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Adenovirus-vectored vaccines played an important role in the global response to SARS-CoV-2. Adenovirus platforms have many advantages including a simple and readily transferred manufacturing process, low cost, and thermostability. Speed of production of an initial Good Manufacturing Practice (GMP)-compliant batch has, however, been viewed as a limitation of adenovirus vectors relative to mRNA platforms. Production of the initial viral starting material and release testing are key rate-limiting steps. Production of viral starting material from DNA, and release testing in accordance with regulatory expectations, for first-in-human trials of adenovirus-vectored vaccines. We describe experience of these stages in the production of the first GMP batches for multiple adenovirus-vectored candidates and the adaptations made for ChAdOx1 nCoV-19 (the Oxford COVID-19 vaccine) in early 2020. We also report development of a streamlined approach to starting material generation, enabling initial GMP batch availability within c. 60 days of publication of a new pathogen sequence. Using a New World arenavirus vaccine construct as a proof of concept, we demonstrate reproducible execution of this pipeline, maintaining acceptable infectivity and other quality attributes. We discuss opportunities for additional time savings in the future. This work demonstrates suitability of an adenovirus platform to contribute to the \"100 Days Mission\" for vaccines against \"Disease X\".

Mycobacterium tuberculosis is the main causative agent of tuberculosis. BCG, the only licensed vaccine, provides inadequate protection against pulmonary tuberculosis. Controlled human infection models are useful tools for vaccine development. We aimed to determine a safe dose of aerosol-inhaled live-attenuated Mycobacterium bovis BCG as a surrogate for M tuberculosis infection, then compare the safety and tolerability of infection models established using aerosol-inhaled and intradermally administered BCG. This phase 1 controlled human infection trial was conducted at two clinical research facilities in the UK. Healthy, immunocompetent adults aged 18–50 years, who were both M tuberculosis-naive and BCG-naive and had no history of asthma or other respiratory diseases, were eligible for the trial. Participants were initially enrolled into group 1 (receiving the BCG Danish strain); the trial was subsequently paused because of a worldwide shortage of BCG Danish and, after protocol amendment, was restarted using the BCG Bulgaria strain (group 2). After a dose-escalation study, during which participants were sequentially allocated to receive either 1 × 103, 1 × 104, 1 × 105, 1 × 106, or 1 × 107 colony-forming units (CFU) of aerosol BCG, the maximum tolerated dose was selected for the randomised controlled trial. Participants in this trial were randomly assigned (9:12), by variable block randomisation and using sequentially numbered sealed envelopes, to receive aerosol BCG (1 × 107 CFU) and intradermal saline or intradermal BCG (1 × 106 CFU) and aerosol saline. Participants were masked to treatment allocation until day 14. The primary outcome was to compare the safety of a controlled human infection model based on aerosol-inhaled BCG versus one based on intradermally administered BCG, and the secondary outcome was to evaluate BCG recovery in the airways of participants who received aerosol BCG or skin biopsies of participants who received intradermal BCG. BCG was detected by culture and by PCR. The trial is registered at ClinicalTrials.gov, NCT02709278, and is complete. Participants were assessed for eligibility between April 7, 2016, and Sept 29, 2018. For group 1, 15 participants were screened, of whom 13 were enrolled and ten completed the study; for group 2, 60 were screened and 33 enrolled, all of whom completed the study. Doses up to 1 × 107 CFU aerosol-inhaled BCG were sufficiently well tolerated. No significant difference was observed in the frequency of adverse events between aerosol and intradermal groups (median percentage of solicited adverse events per participant, post-aerosol vs post-intradermal BCG: systemic 7% [IQR 2–11] vs 4% [1–13], p=0·62; respiratory 7% [1–19] vs 4% [1–9], p=0·56). More severe systemic adverse events occurred in the 2 weeks after aerosol BCG (15 [12%] of 122 reported systemic adverse events) than after intradermal BCG (one [1%] of 94; difference 11% [95% CI 5–17]; p=0·0013), but no difference was observed in the severity of respiratory adverse events (two [1%] of 144 vs zero [0%] of 97; 1% [−1 to 3]; p=0·52). All adverse events after aerosol BCG resolved spontaneously. One serious adverse event was reported—a participant in group 2 was admitted to hospital to receive analgesia for a pre-existing ovarian cyst, which was deemed unrelated to BCG infection. On day 14, BCG was cultured from bronchoalveolar lavage samples after aerosol infection and from skin biopsy samples after intradermal infection. This first-in-human aerosol BCG controlled human infection model was sufficiently well tolerated. Further work will evaluate the utility of this model in assessing vaccine efficacy and identifying potential correlates of protection. Bill & Melinda Gates Foundation, Wellcome Trust, National Institute for Health Research Oxford Biomedical Research Centre, Thames Valley Clinical Research Network, and TBVAC2020.

