Preventive and Therapeutic Vaccination for COVID-19 Using NASVAC

Abstract

Aims and background

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which belongs to the coronavirus family, suddenly emerged in China and neighboring regions towards the end of 2019. It disrupted the entire world within weeks to months, and finally became a pandemic that continued for about 3 years.

Patients and methods

One year after the commencement of infection, a vaccine surfaced on a global basis that imparted its effect on the progression of the infection. At the same time, symptomatic treatment was initiated to treat the patients and prevent progression to severe forms. Subsequently, antiviral drugs of different paradigms were used with mixed outcomes. Finally, immune modulators were mainly used to delay complications and reduce mortality.

Results

Despite all these approaches, there have been nearly one billion infections and almost one million fatalities due to the pandemic of COVID-19. Two years after the containment of the virus, if the entire episode is considered holistically, it becomes evident that more insights are required to develop preventive and therapeutic measures against the sudden appearance of some microbe.

Conclusion

This is the initial theological approach, and more sophisticated drugs may be developed based on this principle.

Introduction

The virus responsible for causing coronavirus disease 2019 (COVID-19) was eventually identified as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This is a positive-sense, single-stranded RNA beta-coronavirus, which is a novel virus. Humans first identified the virus in December 2019. After the initial days, SARS-CoV-2 spread at a massive pace. The pandemic of the virus was declared by the World Health Organization (WHO) within months of its first unconfirmed reporting. Subsequently, the virus underwent mutations, and several subtypes of the virus emerged with specific pathological features. Although a serious type of infection has been contained and mortality has been reduced, it seems that the human race would have to live with one or more forms of the virus for an extended period.^1–5 Even now, the virus is detected in almost all countries worldwide that keep surveillance against COVID-19; however, the severity has been contained. More importantly, the virus has been undergoing constant mutation, and new subtypes are on the rise, indicating the need to pay closer attention.^6–8

From the public health viewpoint, SARS-CoV-2 has now been contained. However, COVID-19 has a wide spectrum of manifestations. On one extreme, there are many patients who remain asymptomatic, while others suffer from extreme forms with consolidation of the lung and an immune anomaly. Most of these cannot be reversed at some point, and the patients ultimately proceed to a downhill and fatal course. Some experience severe forms of the disease with poor prognosis.^9–12 The elderly, obese, and those who have comorbidities experienced most of the fatalities from COVID-19.^13–18 More importantly, infection by SARS-CoV-2 is related to multivariate symptoms that are collectively called post-COVID syndrome. The extent of these pathological scenarios is variable, may last for a long period, and many patients have been suffering from compromised quality of life.

Theological Basis for the Development of Drugs for the Prevention and Treatment of COVID-19

The WHO states that about 773 million people were infected by SARS-CoV-2, and the death toll reached about 7.4 million. Although both the incidence and mortality crossed WHO observations, the severity of COVID-19 has now become a memory. The exact cause as to why a coronavirus became so virulent and led to a worldwide pandemic is still unknown. Many theories and observations have been brought forward, but nothing could cross the boundary of reality and truth. However, another future pandemic is a realistic possibility. During the outbreak of COVID-19, the entire world was unprepared, and the public health measures became the only option to contain SARS-CoV-2 spread. The future pandemic of a similar nature may be more devastating and complex. In fact, many viral infections have been persisting in pandemic form.

Thus, contemporary medical science demands that the spectrum of drug development should be directed in several directions, as has been taught by the SARS-CoV-2 pandemic. Firstly, there should be drugs for the containment of infections like SARS-CoV-2. SARS-CoV-2 is predominantly transmitted via the nasal route. The route of entry of the virus deserves serious concern.

Secondly, the mortality from SARS-CoV-2 is nearly 1%. However, due to considerably low reporting, the actual mortality may be double or even triple. One of the characteristics of SARS-CoV-2-induced severe diseases do not develop overnight, and thus there is considerable time for taking due action. It is also important to develop in-depth knowledge about SARS-CoV-2-induced extra-pulmonary complications, pathologies, and the development of severe forms of COVID-19.^19–21

If the knowledge and know-how of the present day are compiled for the elimination of SARS-CoV-2, this may prevent outbreaks of similar or aberrant pandemics in the future. Thus, the development of drugs for SARS-CoV-2 prevention and treatment should be directed to tackling microbes as a general enemy of mankind. These may be DNA or RNA viruses or microbes of other natures. Considering all these aspects, COVID-19 posed challenges, and on the basis of prevailing insights, we moved to repurpose one formulation for broader usage.

Although several microbes use different routes for entering the human body, this review will mainly discuss the drug development for airborne pathogens.

