**4. SARS-CoV-2 vaccine development in COVID-19 genomic era**

A major advantage in reducing the COVID-19 pandemic was the development of vaccines against SARS-CoV-2 since all the approved COVID-19 vaccines, although based on the initial SARS CoV-2 strain, continue to preserve efficacy against hospitalization and death, especially after administration of a booster dose [90]. Until the appearance of Omicron, all variants had a convergent evolution pattern [91], selecting similar mutations in particularly vulnerable genomic sites and clustering in similar serotypes. Omicron is highly antigenically divergent from the other VOCs [92] and is characterized by a continuous antigenic drift, giving rise to several sublineages, with limited cross-antigenicity. The most recently selected sub-lineages, designated as variants of concern (BA4, BA5) have higher neutralization escape capacity [93], and vaccination efficacy seems to decrease, although not significantly, even in terms of protection against severe forms of the disease.

Currently, according to the WHO, more than 150 vaccines are in clinical development and almost 200 are in preclinical development [94]. Reported studies demonstrated that the benefits of COVID-19 vaccination compensate for the risks that involve rare but serious adverse effects [95]. For example, a study focused on the administration of almost two million first doses of the vaccine Pfizer-BioNTech vaccine in the US reported only 21 cases of anaphylaxis after administration, with no fatalities reported [96].

COVID-19 vaccines developed so far and tested or approved for clinical trials can be classified into inactivated vaccine, live attenuated, viral vector-based vaccine, RNA, DNA, protein subunit, and virus-like particle (VLP) vaccines [97].

**Inactivated vaccines** are obtained from a virus multiplied on cell cultures and then chemically inactivated. This system can sustain stably expressed,

conformationally native antigenic epitopes [98]. The advantages of inactivated vaccines include the capacity of the vaccine to induce an immune response that results in production of antibodies against many epitopes of the SARS-CoV-2, including S protein, N protein, and E protein [99]. On the other hand, the vaccine is well tolerated, the adverse reactions reported are rare, without reported deaths, and the study and development of this type of vaccine are relatively complete [100]. The disadvantages arise mainly from the fact that the living virus must be manipulated in a biosafety level-3 laboratory at least and there is a limitation in vaccine production that depends on viral productivity [97].

**Viral vector-based vaccine** is also considered a classic vaccine since this medical technology was introduced in 1972 by Jackson et al., and uses a secondary virus as a transient gene expression vector. Nonreplicating viral vector-based vaccine, the most commonly utilized, uses viral vectors deficient in replication, to deliver a specific antigen to the host cell in order to induce immunity against the desired antigen. The vector used in the viral vector-based vaccine developed against SARS-CoV-2 infection is adenovirus [101]. Administration of this type of vaccine against SARS-CoV-2 infection seems to induce rapid and complex antibody responses as well as cellular immune responses by activation of Th1 cell responses [102]. Comparing to inactivated vaccines the production of viral vector vaccines is safer as there is no need to manipulate live SARS-CoV-2. However, in the case of adenovirus-based viral vector vaccines, rare but severe reactions have been reported, especially thrombocytopenia, sustaining the need of monitoring platelet levels. The mechanism that triggers these effects is mainly the development of pathological anti-platelet factor 4 (PF4) antibodies, as result of vaccine administration that activates platelets and the coagulation system. Also, the immunogenicity of these vaccines can be reduced in some people that present neutralizing antibodies against several adenoviruses [103].

**Live attenuated vaccines** are developed by a recoding of the virus genome, being a well-known method of immunization against pathogens. Thus, the virus is attenuated by *in vitro* or *in vivo* passage or reverse-genetic mutagenesis, resulting in a weakly pathogenic that is also able to mimic the live virus infection. Usually, this type of vaccine can produce a durable immune response, but the apparition of secondary mutations that can cause reversion into virus wild-type strains, especially in the case of RNA viruses is considered a disadvantage for this type of vaccine [99, 104, 105].

