WO2021243417A1 - Immunogenic formulations - Google Patents

Abstract

Disclosed are methods for inducing and generation of neutralising antibodies against SARS-CoV-2 in a subject, comprising administering to the subject an immunogenic formulation comprising an attenuated Mycobacterium bovis (BCG) strain and at least one antigen from SARS-CoV-2. Specifically, in addition to the attenuated Mycobacterium bovis (BCG) strain and the spike protein from SARS-CoV-2, the immunogenic formulations can also comprise an additional adjuvant such as aluminium hydroxide, delta inulin, or a combination of delta inulin and CpG dinucleotides (Advax).

Description

IMMUNOGENIC FORMULATIONS

Field of the Art

[0001] The present disclosure relates to immunogenic formulations for use against pathogenic infections, including viral infections such as those of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and to methods for treating and preventing such infections, in particular by inducing the generation of neutralising antibodies.

Background

[0002] New approaches are constantly needed to combat the emergence of novel pathogenic infections and diseases representing serious threats to human and animal health. Of particular concern are viruses causing respiratory disease given the potential of rapid mass transmission and of epidemics and pandemics occurring.

[0003] Coronaviruses are a large family of enveloped single-stranded RNA viruses often causing acute disease in the upper and lower respiratory tract. The coronavirus envelope generally includes three membrane proteins: the spike protein (S), the membrane protein (M) and the envelope protein (E). Some coronaviruses also have a hemagglutinin-esterase protein (HE) in their envelope. The spike protein is responsible for binding of the virus particles to their cellular receptors, mediating membrane fusion and virus entry into the cell.

[0004] Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002-2003 causing acute respiratory distress syndrome with 10% mortality (up to 50% mortality in aged individuals). Middle Eastern respiratory syndrome coronavirus (MERS-CoV) emerged in the Middle East in April 2012, manifesting as severe pneumonia, acute respiratory distress syndrome and acute renal failure.

[0005] More recently, in December 2019 a novel coronavirus emerged in China, designated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Extremely infectious, SARS- CoV-2 infected more than six million people globally in less than five months, causing more than 380,000 deaths worldwide, and more than 100,000 in the United States alone. The clinical spectrum of the respiratory disease caused by SARS-CoV-2, COVID-19, varies from asymptomatic to severe clinical manifestations characterized respiratory failure (acute respiratory distress syndrome) necessitating ventilation support in an intensive care unit, acute lung inflammation, sepsis, septic shock and multiple organ dysfunction syndrome.

[0006] In the rush to develop a vaccine against SARS-CoV-2, numerous approaches are being employed, including DNA and RNA vaccines, inactivated/attenuated virus, recombinant vectors and protein in adjuvant. A similarly broad suite of approaches was used for SARS-CoV vaccine development, however in some cases candidates induced severe eosinophilic lung pathology, an effect that was also seen with vaccines for other coronaviruses such as MERS- CoV and is associated with the induction of Th2 immunity. Only two of nine COVID-19 candidate vaccines currently in human trials use technology previously approved for licenced human vaccines. Thus in an attempt to accelerate vaccine development there remains uncertainty over the safety and efficacy of lead candidates. Additionally, the requirement for multiple doses for most approved vaccines is a barrier to rapid, mass vaccination and has necessitated changes in dosing schedules in some countries to ensure sufficient vaccine coverage.

[0007] Currently, two predominant SARS-CoV-2variants of concern, B.1.1.7 and B.1.351, both have increased transmissibility and antibodies from convalescent patients and vaccine recipients have reduced capacity to neutralize B.1.351. Vaccination with one of the most widely deployed vaccines, ChAdOxl nCoV-19, does not protect against mild-to-moderate COVID-19 due to the B.1.351 variant in South Africa.

[0008] There is a clear and urgent need for the development of clinically effective vaccine compositions to combat coronavirus infections such as SARS-CoV-2.

[0009] The ability to induce the generation of neutralising antibodies is an important feature of vaccines against a variety of pathogens, and this is likely to also be a desirable feature in a successful vaccine against SARS-CoV-2. However in the case of many pathogens, it remains a challenge in the art to develop vaccines capable of inducing high levels of neutralising antibody production.

Summary of the Disclosure

[0010] A first aspect of the present invention provides a method for inducing the generation of neutralising antibodies against a pathogen in a subject, comprising administering to the subject an immunogenic formulation comprising an attenuated Mycobacterium strain and at least one antigen from said pathogen.

[0011] The pathogen may be a virus, bacterium, fungus or parasite. The viral pathogen may be, for example, a coronavirus, a flavivirus, human immunodeficiency virus (HIV), respiratory syncytial virus (RSV) or influenza. The bacterial pathogen may be, for example, Chlamydia trachomatis or Pseudomonas aeruginosa. The parasitic pathogen may be, for example, Plasmodium falciparum, fluke or hookworm.

[0012] In an exemplary embodiment, the virus is a coronavirus, such as SARS-CoV-2. [0013] The attenuated Mycobacterium strain may express at least one protein from the pathogen, or an antigenic fragment thereof.

[0014] Typically the attenuated Mycobacterium strain comprises or is derived from the Bacille Calmette-Guerin (BCG) strain.

[0015] The immunogenic formulation may further comprise at least one adjuvant. In exemplary embodiments the adjuvant may comprise aluminium hydroxide, delta inulin or a combination of delta inulin and CpG dinucleotides.

[0016] A second aspect of the present invention provides an immunogenic formulation for inducing an immune response to a coronavirus or for preventing or treating coronavirus infection, the formulation comprising an attenuated Mycobacterium strain and at least one coronavirus antigen.

[0017] In a particular embodiment, the coronavirus is a severe acute respiratory syndrome- related coronavirus. In an exemplary embodiment, the coronavirus is SARS-CoV-2.

[0018] Typically the attenuated Mycobacterium strain comprises or is derived from the Bacille Calmette-Guerin (BCG) strain.

[0019] Typically at least one coronavirus antigen comprises a surface glycoprotein of the coronavirus, or an antigenic fragment thereof. In a particular embodiment the surface glycoprotein is a coronavirus spike protein. In an exemplary embodiment the coronavirus spike protein is the spike protein of SARS-CoV-2. In an exemplary embodiment, the spike protein comprises the amino acid sequence of SEQ ID NO:l, or a sequence having at least or about 85% sequence identity thereto.

[0020] A third aspect of the present invention provides an immunogenic formulation for inducing an immune response to a coronavirus or for preventing or treating coronavirus infection, the formulation comprising a recombinant attenuated Mycobacterium strain that expresses at least one coronavirus protein or an antigenic fragment thereof.

[0021] In a particular embodiment at least one coronavirus protein comprises a surface glycoprotein of the coronavirus. In an exemplary embodiment the surface glycoprotein is a spike protein. In an exemplary embodiment the coronavirus spike protein is the spike protein of SARS-CoV-2. In an exemplary embodiment, the spike protein comprises the amino acid sequence of SEQ ID NO:l, or a sequence having at least or about 85% sequence identity thereto. The coronavirus spike protein or antigenic fragment thereof expressed by the recombinant attenuated Mycobacterium strain may be encoded by the nucleotide sequence of SEQ ID NO:4, or a portion thereof, or a sequence having at least or about 85% sequence identity thereto.

[0022] In accordance with the second and third aspects, the immunogenic formulation may further comprise at least one adjuvant. In exemplary embodiments the adjuvant may comprise aluminium hydroxide, delta inulin or a combination of delta inulin and CpG dinucleotides. [0023] In particular embodiments of the second and third aspects, said inducing of an immune response to the coronavirus comprises inducing the generation of neutralising antibodies against the coronavirus in a subject.

[0024] A fourth aspect of the present invention provides a vaccine composition against coronavirus, comprising an immunogenic formulation of the second or third aspect.

[0025] A fifth aspect of the present invention provides a method for inducing an immune response to a coronavirus in a subject, comprising administering to the subject an attenuated Mycobacterium strain and at least one coronavirus antigen.

[0026] The attenuated Mycobacterium strain and at least one coronavirus antigen may be in the same or different compositions. Where the attenuated Mycobacterium strain and at least one coronavirus antigen are in different compositions, the compositions may be administered simultaneously or sequentially, and the compositions may be administered by the same or different routes.

[0027] A sixth aspect of the present invention provides a method for preventing or treating a coronavirus infection and/or a disease or disorder caused by a coronavirus infection, comprising administering to a subject in need thereof an attenuated Mycobacterium strain and at least one coronavirus antigen.

[0028] The attenuated Mycobacterium strain and at least one coronavirus antigen may be in the same or different compositions. Where the attenuated Mycobacterium strain and at least one coronavirus antigen are in different compositions, the compositions may be administered simultaneously or sequentially, and the compositions may be administered by the same or different routes.

[0029] The disease or disorder caused by the coronavirus infection may be COVID-19.

[0030] A seventh aspect of the present invention provides a method for inducing an immune response to a coronavirus in a subject, comprising administering to the subject an immunogenic formulation of the second or third aspect or a vaccine composition of the fourth aspect.

