Responsible Editor: John Di Battista.
V. Kubyshkin  kubyshkin_av@mail.ru
1 V.I. Vernadsky Crimean Federal University, Simferopol,  Russia

SARS‑CoV‑2 will constantly sweep its tracks: a vaccine containing CpG  motifs in ‘lasso’ for the multi‑faced virus
V. Oberemok1 · K. V. Laikova1 · K. A. Yurchenko1 · N. A. Marochkin1 · I. I. Fomochkina1 · A. V. Kubyshkin1

Received: 13 May 2020 / Revised: 28 June 2020 / Accepted: 6 July 2020
© Springer Nature Switzerland AG 2020

Abstract

During the current COVID-19 pandemic, the global ratio between the dead and the survivors is approximately 1 to 10, which  has put humanity on high alert and provided strong motivation for the intensive search for vaccines and drugs. It is already  clear that if we follow the most likely scenario, which is similar to that used to create seasonal infuenza vaccines, then we  will need to develop improved vaccine formulas every year to control the spread of the new, highly mutable coronavirus  SARS-CoV-2. In this article, using well-known RNA viruses (HIV, infuenza viruses, HCV) as examples, we consider the  main successes and failures in creating primarily highly efective vaccines. The experience accumulated dealing with the  biology of zoonotic RNA viruses suggests that the fght against COVID-19 will be difcult and lengthy. The most efective  vaccines against SARS-CoV-2 will be those able to form highly efective memory cells for both humoral (memory B cells)  and cellular (cross-reactive antiviral memory T cells) immunity. Unfortunately, RNA viruses constantly sweep their tracks  and perhaps one of the most promising solutions in the fght against the COVID-19 pandemic is the creation of ’universal’  vaccines based on conservative SARS-CoV-2 genome sequences (antigen-presenting) and unmethylated CpG dinucleotides  (adjuvant) in the composition of the phosphorothioate backbone of single-stranded DNA oligonucleotides (ODN), which  can be efective for long periods of use. Here, we propose a SARS-CoV-2 vaccine based on a lasso-like phosphorothioate  oligonucleotide construction containing CpG motifs and the antigen-presenting unique ACG-containing genome sequence  of SARS-CoV-2. We found that CpG dinucleotides are the most rare dinucleotides in the genomes of SARS-CoV-2 and other  known human coronaviruses, and hypothesized that their higher frequency could be responsible for the unwanted increased  lethality to the host, causing a ‘cytokine storm’ in people who overexpress cytokines through the activation of specifc  Toll-like receptors in a manner similar to TLR9-CpG ODN interactions. Interestingly, the virus strains sequenced in China  (Wuhan) in February 2020 contained on average one CpG dinucleotide more in their genome than the later strains from the  USA (New York) sequenced in May 2020. Obviously, during the frst steps of the microevolution of SARS-CoV-2 in the  human population, natural selection tends to select viral genomes containing fewer CpG motifs that do not trigger a strong  innate immune response, so the infected person has moderate symptoms and spreads SARS-CoV-2 more readily. However,  in our opinion, unmethylated CpG dinucleotides are also capable of preparing the host immune system for the coronavirus  infection and should be present in SARS-CoV-2 vaccines as strong adjuvants.

Keywords SARS-CoV-2 · COVID-19 pandemic · Phosphorothioate oligonucleotides · CpG motif · Vaccine

Vaccines and drugs help mankind reduce the efects of natu ral selection when meeting and dealing with pathogens. On  the one hand, this slows down the process of adaptation of the human population to negative environmental factors; on  the other hand, we save that which is most valuable—the  life of each individual person. Nevertheless, it must be recognized that drugs and vaccines only delay the fatal meet ing of the causative agent of the disease with those who  are pathogen-sensitive or their genetic descendants. Natural  selection is merciless—only the fttest survives. Since no  one knows for sure which pathogen he or she will be sen sitive to, everyone expects that at the nearest pharmacy a  person will fnd, at the right time, a medicine that will save his or her life. Obviously, this is not what happens. Today,  during the sudden COVID-19 pandemic, we understand how  fragile our habitual way of life has turned out to be. In fact,  we are at the mercy of pure natural selection, and we are  not able to oppose it with any efective vaccine or drug.  Nevertheless, a sufciently large number of research groups  have already joined the race to create vaccines and drugs  against the SARS-CoV-2 virus, which in the next few years  will signifcantly slow down the efect of natural selection.  In turn, weakened by vaccines and drugs, natural selection  will continue to work and COVID-19 will morph into a dis ease with mild symptoms in the years to come. However, at  this moment in time, during the exponential growth of the  pandemic, the virus is aggressive and needs to be restrained  as soon as possible.

