Retrospective View of COVID-19 Pandemic: Treatment, Management and Development of Preventive Measures

SARS-CoV-2 has held the world hostage over the last 3 years, constantly mutating to elude therapeutic measures, with 765,903,278 documented cases and 6,927,378 deaths as of May 17 th , 2023. During this time, biotechnology hastened advancement of preventive measures, with healthcare professionals repurposing


Introduction
Coronavirus disease 2019 (COVID-19), caused by the pathogenic virus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), was first identified in December 2019 [1], ultimately resulting in the largest global health crisis in a century. On March 12, 2020, World Health Organization (WHO) declared COVID-19 a global pandemic [2]. Since then, SARS-CoV-2 has wreaked havoc on global health and economics. At the time of writing (May 17 th , 2023), the virus is responsible for over 765 million infections and 6.9 million deaths worldwide [3]. A timeline of SARS-CoV-2 development is shown in Figure 2. SARS-CoV-2 shares approximately 80% homology with SARS-CoV-1, with the disparity responsible for mutations in the COVID-19 spike protein [4]. SARS-CoV-2 enters through the respiratory tract before attaching to cell surface receptor proteins, like Angiotensin-Converting Enzyme 2 (ACE2), through its homotrimer spike. Following receptor-mediated cell-entry it is directly translated by host ribosomes, particularly in the epithelial linings of the lungs, kidneys, and heart [5].
SARS-CoV-2 mutates rapidly. In February 2020 spike protein mutation A23403G (D614G) emerged, increasing viral fitness and infectivity [6]. The single nucleotide substitution in the spike protein receptor binding domain increased affinity to the ACE2 receptor and heightened its replication in human cells [7]. Rolling genomic mutations continue to affect both broader viral fitness, such as transmissibility and infectivity, and pandemic containment measures such as detection and vaccination [8]. Notable strains include Alpha (B.1.1.7), Beta (B.1.351.), Gamma (P.1) and Delta (B.1.617.2), the latter of which developed a significantly higher viral load (up to 1,200 times greater than the ancestral virus) and a shorter incubation period [9].
The dominant strain Omicron (B.1.1.529), first reported in South Africa in late 2021 [10], was classified as a 'variant of concern' (VOC) by WHO from initial detection [11]. Though health outcomes are less severe, it is highly contagious and carries genetic variations causing increased transmissibility and vaccine evasion [12]. One of these genetic variations also leads to 'S gene dropout' or 'target failure', where one of the genetic sections targeted by polymerase chain reaction (PCR) testing gives a false negative [13]. Omicron (BA.5) and its subvariants have dominated the sequenced cases lodged with GISAID since May 2022 through the end of the year (85%) [14]. WHO's most recent variant of interest is Arcturus (XBB. 1.16), due to its growth advantage and immune evasion [15]. First discovered in January 2023 in India, it has spread to 31 countries [15].
As of May 17 th , 2023, there are 199 vaccines in preclinical development. Eleven vaccines have WHO emergency authorization [16] via four main platforms: mRNA, viral vector, inactivated and protein subunit.
Repurposing pre-existing drugs for the treatment of SARS-CoV-2 is efficient and cost-effective [17].{Singh, 2020 #63} Computational and biological experimental methods can identify approved pharmaceuticals that target specific SARS-CoV-2 mechanisms using molecular docking, signature matching and genome-wide associated studies [18]. Alternatively, experimental methods including assays and phenotypic screening can identify effective drugs [19]. Pre-existing candidates have already undergone risk assessments in both preclinical models and humans, making this significantly more efficient and economical than standard drug development [17,19]. For example, nirmatrelvir was originally developed for the treatment of hepatitis C, it is now packaged alongside ritonavir as Paxlovid by Pfizer. Phase 3 trials showed that among people with COVID-19 who were at high risk of hospitalization or death, those who received Paxlovid had a 90% lower risk of the above than placebo [20].
Immunotherapeutic approaches have shown clinical benefits in patients with Influenza, SARS, and Middle East Respiratory Syndrome (MERS) [21], suggesting potential for SARS-CoV-2 treatment. However, the complexity of COVID-19's immune avoidance mechanisms and associated pro-inflammatory events, such as cytokine storm and T cell exhaustion, have prevented isolating a single effective approach. In response to the pandemic's urgency, the Food and Drug Administration (FDA) authorized emergency use of immunotherapy methods such as recombinant monoclonal antibodies (rMAB) and convalescent plasma (CP) therapy. Alternatively, CP therapy utilizes plasma from recovered COVID-19 patients to impede infection and mitigate hyperactivated immune reaction. Some promising immunotherapeutic approaches are undergoing trials. The most notable are the inhaled interferon β-1α (IFNβ-1α) and peg-interferon λ-1 (peg-IFNλ-1) based immunotherapies, interleukin-6 (IL-6) blockade and Janus kinase (JAK) inhibition [22].
Gene-editing is the most novel technology applied to SARS-CoV-2. It has been tailored to virus degradation, detection, and diagnosis [23][24][25]. The CRISPR/Cas systems have enormous potential due to their precision, efficiency and cost-effectiveness [24]. Figure 3 explains the guide RNA and Cas enzyme process. CRISPR-based diagnostic technologies include endonucleasetargeted CRISPR trans reporter (DETECTR) and Specific Highsensitivity Enzymatic Reporter un-LOCKing (SHERLOCK). As a treatment, CRISPR-Cas13-based strategies such as Prophylactic Antiviral CRISPR in huMAN cells (PAC-MAN) have strong potential for virus degradation in humans [26].

