By Guillaume Trusz and Brian Zabel
As any scary-movie fan will tell you, it is oftentimes what we do not see that we should fear the most. Over the last couple of months, the world has been rocked by an invisible intruder that has become ingrained in our everyday lives. The coronavirus disease (COVID-19) has exposed our vulnerabilities and changed our outlooks on life forever. Nevertheless, outbreaks of severe viral diseases remain common, such as H1N1 in 2009, Ebola in 2014, or even Zika in 2016. In order for these outbreaks to cause disease on a grand scale, several conditions must come together perfectly, or in the case of James Reason’s Swiss Cheese model, all of the holes become ideally aligned with every slice. The ease of viral transmission, delayed manifestation of clinically relevant symptoms, high population density, and interconnectedness amongst major cities have each contributed to the wide spread of these viruses, which would lead one to think that we would be used to these spillover events by now. And yet, it seems that each time we are left scrambling for answers.
For many people (authors included), the last couple of months have been one of the most stressful and purely exhausting times to date but despite all that, these are exhilarating times for scientific researchers and medical professionals alike. Novel molecular entities and numerous drugs stashed away in various pipelines have surfaced, each one carrying with it the hopes and dreams of a solution. In this post, we assess the global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) preventative and therapy landscape to shed some light on some of the most promising treatments comprising small molecules, vaccines whose overall goal is to stimulate the development of virus-specific antibodies, and of course antibodies themselves!
Sanofi – Plaquenil® (hydroxychloroquine) and generic manufacturers (Teva/ Mylan/ Novartis)
Chloroquine and hydroxychloroquine, both discovered at Bayer in the first half of the 20th century in an attempt to synthetically produce quinine-like compounds, are currently used to treat and prevent malaria. Over time, both small molecules made their way into the hands of people suffering from autoimmune diseases, such as lupus and rheumatoid arthritis, and more recently were considered as treatments for COVID-19. Of the two compounds, hydroxychloroquine has shown more potential due to a superior half maximal effective concentration (EC50) value. Although the exact mechanism of action of hydroxychloroquine against malaria is debated, hydroxychloroquine presumably works against COVID-19 by preventing the terminal glycosylation of the angiotensin-converting enzyme 2 (ACE2), a protein located on the outer cell membrane of human lung cells which serves as the receptor for the SARS-CoV-2 spike protein. Unglycosylated ACE2 proteins interact differently with the viral spike protein which may decrease the chance of viral entry into those cells. Without internalization, the viral particles cannot replicate and thus cannot be as virulent. Although hydroxychloroquine has not been approved by the Food and Drug Administration (FDA) for treating COVID-19, it received Emergency Use Authorization (EUA), meaning the FDA authorized its use in a state of emergency. However, a myriad of side effects and contraindications continued to cloud its general application and the EUA was revoked in mid-June 2020.
Gilead Sciences– GS-5734 – Veklury® (remdesivir)
Gilead, the big player when it comes to treating virally transmitted diseases such as HIV as well as hepatitis B and C viruses, seems strategically placed to develop a COVID-19 treatment. Remdesivir was originally developed for a hepatitis C indication, and most recently was tested for the treatment of Ebola but failed to compete against antibody therapies. The small molecule, which mimics a ribonucleic acid (RNA) nucleotide, inhibits transcription and the formation of new RNA sequences. The abnormally shaped and truncated RNA sequences that are produced cannot serve the same purpose as was originally intended. Any RNA-based viruses (such as SARS-CoV-2) are hindered by such a process and simply cannot replicate their genome using the cellular machinery.
This repurposing of drugs from one virus to another seems a little odd at first but having gone through years of research and development, remdesivir has proven to be relatively safe and well tolerated by patients and has allowed the development of a full safety profile; in fact certain biotech companies today are built entirely on the premise of repurposing. Despite also receiving an EUA from the FDA, remdesivir has so far only shown modest improvements in patients who have contracted the virus, allowing some of them to recover in 10 days rather than the usual 14 days.
