Picture source: Dr. Matt Delisa, Locally Sourced Science
The development, production, and manufacturing of large-scale vaccines are necessary for control of the pandemic internationally- to divide the load as well as keep vaccines up to date with the emerging variants- especially in underdeveloped countries. The vaccination has turned into a race between SARS-CoV-2 and scientists, where we saw the emergence of new SARS-CoV-2 variants of concern and variants of interest, and scientists are not far behind with over 320 vaccine candidates developed amongst which 126 are already in the clinical phase, till date. The currently used vaccines render reduced protection against newly emerging SARS-CoV-2 variants and the systemic immunity induced by intramuscular vaccines tends to diminish over time. On the other hand, intranasal vaccines target the primary entry route of SARS-CoV-2 to induce a local mucosal immunity (along with a systemic immune response), gaining an advantage over the currently available intramuscular vaccines. Intranasal vaccines could also potentially prevent further transmission of the virus. Even more, intranasal vaccines are needle-free, cost-effective as well as easy to administer. Recent research propose using bacterial outer membrane vesicles (OMVs) as a platform for vaccines.
OMVs are bacterial outer membrane-enclosed spherical nanoparticles spontaneously released by gram-negative bacteria and are composed of bacterial antigens in native conformation, usually incorporated in their membrane. Although OMVs resemble the bacteria in their membrane and cargo composition, they are not capable of replication or pathogenesis. OMVs carry pathogen-associated molecular patterns (PAMPS) such as lipopolysaccharides, lipoproteins, and polysaccharides which can initiate an immune response from antigen-presenting cells through their pattern recognition receptor (PRR) signaling, finally resulting in a strong adaptive immune response. Altogether, OMVs tend to be a promising vaccine or adjuvant. FDA-approved OMV vaccines include MenB (Neisseria meningitis serogroup B) vaccine and 4CMenB (Bexsero) vaccine, which are already improving healthcare all over the world. OMVs are also demonstrated to be more immunogenic than previously developed vaccines against bacterial infections of N. gonorrhea, Mycobacterium tuberculosis, Klebsiella pneumoniae, and Salmonella. OMVs can be easily engineered and produced in a large scale, and remain extremely stable at 2-8 °C, making the vaccines have a longer shelf-life and ergo, more convenient distribution from the place of manufacture to other countries in the world.
In this blog article, we will shine a light on three preclinical OMV-based vaccines against SARS-CoV-2. In the first study from Bazil, successful cellular and humoral immune response were elicited by Neisseria meningitidis OMVs carrying recombinant receptor-binding domain (rRBD) of the spike protein from SARS-CoV-2. The viral structural glycoprotein called ‘spike’ serves a key role in cellular receptor recognition and cell membrane fusion for entry of the virus into targeted cells. Specifically, RBD of the spike protein binds to a receptor in human cells- angiotensin-converting enzyme 2 (ACE2)- to mediate viral entry, making RBD an attractive vaccine candidate. OMVs from N. meningitidis and aluminum hydroxide were chosen as adjuvants. Aluminum hydroxide is safely administered to humans in various vaccines including Bexsero, and N. meningitidis OMVs have been extensively confirmed to be safe and effective. This vaccine was provided twice intramuscularly, followed by twice intranasally. While all the doses incorporated OMVs as adjuvants, only intramuscular doses contained aluminum hydroxide. Both the adjuvants mixed with rRBD induced significantly higher IFN-gamma and IL-17 producing cells in splenocytes. On 15 and 45 days after the first immunization dose, IgG production increased significantly as compared to rRBD administration alone. IgA was also produced after 37 days, ie, after the first boost of intranasal vaccination.