Vaccines
Vaccines are our most successful tool to prevent severe illness and death from COVID-19 [27]. Their development has been accelerated by partnerships between institutions and governments. Many vaccines were approved quickly under WHO's emergency use criteria, which requires at least 50% efficacy, 6-month protection, usability in older adults and a maximum of 2 doses [28]. Among those to receive emergency use approval from WHO within one year of the outbreak are Pfizer/BioNTech, Covishield, Janssen, Moderna and AstraZeneca.
There are currently 50 vaccines approved by at least 1 country, and 11 approved by WHO [16]. Pfizer/BioNTech's mRNA vaccine, Moderna's mRNA vaccine, Oxford/AstraZeneca's non-replicating viral vector vaccine, Johnson & Johnson's non-replicating viral vector vaccine and Sinopharm's inactivated vaccine are the most widely used and approved globally [16]. Most vaccines in clinical trials are protein subunit (32%) or non-replicating viral vector (14%) [29], with no WHO-approved replicating viral vector or live attenuated. (Table S1, Figure S1 and Figure S2    with BioRender.com. Phase 1 trials test new vaccines in a small group first, to assess safety profiles. Phase 2 trials expand testing to a larger group after a vaccine is found to be safe in Phase 1 trials, to identify any adverse side effects. Phase 3 trials are run on different populations across various regions or countries and are typically run right before a vaccine meets approval. After approval the vaccine is monitored long-term in phase 4 trials, across a broader population to ensure its safety and efficacy. There are many promising vaccines in development; however, the difficulty in developing a safe and effective vaccine is evidenced in the slow progress and abandonment of candidates. For example, CureVac's mRNA vaccine phase I trials showed more than 70% efficacy against moderate-to-severe infection. Efficacy dropped to just 48% in phase III trials [36], hence, it was abandoned in October 2021. Outlines for selected promising candidate vaccines are summarized in Table 1 at the end of the vaccines section. Ultimately, the success of vaccination efforts in pandemic control will continue to depend on widespread distribution and uptake of effective vaccines, as well as adaptation of strategies in response to emerging variants. 70  mRNA Vaccines mRNA vaccines are extremely effective in preventing severe COVID-19 as transfected mRNAs express antigen proteins in vivo, inducing robust humoral and cellular immune responses [37]. mRNA vaccines are manufactured by synthesizing the mRNA sequence that encodes the viral protein of interest, normally the spike protein for SARS-CoV-2. The manufactured mRNA is protected during delivery to the host by advancements such as lipid nanoparticle (LNP) biotechnology [38]. The encapsulated mRNA is then formulated with other necessary components to create the final vaccine product and administered intramuscularly. There are two WHO-approved mRNA vaccines: Pfizer (Oxford/ AstraZeneca) and SpikeVax (Moderna) [16]. mRNA vaccines are WHO-authorized for use in children, with Pfizer BNT162b2 approved for children 5 years and older and Moderna approved for 12 years and older [39]. Trials have shown both vaccines to be safe for their respective age groups, with an overall efficacy of 90.7% for Pfizer in 5 to 11-year-olds [40] and 92.7% for Moderna in adolescents [41].
Moderna, Pfizer and Livzon have targeted specific variants with their mRNA platforms. mRNA vaccines offer high adaptability in responding to emerging strains of SARS-CoV-2 by allowing delivery of a transcript that encodes a specific immunogen or antigen. This versatility has already proven itself in the wake of Omicron. In December 2021 a South African study found that protection against hospitalization from SARS-CoV-2 by Pfizer/BioNTech's vaccine dropped from 93% to 70% across two monitoring periods [42]. The key difference was the emergence of the Omicron variant, highlighting the need for tailored vaccines.
Moderna's first multivalent vaccine contained both the mRNA-1273 (original) and mRNA-1273.351 (Beta) strains [43]. Booster shots using this hybrid indicated strong B cell memory response and increased neutralizing antibody titres compared to the original vaccine alone [43]. Moderna also combined their ancestral formula with the Omicron-tailored booster (mRNA-1273.529) to create bivalent vaccine, 1273.211 [16]. It generates a modestly higher level of antibody response against multiple SARS-CoV-2 Omicron subvariants (approximately 1.6-1.9 times) including BA.1 and BA.4/BA.5, and a similar antibody response against the original virus, compared to Moderna's original booster vaccine [44]. Pfizer's Omicron formula has shown substantially higher neutralizing antibody responses against Omicron BA.1.35. Studies report higher neutralisation titres following a booster dose of Pfizer bivalent BA.4/5 vaccine for BA.4/5 and other subvariants (e.g. BQ.1, XBB) compared to the Pfizer original vaccine [45,46]. One US study showed protection against hospitalisation or death with a bivalent BA.4/5 booster (either Pfizer or Moderna) was 61.8% compared with an original booster of 24.9% [45]. Interestingly, Livzon is also developing an Omicron-tailored protein subunit vaccine (V-01-351/V-01D), though there is limited data available as it is in phase 3 trials [29]. Currently, there is no regulatory consensus on the efficacy or necessity of Omicronspecific vaccines. Early primate studies indicate that Omicronspecific boosters offer no greater protection than a 3-dose schedule of currently available vaccines [47,48].
Pfizer/BioNTech and Moderna vaccines have strong efficacy rates of 95% in preventing severe COVID-19 [49,50] and are globally extremely well-received due to their high safety profile [49,50]. Other mRNA vaccines include Takeda (TAK-919), approved solely in Japan [16]. mRNA vaccines have a strong safety profile, with transient side effects such as redness at injection site or fatigue [49]. They face a major challenge in that they require extremely low temperatures to transport, with the prototypical Pfizer vaccine requiring an environment of −70 °C. They are a logistical challenge, especially in lower income nations [51]. Their stringent cold-chain requirements makes them an unsuitable candidate for global vaccination efforts alone [52], particularly given the importance of global vaccination to stop VOCs generation. Thus, a range of accessible vaccine technologies are needed.