A new and more effective tuberculosis (TB) vaccine is urgently needed, but development is hampered by the lack of validated immune correlates of protection. Bacillus Calmette Guérin (BCG) vaccination by the aerosol (AE) and intravenous (IV) routes has been shown to confer superior levels of protection from challenge with Mycobacterium tuberculosis (M.tb) in non-human primates (NHP) compared with standard intradermal (ID) administration. This finding offers a valuable opportunity to investigate which aspects of immunity are associated with improved control of M.tb and may represent biomarkers or correlates of protection. As TB vaccine research to date has focused largely on cellular immunity, we aimed to better characterize the poorly-understood serum antibody response to BCG administered by different routes of vaccination in NHP. We demonstrate superior M.tb-specific IgG, IgA, and IgM titers in serum following IV BCG vaccination compared to the ID or AE routes. We also observe improved capacity of IgG induced by IV BCG to opsonize the surface of mycobacteria, and report for the first time that M.tb-specific IgG from IV BCG vaccinated animals is of higher avidity compared with IgG from ID or AE BCG vaccinated animals. Notably, we identified a significant correlation between IgG avidity and measures of protection from aerosol M.tb challenge. Our findings highlight a potential role for antibodies as markers and/or mediators of the superior vaccine-induced protection IV BCG confers against TB and suggest that quality, as well as quantity, of antibodies should be considered when developing and evaluating TB vaccine candidates. •Superior serum antibody titers following IV vs. ID or aerosol BCG vaccination.•IgG induced by IV BCG has improved mycobacterial opsonization vs. ID or aerosol BCG.•Higher avidity M.tb-specific IgG following IV vs. ID or aerosol BCG vaccination.•Association between IgG avidity and protection from aerosol M.tb challenge.

Tuberculosis (TB), caused by (MTB), remains a significant global health concern. The existing vaccine, Bacillus Calmette-Guérin (BCG), provides inconsistent protection, highlighting the pressing need for a more effective vaccine. We aimed to identify novel antigens and assess their protective efficacy as TB vaccine candidates. Using immunopeptidomics, we identified 64 and 80 unique mycobacterial antigens derived from BCG and MTB, respectively. We prioritised antigens based on HLA allele coverage through an immunoinformatics approach. The candidates, , , and , delivered as DNA vaccines, were evaluated for efficacy in mice using the ex vivo Mycobacterial Growth Inhibition Assay (MGIA) and was identified as a promising candidate. In vivo murine challenge experiments confirmed the protective efficacy conferred by when formulated as recombinant protein with AS01™ or AddaS03™ adjuvants, compared to the naïve group. The immunogenic profiles of formulated in the two different adjuvants differed, with -AS01™ inducing antigen-specific IFN-γ, TNF-α, IL-2, IL-17, IgG1 and IgG2a-c, while -AddaS03™ induced TNF-α, IL-2, IL-17, IL-4, IgM, IgG1, IgG2b. Our findings highlight as a promising protective antigen for future TB vaccine development.