**DNA vaccines** use a sequential transcription-to-translation process that sustain the production in host cells of a viral antigen that is encoded by a recombinant DNA plasmid, inducing neutralizing antibodies [105]. DNA vaccines present a higher stability compared with mRNA vaccines**,** the production risk of DNA vaccines is relatively low and does not require the presence of an infectious agent. On the other hand, the immunogenicity of the DNA vaccine is low and the efficacy depends on the injection method [97].

**mRNA-based vaccines** comprise mRNA molecules that encode viral protein antigens and the main problem of this type of vaccine is removed by utilization of nanoparticle delivery carriers that overcome mRNA instability. Moreover, these nanoparticles are adjuvants to sustain the activation of the immune response. The method to obtain these vaccines is based on the *in vitro* transcription process for obtaining mRNA and the technology is quite developed today to allow obtaining large doses in a short time for any pathogen [106]. Due to the high vaccination rate with this type of vaccine, it can be clearly demonstrated that SARS-CoV-2 mRNAbased vaccination induces a persistent germinal center B cell response and Th1 cell responses, which allows the development of strong humoral immunity [107]. The main disadvantages are the reported adverse reactions, especially myocarditis and the necessity to preserve the vaccines at low temperatures to avoid mRNA degradation [108].

**Protein subunit vaccines** use key viral proteins or peptides that can be obtain *in vitro* using bacteria, yeast, or mammalian cells [99]. COVID-19 protein subunit vaccine can induce Th1 cell responses and a high titer of neutralizing antibodies but due to the large molecular weight, the *in vitro* synthesis rate of the viral S protein is relatively low [97].

**Virus-like particle vaccines** use viral capsid proteins or replication-defective virus particles without the viral genomes but the technology for obtaining this type of vaccine is more complex [109].

Although studies to date show that the administration of COVID-19 vaccines may reduce the risk of symptomatic infection and decrease mortality, a decline in virus-neutralizing activity with the emergence of new variants has been reported. Therefore, the strong contagious activity of Alpha and Delta variants and the powerful immune escape ability of Beta and Gamma variants were outclassed by the capacity of the Omicron variant to evade the immunity induced by the COVID-19 vaccines [97]. For example, the efficacy of BNT162b2 COVID-19 vaccine (Pfizer BioNTech) against SARS-CoV-2 VOCs starts at almost 90% in the case of B.1.1.7 (Alpha) [110] and decreases (in some reports) to about 35% in the case of B.1.1.529 (Omicron) [111].

Several studies have shown that breakthrough infections including Omicron VOC increase the breadth of the immune response in vaccinated persons [112]. As such, Omicron-specific vaccine candidates have been developed by several pharmaceutical companies and might be administered as booster doses for the recipients of a primary vaccination scheme or for persons already infected with previously circulating variants. In recent press releases, both Pfizer and Moderna [113, 114] announced that a second booster with Omicron-adapted vaccine candidates (either in a monovalent or bivalent formulation with the classic vaccine) increased significantly the magnitude of the immune response against the Omicron sublineages. It is hoped that such broad responses will be preserved for a longer period of time, as shown by data from trials with a previous version of a Moderna vaccine candidate developed against Betaanother highly immune evasive variant. Presently, administration of an Omicronspecific vaccine in unvaccinated persons is not recommended, due to insufficient data on the level of cross-protection against unrelated variants [115].

Future vaccination strategies are envisioned, aimed at finding better administration regimens (using extended intervals between doses, increased antigen concentrations, heterologous prime-boost schemes), or better vaccine formulations (multivalent vaccines, encoding the Spike protein of multiple VOCs, pan-coronavirus vaccines, and mucosal vaccines, administered intra-nasally).

### **5. Antiviral treatment development and emergent SARS-CoV-2 variants**

The development of direct antiviral drugs was rather slow, direct medication being replaced by vaccines in the treatment of COVID-19. However direct antiviral treatment proved to be effective, indifferent to the mutations that have accumulated while SARS-CoV-2 variants have emerged. Currently, the main therapeutic strategies are directed toward (a) direct inhibition of the viral entry, (b) inhibition of viral replication, and (c) immunomodulatory treatment to block the cytokine release storm that underlies COVID-19 severe evolution [116].

