Respiratory syncytial virus (RSV) remains a health threat to young children worldwide. The host immune response plays a key role in disease following infection. Infection models advance our understanding of respiratory viruses, but individual models have gaps, which overlapping complementary systems can fill. We compared disease signatures in mice, adults and children; combining transcriptomic data collected from blood, nasal mucosa and lung biopsy following RSV infection. We identified both shared and species-specific pathways triggered by RSV. While systemic responses in children's blood were more similar to those in RSV-challenged adults, mucosal responses during primary infection in mice more closely resembled those in children. We identified an association between IL-17 pathways and RSV pathogenesis and with over-expression of the downstream effectors S100A8 and S100A9. Inhibiting these with the anti-inflammatory drug Paquinimod reduced disease. Here we demonstrate that integrating mouse and human transcriptomic data can identify novel targets to treat RSV disease.

Self-amplifying ribonucleic acid (saRNA) vaccines are a next-generation RNA vaccine platform with great potential. Systems vaccinology provides a potent tool to interrogate vaccine-induced responses in volunteers and to dissect the mechanisms by which vaccines elicit a protective immune response or cause reactogenicity. In the current study, we performed transcriptomic analysis on blood samples collected from volunteers vaccinated as part of a phase I study of an saRNA vaccine expressing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike antigen. We observed significant gene over-expression following both the prime and boost vaccinations. Over-expressed genes were predominantly associated with type I interferon signaling pathways and innate immune cell recruitment. This transcriptomic signature was reflected by an increase in cytokines in the plasma at the same time points and a significant increase in monocytes in the blood, both of which correlated with the antibody response to the vaccine. When individuals were segregated by the degree of reactogenicity, we also detected differences in gene expression related to immune responses. Overall, results show that saRNA induces a potent, acute inflammatory response with similarities to other RNA vaccines, and it will be important to further dissect the role of the over-expressed genes in immunogenicity and reactogenicity.

How we write scientific papers does not reflect how we do science. Their formal structure infers a pre-ordained linear process rather than reflecting the messy creativity of research. This matters in the AI age because it masks the human in the process.

Background SARS-CoV-2 viral load in the upper respiratory tract (URT) typically peaks and declines within days of infection, even in individuals without prior infection or vaccination. Although this implicates the URT innate immune response in effectively restricting viral replication, the nature of the protective responses and how they are affected by demographic factors is poorly defined. Methods We recruited 54 seronegative household contacts of recently diagnosed COVID-19 cases and prospectively collected URT samples during and after exposure. Among the 39 individuals who became infected, we quantified airway mucosal cytokine and chemokine responses and virus-specific nasal IgA using Meso Scale Discovery assays, and assessed associations with demographic factors, viral load, and symptoms. Findings Participants with higher BMI had higher URT viral loads and more marked symptoms. This was significantly associated with delayed induction of protective inflammatory mediators in the airway mucosa but not in blood. Induction of virus-specific nasal IgA at 1-week post-infection also correlated with lower viral load. Interpretation Elevated BMI retards initial airway mucosal innate immune responses to infection, which may partially explain the pronounced adverse impact of higher BMI on clinical and virological outcomes in COVID-19. Funding This work is supported by the NIHR Health Protection Research Unit in Respiratory Infections, Imperial College London in partnership with the UK Health Security Agency (Grant number: NIHR200927; AL) and the Medical Research Council (Grant number: MR/X004058/1). Infrastructure support for this research was provided by the NIHR Imperial Biomedical Research Centre (BRC).

Abstract Non-canonical amino acids (ncaas) are increasingly used in vaccinology to improve vaccine adaptability and immunogenicity. Cell-free protein synthesis (CFPS) offers a promising route for site-specific ncaa incorporation, but conventional prokaryotic CFPS systems show limitations to produce complex proteins requiring post-translational modifications while eukaryotic systems have historically been difficult to scale and show low protein yields. Here we establish efficient site-specific introduction of ncaas into complex proteins with the high-yielding and scalable eukaryotic tobacco BY-2 CFPS system (BYL), commercialized as ALiCE®. ncaa incorporation yields reached up to 2 mg/ml with linear scalability up to 10 ml. We applied ncaa incorporation in BYL to enable click chemistry bioconjugation of the receptor binding domain (RBD) of influenza hemagglutinin to pre-assembled hepatitis B core (HBc) virus-like particles (VLPs). The resulting VLP-RBD conjugates exhibited hemagglutination activity, unlike the individual components, and protected mice from weight loss after influenza challenge. This research thus enables ncaa introduction for recombinant proteins produced in BYL, constructing a novel plug-and-play vaccine platform and further expanding the capabilities of BYL to produce vaccine candidates and other proteins of interest.