Mycobacteria are known to exert a range of heterologous effects on the immune system. The mycobacteria-based Freund's Complete Adjuvant is a potent non-specific stimulator of the immune response used in immunization protocols promoting antibody production, and Mycobacterium bovis Bacille Calmette Guérin (BCG) vaccination has been linked with decreased morbidity and mortality beyond the specific protection it provides against tuberculosis (TB) in some populations and age groups. The role of heterologous antibodies in this phenomenon, if any, remains unclear and under-studied. We set out to evaluate antibody responses to a range of unrelated pathogens following infection with Mycobacterium tuberculosis (M.tb) and vaccination with BCG or a candidate TB vaccine, MTBVAC, in non-human primates. We demonstrate a significant increase in the titer of antibodies against SARS-CoV-2, cytomegalovirus, Epstein-Barr virus, tetanus toxoid, and respiratory syncytial virus antigens following low-dose aerosol infection with M.tb. The magnitude of some of these responses correlated with TB disease severity. However, vaccination with BCG administered by the intradermal, intravenous or aerosol routes, or intradermal delivery of MTBVAC, did not increase antibody responses against unrelated pathogens. Our findings suggest that it is unlikely that heterologous antibodies contribute to the non-specific effects of these vaccines. The apparent dysregulation of B cell responses associated with TB disease warrants further investigation, with potential implications for risk of B cell cancers and novel therapeutic strategies.

Tuberculosis (TB) is a major global health problem and the only currently-licensed vaccine, BCG, is inadequate. Many TB vaccine candidates are designed to be given as a boost to BCG; an understanding of the BCG-induced immune response is therefore critical, and the opportunity to relate this to circumstances where BCG does confer protection may direct the design of more efficacious vaccines. While the T cell response to BCG vaccination has been well-characterized, there is a paucity of literature on the humoral response. We demonstrate BCG vaccine-mediated induction of specific antibodies in different human populations and macaque species which represent important preclinical models for TB vaccine development. We observe a strong correlation between antibody titers in serum versus plasma with modestly higher titers in serum. We also report for the first time the rapid and transient induction of antibody-secreting plasmablasts following BCG vaccination, together with a robust and durable memory B cell response in humans. Finally, we demonstrate a functional role for BCG vaccine-induced specific antibodies in opsonizing mycobacteria and enhancing macrophage phagocytosis in vitro , which may contribute to the BCG vaccine-mediated control of mycobacterial growth observed. Taken together, our findings indicate that the humoral immune response in the context of BCG vaccination merits further attention to determine whether TB vaccine candidates could benefit from the induction of humoral as well as cellular immunity.