[0031] An eighth aspect of the present invention provides a method for preventing or treating a coronavirus infection and/or a disease or disorder caused by a coronavirus infection, comprising administering to a subject in need thereof an immunogenic formulation of the second or third aspect or a vaccine composition of the fourth aspect.

[0032] In particular embodiments of the methods of the fourth to the eighth aspects, said inducing of an immune response to the coronavirus comprises inducing the generation of neutralising antibodies against the coronavirus in a subject.

[0033] Accordingly, a further aspect of the invention provides a method for inducing the generation of neutralising antibodies against a coronavirus in a subject, comprising administering to the subject an immunogenic formulation comprising an attenuated Mycobacterium strain and at least one coronavirus antigen.

[0034] A further aspect of the invention provides a method for inducing the generation of neutralising antibodies against a coronavirus in a subject, comprising administering to the subject an immunogenic formulation comprising an attenuated Mycobacterium strain that expresses at least one coronavirus protein or an antigenic fragment thereof.

Brief Description of the Drawings

[0035] Aspects and embodiments of the present disclosure are described herein, by way of non-limiting example only, with reference to the following drawings.

[0036] Figure 1. Anti-spike antibodies are generated early after BCG: spike vaccination. Mice were vaccinated intradermally with a mixture of BCG (5x 10^s CFU), spike protein (5 μg) or alum (50 μg Alhydrogel^®). Anti-spike protein IgG antibodies in blood were assessed two weeks later by ELISA.

[0037] Figure 2. SARS-CoV-2-specific cytokine production by PBMCs after vaccination with BCG^SpK. At two weeks post vaccination, PBMCs were restimulated ex vivo with spike protein and flow cytometry was used (A, representative plots) to determine the production of IFN-g (B), IL-17 (C) or IL-2 (D) by CD4⁺ T cells.

[0038] Figure 3. Polarization of antibody responses after BCG^SpK immunisation. Mice were vaccinated as described in Example 3. At 3 weeks post vaccination, mice were boosted with the same vaccine for all Spk vaccines; BCG:Spk^Alm was boosted with Spk^Alm, while BCG:Spk^Adx was boosted with Spk^Adx. At one week post boost the titre of total IgG (A), IgG2a (B) or IgGl (C) in the sera of mice was determined by ELISA. Data shown is from one week after boost. The ratio of IgG2a to IgGl antibody titres is shown in D. Titres were calculated as the dilution of the sample that reaches the average of the control serum +/- 3 standard deviations. [0039] Figure 4. BCG promotes the generation of neutralising antibodies in mice. C57BL/6 mice were vaccinated as described in Example 3 and at one week post boost the sera from mice were preincubated with SARS-CoV-2 pseudovirus. After incubated for 1 hour at 37 °C, the mixture was added to ACE2-transfected 293T cells to detect viral infectivity. The proportion of GFP positive cells was determined using the Opera Phenix High Content Screening System (Perkin Elmer) and the titre determined by comparison to cells treated with sera from unvaccinated mice.

[0040] Figure 5. Single immunisation with BCG:CoVac vaccine induces rapid development of anti-SARS-CoV-2 spike antibodies and IFN-y-secreting T cells, a, C57BL/6 mice were vaccinated subcutaneously with PBS, BCG, BCG^SpK, Alum^SpK or BCG:CoVac and whole blood collected at day 14, 28 and 42. b, c, spike-specific IgGl and IgG2c titres in plasma were determined by ELISA estimated by the sigmoidal curve of each sample interpolated with the threshold of the negative sample ± 3 standard deviations. The dotted line shows the limit of detection, d, At 14 days post-vaccination PBMCs were restimulated ex vivo with 5 μg/mL of SARS-CoV-2 spike and cytokine production determined by flow cytometry. Representative dot plots of CD44⁺ CD4⁺ T cells and CD44⁺ CD8⁺ T cells expressing IFN-g. e, Numbers of circulating CD4⁺ and CD8⁺ T cells expressing IFN-yor CD4⁺ T cells expressing IL-17 or TNF. Data presented as mean ± s.d. Significant differences between groups compared to BCG^Spk *p<0.05, **p<0.01, ***p<0.01 or Alum^Spk †p<0.05, ††p<0.01, †††p<0.001 was determined using one-way ANOVA.

[0041] Figure 6. BCG:CoVac vaccination promotes expansion of spike-specific B cells and T follicular helper cells in mice, a-c, C57BL/6 mice were vaccinated subcutaneously with PBS, BCG, BCG^SpK, Alum^SpK or BCG:CoVac and 7 days after immunization B and T cell response assessed by multicolour flow cytometry in the draining lymph node. Shown are representative dot plots of spike-specific germinal centre B cells (a, CD19⁺MHCII⁺GL7⁺CD38^" ), plasma cells (b, CD19⁺MHCII⁺CD138⁺) and T follicular helper T cells (c, CXCR5⁺BCL6⁺). d-f, The total number of d, spike⁺ GC B cells, e, spike⁺ plasma cells and f, T follicular helper cells. Data presented as mean ± s.d. Significant differences between groups *p<0.05, **p<0.01, ***p<0.01 were determined by one-way ANOVA.

[0042] Figure 7. BCG:CoVac induces high titre neutralizing antibodies against live SARS- CoV-2 that correlate with the production of antigen- specific IgG2c. a-e, Plasma from vaccinated mice (from Fig. 5) were tested for neutralizing activity against live SARS-CoV-2 infection of VeroE6 cells, a, Neutralizing antibody (NAb) titres (IC50) were calculated as the highest dilution of plasma that still retained at least 50% inhibition of infection compared to controls. NAb titres from PCR confirmed SARS-CoV-2-infected individuals (COVID) were determined using the same method. b,c, Spearman correlations of spike- specific IgG2c or IgGl titres and NAbs after Alum^SpK or BCG:CoVac vaccination. d,e, Correlation of IgG2c or IgGl titres and NAbs after vaccination with BCG^SpK. The dotted line shows the limit of detection. Data presented as mean ± s.d. Significant differences between groups compared to BCG^Spk **p<0.01, ***p<0.0 or Alum^Spk †p<0.05, †††p<0.001 was determined by one-way ANOVA.

[0043] Figure 8. A single dose of BCG:CoVac protects against severe SARS-CoV-2 infection, a, Mice were immunised with sham (PBS), BCG or BCG:CoVac 21 days prior to challenge with 10³ PFU SARS-CoV-2. Disease outcomes were assessed 6 days later, b, Percentage of initial body weight loss in hemizygous male K18-hACE2 mice (n=4/group). c, Cytokine/chemokine quantification in lung homogenates, d, Total number of inflammatory cells in stained histological sections of lungs, e, Viral titres in lung homogenates determined using plaque assay. The dotted line represents the limit of detection, f, Six weeks after immunization mice were challenged with M. tuberculosis H37Rv by aerosol (-100 CFU) and four weeks later the bacterial load was assessed in the lungs and presented as logio of the mean CFU ± SEM. Significant differences between groups *p<0.05, **p<0.01 were determined by one-way ANOVA.

[0044] Figure 9. Heterologous boosting of BCG:CoVac -primed mice results in augmented SARS-CoV-2-specific IgG2c titres and neutralizing antibodies, a, C57BE/6 mice were vaccinated (as in Fig. 5) and at day 21 mice were boosted with Alum^Spk. b, Spike-specific IgG2c titres in plasma were determined by EFISA estimated from the sigmoidal curve of each sample interpolated with the threshold of the negative sample ± 3 standard deviations, c, Neutralizing antibody (NAb) titres (ICso) were calculated as the highest dilution of plasma for all groups that still retained at least 50% inhibition of infection compared to controls. The dotted line shows the limit of detection. d,e, NAb titres against the B.1.1.7 or B.1.351 SARS-CoV-2 variants were also determined using plasma from either d, Alum^SpK (D) or e, BCG:CoVac-vaccinated mice. Data presented as mean ± s.d. Significant differences between groups compared to BCG^Spk *p<0.05, **p<0.01, ***p<0.01 or Alum^Spk †p<0.05, ††p<0.01, †††p<0.001 were determined by one-way ANOVA.

Detailed Description

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the disclosure belongs. All patents, patent applications, published applications and publications, databases, websites and other published materials referred to throughout the entire disclosure, unless noted otherwise, are incorporated by reference in their entirety. In the event that there is a plurality of definitions for terms, those in this section prevail. Where reference is made to a URF or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference to the identifier evidences the availability and public dissemination of such information. [0046] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0047] In the context of this specification, the term "about," is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.

[0048] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0049] In the context of the present specification, the terms “protein” and “polypeptide” may be used interchangeably herein.

[0050] The term "optionally" is used herein to mean that the subsequently described feature may or may not be present or that the subsequently described event or circumstance may or may not occur. Hence the specification will be understood to include and encompass embodiments in which the feature is present and embodiments in which the feature is not present, and embodiments in which the event or circumstance occurs as well as embodiments in which it does not.