Moderna Therapeutics – mRNA-1273 – Liposome-based mRNA vaccine – Phase III Clinical Trial Recruiting
Most people (scientists included) had not heard of Moderna (or Mode-RNA as the name suggests) prior to recent events. However, Moderna is well-known in the world of finance, previously being designated as a “unicorn” (over $1 billion in private funding) and eventually launching the largest initial public offering (IPO) for a pre-commercial biotech company yet. Despite all that, Moderna has operated in the shadows, oftentimes appearing elusive in terms of performance.
On to the science. Liposomes are spherical vesicles consisting of at least one lipid bilayer. They can encapsulate and transport almost anything from synthetic drugs to larger macromolecules and have played a major role in both medicine and research for years. Doxil, which received FDA approval in 1995, became the first approved chemotherapeutic nanoparticle and lipofection remains one of the most common techniques in genetic transfection. The power of liposomes lies not only in their ability to be optimized in terms of choice of lipid composition but also having the option to decorate their outer surface with various other entities ranging from antibody fragments for active targeting to polyethylene glycol (PEG) tails to increase their serum half-life (t1/2), all the while maintaining the integrity of said cargo.
Moderna’s vaccine idea is built on the premise that cells that are transfected by their liposome will express the protein encoded by the carried mRNA, which in the case of mRNA-1273 encodes the spike protein of SARS-CoV-2. The protein will eventually be translated in the cell cytoplasm and expressed on the outer surface of the cells, waiting to be recognized by the host’s immune system. Over time the body will recognize the spike protein as foreign and mount an immune reaction against it. Antigen presenting cells (APCs), such as dendritic cells, can acquire and detect the foreign material both directly and indirectly, and will stimulate the necessary immune cells. Antibodies targeting various epitopes of the spike protein will eventually be produced by terminally differentiated B cells and hopefully provide continued immunity through long-lived memory B cells.
Inovio Pharmaceuticals – INO-4800 – DNA vaccine – Phase II Clinical Trial Planned
Inovio seems poised to tackle this pandemic as another vaccine in their pipeline, INO-4700, has recently been investigated for use against Middle East respiratory syndrome (MERS), a disease that stems from a coronavirus of the same family as SARS-CoV-2. The idea behind INO-4800 is similar to Moderna’s in that this novel vaccine is nucleotide based. Inovio’s vaccine consists of a DNA plasmid that encodes the spike protein of SARS-CoV-2 that is administered intradermally, followed by the use of a proprietary hand-held electroporation device called CELLECTRA™. CELLECTRA uses electrical pulses to open small pores on the cells which are targeted, allowing entry points for the plasmids. Following expression of the plasmid, spike proteins are produced and expressed on the cell surface and wait to be recognized by the patient’s immune system.
One growing challenge behind this rapid development of novel therapeutics and vaccines remains the safety measures that need to be implemented. Most novel therapeutics take anywhere from 8 to 10 years to come to fruition, going through extensive in vitro experiments, various preclinical assessments, and studied in hundreds of human participants. Finding the right preclinical models to test experimental therapies has proven quite challenging. Due to structural differences between the human and mouse ACE2 proteins, SARS-CoV-2 has proven quite inefficient at infecting wild type mice (the go to animal for many preclinical studies). There are however clever workarounds for this; for example, human ACE2 transgenic mice and mice transduced with human ACE2-expressing adenovirus are both susceptible to SARS-CoV-2 infection. Inovio has recently started working with ferrets, a relatively common animal model for studying viral respiratory infections in humans. Although ferrets are not the perfect model either (due to the limited nature of viral replication in vivo), the efficacy of the experimental vaccine can be determined by assessing respiratory transmission amongst ferrets via coughing and sneezing (infecting each other much like people do).