SARS-CoV-2 is positive-sense, single-stranded RNA  betacoronavirus from the Coronaviridae family. Genomic  alignments suggest that the SARS-CoV-2 from the genus  Betacoronavirus may be the result of a recombination of  genetic material from two diferent viruses, one similar  to the Chinese horseshoe bat virus and the other closer  to the pangolin virus [1]. In fact, only two scenarios will  occur simultaneously in the very near future: people who  are genetically resistant to the virus will get sick, recover,  and develop immunity, while people who are sensitive to  the virus will need drugs and vaccines, which will have to  be researched and developed if they are to recover. Since  SARS-CoV-2 mutates constantly and more frequently  than other RNA viruses (coronaviruses possess the longest  genomes of all known RNA viruses, so more errors are made  when they are copied; also, viral RNA-dependent RNA poly merases do not have a proofreading nuclease activity), it is  likely that many new strains of the virus will appear and  this will be a problem for creating vaccines. Still, vaccines  are arguably the most powerful medical intervention in the  fght against infectious diseases [2]. Generally, all vaccines  work on the same basic principle. They present part or all  of the pathogen to the human immune system, usually in the  form of injections and in low doses, to induce the system to  produce antibodies to the pathogen and activate induction  of cross-reactive antiviral T cells, particularly the cytotoxic  T lymphocytes that can kill virus-infected cells. Specifc  T lymphocytes and antibody-producing plasma cells (B  cells) represent a kind of immune memory, which, once a  virus is detected, can be quickly mobilized again if a per son is exposed to the virus in its natural form. Traditionally,  adjuvants (aluminum salts, squalene-in-water emulsions,  imidazoquinoline derivatives, saponins, synthetic pho phorothioate-linked DNA oligonucleotides with optimized  CpG motifs, etc.) have been used in vaccines to increase the  magnitude of an adaptive response to a vaccine, based on  antibody titer or ability to prevent infection. However, a sec ond role for adjuvants has become increasingly important: guiding the type of adaptive response to produce the most  efective forms of immunity for each specifc pathogen [3,  4]. Previous studies on several animal coronaviruses also  suggest that not only humoral but also cellular, immunity  plays a role in protecting against virus infection [5, 6]. Classic methods involve taking a whole virus, weakening or kill ing it, then injecting its remains into a person’s body. For the human population, COVID-19 is the third signif cant coronavirus infection to occur in the twenty-frst cen tury, following severe acute respiratory syndrome (SARS) in  2002–2003 and Middle East respiratory syndrome (MERS)  in 2012, which have not stopped yet. Since 2012 until 29  February 2020, the total number of laboratory-confrmed  MERS-CoV infection cases reported globally to WHO is  2538 with 871 associated deaths. But while those outbreaks  alerted the world to the virulent potential of the coronavirus  family, SARS fzzled out faster than a vaccine could make  its way through clinical trials, and MERS was not viewed  seriously enough to generate sustained funding for vaccine  developers. This despite the fact that the 2002 SARS and  2012 MERS outbreaks, both caused by close relatives of the  new coronavirus, were warning shots that claimed approxi mately 774 and 871 lives, respectively.

One major hitch in developing a COVID-19 vaccine is  that no medically proven predecessor exists for any type of  human coronavirus. Only a small number of SARS-CoV  vaccines made it to Phase I clinical trials before funding  dried up because the virus was eradicated from the human  population through non-pharmaceutical interventions when  the case numbers were still small. The frst vaccine tested in  a clinical trial is made from the inactivated form of SARS CoV. Several live attenuated, genetically engineered, or  vector vaccines encoding the SARS-CoV spike (S) protein  have been tested in pre-clinical studies. These vaccine can  didates are efective in terms of eliciting protective immu nity in the vaccinated animals. However, the use of whole  virus or full-length S protein-based immunogens in humans  should be approached with caution, because their use could  induce harmful immune or infammatory responses. Some  authors have proposed the use of the receptor-binding  domain (RBD) of SARS-CoV S protein (residues 318–510)  in the development of a safe and efective subunit SARS  vaccine. This functional domain, responsible for mediating virus-receptor binding, is also a major neutralization  determinant of SARS-CoV [7]. These trials, performed  using an inactivated virus vaccine and a spike-based DNA  vaccine, have produced encouraging results; in addition to  being safe, the vaccines induced neutralizing antibody titers  [8, 9]. The structure of the S protein of SARS-CoV-2 was  solved in record time at high resolution, contributing to our  understanding of this vaccine target [10, 11]. It has been  shown that some of the neutralizing monoclonal antibod ies isolated against SARS-CoV, such as CR3022 [12, 13],  can cross-react with the receptor-binding domain of SARS CoV-2. This suggests that SARS-CoV vaccines might cross protect against SARS-CoV-2. However, because these vac cines have not been developed further than Phase I, they are currently not available for use. MERS-CoV vaccines  are unlikely to induce strong cross-neutralizing antibod ies to SARS-CoV-2 because of the phylogenetic distance  between the two viruses. Nevertheless, we can still learn  a lot from these vaccines about how to move forward with  SARS-CoV-2 vaccine design [14].