[0051] As used herein the terms "preventing”, “prevention”, "treating", "treatment" and grammatical equivalents refer to any and all uses which remedy, prevent, retard or delay the establishment of a coronavirus infection or a condition, symptom or clinical manifestation associated with a coronavirus infection, or otherwise prevent, hinder, retard, or reverse the progression of such an infection, condition, symptom or clinical manifestation. Thus, the terms “preventing” and "treating" and the like are to be considered in their broadest context. For example, treatment does not necessarily imply that a patient is treated until total recovery. For example, where a condition displays or is characterized by multiple symptoms or manifestations, the treatment or prevention need not necessarily remedy, prevent, hinder, retard, or reverse all of said symptoms or manifestations, but may prevent, hinder, retard, or reverse one or more of said symptoms or manifestations.

[0052] The term "subject" as used herein refers to mammals and includes humans, primates, livestock animals (e.g. sheep, pigs, cattle, horses, donkeys), laboratory test animals (e.g. mice, rabbits, rats, guinea pigs), performance and show animals (e.g. horses, livestock, dogs, cats), companion animals (e.g. dogs, cats) and captive wild animals. Preferably, the mammal is human or a laboratory test animal. Even more preferably, the mammal is a human. [0053] One strategy for accelerating the development of vaccines against specific viral infections is the repurposing of existing licensed vaccines. The present invention is predicated on this strategy.

[0054] Bacille Calmette-Guerin (BCG) is an attenuated Mycobacterium bovis strain that is used as a vaccine against Mycobacterium tuberculosis in newborns. From the approval of BCG as a vaccine against tuberculosis, it has been administered to more than 3.3 trillion people worldwide. Its widespread uptake has been facilitated by many advantageous characteristics, including high thermostability in freeze-dried form. Moreover, the immunization of newborn children with BCG is regarded as risk-free and only produces minimal side effects. BCG is highly immunogenic and only one dose is able to generate an immune response that is maintained for a long period of time. BCG induces a potent Thl-type immune response both in adults and children. However a limitation is that BCG lacks specificity for coronaviruses such as SARS- CoV-2 and thus will not induce immune memory.

[0055] As exemplified herein, the present inventors have surprisingly found that coadministration of BCG and the SARS-CoV-2 spike protein resulted in increased levels of specific neutralising antibodies, compared to levels produced by administration of either component alone, indicating that BCG can serve as an effective vehicle to induce specific immunity against the spike protein, a major SARS-CoV-2 antigen. Moreover, co-administration of BCG and the SARS-CoV-2 spike protein resulted in elevated production of all cytokines, in particular IL-17, with data indicative of induction of a Thl immune response. Without wishing to be bound by theory, the inventors suggest this is of significance as previously reported experiences with SARS-CoV vaccination efforts, and thus of concern in the development of SARS-CoV-2 vaccines, has been the development of detrimental Th2 responses that lead to enhanced disease after vaccination and challenge in animal models (Honda-Okubo et al., 2015; Peeples et al., 2020). Furthermore, it has been shown that in patients recovering from COVID-19, T cell responses are predominately Thl, and expression of IFN-g is lower in severe cases compared to mild ones (Chen et al., 2020). That the co-administration of BCG vaccine and the SARS-CoV- 2 spike protein produces a favourable immune profile is also supported by the reduced expression observed herein of IL-17 and IL-2; heightened expression of IL-17 and IL-2 receptor has been associated with severe COVID-19 disease (Xu et al., 2020) and blocking IL-17 has been suggested to be a plausible therapy to treat acute respiratory distress syndrome (ARDS) in COVID-19 (Pacha et al, 2020).

[0056] in particular, described herein is the development of a SARS-CoV-2 vaccine,

BCG:CoVac, that combines a stabilized, trimeric form of the spike protein with the alum adjuvant. BCG:CoVac stimulates SARS-CoV-2-specific antibody and T cell responses in mice after a single vaccination, including the elicitation of high-titre neutralizing antibodies (Nabs). Critically, a single dose was shown to protect mice against severe SARS-CoV-2, demonstrating that BCG:CoVac is a highly immunogenic and promising vaccine candidate. The level of immune response observed (particularly the generation of neutralizing antibodies) is equivalent to or exceeds that elicited by approved COVID-19 vaccines, when these candidates were tested in the murine model.

[0057] As demonstrated herein, the BCG:CoVac vaccine can neutralize two of the key SARS-CoV-2 variants of concern that are circulating globally, B.1.1.7 and B.1351, thus suggesting that methods and immunogenic formulations of the present disclosure could afford protection against SARS-CoV-2 escape mutants or new pandemic coronaviruses that may emerge in the future. The inventors also suggest that methods and immunogenic formulations of the present disclosure may induce protection against other respiratory infections where BCG is known to induce some level of protective immunity, including future pandemic viruses. [0058] One aspect of the present invention provides a method for inducing the generation of neutralising antibodies against a pathogen in a subject, comprising administering to the subject an immunogenic formulation comprising an attenuated Mycobacterium strain and at least one antigen from said pathogen.

[0059] Typically, the immunogenic formulation induces the generation of higher levels of neutralising antibodies against the pathogen than are generated in the absence of the immunogenic formulation.

[0060] The pathogen may be any pathogen, such as a virus, a bacterium, a parasite or a fungus, against which the generation of neutralising antibodies is considered important in the development of immunogenic formulations and vaccines. Exemplary viral pathogens include those for which effective and reliable vaccines are still sought, such as coronaviruses including MERS-CoV, SARS-CoV and SARS-CoV-2, human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), influenza, and members of the flaviviruses including dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV). Exemplary bacterial pathogens include those for which effective and reliable vaccines are still sought, such as Chlamydia trachomatis, Pseudomonas aeruginosa and Mycobacterium tuberculosis. Exemplary parasitic pathogens include those for which effective and reliable vaccines are still sought, such as Plasmodium falciparum, flukes (including Schistomsomajaponicum, S. mansoni and S. haematobium, and hookworm (including Ancylostoma duodenale and Necator americanus). [0061] In another aspect, the present invention provides an immunogenic formulation for preventing or treating coronavirus infection, the formulation comprising an attenuated Mycobacterium strain and at least one coronavirus antigen.

[0062] Typically in accordance with the present invention the coronavirus is a betacoronavirus, optionally a sarbecovirus. In particular embodiments the coronavirus is a severe acute respiratory syndrome -related coronavirus such as SARS-CoV or SARS-CoV-2. In exemplary embodiments the coronavirus is SARS-CoV-2.

[0063] Immunogenic formulations of the invention may comprise one or more coronavirus antigens. Such antigens may comprise, for example, any coronavirus surface protein including the spike protein (S), the membrane protein (M), the envelope protein (E) or the hemagglutinin- esterase protein (HE), or an antigenic fragment thereof. Embodiments of the invention contemplate combinations of two or more of said proteins or antigenic fragments thereof.

[0064] In exemplary embodiments, immunogenic formulations comprise at least a coronavirus spike protein (S) or an antigenic fragment thereof. The spike protein may be derived from SARS-CoV-2. The spike protein may comprise the amino acid sequence of SEQ ID NO:l or a sequence having at least or about 85% sequence identity thereto. For example, a suitable spike protein may have at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:l.

[0065] Alternatively, or in addition, an immunogenic formulation may comprise a coronavirus envelope protein (E) or an antigenic fragment thereof. The envelope protein may be derived from SARS-CoV-2. The envelope protein may comprise the amino acid sequence of SEQ ID NO:2 or a sequence having at least or about 85% sequence identity thereto. For example, a suitable spike protein may have at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:2. [0066] Alternatively, or in addition, an immunogenic formulation may comprise a coronavirus membrane protein (M) or an antigenic fragment thereof. The membrane protein may be derived from SARS-CoV-2. The membrane protein may comprise the amino acid sequence of SEQ ID NO: 3 or a sequence having at least or about 85% sequence identity thereto. For example, a suitable spike protein may have at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:3.

[0067] In exemplary embodiments, an immunogenic formulation comprises a coronavirus spike protein, optionally a SARS-CoV-2 spike protein, or antigenic fragment thereof in combination with one or more of a coronavirus envelope protein or antigenic fragment thereof, a coronavirus membrane protein or antigenic fragment thereof and a coronavirus hemagglutinin- esterase protein or antigenic fragment thereof. Where two or more antigens are employed, they may or may not be from the same coronavirus species or strain.

[0068] The attenuated Mycobacterium strain is typically a M. bovis strain, more typically the Bacille Calmette-Guerin (BCG) strain. A variety of BCG strains are available (see, for example, Grange et al., 1983), including for example the Pasteur BCG strain (strain BCG / Pasteur 1173P2), the Danish BCG strain 1331, the Tokyo BCG substrain 172, and strains available from the American Type Culture Collection (ATCC) under accession numbers 357333 and 35734. There are also numerous commercial sources of BCG strains. The scope of the present disclosure is not limited by reference to any specific BCG strain or source thereof. In an exemplary embodiment the BCG strain is the BCG Pasteur 1173P2 strain.

[0069] The attenuated Mycobacterium strain employed in an immunogenic formulation of the present invention may be derived from a Mycobacterium bacterial culture in exponential growth or stationary phase in buffered saline solution.

