The world has recently seen immense activity in the field of vaccine development due to the coronavirus disease 2019 (COVID-19) pandemic, as the causative virus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to emerge in new variants with increased transmissibility, immune escape capability and virulence attributes.
A new paper describes a novel route of prevention using messenger ribonucleic acid (mRNA) molecules that encode protective antibodies within the recipient.
The use of mRNA to encode vaccines in the form of SARS-CoV-2 spike protein was pioneered by the Pfizer/BioNTech and Moderna vaccines. These were among the many vaccine technologies pressed into use to counter the relentless spread and the rising death toll of the COVID-19 pandemic.
In addition, monoclonal antibodies were also isolated for their neutralizing activity against the virus, both pre-and post-exposure. Emergency use authorization (EUA) was obtained for four mAb protocols: the therapeutic mAbs casirivimab + imdevimab, bamlanivimab + etesevimab, and sotrovimab and the prophylactic mAbs tixagevimab + cilgavimab. Of these, the first two are no longer effective, and their EUA has been withdrawn.
These antibodies need to be given intravenously, necessitating a medical setting. They must be delivered in relatively large doses of 10–100 mg kg−1 to compensate for the fact that only a small fraction reaches the site of interest. This drives up the cost of treatment, limiting their availability, especially in low- and middle-income countries (LMIC).
Alternative methods of mAb production need to be explored. The current paper, published in Advanced Science, discusses such an alternative, where a formulation amenable to nebulization was designed to allow the introduction of antibody-encoding mRNA into the lungs to neutralize the disease. Both in vitro and in vivo results support the use of this novel technology to fight not only COVID-19 but also other respiratory virus infections.
The use of mRNA is safe in that it does not enter the nucleus, unlike DNA or viral vectors carrying DNA, which relies on their effect on nuclear entry and integration with the host DNA genome. Secondly, the half-life of mRNA in circulation is relatively short, avoiding long-term consequences. By reducing the required dose to a hundredth of the original amount, when delivered directly to the respiratory tract rather than systemically, the overall cost of therapy is significantly decreased.
Prior research showed that this approach was feasible, using intravenous lipid nanoparticle (LNP)-encapsulated mRNA encoding a neutralizing antibody against the chikungunya virus, which would be expressed in the liver.
Earlier research by the same authors showed the capability to direct mRNA to the lungs to produce mAbs there. By avoiding the need to introduce the recombinant spike protein, the researchers encoded a membrane anchor in the heavy chain of the immunoglobulin G (IgG) antibody molecule. This allowed the tissue to retain the antibody for several weeks.
The current study moved a step further by shifting from the earlier intratracheal administration to nebulization, which can be performed by the individual outside a medical setting. This would allow the mAbs to be expressed at high concentrations at the mucosal surface, the principal site of entry of respiratory viruses, while avoiding the need to administer large doses systemically.
What did the study show?
The study findings show the ability of nebulized mRNA-encoded neutralizing antibody (nAb) to counter SARS-CoV-2 infection in the hamster lung, reducing the viral count and mitigating lung disease signs as well as infection-related weight loss in the hamsters.
The glycosylphosphatidylinositol (GPI) membrane anchoring molecule allowed the antibodies to remain linked to the cell membrane. The antibodies tested here comprised six mAbs isolated from B cells taken from SARS-CoV-2-infected individuals.
Direct stochastic optical reconstruction microscopy (dSTORM) enhanced the ability to view the antibodies once expressed within the cell culture. One formed long strings on the cell surface and was thus disqualified from further testing in vivo.
All the bound and anchored mAbs retained neutralizing capacity, as shown by their cytopathic effect (CPE) in a monolayer cell culture. Though most of them were able to neutralize the original variant and the B.1.1.7 variant, one failed to offset the latter. The protective capability was linked to the specific mRNA-encoded antibodies and not to the GPI anchor, as shown by a control antibody with an attached GPI.
All the mAbs could neutralize the virus at low concentrations, with low nanomolar half-maximal inhibitory concentration (IC50). Further testing was carried out using two selected mAbs, COV2-2832 and DH1041.
Following these promising in vitro findings, further testing was carried out in Syrian golden hamsters, which provide a robust animal model for human COVID-19 infection.
Longer retention in lungs
This demonstrated that the antibodies were, as expected, anchored to the cell membrane in the lung by the GPI molecule. Compared with the secreted or non-anchored form, it was found to remain in the lung tissue. At the same time, the latter led to increased serum concentrations, the difference in post-transfection serum levels being 27-fold in favor of the secreted form. This occurred despite the efficient translation of both types in lung tissue following nebulization, peaking at 24-48 hours.
The encoded anchor enhanced lung retention from just over one day to seven days, which could mean a single-dose approach to the post-exposure treatment of COVID-19.
This approach would not be onerous compared with other nebulizer-based treatments, which typically consist of multiple doses a day or a single dose for up to 22 h.”
The nebulized formulation reached all parts of the lung uniformly and richly, both the alveolar space and the airway epithelium.
Since the virus is mostly found within the alveolar space, this finding indicates the potential for transfected antibodies to prevent COVID-19 and severe disease. The effective dose delivered to the lungs can be enhanced by increasing the concentration of the formulation.
The ratio of mRNA delivered to the lung to total delivered mRNA is about 12% in hamsters but is likely to be much higher, nearer to 30-50%, in larger mammals. This would mean that a much smaller amount of the nebulized formulation would be required to achieve the required dosage.
These data indicate that a low dose of mRNA can achieve high expression of durable mAb constructs across much of the hamster lung alveolar and airway epithelial compartments, with minimal pulmonary toxicity.”
Moreover, the hamsters treated with the mRNA-encoded mAbs before being inoculated two days later with the virus showed 1% weight gain by day 5, starting from the second day of treatment. At the same time, untreated exposed controls lost 5% of their body weight by the fifth-day post-inoculation.
Examination of the lung tissue from treated hamsters and controls showed lower viral titers by over 80% in the former, compared to controls, with an ~80% decrease in the viral RNA load as measured by quantitative polymerase chain reaction (qPCR). Lung pathology was also largely mitigated in treated hamsters.
The study also suggested that weight loss was the most reliable correlate of the animal’s health, while lung pathology reflects the strength of the immune response rather than the intensity of viral replication. The delivery of the mRNA did not cause lung inflammation or damage to any lung tissue.
What are the implications?
Despite the use of human mAbs in a hamster model, which shows sub-optimal Fc-mediated immune functions, the current study demonstrated the ability of these antibodies, when expressed by nebulized mRNA within the lung tissue, to induce an encouraging degree of protection against the virus. This appears to be a fruitful option for passive immunization that cuts short the time required for the host to neutralize the virus following infection while overcoming any deficiency of the host immune system itself.
The nebulization approach allows for self-administration, ensuring wider distribution in low-resource settings.
Both mRNA-expressed COV2-2832 and DH1041 are a clear complementary prophylactic strategy to the therapies currently in use.”