Vaccines are usually developed on the basis of certain  virus strains, as follows. The most well-known example is  infuenza vaccines. Infuenza viruses belong to the Ortho myxoviridae family and are characterized by segmented,  negative-strand RNA genomes. Infection with infuenza  viruses is a constant challenge, burdening people’s health  and economies worldwide every year. These infections cause  epidemics that can become pandemics, afecting 5–30% of  the global population annually, and are responsible for millions of hospitalizations and hundreds of thousands of deaths  each year [15]. Natural reservoirs exist for infuenza, infect ing a wide range of animal hosts. These include wild aquatic  birds, the natural reservoir for the infuenza A virus, and  pigs. Since pigs can become infected with infuenza B virus,  they may provide a natural reservoir for the virus [16]. While  infuenza, like COVID-19, causes morbidity among all ages,  those most vulnerable to its serious complications, such as  pneumonia, are the elderly [17, 18]. At the present time, the  two available types of infuenza vaccines are administered  by diferent routes: parenteral inactivated infuenza vaccines  (IIV), delivered by injection, and intranasal live attenuated  infuenza vaccines (LAIV). LAIV mimic the course of natural infections and thus induce a stronger immune response  than IIV [19]. Intransal administration of LAIV produces a  variety of adaptive immune responses, including serum anti bodies and mucosal and cell-mediated immunity [20]. Based  on data gathered by the Global Infuenza Surveillance and  Response System (GISRS), a partnership of 141 national  infuenza centers in 111 countries, six WHO collaborating  centers, and four WHO essential regulatory laboratories,  WHO updates the composition of infuenza vaccines (both  IIV and LAIV) annually. The WHO GISRS continually collects and analyses global infuenza virus samples. By moni toring circulating infuenza A and B viruses for how recently  they have occurred and with what frequency, changes can  be made to the components in the vaccines slated for the  upcoming infuenza season in both the northern and south ern hemispheres [21]. Current infuenza vaccination strategy  induces cells to produce antibodies directed against the sur face antigens of these viruses, with the main targets being  the surface glycoprotein antigens hemagglutinin (HA) and  neuraminidase (NA). The amino acid sequences encoding  HA and NA are hypervariable, as a consequence of antigenic drift or shift, respectively. This characteristic makes them  responsible in large part for both epidemic and pandemic  infuenza outbreaks [15]. In addition, these frequent changes  to the surface antigens of infuenza viruses allow them to  avoid antibody-mediated immunity. On the other hand, it has  been shown that cytotoxic T-lymphocyte populations, when  directed against the internal antigens of infuenza A virus,  demonstrate a broad cross-reactivity with the infuenza virus  subtypes [22].

Both IIV and LAIV vaccines have a history  of safe use in children and adults. However, both can cause  adverse events and/or rare adverse events, some more preva lent in children, others more likely to occur in adults [23].  Each year, necessary changes are made to the formulation of  infuenza vaccines and when they are administered. Factors  that afect those being vaccinated, both genetic and environmental, are difcult, if not impossible, to control. For  these reasons, even though vaccines undergo strict monitor ing before authorization, the possible occurrence of adverse  efects, such as narcolepsy [24], cannot be completely elimi nated. Consequently, annual post-licensure vaccine safety  surveillance is fundamental [25]. For all of these reasons,  no universal infuenza vaccine is yet available against all  infuenza virus subtypes and seasonal infuenza vaccines  require annual updates. The lack of preventive vaccines for  clinical use in humans against such viruses makes emerg ing infuenza and coronaviruses a serious global threat [26].  Currently, available vaccines keep deaths from infuenza  relatively under control. However, when any infuenza virus  lineage emerges in a novel mammalian host population, the threat of a direct or indirect pandemic is a real risk [27]. The creation of vaccines for seasonal infuenza viruses  is justifed because they work, but for some other RNA  viruses, this is not so. The human immunodeficiency  virus, types 1 and 2 (HIV-1 and HIV-2), is an example of a  zoonotic infection that has spread all over the world with out being stopped. At the moment, approximately 38 mil lion people have been infected with HIV, but there is not  a single successful vaccine able to efectively contain the  HIV pandemic [28]. Both types of HIV are the result of  multiple cross-species transmissions that occurred when  simian immunodefciency viruses (SIV) naturally infected  African primates [29]. The human immunodefciency virus  (HIV), which consists of two identical negative-sense single stranded RNA molecules, belongs to the genus Lentivirus  in the Retroviridae family, subfamily Orthoretrovirinae [28].  Attempts to develop a safe and efective HIV vaccine have  been hindered by the difculty in clearly defning the spe cifc immune responses responsible for preventing infection  and limiting disease progression. Since early attempts to use  vaccines to induce neutralizing antibodies ended in disap pointment, recent research eforts have focused on vaccines  that induce T-cell immunity. The belief that the specifc  antiviral CD8 T lymphocyte response is crucial for immune control of viral replication has provided an important facet  for ongoing eforts to develop efective vaccines against  HIV. Unfortunately, while the actions of T cells are known  to limit HIV and other viral infections after the infection has  already occurred, their role, if any, in protecting against initial infection is much less clear [2]. HIV-1 is highly mutable.  This, combined with its complex structure and life cycle,  has placed further obstacles in the path to developing an  efective vaccine. The virus is a master of obfuscation.

