The present invention relates to immunogenic compositions for use in aerosol delivery to the respiratory tract of a human. The compositions comprise an expression vector, which comprise one or more exogenous nucleotide sequences from one or more pathogens. The compositions are able to elicit a protective immune response and, in particular, elicit both a local mucosal and systemic immune response, which is protective.

Traditionally, vaccines have been based on whole inactivated or attenuated pathogens. However, for many infectious diseases such as malaria, this approach is impractical and the focus of research has changed to the development of 'subunit vaccines' expressing only those pathogen-derived antigens that induce immune correlates of protection.

Subunit vaccines present an antigen to the immune system without introducing a whole infectious organism unless the organism is a viral vector, where the vector is a whole organism. One such method involves the administration of a specific, isolated protein from an infectious organism. However, this technique often induces only a weak immune response and the isolated proteins may have a different three-dimensional structure than the protein in its normal context, resulting in the production of antibodies that may not recognize the infectious organism.

Thus, alternative methods have been considered for the delivery of antigens, such that stronger, protective immune responses can be elicited. Thus, it has been proposed to deliver antigens by means of viral or bacterial vectors, which carry nucleotide sequences encoding the antigen of interest. The antigen is thus expressed in the vector and is released in the subject from the vector, thereby eliciting an immune response.

Delivery of immunogenic compositions by means of the respiratory tract has also been proposed. Such an approach can make use of the mucosal immune system and thereby elicit a mucosal immune response. For instance, US2011/0081377 discloses viral vector constructs for delivery of vaccine antigens via the respiratory tract. The data provided in that disclosure all relates to a range of animal models and in general shows that such a mode of delivery generates a strong immune response in the lung.

However, there still exists a need for vaccines which can be delivered via the respiratory tract. We have now found that aerosol delivery of virus-based expression vectors to a subject via the respiratory tract elicits both a strong local and systemic protective effect and this has been confirmed in human subjects.

Thus, in a first aspect, the present invention provides an immunogenic composition for use in aerosol delivery to the respiratory tract of a human subject comprising a viral expression vector, wherein said viral expression vector comprises at least one exogenous nucleotide sequence, encoding one or more epitopes or a protein or polypeptide antigen, derived from a pathogen, and wherein said immunogenic composition elicits both a local, mucosal, immune response and a systemic immune response against the one or more protein or polypeptide antigens. As discussed herein, the antigen is a pathogen-derived antigen. Preferably, the pathogen is selected from the group consisting of bacteria, viruses, prions, fungi, protozoa and helminths. Preferably, the antigen is derived from the group consisting of M. tuberculosis, Plasmodium sp, influenza virus, HSV, HIV, Hepatitis B or C virus, Cytomegalovirus, Human papilloma virus, chlamydia species, malaria parasites, leishmania parasites or any mycobacterial species. Preferred antigens include TRAP, MSP- 1 , AMA- 1 , RH5, Pfs25 and CSP from Plasmodium, influenza virus antigens and ESAT6, TBI 0.4 85 A and 85B antigens from Mycobacterium tuberculosis. Particularly preferred antigens include Ag85 A from

Mycobacterium tuberculosis and nucleoprotein (NP) and matrix protein 1 (Ml) from influenza A virus. As used herein, the term 'antigen' encompasses one or more epitopes from an antigen and includes the parent antigen, and fragments and variants thereof. These fragments and variants retain essentially the same biological activity or function as the parent antigen. Preferably, they retain or improve upon the antigenicity and/or immunogenicity of the parent antigen. Generally, "antigenic" is taken to mean that the protein or polypeptide is capable of being used to raise antibodies or T cells or indeed is capable of inducing an antibody or T cell response in a subject. "Immunogenic" is taken to mean that the protein or polypeptide is capable of eliciting a potent and preferably a protective immune response in a subject. Thus, in the latter case, the protein or polypeptide may be capable of generating an antibody response and a non-antibody based immune response. Preferably, fragments of the antigens comprise at least n consecutive amino acids from the sequence of the parent antigen, wherein n is preferably at least, or more than, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 57, 58, 59, 60, 70, 80, 90 or 100. The fragments preferably include one or more epitopic regions from the parent antigen. Indeed, the fragment may comprise or consist of an epitope from the parent antigen. Alternatively, the fragment may be sufficiently similar to such regions to retain their antigenic/immunogenic properties.

The antigens discussed herein include variants such as derivatives, analogues, homologues or functional equivalents of the parent antigen. Particularly preferred are derivatives, analogues, homologues or functional equivalents having an amino acid sequence similar to that of the parent antigen, in which one or more amino acid residues are substituted, deleted or added in any combination. Preferably, these variants retain an antigenic determinant or epitope in common with the parent antigen.

Preferably, the derivatives, analogues, homologues, and functional equivalents have an amino acid sequence substantially identical to amino acid sequence of the parent antigen. The exogeneous nucleotide sequence may encode more than one antigen. The viral vector may be designed to express the one or more antigen genes as an epitope string. Preferably, the epitopes in a string of multiple epitopes are linked together without intervening sequences such that unnecessary nucleic acid and/or amino acid material is avoided. The creation of the epitope string is preferably achieved using a recombinant DNA construct that encodes the amino acid sequence of the epitope string, with the DNA encoding the one or more epitopes in the same reading frame. An exemplary antigen, TIPeGFP, comprises an epitope string which includes the following epitopes: E6FP, SIV-gag, PyCD4 and Py3. Alternatively, the antigens may be expressed as separate polypeptides.

One or more of the antigens or antigen genes may be truncated at the C-terminus and/or the N-terminus. This may facilitate cloning and construction of the vectored vaccine and/or enhance the immunogenicity or antigenicity of the antigen. Methods for truncation will be known to those of skill in the art. For example, various well-known techniques of genetic engineering can be used to selectively delete the encoding nucleic acid sequence at either end of the antigen gene, and then insert the desired coding sequence into the viral vector. For example, truncations of the candidate protein are created using 3' and/or 5' exonuclease strategies selectively to erode the 3' and/or 5' ends of the encoding nucleic acid, respectively. Preferably, the wild type gene sequence is truncated such that the expressed antigen is truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids relative to the parent antigen. Preferably, the antigen gene is truncated by 10 - 20 amino acids at the C- terminus relative to the wild type antigen. More preferably, the antigen gene is truncated by 13 - 18 amino acids, most preferably by 15 amino acids at the C- terminus relative to the wild type antigen. Preferably, the Ag85 A antigen is C-terminally truncated in this manner.