The changes announced by a major UK science funder are putting scientists — and the future of research — in a difficult position. The changes announced by a major UK science funder are putting scientists — and the future of research — in a difficult position.

Since its use during the COVID-19 pandemic, mRNA has emerged as a leading candidate vaccine platform for pandemic infections. A critical difference between RNA-encoded antigen and protein vaccines is that RNA-based vaccines require the antigen to be translated in the body, adding an important variable. Much of the research focus in the field has been on ways to increase expression, but inflammation plays a critical role. The vaccine delivered is a combination of the RNA and the formulation, so both elements need to be considered. Formulated RNA can act as a form of adjuvant but can also activate cellular pathways that inhibit expression. Expression and inflammation are interlinked, but independent-a deeper understanding of the quality and quantity of immune induction will help to develop more efficient RNA vaccines. Here, we discuss factors that shape responses to RNA-based vaccines. These include the composition of the vaccine (the use of modified RNA bases, whether self-replicating or traditional mRNA and, critically, the formulation) and the type of cells that take up and translate the RNA. We then consider challenges presented by current generation RNA vaccines including clinical impact and how improved immunological understanding can inform the development of improved RNA vaccine platforms.

Vaccines are central to the strategy to control antimicrobial resistant (AMR) bacterial infections; one multidrug resistant pathogen of particular concern is Acinetobacter baumannii. In this study we identified two novel A. baumannii antigens using mass spectrometry and phage expression: Oxa23 and PAL. These genes are highly conserved between different isolates of A. baumannii and recognised by convalescent human sera. We explored their protective immunity using two different vaccine platforms, recombinant outer membrane vesicles (rOMV) and mRNA. RNA vaccine immunised mice had significantly reduced bacterial load in their lower airways following challenge with carbapenem resistant A. baumannii, with Oxa23 providing better protection than PAL. We then compared routes of delivery and RNA vaccine platforms, demonstrating that intranasally delivery of mRNA encoding OXA-23 (formulated with GL67A) significantly reduced disease severity and enhanced bacterial clearance. These studies validate in silico identified antigens through challenge studies and novel mucosal vaccine delivery approaches.

Challenges with vaccine reactogenicity, stability, and access have highlighted the need to develop alternative strategies for formulation and delivery. We explored the incorporation of cucurbit[n]urils (CBs), as supramolecular "hosts," into nucleic acid-polymer polyplexes. CBs are small, non-toxic, barrel-shaped molecules that transiently crosslink polymers containing supramolecular "guests," thereby increasing molecular weight (MW) of the complex, a correlate of transfection efficiency. We tested whether the supramolecular interactions of CB[8] impact polyplex function. We generated a library of different CB[8] polyplexes using plasmid DNA (pDNA), varying N/P (the ratio of polymer to plasmid), the length, and guest (phenylalanine [Phe]) group frequency of the polyethylenimine (PEI) polymer backbone. We found that N/P 32 and the 20Phe1 (20kDa PEI with 1 mol% Phe) gave optimal gene expression and that incorporating CB[8] in polyplex formulations improved gene expression, both in vitro and in vivo. Despite increases in gene expression, inclusion of CB[8] in formulations with higher guest-binding capacity led to decreased immunogenicity, possibly as a result of dampened innate immune responses. Our data show that CB[8] polyplexes increase gene delivery and expression but alter inflammatory responses. These findings highlight that rational design of the CB[8] polymer system can enable nucleic acid delivery for both vaccine and therapeutic applications.