[0070] The attenuated Mycobacterium strain, optionally the BCG strain, may be a recombinant strain. The recombinant attenuated Mycobacterium strain may contain one or more genes that encode at least one coronavirus antigen as described herein. Accordingly, an aspect of the invention provides an immunogenic formulation for preventing or treating coronavirus infection, the formulation comprising a recombinant attenuated Mycobacterium strain that expresses at least one coronavirus protein or antigenic fragment thereof.

[0071] Nucleotide sequences encoding at least one coronavirus protein or antigenic fragment may be integrated into the genome of the attenuated Mycobacterium strain, or may be inserted into an extrachromosomal plasmid, in one or more copies, such that the coronavirus protein or antigenic fragment is expressed by the attenuated Mycobacterium. Introduction of exogenous coronavirus nucleotide sequences into the Mycobacterium genome and/or integration into the Mycobacterium genome can be achieved by a variety of techniques that are well known to those skilled in the art. The coronavirus nucleotide sequences can be present in single or multiple copies, and expression may be controlled, constitutively or inducibly, by endogenous Mycobacterium promoters or exogenous promoters. The coronavirus immunogenic proteins or fragments can be expressed by the attenuated Mycobacterium strain in a variety of forms, for example soluble cytoplasmic form, extracellularly secreted form or membrane bound form. [0072] Coronavirus proteins or antigenic fragments expressed by the attenuated Mycobacterium strain may comprise or be derived from, for example, any coronavirus surface protein including the spike protein (S), the membrane protein (M), the envelope protein (E) or the hemagglutinin-esterase protein (HE), or an antigenic fragment thereof. Embodiments of the invention contemplate attenuated Mycobacterium strains expressing combinations of two or more of said proteins or antigenic fragments.

[0073] The spike protein (S) may be derived from SARS-CoV-2. The spike protein may comprise the amino acid sequence of SEQ ID NO:l or a sequence having at least or about 85% sequence identity thereto. For example, a suitable spike protein may have at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:l. The coronavirus spike protein or antigenic fragment thereof expressed by the recombinant attenuated Mycobacterium strain may be encoded by the nucleotide sequence of SEQ ID NO:4, or a portion thereof, or a sequence having at least or about 85% sequence identity thereto. For example, a suitable nucleotide sequence encoding the spike protein may have at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:4.

[0074] The envelope protein (E) may be derived from SARS-CoV-2. The envelope protein may comprise the amino acid sequence of SEQ ID NO:2 or a sequence having at least or about 85% sequence identity thereto. For example, a suitable envelope protein may have at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:2. The coronavirus envelope protein or antigenic fragment thereof expressed by the recombinant attenuated Mycobacterium strain may be encoded by the nucleotide sequence of SEQ ID NO: 5, or a portion thereof, or a sequence having at least or about 85% sequence identity thereto. For example, a suitable nucleotide sequence encoding the envelope protein may have at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:5.

[0075] The membrane protein (M) may be derived from SARS-CoV-2. The membrane protein may comprise the amino acid sequence of SEQ ID NO:3 or a sequence having at least or about 85% sequence identity thereto. For example, a suitable membrane protein may have at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:3. The coronavirus membrane protein or antigenic fragment thereof expressed by the recombinant attenuated Mycobacterium strain may be encoded by the nucleotide sequence of SEQ ID NO:6, or a portion thereof, or a sequence having at least or about 85% sequence identity thereto. For example, a suitable nucleotide sequence encoding the membrane protein may have at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:6. [0076] In particular embodiments, an immunogenic formulation for inducing an immune response against coronavirus induces the generation of higher levels of neutralising antibodies against the coronavirus than are generated in the absence of the immunogenic formulation. [0077] Immunogenic formulations and vaccine compositions in accordance with the present invention typically comprise one or more adjuvants, diluents, excipients, carriers, or other pharmaceutically acceptable substances. The term “pharmaceutically acceptable” is used to refer to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism.

[0078] Suitable exemplary adjuvants include, but are not limited to, aluminium salt-based adjuvants, squalene-based adjuvants, CpG dinucleotide-based adjuvants, saponin-based adjuvants, N-acetylmuramyl-L-alanyl-D-glutamine, DC-chol, poly[di(carboxy!atophenoxy)phosphazene], monophoshoryl lipid A, QS-21, delta-inulin, cholera toxin and formyl methiony! peptide, and other adjuvants known to those of ordinary skill in the art (see, e.g., Vaccine Design, the Subunit and Adjuvant Approach, 1995, M. F. Powell andM. J. Newman, eds., Plenum Press, N. Y.; Del Guidice etal., Seminars in Immunology, 2018, 39:14-21). Suitable aluminium salt-based adjuvants include, for example aluminium hydroxide, aluminium phosphate and derivatives thereof. Suitable squalene-based adjuvants include, for example, MF59 (oil-in-water emulsion comprising squalene and surfactants Tween 80 and Span 95) and AS03 (oil -in- water emulsion comprising squalene, DL-oc-tocopherol and polysorbate 80). A suitable CpG dinucleotide-based adjuvant is, for example, CpGlOlB (TLR9-targeting adjuvant). A suitable saponin-based adjuvant is, for example, Matrix-M (comprising nanoparticles composed of Quillaja saponins, cholesterol and phospholipid). Those skilled in the art will appreciate that suitable adjuvants may be obtained from a variety of commercial sources. In exemplary embodiments of the present invention, a suitable adjuvant is aluminium hydroxide (also referred to as alum). By way of example, a suitable aluminium hydroxide adjuvant is a wet gel suspension marketed as Alhydrogel^®. By way of example, suitable inulin- based adjuvants are marketed as Advaxl, comprising delta inulin microparticles, and Advax2, comprising delta inulin microparticles in combination with CpG dinucleotides.

[0079] The immunogenic formulations and vaccine compositions of the present invention may be administered by any suitable route such as, for example, intradermally, intramuscularly, subcutaneously, intravenously, intrapulmonary, orally or intranasally. The immunoprotective amount of immunogenic formulation or vaccine composition to be administered is typically determined on a case by case basis and may be determined by the skilled person without undue burden or the need for further invention. [0080] Immunogenic formulations and vaccine compositions of the invention are typically administered so as to produce an immunoprotective effect in a subject. The immunoprotective effect represents the combined effect of the attenuated Mycobacterium strain and the coronavirus antigen. Thus, in this context one can consider immunoprotective amounts of both the attenuated Mycobacterium strain and the coronavirus antigen. By way of example only, where the subject is a human, the immunoprotective amount of the attenuated Mycobacterium strain may range from about 1 x 10⁴ CFU per dose to about 1 x 10¹⁰ CFU per dose. For example, the immunoprotective amount may be about 1 x 10⁴, 1 x 10⁵, 1 x 10⁶, 1 x 10⁷, 1 x 10^s, 1 x 10⁹, or 1 x 10¹⁰ CFU of attenuated Mycobacterium strain per dose. By way of example only, where the subject is a human, the immunoprotective amount of a coronavirus protein or antigenic fragment thereof, may comprise between about 0.5 μg and about 200 μg, or between about 2 μg and about 100 μg, or between about 5 μg and about 50 μg per dose. For example, the immunoprotective amount may be about 0.5 μg, 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 pg, about 70 μg, about 80 μg, about 90 pg, about 100 pg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, about 160 μg, about 170 μg, about 180 μg, about 190 μg, or about 200 μg of coronavirus protein or antigenic fragment thereof per dose.

[0081] Immunogenicity can be assessed and monitored by a range of techniques available to those skilled in the art, including the production of antibodies in the subject subsequent to administration of the immunogenic formulation or vaccine composition.

[0082] In order to produce an immunoprotective effect the immunogenic formulations and vaccine compositions of the present invention may be administered in a single dose or in a series of doses. Where more than one dose is required, the doses may be administered days, weeks or months apart, such as for example, 1, 2, 3, 4, 5, 6 or 7 days apart, 1 , 2, 3, 4, 5 or 6 weeks apart, or 1, 2, 3, 4, 5 or 6 months apart. The immunogenic formulations and vaccine compositions can be administered in a series including one or more boosters.

[0083] The dosage of the immunogenic formulation or vaccine composition to be administered a subject and the regime of administration can be determined in accordance with standard techniques well known to those of ordinary skill in the pharmaceutical and veterinary arts, taking into consideration such factors as the intended use, the particular coronavirus antigen, the adjuvant (if present), the age, sex, weight, species and general condition of the subject, and the route of administration. Preliminary doses can be determined according to animal tests, and the scaling of dosages for human administration is performed according to art-accepted practices such as standard dosing trials. Appropriate dosages can be readily determined by routine experimentation .

[0084] Immunogenic formulations and vaccine compositions of the present invention are typically sterile and may contain one or more pharmaceuticaUy-acceptable carriers, such as one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration to a subject. Formulation of vaccines of the present disclosure into pharmaceutical compositions can be accomplished using methods known in the art.