One or more of the antigen genes may also comprise a leader sequence. The leader sequence may affect processing of the primary transcript to mRNA, translation efficiency, mRNA stability, and may enhance expression and/or immunogenicity of the antigen. Preferably, the leader sequence is tissue plasminogen activator (tPA). Preferably, the tPA leader sequence is positioned N-terminal to the one or more antigens.

The leader sequence such as the tPA leaders sequence may be linked to the sequence of the antigen via a peptide linker. Peptide linkers are generally from 2 to about 50 amino acids in length, and can have any sequence, provided that it does not form a secondary structure that would interfere with domain folding of the fusion protein.

One or more of the antigen genes may comprise a marker such as the Green Fluorescent Protein (GFP) marker to facilitate detection of the expressed product of the inserted gene sequence. One or more of the antigen genes may comprise a nucleic acid sequence encoding a tag polypeptide that is covalently linked to the antigen upon translation. Preferably the tag polypeptide is selected from the group consisting of a PK tag, a FLAG tag, a MYC tag, a polyhistidine tag or any tag that can be detected by a monoclonal antibody. The nucleic acid sequence encoding the tag polypeptide may be positioned such that, following translation, the tag is located at the C-terminus or the N-terminus of the expressed antigen or may be internal to the expressed antigen. Preferably, the tag is located at the C-terminus of the expressed antigen. In a preferred embodiment, one or more of the antigen genes encode a PK tag. A tag of this type may facilitate detection of antigen expression and clones expressing the antigen, and/or enhance the immunogenicity or antigenicity of the antigen. The exogeneous nucleotide sequence is preferably operably linked to regulatory sequences necessary to drive translation, transcription and/or expression of the exogeneous nucleotide sequence /transgene in a host cell, for example a mammalian cell. As used herein, the phrase "operably linked" means that the regulatory sequences are contiguous with the nucleic acid sequences they regulate or that said regulatory sequences act in trans, or at a distance, to control the regulated nucleic acid sequence. Such regulatory sequences include appropriate expression control sequences such as transcription initiation, termination, enhancer and promoter sequences, efficient RNA processing signals, such as splicing and polyadenylation signals, sequences that enhance translation efficiency and protein stability and sequences promote protein secretion. Additionally they may contain sequences for repression of transgene expression, for example during production in cell lines expression a transactivating receptor. Promoters and other regulatory sequences which control expression of a nucleic acid have been identified and are known in the art. Preferably, for adenoviral vectors or plasmid DNA vectors the promoter is selected from the group consisting of human CMV promoters, simian CMV promoters, murine CMV promoters, ubiquitin, the EF1 promoter, frog EF1 promoter, actin and other mammalian promoters. Most preferred are human CMV promoters and in particular the human CMV major immediate early promoter. For MVA vectors any internal promoter may be used, for example the p7.5 promoter or the Fl 1 promoter and many others are known. In addition strong synthetic promoters may be used such as the SSP sequence.

The exogeneous nucleotide sequence(s) of interest may be introduced into the viral vector as part of a cassette. As used herein, the term "cassette" refers to a nucleic acid molecule comprising at least one nucleotide sequence to be expressed, along with its transcriptional

5

SUBSTITUTE SHEET RULE 26 and translational control sequences to allow the expression of the nucleotide sequence(s) in a host cell, and optionally restriction sites at the 5' and 3' ends of the cassette. Because of the restriction endonuclease sites, the cassettes can easily be inserted, removed or replaced with another cassette. Changing the cassette will result in the expression of different sequence(s) by the vector into which the cassette is incorporated. Alternatively, any method known to one of skill in the art could be used to construct, modify or derive said cassette, for example PCR mutagenesis, In-Fusion ^® , recombineering, Gateway ^® cloning, site-specific recombination or topoisomerase cloning.

The expression control sequences preferably include the viral elements necessary for replication and virion encapsidation. Preferably, the elements flank the exogeneous nucleotide sequence.

Examples of preferred viral vectors include Vaccinia virus vectors or Adenovirus vectors. The viral vectors can be of human or simian origin.

In one embodiment, the viral vector is non-replicating or replication-impaired. As used herein, the term "non-replicating" or "replication-impaired" means not capable of replicating to any significant extent in the majority of normal mammalian cells, preferably normal human cells. It is preferred that the viral vector is incapable of causing a productive infection or disease in the human patient. However, the viral vector is preferably capable of stimulating an immune response. Viruses which are non-replicating or replication-impaired may have become so naturally, i.e. they may be isolated as such from nature. Alternatively, the viruses may be rendered non-replicating or replication-impaired artificially, e.g. by breeding in vitro or by genetic manipulation. For example, a gene which is critical for replication may be functionally deleted.

The immunogenic and/or antigenic compositions as described herein may be prophylactic (to prevent infection), post-exposure (to treat after infection but before disease) or therapeutic (to treat disease). Preferably, the composition is prophylactic or post-exposure. Preferably, the composition is a vaccine.

It is also contemplated that the compositions described herein can be administered in combination with adjuvants or other treatments. For example the compositions could be administered with one or more additional active agents. Alternatively, they can be coadministered with one or more antimicrobial compounds, such as suitable antimicrobial compounds including antituberculous chemotherapeutics such as rifampicin, isoniazid, ethambutol and pyrizinamide.

Suitable carriers and/or diluents are well known in the art and include pharmaceutical grade starch, mannitol, lactose, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, (or other sugar), magnesium carbonate, gelatin, oil, alcohol, detergents, emulsifiers or water (preferably sterile).

Suitable adjuvants for human use are well known in the art and include incomplete Freund's adjuvant, Freund's adjuvant with MDP (muramyldipeptide), alum (aluminium hydroxide), alum plus Bordatella pertussis and immune stimulatory complexes (ISCOMs, typically a matrix of Quil A containing viral proteins), MF59 (Novartis Vaccines, Siena) and matrix M (Isconova, Uppsala). Alternatively, adjuvants may be encoded in the viral vector rather than mixed with the vector. Examples of encoded adjuvants include CD74 (invariant chain), IMX- 313 which is homologous to a fragment of human C4-binding protein, and co-stimulatory molecules such as 4-1BBL.

The compositions described herein are formulated as aerosols. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomising device. Alternatively the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve, which is intended for disposal once the contents of the container have been exhausted. Where the dosage form comprises an aerosol dispenser, it will contain a pharmaceutically acceptable propellant. The aerosol dosage forms can also take the form of a pump-atomiser.