For  instance, it uses variable glycosated residues to camoufage  the conserved receptor- and coreceptor-binding sites on the  viral envelope (Env) glycoproteins engaged in the virus’s  attachment and fusion to host membrane receptors [30]. In  addition, the conserved sites are thermostably concealed  [31]. These are just two of the features that allow the virus  to nimbly evade detection by cross-reactive antibody (Ab)- producing B cells that recognize conserved sites. As a part  of its life cycle, HIV-1 is able to integrate into its host’s  genome, forming a latent provirus that escapes immune recognition owing to the absence of signifcant protein expres sion [32]. Neutralizing antibodies (NAb) typically play a  key role in controlling viral infections and contribute to the  protective efect of many successful vaccines. Compelling  data gathered using experimental animal models have dem onstrated that NAb can prevent HIV-1 acquisition; however,  no similar data exists for humans and the role of NAb in  controlling established infection in humans is also limited.  It is clear that HIV-specifc NAb drives the evolution of the  HIV-1 envelope glycoprotein within an infected individual.  The ability of the virus to evade immune selection may be  the main reason HIV-1 NAb exert limited control during  infection. Attempts to defne NAb capable of providing a  broad spectrum of protection against diverse circulating  HIV-1 strains have encountered the difculties posed by the  extraordinarily antigenic diversity of HIV-1 [33]. Reports  that the breadth and potency of humoral responses are higher  in subtypes C and A than in subtype B infections provides  evidence that viral genetic subtype may also be important  [34, 35]. In addition to its talents for concealment, the virus  can also make mistakes. The high error rate of the reverse  transcriptase [36], combined with the rapid turnover of  plasma virions [37], further hinders the development of  an efective vaccine by providing a dizzying range of variants from which to select and escape from both cellular and  humoral immune responses [38, 39]. At present, at least 12  recognized HIV-1 genetic subtypes are known to exist in  humans. Each of these subtypes has rapidly diversifed to  yield intersubtype recombinants [40, 41] that present an  even greater challenge in the development of a universal  HIV vaccine.

Another ’multi-headed beast’ sweeping its tracks is  the hepatitis C virus (HCV). HCV is a positive-sense sin gle-stranded RNA virus of the Flaviviridae family [42].

Although the zoonotic origins of HCV in humans remain  unclear, recent and direct cross-species transmission from  horses to dogs appears plausible [43]. Highly diverse HCV related viruses have also been detected in bats and rodents  [44], in cattle [45], and in black-and-white colobus monkeys  [46]. There is good news in the form of direct-acting anti virals (DAA) against the hepatitis C virus (HCV) infection,  which have been shown to achieve a complete cure in>95%  of cases [47, 48]. It has been suggested that DAA have the  potential to eliminate an infection that afects more than  71 million individuals worldwide [49]. This is particularly  important because many individuals do not seek treatment  for constant hepatitis C because they are unaware that they  are infected. Sadly, it is often a long time after the initial  infection, when hepatitis C has precipitated serious health  issues, that these individuals look for help. It is estimated  that only 5% of HCV cases worldwide are diagnosed [50,  51]. This is one reason that the development of vaccines  that protect against persistent HCV infection remain a public  health priority. Without access to vaccines that can limit  viral transmission, even the broad use of highly efective  DAA is unlikely to achieve HCV elimination. In addition,  DAA treatment does not protect against reinfection, further  underscoring the need for an efective vaccine [52]. Cur rent preclinical and clinical trials are being carried out for  two vaccines that target either the antibody or the T cell  response. The next generation of vaccines is likely to involve  a combination of these two strategies. The frst vaccine contains a recombinant form of the virus envelope glycoproteins  gpE1 and gpE2, aimed at inducing neutralizing antibodies  and CD4 helper T cells [53, 54]. The second vaccine is vec tor-based and encodes nonstructural (NS) proteins of the  virus (NS3-NS5) using chimpanzee adenovirus priming and  a modifed vaccinia Ankara (MVA) boost. These represent  two of the most promising strategies: production of broadly  neutralizing antibodies (bNAb) to neutralize the infectivity  of the virus and generation of potent virus-specifc CD4 and  CD8 T cells to eliminate infected hepatocytes [55]. Multiple  studies of HCV in humans and chimpanzees have provided  strong evidence for the kinetic association between spontaneous viral clearance and the induction of a broad, sustained  group of HCV-specifc CD4 and CD8 T cells [56]. As it  turns out, these T cells are polyfunctional, producing mul tiple cytokines and efector functions [57]. For instance, as  the infection is being cleared, virus-specifc T cells develop  a memory T cell phenotype that upregulates cell surface  expression of the IL-7 receptor CD127 [58–60]. If the  virus persists, CD8 T cells that recognize intact epitopes  (i.e., epitopes that have not mutated) become exhausted and  express exhaustion markers such as programmed death 1  (PD1), T-cell immunoglobulin and mucin domain-containing-3 (Tim-3), cytotoxic T-lymphocyte protein 4 (CTLA4),  2B4, CD160, KLRG1, T-cell immunoreceptor with Ig and ITIM domains (TIGIT), and CD39 [56, 61]. There is  an increasing amount of evidence that the specifcity and  potency of the early antibody response can infuence the  acute infection outcome and that neutralizing antibodies  seem to play a part in controlling HCV during chronic infection [62]. In particular, it is worth noting the data from stud ies showing that bNAb isolated from chronically infected  and spontaneously resolved individuals have the capacity to  block infectivity in mouse models of HCV infection, further  emphasizing the important protective efect of the antibody  response [63–65].