Both Inovio’s and Moderna’s potential nucleic acid-based vaccines are novel and remain largely hypothetical as no nucleic acid-based vaccines have been approved to date, but both may soon show the world that these platforms are the future of vaccinology. Each company has been primarily touting their platform’s simplicity, stability as well as manufacturability, and with additional aids (i.e. encompassing liposomes or electroporation machines) the nucleic acids are less prone to degradation from exonucleases, or nucleic acid degrading enzymes. Most concerns regarding nucleic acid-based vaccines are centered around the possibility of nucleic acid uptake into cells that were not the intended target, the fate of the administered entities when and if they fail to be taken up by their target cells, and in the case of the DNA platform, the risk of oncogenic mutagenesis from unintended and untargeted genomic integration still looms.
Sorrento Therapeutics – STI-6991 – Cell-based vaccine – Phase I Clinical Trial Planned
Subunit vaccines are based on simple recombinant proteins or protein fragments (such as a naked spike protein of SARS-CoV-2) that can trigger an immune response. However, subunit vaccines can suffer from a lack of immunogenicity, predominantly caused by poor subunit choice, incorrect or abnormal protein folding, or simply poor presentation to the recipient’s immune system. Moderna and Inovio attempt to circumvent this issue by having a patient’s own cells express vaccine targets, which in this case may enable expression of a properly folded spike protein quite similar to the endogenous viral protein, but the aforementioned possible problems of how many and which cells in the patient will take up the spike protein encoding nucleic acids could prove intractable.
To overcome this issue, Sorrento Therapeutics has developed STI-6991, an I-Cell™ COVID-19 cellular vaccine consisting of replication-deficient human erythroleukemia K562 cells expressing membrane-bound spike proteins of SARS-CoV-2. The spike proteins, which give the virus the appearance of a crown shape when looked at under an electron microscope, hence corona or Latin for crown, are single pass type 1 transmembrane proteins. Expression of spike proteins on the cell membranes of K562 cells would, in theory, stabilize their three-dimensional structure and using a human-derived cell line should help the patient’s immune system focus on the foreign spike protein. Control over the amount of protein expression, or overexpression, on the K562 cells could prove instrumental in proper immune activation in the long run.
Novavax – NVX-CoV2373 – Nanoparticle vaccine – Phase I Clinical Trial Recruiting
Novavax, a company entirely dedicated to developing next-generation vaccines has also rolled out a promising product. Rather than using the traditional attenuated or inactivated virus for their vaccine, Novavax has created a nanoparticle, NVX-CoV2373, which contains the trimeric form of the SARS-CoV-2 spike protein (homotrimerization being essential for viral entry into host cells), along with their proprietary saponin-based Matrix-M™ adjuvant.
NVX-CoV2373 is a virus-like-particle (VLP), a self-assembling nanostructure that mimics the conformation and protein organization of the native virus. These multi-protein structures, which can be composed of all or some of the viral surface proteins, lack the viral genome and are unable to replicate. As a result, attenuation or inactivation of the VLP is unnecessary. This facilitates the conservation of the surface protein integrity, which can be severely altered during the viral attenuation or inactivation, and exposes conformational epitopes on the protein similar to those found on the original virus.
Often referred to as an immunologist’s “dirty little secret”, finding the right adjuvant for a vaccine is key as it not only improves the duration but also the quality of the recipient’s immune response by stimulating it at various levels. Well known vaccine adjuvants include thimerosal (a mercury salt which is no longer in use) and the much more common aluminum-based adjuvants. Numerous adjuvants have come forward in the previous weeks including AS03 (GlaxoSmithKline) and MF59 (Novartis), both of which are squalene-based, and have been used in the past for H1N1 and seasonal influenza vaccines respectively. The exact mechanism of action of Novavax’s Matrix-M™, and other saponin-based adjuvants, is still greatly debated, with theories such as integration of saponin particles within cell membranes, immunogenicity of its functional groups, and even induction of cytokines such as interferons (IFNs) and interleukins (ILs) being released from APCs, leading the way. Choosing the right adjuvant and thus optimizing an individual’s immune response at various levels (both cellular and humoral) will be key in providing a long-term and robust immunity, especially as we anticipate the next wave of this pandemic.