Viral vector vaccines (non-replicating)
Non-replicating viral vectors stimulate both the cellular and humoral immune systems, commonly using adenoviruses [53]. They are manufactured by modifying a harmless virus to remove its replication capacity and inserting genetic material encoding the target antigen, in this case SARS-CoV-2. This modified viral vector is then grown, purified, and formulated to create the final vaccine product, which is administered intramuscularly. There are four WHO-approved vaccines: Janssen (Johnson & Johnson), Vaxzevria (Oxford/AstraZeneca) and Convidecia (Cansino) and (Covishield), though the latter 2 are far less widely used [16].
Neither 'Janssen' [54] nor 'Vaxzevria' [55] are safe for use in children as there is limited data available. 'Janssen' studies show that a second dose 2 months after the initial dose substantially increases efficacy, especially against symptomatic infections, including when caused by VOCs. Vaccine efficacy of 2 doses, 2 months apart, was 94%. In comparison, the single dose vaccine efficacy was 72% [56]. Vaxzevria has an efficacy of 74% in individuals without prior infection [55].
One notable non-replicating vaccine is the nasal spray variant of the Ad5-nCoV vaccine by CanSino and the Beijing Institute of Technology (currently in phase IV trials). No aerosol vaccines have been approved, but early studies show promising immunogenic responses from a simple, painless, and widely-tolerated process [57]. Unlike intramuscular administration, nasal inhalation provides better respiratory mucosal immunity, while offering similar immune responses in the blood. One aerosol-delivered Advectored tuberculosis vaccine significantly increased airway-tissue resident memory cells (CD4 and CD8) [58] and Ag85A-specific T cell responses in the blood [58] A second Ad-vectored intranasal vaccine was tested on mice expressing the ACE2 receptor. A single dose induced high levels of neutralizing antibodies, prompted strong systemic and mucosal immunoglobulin A (IgA) and T cell responses and almost entirely prevented SARS-CoV-2 infection in the upper and lower respiratory tracts [59]. These findings are particularly potent given that Omicron replicates significantly more quickly in human nasal epithelial cells and broader respiratory epithelial cells than the Delta variant, contributing to transmissibility [60].
These vaccines pose a challenge as subsequent/booster vaccines can result in immunity to the vector virus, while preexisting immunity can neutralize the initial vaccine. For example, an adenovirus type-5 vectored vaccine trialled in China induced a stronger response for younger people than for those aged 55 years or older, who already had higher baseline neutralizing antibody to adenovirus type-5 [53]. To combat this baseline immunity problem, Oxford/AstraZeneca used a modified chimpanzee adenovirus, ChAdOx1, in their nCoV-19 vaccine [61]. Russia's Gameleya Research Institute used both Ad26 (also in the 'Janssen' vaccine) and Ad5 in their Sputnik V vaccine, finding an efficacy of 91.6% [62].
AstraZeneca/Oxford and Johnson & Johnson vaccines have raised concerns due to rare thrombotic adverse reactions, but multiple advisory boards have deemed these vaccines safe with benefits outweighing any risks [63,64]. The mechanism behind vaccine-induced blood clotting is currently unknown, although unintended splice protein variants may play a role. Transcription of S protein variants may enable binding to ACE-2 expressing endothelial cells in blood vessels [65]. Some national governments (e.g. Denmark) ceased use, and there has been a broader loss of public trust [52]. Modifying the spike protein open reading frame to avoid these spike protein variants may increase vaccine safety and restore public trust [65].