Concept of Development of Drugs for COVID-19 Prophylaxis and Treatment or COVID-19-like Pathogenesis

The authors and the laboratories where they work are not dedicated to research regarding COVID-19 or other airborne viruses. In fact, these individuals and laboratories have been working on almost all aspects of hepatitis viruses (epidemiology, public health spectrum, prophylaxis and drug development against hepatitis viruses), especially the hepatitis B virus (HBV).

Lessons Learned from HBV Infection and Drug Development for Chronic Hepatitis B

In the course of drug development for COVID-19 and also for the prevention of SARS-CoV-2, we dissected the know-how of our team for a treatment development approach against chronic hepatitis B (CHB). Hepatitis B virus infection represents a complex public health issue globally. According to WHO estimates, approximately 2 billion out of the 7.6 billion total world population have been infected by HBV at some point in their life. This varies from region to region. Epidemiological data endorsed by the WHO indicate that approximately 12–25% of chronic HBV-infected subjects would develop evidence of liver damage, and they may also develop progressive liver disease. The mortality due to HBV-related pathogenesis has reached nearly 1 million per year.

There has been a prophylactic vaccine against HBV for about 60 years. Also, we now have several antiviral drugs for the treatment of CHB.^22–25 Among these drugs, there are interferons (IFNs; standard and their pegylated forms) and nucleoside analogs (NUCs).^26–32 Interferons did not become preferred modalities for CHB even in the developed world due to safety issues, high costs, parental routes of administration and limited efficacy. Nucleoside analogs led to considerable optimism among both patients and physicians for the treatment of CHB as they are orally administered and can lead to a reduction of HBV load and even HBV DNA negativity. Approximately 25 years have passed since the entry of NUCs for the treatment of CHB. It is now apparent that NUCs cannot be the treatment of choice for CHB, as there are notable limitations of NUCs.

As an antiviral approach was not effective in CHB patients, several investigators used immunotherapy for CHB patients. This was supported by the concept that CHB is an immune-mediated disease induced by HBV. However, the use of polyclonal immune modulators did not bring any notable benefit for CHB patients, and these have been extensively discussed in many publications around the world.^33–36

Because of these limitations, we and others developed a strategy of treating CHB patients with an antigen-specific immune modulator, and hepatitis B surface antigen (HBsAg) was used as the first mode of antigen-specific drug for CHB in 1984. Critical analysis led to the understanding that we need some modification of the nature of the antigen and the route of administration for antigen-specific immune therapy. This led to the development of NASVAC by the Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba, a drug with two antigens of HBV [HBsAg and hepatitis B core antigen (HBcAg)]. Also, it was prepared by CIGB in such a manner that it can be administered via the nasal route. The efficacy of NASVAC was first assessed in HBV transgenic mice that expressed all HBV-related antigens and HBV Dane particles with HBV DNA. The inspiring data from animal studies led us to have clinical trials with NASVAC against CHB patients around the world. The development of NASVAC follows the recommended pathway. At first, the safety and efficacy of NASVAC were evaluated in healthy human volunteers. Then, phase I, II, and III clinical trials in CHB patients followed. At the same time, we showed that NASVAC can also be used only through the nasal route. Moreover, in addition to the therapeutic potential of NASVAC in CHB patients, NASVAC exhibited potent prophylaxis potential in normal human subjects.^37–41

Marking the Action of Adjuvant and the Route of Administration from the Trials with NASVAC in CHB Patients

It is a natural query why HBsAg-based vaccines could not show their efficacy in CHB patients after using different types of vaccines in different combinations with other materials. Ultimately, it was found that the adjuvant in NASVAC, HBcAg, is the mastermind of all important potentials of NASVAC. Evidence was retrieved to support this consensus.

HBcAg as a Potent Adjuvant in Various Systems

Stimulation of the HepaRG cell line with HBcAg in vitro is associated with multi-TLR agonist effect.^42–44 This is an important finding as toll-like receptors (TLR) and their receptors are regulators of immunity during different viral infections. This has been shown in patients with CHB in a Cuban study in which an increase in innate immune receptors, namely RNA-sensing TLR3, TLR7, and TLR8 on the tonsils of the patients was documented following NASVAC administration. Besides, dramatic survival benefits have been seen in lethal SARS-CoV-2 infected mice model following administration of TLR3, TLR7/8, and TLR9 agonists.^45,46 Transcriptomic profiling studies involving SARS-CoV-2 infected cells in vitro also support the role of TLR3 and TLR7/8 agonists for early immune stimulation.⁴⁷ Furthermore, TLR3 gene downregulation is more pronounced in bovine coronavirus compared to bovine rotavirus.⁴⁸ This suggests that TLR3 is the target of coronavirus infection. Su et al. used particulate HBcAg, which is a potent immunogen used as a vaccine carrier platform. Hepatitis B core antigen produced in E. coli encapsidates random bacterial RNA (bRNA). They provided evidence that the adjuvant property of HBcAg is mainly responsible for the improved properties of this vaccine. Also, cross-protective properties and adjuvant activity have been shown when flu vaccines were combined. The HBcAg has also been used in other vaccine development by combining with CPGODN. Hepatitis B core antigen has also been shown to be a potent adjuvant in the course of development of therapy for glioblastoma. The adjuvant activity of HBcAg has also been found in Chlamydia infection. Taken together, the adjuvant properties of HBcAg have been elucidated in various systems, and this can be used for the development of prophylactic drugs and drugs for treatment.