Thus, in one way or another, all vaccines aimed at pre venting zoonotic RNA viruses are focused on producing spe cifc antibodies and activating cytotoxic T lymphocytes that  can cause the death of virus-infected cells. In general, the  lack of efective vaccines against some RNA viruses, such as  coronaviruses, HIV, and HCV, can be explained by the large  number of mutations that occur during replication, as well  as weak antibody activity. To some extent, the slow progress  in the development of HCV and SARS coronavirus vaccines  is also due to the lack of sufcient funding and the advent of  highly efective DAA in the case of HCV. Nevertheless, a  large number of RNA viruses of zoonotic origin will always  carry with them the danger of new pandemics; the crea tion of vaccines can help humans be better prepared to meet  them. Ideal vaccines are those that will be efective for the  vast majority of people and against most of the strains of  the target circulating RNA virus. One promising strategy is  to create vaccines based on conservative sequences of viral  genomes using oligonucleotides. Despite the impressive suc cess of currently approved adjuvants for generating immu nity to viral and bacterial infections, there remains a need  for improved adjuvants that enhance protective antibody  responses, especially in populations that respond poorly to  current vaccines. Today, the larger challenge is to develop  vaccines that generate strong T cell immunity with purifed  or recombinant vaccine antigens. Oligonucleotides can be  used as both antigens and adjuvants.

Several studies have shown that oligonucleotides can  improve the efcacy of vaccines, either by inducing antigen  modifcation (i.e., by enhancing expression of immunogenic  molecules) or by targeting certain components of the host  immune system to achieve the desired immune response.  However, despite their extensive use, problems such as  insufcient stability and low cellular delivery have not been  sufciently resolved to achieve efective and safe oligonucleotide-based vaccines [66]. For many years, nucleic acids  and short nucleotide molecules have been used as vaccine  components. Their use has ranged from DNA [67, 68] or  RNA vaccines [69, 70], to oligonucleotide sequences containing unmethylated cytidine phosphate guanosine (CpG)  motifs with signifcant immunostimulatory (adjuvant) prop erties [71, 72]. More recently, strategies that manipulate the expression of genes controlling the immune response or the  expression of antigens of interest are being used to improve  immunogenicity and vaccine efcacy. This line of research  has a history of more than 30 years; in one of the frst experiments, when spleen cells from BALB/c mice were incubated  with the nucleic acid fraction from diferent bacteria, the nat ural killer (NK) activity of the cells was remarkably elevated  and the cells produced factors that activated macrophages  and inhibited viral growth. The factor that activated the mac rophages was interferon (IFN)-gamma and the other factor,  which inhibited viral growth, was IFNalpha/beta. However,  the nucleic acid fraction from neither of the vertebrate cells  showed any such activity. This report provided evidence for  the frst time that, while DNA from microorganisms and  invertebrates could induce IFN, the DNA from vertebrates, and probably from plants as well, could not [73]. In 1995, Krieg et al. reported that unmethylated CpG  dinucleotides (CpG ODN) within bacterial DNA activated  host defense mechanisms that led in turn to innate and adap tive immune responses [74]. CpG ODN is a ligand of Toll like receptor 9 (TLR-9) in antigen-presenting cells. Toll-like  receptors (TLR) are a family of non-clonal pattern-recogni tion receptors that recognize conserved pathogen-associated  molecular patterns (PAMP). Under homeostatic conditions,  TLR discrimnate ‘non-self’ from ‘self’ in that they do not  sense host-derived ‘self-molecules’ but become activated  by PAMP during infection [75]. Ligand binding by TLR3,  TLR7, TLR8, and TLR9 occurs in the acidic environment  of the endosome [76] and causes cellular activation, pre sumably by stabilizing preformed TLR dimers or inducing  TLR-dimer formation. CpG ODN/TLR-9 interaction induces  an innate immune response that promotes the subsequent  development of adaptive immunity [71, 77]. Their utility  as vaccine adjuvants has been evaluated in diferent clinical  trials and the results indicate that CpG ODN augments the  induction of vaccine-specifc cellular and humoral responses  [72]. In 2017, the FDA approved HEPLISAV-B, the frst  vaccine with a CpG ODN as an adjuvant, for hepatitis B  vaccines [78]. However, it has also been reported that CpG  ODN can induce high levels of pro-infammatory cytokines,  with the potential risk of developing or worsening autoim mune diseases and systemic infammatory response syn drome (SIRS) [79, 80].

TLR9 agonists have demonstrated a substantial potential  as vaccine adjuvants, and as mono- or combination thera pies for the treatment of cancer and infectious and allergic  diseases. Commonly referred to as CpG oligodeoxynu cleotides (ODN), TLR9 agonists directly induce the acti vation and maturation of plasmacytoid dendritic cells and  enhance diferentiation of B cells into antibody-secreting  plasma cells. Preclinical and early clinical data support the  use of TLR9 agonists as vaccine adjuvants, where they can  enhance both the humoral and cellular responses to diverse antigens [77]. When used as vaccine adjuvants, CpG ODN  improve the function of professional antigen-presenting cells  and boost the generation of humoral and cellular vaccine specifc immune responses. These efects are optimized by  maintaining the ODN and the vaccine in close proximity.  The adjuvant properties of CpG ODN are observed when  administered either systemically or mucosally, and persist  in immunocompromised hosts. Preclinical studies indicate  that CpG ODN improve the activity of vaccines targeting  infectious diseases and cancer. Clinical trials demonstrate  that CpG ODN have a good safety profle and increase the  immunogenicity of co-administered vaccines [71].