Inactivated Vaccines
Inactivated vaccines are one of the most common immunization methods [66]. They are manufactured by growing the target microorganism, SARS-CoV-2, in large quantities under controlled laboratory conditions. The microorganism is then treated with chemicals, heat, or radiation to render it inactive while maintaining its structural integrity, before being administered intramuscular. There are three WHO-approved inactivated vaccines: Covaxin (Bharat), Covilo (Sinopharm Beijing) and Coronavac (SinoVac) [16]. There are 12 vaccines approved in a limited number of countries undergoing clinical trials [29].
Inactivated vaccines are well-established in the COVID-19 vaccine landscape (27% of WHO approved vaccines and 13% of candidate vaccines in clinical trials [29]); however, there are concerns around waning immunity and the need for additional vaccine doses to maintain protection. COVID-19 immunity can wane relatively quickly following a genuine infection: researchers following recovered SARS-CoV-1 patients for 3 years found that immunity dropped significantly within 18 months [67]. The first inactivated COVID-19 vaccine, 'CoronaVac' was developed by SinoVac from July 2020 and received WHO EUL approval on June 1, 2021 [16]. However, concerns around CoronaVac's lasting immunity, particularly in the immunocompromised, led WHO to formally recommend booster programs for 'CoronaVac' [68].
Studies show that mRNA vaccines and the Novavax protein subunit vaccine elicit stronger antibody responses than inactivated viral vaccines and viral vector vaccines [67,69]. Inactivated viruses take longer to develop and may be more suitable for booster vaccinations or as the final vaccine after an mRNA vaccine has primed the immune system [52]. Heterologous vaccination schemes have proven to be as effective as a full mRNA program and provide strong humoral/cellular immune responses [70,71], suggesting this mosaic approach may be useful where vaccine supply is scarce.

Protein Subunit Vaccines
Protein subunit vaccines contain specific isolated virus proteins so have a strong safety profile. They are produced by selecting the specific proteins from the virus that can elicit a potent immune response. These proteins are then manufactured using recombinant DNA technology or protein expression systems and formulated into a vaccine to stimulate an immune response against the virus, before being administered intramuscularly. There are 59 protein subunit vaccine candidates and 18 approved in various countries [16]. Novavax and COVOVAX (Novavax formulation) are the only WHO-approved vaccines of this type [16]. However, against the B.1.351 (Beta) strain efficacy was limited at 51%. When comparing efficacy and neutralizing antibodies in vivo, the Novavax protein subunit vaccine gives a more robust response than viral vector and inactivated vaccines [67,69].
Novavax is relatively simple to store and transport with a shelf life of 9 months at 2 to 8°C [76]. Given its extremely high efficacy in comparison to other vaccine types and its ease of transport, particularly in the context of Pfizer's extremely low temperature requirements, protein subunit vaccines have excelled in the vaccine space.

Viral Vector Vaccines (Replicating) and Live Attenuated Vaccines
Replicating viral vector vaccines use a modified virus as a delivery system to produce viral antigens within cells, without replicating the target pathogen. Live-attenuated vaccines (LAV) use a live, weakened form of the target pathogen, providing strong immune protection [77]. There are currently no WHO-approved replicating viral vector or live-attenuated vaccines [16], though both have candidates up to phase III trials [29].
Replicating viral vector vaccines stimulate a comprehensive immune response [66].

Repurposing Drugs
Ongoing clinical trials are assessing the treatment efficacy of pre-existing drug therapies for SARS-CoV-2, including antiviral, antiparasitic and antibiotic candidates. The following overview of these candidates is based on therapeutic utility for SARS-CoV-2, sample size and accessibility of their clinical trials and results, and their status as recommended, novel or controversial treatments. (1) SARS-CoV-2 enters the cell via binding of its spike proteins with the host cellular ACE2 receptor. Lopinavir-Ritonavir, and Umifenovir (Arbidol) act to inhibit the early stage of viral pathogenesis. (2) Fusion and endocytosis of the virus is aided by a low endosomal pH, therefore disruption of this acidic environment by Chloroquine/Hydroxychloroquine can create an antiviral effect. Ivermectin is hypothesized to inhibit viral nucleic transport. (3, 4) Viral RNA is released into the cytoplasm and translated by host ribosomal mechanisms. Azithromycin inhibits bacterial protein synthesis, however, may only serve as an anti-inflammatory for COVID-19. (5) Functional, non-structural proteins such as RNA dependent RNA polymerase (RdRp) and viral protease are synthesized. Inhibition of these proteins by Darunavir, Remdesivir, Favipiravir, Ribavirin and Sofosbuvir prevents viral RNA replication and further protein synthesis. (6) Functional proteins enable replication, transcription, and translation of SARS-CoV-2, creating structural proteins. Molnupiravir creates defective RNA nucleosides which then cannot be used to make new viruses. (7,8,9) Assembly and packaging of proteins make intact virions which are then released from the cell via exocytosis which can potentially be inhibited by Chloroquine/Hydroxychloroquine [17,105,106].

