Lower respiratory infection is one of the top four causes of human deaths globally. Currently, the world is still reeling from the effects of the Coronavirus, and sadly, the virus has contributed to the nearly 3 million deaths recorded annually as a result of respiratory infections. Some of the pathogens responsible for these infections and the deaths include pneumococcus bacteria, respiratory syncytial virus (RSV), and influenza virus. The year 2018 marked the 100th anniversary of one of the deadliest and the largest public health crisis in modern times – the 1918 Spanish Flu. The pandemic was responsible for killing nearly 2% of the global population and in less than two years after the 100th anniversary of the flu, the world is dealing with yet another flu that is threatening to wipe out a significant part of the population – Covid-19.
According to the World Health Organization, the seasonal flu is responsible for nearly half a million deaths every year. Of all these deaths, it is estimated that respiratory syncytial virus (RSV) is responsible for nearly 78% of them, with the most affected group being the pediatric population. RSV was responsible for nearly 34 million cases of lower respiratory infections in children under five years in 2006 globally. This resulted in about 100,000 deaths. RSV is not responsible for a higher mortality rate in the developed countries, but it is a significant scare and a real burden to the healthcare systems of such countries. This is because a huge number of children under the age of 5 years will always require hospitalization whenever they catch the virus. In the United States alone, it is estimated that nearly 86,000 children are hospitalized every year as a result of this virus, and this usually results in nearly $394 million in total costs.
To reduce the burden on the healthcare systems as well as the potential of another influenza pandemic, there is a need for an efficient vaccine. The current vaccines being used to contain flu have representative hemagglutinins of H1N1, H3N2, and various type B viruses. The vaccines have had higher efficacy, however, it usually takes time for the immunity to develop and be in a position to effectively fight off the viruses, especially after someone is affected. Also, there is always a need for the vaccines to be reformulated every year due to the antigenic drifts of the viruses. The virus strains will be different every year, and a vaccine used last year will not be effective on the new strains present in another year. Also, if the vaccines are not reformulated, they will not be effective against an epidemic virus because of the higher chances that the virus used in the formulation of the vaccine will not match the strain of the epidemic virus.
Due to these reasons, there is a need for a universal vaccine to replace the seasonal vaccines and to provide long-lasting protection against both the seasonal viruses, as well as the pandemic strains. For RSV for example, there has been no successful licensing of a vaccine, despite decades of thorough research. The other types of flu may already have vaccines, but as noted earlier, such must be reformulated each year to march the prevailing strain. Since there has been no significant success in coming up with a vaccine to eradicate the seasonal flu, the research world is now turning to antiviral molecules as a treatment to the already affected persons. The current standard care for flu focuses on two proteins – the matrix-2 (M2) or the neuraminidase (NA) protein. M2 is a proton-selective ion channel protein that makes the migration of H+ ions possible in the interior of virus particles. This process usually takes place during endosome acidification and it is also a vital process for the uncoating of viruses. NA attaches itself to the sialic acid which is normally used by the virus to attach to the host receptor. This makes it possible for the virus to be released right from the infected cells and stop its further spread in the host. The current drugs licensed to target M2 are amantadine and rimantadine. They both belong to adamantane derivatives. Those that target NA on the other hand are peramivir and zanamivir. In principle, these drugs are considered as universal antivirals and they can be used in the treatment of all kinds of influenza virus strains. However, resistance strains have been reported in the last two decades and such have become serious issues.
Due to the perennial challenges that have been encountered in the process of developing universal vaccines for flu, recent reports suggest a shift towards the use of peptide-based strategies for designing peptidic macrocyclic compounds that can inhibit the fusion of influenza A group 1 viruses. In a similar manner to broadly neutralizing antibodies – bnAbs, the peptidic macrocyclic compounds target to bind to the conserved HA stem. This approach has the potential of reducing the chances of generating escape mutants. HA is considered as a trimeric metastable protein, with each subunit containing HA2 and HA1 subdomains which are linked with the help of a disulfide bond.
Due to the binding of the cell surface receptors, the actions of both HA1 and HA2 causes the virus to be internalized through the process of endocytosis. Endosomes are known to have a very low pH of 5 and 6, and this is sufficient to trigger a major structural rearrangement of the HA2. Every HA2 has a subunit monomer that also has a long helix connected using an extended loop to a shorter helix. The shorter helix usually has its C-terminus anchored to the viral membrane, while the N-terminus of the short helix is where we find the fusion peptide. As a result of this structure, there is a six-helix bundle formed at the trimeric HA2. Since the short helix is usually antiparallel to the long helix, in the prefusion form, the fusion peptide is normally located next to the viral membrane.
When the pH changes, the extended loop will rotate dramatically to fuse with the long helix such that the new position of the fusion peptide now becomes 1800 relative to the viral membrane and the host cell endosomal membrane. This rearrangement of the structure is the force responsible for the fusion between the host cell endosomal membrane and the viral membrane. It is what leads to the neutralization of the virus just like it is usually the case with bnAbs.
