3 mediated IL-13 regulation detected in lung conventional dendritic cells, 24 h post viral vector vaccination.

This study demonstrates that 24 h following viral vector-based vaccination IL-13Rα2 functions as a master sensor on conventional dendritic cells (cDCs), abetted by high protein stability coupled with minimal mRNA expression, to rapidly regulate DC mediated IL-13 responses at the lung mucosae, unlike IL-13Rα1. Under low IL-13, IL-13Rα2 performs as a primary signalling receptor, whilst under high IL-13, acts to sequester IL-13 to maintain homeostasis, both in a STAT3-dependent manner. Likewise, we show that viral vector-derived IL-13 levels at the vaccination site can induce differential STAT3/STAT6 paradigms in lung cDC, that can get regulated collaboratively or independently by TGF-ß1 and IFN-γ. Specifically, low IL-13 responses associated with recombinant Fowlpox virus (rFPV) is regulated by early IL-13Rα2, correlated with STAT3/TGF-ß1 expression. Whilst, high IL-13 responses, associated with recombinant Modified Vaccinia Ankara (rMVA) is regulated in an IL-13Rα1/STAT6 dependent manner associated with IFN-γR expression bias. Different viral vaccine vectors have previously been shown to induce unique adaptive immune outcomes. Taken together current observations suggest that IL-13Rα2-driven STAT3/STAT6 equilibrium at the cDC level may play an important role in governing the efficacy of vector-based vaccines. These new insights have high potential to be exploited to improve recombinant viral vector-based vaccine design, according to the pathogen of interest and/or therapies against IL-13 associated disease conditions.

Is host heparanase required for the rapid spread of heparan sulfate binding viruses?

Vaccinia virus (VACV), like many other viruses, binds to cell surface heparan sulfate (HS) prior to infecting cells. Since HS is ubiquitously expressed extracellularly, it seemed likely that VACV-HS interaction may impede virus spread, with host heparanase, the only known mammalian endoglycosidase that can degrade HS, potentially overcoming this problem. In support of this hypothesis, we found that, compared to wild type, mice deficient in heparanase showed a 1-3 days delay in the spread of VACV to distant organs, such as ovaries, following intranasal inoculation, or to ovaries and spleen following intramuscular inoculation. These delays in spread occurred despite heparanase deficiency having no effect on VACV replication at inoculation sites. Subsequent in vitro studies revealed that heparanase treatment released VACV from HS expressing, but not HS deficient, infected cell monolayers. Collectively these data suggest that VACV relies on host heparanase to degrade HS in order to spread to distant sites.

Mucosal and systemic SIV-specific cytotoxic CD4+ T cell hierarchy in protection following intranasal/intramuscular recombinant pox-viral vaccination of pigtail macaques.

A HIV vaccine that provides mucosal immunity is urgently needed. We evaluated an intranasal recombinant Fowlpox virus (rFPV) priming vaccine followed by intramuscular Modified Vaccinia Ankara (rMVA) booster vaccine, both expressing SIV antigens. The vaccination generated mucosal and systemic SIV-specific CD4+ T cell mediated immunity and was associated with partial protection against high-dose intrarectal SIVmac251 challenge in outbred pigtail macaques. Three of 12 vaccinees were completely protected and these animals elicited sustained Gag-specific poly-functional, cytotoxic mucosal CD4+ T cells, complemented by systemic poly-functional CD4+ and CD8+ T cell immunity. Humoral immune responses, albeit absent in completely protected macaques, were associated with partial control of viremia in animals with relatively weaker mucosal/systemic T cell responses. Co-expression of an IL-4R antagonist by the rFPV vaccine further enhanced the breadth and cytotoxicity/poly-functionality of mucosal vaccine-specific CD4+ T cells. Moreover, a single FPV-gag/pol/env prime was able to induce rapid anamnestic gp140 antibody response upon SIV encounter. Collectively, our data indicated that nasal vaccination was effective at inducing robust cervico-vaginal and rectal immunity, although cytotoxic CD4+ T cell mediated mucosal and systemic immunity correlated strongly with 'complete protection', the different degrees of protection observed was multi-factorial.