Antibody production is related to vaccine efficacy and to pandemic remediation, such as COVID-19. The spike molecule has been the chief protein target for the COVID-19 vaccine. The concentration of antibodies for SARS-CoV-2 has been reported to be between 5–80 µg/mL and, depending upon the degree of infection and/or disease, within people prior, during, and post vaccination. However, the nucleocapsid protein, could also be the target of COVID-19 antibodies, has not been used for the COVID-19 vaccine production yet. Therefore, we highlight here how the nucleocapsid protein can be targeted by the antibodies produced in response to nucleocapsid antigen, In vitro conditions.
This was demonstrated by assembling a biomarker using natural and synthetic genetic sequences in yeast to produce recombinant proteins, including spike and nucleocapsid. This genetic assemblage was carried out using pYES2 (INVSc1 Saccharomyces cerevisiae) which was grown under different conditions, such as external application of micro-current during cDNA yeast culture growth, using synthetic biology. Cell-free lysates from these cloned yeasts were used as the source of antigens which contain the recombinant spike and/or nucleocapsid proteins. cDNA yeast grown under micro-current produced a lysate/antigen which induced higher antibody expression when they were mixed at different concentrations, as compared to the lysate/antigen produced without micro-current, which fluorescence of the antibody was lower when both were mixed. Hence, N antibody showed higher fluorescence when mixed with its respective lysate/antigen, under micro-current. The results of the investigation also corroborate the importance of nanoparticle sizes. The presence of these proteins was confirmed through particle size and molecular weight using the Zeta-sizer light-scattering method, as well using a theoretical mathematical formula. The interaction of the cell-free lysate with the commercial antibodies against spike and nucleocapsid was recorded by using a photonic detection method, after non-radioactive labeling through molecular conjugation.
The results show that the nucleocapsid antigen was targeted by the COVID-19 antibody similarly to, and sometimes stronger than, the spike antigen, depending upon the ratio (i.e. lysate: antibody= 1:1, 1:2, 1:3, and vice versa) between the antigen lysate and the antibody concentrations; 1:2 of lysate: antibody yields the best result. Different concentrations of commercial antibody were tested, ranging from 0.5–100 µg/mL. Our results show a higher antibody binding when combined with N lysate/antigen. This affinity was enhanced when each of the antibodies were mixed with the two lysate/antigens, in which nucleocapsid showed the higher antibody binding. This suggests a possible molecular compensation between spike and nucleocapsid proteins. Results indicate that nucleocapsid could be a potential and/or complement protein for vaccine development, and blood testing will be done to corroborate the denoted technology.
COVID-19, spike, nucleocapsid, particle size, micro-current, vaccine, molecular compensation
The COVID-19 pandemic affected the entire world at different population levels, producing different reactions from the global scientific community (Cuero, 2021; Johns Hopkins Coronavirus Resource Center 2020). It was caused by severe acute respiratory syndrome SARS-CoV-2 (Coronavirus 2 or COVID-19) and the large number of people who were affected and/or lost their lives due to COVID-19 has been well-reported (Johns Hopkins Coronavirus Resource Center). Hence, scientists have made a global collaboration to characterize, study and generate mechanisms to identify and control it (Cuero, 2021; Dutta et al., 2020).
The SARS-CoV-2 virus’ RNA genome is approximately 30 kb, which includes various reading frames and nonstructural and structural proteins which include Spike (S), Nucleocapsid (N), Membrane (M), and Envelope (E) (Cuero, 2021; Dutta et al., 2020). The two proteins, S and N, have been the most used to identify the SARS-CoV-2 virus. However, the S protein is most commonly used for developing an anti-COVID vaccine, despite its higher mutagenesis, which could make mitigation of the disease more complex, as compared to the N protein which exhibit high conservative regions and fewer mutations over time (Dutta et al., 2020).
For instance, S protein requires higher concentration for mitigation of the COVID-19 disease. Furthermore, reports have shown that the increase of the infection rate of COVID-19 was related to the decrease of concentration of antibodies at a particular time (Williams, 2022). Other reports have demonstrated that the interactive effect between antigen and antibody depends also on the concentrations of these proteins, which in COVID-19 are S or N proteins, to target the igG antibodies (Dutta, 2020). Also, the N protein is more conserved, and exhibits more stability and less mutations, thus possibly making it a more dependable target over time compared to the S protein (Dutta et al., 2020; Kang et al., 2020). Hence, it suggests that N protein could be considered as a candidate for a SARS-CoV-2 vaccine (Dutta et al., 2020).
The two proteins also exhibit some distinctly different physical-chemical characteristics – the two main proteins have different sizes – S is approximately180 kDa, and N is approx. 114.07 kDa. In addition, S protein is hydrophobic, and N protein is able to interact with the genome through both hydrophobic and hydrophilic interaction (Muriaux & Darlix, 2010). This confirms the scientific evidence of the dielectric properties of microbial and mammalian cells (Edmonds, 2001). The N protein also has an advantage of being multivalent and an adaptable RNA-binding protein (Cubuk et al., 2021) – perhaps this is what allows the N protein the ability to protect the SARS-CoV-2 RNA.
The electrical properties of both S and N proteins also determine their molecular binding capacities. For instance, S protein has a unit that binds to the negatively-charged nitrogen and oxygen active points of the ACE2 receptor in human cells. The S protein also possesses a receptor binding domain (RBD) that has active points which are positively charged (Huang et al., 2020). This may allow the S protein to be an electron acceptor. While the N protein, which binds and protects the RNA of the virus, is both hydrophobic and hydrophilic, hence the external part of the protein (which is hydrophobic and makes it less reactive) and the internal part (which is hydrophilic and nucleophile), enabling it to react and protect the RNA of the virus (Cubuk et al., 2021).