To create such a peptide, researchers used a discontinuous segment found at the complementary determining regions – CDRs as well as Framework Regions – RFs of the bnAbs. A similar approach has been used by other researchers in the past. This is because CDRs are hypervariable loops of the antibodies that bind to the antigen while RFs are variable domains responsible for assisting CDRs to bind more tightly to the antigens. Also, during the design of the peptides, several x-ray structures of the bnAbs attached to the HA stem region were used.
For potential RSV therapies, it is believed that resistance will become a problem with regards to the neutralizing antibodies that are currently in development. The standard care currently being used features prophylactic treatment of at-risk children using palivizumab. Palivizumab is a monoclonal antibody that has been designed to work against RSV F protein. This particular protein is usually administered monthly via injection during the peak flu season. Sadly, low efficacy as well as high costs of production of this remedy has limited the use of this therapy for preterm infants suffering from bronchopulmonary dysplasia and other chronic respiratory diseases. Consequently, 60% of at-risk infants usually go untreated, and sadly, there is still no efficient therapy that is available for treating the adult demographics. Recombinant antibodies that target that aims at targeting the antigenic sites of preF which have been designed to have longer lifespans than palivizumab are currently under development to be used as a way the number of injections that are being used with the present standard care.
In principle, there should be no worries about resistance when it comes to treating young infants using antiviral drugs given that conditions such as RSV are not chronic. In most cases, the viral load will usually go down significantly following the administration of the antiviral, leaving little to no time for the virus to mutate. However, during the clinical trials of presatovir, escape mutants T400I and F140L were identified, suggesting potential future resistance to RSV. Also, resistance is likely to become a key issue for hematopoietic cell transplant – HCT patients. This is because such patients usually have to be on therapy for about six months. During recent clinical settings, some of the most advanced molecules to be identified include Ziresovir, Lumicitabane, and Presatovir. During these trials, Lumicitibane is considered a pro-drug nucleoside polymerase inhibitor while Presatovir is small molecule fusion inhibitors that have been designed to target preF.
It is worth noting that all of the small molecule infusion inhibitors that have been discovered to work against RSV up to the present times are highly likely to interact and bind together with the same binding pocket present in preF. These pockets feature a threefold symmetric cavity which is usually formed at the intersection of the three protomers of preF. When the small molecules are bound to this site, they become tethered to the fusion peptide as well as the heptad repeat 2 – HR2 of F. Consequently, this leads to the stabilization of the metastable preF and also altering the conformational rearrangement of the fusion peptide and HR2. This conformational rearrangement is usually necessary for the fusion of the host cell membrane and the virus. You can think of the inhibitors as a hand fidget spinner toy where each ring of the toy comprises of one aromatic moiety of the inhibitor.
Some of the peptides obtained from the F and HR2 domains may be successful in being used as fusion inhibitors. These peptides can be used to target the transition that happens between postF and preF, with the small molecule escape mutants not having any effects in the inhibitory activity. Currently, RH2 is mainly an unstructured peptide present in an aqueous solution that turns into a-helix when it is bounded to a trimeric HR1 coiled-coil. When a scan of synthetic peptides obtained from 35 amino acids drawn from HR2 wild-type sequence, it led to the identification of T118 and T108 peptides.
These two peptides were proven to have the ability to stop syncytia formation occasioned by a virus. There were attempts to reduce the length of the T108 peptide sequence, but those attempts did not bear any fruits since when the length of the peptide is reduced to less than 30 amino acids, this would effectively abrogate its activities. To improve and enhance the pharmacological properties of the peptide, it was necessary to modify T118 into a series of stabled peptides. The technology for stabling peptides heavily depends on the incorporation of unnatural olefinic amino acids – UUA in precise locations or positions where they will not interfere with the expected binding of the peptides to its target.
It also depends on the subsequent cross-linking of the non-natural amino acids with the aid of Grubbs mediated ruthenium metathesis. The result is a side-chain to side-chain incorporation of a hydrocarbon macrocyclic bridge that increases the affinity of the peptide to the target, as well as the cellular permeability of the peptides through endocytosis uptake mechanism.
The peptides derived from the HR2 domains of F can also be used in the development of pan inhibitors targeting other lower respiratory pathogens. For example, 36-mer peptide obtained from HR2 domain of the human parainfluenza 3 virus (HIPV3) showed the ability to inhibit HIPV3, Nipa Virus (Ni), and Hendra virus (Hev) – very highly pathogenic viruses.
The above is just a subtle look at how antiviral peptides can be used and are being used as remedies for treating lower respiratory viral infections. With the Covid-19 pandemic, the studies have become even more relevant. Though there is still a long way to go, the research world is optimistic that there will be a breakthrough in the formulation of a universal flu vaccine to tackle the seasonal strains as well as the pandemic strains.
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