#### **5.1 Direct inhibition of the viral entry**

In the first category, there are several anti-spike protein monoclonal antibodies (MAB) such as bebtelovimab, sotrovimab, casirivimab and imdevimab, bamlanivimab and etesevimab used for the treatment of mild-to-moderate COVID-19 in adults and pediatric patients (12 years of age and older weighing at least 40 kg). Additionally, tixagevimab and cilgavimab were authorized for emergency use as pre-exposure prophylaxis for prevention of COVID-19 in adults and pediatric individuals. However, in the context of high frequency of the Omicron BA.2 sub-variant, authorizations for all of these monoclonal antibodies were revoked, except for Bebtelovimab that has a broad neutralizing activity, unaffected by the most common mutations present in all of the known variants of concern of SARS-CoV-2, including the Omicron subvariants BA1/BA2 [117, 118].

The administration of MABs has several significant drawbacks, including the need for intravenous administration in healthcare units by qualified healthcare personnel who have access to emergency medications to treat severe reactions, including anaphylaxis. Common side effects include hypersensitivity, with anaphylaxis and infusion-related reactions, nausea, vomiting, pruritus, and rash. Among advantages, we can count no drug-drug interactions.

This strategy can be used against the receptor binding domain only as long as no mutations in the spike glycoprotein occur. One possible way around this problem would be administration of several MAB antibodies that could simultaneously bind to different parts of the receptor binding domain of the SARS-CoV-2 spike protein.

#### **5.2 Inhibition of viral replication**

To date, several antivirals received conditional authorizations for usage in the interest of public health because they address an unmet medical need and the benefit of immediate availability outweighs the risk from less comprehensive data than normally required (**Table 1**) [119]. The oldest is remdesivir, a drug that has to be intravenously administrated, with plenty of adverse effects, which drastically reduce its utility, conditioning its administration by hospitalization. The newest molnupiravir and nirmatrelvir – ritonavir are both oral antivirals, less expensive, with huge advantage that they can be administered in patients isolated at home. However, the treatment has to be started early, and Paxlovid came with several drug–drug interactions that can complicate its use in patients taking other medications. Moreover, there is a concern about molnupiravir regarding a potential impairment of bone and cartilage growth, being restricted for usage in children [120].

**Molnupiravir** is a slightly modified small-molecule drug developed from a ribonucleoside known as NHC (β-d-N4 -hydroxycytidine) by a research team at Emory University in Atlanta, Georgia. Intended initially to enter clinical trials against influenza, tested it as a treatment for COVID-19 by Ridgeback Biotherapeutics Company, which partnered later with Merck in May 2020, for large-scale clinical trials [121].

After oral administration, molnupiravir breaks down to form NHC that is further phosphorylated to NHC triphosphate. Under this form, NHC is linked by SARS-CoV-2 RNA-dependent RNA polymerase (RdRP) and used for RNA chain elongation during viral replication instead of guanosine or adenosine. This leads to an accumulation of errors in the viral genome that ultimately render the virus noninfectious and unable to replicate [122–124].

*SARS-CoV-2 Variant Surveillance in Genomic Medicine Era DOI: http://dx.doi.org/10.5772/intechopen.107137*


#### **Table 1.**

*Anti-viral drugs currently used for COVID-19 therapy.*

Efficacy and safety were evaluated in phase 3 double-blind, randomized, placebocontrolled trial MOVe-OUT clinical trials [NCT04575597] on unvaccinated and seronegative subjects, with final results published on February 10, 2022, reporting a relative risk reduction of 30% for hospitalization or death at 29 days [122]. Moreover, new results in evaluating virological outcomes presented at the 2022 European Congress of Clinical Microbiology and Infectious Diseases (ECCMID) (Abstract #4514) showed that molnupiravir was associated with more rapid elimination of infectious viruses than placebo. Thus, at 3 and 5 days of treatment, no infectious virus was detected in patients who received LAGEVRIO compared with 21.8%, and 2.2% respective, of patients who received placebo [125]. The risk for adverse effects was 30% with molnupiravir vs. 33% with placebo. The most common adverse effects reported were diarrhea (3%) and nausea (2%) classified as mild or moderate. Also, no drug–drug interactions have been identified [122].