Antiviral
Protease inhibitor Phase IV S17

Remdesivir
Remdesivir (VEKLURY®) is an antiviral prodrug initially developed to treat RNA viruses, such as Ebola [107], and the first drug approved by the U.S. Food and Drug Administration (FDA) for SARS-CoV-2 [108]. Remdesivir is a broad-spectrum antiviral that was applied to SARS-CoV-2 given its success with previous coronaviruses. An adenosine nucleotide, it works by being metabolized into remdesivir triphosphate (RDV-TP) [109]. RDV-TP is a highly selective (3.65-fold) competitive inhibitor of adenosine triphosphate (ATP). It targets the RNA-dependent RNA polymerase (RdRp) enzyme of the SARS-CoV-2 virus, incorporating itself into the viral RNA chain during replication to prevent addition of further nucleotides.
Side-effects for Remdesivir are typically mild, but RDV-TP also affects DNA and RNA polymerases in humans which risk injuries to organs. Intravenously administered Remdesivir may cause elevated liver enzymes, harmful for patients with liver conditions [110]. It may be used in renally-impaired patients with estimated globular filtration rates (eGFR) < 30 ml/min [111].
Both placebo and standard of care (SOC) trials show promising results. A 10 day Remdesivir treatment reduced median recovery time from 15 to 10 days in severely ill patients [112]. At the end of the trial, 46% of Remdesivir patients were not hospitalized compared to 36% of placebo. In SOC comparisons, patients treated with Remdesivir for 5 days showed significant improvement over SOC patients on Day 11, with 70% of Remdesivir patients not hospitalized compared to 60% of SOC [113,114]. One significant trial measured the clinical status of outpatients at 28 Days, comparing a 3-day treatment to placebo [115]. 0.7% of Remdesivir patients had COVID-19, related hospitalizations or all-cause deaths compared to 5.4% of placebo. Remdesivir treatment reduced the rate of hospitalization or death due to COVID-19 by 87% [116].
Aerosolized nano-liposomal carriers of Remdesivir may improve direct drug delivering to lung cells, increase metabolization to RDV-TP and reduce side-effects [117]. Remdesivir nanoliposomes had minimal cytotoxicity in A549 lung carcinoma cells, sustained release up to 50 hours, and significant lung deposition [118]. This aerosol delivery is a future avenue for greater efficacy and self-administration.
Molnupiravir side effects include gastro-intestinal symptoms, dizziness and nausea. It has a clean safety profile in general [122,123]. Molnupiravir has been found to significantly reduce SARS-CoV-2 load and disease transmission in ferret models [124]. In humans, a phase I trial found no significant increase in adverse effects compared to placebo [122,123]. A phase II trial in hospitalized patients compared the efficacy of placebo treatment and 200 mg, 400 mg, and 800 mg dosages of Molnupiravir twice per day for a 5-day course [125,126]. The trial found no significant increase in adverse effects, median recovery or all-cause mortality with higher dosages.
A phase III trial found that Molnupiravir (800mg over a 5-day course) significantly reduced percentage of all-cause hospitalization or death (7.3%) compared to placebo (14.1%) to placebo in non-hospitalized, non-vaccinated participants with mild-moderate COVID-19 [127,128]. Treatment reduced risk of hospitalization or death by 31% compared to placebo.

Favipiravir
Favipiravir (Avigan®) is an broad-class antiviral initially approved for novel influenza [129]. In vitro and in vivo studies demonstrate its efficacy against Ebola, arenavirus, yellow fever virus and foot-and-mouth disease [130]. Favipiravir-RTP, the active form of this drug (a purine analogue), acts as a substrate for the viral RdRp, inhibiting its activity and ultimately terminating protein synthesis [130,131]. Just like with other viruses, for SARS-CoV-2 it hinders replication of the virus and thus viral load in the patient.
Favipiravir has no optimal dosage established for SARS-CoV-2 [130]. It's oral administration allows for easy at-home treatment of mild-to-moderate COVID-19 [132]. Side effects are mild, including nausea, vomiting, diarrhea and reversible elevation of bilirubin and uric acid. Favipiravir is teratogenic so not administered to pregnant patients [130].
The limited Favipiravir trials for SARS-CoV-2 treatment show reasonable efficacy. One phase III trial showed reduction in median time to clinical cure from 5 to 3 days [133]. In a second trial, by day 10 98% of patients, compared to79% of control, returned negative PCR testing [134].