COVID-19 vaccine supply shortages are causing concerns about compromised immunity in some countries as the interval between the first and second dose becomes longer. Conversely, countries with no supply constraints are considering administering a third dose. We assessed the persistence of immunogenicity after a single dose of ChAdOx1 nCoV-19 (AZD1222), immunity after an extended interval (44–45 weeks) between the first and second dose, and response to a third dose as a booster given 28–38 weeks after the second dose. In this substudy, volunteers aged 18–55 years who were enrolled in the phase 1/2 (COV001) controlled trial in the UK and had received either a single dose or two doses of 5 × 1010 viral particles were invited back for vaccination. Here we report the reactogenicity and immunogenicity of a delayed second dose (44–45 weeks after first dose) or a third dose of the vaccine (28–38 weeks after second dose). Data from volunteers aged 18–55 years who were enrolled in either the phase 1/2 (COV001) or phase 2/3 (COV002), single-blinded, randomised controlled trials of ChAdOx1 nCoV-19 and who had previously received a single dose or two doses of 5 × 1010 viral particles are used for comparison purposes. COV001 is registered with ClinicalTrials.gov, NCT04324606, and ISRCTN, 15281137, and COV002 is registered with ClinicalTrials.gov, NCT04400838, and ISRCTN, 15281137, and both are continuing but not recruiting. Between March 11 and 21, 2021, 90 participants were enrolled in the third-dose boost substudy, of whom 80 (89%) were assessable for reactogenicity, 75 (83%) were assessable for evaluation of antibodies, and 15 (17%) were assessable for T-cells responses. The two-dose cohort comprised 321 participants who had reactogenicity data (with prime-boost interval of 8–12 weeks: 267 [83%] of 321; 15–25 weeks: 24 [7%]; or 44–45 weeks: 30 [9%]) and 261 who had immunogenicity data (interval of 8–12 weeks: 115 [44%] of 261; 15–25 weeks: 116 [44%]; and 44–45 weeks: 30 [11%]). 480 participants from the single-dose cohort were assessable for immunogenicity up to 44–45 weeks after vaccination. Antibody titres after a single dose measured approximately 320 days after vaccination remained higher than the titres measured at baseline (geometric mean titre of 66·00 ELISA units [EUs; 95% CI 47·83–91·08] vs 1·75 EUs [1·60–1·93]). 32 participants received a late second dose of vaccine 44–45 weeks after the first dose, of whom 30 were included in immunogenicity and reactogenicity analyses. Antibody titres were higher 28 days after vaccination in those with a longer interval between first and second dose than for those with a short interval (median total IgG titre: 923 EUs [IQR 525–1764] with an 8–12 week interval; 1860 EUs [917–4934] with a 15–25 week interval; and 3738 EUs [1824–6625] with a 44–45 week interval). Among participants who received a third dose of vaccine, antibody titres (measured in 73 [81%] participants for whom samples were available) were significantly higher 28 days after a third dose (median total IgG titre: 3746 EUs [IQR 2047–6420]) than 28 days after a second dose (median 1792 EUs [IQR 899–4634]; Wilcoxon signed rank test p=0·0043). T-cell responses were also boosted after a third dose (median response increased from 200 spot forming units [SFUs] per million peripheral blood mononuclear cells [PBMCs; IQR 127–389] immediately before the third dose to 399 SFUs per milion PBMCs [314–662] by day 28 after the third dose; Wilcoxon signed rank test p=0·012). Reactogenicity after a late second dose or a third dose was lower than reactogenicity after a first dose. An extended interval before the second dose of ChAdOx1 nCoV-19 leads to increased antibody titres. A third dose of ChAdOx1 nCoV-19 induces antibodies to a level that correlates with high efficacy after second dose and boosts T-cell responses. UK Research and Innovation, Engineering and Physical Sciences Research Council, National Institute for Health Research, Coalition for Epidemic Preparedness Innovations, National Institute for Health Research Oxford Biomedical Research Centre, Chinese Academy of Medical Sciences Innovation Fund for Medical Science, Thames Valley and South Midlands NIHR Clinical Research Network, AstraZeneca, and Wellcome.

A variety of Good Manufacturing Practice (GMP) compliant processes have been reported for production of non-replicating adenovirus vectors, but important challenges remain. Most clinical development of adenovirus vectors now uses simian adenoviruses or rare human serotypes, whereas reported manufacturing processes mainly use serotypes such as AdHu5 which are of questionable relevance for clinical vaccine development. Many clinically relevant vaccine transgenes interfere with adenovirus replication, whereas most reported process development uses selected antigens or even model transgenes such as fluorescent proteins which cause little such interference. Processes are typically developed for a single adenovirus serotype – transgene combination, requiring extensive further optimization for each new vaccine. There is a need for rapid production platforms for small GMP batches of non-replicating adenovirus vectors for early-phase vaccine trials, particularly in preparation for response to emerging pathogen outbreaks. Such platforms must be robust to variation in the transgene, and ideally also capable of producing adenoviruses of more than one serotype. It is also highly desirable for such processes to be readily implemented in new facilities using commercially available single-use materials, avoiding the need for development of bespoke tools or cleaning validation, and for them to be readily scalable for later-stage studies. Here we report the development of such a process, using single-use stirred-tank bioreactors, a transgene-repressing HEK293 cell – promoter combination, and fully single-use filtration and ion exchange components. We demonstrate applicability of the process to candidate vaccines against rabies, malaria and Rift Valley fever, each based on a different adenovirus serotype. We compare performance of a range of commercially available ion exchange media, including what we believe to be the first published use of a novel media for adenovirus purification (NatriFlo® HD-Q, Merck). We demonstrate the need for minimal process individualization for each vaccine, and that the product fulfils regulatory quality expectations. Cell-specific yields are at the upper end of those previously reported in the literature, and volumetric yields are in the range 1 × 1013 – 5 × 1013 purified virus particles per litre of culture, such that a 2–4 L process is comfortably adequate to produce vaccine for early-phase trials. The process is readily transferable to any GMP facility with the capability for mammalian cell culture and aseptic filling of sterile products.