The COVID-19 pandemic highlighted the need for rapidly deployable, flexible vaccine platforms; particularly RNA which is now being explored for several other pathogens. DNA vaccines have potential advantages over RNA, including cost of manufacture, ease of storage and potentially lower reactogenicity. However, they have historically underperformed in large animals and human trials due to low immunogenicity. The interplay between antigen expression and the innate immune response impacts the overall immune response to DNA vaccines. Here, we explore the effect of altering non-coding 5’ regions, on the immunogenicity of a closed linear DNA platform, Doggybone™ DNA (dbDNATM), produced by a rapid and scalable cell-free method. Using a mouse model, we found that enhancer sequences and DNA targeting sequences (DTS) increased influenza virus hemagglutinin (HA) expression and improved immune responses. Additional CpG motifs did not provide any immune benefit. We also found that the effect of non-coding sequences was target specific, with differing effects in influenza HA, SARS-CoV-2 Spike and eGFP constructs. To separate the effects of immune sensing of the DNA construct and the expression of the encoded antigen, we combined a separate CpG oligodeoxynucleotide (ODN) with the highest expressing DNA vaccine; we observed reduced expression, but higher inflammation resulting in equivalent immunogenicity. Further refinement is required to fully understand the interplay of factors required for the induction of protective immunity by DNA vaccines.

Background: Group B streptococcus (GBS) is a commensal bacterium of the digestive and genital tracts that can occasionally cause maternal and neonatal disease. GBS is particularly a burden in low-resource settings, where infections with HIV are also highly prevalent. This study investigates the impact of HIV status on GBS colonization and antibody levels. Methods: In Uganda, 90 nonpregnant women of childbearing age were followed for 3 months. Every 2 weeks, rectal and vaginal swabs were tested for GBS, and vaginal cups and blood were collected for measurement of GBS capsular polysaccharides (CPS) IgG using standardized assays. Results: Twenty-six of 90 women were living with HIV. Almost 51/90 women were GBS colonized at 1 or several visits. GBS colonization fluctuated in the rectal and vaginal sites. Most prevalent serotypes were Ia and III, with 33 individuals carrying different serotypes over time. Serum and vaginal CPS-IgG levels were stable over 12 weeks. In serum, for serotypes Ib-V, the geometric mean concentration (GMC) of CPS-IgG did not differ between HIV+ and HIV- participants. However, the GMC for serum CPS-Ia-IgG in the HIV+ group was 2.5 times lower than in the HIV- group (P = 0.038). Vaginal CPS-IgG was measurable in 5/26 (19%) HIV+ participants, and 32/64 (50%) HIV- participants. Conclusions: Despite fluctuating GBS colonization, antibody levels remained stable over 12 weeks. The level of CPS-Ia-specific IgG in serum was lower in women with HIV than in those without HIV. Vaginal CPS-specific IgG was not measurable in 81% of individuals with HIV.

BACKGROUND:A proportion of individuals exposed to respiratory viruses avoid contracting detectable infection. We tested the hypothesis that early innate immune responses associate with resistance to detectable infection in close contacts of COVID-19 cases. METHODS:48 recently-exposed household contacts of symptomatic COVID-19 cases were recruited in London, UK between May 2020 and March 2021 through a prospective, longitudinal observational study. Blood and nose and throat swabs were collected during the acute period of index case viral shedding and longitudinally thereafter. Magnitude of SARS-CoV-2 exposure was quantified, and serial PCR and serological assays used to determine infection status of contacts. Whole-blood RNA-seq was performed and analysed to identify transcriptomic signatures of early infection and resistance to infection. FINDINGS:24 highly-exposed household contacts became PCR-positive and seropositive whilst 24 remained persistently PCR-negative and seronegative. A 96-gene transcriptomic signature of early SARS-CoV-2 infection was identified using RNA-seq of longitudinal blood samples from PCR-positive contacts. This signature was dominated by interferon-associated genes and expression correlated positively with viral load. Elevated expression of this 96-gene signature was also observed during exposure in 25% (6/24) of persistently PCR-negative, seronegative contacts. PCR-negative contacts with elevated signature expression had higher-magnitude SARS-CoV-2 exposure compared to those with low signature expression. We validated this signature in SARS-CoV-2-infected individuals in two independent cohorts. In naturally-exposed healthcare workers (HCWs) we found that 7/58 (12%) PCR-negative HCWs exhibited elevated signature expression. Comparing gene-signature expression in SARS-CoV-2 Controlled Human Infection Model (CHIM) volunteers pre- and post-inoculation, we observed that 14 signature genes were transiently upregulated as soon as 6 hr post-inoculation in PCR-negative volunteers, while in PCR-positive volunteers gene-signature upregulation did not occur until 3 days later. INTERPRETATION:Our interferon-associated signature of early SARS-CoV-2 infection characterises a subgroup of exposed, uninfected contacts in three independent cohorts who may have successfully aborted infection prior to induction of adaptive immunity. The earlier transient upregulation of signature genes in PCR-negative compared to PCR-positive CHIM volunteers suggests that ultra-early interferon-associated innate immune responses correlate with, and may contribute to, protection against SARS-CoV-2 infection. FUNDING:This work was supported by the NIHR Health Protection Research Unit in Respiratory Infections, United Kingdom, NIHR Imperial College London, United Kingdom (Grant number: NIHR200927; AL) in partnership with the UK Health Security Agency and the NIHR Medical Research Council (MRC), United Kingdom (Grant number: MR/X004058/1). Support for sequencing was provided by the Imperial BRC Genomics Facility which is funded by the NIHR, United Kingdom. The development of the hybrid DABA assay used for quantification of SARS-CoV-2 anti-Spike RBD antibodies was supported by the MRC (MC_PC_19078).