[0085] Also provided by the present invention are methods for inducing an immune response to a coronavirus, methods for preventing or treating a coronavirus infection in a subject, and methods for preventing or treating a disease or disorder caused by a coronavirus infection. Methods of the invention typically result in the induction or generation of a Thl immune response in the subject. Typically, the methods of the invention result in the increased expression of IFN-g.

[0086] The methods may comprise administering to a subject an immunogenic formulation or vaccine composition of the invention. Alternatively, the method may comprise administering a composition comprising an attenuated Mycobacterium strain as hereinbefore described and a composition comprising at least one coronavirus antigen as hereinbefore described. Administration of separate compositions may be simultaneous or sequential and may be by the same or different routes. In this context, by “sequential” administration is meant a time difference of from seconds, minutes, hours or days between the administration of the compositions.

[0087] In order to increase the effectiveness of the immunogenic formulations and vaccine compositions of the present invention, it may be desirable to combine the administration of said formulations and compositions with one or more additional agents effective in the induction of immune responses, in particular Thl immune responses. Such additional agents may be administered in the same formulation as the immunogenic formulation or vaccine composition of the invention, or in a different formulation(s), administered via the same or different routes. Such administration may be simultaneous or sequential. By “sequential” administration is meant a time difference of from seconds, minutes, hours or days between the administration of the components.

[0088] While exemplary embodiments of the invention are described herein with reference to the SARS-CoV-2 coronavirus, those skilled in the art will appreciate that the scope of the invention is not limited thereto. Embodiments of formulations, compositions and methods of the invention may be employed in the rapid development of efficacious vaccine treatments against novel emerging coronaviruses. In such instances one or more antigens from a novel emerging coronavirus may be used in immunogenic formulations as described herein in addition to the specific coronavirus antigens described herein or in place of such antigens.

[0089] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0090] The present disclosure will now be described with reference to the following specific examples, which should not be construed as in any way limiting the scope of the disclosure.

Examples

[0091] The following examples are illustrative of the disclosure and should not be construed as limiting in any way the general nature of the disclosure of the description throughout this specification.

Examples 1 - 3: General methods

[0092] Animals: C57BL/6 mice (6-8 weeks of age) were housed at the Centenary Institute (Sydney NSW, Australia) in pathogen-free conditions. All mouse work was performed according to ethical guidelines as set out by the Sydney Local Health District Animal Welfare Committee.

[0093] Vaccination: Mice were anaesthetised with Ketamine/Xylasine and vaccinated intradermally in each ear pinna with 5x10^s CFU of the M. bovis BCG vaccine (Pasteur; hereafter 'BCG') and/or 5 μg of SARS-CoV-2 spike protein (RayBiotech or prepared in house) with or without 50 μg of Alhydrogel^® (InvivoGen), using a 31g ultrafine insulin syringe. Co-injected BCG and SARS-CoV-2 spike protein is herein referred to as 'BCG^SpK'.

[0094] Sample collection: At regular 2- week intervals mice were tail bled in heparin coated Eppendorf tubes. Blood was centrifuged at 2000 rpm at room temperature and plasma collected. The remaining blood was diluted to lmL in PBS heparin 20U/mL and stratified on Histopaquel083 (Sigma- Aldrich). After 30 min centrifugation, the peripheral blood mononuclear cell (PBMC) layer was collected, washed with complete media and used in restimulation assays.

[0095] Re -stimulation assay: PBMCs were resuspended at a concentration of 2-3 x 10⁶ cells/mL and restimulated at 37°C in 96 well plates with SARS2-CoV-2 spike protein (10 mg/mL), or left unstimulated. After 4-5 hours incubation, protein transport inhibition cocktail (eBioscience) was added to each well and incubated for further 10-12 hours. Cells were then washed and stained for cell surface and intracellular markers.

[0096] IgG ELISA. Ninety-six well ELISA plates (Costar) were coated overnight with 1 mg/mL of SARS2-CoV-2 spike protein in carbonate buffer. Blocking was performed for at least 2 hours with 3% BSA. Titres of total IgG, or IgGl and IgG2c were measured by ELISA in the serial diluted plasma.

Example 1 - Induction of SARS-CoV-2-specific antibody responses after BCG^SpK vaccination

[0097] In order to determine if BCG could present the SARS-CoV-2 spike protein in a form that results in humoral immunity, BCG, SARS2-CoV-2 spike protein and/or alum were combined and injected intradermally (i.d.) into mice. Two weeks later mice were bled and the level of IgG antibodies in plasma determined. Background level of IgG was detected in mice vaccinated with PBS vehicle or BCG alone. The co-administration of spike protein and BCG, and of spike protein, BCG and alum, resulted in elevated levels of IgG compared to BCG alone (Figure 1). Spike protein alone or spike with alum (without BCG) led to intermediate levels of anti-spike IgG.

[0098] These results demonstrate that increased levels of specific antibodies appeared after vaccination with BCG^SpK, thus indicating that BCG can serve as an effective vehicle to induce specific immunity against a major SARS-CoV-2 antigen. The data suggests that BCG possesses adjuvant properties that can effectively activate B cells and induce class switching of immunoglobulin isotypes.

Example 2 - BCG^SpK vaccination results in favourable T cell immunity after intradermal vaccination

[0099] To determine if virus-specific T cell responses were apparent after vaccination with combinations of BCG and spike protein, mice were vaccinated as described above, and two weeks later PBMCs were isolated, stimulated with SARS2-CoV-2 spike protein and cytokine secretion by CD4⁺ T cells determined. Cells from mice that received the combination of BCG and spike protein expressed high levels of IFN-g after ex vivo restimulation, but relatively low levels of IL-17 and IL-2 (Figure 2). The addition of alum to either spike protein or BCG and spike resulted in elevated production of all cytokines, in particular IL-17. Mice vaccinated with PBS vehicle or BCG alone did not produce appreciable cytokine-expressing cells after antigen stimulation.

[00100] The data obtained is indicative that the immune response induced by the BCG^SpK combination is predominately a Thl (IFN-g) response. Reduced IL-17 and IL-2 was noted in mice administered BCG^SpK compared to other vaccinated group (Figure 2). This suggests a favourable immune profile is produced by BCG^SpK, as heightened expression of IL-17 and IL-2 receptors has been associated with severe COVID-19 disease (Xu et al., 2020).

Example 3 - BCG^SpK vaccination produces neutralising antibodies [00101] Mice (as described above in the General Methods) were anaesthetised with Ketamine/Xylasine and vaccinated subcutaneously in each ear pinna with 5x10^s CFU of M. bovis BCG (Pasteur) and/or 2 μg of SARS-CoV-2 spike protein (prepared in house) with or without 500 μg of Alhydrogel^® (Alum; Invivogen) or lmg of Advax2 (Vaxine) adjuvant. At 3 weeks, mice were boosted with the same antigen/adjuvant vaccine without BCG.

[00102] The following groups were used:

Table 1: Immunisation groups used for experiments

[00103] Sample collection, T cell restimulation and IgG ELISA were carried out as described above in the General Methods.

[00104] One week post boost the sera from mice were preincubated with SARS-CoV-2 pseudovirus. The SARS-CoV-2 SpK pseudovirion neutralisation assay was developed in house and uses a combined GFP-luciferase reporter to directly measure viral entry into host cells. After incubation for 1 hour at 37 °C, the mixture was added to ACE2-transfected HEK293T cells to detect viral infectivity. The proportion of GFP positive cells was determined using the Opera Phenix High Content Screening System (Perkin Elmer) and the titre determined by comparison to cells treated with sera from unvaccinated mice.

[00105] Background levels of total IgG (Figure 3A), IgGl (Figure 3B) and IgG2a (Figure

3C) were detected in mice vaccinated with the PBS vehicle or BCG alone. Spike protein or

Spk^Alm elicited IgGl (Th2 immunity) but no IgG2a (Thl immunity), indicative of skewing towards a Th2-like immunity (Figure 3D). However, the addition of BCG led to a much greater increase in the production of IgG2a and skewing towards Thl immunity (Figure 3D). Use of the Advax2 adjuvant in the place of Alum led to an increase in the amount of total IgG, in particular IgG2a (Figure 3B) and thus greater skewing towards Thl immunity (Figure 3D). Thus BCG can potentiate Thl -like immunity when delivered with the SARS-CoV-2 spike protein.

[00106] The development of high level neutralising antibodies is considered crucial for protection against COVID-19. To assess this, sera from the mice vaccinated as described above (for Figure 3) were tested for the presence of neutralising antibodies. Antibodies from mice vaccinated with BCG or spike protein alone were unable to neutralise pseudovirus entry, while low neutralising antibody activity was seen with Spk^™ (Figure 4). Strikingly, BCG:Spk^Alm induced high titre neutralising antibodies, with BCG potentiating neutralising activity by -10- fold (BCG:Spk^Alm vs SpK^Alm, Figure 4). The addition of Advax to BCG:spike (BCG:Spk^Adx) also led to an increase in neutralising antibodies, although the antibody level was lower than that seen with BCG:Spk^Alm (Figure 4). Thus BCG is able to lead to a marked increase in anti-spike neutralising antibodies.