The majority of studies carried out previously in SARS CoV-infected humans and animals pointed to a dysregu lated/exuberant innate response as the leading contributor  to SARS-CoV-mediated pathology. Almost two decades  after the 2002–2003 SARS epidemic, we still do not have  any approved preventive or therapeutic agents available with  which to respond to the re-emergence of SARS-CoV or other  related viruses. A strong neutralizing antibody response  generated against the spike (S) glycoprotein of SARS-CoV  is completely protected in the susceptible host. However,  neutralizing antibody titers and the memory B cell response  are short lived in SARS-recovered patients and the antibody  will only target the primary homologous strain. Follow-up  studies from patients who recovered from SARS suggest  that the SARS-CoV-specifc antibody response is feeting.  In these patients, the SARS-CoV-specifc IgM and IgA  response lasted less than 6 months, while the virus-specifc  IgG titer peaked 4 months post-infection and declined mark edly after 1 year. Despite the lack of virus-specifc memory  B cell response, SARS-CoV-specifc memory T cells persist  in SARS-recovered patients for up to 6 years post-infection.  Consistent with the results from these human studies, results  from animal studies also suggest that strong virus-specifc  T cell responses are required to protect mice from lethal  SARS-CoV-MA15 infection. Future vaccine interventions  should, therefore, consider strategies that enhance the T  cell response to provide a robust long-term memory. Since  tissue-resident memory T cells provide better protection,  boosting both local and systemic memory T cell responses  would be a more useful strategy than either of these interventions alone. Although the amount of data available on  virus-specifc memory T cell responses to emerging cor onaviruses is limited, existing studies indicate that lung  resident virus-specifc memory CD8  T cells provide sub stantial protection following SARS-CoV challenge. These  studies suggest a potential role for virus-specifc memory T  cells in broad and long-term protection against SARS-CoV  infection. This is important as the response of neutralizing  Abs and memory B cells to SARS-CoV declines signif cantly 1–2 years post-infection; in addition, these responses  are strain specifc [81]. Taken together, these data clearly suggest that the CpG ODN/TLR-9 interaction induces a  strong innate immune response that promotes the subse quent development of an adaptive immunity triggered by  the production of T helper cells one and pro-infammatory  cytokines.

With advancing age, both humans and animals become  highly susceptible to SARS-CoV and other respiratory virus  infections. Such an age-dependent increase in susceptibility  is associated with a signifcant reduction in the magnitude of  the virus-specifc T cell response [82–84]. Young C57BL/6  mice (6 weeks of age) generate a SARS-CoV-specifc CD8 T  cell response that is approximately eightfold greater than that  observed in 12-month-old mice [85]. Virus-specifc CD8 T  cells play a critical role in viral clearance; CD4 T cells pro vide the necessary support for the antiviral function of the  CD8 T cells [86]. Generally, results suggest that the role of  virus-specifc primary CD4 and CD8 T cell responses to  respiratory or other coronavirus infections are both virus and  mouse strain dependent [81]; in other words, they depend on  the genetic component of the host-virus relationship. Even tually, in the absence of vaccines and drugs, natural selection  will rapidly lead to the survival of only genetically resistant  individuals in a host population.

At this juncture, we propose a SARS-CoV-2 aerosol based vaccine based on a lasso-like phosphorothioate oli gonucleotide construction containing CpG motifs and anti gen-presenting unique sequence (APUS) (Fig. 1). Aerosol immunization seems to be a promising method of vaccina tion [87, 88], including as it does aerosolized delivery of  nucleic acids optimized for lung uptake [89] and nasal spray  [90]. Several independent studies have now demonstrated  that the dependence on CpG motifs for TLR9 activation is restricted to synthetic phosphorothioate oligodeoxynucleo tides (PS-ODN), and that natural phosphodiester oligode oxynucleotides (PD-ODN) bind and activate TLR9 via the  2′ deoxyribose backbone in a sequence-independent manner  [91–95]. Indeed, further control can be achieved through  recognition of cytosine methylation, which acts as a negative  determinant, i.e., its presence in mammalian DNA prevents  the triggering of TLR9.

In the proposed construction, the main role of the antigen presenting part of the vaccine will be played by the phos phorothioate oligonucleotides found on the APUS region  (Fig. 1). Phosphorothioate (PS) oligonucleotides, which  contain a non-bridging sulfur, are the most widely studied  oligonucleotides; compared to unmodifed oligonucleo tides, they possess higher solubility and nuclease stability,  with improved membrane penetration. Nuclease stability is  important because human lung fuid possesses DNase activ ity. This activity can be inhibited by the use of direct nucle ase inhibitors, which enhance the uptake of oligonucleo tide-based vaccines by cells [96] or through oligonucleotide  modifcation. Therapeutic use of oligonucleotides is highly  dependent upon their efcient uptake by cells. The literature  contains many reports of experiments both in tissue culture  and in vivo using a phosphorothioate backbone to gener ate antisense efects mediated by an RNase H-dependent  mechanism. These data have led to the investigation of phos phorothioate oligonucleotides in therapeutic clinical trials  [97]; it has also been demonstrated that CpG motifs within  phosphorothioate (PS)-modifed DNA efectively drive Toll like receptor 9 (TLR9) activation [98].

