Detection of SARS-CoV-2 and Its Immune Response: Comparison
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Coronaviruses are a large family of well-established pathogens of various hosts, including domestic animals, wildlife, and humans.

  • lateral flow immunoassay
  • biosensors
  • COVID-19

1. Overview of SARS-CoV-2

Coronaviruses are a large family of well-established pathogens of various hosts, including domestic animals, wildlife, and humans [1]. The viruses that caused previous outbreaks in humans, causing severe respiratory illness, lung injury, and death, are SARS-CoV (severe acute respiratory syndrome coronavirus) in 2003 and MERS-CoV (Middle East respiratory syndrome coronavirus) in 2012 [2]. A recent genomic analysis with several bioinformatics tools showed that SARS-CoV-2 has a genome very similar to the Bat coronavirus and the receptor binding domain (RBD) of the spike glycoprotein like the Malayan pangolin coronavirus [3]. This evidence indicates that the horseshoe bat is the natural reservoir, and the main evidence suggests that the Malayan pangolin is an intermediate host [3].
SARS-CoV-2 is an enveloped virus with a single-stranded positive-sense RNA. The genome size of this pathogen ranges from 29.8 kb to 29.9 kb [4]. The virus encodes at least 29 proteins. The structural proteins are spike (S), membrane (M), envelope (E), and nucleocapsid (NP) proteins [5]. Nonstructural proteins (nsps) have functions necessary for replication and transcription in the viral life cycle [6]. Viral particle size ranges from 80 to 120 nm [7].
The mechanism of viral infection in humans is through droplets and aerosols, which can travel through the air [8]. Infection occurs in cells expressing ACE2 (angiotensin-converting enzyme 2) and TMPRSS2 (transmembrane serine protease 2) [9]. Coronavirus S protein binds to ACE2, the main SARS-CoV-2 receptor that mediates viral entry into cells, and TMPRSS2 cleaves the S protein (at the S1 and S2 subunits) of SARS-CoV-2, which facilitates the fusion of SARS-CoV-2 and the cell membrane [9][10][11]. In addition, it has been shown that the cysteine proteases cathepsin B and endosomal cathepsin L may also contribute to this process [10][12][13]. In the respiratory tract, ACE2 and TMPRSS2 are expressed in the secretory and hair cells of the nose, the secretory and hair cells of the conducting airways, in type II alveolar cells in the lungs, and in the corneal conjunctiva of the eye [14][15][16][17].
The etiological virus of the pandemic has continuously evolved, with many variants emerging worldwide. Variants are categorized as the variant of interest, variant of concern, and variant under monitoring [18]. There are five SARS-CoV-2 lineages designated as the variant of concern alpha, beta, gamma, delta, and omicron variants [19]. These variants increase transmissibility compared to the original virus and potentially increase disease severity [20].

2. Immune Response against SARS-CoV-2 in Brief

The SARS-CoV-2 infection involves diverse stages in the individual: start of infection, disease development, recovery, or systemic compromise. Each infection stage triggers and modulates innate and adaptative immune system mechanisms. Although SARS-CoV-2 is a virus that humanity is learning about, the immune response is equipped with mechanisms capable of dealing with this new threat. In the initial phase of SARS-CoV-2 infection, the individual presents a presymptomatic phase lasting up to 5 days, in which a high viral load is present [21]. In these early days of infection, antibodies may not have been produced. Therefore, innate immunity is the first activated. The innate immune response comprises soluble and cellular components that respond nonspecifically against the virus. The cellular compounds include dendritic cells (DC), monocytes, macrophages, neutrophils, natural killer (NK) cells, and other innate lymphoid cells (ILCs) [22]. Whereas soluble components include complement systems, soluble proteins, interferons, chemokines, and naturally occurring antibodies [23]. Immune response cells recognize pathogen-associated molecular patterns (PAMPs) of SARS-CoV-2 through pattern recognition receptors (PRRs) such as Toll-like receptors (TLR), RIG-I-like receptors (RLR), and melanoma differentiation-associated protein 5 (MDA5). The viral sensing triggers the activation of signaling pathways which induce the production of immune mediators to generate an antiviral state mainly mediated by type I (IFN-α/β) and type III (IFN-λ) interferons (IFNs) [24]. Reports have described that robust IFNs production during the early stage of infection is required to have a protective innate immune response against the virus [25]. On the contrary, an inadequate and slow response to type I and type III IFNs due to virus evasion mechanisms, host comorbidities, or genetic defects cause an exacerbated immune response. This inadequate response induces elevated levels of chemokines (CCL2, CCL8, CXCL2, CXCL8, CXCL9, and CXCL16), high expression of proinflammatory cytokines such as IL-6, IL-10, IL-1, and TNF, in addition to activation, and recruitment of immune cells [26][27]. The called “cytokine storm” leads to unbalanced levels of proinflammatory and antiviral mediators that remain the leading cause of ARDS and multi-organ failure [25][26][28].
On the other hand, the adaptive immune response is orchestrated by CD8+ T lymphocytes, TCD4+, and B lymphocytes, responsible for immunological memory. In response to SARS-CoV-2 infection, it has been shown that non-severe patients or patients with mild symptoms have a low viral load and may not have produced antibodies [29][30]. In contrast, antibodies have been detected by immunoassay tests and biosensors in patients with severe symptoms or cases [29][31]. Patients with a high viral load activate the humoral immune response in the first two weeks of infection [32]. The first seroconversion of antibodies is against protein N, followed by protein S of SARS-CoV-2 in patients with disease symptoms [33]. Immunoglobulins IgA and IgM begin to be detected within the first ten days of infection; however, both antibodies can cross-react with protein N, which is highly conserved among coronaviruses [34][35]. Moreover, high levels of IgG1 and IgG3 are expressed ten to fourteen days after infection in patients with disease symptoms [36][37]. Older adults and seriously ill individuals reach high specificity antibodies concentrations against SARS-CoV-2 S protein.
Due to the urgency of reducing thousands of people’s cases and deaths, scientists have developed several vaccines against COVID-19. Efforts are being made to apply vaccines with emergency use authorization to the world population. Vaccination elicits immune responses capable of potently neutralizing SARS-CoV-2. However, the available data show that most approved COVID-19 vaccines protect against severe disease but do not prevent the clinical manifestation of COVID-19 [38]. Instead, it has been demonstrated that new variants with mutations in the spike, the main target of neutralizing antibodies, can escape the neutralization of humoral immunity [39][40].

