Feasibility of the Vaccine Development for SARS-CoV-2 and Other Viruses Using the Shell Disorder Analysis

Gerard Kian-Meng Goh(1),* A. Keith Dunker(2), James A. Foster(3), and Vladimir N. Uversky(4)

1)Goh's BioComputing, Singapore 548957, Republic of Singapore (gohsbiocomputing@yahoo.com)

2)Center for Computational Biology and Bioinformatics, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN46202, USA

3)Department of Biological Sciences University of Idaho, Moscow, ID 83843, USA

4)Department of Molecular Medicine,Morsani College of Medicine, University of South Florida, Tampa, FL, USA

*Corresponding author: Gerard Goh, gohsbiocomputing@yahoo.com

Abstract

Several related viral shell disorder models were built using AI predictors of protein disorder. The parent model detected the presence of high levels of disorder in the outer shells of viruses that lack vaccines. Another model found correlations between inner shell disorder and viral virulence. A third model was able to positively correlate the levels of respiratory transmission of coronaviruses (CoVs). These models are linked together by the fact that the models have uncovered two novel immune evading strategies employed by the various viruses. The first involves the use of highly disordered “shapeshifting” outer shell to prevent antibodies from binding tightly to the virus, thus likely accounting for the failure to develop a successful vaccine. The second usually involves a more disordered inner shell that provides for more efficient binding in the rapid replication of viral particles before any host immune response. This “Trojan horse” immune evasion often backfires on the virus when the viral load becomes too great in a vital organ, which leads to death of the host. Just as such virulence entails the viral load to exceed at a vital organ, a minimal viral load in the saliva/mucus is necessary for respiratory transmission to be feasible.. As for the SARS-CoV-2 no high levels of disorder can be detected at the outer shell M protein but some evidence of correlation between virulence and inner shell (N) disorder has been observed. This suggests that, unlike HIV, HSV and HCV, the development of vaccine for SARS-CoV-2 is likely feasible and that an attenuated vaccine strain can either be found in nature or developed by appropriate genetic modification of N, the inner shell.

Goals

A large number of COVID-19 vaccine candidates are currently in development or in clinical trials. While many of the vaccine candidates seem promising, the historical potentials for successes or failures in the search for vaccines in general have been mixed. While effective vaccines for rabies, polio and yellow fever virus (YFV) have been found, vaccines for HIV, HSV (herpes simplex virus) and HCV (hepatitis C virus) have never been found despite approximately 40, 30 and 100 years of search respectively. We present data using shell disorder models to show that SARS-CoV-2 is different from HIV, HSV and HCV. Furthermore, the shell disorder models provide suggestions for for vaccine development and therefore the development of a COVID-19 vaccine is feasible.

Protein Intrinsic Disorder and the Shell Disorder

Models

An important concept used is protein intrinsic disorder, which describes situations where the entire or a part of a protein is devoid of structure. Figure 1 shows an example of a protein that is partially disordered. The regions colored in red are those predicted to be disordered by PONDR(r)-VLXT. PONDR(r)-VLXT was built using neural networks (AI) that recognize disordered regions given the sequence inputs.

The Shell Disorder Models

Figure 1 3D structure of the SARS-CoV-2 N (Nucleocapsid) with disorder regions colored in red.

Three closely related projects were initiated sequentially during the last 15 years. They involved the use of PONDR-VLXT(r) to study disorder of the various shell proteins of a variety of viruses.

(1) Parent Shell Disorder Model (2005)that correlates outer shell disorder with absence/presence of vaccines for various viruses: HIV vaccine mystery and the viral shapeshifters.

(2) Spinoff Project #1 (2011) CoV (Coronavirus) Shell Disorder Model that correlates shell disorder with transmission mode potentials

(3) Spinoff Project #2 (2014) Virulence-Inner Shell Disorder Model that correlates inner shell disorder with virulence of a large number of viruses such as flaviviruses (e.g. YFV, dengue (DENV)) and Nipah virus.

Parent Project: HIV Vaccine Mystery and the Viral Shapeshiftes

Project began in 2005: The failures in the search for HIV, HSV, HCV vaccines can be linked to their highly disordered outer shells (First published in VirologlgyJ 2008).

Figure 1 Virion Physiology of HIV and Coronavirus (CoV). Note the similarities in shells. Our models focus on the disorder of these shell proteins

Table 1-2. Shell proteins of various viruses. Feline Immunodeficiency Virus (FIV). Equine Infectious Anemia Virus (EIAV).. Effective vaccines for poliovirus, rabies, YFV, rotavirus, EIAV (HIV's horse cousin) and smallpox have been discovered. Vaccines for HIV, HSV and HCV have never been discovered despite a search that spanned approximately 40, 30 and 100 years respectively.

Percentage of Intrinsic Disorder (PID) is defined as the number of amino acid residues predicted to be disordered divided by the total number of residues in the protein chain. We can notice the abnormally high disorder levels (PID) of the outer shells of HSV, HCV and HSV compared to viruses (e.g. rabies, YFV, rotavirus) which have effective vaccines.