Ivermectin
Ivermectin (Stromectol®) is an antiparasitic, semisynthetic, anthelmintic agent with potent antiviral and anti-inflammatory properties. Derived from the soil bacterium Streptomyces avermitilis, it was originally used in animals for heartworm and parasites. It is WHO-approved for human treatment of river blindness, scabies and lice [135]. Ivermectin shows antiviral activity against a range of RNA and some DNA viruses, including Zika, dengue and yellow fever [136], though its exact mechanism is unclear. In parasites, ivermectin selectively binds to glutamategated chloride ion channels in invertebrate muscle and nerve cells, resulting in hyperpolarization and paralyzing the parasite. In viruses, it may have antiviral action via inhibition of IMPα/β1mediated nuclear import of viral proteins into host cells [136]. By disrupting this process, it may impede the virus' ability to replicate and spread in the body.
Potential side effects including headache, muscle pain, or nausea [135,137], as well as sudden drop in blood pressure and facial/limb swelling [138]. Laboratory test abnormalities include decrease in white cell count and elevated liver tests [138].
In vitro studies show that SARS-CoV-2 infected cells treated with 5 μM of Ivermectin result in a 99.98% reduction in viral RNA [137]. In a phase III trial, patients treated with Doxycycline and Ivermectin recovered more quickly (60.7% vs. 44.4%), were less likely to progress to more serious disease (8.7% vs. 17.8%), and were more likely to be COVID-19 negative by PCR on day 14 (7.7% vs. 20.0%) [139]. Also, topical Ivermectin and Carrageenan prevented COVID-19 spread in an observational trial, with 0% positive cases after 28 days compared to 11.2% in the control [140]. Following a trend of patients self-administering Ivermectin, the FDA advised against its usage and restricted prescriptions by general practitioners. Serious harm may occur in self-medicating without proper guidance [138]. However, reliable results from ongoing trials could lead to its repurposing in combination with other therapeutics for COVID-19 treatment, potentially changing public perception and usage.

Paxlovid
Pfizer's Paxlovid was the first oral antiviral to receive an EUA, approved by the FDA in December 2021 [141]. It contains nirmatrelvir and ritonavir, co-packaged for oral use. Nirmatrelvir is a SARS-CoV-2 main proteases inhibitor, designed especially for SARS-CoV-2, which prevents the protease from cleaving viral polyproteins into functional components necessary for viral replication. Ritonavir is an HIV-1 protease inhibitor and CYP3A inhibitor, originally designed for treatment of HIV/AIDS. In Paxlovid, it increases the concentration and duration of nirmatrelvir in the body by inhibiting the enzyme responsible for metabolizing nirmatrelvir. Together, the two drugs work toward reducing viral replication and therefore viral load in patients. Side effects are typically mild, including nausea, fatigue and altered taste [142].
Treating high-risk patients with only nirmatrelvir showed a 67% reduction in hospitalizations and an 81% reduction in mortality in patients 65 years and above. No significant benefit in protection from severe COVID-19 was seen in younger adults [143]. Early administration of Paxlovid significant reduces risk of all-cause mortality and disease progression, and reduces viral burden faster than non-use [144]. A 2023 US study found that high-risk patients receiving Paxlovid within five days of a positive COVID-19 test had 26% lower risk of long COVID symptoms after 90 days compared to placebo [145]. It was initially aimed at those with moderate COVID-19 at extreme risk of severe disease progression [142], but is now indicated for non-hospitalized patients at risk of disease progression [144].