Two-photon polymerization (2PP) 3D printing enables top-down biomaterial synthesis with nanoscale spatial resolution for de novo design of monodisperse injectable drug delivery systems. Spatiotemporal Controlled Release Inks of Biocompatible polyEsters (SCRIBE) is a novel poly(lactic-co-glycolic acid)-triacrylate resin family with sub-micron resolution and tuneable hydrolysis that addresses the limitations of current 2PP resins. SCRIBE enables the direct printing of hollow microparticles with tuneable chemistry and complex geometries inaccessible to molding techniques, which are used to engineer controlled protein release in vitro and in vivo. SCRIBE microparticles are used to modulate antibody titers and class switching as a function of antigen release rate and extend these findings to enable a single-injection vaccine formulation with extended antibody induction kinetics. Demonstrating how the chemistry and computer-aided design of 2PP-printed microparticles can be used to tune responses to biomacromolecule release in vivo opens significant opportunities for a new generation of drug delivery vehicles.

Self-amplifying RNA (saRNA) is an extremely promising platform because it can produce more protein for less RNA. We used a sort and sequence approach to identify host cell factors associated with transgene expression from saRNA; the hypothesis was that cells with different expression levels would have different transcriptomes. We tested this in CDK4/hTERT immortalized human muscle cells transfected with Venezuelan equine encephalitis virus (VEEV)-derived saRNA encoding GFP. Cells with the highest expression levels had very high levels of transgene mRNA (5%-10% total reads); the cells sorted with low and negative levels of GFP protein also had detectable levels of both VEEV and GFP RNA. To understand host responses, we performed RNA sequencing. Differentially expressed gene (DEG) patterns varied with GFP expression; GFP high cells had many more DEGs, which were associated with protein synthesis and cell metabolism. Comparing profiles by an unsupervised approach revealed that negative cells expressed higher levels of cell-intrinsic immunity genes such as IFIT1, MX1, TLR3, and MyD88. To explore the role of interferon, cells were treated with the Jak inhibitor ruxolitinib. This reduced the number of DEGs, but differences between cells sorted by expression level remained. These studies demonstrate the complex interplay of factors, some immune related, affecting saRNA transgenes.