Suitably, the compositions are administered as aerosols by means of a nebuliser. There are many examples of such nebulisers available on the market. Preferably the nebuliser is a hand held device which may be held and / or operated by the subject being immunised. One such nebuliser is that supplied by Omron, under the name MicroAir U22, which is a commercially available battery-operated hand-held lightweight ultrasonic mesh nebuliser (Omron, 2012). This device is silent and generates a mist with a particle size of between 2 and 4 μιη from fluid placed within the nebuliser chamber. A patient inhales the aerosol through a mouthpiece at their normal relaxed respiratory rate and tidal volume. The composition is suitably diluted into a total volume of 1 mL with 0.9% saline which aids nebulisation. Other examples of nebulisers are well known to the skilled person.

The composition is preferably pyrogen-free. It is preferably buffered e.g. at between pH 6 and pH 8, generally around pH 7. Preferably, the composition is substantially isotonic with humans.

The compositions described herein deliver an immunogenically effective amount of the viral vector to a patient. As used herein 'immunogenically effective amount' means that the administration of that amount to an individual, either as a single dose or as a series of doses, is effective for prevention or treatment of a disease or condition. In particular, this phrase means that a sufficient amount of the viral vector is delivered to the patient over a suitable timeframe such that a sufficient amount of the antigen is produced by the patient's cells to stimulate an immune response which is effective for prevention or treatment of a disease or condition. This amount varies depending on the health and physical condition of the individual to be treated, age, the capacity of the individual's immune system, the degree of protection desired, the formulation of the vaccine, the doctor's assessment of the medical situation and other relevant factors. In general, a pharmaceutically effective dose comprises 1 x 10 to 1 x 10 viral particles. For Vaccinia viral vectors, for example, the dose will be at the lower end of this range, typically 1-10 x 10 plaque forming units whilst for Adenovirus vectors it will tend to be towards the upper end of the range. The treatment and prevention of many diseases, including liver stage malaria, tuberculosis and influenza, are associated with the maintenance of a strong cell-mediated response to infection involving both CD4+ and CD8+ T cells and the ability to respond with Thl-type cytokines, particularly IFN-γ, TNF-a, IL-2 and IL-17. Although many subunit vaccine platforms effectively generate human immunity, the generation of robust cell-mediated immune responses, particularly CD4+ and CD8+ T cell immune responses, has been much more challenging. The viral vectors compositions as discussed herein stimulate both cellular and humoral immune responses against the encoded antigen.

In particular, the administration of the compositions by the aerosol route results in

surprisingly strong mucosal and systemic responses, in particular a particularly strong IL-17 response. Also, most remarkably, the aerosol administration is surprisingly found to induce stronger systemic (blood) T cell responses of particular types, e.g. interferon-gamma- secreting CD8 T cell responses, than intradermal administration. This induction of very potent blood T cell responses by use of an aerosol route was unexpected and has considerable utility for vaccination.

IL-17-secreting T cells have been found to be of importance in protective immunity to tuberculosis and several other bacterial and non-bacterial infectious diseases, but little has been known about how to induce these effectively in humans. The induction of substantial IL-17-secreting T cells in humans in both blood and mucosal surfaces here is therefore an important advance.

CD8+ T cells are known to be important in protective immunity in humans to both infectious pathogens and cancer. Examples of the former include influenza, HIV, HCV and malaria and examples of the latter include melanoma, prostate cancer, breast cancer and colon cancer. A means of improving on CD8 T cell induction systemically (here in blood) by using aerosol administration of viral vectors therefore has clear utility for both prophylactic and therapeutic immunisation. It is also desirable to induce a memory immune response. Memory immune responses are classically attributed to the reactivation of long-lived, antigen-specific T lymphocytes that arise directly from differentiated effector T cells and persist in a uniformly quiescent state. Memory T cells have been shown to be heterogeneous and to comprise at least two subsets, endowed with different migratory capacity and effector function; effector memory T cells (TEM) and central memory T cells (CTM). TEM resemble the effector cells generated in the primary response in that they lack the lymph node-homing receptors L-selectin and CCR7 and express receptors for migration into inflamed tissues. Upon re-encounter with antigen, these TEM can rapidly produce IFN-γ or IL-4 or release pre- stored perform. TCM express L- selectin and CCR7 and lack immediate effector function. These cells have a low activation threshold and, upon restimulation in secondary lymphoid organs, proliferate and differentiate to effectors. The invention will now be described by means of the following examples, which should not be construed as in any way limiting. The examples refer to the figures in which:

Figure 1: shows a comparison of Ex- vivo summed 85 A peptide pool PBMC Elispot responses From subjects vaccinated with MVA85A vs the aerosol or intra dermal route;

Figure 2: shows a comparison of the BAL ICS Antigen 85A CD4+ T cell responses between two human patient groups vaccinated by the aerosol or intradermal route with the MVA85A vaccine. Levels of Antigen-85A specific CD4+ T cells containing interferon gamma (IFNy), tumour necrosis factor (TNF), interleukin (IL) 2 and IL17 were all higher in the

bronchoalveolar lavage fluid in subjects immunised by the aerosol route than in subjects immunised by the intradermal route. All these cytokines are considered important in vaccine- induced protection against TB.

Figure 3: shows a comparison of the Whole blood ICS Antigen 85 A CD4+ T cell responses between two human patient groups vaccinated by the aerosol or intradermal route with the MVA85A vaccine. Levels of Antigen-85A specific CD4+ T cells containing interferon gamma (IFNy), interleukin (IL) 2 and IL17 were all higher in whole blood in subjects immunised by the aerosol route than in subjects immunised by the intradermal route. All these cytokines are considered important in vaccine-induced protection against TB.

Figure 4: : shows a comparison of several multifunctional CD4+ T cell subsets detected in BAL after aerosol or intra dermal vaccination with MVA85A. Higher levels of several multifunctional T cell subsets considered important in protection against M.tb were higher in the BAL after aerosol than intradermal vaccination

Figure 5: shows a comparison of several multifunctional CD4+ T cell subsets detected in Whole blood after aerosol or intra dermal vaccination with MVA85A. Higher levels of several multifunctional T cell subsets considered important in protection against M.tb were higher in the blood after aerosol than intradermal vaccination

Figure 6: shows a comparison of the BAL and whole blood Antigen 85A CD8+ T cell responses between two groups of human subjects vaccinated by the aerosol or intradermal route with the MVA85A vaccine. Seven days after vaccination, levels of IFNy+ CD8+ T cells were higher in the BAL than in whole blood after both aerosol and intradermal

administration. In the whole blood, levels were higher after aerosol than intradermal administration.

Figure 7: shows a comparison of anti- Vaccinia IgG levels measured in the serum samples between two human patient groups vaccinated by the aerosol or intradermal route with the MVA85A vaccine. Figure 8: shows a comparison of serum IgG levels measured in the serum samples between two human patient groups vaccinated by the aerosol or intradermal route with the MVA85A vaccine.