Another OMV-based intranasal vaccine (OMV-mC-Spike) from Neisseria meningitidis is being developed by Intravacc B. V. scientists in the Netherlands to meet the demand for vaccination against SARS-CoV-2. In their tailored bacteria, they reduced endotoxicity of the OMVs by genetically detoxifying LPS into lpxL1 and deleting the immunogenic porA gene. Then they increased bacterial blebbing for OMV release by deleting the rmpM gene. And here’s how they engineered OMVs to carry SARS-CoV-2 antigens- infectious virions consist of prefusion conformation of spike protein which gets converted into postfusion form following ACE2-RBD interaction. The prefusion form of the spike protein presents the neutralizing epitopes of the virus to targeted antibodies- hence most vaccine formulations (S-2P in mRNA vaccines, Hexapro in NDV-HXP-S) use prefusion stabilized spike protein. The scientists in this study fused Hexapro spike protein with OMVs by the interaction between the positively charged region of mCRAMP (mC- a recombinant amphipathic peptide in the spike molecule) to negatively charged phosphate groups of LPS present on OMVs. This interaction can facilitate the membrane insertion of Hexapro spike protein. After the OMV-mC-Spike vaccine preparation, the immunogenicity of this vaccine with intranasal delivery was shown in mouse and subsequently, its throat and lung protection were demonstrated in a Syrian hamster model. This intranasal vaccine produced high IgA titers in mouse serum, nose, and lungs. In the hamster model, challenging with SARS-CoV-2 after intranasal vaccination with OMV-mC-Spike showed that vaccinated lungs had almost no damage, viral loads were lower, and there was no significant weight loss- showing that the vaccine confers protection. No adverse effects were observed in either of the models. However, unlike the previous study from Brazil, this study showed that the presence of an mCRAMP tag is better to generate an efficient immune response; and that only intranasal delivery of OMV+Spike vaccine can induce sufficient protection.
Researchers from Italy have designed a vaccine based on Escherichia coli OMVs. The main principle behind the vaccines is: OMVs get phagocytosed by antigen-presenting cells (APCs) and the antigens which were being carried by the OMVs are presented on these APCs- resulting in Th1 and antibody response to neutralize those antigens. Previous research showed that faster induction of Th1 response led to less severe COVID-19 and stronger memory T cells, indicating great potential in OMV vaccines. The E. coli OMVs were engineered to carry receptor binding motif (RBM- the portion of spike protein which interfaces the host ACE2 receptor) and were administered intraperitoneally with aluminum hydroxide (adjuvant) in K18-hACE2 transgenic mice (can succumb to severe SARS-CoV-2 infection). The recombinant RBM polyproteins were fused with FhuD2 lipoprotein from Staphylococcus aureus which would assist the delivery of the protein to the surface and inner cargo of the OMVs derived from a hypervesiculating mutant of E. coli. Sera obtained from the mice showed the presence of a high titer of neutralizing anti-spike antibodies. When the immunized mice were challenged with SARS-CoV-2 infection, vaccinated mice developed a much milder disease and lesser inflammation as compared to control mice and viral titer was almost undetectable in the lungs, indicating that vaccination prevented virus replication in the respiratory tract. Moreover, replacing the RBM in the OMV with RBM derived from other SARS-CoV-2 variants also induced protection against those variants. It should be kept in mind that this study was different than the aforementioned studies in a few ways: 1. The vaccine was delivered intraperitoneally instead of intranasally, 2. E. coli OMVs were used in place of N. meningitidis OMVs, 3. The virus antigen was incorporated in the OMV surface, as well as intra-vesicular compartment, 4. PBS was used as a control in place of vector, adjuvant, or viral antigen only, and 5. Only this study showed the effectiveness of the vaccine against other SARS-CoV-2 variants.
Positive outcomes of intranasal delivery of vaccines have been demonstrated already where intranasal administration of ChAdOx1 nCoV-19 vaccine reduced virus shedding in rhesus macaque models. Moreover, IgA antibodies do not contribute to inflammation because unlike IgG, they do not activate the complement pathway. We are even more hopeful, as previous studies confirmed that IgA antibodies induced by influenza are cross-protective across different strains and can also generate resident memory T and B cells. Extensive studies on the role of mucosal immunity and IgA antibodies will expand our knowledge on the positive outcomes or possible side effects of these intranasal vaccines.
Peter A. van der Ley, Afshin Zariri, Elly van Riet, Dinja Oosterhoff, Corine P. Kruiswijk. An intranasal OMV-based vaccine induces high mucosal and systemic protecting immunity against a SARS-CoV-2 infection. bioRxiv 2021.08.25.457644; doi: https://doi.org/10.1101/2021.08.25.457644
Alberto Grandi, Michele Tomasi, Cinzia Bertelli, et al. Immunogenicity and pre-clinical efficacy of an OMV-based SARS-CoV-2 vaccine. bioRxiv 2021.07.12.452027; doi: https://doi.org/10.1101/2021.07.12.452027
Article author: Sutonuka Bhar. Sutonuka is a PhD candidate majoring in Medical Microbiology and Bacteriology at the University of Florida. Her work focuses on host immune responses against viruses and bacterial membrane vesicles.
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