3. SARS-CoV-2 Detection

Molecular tests or biosensors are the tools for detecting SARS-CoV-2 nucleic acids/ antigens/antibodies against the virus (Figure 1). In the early part of the illness, viral particles and their subunits can be detected; beyond the first two weeks of illness onset, antibodies against the virus could be detected [41]. The SARS-CoV-2 infection stage is highly correlated to the diagnostic technique recommended for the pandemic. Early diagnosis of the disease and isolation of infected people is key to controlling the transmission of SARS-CoV-2 [42][43]. In the initial phase of SARS-CoV-2 infection, the individual presents a presymptomatic phase lasting up to 5 days, in which a high viral load is present [21]. During these early days of infection, antibodies may not be detected. Therefore, since the pandemic began, the diagnostic method has been based on detecting viral genes using the molecular PCR technique, the gold standard worldwide [44][45][46]. The pandemic has exceeded the ability to identify the virus in laboratories using molecular techniques; this has motivated the development of new technologies for the rapid detection of SARS-CoV-2 that are easy to perform compared to molecular tests in clinical laboratories. LFIA has been the unique device approved and available to use in mass worldwide. Biosensors with transducers are developing in SARS-CoV-2 diagnostic. However, most nanomaterials used in these biosensors present interferences with contaminants in human samples compared to performance under experimental conditions. It is important to emphasize that LFIAs have the unique properties of availability, accessibility, economy, and POC (including home use), these characteristics that are not shared by all biosensors with a transducer. In addition, biosensors with transducers require exclusive handling in laboratories certified under the Clinical Laboratory Improvement Amendments of 1998 [47][48]. The FDA have to date approved only one piezoelectric biosensor [47] (Figure 1).
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Figure 1.  Principio de la prueba LFIA. La pruebale of LFIA test. LFIA detecta la molécula objetivo en una membranatest detects the target molecule on an absorbente con anticuerpos alineados para formar las líneas de prueba y membrane with antibodies aligned to form the test and control. La muestra se coloca en una almohadilla de muestra, luego lines. The sample is placed on a sample pad, then migra a la almohadilla dtes to the conjugado, que contiene el conjugado inmovilizado, generalmente hecho de te pad, which contains the immobilized conjugate, usually made of nanopartículas (oro coicles (colloidal, látex gold, coloreado od or fluorescente, celulosa coloreada latex, colored cellulose) conjugadas con anticuerpos o antígenos. La muestrated to antibodies or antigens. The sample interactúa con els with the conjugado y ambosate, and both migran a la siguiente sección de la tira, donde se inmovilizan loste to the next section of the strip, where the biological componentes biológicos del ensayos of the assay (proteínains/anticuerpos/antígenos). En esta sección, se capturan el analito y el bodies/antigens) are immobilized. In this section, the analyte and conjugado. El exceso de reactivo pasa a través de las líneas dte are captured. Excess reagent passes through the captura y se ae lines and accumula en la almohadillates on the absorbente. Los pad. The resultados ss are interpretan en la membrana ded on the nitrocelulosa como la llulose membrane as the presencia o ausencia de las líneas de prueba y e or absence of the test and control lines.

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