Figure 2. Disorder levels of some retroviruses. Effective vaccines for EIAV (HIV's horse cousin) have been found in 1973 or before. No effective vaccine is available for HIV and FIV. A vaccine for FIV was introduced in 2002 but was later withdrawn from the USA partly because of the lack of efficacy. Note the high outer shell PID (disorder) for HIV and FIV and the low outer shell disorder of EIAV.

Highly disordered outer shell provides for a highly effective immune evasion strategy: Viral Shapeshifters This is seen in Figure 3. It is for this reason that viruses in which effective vaccines have been found have hard (low M PID) outer shells, while HIV, HCV and HSV have no effecove vaccines because of their soft outer shells.

Figure 3. Mechanism of Viral "Shapeshifting" Immune Evasion. Disorder at the outer shell (matrix) allows for motions that affect the glycoprotein surface. As a result of the motions, neutralizing antibodies are unable to bind tightly to the virus.

A Soft Disordered Outer Shell is Associated with Sexual Transmission as in the Case of HIV, HSV and HCV

HCV, HSV and HCV do not have to remain in the environment for long because of their sexually related modes of transmission. They can therefore afford a soft outer shell as seen.

Spinoff Project #1: CoV Mode of Transmission Shell Disorder Model

CoV Shell Disorder Model: Correlation Between Modes of Transmission and (Inner) Shell Disorder

Before the outbreak of MERS-CoV (Middle Eastern Respiratory Syndrome), we designed the CoV Shell Disorder model based on measurements of shell PID (disorder of CoV shell proteins) and the behaviors of animal CoVs especially porcine CoVs. CoVs easily clustered into 3 groups (see Table 3). Group A consists CoVs with high respiratory transmission but lower fecal oral transmission potentials. Group B is made up of CoVs with intermediate fecal oral and respiratory modes. CoVs in Group C are those with lower respiratory but higher fecal oral transmission potentials. Strong correlations between modes of transmission and mainly N PID (inner shell disorder) are found (r2=0.8, p<0.01).

SARS-CoV: Intermediate Fecal-Oral and Respiratory Transmission Potentials

SARS-CoV ( N PID - 50%, M PID =9%) was found to be in Group B i.e. intermediate fecal oral and respiratory transmission potentials. The model and results was published in 2012 (J Pathog).

MERS-CoV: Lower Respiratory but Higher Fecal Oral Transmission Potentials

In 2012, the MERS-CoV outbreak provided an opportunity to validate the model. Given the N & protein sequences, MERS-CoV had to fall into Group C i.e. higher fecal-oral but lower respiratory potentials. transmission potentials (PLoS Curr 2013). Clinical evidence supports this.

SARS-CoV-2: Intermediate Fecal-Oral and Respiratory Transmission Potentials with Something Else Very Odd

Figure 4. SARS-CoV has one of the hardest outer shell in the CoV family as seen by its low M PID.

The 2019-20 COVID-19 pandemic provided yet another opportunity for the validation of the model. This time, the N PID placed SARS-CoV-2 (N PID = 28%) in Group B alongside 2003 SARS-CoV. The model, however, detected something strange about the virus. Its outer shell is one of the hardest in the CoV family as seen in Figure 4.

SARS-CoV-2 has One of the Hardest Outer Shell among CoVs: Contagiousness

This hard outer shell along with its intermediate fecal-oral and respiratory transmission potentials may account for SARS-CoV-2 high contagiousness as the hard outer shell provides greater resistance to the antimicrobial enzymes found in the saliva and mucus. Recently clinical evidence does show that large quantities of the virus are shed at the first sign of symptoms. A later study has shown that such hard shell is commonly associated with CoVs of burrowing animals such as rabbits and pangolins that often come in contact with buried feces.

Spinoff #2: "Trojan Horse" Immunee Evasion-- Correlation of Inner Shell Disorder and Virulence

Ebola Virulence and Inner Shell(NP) Disorder

In 2015, we published a paper (Moleculat Biosystems) that reported correlations (r2=0.92, p<0.01, Figure 5) between virulence (Case-fatality rate(CFR)) and NP (nucleoprotein, inner shell) disorder(PID) among filoviruses/ebolaviruses.

Flavivirus/Dengue Virulence and Inner Shell(C) Disorder

A similar correlation (r2=0.8, p<0.01) was found among a wide variety of flaviviruses (e.g. dengue virus (DENV), Yellow Fever virus (YFV), Zika) and inner shell(C, capsid) disorder as seen in Figure 6. Strong correlation has also been found among DENV types (r2=0.9, p<0.01)

Nipah Virulence and Inner Shell Disorder With Mode of Transmission Linkages

In 1998-9 the Nipah virus (NiV) was discovered during an outbreak in Malaysia . The outbreak involved spread of the virus from bats to pigs and then humans with CFR and N PIDs of 38% and 41.73+0.1 respectively. Outbreaks in Bangladesh and India have higher virulence and N PIDs (2001-7, CFR=71%, N PID=42.67+0.26, 2008-12, CFR=91%, N PID=43.23+0.24). High correlation between CFR and N disorder was found (r2=0.93, p<0.01). As the 1999 outbreak involved pigs as intermediary but the Bangladesh-India cases did not, hints of relationships among modes of transmission, virulence and inner shell disorder can be found. Figure 7 reiterates this link using NiV relatives and HIV.