Examples 4-7: General methods

[00107] Bacterial culture : M. bovis BCG (strain Pasteur) was grown at 37°C in Middlebrook 7H9 media (Becton Dickinson, BD, New Jersey, USA) supplemented with 0.5% glycerol, 0.02% Tyloxapol, and 10% albumin-dextrose-catalase (ADC) or on solid Middlebrook 7H11 media (BD) supplemented with oleic acid-ADC. To prepare single cell suspensions, cultures in exponential phase (OD₆oo=0.6) were washed in PBS, passaged 10 times through a 27G syringe, briefly sonicated and centrifuged at low speed for 10 min to remove residual bacterial clumps. BCG suspensions were frozen at -80° C in PBS supplemented with 20% glycerol, and colony forming units (CFU) for vaccination enumerated on supplemented Middlebrook 7H11 agar plates.

[00108] Immunization : Female C57BL/6 (6-8 weeks of age) purchased from Australian BioResources (Moss Vale, Australia) or hemizygous male K18-hACE2 mice bred in-house (McCray et al., 2007) were housed at the Centenary Institute in specific pathogen-free conditions. SARS-CoV-2 full-length spike stabilized, trimeric protein (SpK) was expressed in EXPI293F™ cells and purified as described previously (Xi et al., 2020). Mice (n=3-4) were vaccinated subcutaneously in the footpad (s.c) with 5x10^s CFU of BCG alone, 5 μg of SpK combined with either BCG (BCG^SpK) or 100 μg of Alhydrogel (Alum) (Invivogen, California, USA, Alum^SpK), or a combination of BCG (5x10^s CFU), SpK (5 μg) and Alyhydrogel (100 μg) (BCG:CoVac). Some mice were boosted three weeks after the first vaccination with 5 μg of SpK combined with 100 μg of Alhyhdrogel. Mice were bled fortnightly after the first immunisation (collected in 10 pi of Heparin 50000 U/mL). Plasma was collected after centrifugation at 300 x g for 10 min and remaining blood was resuspended in 1 mL of PBS Heparin 20 U/mL, stratified on top of Histopaque 10831 (Sigma- Aldrich, Missouri, USA) and the PBMC layer collected after gradient centrifugation.

[00109] Flow cytometry assays: Popliteal lymph nodes were collected at day 7 post immunization, and single cell suspensions were prepared by passing them through a 70 pm sieve. To assess specific B cell responses, 2x10⁶ cells were surface stained with Fixable Blue Dead Cell Stain (Life Technologies) and Spike-AF647 (1 μg), rat anti-mouse GL7-AF488 (clone GL7, 1:200, Biolegend), rat anti-mouse MHC-II-AF700 (clone M5/114.15.2, 1:150), rat anti-mouse IgD-PerCP5.5 (clone ll-26c.2a, 1:200, BD), rat anti-mouse IgM-BV421 (clone RMM-1, 1:200, Biolegend), rat anti-mouse CD138-BV605 (clone 281-2, 1:200, Biolegend), rat anti-mouse CD19-BV785 (clone 1D3, 1:200, BD), rat anti-mouse CD38-APCy7 (clone 90, 1:200, Biolegend). To assess T cell responses, 2xl0⁶ lymph node cells were stained with the following monoclonal antibodies: rat anti-mouse CXCR5-biotin (clone 2G8, 1:100, BD), streptavidin PECy7, rat anti-mouse CD4-AF700 (clone RM4-5, 1:200, BD), rat anti-mouse CD8-APCy7 (clone 53-6.7, 1:200, BD), rat anti-mouse CD44-BV605 (clone IM7, 1:300, BD). Cells were then fixed and permeabilized using the eBioscience fixation/permeabilization kit (ThermoFischer) according to the manufacturer’s protocol and intracellular staining was performed using anti- BCL-6-AF647 (clone K112-91, 1:100, BD).

[00110] To assess SpK-specific cytokine induction by T cells, murine PBMCs were stimulated for 4 hours with SpK (5 μg/mL) and then supplemented with Protein Transport Inhibitor cocktail (Life Technologies, California, USA) for a further 10-12 hours. Cells were surface stained with Fixable Blue Dead Cell Stain (Life Technologies) and the marker-specific fluorochrome-labeled antibodies rat anti-mouse CD4-AF700 (clone RM4-5, 1:200, BD), rat antimouse CD8-APCy7 (clone 53-6.7, 1:200, BD), rat anti-mouse CD44-FITC (clone IM7, 1:300, BD). Cells were then fixed and permeabilized using the BD Cytofix/Cytoperm™ kit according to the manufacturer’s protocol. Intracellular staining was performed using rat anti-mouse IFN- □ -PECy7 (clone XMG1-2, 1:300, BD), rat anti-mouse IL-2-PE (clone JES6-5H4, 1:200, BD), rat anti-mouse IL-17-PB (clone TC11-18H10.1, 1:200, BioLegend California, USA), rat antimouse TNF-PErCPCy5.5 (clone MP6-XT22, 1:200). All samples were acquired on a BD LSR- Fortessa (BD) or a BD-LSRII, and assessed using FlowJo™ analysis software vl0.6 (Treestar, USA).

[00111] Antibody ELISA: Microtitration plates (Corning, New York, USA) were incubated overnight with 1 μg/mL SpK at room temperature (RT), blocked with 3% BSA and serially diluted plasma samples were added for 1 hour at 37°C. Plates were washed and biotinylated polyclonal goat anti-mouse IgGl (1 :50,000, abeam Cambridge, UK), polyclonal goat anti-mouse IgG2c (1:10,000, abeam), or polyclonal goat anti-mouse IgG (1:350,000, clone abeam) added for 1 hour at RT. After incubation with streptavidin-HRP (1:30,000, abeam) for 30 minutes at RT, binding was visualized by addition of tetramethyl benzene (Sigma- Aldrich). The reaction was stopped with the addition of 2N H2SO4 and absorbances were measured at 450 nm using a M1000 pro plate reader (Tecan, Mannedorf, Switzerland). Endpoint titres were calculated as the dilution of the sample that reached the average of the control serum ± 3 standard deviations. [00112] High content fluorescent live SARS-CoV-2 neutralization assay : A high-content fluorescence microscopy approach assessed the ability of patient sera to inhibit SARS-CoV-2 infection and the resulting cytopathic effect in live permissive cells (VeroE6). Sera were serially diluted and mixed in duplicate with an equal volume of 1.5xl0³ TCIDso/mL virus solution (B.1.319) or 1.25xl0⁴ TCIDso/mL virus solution (A2.2, B.1.1.7, B.1.351). After 1 hour of virus- serum coincubation at 37°C, 40 μL were added to equal volume of freshly-trypsinised VeroE6 cells in 384-well plates (5xl0³/well). After 72 hours, cells were stained with NucBlue (Invitrogen, USA) and the entire well surface was imaged with InCell Analyzer 2500 (Cytiva). Nuclei counts were obtained for each well with InCarta software (Cytiva), as proxy for cell death and cytopathic effect resulting from viral infection. Counts were compared between convalescent sera, mock controls (defined as 100% neutralization), and infected controls (defined as 0% neutralization) using the formula; % viral neutralization = (D-(1-Q))x100/D, where Q = nuclei count of sample normalized to mock controls, and D = 1-Q for average of infection controls. The cut-off for determining the neutralization endpoint titre of diluted serum samples was set to >50% neutralization.

[00113] SARS-CoV-2 challenge experiments'. Male hemizygous K18-hACE2 mice were transported to the PC3 facility in the Centenary Institute for SARS-CoV-2 infection. Mice were anaesthetised with isoflurane followed by intranasal challenge with 10³ PFU SARS-CoV-2

(VICOl/2020) in a 30 μL volume. Following infection, mice were housed in the IsoCage N biocontainment system (Tecniplast, Italy) and were given access to standard rodent chow and water ad libitum. Mice were weighed and monitored daily, with increased monitoring when mice developed symptoms. At day 6 post-infection, mice were euthanised with intraperitoneal overdose of pentobarbitone (Virbac, Australia). Blood was collected via heart bleed, allowed to coagulate at RT and centrifuged (10,000 xg, 10 min) to collect serum. Multi-lobe lungs were tied off and BALF was collected from the single lobe via lung lavage with 1 mL HANKS solution using a blunted 19-gauge needle inserted into the trachea. BALF was centrifuged (300 xg, 4°C,

7 min), and supernatants collected and snap frozen. Cell pellets were treated with 200 μL Red

Blood Cell Lysis Buffer (ThermoFisher, USA) for 5 min, followed by addition of 700 μL

HANKS solution to inactivate the reaction and then centrifuged again. Cell pellets were resuspended in 160 μL HANKS solution and enumerated using a haemocytometer (Sigma- Aldrich, USA). Multi-lobe lungs were collected and cut into equal thirds, before snap freezing on dry ice. Lung homogenates were prepared fresh, with multi-lobe lungs placed into a gentleMACS C-tube (Miltenyi Biotec, Australia) containing 2 mL HANKS solution. Tissue was homogenised using a gentleMACS tissue homogeniser, after which homogenates were centrifuged (300 xg, 7 min) to pellet cells, followed by collection of supernatants for plaque assays and cytokine/chemokine measurements. The single lobe lung was perfused with 0.9% NaCl₂ solution via the heart, followed by inflation with 0.5 mL 10% neutral buffered formalin through the trachea, and placed into a tube containing 10% neutral buffered formalin. Following fixation for at least 2 weeks, single lobes were transported to a PC2 facility where they were paraffin-embedded, sections cut to 3 μm thickness using a Leica microtome (Leica, Germany) and then stained using Quick Dip Stain Kit (Modified Giemsa Stain) protocol as per manufacturer’s instructions (POCD Scientific, Australia). Inflammatory cells in single lobe lungs were counted using a Zeiss Axio Imager.Z2 microscope with a 40X objective (Zeiss, Germany).