LakePharma – LP-151 – Protein subunit vaccine (in the spirit of transparency, please note that both authors are LakePharma employees)
The SARS-CoV-2 spike protein initiates viral attachment via binding of the receptor binding domain (RBD) of the spike protein to human ACE2, thus presenting the RBD as a crucial subunit candidate for COVID-19 vaccines. SARS, MERS, and COVID-19 research suggest that an RBD-focused approach can elicit robust protective immunity through neutralizing antibody generation. The low neutralizing titers frequently seen in COVID-19 convalescent patients suggests limitations of inactivated SARS-CoV-2 or full spike protein as vaccine antigens because, similarly to the natural virus, they contain the same non-neutralizing epitopes that may not elicit protective neutralizing antibodies. Further, mutagenic escape is theoretically less likely to occur with RBD compared to other spike protein domains due to fitness reduction, making RBD a relatively more stable vaccine target than other SARS-CoV-2 viral proteins. Importantly, based on our experience in supplying serological assay material, RBD production yields are roughly ten times higher than that of the full spike protein ectodomain, which significantly reduces scale-up timelines.
Mouse models are commonly used to guide early-stage vaccine development and provide key insight into safety, immunogenicity, efficacy, and adjuvant selection. Although useful for minimizing the effects of genetic variation on vaccine-driven immunity in vivo, single strains of inbred mice do not mimic the diversity of human immune responses. Vaccine failure rates of up to 30% have been reported and in some cases are linked with specific major histocompatibility complex (MHC) class II human leukocyte antigen (HLA) haplotypes. We hypothesize that more expansive preclinical testing in immunologically diverse mice can mitigate the shortcomings of testing in single inbred strains. We therefore evaluated our vaccine candidate, formulated with different adjuvants, in LakePharma’s PentaMice™ Platform. PentaMice are a diverse set of wild type mice generated via in-house breeding that comprise five strains of F1 and outbred wild type mice that together cover nine distinct MHC class II haplotypes. In terms of MHC class II alone, PentaMice provide greater than forty-fold diversity versus the commonly used inbred C57Bl/6 mouse strain. We hypothesize that by identifying an optimal adjuvant that leads to robust spike protein-ACE2 binding neutralization activity in all five PentaMice strains, we improve our chance for widespread efficacy across immunologically diverse human patients. PentaMice thus provides an ideal platform for assessing vaccine safety and efficacy in pandemic scenarios. LakePharma is currently completing investigational new drug (IND) enabling studies and beginning manufacturing process development for the RBD subunit vaccine.
AstraZeneca – AZD1222 – Adenovirus vaccine – Phase III Clinical Trial Recruiting
Previously known as ChAdOx1 nCoV-19, AZD1222 uses the ChAdOx1 weakened adenovirus construct developed at the University of Oxford that has been engineered to express the SARS-CoV-2 spike protein. The use of weak virus strains to stimulate one’s immune system is one of the pillars of vaccinology. Using less virulent strains dates back to the late 1700’s when country doctor Edward Jenner used cowpox lesions from the hands of local dairymaids to inoculate (or in this case variolate) people so as to protect them if infected with the actual smallpox virus. Jenner’s experiment proved successful, and although he was not the first person to try such proceedings, he is now hailed as the father of immunology and the word vaccine, which stems from the Latin vacca, for cow, is forever associated with these ruminants. AZD1222, a replication-deficient engineered virus, cannot cause an ongoing infection in patients and should prove to be a less formidable opponent for the human immune system than SARS-CoV-2. AstraZeneca has already designated Catalent as a key player in the large-scale manufacturing of this potential vaccine.
