The course of the corona pandemic will strongly depend on how quickly medications or vaccines against the SARS co-virus 2 can be developed.
In at least one Phase III study, researchers want to investigate whether the vaccine candidate VPM1002, originally developed against tuberculosis by scientists at the Max Planck Institute for Infection Biology, is also effective against infection with SARS-CoV-2.
The large-scale study is to be carried out at several hospitals in Germany and will include older people and health care workers.
Both groups are particularly at risk of the disease. VPM1002 could thus help bridge the time until a vaccine specifically effective against SARS co-virus 2 is available.
VPM1002 is based on a vaccine called BCG, which was developed at the beginning of the 20th century. Studies on mice show that the BCG vaccine can protect not only against tuberculosis but also against viral infections of the respiratory tract.
Accordingly, mice suffering from influenza have fewer influenza A viruses in their blood if they had previously been vaccinated with BCG. The animals thus showed less damage to the lungs.
According to further studies, vaccination with BCG also increases the animals’ resistance to other viruses (e.g. herpes type 1 and 2). Apparently, a vaccination with BCG also activates the immune system against a viral infection. In this way, the vaccine reduces the risk of severe disease progression and thus lowers the death rate.
VPM1002 is safe and more effective than standard vaccination
VPM1002 contains weakened tuberculosis-like bacteria. These are genetically modified in such a way that immune cells can better recognize them.
The vaccine candidate, originally developed at the Max Planck Institute for Infection Biology in Berlin by the group of Stefan H.E. Kaufmann, thus provides more effective protection against tuberculosis than the old vaccine and is intended for use in new-borns as well as for boosting a vaccination in adults.
Recent studies have shown that VPM1002 can also be effective against cancer and prevent the recurrence of bladder tumours.
Scientists have investigated this further development of the BCG vaccine in a series of studies in mice as well as in several clinical trials. In 2018, a Phase II study confirmed that VPM1002 is well tolerated by new borns and is effective.
The vaccine is currently being tested in a further Phase III study on adult volunteers in India. It should be completed by mid 2020.
“Results to date show that vaccination with VPM1002 is safe and more effective than standard vaccination with BCG”, says Stefan H.E. Kaufmann.
Studies on mice show that the BCG vaccine can protect not only against tuberculosis but also against viral infections of the respiratory tract.
The higher safety profile of VPM1002 and the improved effectiveness give reason to hope that the new vaccine will also be better able to alleviate the symptoms of an infection with the SARS co-virus 2 than the BCG vaccine.
“In addition, VPM1002 can be manufactured using state-of-the-art manufacturing methods which would make millions of doses available in a very short time”, says Adar C. Poonawalla, CEO and Executive Director, Serum Institute of India.
Discussions with authorities
The partners involved, Vakzine Project Management (VPM) and the Serum Institute of India, have already held promising discussions with the authorities regarding the implementation of a Phase III study with VPM1002 in Germany in order to investigate the effectiveness of the vaccine in elderly people and healthcare workers.
“These population groups are particularly affected by the current pandemic”, says Leander Grode, Managing Director of VPM “and could therefore particularly benefit from a vaccination with VPM1002”.
If the results are positive, VPM1002 could help ease the burden on healthcare systems until a vaccine specifically effective against SARS-CoV-2 becomes available.
In 2004, the Max-Planck-Gesellschaft granted the license for the vaccine to the company Vakzine Projekt Management (VPM).
In 2012, the company began to further develop the vaccine together with the Serum Institute of India, one of the largest vaccine manufacturers worldwide. The company has now acquired a majority stake in VPM.
Infection with Mycobacterium tuberculosis (Mtb) led to 10.4 million recorded cases of tuberculosis (TB) in 2015, with 1.8 million recorded deaths [World Health Organization (WHO) report 2016].
The current therapy involves 6–9 months of antibiotics, with the emergence of multiple drug resistant strains being a continuing obstacle. An attenuated form of the bovine Mycobacterium species, Mycobacterium bovis bacille Calmette–Guerin (BCG) has been in clinical use since 1921 and remains the only licensed vaccine against TB.
BCG partially protects against TB meningitis and disseminated TB in infants and has non-specific immunostimulatory effects (1), which reduce general infant mortality by enhancing responses to other infectious diseases (2, 3).
However, in all age groups, BCG does not adequately protect against pulmonary TB, the most prevalent form of disease and the route of disease transmission.
In addition, BCG can cause severe adverse effects in immunocompromised individuals (4) and hence is contraindicated in HIV-infected individuals, the group that is most vulnerable to TB.
However, in the absence of an alternative, BCG continues to be used in the immunization programs of several countries. To overcome these issues, several TB vaccine candidates are under development (5). One of the most advanced among them is BCG ΔureC::hly (VPM1002) (6).
VPM1002 is a recombinant BCG (rBCG) in which the urease C gene has been replaced by the listeriolysin O (LLO) encoding gene (hly) from Listeria monocytogenes (7). Urease C drives neutralization of phagosomes containing mycobacteria by generation of ammonia, thereby inhibiting phagolysosomal maturation and contributing to the survival of mycobacteria inside the macrophage (8, 9).