Antibodies, or immunoglobulin, are proteins which are produced by the immune system in response to a foreign substance called an antigen, and the antibody-antigen interaction is the foundation for vaccine development. For instance, in SARS-CoV-2, there are specific antibodies for S or N proteins (Cuero, 2021; Barnes et al., 2020). The antibody-antigen interaction, which is based on chemical bonding, also follows electrical principles. This interaction is based on nucleophile union points influenced by hydrogen, positively-charged nitrogen, and negatively-charged nitrogen and oxygen (Huang et al., 2020). The antibody-antigen interaction also resembles the lock and key principle (Branden, et al. 1995). The technique for effective production of antibody has improved markedly (Ozturk & Palsson, 1991).
Immunoglobulin antibody (IgG) are induced by the S and N proteins from CoV-SARS-2. However, the most common diagnosis of COVID-19 is based on RT-qPCR which, at times, has shown inconsistency due to inadequate sensitivity and/or specificity, low throughput, and other factors (Chen et al., 2021). This suggests the need for more reliable COVID-19 tests, and perhaps the use of N protein as a complementary vaccine to strengthen protection against the disease. Also, one of the most used methods to diagnose this disease is by using the ELISA technique to identify the proteins related to CoV-SARS-2 (Cuero, 2021; Liu et al., 2020).
Currently, in vitro transcription (IVT) is a technique used today for making messenger RNA (mRNA) for encoding different proteins including S and N (Kim, 2021). Therefore, we herein are proposing the use of complementary DNA (cDNA) to produce recombinant proteins including nucleocapsid as an alternative to, or complement of, COVID-19 vaccines, influenced by antibody concentrations, using synthetic biology.
We used a molecular and synthetic biology approach by assembling genes to codify for nucleocapsid or spike proteins in Eukaryotic yeast cells. This was done to study antibody-antigen interaction In vitro towards a possible use of the nucleocapsid protein as an alternative or complementary to spike protein to develop a vaccine. In addition, this investigation was to demonstrate the use of a biophotonic test to effectively confirm the interaction between the antigen/lysate-antibody based on concentrations.
In general, the sizes of nanoparticles span the range between 1 and 100 nm. However, how a nanoparticle is viewed and defined depends very much on the specific application and on its electro-conductivity. Nevertheless, regardless of the application of nanoparticles, they all must exhibit high-energy capacity, which can also depend on the shape of the nanoparticle (Cuero, 2016).
Applied external micro-currents have been used for stimulating cell division and/or molecular expression, especially for microbial growth culture. Microorganisms, such as bacteria and yeast have been used to carry out electrochemical investigations since they can be either producers, donors, receptors, or conductors of electricity (Noble, 1978; Tondera et al., 2019).
Photonity tests are based on fluorescence induced by fluorophores or fluorochromes that mark cells and identify the wavelengths in which energy will move in the form of photons, expressed as a fluorescence (Tario and Wallace, 2014; Cuero, 2023). Cyanines dyes are commonly used as fluorescent molecules, which are composed of odd methine chains – two nitrogen atoms at their ends which play the roles of both electron acceptors and donors, making them key points in the marking and expression of molecules such as proteins (Ziarani et al., 2018). The wavelength in the methine chain will influence the absorption by cyanine within the electromagnetic spectrum. The sulfo-Cyanine3 and sulfo-Cyanine5 dyes (S-Cy3 and S-Cy5) consist of two indole rings and a sulfo group, which allows solubility in water, greater versatility, and greater photostability (Black et al., 2017). Both Cy3 and Cy5 fluorophores exhibit different absorbencies, hence the Cy3 approaches the visible light range while the Cy5 approaches in an affinity toward infrared wavelengths (Moreira et al., 2015). During the process of conjugation, the fluorophore dyes bind to the targeted proteins, antigens, or antibodies. Thus, the dyes absorb the photon inducing changes in the protein, and expresses this energy in the form of fluorescence that is recognized as the photosensitization process (Cuero, 2023). Our results show that photonity could be used as a complementary test to reconfirm current, standard COVID-19 tests such as PCR, that sometimes exhibit ambivalent outcomes (Chen et al., 2021). Thus, we deployed sensitive photonic technology to test and confirm our approach.
Photonity is a natural, physical, inherent characteristic expressed in all systems. This photonic characteristic influences all biological systems. Photons which are emitted by all biomolecules are referred to as ‘biophotons’ due to the common expression of photonity in all biomolecules, and it has effectively been used as a sensitive technique for detection of biomolecules (Freifelder 1985; Cuero, 2022; Cuero et al., 2020). Also, a light scattering technique, Zeta-sizer, which is a very sensitive method for detecting the size of molecules such as proteins, was effectively used in our investigation (Cuero, 2022; Cuero et al., 2020; Nobbmann, et al., 2007). For instance, the use of Zeta-sizer provides an advantage to confirm the presence of both S and N proteins based on the molecular size, which were produced in this present investigation throughout the assemblage of genes codifying for S and N proteins. The advantage of using the Zeta-sizer technique, which can characterize biomolecules in solution, is to obtain precise results in a short period of time (i.e. within one minute) with precision (Nobbmann, et al., 2007). Therefore, the benefit of using these two detection technologies, photonity and Zeta-sizer, in the denoted investigation was to enhance the reliability of our results.
Under Process (Review Completed)