Monoclonal Antibodies
There are three main recombinant monoclonal antibodies (rMABs): Casirivimab/Imdevimab, Bamlanivimab/Etesevimab and Sotrovimab. The three rMABs therapies work in a similar manner, reducing a patient's viral load to accelerate recovery. Casirivimab/Imdevimab is a combination of rMABs, sold under the name REGEN-COV TM or Ronapreve TM . Both Casirivimab and Imdevimab are immunoglobulin gamma-1 proteins which attach to the spike protein of SARS-CoV-2 [146]. REGEN-COV TM is effective as both a preventative measure and a treatment. Of patients who had never been infected with SARS-CoV-2, 4.8% of a REGEN-COV TM treatment group developed COVID-19 after a 4 week period in comparison to 14.2% of the placebo [147]. As a treatment, REGEN-COV TM significantly reduces hospitalization and death due to COVID-19 and results in a shorter recovery period (10 days compared to 14 days for placebo) [148]. REGEN-COV TM is promising for both treating and preventing COVID-19 and could be an alternative to vaccination for immunocompromised and other unvaccinated people.
Bamlanivimab/Etesevimab are similar rMABs which neutralize the virus, prevent binding to ACE2 receptors and entry to host cells (Graphic representation found in appendix under Figure 8 and 9) [149, 150]. Bamlanivimab/Etesevimab has reduced hospitalization and death by COVID-19 [151]. After 28 days, hospitalization was 2.1% in treatment group vs. 7.0% in placebo. 10 deaths occurred in placebo, while the treatment group also showed lower rates of high viral load (9.8% vs 29.5% in the placebo group). Bamlanivimab/Etesevimab reduced viral load but not recovery time, suggesting need for further research and trials.
Sotrovimab is another rMAB that was originally used against SARS-CoV-1 as well as other sarbecoviruses [152]. It binds to a highly conserved epitope of the SARS-CoV-2 spike protein. Sotrovimab contains two modifications in the Fc binding region to increase its half-life, therapeutic duration and concentration [153]. Sotrovimab shows promising trial results with hospitalization rates of 1% in the treatment group compared to 7% in placebo. Only 2% of the treatment group experienced serious adverse effects compared to 6% of placebo [152]. Sotrovimab shows promising results as broader therapy for all sarbecoviruses including SARS-CoV-2.

Convalescent Plasma Therapy
Convalescent plasma (CP) therapy was recommended as a possible treatment early in the pandemic due to its antiviral properties and success in prior infection outbreaks, endemic situations like SARS-CoV-1, H1N1, and Ebola, when treatments were scarce [154]. The antibodies present in convalescent plasma, taken from a recovered patient for the target virus, recognize and bind to specific viral components, neutralizing the virus and triggering an immune response. Enhancing the antiviral response and reducing hyperinflammation are key in treating COVID-19 [21]. CP from recovered COVID-19 patients contains neutralizing IgM and IgG antibodies which limit viral growth [21]. It also encompasses immune-modulatory cytokines that regulate the cytokine storm.
The FDA authorized CP therapy for emergency use and allowed the use of high-titre COVID-19 CP based on a large observational study of hospitalized patients, which showed that the risk of death was 7% lower in a high-titre CP infusion subgroup compared to a low-titre group [155].
CP trials have yielded mixed results, due to factors such as therapy commencement, the quality and number of antibodies transfused, and donor characteristics. To properly select an appropriate donor, conduct serological tests via ELISA prior to trial to measure spike and RBD specific antibody present in the serum. The functionality and quality of these antibodies should be ascertained by neutralization assays [156].
The RECOVERY trial tested CP efficacy in patients that entered the hospital with COVID-19. CP had no influence on length of hospital stay while 24% of both groups died within 28 days. Thus, the largest clinical trials for CP concluded that hightitre CP was ineffective in improving survival or other clinical outcomes [157]. A Bayesian re-evaluation of the RECOVERY trial found that the subgroup of patients who had not yet developed anti-COVID-19 antibodies benefited from CP [158]. This was supported by a randomized, placebo-controlled trial of CP with high IgG titres given within 72 hours of mild symptoms onset. 3% of CP patients developed serious respiratory disease compared to 16% of placebo [159].
The PLACID trial had multiple limitations including bias due to its open label design and no pre-commencement computation of neutralizing antibody titre in donors and patients. It found that CP did not alleviate development of severe COVID-19 nor all deaths.
CP should clinically benefit COVID-19 patients; however, trials have yielded heterogenous results in reducing mortality and clinical improvement. Most CP data originates from uncontrolled cohort studies or case series, thus more controlled studies are needed.

CRISPR/Cas Technology for Elimination and Treatment
As various vaccine platforms were being developed and refined as preventive measures, other biotechnological tools to eliminate the virus were explored, with CRISPR on the forefront. The gene-editing technology, CRISPR/Cas showed promise in vitro and in vivo models, in preventing viral replication of SARS-CoV-2 due to its high precision nature and versatility. Previous successful reprogramming of CRISPR-Cas13 to detect and inhibit RNA viruses suggest its potential against any virus, as demonstrated in studies performed by the Broad Institute (MIT) and Harvard University [160].
Reprogrammed CRISPR-Cas systems have shown promise in silencing the efficiency of SARS-CoV-2 [161]. Accessible regions of spike and nucleocapsid transcripts were targeted to suppress replication-component SARS-CoV-2 in Green African monkey kidney cells and human embryonic kidney cells, achieving levels of up to 95% efficiency. The CRISPR-Cas system also inhibited nucleocapsid expression of the virus by 90% for both the ancestral virus and the Delta variant, achieved through CRISPR RNA (crRNA) redesign.
Furthermore, Prophylactic Antiviral CRISPR in huMAN cells (PAC-MAN) effectively degrades SARS-CoV-2 sequences in human lung epithelial cells [26]. This system uses repurposed Cas13d RNA-guided RNA endonuclease activity to target and inhibit viral replication by targeting specific genomic regions such as the RNA-dependent RNA polymerase (RdRP) and nucleocapsid proteins, which would halt viral replication.
CRISPR technology's adaptability enables rapid cRNA design and production, making it extremely adaptable to ongoing SARS-CoV-2 mutations. PAC-MAN's RNA-targeting approach suggests potential efficacy against all coronavirus strains. However, the success of CRISPR therapeutics depends on effective delivery to infected cells and minimizing off-target expression and cleavage in the host.