Figure 9: shows a comparison of various vaccination strategies on CFU in lungs and spleen of mice. Boosting BCG with intranasal ChAdOxl .85A followed by intranasal MVA85A is significantly more protective than BCG alone in mice. Example 1: Immunogenicitv of a candidate TB vaccine, MVA85A, delivered by aerosol to the lungs of macaques

Tuberculosis (TB) is a re-emerging disease. The only available vaccine, BCG, is delivered intradermally and confers highly variable efficacy against pulmonary disease. There is an urgent need for improved vaccination strategies. Murine studies suggest immunisation delivered directly to the respiratory mucosa may be a more effective route of vaccination.

This study compared the immunogenicity of a leading candidate TB vaccine MVA85A in rhesus macaques, delivered either as an aerosol or as an intradermal boost immunisation 12 weeks after an intradermal BCG prime. Aerosol vaccination was well tolerated. MVA85A delivered by aerosol or by intradermal injection induced antigen-specific immune responses, in the periphery and the lung, with a trend for the highest response when the compartment and route of delivery were matched. The ability of poxvirus-vectored vaccines delivered by the systemic route to induce responses in the mucosal immune compartment in macaques is in contrast to the independent compartmentalisation of mucosal and systemic immune systems described in mice. Unlike intradermal vaccination, aerosol vaccination did not induce a detectable serum anti-vector antibody response.

Introduction

Tuberculosis (TB) in humans is caused by infection with Mycobacterium tuberculosis (M. tb), and is one of the leading global causes of death from a single infectious agent, with an estimated 8.8 million new cases worldwide and 1.77 million deaths in 201 1. The only licensed vaccine against TB is Bacille Calmette Guerin (BCG), a live attenuated strain of Mycobacterium bovis, which was first introduced in 1921 and remains in routine use around the globe. During this time it has been administered to several billion people, predominantly delivered by intradermal injection. Whilst BCG is well tolerated, it is not effective in all populations, does not prevent infection in high burden settings, and is contra- indicated in immunocompromised patient ^s .

One leading approach for the development of a more effective vaccination regimen is to incorporate BCG or a BCG replacement vaccine into a heterologous prime-boost strategy with a subsequently administered booster vaccine. Modified vaccinia Ankara (MVA) is a safe, replication-defective, viral vector, capable of inducing both cellular and antibody immunity to target antigens with protective potential. The most clinically advanced "boost" candidate is the viral-vectored subunit TB vaccine, MVA85A, which expresses the highly conserved, immunodominant mycobacterial antigen, 85A. Experiments in guinea pigs, cattle, and non-human primates have shown that a prime-boost schedule of vaccination with BCG followed by systemically administered (intradermal) MVA85A-boost induces cellular immunity and can improve protective efficacy against subsequent challenge with M.tb or M. bovis, compared to BCG alone. It is well tolerated and highly immunogenic in healthy adults, adolescents, children and infants and in HIV- and M. tb- infected adults, and is currently being evaluated in a BCG-prime MVA85A-boost regimen in a large phase lib efficacy trial in infants in South Africa.

Conventionally, both BCG and MVA85A have been administered by intra-dermal (i.d.) injection, a route of inoculation that has been shown to induce strong mycobacterial-specific immune responses in the periphery. However, the profile of responses induced at mucosal surfaces has yet to be defined. Furthermore, there is now a general recognition that the mucosal associated lymphoid tissue (MALT) should be considered as an immune

compartment separate from the peripheral system, and that triggering immune responses in the MALT may lead to increased protection against pulmonary TB. Vaccine delivery directly to the lung by aerosol also brings the advantages associated with needle-free delivery that include speed and simplicity of immunisation, which allows application by non-medical personnel and, as delivery is non invasive and pain free, it brings great social acceptance.

The route of vaccine delivery determines the location of antigen-specific cells and thus the protective efficacy of the vaccine. Evidence from animal studies suggests that the prevalence of antigen-specific cells in the airway lumen is important for optimal protection against TB. Replication-deficient vaccinia and adenoviral vectors encoding antigen 85A demonstrated better protection when delivered to the murine respiratory mucosa than when administered systemically.

Protection from TB is critically dependent on the cellular immune response, in particular CD4+ and also probably CD8+ T cell mediated cellular responses, the mechanisms of which are not fully elucidated. The main, immunological readout used in TB vaccine studies to date has been the measurement of antigen-specific interferon-gamma (IFNy) in one of several immunological assays. Whilst there is strong evidence to support an essential role for IFNy in protective immunity against M.tb , it may not be sufficient alone. There are other cytokines, such as TNFa, which are known to be important, and there are also other functions of T cells which may be involved. Studies investigating viral and other intracellular pathogens have highlighted the importance of T-cell functionality in controlling these infections. Moreover, the presence of antigen-specific polyfunctional CD4 and CD8 T-cells, expressing

combinations of IFN-γ, tumour necrosis factor alpha (TNFa), and interleukin 2 (IL-2), has been correlated to a positive clinical outcome in HIV/TB co-infection and murine Leishmania major infection. In addition, it has been demonstrated that these polyfunctional cells are induced following vaccination with BCG and the novel candidate TB vaccine, MVA85 A

MATERIALS AND METHODS

Verification of viral viability following aerosolisation by mesh nebuliser

Studies were performed to assess the viability of MVA85A following aerosolisation with the Omron MicroAIR hand-held, vibrating mesh nebuliser. The mouthpiece attached to the nebuliser was connected by a sealed system to a collection vessel in which aerosolised MVA85A was condensed. The viability of the batch of clinical grade MVA85 A selected for use in the macaque study after aerosolisation was compared alongside an identical non- aerosolised control sample. 2.7x10 ⁷ plaque forming units (pfu) of MVA85A were diluted in 1ml PBS, nebulised using the MicroAIR device and collected using an impinger. The viability of MVA85A after aerosolisation was evaluated by plaque assay, using chicken embryo fibroblast (CEF) cells.

The reproducibility of different mesh nebulisers was determined by comparison of the time taken to aerosolise 1 ml of sterile PBS. Plaque assay for MVA85A pfu determination method for biodistribution and in-vitro studies

Briefly, tissue samples were thawed and homogenised in 2ml 2% FCS DMEM (Invitrogen, D6546) medium using GentleMacs M tubes on a Dispomix (programme 14), followed by 2 cycles of snap freeze/thaw in dry ice/isopropanol to release any viral particles from within the organ tissue. Samples were spun to remove as much tissue debris as possible before being plated out on sub-confluent low passage (P3) CEF cells in a 96 well plate and incubated for 96 hours. Supernatant was then plated onto a second set of CEF cells in poly-lysine coated 96 well plates to allow further viral replication for 2 days before fixing cells with methanol. Cells were immunostained with rabbit anti-vaccinia primary antibody (ISL 126-1063) and anti-rabbit HRP conjugate secondary antibody (Donkey anti-rabbit-HRP conj antibody, Amersham NA934V), for the presence of MVA. Detection was by DAB chromogenic staining (Vector laboratories ImmPACT DAB chromogenic substrate, SK-4105). Reference MVA85A virus at a low pfu concentration (2 x 10 ³ pfu/ml) was used as a positive control in both rounds of amplification, with non-infected cell supernatants and cells used as negative controls. An additional control of inguinal lymph node material from each test subject, spiked with a known amount of MVA85 A (80 μΐ at 2 x 10 ⁴ pfu/ml added to 720 μΐ INL sample, 100 μΐ per well) was also used.