Molnupiravir received an Emergency Use Authorization (EUA) issued by the Food and Drug Administration (FDA) on December 23, 2021 for LAGEVRIO as treatment of mild-to-moderate COVID-19 in adults with positive results of direct SARS-CoV-2 viral testing, who are at high-risk for progression to severe COVID-19, including hospitalization or death [126]. Also, received authorization in the United Kingdom (U.K.) for molnupiravir (MK-4482, EIDD-2801), and currently is under review for authorization by European Medicines Agency (EMA).

**Nirmatrelvir – Ritonavir** (Paxlovid) is a combination of two drugs, ritonavir (a pharmacokinetic enhancer) and newly developed protease inhibitor nirmatrelvir (PF-07321332), a structure-based potent inhibitor of SARS-CoV-2 3-chymotrypsin– like cysteine protease enzyme (Mpro) [127]. Mpro protease is involved in processing viral polyproteins into functional units, and since essential to viral replication. Within the absence of recognized human analog, Mpro is an attractive antiviral target across a wide spectrum of coronaviruses. Nirmatrelvir is administrated with ritonavir, an inhibitor of cytochrome P450 (CYP) 3A4, that blocks the metabolism of nirmatrelvir increasing and maintaining its plasma concentration approximately five to six times higher than the in vitro 90% effective concentration [127, 128].

Evidence for efficacy and safety comes from a phase 2–3 double-blind, randomized, and controlled trial, which enrolled unvaccinated, non-hospitalized adults with high risk for progression to severe COVID-19. Subjects that were treated within 3 days after symptom onset with 300 mg of nirmatrelvir plus 100 mg of ritonavir had an 89% lower risk of progression to severe COVID-19 than placebo group [NCT04960202]. The risk for adverse effects was 22.6% with nirmatrelvir plus ritonavir vs 23.9% with placebo. The most common adverse effects were dysgeusia (5.6%) and diarrhea (3.1%) [129].

Paxlovid is associated with several drug–drug interactions that could complicate its use in the community due to ritonavir association that inhibits CTP3A and therefore may increase plasma concentration of drugs that may be associated with serious, life-threatening events (e.g., colchicine, clozapine, diazepam, simvastatin, etc.). Conversely, products that may increase the metabolism of nirmatrelvir/ritonavir and reduce their concentrations may be associated with a loss of antiviral effect (e.g., rifampicin, carbamazepine) [130]. Thus, a careful assessment of patients' medication is needed before administrating Paxlovid. A web tool developed by the University of Liverpool, which monitors drug interactions with current anti-COVID-19 therapies, may be helpful [131].

Nirmatrelvir – Ritonavir received a EUA issued by the FDA on December 22, 2021, for PAXLOVID for the treatment of mild-to-moderate COVID-19 in adults and pediatric patients (12 years of age and older weighing at least 40 kg) with high risk for progression to severe COVID-19 [132]. It is also authorized for use in European Union [133] since January 2022, under a conditional marketing authorization.

Both molnupiravir and nirmatrelvir, effectively inhibited viral replication of the Delta variant and the Omicron variant however, slight differences in antiviral response among wild-type, Delta, and Omicron variants were observed [134].

**Remdesivir** is a broad-spectrum antiviral medication administered via intravenous injection. The compound is a prodrug whose metabolizing leads to the release of a nucleoside analog GS-441524 monophosphate with subsequent biotransformation into GS-441524 triphosphate. Under this form, it acts as an inhibitor of the viral RNA-dependent RNA polymerase (RdRp) with potent activity against an array of RNA virus families including Filoviridae, Paramyxoviridae, Pneumoviridae, and Orthocoronavirinae [135]. Various studies have documented its inhibitory activity against SARS-CoV-1, Middle East respiratory syndrome (MERS-CoV), and SARS-CoV-2 in vitro [136, 137].