We found that CpG dinucleotides are the most rare dinu cleotides in the genomes of SARS-CoV-2 and other known  human coronaviruses (Table 1), and hypothesized that their  higher frequency could cause the unwanted increased lethal  efect on the host, resulting in a ’cytokine storm’ in people  prone to overexpress cytokines through the activation of  specifc Toll-like receptor interactions with single-stranded  RNA in a manner similar to those of TLR9-CpG ODN inter actions. Most RNA viruses that infect mammals and other  vertebrates profoundly suppress CpG dinucleotide frequen cies [99]. The functional nature of this apparent selection against CpG dinucleotides operating on both host and viral  cytoplasmic RNA sequences remains unexplained. However, it has been frequently hypothesized that rec ognition of CpG motifs is part of an as yet uncharacter ized ‘self-non-self’ recognition system coupled to an innate  immunity [100]. This may be functionally and perhaps evo lutionarily related to the Toll-like receptor 9 that recognizes  non-methylated CpG dinucleotides in DNA sequences [101,  102]. Atkinson et al. decided to investigate the roles played  by the various components of the innate immune system in  recognizing dinucleotides and recruiting antiviral defenses.  Their interest was caught when they observed that elevated  frequencies of both UpA and CpG attenuated viral replica tion, whereas frequencies lower than those of native viral  sequences enhanced replication [99]. However, our think ing goes in the other direction: natural selection aims to  strengthen the genotypes of RNA viruses with fewer CpG  dinucleotides, because otherwise, a strong ’cytokine storm’  is unleashed in people prone to overexpress cytokines,  causing the death of the host, which for the virus presents  a developmental impasse. Any virus that completely elimi nates its host population fnds itself in an evolutionary dead  end since viruses are obligate intracellular parasites. A cer tain balance of power exists in the virus-host relationship;  to an extent, all of its participants remain ‘gentlemen’ and  provide their opponents with the opportunity to survive and  reproduce. The system sacrifces units, but the community  is saved. Dinucleotides appearing and disappearing dur ing the microevolution of the coronavirus CpG apparently  help to adjust (more or less) the optimal level at which the  parasite disseminates when the virus replicates, resulting  in a moderate number of deaths of individuals in the host  population. Since SARS-CoV-2 appeared only recently in  the human population, in the near future we will see just  such an adjustment as the number of CpG oligonucleotides  changes in the direction optimal for the virus to replicate.  Interestingly, the genome of virus strains sequenced in China  (Wuhan) on February 11, 2020 contained on average 1 CpG  dinucleotide more than was observed in later strains from  the USA sequenced on May 6, 2020 (Table 2). Obviously,  at the beginning of the microevolution of SARS-CoV-2 in the human population, natural selection tends to favor viral  genomes containing fewer CpG motifs so as not to trigger  a strong innate immune response. Apparently, people who  become infected with the virus have moderate symptoms  and spread SARS-CoV-2 more readily. It is worth noting that  the infuenza A virus, which originated from an avian reservoir, has undergone a signifcant reduction in the number of  CpG nucleotides since its introduction into humans [102].  This hypothesis is further reinforced by the fact that the  CpG motif in an AU-rich oligonucleotide can signifcantly  stimulate the immune response of plasmacytoid dendritic  cells [100, 103].

In our ‘lasso’ construct, we placed the CpG motifs in  the ‘tail’ region in the to 5′-purine-purine-unmethylated  deoxycytosine-deoxyguanosine-pyrimidine-pyrimidine-3′ position [104]. The ‘neck’ region of the construct features  a double-stranded DNA region stabilized by 30 hydrogen  bonds between 10 cytosines and 10 guanines. The double stranded DNA ‘neck’ makes it possible to form a loop in  the region of the antigen-presenting ‘head’. The presence  of such a loop allows us to take advantage of the frequent  formation of RNA loops in cells and their possible inter action with the corresponding Toll-like receptor interact ing with single-stranded RNA. Only adenines, cytosines,  and guanines are present in the construct, which makes it  possible to activate pattern recognition receptors interact ing with both ‘non-self’ DNA and ‘non-self’ RNA. Since  the PS backbone is the basis of the construct, the presence  of deoxyribose will be of little importance and everything  will depend on the nucletide sequence of the construct [98].  CpG motifs in nuclease-resistant PS backbones have been  found to dramatically enhance B cell stimulatory proper ties [105, 106]. In almost all vertebrates, the optimal PS  CpG ODN are extraordinarily strong mitogens for B cells  [107–110]. While the inhibitory efect observed for poly G  motifs of CpG ODN reduces the level of B cell activation  [111], in our construct, this motif is found in the double stranded ’neck’ and may have a positive inhibitory efect  during possible acute infammation that occurs after vacci nation. As the body temperature rises, the double-stranded  DNA ‘neck’ melts to produce a single-stranded oligonu cleotide sequence containing two poly G motifs. Of note, in vertebrate genomes, CpG dinucleotides occur only~25%  as frequently as would be predicted if base utilization were  random [112]. Furthermore, the bases fanking CpGs in ver tebrate genomes are not random: the most common base pre ceding a CpG is a C and the most common base following a  CpG is a G [113]. These types of CpG motifs do not support  strong immune stimulation. In addition to these diferences  in CpG content, methylation of CpG dinucleotides difers  between virus and host. These dinucleotides, which are not  methylated in the pathogen DNA, are routinely methylated in  vertebrate DNA at the 5th position of~70% of the cytosines  [112]. If immune recognition of CpG DNA has evolved as an  efective defense, then it seems likely that pathogens would  evolve counter strategies to block or evade the defense. One  potential strategy would be for a pathogen to reduce the level  of CpG in its genome to decrease its immune-stimulatory  efects. In principle, this should be much easier for patho  gens having small genomes and many of them were shown  to have very low CpG content [114, 115].