The development of an effective vaccine against  Mycobacterium tuberculosis is hampered by an incomplete understanding of immunoprotective mechanisms. We utilise an aerosol human challenge model using attenuated Mycobacterium bovis BCG, in BCG-naïve UK adults. The primary endpoint of this study (NCT03912207) was to characterise the early immune responses induced by aerosol BCG infection, the secondary endpoint was to identify immune markers associated with in-vitro protection. Blinded volunteers were randomised to inhale 1 × 10 7 CFU aerosolised BCG or 0.9% saline (20:6); and sequentially allocated to bronchoscopy at day 2 or 7 post-inhalation (10 BCG, 3 saline each timepoint). In the bronchoalveolar lavage post-aerosol BCG infection, there was an increase in frequency of eosinophils, neutrophils, NK cells and Donor-Unrestricted T cells at day 7, and the frequency of antigen presenting cells decreased at day 7 compared with day 2. The frequency of interferon-gamma+ BCG-specific CD4+ T cells increased in the BAL and peaked in the blood at day 7 post-BCG infection compared to day 2. BAL cells at day 2 and day 7 upregulated gene pathways related to phagocytosis, MHC-II antigen loading, T cell activation and proliferation. BCG’s lack of key virulence factors and its failure to induce granulomas, may mean the observed immune responses do not fully recapitulate Mycobacterium tuberculosis infection. However, human infection models can provide unique insights into early immune mechanisms, informing vaccine design for complex pathogens. In this study, Marshall et al. studied early immune responses in the lung and blood after an aerosol infection with BCG. Understanding which cells respond to this infection can help us understand how to design new tuberculosis vaccines.

The decline in protective antibody titers and efficacy over time of the circumsporozoite protein (CSP)-based RTS,S/AS01 and R21/Matrix-M vaccines highlights the need for improved vaccines. Sporozoite microneme protein essential for cell traversal-1 (SPECT-1) is a conserved ( ) antigen that plays a key role in parasite movements while traversing host cells. Targeting SPECT-1 as an addition to the CSP in the same vaccine may enhance immune response and protection. We engineered a series of recombinant Hepatitis B surface antigen (HBsAg) virus-like particles (VLPs) displaying SPECT-1 alone or in combination with NANP repeats and C-terminal region of CSP. These monovalent and bivalent VLP vaccine candidates were evaluated alongside R21 in a prime-boost regimen which was followed by challenge with transgenic parasite expressing the same antigens. We also assessed the quality of the antibody response following vaccination. Based on the antibody titers and protective efficacy, one bivalent VLP construct was selected for further evaluation with different adjuvant formulations. All bivalent VLPs formed to display antigenic epitopes that were accessible to antigen-specific antibodies. Bivalent VLPs induced IgG responses against both NANP and SPECT-1 epitopes, confirming co-display of the target antigens. One of the bivalent candidates (N4) provided similar efficacy to R21 in formulation with Matrix-M adjuvant in mice, whereas addition of SPECT-1 significantly reduced efficacy of another candidate (N2). NANP-specific IgG response negatively correlated with blood stage parasite load. When N4 was formulated with LMQ or SMNP adjuvants, measured humoral responses or protective efficacy did not improve. Further investigation is needed to determine whether the immune response persists over time. Our findings show that SPECT-1 can be effectively incorporated into the HBsAg VLPs and co-displayed with CSP epitopes without impairing immunogenicity. Although protection was evaluated using transgenic parasites expressing antigens, the modular HBsAg VLP platform theoretically allows incorporation of antigens from other species such as .