[00114] Plaque assays: VeroE6 cells (CellBank Australia, Australia) were grown in Dulbecco’s Modified Eagles Medium (Gibco, USA) supplemented with 10% heat-inactivated foetal bovine serum (Sigma-Aldrich, USA) at 37°C/5% CO₂. For plaque assays, cells were placed into a 24-well plate at 1.5x10⁵ cells/well and allowed to adhere overnight. The following day, virus-containing samples were serially diluted in Modified Eagles Medium (MEM), cell culture supernatants removed from the VeroE6 cells and 250 μL of virus-containing samples was added to cell monolayers. Plates were incubated and gently rocked every 15 min to facilitate viral adhesion. After 1 hr, 250 μL of 0.6% agar/MEM solution was gently overlaid onto samples and placed back into the incubator. At 72 hr post-infection, each well was fixed with an equal volume of 8% paraformaldehyde solution (4% final solution) for 30 min at RT, followed by several washes with PBS and incubation with 0.025% crystal violet solution for 5 min at RT to reveal viral plaques.

[00115] Cytometric bead arrays (CBAs): CBAs were performed as per the manufacturer’s instructions (Becton Dickinson, USA). Briefly, a standard curve for each analyte was generated using a known standard supplied with each CBA Flex kit. For each sample, 10 μL was added to a well in a 96- well plate, followed by incubation with 1 μL of capture bead for each analyte (1 hr, RT, in the dark). Following capture, 1 μL of detection bead for each analyte was added to each well, followed by incubation (2 hr, RT, in the dark). Samples were then fixed overnight in an equal volume of 8% paraformaldehyde solution (4% final solution). The following day, samples were transferred to a new 96-well plate and then transported to the PC2 facility for a second round of fixation. Samples were examined using a BD LSR Fortessa equipped with a High-Throughput Sampler (HTS) plate reader.

[00116] Mycobacterium tuberculosis aerosol challenge : Eight weeks after the last vaccination mice were infected with M. tuberculosis H37Rv via the aerosol route using a Middlebrook airborne infection apparatus (Glas-Col, IN, USA) with an infective dose of -100 viable bacilli. Four weeks later, the lungs and spleen were harvested, homogenized, and plated after serial dilution on supplemented Middlebrook 7H11 agar plates. Colonies forming units (CFU) were determined 3 weeks later and expressed as logio CFU.

[00117] Statistical analysis: The significance of differences between experimental groups was evaluated by one-way analysis of variance (ANOVA), with pairwise comparison of multi- grouped data sets achieved using Tukey’s or Dunnett's post-hoc test. Differences were considered statistically significant when p < 0.05.

Example 4 - BCG vaccination promotes SARS-CoV-2 specific antibody and T cell responses in mice

[00118] The inventors subcutaneously (s.c) vaccinated mice with a single dose of BCG formulated with a stabilized, trimeric form of the SARS-CoV-2 spike protein (Amanat et al., 2020) and the titre of IgG2c or IgGl anti-SpK antibodies was determined at various timepoints post-immunization (days 14, 28 and 42; Fig. 5a). While BCG- vaccination resulted in background levels of anti-SpK antibodies, titres were approximately 100-fold higher for both isotypes after BCG^Spk vaccination, and similar to levels achieved with Alm^SpK (Fig. 5b, 5c). Addition of alum to BCG^Spk (termed BCG:CoVac) further increased antibodies titres, particularly IgG2c, which were significantly greater after BCG:CoVac vaccination compared to mice immunised with either CG^Spkor Alm^SpK, at all timepoints examined (Fig. 5b, 5c).

[00119] The IgG2c Ab isotype correlates with Thl-like immunity in C57BF/6 mice, and such responses are considered necessary for effective protection against SARS-CoV-2 infection. The inventors therefore examined the frequency of IFN-y-expressing T cells after a single dose of BCG:CoVac at 2 weeks post-vaccination. BCG^SpK and BCG:CoVac induced the generation of SpK-specific CD4⁺ and CD8⁺T cells secreting IFN-y(Fig. 5d, 5e), consistent with Thl immunity observed after BCG vaccination. The greatest response was observed after vaccination with BCG:CoVac, with the numbers of IFN-y-secreting T cells significantly increased compared to vaccination with either BCG or Alum^SpK. Fow levels of the inflammatory cytokines IF- 17 and TNF were observed after BCG:CoVac vaccination (Fig. 5d), consistent with the results shown in Example 2.

[00120] The inventors further dissected vaccine-induced immunity by defining the cellular makeup in draining lymph nodes 7 days after vaccination. Both Alum^SpK and BCG:CoVac induced appreciable expansion of SpK-specific germinal centre (GC) B cells (CD19⁺MHCII⁺GL7⁺CD38 ;Fig. 6a) and plasma B cells (CD19⁺MHCII⁺CD138⁺;Fig. 6b). Cells with a T follicular helper cell (Tfh) phenotype (CD4⁺CXCR5⁺BCL6⁺) were apparent after vaccination with Alum^SpK or BCG:CoVac, with Tfh frequency greatest in the latter group (Fig. 6c). The total numbers of GC B cells (Fig. 6d), plasma B cells (Fig. 6e) and Tfh cells (Fig. 6f) were all significantly increased in BCG:CoVac-vaccinated mice compared to immunisation with Alum^SpK.

[00121] Overall, these data show that BCG vaccination promotes early and pronounced anti- SARS-CoV-2 immunity when co-delivered with the trimeric SpK antigen, which can be further enhanced with the addition of alum.

Example 5 -High titre, SARS-CoV-2 neutralizing antibodies after a single immunization with BCG.CoVac

[00122] The elicitation of GC B cell and Tfh responses after immunisation with experimental SARS-CoV-2 vaccines correlate strongly with the induction of neutralizing antibodies (NAbs) (Lederer et al., 2020) and such NAbs are a key determinant of protection induced by current vaccines used in humans. The inventors therefore measured NAb levels after a single dose of BCG:CoVac. No NAbs were detected in the plasma of mice vaccinated with BCG (Fig. 7a). Surprisingly, NAb titres were at near background levels for mice vaccinated with BCG^SpK (Fig. 7a), despite the high levels of IgG Ab isotypes detected in these same animals (Fig. 5). High NAbs titres were detected as early as 2 weeks post-immunisation upon vaccination with BCG:CoVac, and titres were significantly increased compared to vaccination with Alum^SpK (approximate 10-fold increase). The mean NAbs titres in the plasma of BCG:CoVac-vaccinated mice were approximately 10-fold greater than those seen in SARS-CoV-2 infected humans (Fig. 7a). Although the levels of NAbs peaked at 2 weeks post-vaccination with BCG:CoVac, they remained significantly elevated up to day 42 post-immunization unlike in other immunized groups.

[00123] Since previous work suggests that the level of IgG antibody correlates with NAbs titres after SARS-CoV-2 infection (Suthar et al., 2020), the inventors examined if a similar phenomenon was observed after vaccination with BCG:CoVac. Strong correlation (r> 0.9) was observed between IgG2c isotype and NAbs in groups vaccinated with BCG:CoVac or Alum^SpK (Fig. 7b), with a significant yet less robust correlation between IgGl and NAbs for these groups (Fig. 7c). There was no correlation between NAbs and either IgGl or IgG2c Ab for mice vaccinated with BCG^SpK alone (Fig. 7d, 7e). [00124] These data suggest alum is required for the optimal generation of NAbs after BCG:CoVac vaccination. This is a significant advantage for implementation of this vaccine candidate, due to the low cost and long standing safety record of alum. Importantly, the potential risk of vaccine-associated enhanced respiratory disease (VAERD) due to the selective induction of Th2 by alum is offset by the strong Thl immunity induced by BCG:CoVac, driven by BCG.