Its depletion allows for rapid phagosome acidification, which promotes phagolysosome fusion and provides the optimal pH for LLO stability (10). LLO is a cholesterol-dependant cytolysin that forms transmembrane β-barrel pores in the phagolysosome membrane, allowing escape of L. monocytogenes into the cytosol (10, 11).
Its expression in VPM1002 results in the release of antigens and bacterial DNA into the cytosol, triggering autophagy, inflammasome activation, and apoptosis. VPM1002 has demonstrated substantially increased immunogenicity, efficacy, and safety in preclinical studies, successfully passed Phase I and II clinical trials, and will now enter a Phase II/III clinical trial in India in 2017.
This review summarizes the development, preclinical, and clinical testing of VPM1002 (Figure (Figure1).
Design and Generation of VPM1002
The attenuation of BCG was achieved by passaging virulent M. bovis in bile-containing medium for 13 years in the laboratory (12), during which time several genome segments were lost, including a segment known as Region of Difference 1 (RD1) which encodes the unique mycobacterial ESX-1 type VII secretion system (13, 14).
ESX-1-dependent perturbation of host cell membranes requires direct contact with pathogenic mycobacteria such as Mtb, allowing the bacilli or their antigens to egress the phagosome into the cytosol (15).
Mtb antigens are thus accessible to both the endocytic major histocompatibility complex (MHC) class II antigen presentation pathway and the MHC I antigen presentation pathway in the cytosol, and consequently can stimulate CD4+ and CD8+ T-cell subsets, respectively, both of which are required for optimal protection against TB (16–21).
In addition, ESX-1 dependent release of Mtb DNA into the cytosol can be detected by host sensors, leading to activation of NLR family pyrin domain-containing 3 (NLRP3) and absent in melanoma 2 inflammasomes, release of interferons, increased autophagy and apoptosis (22–25).
Induction of apoptosis in infected host cells generates vesicles carrying mycobacterial antigens that can be phagocytosed by bystander antigen presenting cells, mainly dendritic cells (DCs) and trafficked through MHC I antigen processing pathways to stimulate CD8+ T cells in a process known as cross-priming (26, 27).
Mice with deficient cross-presentation due to the absence of annexin 1 show impaired Mtb-specific CD8+ T cells and are highly susceptible to TB (28). Lacking the ESX-1 secretion system, BCG is restricted to the phagosome of host cells, therefore its antigens and bacterial DNA do not enter the cytosol and the antigens are primarily processed by MHC class II pathways, stimulating CD4+ T cell responses (13, 14, 29, 30).
BCG induces only weak apoptosis and CD8+ T cell responses (26). Furthermore, both BCG and Mtb inhibit surface MHC II expression, as urease-dependent alkalinization of the phagosome causes intracellular sequestration of MHC II dimers, resulting in suboptimal CD4+ T cell responses (31–33). Phagosomal biology is therefore a clear target for interventions aimed at enhancing T cell responses against mycobacteria.
Originally, VPM1002 was designed to improve accessibility of mycobacterial antigens to the MHC I pathway via cytosolic egression of antigens mediated by LLO perturbation of phagosomal membranes in order to improve induction of CD8+ T cells by the parental BCG strain (34, 35).
In addition, leakage of phagolysosomal proteases such as cathepsins into the cytosol could activate caspases, leading to apoptosis and subsequent cross-presentation of mycobacterial antigens, which promotes both MHC I and MHC II restricted T cell stimulation (36).
Studies with L. monocytogenes have shown that pore formation by LLO also triggers many downstream effects such as activation of the NLRP3 inflammasome, induction of cytokine expression, activation of kinases, triggering of endocytosis, histone modification and release of calcium from intracellular stores (37).
An Hly recombinant strain, hly+ rBCG+, was generated by integrating the hly gene into BCG using the mycobacteria-Escherichia coli shuttle vector pMV306 (34). LLO was detected in the membrane structures, phagosomal space, and cytoplasmic vacuoles of macrophages infected with BCG pMV306::hly, and intracellular persistence of this strain was reduced compared with the parental BCG strain.
MHC I presentation of co-phagocytosed soluble protein was improved in macrophages infected with this strain compared to BCG (34) and an in vitro human cytotoxic T lymphocyte (CTL) assay using cultured DCs and T cells from healthy human donors demonstrated that hly+ BCG infection was better at inducing CTL responses than BCG infection (38).
In the next generation strain, deletion of ureC was performed to ensure an optimal (acidic) pH for LLO stability; however, absence of ureC also promotes MHCII trafficking to the macrophage surface (31), which would also stimulate CD4+ T cell responses. To generate ΔureC hly+ BCG, the chromosomal integrative shuttle vector pMV306hyg-hly (8) was used to transform M. bovis BCG ΔureC::aph, and hygromycin-resistant clones were selected (35). The vaccine was licensed to Vakzine Projekt Management, and named “VPM1002.”