Experimental animals

The animals used in this study were rhesus macaques of Indian origin obtained from an established UK breeding colony. All animals were 4.5 to 5 years old at the time of vaccination and naive in terms of prior exposure to mycobacterial antigens (M tb infection or environmental mycobacteria) as demonstrated by a negative tuberculin test whilst in their original breeding colony and by the IFN-γ based Primagam™ test kit (Biocor, CSL, USA) just prior to study start. Monkeys were housed according to the Home Office (UK) Code of Practice for the Housing and Care of Animals Used in Scientific Procedures (1989) and the National Committee for Refinement, Reduction and Replacement (NC3Rs) Guidelines on Primate Accommodation, Care and Use, August 2006. They were sedated by intramuscular (i.m.) injection with ketamine hydrochloride (lOmg/kg) (Ketaset, Fort Dodge Animal Health Ltd, Southampton, UK) for all procedures requiring removal from their cages. All protocols involving animals were approved by the Ethical Review Committee of the Health Protection Agency, Porton, UK. None of the animals had been used previously for experimental procedures

Vaccination

Eight animals (groups A and B) were immunised intradermally in the upper left arm with 100 μΐ BCG, Danish strain 1331 (SSI, Copenhagen, Denmark). Twelve weeks after immunisation with BCG, four animals (Group A) were immunised intradermally in the upper right arm with

100 μΐ (1 x 10 pfu) MVA85A. Four animals (Group B) were exposed to an aerosol containing MVA85A created by nebulisation of 10 ⁸ pfu in 1 ml sterile PBS using an Omron MicroAir mesh nebuliser (Omron Healthcare UK Ltd, Milton Keynes, UK) and delivered to the nose of each sedated primate via a modified paediatric anaesthesia mask.

15

SUBSTITUTE SHEET RULE 26 Skin vaccination sites were monitored and assessed for local reactions after vaccination with BCG and MVA85A.

Clinical assessment

The primary purpose of the study was to assess tolerability using the following readouts. Animal behaviour was observed daily throughout the study for contra-indicators such as depression, withdrawal from the group, aggression, reduced food and water intake, changes in respiration rate, or cough. Animals were sedated every two weeks to measure weight, body temperature, blood haemoglobin levels and erythrocyte sedimentation rate (ESR), and to collect blood samples for immunology. Blood cell haemoglobin was measured using a HaemaCue haemoglobinometer (Haemacue Ltd, Dronfield, UK) and ESR was measured using the Sediplast system (Guest Medical, Edenbridge, UK). Chest X-rays were collected prior to BCG vaccination, 2 weeks prior to vaccination with MVA85A, and 1 and 8 weeks after MVA85A vaccination.

Bronchoalveolar Lavage (BAD

To determine the immune response in the lung, BAL samples were collected using a bronchoscope (All-Scope XE30 4mm flexible bronchoscope, VES, Southend on Sea, Essex. SS2 5BZ ) on two occasions four weeks apart prior to BCG vaccination and at weeks 4 and 8 after BCG vaccination and 1, 3, and 7 weeks after the MVA85A booster vaccination. On each occasion three consecutive washes were performed, each using 20 ml volumes of Hanks balanced salts (Sigma- Aldrich, Dorset, UK) instilled into the lungs, and collected.

Interferon-gamma (IFN-γ) ELISpot Assay

Peripheral blood mononuclear cells (PBMC) were isolated from heparin anti-coagulated blood, and lymphoid mononuclear cells from tissue samples, by Ficoll-Hypaque Plus (GE Healthcare, Buckinghamshire, United Kingdom) density gradient separation using standard procedures. An IFN-γ ELISpot assay was used to estimate the numbers and IFN-γ production capacity of mycobacteria-specific T cells in PBMC using a human/monkey IFN-γ kit (MabTech, Nacka. Sweden) as previously described ⁽²⁹⁾ In brief, PBMCs were cultured with 10 μg/ml PPD (SSI, Copenhagen, Denmark), or a pool containing overlapping 15mer peptides spanning Ag85A (Peptide Protein Research Ltd, Wickham, UK), or without antigen, in triplicate, and incubated for 18 hours. Phorbol 12-myristate (Sigma- Aldrich Dorset, UK) (100 ng/ml) and ionomycin (CN Biosciences, Nottingham, UK) (1 μg/ml) were used as a positive control. After culture, spots were developed according to the manufacturer's instructions. Determinations from triplicate tests were averaged. Data were analysed by subtracting the mean number of spots in the cells and medium-only control wells from the mean counts of spots in wells with cells and antigen or peptide pools.

Intracellular cytokine staining

PBMCs were thawed, washed, re-suspended in medium (RIO) consisting of RPMI 1640 supplemented with L-glutamine (2 mM), penicillin (50 U/ml)/streptomycin (50 μg/ml) and 10% heat-inactivated foetal bovine serum with 1 U/ml of DNase (Sigma, Poole, UK) and incubated at 37°C for 2 hours. BAL cells were isolated from BAL fluid by centrifugation (400g for 5 minutes) and assessed directly following isolation. Cell concentrations were adjusted to 1 x 10 ⁶ cells/ml in R10 and stimulated with 10 μg/ml PPD (SSI, Copenhagen, Denmark). Intracellular cytokine staining to evaluate the production of the cytokines, IFNy, TNFa, and IL-2, was performed as previously described

Flow cytometric acquisition and analysis.

Cells were analysed using a 4b SORP LSRII (BD Biosciences, Oxford, UK). Cytokine- secreting T cells were identified using a forward scatter-height (FSC-H) versus side scatter- area (SSC-A) dot plot to identify the lymphocyte population, to which appropriate gating strategies were applied to exclude doublet events, non- viable cells, monocytes (CD14 ⁺ ) and B cells (CD20 ⁺ ) prior to sequential gating through CD3 ⁺ , CD8 ^" and CD4 ⁺ versus IFNy, and CD3 ⁺ , CD8 ⁺ and CD4 ^" versus IFNy histograms. Data were analysed using FlowJo v8.8.6 before further manipulation using the programs PESTLE (vl.6.2) and SPICE (v5.0) to generate graphical representations of T-cell responses using background-subtracted flow data (Mario Roederer, Vaccine Research Centre, NIAID, NIH).