Example 6 - BCG:CoVac affords sterilizing immunity against SARS-CoV-2 infection in

K18-hACE2 mice

[00125] Wild-type mice are not permissive to SARS-CoV-2 infection, due to incompatibility in the receptor binding domain of the viral spike protein with the murine angiotensin-converting enzyme 2 (ACE2). Transgenic mice expressing the human (h)ACE2 such as the K18-hACE2 mouse, are highly susceptible to SARS-CoV-2 infection, succumbing to lethal infection within 7 days post-infection. The inventors next assessed the protective role of BCG or BCG:CoVac vaccination in SARS-CoV-2 infection in K18-hACE2 mice. Mice were vaccinated 21 days prior to inoculation with 10³ PFU SARS-CoV-2 (Fig. 8a). Mice sham vaccinated with PBS succumbed to infection within 6 days with substantial deterioration in condition with high clinical scores (Fig. 8b) and 20% weight loss (Fig. 8c). This was associated with high viral titres in the airways (bronchoalveolar lavage fluid, BAFF) (Fig. 8d) and lung tissues (Fig. 8e). These events led to extensive lung inflammation with substantial increases in inflammatory cells in the airways (Fig. 8f) and lung tissue (Fig. 8g), and pro-inflammatory cytokine, IF-6, KC (murine equivalent of IF- 8) and MCP-1 mRNA levels in the lung tissues (Fig. 8h) and airways (data not shown). MCP-1 was increased in serum (data not shown). These are the archetypal cytokines associated with human COVID-19 (Yang et al., 2020). Vaccination with BCG showed some beneficial effects and partially protected against weight loss (-10%) and lung IF-6 and KC responses but not other disease features. Remarkably, vaccination with BCG:CoVac 21 days prior to infection completely protected against infection, with no observable weight loss or any clinical scores throughout the duration of the experiment (Fig. 8b, 8c). These mice had no detectable virus in the airways or lungs (Fig. 8d, 8e). They had few signs of lung inflammation with moderate levels on inflammatory cells in the airways and virtually none in the lung tissue (Fig. 8g), and only baseline levels of all pro-inflammatory cytokines in the airways, lung and serum (Fig. 8h). Additionally, combination of spike and alum with BCG did not alter efficacy of the BCG vaccine against aerosol M. tuberculosis in mice (Fig 8i).

[00126] Collectively, these findings demonstrate that single dose administration of BCG:CoVac is sufficient to completely protect mice from the development of COVID-19 manifestations, neutralizing infectious SARS-CoV-2 and preventing pathogenic inflammation in the lung.

Example 7 - Broadening of BCG.Co Vac immunity against SARS-CoV-2 by heterologous vaccine boosting

[00127] COVID-19 subunit vaccines typically display poor immunity after a single dose and require a booster to induce sufficient generation of NAbs. Whilst high-titre NAbs were observed as early as two weeks post-BCG:CoVac vaccination (Fig. 7), the inventors sought to determine if responses could be further augmented by boosting with a prototype subunit vaccine (Alum^SpK) (Fig. 9a). At 7 days post-boost (day 28), IgG2c titres in plasma from mice primed with BCG^SpK or BCG:CoVac were increased and remained elevated up to day 42 (Fig. 9b). Corresponding augmentation of NAbs was also seen in these boosted groups, with significantly elevated responses in BCG:CoVac primed mice boosted with Alum^SpK (Fig. 9c). Boosting Alum^SpK vaccination with a second dose led to a greater than 10-fold increase in NAbs in boosted mice, however responses were significantly higher in those with the BCG:CoVac -prime, Alum^SpK" boost combination (Fig. 9c). Strikingly, plasma from BCG: Co Vac- vaccinated mice was able to neutralize both the B.1.1.7 variant (1.3-fold decrease compared to wild-type virus) and B.1.351 variant (2.7-fold decrease) (Fig. 9d). Neutralization capacity against B.1.1.7 and B.1.351 was maintained to some extent after prime-boost with Alum^SpK only, however titres were approximately 10-fold less than seen with the BCG:CoVac prime, Alum^SpK combination (Fig. 9e).

[00128] Taken together, these data indicate that the antigen-specific immunity imparted by BCG:CoVac can be further enhanced by heterologous boosting with a second SARS-CoV-2 vaccine, with this vaccination regime able to induce antibodies that can neutralize key VOCs.

References

Amanet et al., 2020, Nature Medicine 26:1033-1036

Chen et al., 2020. J Clin Invest 130:2620-2629

Del Guidice et al., 2018, Seminars in Immunology 39:14-21

Grange et al., 1983, Tubercle 64:129

Honda-Okubo et al., 2015,. J Virol 89:2995-3007

Lederer et al., 2020, Immunity 53:1281-1295

McCray et al, 2007, J Virol 81:813-821

Peeples, 2020, Proc Natl Acad Sci USA 117:8218-8221

Xi et al., 2020, Molecules 25:5392

Xu et al., 2020, Lancet Respir Med 8:420-422

Yang et al., 2020, J Allergy Clin Immunol 146:119-127

Claims

Claims

1. A method for inducing the generation of neutralising antibodies against a pathogen in a subject, comprising administering to the subject an immunogenic formulation comprising an attenuated Mycobacterium strain and at least one antigen from said pathogen.

2. A method according to claim 1, wherein the attenuated Mycobacterium strain expresses at least one protein from the pathogen, or an antigenic fragment thereof.

3. A method according to claim 1 or 2, wherein the attenuated Mycobacterium strain comprises or is derived from the Bacille Calmette-Guerin (BCG) strain.

4. A method according to any one of claims 1 to 3, wherein the immunogenic formulation further comprises at least one adjuvant.

5. A method according to claim 4, wherein the adjuvant comprises aluminium hydroxide, delta inulin or a combination of delta inulin and CpG dinucleotides.

6. A method according to any one of claims 1 to 5, wherein the pathogen is a coronavirus.

7. An immunogenic formulation for inducing an immune response to a coronavirus or for preventing or treating coronavirus infection, the formulation comprising an attenuated Mycobacterium strain and at least one coronavirus antigen.

8. An immunogenic formulation according to claim 7, wherein the coronavirus is a severe acute respiratory syndrome -related coronavirus.

9. An immunogenic formulation according to claim 7 or 8, wherein the coronavirus is SARS-CoV-2.

10. An immunogenic formulation according to any one of claims 7 to 9, wherein the attenuated Mycobacterium strain comprises or is derived from the Bacille Calmette-Guerin (BCG) strain.

11. An immunogenic formulation according to any one of claims 7 to 10, wherein the at least one coronavirus antigen comprises a surface glycoprotein of the coronavirus, or an antigenic fragment thereof.

12. An immunogenic formulation according to any one of claims 7 to 11, wherein the surface glycoprotein is a coronavirus spike protein.

13. An immunogenic formulation according to claim 12, wherein the coronavirus spike protein is the spike protein of SARS-CoV-2.

14. An immunogenic formulation according to claim 13, wherein the spike protein comprises the amino acid sequence of SEQ ID NO:l, or a sequence having at least or about 85% sequence identity thereto.

15. An immunogenic formulation for inducing an immune response to a coronavirus or for preventing or treating coronavirus infection, the formulation comprising a recombinant attenuated Mycobacterium strain that expresses at least one coronavirus protein or an antigenic fragment thereof.

16. An immunogenic formulation according to claim 15, wherein the coronavirus is a severe acute respiratory syndrome -related coronavirus.

17. An immunogenic formulation according to claim 15 or 16, wherein the coronavirus is SARS-CoV-2.

18. An immunogenic formulation according to any one of claims 15 to 17, wherein the attenuated Mycobacterium strain comprises or is derived from the Bacille Calmette-Guerin (BCG) strain.

19. An immunogenic formulation according to any one of claims 15 to 18, wherein at least one coronavirus antigen comprises a surface glycoprotein of the coronavirus, or an antigenic fragment thereof.

20. An immunogenic formulation according to any one of claims 15 to 19, wherein the surface glycoprotein is a coronavirus spike protein.

21. An immunogenic formulation according to claim 20, wherein the coronavirus spike protein is the spike protein of SARS-CoV-2.

22. An immunogenic formulation according to claim 21, wherein the spike protein comprises the amino acid sequence of SEQ ID NO:l, or a sequence having at least or about 85% sequence identity thereto.

23. An immunogenic formulation according to claim 21, wherein the spike protein or antigenic fragment thereof is encoded by the nucleotide sequence of SEQ ID NO: 4, or a portion thereof, or a sequence having at least or about 85% sequence identity thereto.

24. An immunogenic formulation according to any one of claims 7 to 23, wherein the immunogenic formulation further comprises at least one adjuvant.

25. An immunogenic formulation according to claim 24, wherein the adjuvant comprises aluminium hydroxide, delta inulin or a combination of delta inulin and CpG dinucleotides.

26. A vaccine composition against coronavirus, comprising an immunogenic formulation according to any one of claims 7 to 25.

27. A method for inducing an immune response to a coronavirus in a subject, comprising administering to the subject an immunogenic formulation according to any one of claims 7 to 25 or a vaccine composition according to claim 26.

28. A method for preventing or treating a coronavirus infection and/or a disease or disorder caused by a coronavirus infection, comprising administering to a subject in need thereof an immunogenic formulation according to any one of claims 7 to 25 or a vaccine composition according to claim 26.

29. A method according to claim 28, wherein the disease or disorder caused by the coronavirus infection is COVID-19.