Figure 5. Correlation between Ebola (EBOV)/Filovirus virulence and inner shell (NP, Nucleoprotein) disorder (r2=0.92, p<0.01). Filovirus and ebolavirus types: MARV (Marburg), ZEBOV (Ziare), SEBOV (Sudan), BEBOV (Bundigugyo), REBOV (Reston). CFR: Case-Fatality Rate. VP30 is the matrix or membrane protein.

Figure 6. Correlation Between Virulence and Inner Shell (C, capsid) Disorder (r2=0.8, p <0.05) Among Flaviviruses e.g Tick-borne encephalitis virus (TBEV), West Nile virus (WNV), TBEV-FE(Far East), TBEV-Si(Siberia), TBEV-Eu (Europe)

"Trojan Horse" Immune Evasion Strategy.

The "Trojan Horse" immune evasion strategy involves quick replication of virus before the host immune system recognizes its presence. The inner shell proteins are crucial to viral replication and disorder makes it more efficient. The problem is that this strategy often backfires by killing the host when the viral load overwhelms vital organs.

Figure 7. Links between Mode of Transmission and Inner Shell PID especially among NiV (Nipah) and its Relatives. HeV (Hendra Virus), MeV (Measles Virus) and NIV are related. HIV is a retrovirus.

Shell Disorder Models Re-affirm Feasibility of COVID-19 Vaccine Development

Shell Disorder Models: SARS-CoV Vaccine Development is Feasible

According to the parent shell disorder model, the search for HIV, HCV and HSV have failed because of their modes of transmission that allows for their soft outer shells. None of these can been oberved in SARS-CoV or SARS-CoV2 as seen in Figure 8. Therefore, feasibility is seen as likely in the development of vaccine for SARS-CoV-2.

Links Among Virulence, Modes of Transmissions and Inner Shell Disorder: Viral Loads in Vital Organs Vs. Body Fluids

We have seen disorder of the inner shell proteins allows for more efficient replication of the virus. Inner shell proteins play vital roles in the replication process. Therefore, inner shell disorder is instrumental in the "Trojan Horse" immune strategy i.e. rapid production of viral particles before the host immune system is able to detect the presence of the virus. Oftentimes, this backfires when the viral loads at vital organs overwhelm the host and lead to death. Likewise, inner shell disorder plays important roles in modes of transmission as respiratory transmission may not be feasible unless the viral loads in body fluids exceed certain thresholds.

Links between SARS Virulence and N PID Suggest Vaccine Strategy

Evidence that SARS-CoV-1/2 employ the "Trojan Horse" immune evasion strategy can be seen in Figure 9, which also suggests a unique strategy for vaccine development that involves attenuating the virus by mutating the N protein such that greater order is provided.

Shell Disorder Models Suggest that SARS-CoV-2 Entered the Human Population as a Vaccine Strain Several Years Ago

A study of pangolin-CoV from 2017-9 using the disorder models suggests that SARS-CoV-2 entered the human population a few years ago as an attenuated strain via pangolins before mutating into its current virulent form just as the Sabin polio vaccine did (Figure 10). This may be the reasons that the medical community was not aware of its presence in the human population until recently since COVID-19 existed for several years before as a mild cold. This also explains how SARS-CoV-2, unlike SARS-CoV, was able to remain and adapt in the human population for long period of time.

Figure 8. SARS-CoV-2 Outer Shell Disorder Does Not Resemble That of HIV or HSV or HCV. It resembles those of viruses in which effective vaccines have been found such as rotavirus, YFV and rabies.

Figure 9. Some Evidence of "Trojan Horse" Immne Evasion Strategy Seen in SAR-CoV-1/2. Strong Correlations between flavivirus virulence and inner shell (C) disorder have been seen. Current evidence suggests likewise for COVID-19 since SARS-CoV-1/2 have N PIDs of 50% and 48% with CFRs of 10 and 0.5-3% respectively.

Figure 10. A Precursor SARS-CoV-2 Entered Humans as an Attenuated Strain via Pangolins in 2017 or Before. Several pangolins-CoV strains have been analyzed using the shell disorder models. A 2017 strain has been identified as an attenuated strain with high fecal-oral but low respiratory transmission potentials that provides for a slow but silent spread. Based on disorder analysis of M and N PIDs of pangolin-CoV, pangolins are able to attenuate the virus because of their behaviors associated with fecal-oral transmission in nature. Such is not detected in the case of civet cats and the 2003 SARS-CoV.

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