Vaccinia antibody ELISA

Anti- vector IgG titres were measured in serum samples by vaccinia-specific direct ELISA assay. In brief, Nunc maxisorb 96 well plates were coated with inactivated Lister strain vaccinia (Autogen Bioclear). After blocking, samples were diluted 1/25 in assay diluent/blocking buffer (PBS + 5% skimmed milk powder + 0.1% tween-20) and added in duplicate. A sample dilution series was created by 4 fold serial dilution. A reference curve was created by inclusion of a polyclonal anti- vaccinia virus human immunoglobulin (VIG) control sample (NR-650, Biodefense and Emerging infections research resource repositories, USA), serial diluted in parallel. Serum and VIG control samples were incubated at room temperature for 2 hours. After washing, plates were incubated for 2 hours with anti-monkey IgG peroxidise (Insight Biotechnologies), washed, and then developed using ABTS peroxidise substrate system (Insight Biotechnologies). Absorbance at 405nm was then measured using a Versamax plate reader with Softmax Pro software. A VIG control curve was plotted for each plate and the inflection point used to interpret the serum sample IgG titre.

Necropsy

Before necropsy, animals were sedated with ketamine (15 mg/ml, i.m.), weighed,

photographed, chest X-rays taken, clinical data collected and ex-sanguination was effected via the heart, before termination by injection of a lethal dose of anaesthetic (Dolelethal, Vetoquinol UK Ltd, 140 mg/kg). A full necropsy was performed and gross pathology assessed. Samples of spleen, liver, kidneys and hilar, inguinal and axillary lymph nodes, tonsil, brain and olfactory bulb, heart & pericardium, samples from the small intestine (ileum, jejunum, duodenum), colonic lymph nodes, transverse colon, mesenteric lymph nodes, eye, nasal turbinate (right side) and the upper left lung lobe were removed, dissected on sterile trays and placed into formalin buffered saline for histology. All other lung lobes were collected for assessment of immune responses.

Pathology / Histology

Representative sections from all tissues described above, were processed to paraffin wax, sections cut at 5 μπι, and stained with haematoxylin and eosin (H&E) for examination microscopy.

Statistical analyses

To compare the immune responses measured by ex-vivo Elispot assay in animals by vaccination group, the area under the curve (AUC) for each response was calculated using Sigmaplot version 10 (Systat Software Inc, Hounslow, UK) for each animal. The area under the curves calculated for the animals in each test group were compared to those for the animals in the other test group with a Mann Whitney test using Minitab, version 15 (Minitab Ltd, Coventry, UK).

To compare T-cell functional profiles measured by polyfunctional flow cytometry between vaccination groups, the frequency of each functional subset (triple, dual and monofunctional cytokine producing CD4 or CD8 T-cells) was compared using a Mann- Whitney test at each analysis time point.

Similarly, vaccine-induced changes in the T-cell functional profile, within each vaccination group, were assessed by comparing the frequency of functional subsets at each analysis time point with median baseline values.

RESULTS

In vitro studies to optimise delivery of MVA85A through the mesh nebuliser

Initial studies demonstrated that the aerosolisation process did not adversely affect the viability of MVA85A; and showed the level of viable MVA85A collected post-aerosolisation was not significantly different from that determined in the vaccine preparations prior to aerosolisation. Nebulisation rates were found to be highly reproducible between consecutive runs and between four different mesh caps and different devices. To ensure optimal delivery of the aerosol, a range of paediatric masks were tested for use with rhesus macaques. The best fit type and make was enhanced using a spacer to fill the gap under the chin. Based on the results from the optimisation studies, the strategy adopted for vaccination of rhesus macaques in the immunogenicity study used separate, pre-prepared, mesh caps and the same device for all vaccinations.

Safety of aerosol delivered MVA85A

Vaccination did not lead to perturbations in any of the clinical parameters measured. Chest radiographs collected through the study demonstrated normal pulmonary structure which remained unchanged post- vaccination. Seven of the eight animals developed local reactions at the site of BCG vaccination 2 and 4 weeks post- vaccination, with reactions still visible in 3 animals at the end of the study. Intradermal vaccination with MVA85A induced reactions at the vaccination site one week post-immunisation in all 4 animals which resolved 3 weeks later. At the end of the study, 9 weeks after vaccination with MVA85A, gross pathology and histological assessments did not identify any adverse effects induced by vaccination. Overall, aerosol vaccination was well tolerated by all animals and caused no adverse effects.

Distribution of MVA in tissues No viable virus was detected in any of the NHP tissue samples provided, whether

administered by id or aerosol route

Evaluation of the T-cell response induced by each route of vaccination in peripheral blood The systemic profiles of immune responses induced following immunisation with BCG and MVA85A by aerosol and intradermal injection were characterised. An increased frequency of PPD-specific IFNy secreting CD4+ T cells was detected by ex-vivo ELIspot in all 8 animals following vaccination with BCG. After intradermal boosting with MVA85A, an increased response was seen in one animal but not in any of the animals after aerosol MVA85A administration. Responses to the Ag85A peptides were not detectable after BCG vaccination. After boosting with MVA85 A, high responses were detected in two of the four animals in each immunisation group, one week post-MVA85A vaccination, which contracted over the subsequent 3 weeks as expected. Systemic immune responses in the 2 responding animals receiving intradermal MVA85A were higher than responses in the two responding animals in the aerosol administered group, these differences did not reach statistical significance (P=0.665). Multiparameter flow cytometry was performed to evaluate the production of the cytokines ΓΡΝγ, TNFa and IL-2, in stimulated PBMCs isolated throughout the in vivo time- course. After intradermal MVA85A vaccination, there was an increase in the double

(TNFa+IFNy+) and triple positive (TNFa+IFNy+IL-2+) populations of antigen specific CD4+ T cells which was not seen in the aerosol administered group. This population contracted but was still detectable 4 weeks after vaccination. Differences between the groups were not statistically significant. Interestingly, at 8 weeks post MVA85A vaccination, there was a population of double positive antigen-specific CD4+ T cells in both aerosol and intradermally vaccinated animals.

Dual and triple positive CD8 T cell populations were not induced following BCG or

MVA85A vaccination, and single CD8+ T cell populations were only detected sporadically and at low frequencies.