Data on its efficacy on COVID-19 infected patients came from ACTT-1 clinical trial [NCT04280705], which showed that remdesivir treatment shorten recovery times in hospitalized patients with COVID-19 (median recovery time of 10 days (95% confidence interval [CI], 9 to 11), in remdesivir treated patients as compared with 15 days (95% CI, 13 to 18) among those who received placebo) [138] and reduces chances of hospitalization or death with 87% for patients at high risk of severe disease [139].

The most common adverse effects that occurred in 5% of patients treated with remdesivir were respiratory failure, decreased glomerular filtration rate with increased creatinine in the blood, decreased lymphocyte and hemoglobin counts, anemia, and increased blood sugar levels [138]. Also, 5% of patients experienced nausea, headache, and cough.

In October 2020, remdesivir received the first FDA EUA as a treatment for COVID-19, which was extended in April 2022, for the first time for pediatric patients under 12 years of age including those who are older than 28 days, weighing at least 3 kg [140]. Also, in July 2020 received a conditional marketing authorization from EMA.

Remdesivir showed similar antiviral activity against the wild-type virus and the VOCs Alpha, Beta, Gamma, Delta, and Omicron variants. These findings are justified by the fact that the target proteins of these antivirals, viral RNA dependent RNA polymerase, and the viral main protease Mpro, respectively, are highly conserved. These results indicate that is a high probability that VOC that might emerge in the future will remain susceptible to antivirals that do not target the spike protein [141].

#### **5.3 Immunomodulatory treatment**

Many of the complications associated with COVID-19 are due to an augmented host immune response, which contributes to the increased severity of COVID-19 and death. Several immunomodulatory drugs can be administered in-hospital to severely ill patients to reduce inflammation and prevent a cytokine storm. These include corticosteroids, monoclonal antibodies that block the IL-6 receptor (tocilizumab, sarilomab, and siltuximab), monoclonal antibodies that block the IL-1 beta receptor (anakinra), and selective Janus kinase 1 and 2 inhibitors (JAK1 and 2) (baricitinib and ruxolitinib).

Immunomodulatory treatment leads to an improvement in clinical outcome. Among positive results, a decrease in hospitalization lengths, duration of mechanical ventilation, and mortality with 8.7% in the critically ill, and 6.7% in patients with severe COVID-19, are the most notable ones. Coadministration with anti-viral such as remdesivir, improved the clinical outcome, reducing the number of patients who experience progression to severe respiratory failure or death [142].

However, reported side effects are major. For example, anakinra treatment induces a decrease in hematological parameters, headache, diarrhea, an increase of liver function tests, and hyperglycemia [143]. Treatment with tocilizumab also caused neutropenia with severe infections, thrombocytopenia, and increased the liver enzyme levels. Moreover, several cases of bowel perforation were also reported [144, 145].

Another strategy that has been used in COVID-19 treatment included the use of COVID-19 convalescent plasma or ultrapotent antibodies isolated from SARS-CoV-2 elite neutralizers. These are individuals that displayed a highly potent neutralizing response with IgG 50% inhibitory concentration (IC50) values of <20 μg/mL. The ultrapotent antibodies are directed against conserved viral epitopes with broad spectrum activity against ancestral variant and the variant that emerged lately: B.1.1.7, B.1.351, B.1.429, B.1.617, and B.1.617.2 variants [146].

Convalescent plasma administration is nevertheless limited to high-titer products. It was associated with allergic and anaphylactic reactions febrile nonhemolytic reactions, hemolytic reactions, metabolic complications, transfusion-transmitted infections, and thrombotic events. Moreover, there is a theoretical risk of antibodymediated enhancement of infection and suppressed long-term immunity [147].

Currently, due to the intense studies carried out during the SARS-CoV-2 pandemic, several treatment modalities are available for COVID-19. There are both

molecules that block the virus from entering the cell, and molecules that interfere with and block viral replication. In addition, there are immunological modulators that can prevent severe development and even death. These therapeutic strategies are supported by prophylactic ones (e. g. vaccines), all in conjunction with aim of avoiding the disruption of social and economic calm.