Influenza virus represents a challenge for traditional vaccine approaches due to its seasonal changes and potential for zoonotic transmission. Nucleic acid vaccines can overcome some of these challenges, especially through the inclusion of multiple antigens to increase the breadth of response. RNA vaccines were an important part of the response to the COVID-19 pandemic, but for future outbreaks DNA vaccines may have some advantages in terms of stability and manufacturing cost that warrant continuing investigation to fully realize their potential. Here, we investigate influenza virus vaccines made using a closed linear DNA platform, Doggybone™ DNA (dbDNA), produced by a rapid and scalable cell-free method. Influenza vaccines have mostly focussed on Haemagglutinin (HA), but the inclusion of Neuraminidase (NA) may provide additional protection. Here, we explored the potential of including NA in a dbDNA vaccine, looking at DNA optimization, mechanism and breadth of protection. We showed that DNA targeting sequences (DTS) improved immune responses against HA but not NA. We explored whether NA vaccine-induced protection against influenza virus infection was cell-mediated, but depletion of CD8 and NK cells made no impact, suggesting it was antibody-mediated. This is reflected in the restriction of protection to homologous strains of influenza virus. Importantly, we saw that including both HA and NA in a single combined vaccine did not dampen the immune response to either one. Overall, we show that linear dbDNA can induce an immune response against NA, which may offer increased protection in instances of HA mismatch where NA remains more conserved.

Respiratory viral infections remain a major cause of hospitalization and death worldwide. Patients with respiratory infections often lose weight. While acute weight loss is speculated to be a tolerance mechanism to limit pathogen growth, severe weight loss following infection can cause quality of life deterioration. Despite the clinical relevance of respiratory infection-induced weight loss, its mechanism is not yet completely understood. We utilized a model of CD 8+ T cell-driven weight loss during respiratory syncytial virus (RSV) infection to dissect the immune regulation of post-infection weight loss. Supporting previous data, bulk RNA sequencing indicated significant enrichment of the interleukin (IL)-1 signaling pathway after RSV infection. Despite increased viral load, infection-associated weight loss was significantly reduced after IL-1 alpha (but not IL-1 beta) blockade. IL-1 alpha depletion resulted in a reversal of the gut microbiota changes observed following RSV infection. Direct nasal instillation of IL-1 alpha also caused weight loss. Of note, we detected IL-1 alpha in the brain after either infection or nasal delivery. This was associated with changes in genes controlling appetite after RSV infection and corresponding changes in signaling molecules such as leptin and growth/differentiation factor 15. Together, these findings indicate a lung-brain-gut signaling axis for IL-1 alpha in regulating weight loss after RSV infection.

Controlled human infection models (CHIMs) are a critical tool for the understanding of infectious disease progression, characterising immune responses to infection and rapid assessment of vaccines or drug treatments. There is increasing interest in using CHIMs for vaccine development and an obvious need for widely available and fit-for-purpose challenge agents. Inno4Vac is a large European consortium working towards accelerating and de-risking the development of new vaccines, including development of CHIMs for influenza, respiratory syncytial virus and Clostridium difficile. This report (in two parts) summarises a workshop held at the MHRA in 2021, focused on how to select CHIM candidate strains of influenza and respiratory syncytial virus (RSV) based on desirable virus characteristics and which immune assays would provide relevant information for assessing pre-existing and post-infection immune responses and defining correlates of protection. This manuscript (part 2) summarises presentations and discussions centred around RSV CHIMs and immune assays (an additional manuscript summarises influenza CHIM and immune assays: Inno4Vac workshop report Part 1: Controlled human influenza virus infection model (CHIVIM) strain selection and immune assays for CHIVIM studies, November 2021, MHRA, UK).

Controlled human infection models (CHIMs) are a critical tool for the understanding of infectious disease progression, characterising immune responses to infection and rapid assessment of vaccines or drug treatments. There is increasing interest in using CHIMs for vaccine development and an obvious need for widely available and fit-for-purpose challenge agents. Inno4Vac is a large European consortium working towards accelerating and de-risking the development of new vaccines, including the development of CHIMs for influenza, respiratory syncytial virus and Clostridioides difficile. This report (in two parts) summarises a workshop held at the MHRA in 2021, focused on how to select CHIM candidate strains of influenza and respiratory syncytial virus (RSV) based on desirable virus characteristics and which immune assays would provide relevant information for assessing pre-existing and post-infection immune responses and defining correlates of protection. This manuscript (Part 1) summarises presentations and discussions centred around influenza CHIMs and immune assays (a second manuscript summarises RSV CHIM and immune assays: Inno4Vac workshop report Part 2: RSV CHIM strain selection and immune assays for RSV CHIM studies, November 2021, MHRA, UK).