20

SUBSTITUTE SHEET RULE 26 Evaluation of the T-cell response in bronchoalveolar lavage (BAL) induced by vaccination

BCG vaccination induced significant increases in both the proportion of PPD-specific CD4+ T cells in the BAL capable of producing more than one cytokine and the frequency of specific triple, dual, & single positive CD4+ T-cells from 4 weeks after BCG vaccination compared to pre-vaccination levels. Similarly the frequency of PPD-specific CD4 T cell populations peaked 1 week after vaccination with MVA85A (week 13), then declined.

Vaccination induced a number of differences in population frequency and proportion although none significantly different. An increase in the proportion of double (TNFa+IFNy+) and triple positive (TNFa+IFNy+IL-2+) PPD-specific cells compared to pre-vaccination levels was detected 4 weeks after BCG vaccination and was maintained throughout the study. One week after MVA85A vaccination, a higher frequency of PPD-specific single positive IFNy-producing CD4 T cells, as well as a larger proportion of double (TNFa+IFNy+) and triple positive (TNFa+IFNy+IL-2+) PPD-specific cells, was seen in the group that received MVA85A by aerosol compared to the intradermally vaccinated group (Figure 3D). An increase in the frequency of single positive IFNy-producing CD8+ T cells was detected 8 weeks post-BCG that further increased 1 week post-MVA85A (week 13 in both aerosol and intradermally vaccinated animals). Vaccination did not alter the proportion of triple, dual or single positive PPD-specific CD8+ T cells. A small dual positive (IFNy+; TNFa+) population was seen in the aerosol vaccinated group at weeks 8 and 13 that was not detectable in the intradermally vaccinated group.

Evaluation of the T-cell response induced by each route of vaccination in lymphoid tissues

The frequencies of PPD and Ag85A-specific IFNy secreting cells were measured by ELIspot in a range of lymphoid tissues (spleen, bone marrow lung, hilar, and axillary inguinal lymph nodes) collected at necropsy. Responses detected in the tissues from animals that received the MVA85A by aerosol were comparable to those vaccinated intradermally (data not shown). However, there was a non-significant trend for the Ag85 A-specific responses in the lungs from the aerosol group to be higher than those detected in the lungs of the intradermal group (p = 0.6625). Furthermore, the median response in the lung of animals vaccinated by aerosol was approximately 6 fold higher than the response in the peripheral blood. In contrast, the response in the peripheral blood of the animals vaccinated intradermally was almost 6 fold higher than in the lung. Evaluation of the humoral response to the vaccinia vaccine vector induced by each route of vaccination in peripheral blood.

Vector-specific IgG responses were detected in the serum collected from animals after intradermal vaccination with MVA85A. Responses were first detected 2 weeks after vaccination with the peak increase in response from pre- vaccination levels detected 4 weeks post vaccination. In contrast, an anti-vaccinia antibody response was not detected in the serum from animals after aerosol vaccination with MVA85A. (Figure 7)

DISCUSSION

It would be advantageous for a vaccine to induce immune responses at the site where the pathogen is first encountered, to allow a faster local response at the site where it can be most effective. Importantly, in this study the MVA85 A was well tolerated by all the animals, with no adverse effects on any of the clinical parameters tested and thus aerosol vaccination with MVA85A was shown to be similar to that previously reported for other MVA- and NYVAC- vectored vaccines. This study has shown that aerosol vaccination with MVA85A was well tolerated and immunogenic. MVA85A delivered by aerosol or by intradermal injection induced antigen- specific immune responses both in the periphery and the lung, with a trend to the highest response when the compartment and route of delivery were matched. The induction of responses in both the systemic and mucosal immune compartments following vaccination by either the systemic, or mucosal route, is in line with the report from Corbett et al , where aerosol delivery of a NYV AC -vectored HIV vaccine to rhesus macaques induced both systemic and mucosal-associated immune responses in the vaginal and rectal tissues that were comparable to those induced by intramuscular immunisation. The ability of poxvirus- vectored vaccines delivered by the systemic route to induce responses in the mucosal immune compartment in macaques is in contrast to the observations made in mice, where the independent compartmentalisation of the mucosal versus systemic immune systems has been clearly described In the mouse study, systemic immunisation with vaccinia induced cellular and antibody responses only in the systemic lymphoid tissue and not in the mucosal sites.

Live viruses such as vaccinia and adenovirus are widely used as delivery systems in the development of new vaccines; however, their use can be limited by pre-existing immunity induced either by prior natural infection or by previous vaccination. Approaches that could circumvent previous immunity would be extremely useful in the development of improved vaccine strategies. In this study an antibody response to the MVA vector was detected in the serum of animals that received the MVA85A by intradermal injection, but not in the serum of animals which were vaccinated with MVA85A by aerosol. This suggests that delivery of vaccines to the lung provides an immunisation strategy which limits the induction of systemic anti-vector immunity and provides the potential for multiple vaccinations with poxvirus based strategies using mucosal followed by systemic delivery. This study demonstrated the feasibility of delivering a booster vaccination to the mucosal immune system by aerosol and, as mucosal administration of vaccinia vectored vaccines has been previously shown to improve the immunogenicity of vaccinia-vectored vaccines , this also provides the potential to enhance the immunogenicity induced by a prime / boost vaccine regimen. Repeated vaccination with the same vaccine in multiple boost regimens would in turn reduce the need for multiple formulations of the same vaccine antigens for prime/ boost vaccination regimens which would reduce the time and cost of development. Use of the same vector system to deliver vaccines against different diseases would also become feasible without one vaccine limiting the immunity induced by the other. In addition, this approach could also allow the use of vaccine vectors such as AdHu5, whose use has previously been limited because they are commonly encountered and induce immunity in large sectors of the population.

Example 2: Human study of aerosol delivery of MVA85A vaccine as compared to intradermal vaccination

Inhaled aerosol delivery was performed using the MicroAir U22 nebuliser (Omron) which is a commercially available battery-operated hand-held lightweight (140 g including batteries) ultrasonic mesh nebuliser (Omron, 2012). This device is silent and generates a mist with a particle size of between 2 and 4 μιτι from fluid placed within the nebuliser chamber.

Participants hold the device and inhale the aerosol through a mouthpiece at their normal relaxed respiratory rate and tidal volume, effectively self-administering the vaccine. Alternatively if the subject is unable to self-administer the vaccine the device may be held by an assistant while the (vaccinated) subject inhales the aerosol. The vaccine dose is diluted into a total volume of 1 mL with 0.9% saline which aids nebulisation. Samples:

Bronchoalveolar Lavage (BAL):

Cells separated and stimulated for flow Cytometry analysis (Day 7).

Whole Blood (WB): Blood stimulated, fixed and frozen for later flow Cytometry analysis (Days: 0, 7, 14,

28 & 168).