Thus, in the proposed construct, the CpG ’tail’ will  trigger cells that express Toll-like receptor 9 (including  human plasmacytoid dendritic cells and B cells) to mount  an innate immune response characterized by the production  of T helper cells 1 and pro-infammatory cytokines, which  will improve the function of the professional antigen-pre senting cells and boost the generation of humoral and cel lular vaccine-specifc immune responses. Also CpG ‘tail’  is supposed to trigger Toll-like receptors that interact with  the single-stranded RNA containing CpG motif in a manner  similar to interactions with TLR9-CpG ODN. Generally, the  unmodifed ’tail’ will serve as an adjuvant and will prepare  the immune system for the meeting with the antigen found  on the ’head’. The sequence of the ‘head’ (5′-GCAGAGACA GAAGAAACAGCAAAC-3′) is well conserved. It is found  in every SARS-CoV-2 genome located in GenBank at this  moment and belongs to a nucleocapsid phosphoprotein gene  [protein_id=”QJC2104QJC21049.1”) that has 87% query  cover with Homo sapiens protein kinase C eta (PRKCH)  (NG_011514.1). 9.1] that has 87% query cover with Homo sapiens protein kinase C eta (PRKCH) (NG_011514.1). Summing up, the proposed design contains CpG dinu cleotides, important for in the activation of innate immunity,  and a unique genomic sequence of the virus, the antigen presenting component that activates adaptive immunity.  Similar vaccine constructs based on phosphorothioate oli gonucleotides can have a wide variety of unique sequences  based on the circulating SARS-CoV-2 strains, and each vac cine may contain a dozen of these lasso-like constructs. In  the very near future, we plan to test such vaccines on the  murine coronavirus, taking care not to miss any possible  negative efects, and remaining aware that the PS backbone  dramatically increases the non-specifc ODN binding to a  wide variety of proteins [116, 117].

The mechanisms of the ‘cytokine storm’ and how they  contribute to fatalities reported for COVID-19 are still under  investigation. Currently, it is believed that SARS-CoV-2  dampens antiviral interferon responses, which results in  uncontrolled viral replication. For the other coronaviruses,  SARS-CoV and MERS-CoV, the response to viral infection  by type I IFN is suppressed. The infux of neutrophils and  monocytes/macrophages results in hyperproduction of pro infammatory cytokines [118]. In a study of 41 patients hospitalized with COVID-19, high levels of pro-infammatory  cytokines were found, including IL-2, IL-7, G-CSF, IP-10,  MCP-1, MIP-1A, and TNF-α, in the most severe cases [119].  This overproduction of a large number of pro-infammatory  cytokines dubbed a ’cytokine storm,’ begins a cascade of  events that is hard to stop. For instance, the cytokine storm  can initiate viral sepsis and infammatory-induced lung  injury, which leads to other complications including pneumonitis. While ‘cytokine storms’ can overwhelm people of  any age, some scientists believe that this phenomenon may  explain why otherwise healthy young people died during  the 1918 Spanish flu pandemic and more recently during the  SARS, MERS, and H1N1 epidemics. One of the most frequently studied organisms known to cause cytokine storms  is infuenza a virus, particularly the pandemic subtypes. It  has been shown that the H1N1 strain responsible for the  1918 pandemic induced higher levels of pro-infammatory  immune cells and cytokines in the lungs than the seasonal  infuenza viruses. This contributed to its high virulence  and may account for the unusually high mortality rate seen  among young adults during the outbreak [120]. Severe infuenza infections caused by highly virulent subtypes such  H1N1 and H5N1 are characterized by over induction of the  pro-infammatory cytokines TNF-α, IL-1β, IL-6, IL-8, and  monocyte chemoattractant protein-1 (MCP-1) [121, 122],  which eventually results in multiple organ dysfunction and  failure and increased vascular hyperpermeability [122].

With respect to the tendency of the body to overexpress  cytokines and let loose a ‘cytokine storm’ during corona virus infection in some humans, we are proposing vaccine  construct having the CpG motifs which will help people’s  immune systems train for this infammation reaction in  the absence of a real pathogen and real danger. In a broad  sense, the immune system copes with the virus much more  efectively if it has already encountered it. The unmethyl ated CpG motif is certainly that portion of the virus genome  for which the immune system of sensitive people needs to  be prepared. Generally, CpG ODN boosted the humoral  immune response induced by vaccines against a large num ber of pathogens, including infuenza virus, measles virus,  lymphocytic choriomeningitis virus, orthopoxviruses, and  other viruses [71]. At the height of the epidemic, when  more than 500,000 people have already died, apparently it  is time to try this approach when creating a vaccine against SARS-CoV-2. Even though SARS-CoV-2 will constantly  sweep its tracks, an efective vaccine should contain a CpG  motif as a conservative fngerprint of the pathogen to train  the human immune response. The fght against COVID-19  will be difcult and lengthy. Nevertheless, the current situation will help to gain experience and the ability to prevent  future coronavirus pandemics. Life goes on.

Acknowledgements This work was supported by state program No  FZEG-2020-0060 of the Ministry of Science and Higher Education of  Russian Federation.

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