The resistance cassette was subsequently successfully removed, although VPM1002 is equally sensitive to the antimycobacterial agents isoniazid, rifampicin, and ethambutanol in the presence or absence of the hygromycin resistance gene (39).Go to:
Host Cell Responses to VPM1002 In Vitro
Increased quantities of mycobacterial antigen were detected in VMP1002 infected macrophages compared to BCG infected macrophages (35), and mycobacterial DNA was detected only in the cytosol of VPM1002 infected but not BCG infected macrophages (29), indicating that expression of LLO in BCG ΔureC::hly allows the escape of bacterial products to the cytosol, presumably by perturbation of the phagosomal membrane. The bacteria themselves do not escape to the cytosol, unlike Mtb bacilli (29, 35).
Infection of primary human and mouse macrophages demonstrated increased apoptosis after infection with VPM1002 compared to both BCG and BCG::hly, demonstrating the additional benefit of urease C deletion (35).
Membrane disruption can facilitate the release of phagolysosomal proteases such as cathepsins into the cytosol, which are known to induce apoptosis (36, 40). Both the presence of mycobacterial proteins in the cytosol and the induction of apoptosis by perforation of the phagosomal membrane could cause increased trafficking of antigens to MHC I pathways (35). Apoptosis results in an increase in both CD8+ and CD4+ T cell responses in mycobacterial infection, suggesting that DCs may transfer efferocytosed antigens to the endocytic system (27, 36).
The priming potential of apoptotic vesicles isolated from BCG and VPM1002 infected mouse macrophages was investigated in a co-culture system with splenic DCs and T cells, and VPM1002-infected apoptotic vesicles induced more profound CD4+ and CD8+ T cell responses compared to those infected with BCG (41).
Vesicles from VPM1002 infected macrophages also induced higher production of the T helper type (Th)17-polarizing cytokines interleukin (IL)-6 and IL-23, and the immunoregulatory cytokine IL-10 by bone marrow-derived DCs.
Experiments in THP1 macrophages demonstrated that VPM1002 infection leads to activation of multiple caspases (29). The apoptotic effector caspases 3 and 7 were highly activated by VPM1002 in comparison to BCG, as well as caspase 1, which mediates pyroptosis, an inflammatory form of cell death and is an important regulator of the inflammatory response (42).
Inflammasomes are multi-protein complexes composed of intracellular sensors and caspase 1. They control activation of caspase 1, which in turn cleaves the precursors of the cytokines IL-1β and IL-18 into their active forms (43).
VPM1002 infection increased production of IL-1β and IL-18, which was dependent on AIM2 inflammasome activation but not on NLRP 1 and 3 inflammasome activation.
Furthermore, VPM1002 induced increased levels of the autophagy marker microtubule-associated protein light chain 3 in an AIM2- and stimulator of interferon genes (STING)-dependent manner.
The AIM2 inflammasome senses cytosolic DNA and is involved in the induction of caspase 1-dependent pyroptosis (44, 45), while STING acts as an essential adaptor protein in the induction of autophagy by cytosolic DNA (25).
Autophagy, a protein degradation process induced by stress conditions such as infection, promotes the delivery of cytosolic antigens to MHC trafficking pathways (46, 47). It has also been shown to contribute to innate immunity against mycobacteria and other intracellular pathogens (48, 49).
While autophagy was originally thought to be non-specific, it is now known that it can selectively target intracellular pathogens in a process known as xenophagy that involves ubiquitination of pathogen proteins or pathogen-containing endosomes (50).
Intriguingly, gene expression of guanylate-binding proteins (GBPs) was also elevated in VPM1002 infected THP-1 macrophages compared to BCG infected macrophages. Interferon-inducible GBPs have multiple roles in inflammasome activation, autophagy, and lysis of pathogen-containing vacuoles and can even directly target the pathogens themselves (51–54). Whether they play a role in the translocation of mycobacterial components from the phagosome into the cytosol during VMP1002 infection remains to be determined.
Disruption of the VPM1002-containing phagosome membrane by LLO and release of mycobacterial DNA into the cytosol appears to have effects in inducing immune responses that are similar to the effects of ESX-1 activity in Mtb or M. marinum. ESX-1 of M. marinum stimulates autophagosome formation and recruitment to the vacuole; however, unlike LLO it also inhibits autophagic flux, thereby preventing bacterial degradation (49). T
esting of vaccine candidates expressing ESX-1 such as Mtb Δppe25-pe19 (55) and BCG expressing ESX-1 of M. marinum (BCG:ESX-1Mmar) (56) demonstrated that ESX-1 was critical for enhancing innate immune responses via phagosome rupture.
BCG:ESX-1Mmar induced the cGas/STING/TBK1/IRF-3/type I interferon axis and promoted AIM2 and NLRP3 inflammasome activation, resulting in increased frequencies of antigen-specific CD8+ and CD4+ T cells and increased protection against Mtb compared to BCG (56), while Mtb Δppe25-pe19 also led to enhanced protection.
ESX-1 may induce protective immunity by an additional mechanism, as ESAT6 is required for rapid, non-cognate IFN-γ production by CD8+ T cells, mediated by the NLRP3/caspase-1/IL-18 axis (57).
Max Planck Institute
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