Of general concern, which is not exclusive to AstraZeneca’s vaccine platform, is the potential for antibody dependent enhancement (ADE) of SARS-CoV-2. ADE can occur when non-neutralizing virus-specific antibodies are generated upon vaccination or following an initial viral infection. Upon subsequent viral challenge, these non-protective antibodies enable amplification of viral infection by providing the virus with another set of host cells to infect: immune cells with antibody-binding receptors. Non-neutralizing antibodies can also trigger immunopathology via induction of T helper 2 (Th2) immune responses, which can lead to allergic lung inflammation (including influx of eosinophils). So, what could have been a mild viral infection can suddenly transform into a ramped-up life-threatening event. Although ADE remains a theoretical concern for SARS-CoV-2 at this point (due to the newness of the virus and lack of study), ADE has been reported for other coronaviruses and tragically for Denguevaxia, Sanofi’s dengue fever vaccine. Children vaccinated against dengue fever were significantly more likely to develop severe clinical outcomes following exposure to the dengue virus than unvaccinated children due to ADE. As COVID-19 vaccines and therapeutics advance through clinical trials, careful analysis of safety in humans is required to detect and protect people against possible ADE.
Takeda – TAK-888 – Convalescent serum therapy
Serum therapies date back to the latter part of the 19th century. In 1890, Emil von Behring and Shibasaburo Kitasato demonstrated that serum from infected horses could be used to treat pediatric cases of diphtheria. The same idea would then be carried out on a grand scale during the Great War, where horse serum was used to combat tetanus. All of this took place before the advent of monoclonal antibodies, or even knowledge of antibodies in general, and use of serum therapies has gradually declined since then. Nevertheless, extreme times call for extreme measures, and Takeda, Japan’s largest biopharmaceutical company, has halted numerous ongoing projects to combat the virus head-on with this approach.
Individuals who have contracted and recovered from SARS-CoV-2 may have antibodies that have gone through in vivo affinity maturation and optimally neutralize the virus. While the idea of using a previously infected individual’s serum is not novel, the practicality and general applicability of this therapy seems limited from the get-go. However, this polyclonal therapy could prove quite beneficial in the treatment of high-risk individuals who have contracted the virus and need immediate assistance in developing immunity. The antibodies found in convalescent serum will facilitate the recognition and neutralization of SARS-CoV-2 virions in patients whose immune system may be weaker due to old age or an underlying medical condition. Moreover, as opposed to the horse serum therapies of the past, the risk of suffering from anaphylactic shock is quite limited, as the serum proteins are of human origin.
University of Texas, Austin – Nanobody® or single-domain antibody (VHH) llama therapy
UT Austin scientists published a critical paper early in the pandemic regarding the use of cryogenic electron microscopy (cryo-EM) to determine and characterize the structure and possible epitopes of the trimeric spike protein for SARS-CoV-2. More recently, they partnered with scientists at Ghent University in Belgium to develop a llama “antibody” or nanobody targeting that spike protein.
The general interest in llama or camelid antibodies is centered around the fact that these single monomeric variable antibody domains offer the same specificity and selectivity as a regular (or human) antibody but are much smaller. Smaller entities are theoretically more prone to accumulate in greater concentration inside tight and highly pressurized settings, such as tumor microenvironments, where they are expected to be the most beneficial. Although there is presently only one FDA approved nanobody, Cablivi™ (caplacizumab-yhdp), numerous are in clinical development. Moreover, as opposed to horse derived antibodies (see convalescent serum therapy) and antibodies derived from wild type mice, nanobodies remain weakly immunogenic in humans, predominantly due to extensive sequence similarity to human antibodies. Beyond these advantages, increased stability was a key deciding factor for the choice of this route of antibody discovery, and the development of neutralizing nanobodies could prove beneficial for this current pandemic as well as any future outbreaks. Hook’em Horns!
Which of these vaccines and treatments will receive the highly coveted FDA approval and make it to market remains an ongoing question. Nevertheless, today we can only be sure of four things: first, the humongous wave of initiative and investments in the scientific fields has left tech companies reminiscing about the dotcom days; second, no one can say how many more companies will wade into the fray to have their chance of being in the limelight; third, as the human population keeps expanding and encroaching on sparsely populated areas, we are likely to experience more frequent and severe zoonotic outbreaks; and finally, and possibly worst of all, all of this anticipation and apprehension has almost led us to completely forget about Theranos…