Peripheral Blood Mononuclear Cells (PBMC):

Ex-vivo ELISpot and cells frozen for later analysis (Days: 0, 7, 14, 28, 84 & 168).

HUMAN ELISA methods:

Insert- and vector-specific humoral antibody (IgG, IgA and IgM) responses were assessed by Enzyme Linked Immunosorbent Assay (ELISA) in serum samples. Nunc Immunoplates were coated with r85A or MVA and incubated overnight for adsorption. The next morning samples were prepared by diluting test serum 1:10 in Casein blocking buffer. Coating solution was discarded, plates washed 6 times with PBS/Tween and blocked with 200ul of Casein per well for 1 hour at room temperature. Blocking solution was discarded; samples were added at 50ul per well and incubated at room temperature for 2 hours. Contents were discarded and plates washed 6 times with PBS/Tween. 50ul of 1/100 diluted goat anti-human secondary antibody conjugated to alkaline phosphatase (Sigma) was added and incubated for 1 hour. Plates were washed with PBS/Tween and substrate: 4-nitrophenylphosphate tablet in diethanolamine buffer was added. OD values were read at 405 run at 30 minutes for antigen 85 A and 10 minutes for MVA.

Results : 1. Aerosol and intradermal administration induce comparable levels of antigen 85 A specific T cell responses as measured by ex-vivo ELISpot assay (Fig 1)

2. Aerosol administration induces significantly higher levels of antigen 85A specific T cells in the BAL containing IFNy, IL2, TNF and IL17, compared with intradermal administration (Fig 2) 3. Aerosol administration induces higher levels of antigen 85 A specific CD4+ T cells containing IL2, TNF and IL17 in the blood, compared to intradermal vaccination. (Fig 3)

4. Aerosol administration induces higher levels of Antigen 85A specific multifunctional CD4+ T cells in the BAL compared with intradermal administration (Figure 4)

5. Aerosol administration induces higher levels of Antigen 85A specific multifunctional CD4+ T cells in the blood compared with intradermal administration (Figure 5)

6. Aerosol administration induces higher levels of antigen 85A specific CD8+ T cells in the blood than intradermal administration. (Figure 6)

7. After intradermal administration of MVA85A, a significant rise in serum IgG to the MVA vector was demonstrated. In contrast, after aerosol administration, no induction of MVA IgG was detected. (Figure 8)

Both aerosol and ID MVA-85A (lxl0 ⁷ pfu) vaccination of BCG- vaccinated humans induces a significant Antigen 85A specific T cell response as detected in ex-vivo ELISpot assay, when compared to baseline, prevaccination samples. The magnitude of this response is comparable between the two routes. Aerosol-induced response may contract more quickly than those induced by the ID route of vaccination, as measured with this assay.

25

SUBSTITUTE SHEET RULE 26 Week-1 Antigen &5 i-specific IFN-g made by PBMC from volunteers vaccinated by inhalation of 1x10 pfu MVA-85A (Figure 1) is comparable to that made by cells from individuals vaccinated intradermally by a higher dose (1x10 pfu) of MVA-85A. The immunogenicity of the intradermal administration of MVA85A at 1x10 pfu has been reported (Pathan et al. Vaccine. 2012;30:5616-24) Furthermore, it is observed (figures 2 - 5) that

• In the aerosol group, more BAL antigen 85A specific CD4+ T cells make IFN-γ, TNF-a, IL-2 and IL-17 as compared to the ID group.

• Multiple cytokines are made by CD4+ T cells from BAL and WB of volunteers vaccinated intradermally or by inhalation of MVA-85A.

• More CD8+ IFN-g is detected in BAL cells compared to peripheral blood.

These results suggest aerosol administration of MVA85A and therefore other viral vectors may not induce significant levels of anti- vector immunity, which has utility in reusing the same vector and homologous boosting

Example 3; Comparison of immunisation strategies in mice

ChAdOX1.85A was constructed as described in Dicks et al (A Novel Chimpanzee

Adenovirus Vector with Low Human Seroprevalence: Improved Systems for Vector

Derivation and Comparative Immunogenicity2012; PLoS One 7(7) pg e40385)

Six-week old female Balb/c mice were allocated to the following groups:

N- Control

B - BCG prime only

BC - BCG prime followed by boost with ChAdOxl .85 A

BM - BCG prime followed by boost with MVA.85A BCM - BCG prime followed by a first boost with ChAdOxl .85 A, and then a second boost with MVA.85A

BMC - BCG prime followed by a first boost with MVA.85A and then a second boost with ChAdOxl.85 A

C - No BCG prime followed by ChAdOxl .85 A

M - No BCG prime followed by MVA.85A

MC - No BCG prime followed by a first boost with MVA.85A and then a second boost with ChAdOxl.85 A

CM - No BCG prime followed by a first boost with ChAdOxl .85 A, and then a second boost with MVA.85A

The mice were vaccinated with BCG-Pasteur intradermally {Id.) at 4xl0 ⁵ cfu/dose. All groups were boosted 10 weeks after BCG priming. Both MVA.85A and ChAdOxl.85 A were administered intranasally, 25μ1/ηο8ΐπ1, at 5x10 pfu and 1x10 ifu respectively. The BCM and BMC group had a four week interval between the first boost and second boost. Four weeks after the last immunisation (apart from the BCG group who had 16 weeks between the last vaccination and challenge), mice were challenged with M.tb. Mice were challenged with 50-100 cfu of aerosolised M.tuberculosis Erdman K01 strain using Biaera AeroMP- controlled nebuliser. Four weeks after challenge lungs and spleens were removed, homogenised, and plated on Middlebrook 7H11 plates with glycerol and enriched with OADC. Statistical test used: Mann- Whitney, *p<0.05.

As shown in Figure 9, without a prior BCG prime, all groups (MC, CM, M, C) significantly decreased the bacterial load in the lung compared to naive animals. In contrast, when used as boosts to BCG, ChAdOxl.85 A (BC) and MVA85A (BM) did not improve the 2-loglO protection of BCG. Although BMC decreased the bacterial load, compared to BCG, this did not reach significance. The best protection was provided by the BCM group, significantly decreasing the bacterial load by 0.61ogl0, compared to the BCG group. In the spleen, without a prior BCG prime, only the CM group has significantly lower bacterial load compared to naive animals. Similar to the lung data, ChAdOxl.85 A (BC) and MVA85A (BM) did not improve BCG protection, however both BCM and BMC groups had significantly lower bacterial loads in the spleen compared to the BCG vaccinated group. In conclusion, the B-C-M regime was the most promising vaccination regime, significantly improving BCG protection in both lungs and spleens.