CRISPR-based Diagnosis
Fast and accurate diagnostics are crucial to controlling transmission of SARS-CoV-2. PCR testing, while accurate, can take up to 72 hours for result. Rapid antigen tests give results in 15 to 20 minutes but are less accurate and susceptible to user errors and inaccuracies [162] CRISPR-based assays for SARS-CoV-2 detection can take only 40 minutes while maintaining accuracy [163].
DETECTR is a CRISPR/Cas12 based detection technique utilizing a lateral flow assay. For a COVID-positive patient, Cas12 accurately targets and cleaves a reporting molecule confirming the virus' presence in the cell ( Figure 6). This technology has several advantages over current PCR testing: rapid results, no need for intricate laboratory infrastructure, and the integration of simple, accessible reporting formats. CRISPR diagnosis is a cheaper, quicker option for viral detection [163]. The SHERLOCK (STOPCovid) test provides accurate results in under an hour with only two key steps: liquid handling and tube opening [165]. This makes testing simpler and decreases sample cross-contamination. Clinical trials show extremely high accuracy for both negative and positive detection [163]. American trials involved 12 SARS-CoV-2 positive and 5 negative patients. Results successfully revealed SARS-CoV-2 detection in 3/3 trials for 11/12 patient samples with only one misrepresented answer in the three rounds. In negative patients, STOPCovid accurately provided negative results [165].
In May 2020, SHERLOCK became the first CRISPR SARS-CoV-2 rapid diagnostic tool approved by the FDA [166], followed by Mammoth Bioscience's DETECTR in August. These diagnostic tools outweigh all current competitors in SARS-CoV-2 detection. They will only be refined to be more cost-effective and accurate. Indeed, on January 21 st 2022, Mammoth Bioscience's high-throughput assay, DETECTR Boost, was given emergency approval for laboratory use [167]. In January 2022, pharmaceutical giant Bayer paid Mammoth Biosciences $US 40 million to work on gene-editing technologies in four different diseases using their extensive CRISPR toolbox [168]. SARS-CoV-2 technologies based on alternative CRISPR-Cas systems are likely to lead to new and improved methods for diagnosis, treatment and prevention.

Conclusions
Global case numbers of SARS-CoV-2 infections have passed 765 million as recorded in May 2023. The rapid advances in biotechnology during the pandemic has shown the power of global medical cooperation in combating SARS-CoV-2. Among the available treatment and management regiments, vaccination platforms such as mRNA, as well as pre-existing viral vectors, and inactivated vaccines were the best defence against severe illness and death caused by SARS-CoV-2 pandemic. Due to the ease of production and enormous efficacy, mRNA vaccines have been heralded as the future of vaccine technology. The success of mRNA platform in preventing the SARS-CoV-2 infection have led to further explorations and development of mRNA vaccines for other infectious diseases as well as cancer. Furthermore, in tandem with development of vaccine platforms, there is hope for alternative ways of administration of vaccines, transitioning from intramuscular (IM) to the non-invasive intranasal, which may ally fears for many. Unfortunately, at this time, only the viralreplicating platforms are using this approach in clinical trials.
At the height of the pandemic, while waiting for EUA and large-scale production of the various vaccines, healthcare returned to basics in treatment and management, that is, utilizing repurposed medications and monoclonal therapies. Most of these broad-class drugs were able to manage mild to moderate symptoms of SARS-CoV-2 at home; however, combinatory use with immunotherapies improved their therapeutic efficacy. On the other hand, convalescent plasma may have potential but presented logistical challenges and mixed results. Nevertheless, due to limited resources, infrastructure, and economics, not all were privy to these treatments.
Aside from the refinement of various vaccines platforms, one of the most significant biotechnological breakthroughs lie in the redesigning of CRISPR-technology for both detection and treatment purposes. Repurposing of CRISPR technology such as PAC-MAN would represent an additional treatment for combatting mutating variants. Current mRNA vaccines together with putative CRISPR technology, we should be able to mount an agile response not only to coronaviruses, but to other future viral threats. The remarkable scale of global cooperation seen during the SARS-CoV-2 pandemic has reduced vast human suffering and mortality. Furthermore, biotechnological breakthroughs will continue to evolve, thus preparing us for the future pandemics.