Utility of Mucosal Immunity for Drug Development

The mucosal surface is the moist lining found in areas of the human body that connect to the exterior, such as the gastrointestinal, reproductive, urinary, and most importantly, the respiratory tract. The entire surface is covered by epithelial cells and connective tissues. Their main action is to act as a physical barrier and protect human beings from noxious materials. The mucosal immunity has been employed for both preventive and curative purposes. It has also been used for the management of tuberculosis.

Progressing to Develop Prophylactics and Therapeutic Maneuvers for SARS-CoV-2 and COVID-19

History of Vaccine and Drug Usage for Treating COVID

SARS-CoV-2, the virus that is the causative agent of COVID-19, emerged in China in 2019. On 30 January 2020, the WHO declared the emergence of the novel coronavirus (2019-nCoV) a public health emergency of international concern (PHEIC). During this time, the WHO mentioned that “the potential for the virus to spread to countries with weaker health systems, and which are ill-prepared to deal with it” is one major concern and the importance of “preventing the spread of the virus and ensuring a measured and evidence-based response.” WHO declared the COVID-19 outbreak a pandemic on March 11, 2020.

Initially, different public health measures were applied to contain the virus. Several measures, even sealing off regions, were implemented to get rid of it. Finally, a vaccine emerged by the end of 2020 and subsequently several other vaccines were developed around the world. The effect of vaccines reduced the impact of COVID-19 around the world.^49–51

Drug Usage for Treating COVID-19

There is currently no drug that has been proven as an effective therapy for COVID-19. In fact, no drug has been developed on the basis of the life cycle and potential of pathogenesis. Several candidate drugs have been considered and evaluated for treatment. These include clinically available drugs, such as chloroquine, hydroxychloroquine, and lopinavir/ritonavir. These drugs were developed for other pathological conditions, but were later repurposed for use in COVID-19. Sometimes, there was some scientific logic for their usage. In other cases, the drugs were used without any scientific support. However, this may be a usual picture during a pandemic. Novel experimental therapies, such as remdesivir and favipiravir, both with antiviral properties, were used and are also actively being investigated for antiviral efficacy against SARS-CoV-2. However, these drugs were not found to be as effective as was primarily assumed for most of the patients. Clinically available and investigational immunomodulators, such as the interleukin-6 inhibitors tocilizumab and sarilumab, and the anti-granulocyte-macrophage colony-stimulating factor lenzilumab, have also been used and tested for their anticipated effect in counteracting the pro-inflammatory cytokine environment that characterizes severe and critical COVID-19.^3,52–56

Insight Regarding the Possible Usage of NASVAC for the Prevention and Treatment of COVID-19

NASVAC as a Preventive Weapon Against SARS-CoV-2

The SARS-CoV-2 virus, which causes COVID-19, enters the human body by the nasal route. If there are any other exceptions about the route of administration of the virus that need to be explored. However, diagnosis of SARS-CoV-2 is made by collecting a nasal swab and usually SARS-CoV-2 is detected at the early stage of infection, if highly sensitive methods are employed.

Now, it is a million-dollar question after entering the virus, why someone remains asymptomatic, whereas others develop symptoms of mild or moderate pathologies. Finally, a group of patients develop different immune anomalies and ultimately take a downhill course, including a fatal outcome.

To develop some insights about these variabilities, we conducted a clinical trial in 20 apparently healthy but high-risk individuals. The participants were negative for SARS-CoV-2 before entering the trial. The study population was 20, and they were mostly related to the medical field, with a considerable risk of transmitting the infection during the peak of the pandemic. They were given 3 intranasal administrations of 1.0 mL of NASVAC (on day 0, 7, and 14). At the same time, they received sublingual administrations of NASVAC daily for 14 days. The data indicated that none of the volunteers was infected with SARS-CoV-2 during 14 days after the end of NASVAC administration by the nasal route. During a follow-up of 6 months, only four of the 20 volunteers became SARS-CoV-2 positive. Out of these, three remained asymptomatic and only one, who was elderly, needed hospitalization. However, there was no problem of further disease progression.^37,57

NASVAC as a Drug for Treating COVID-19

A phase I/II, open-label, controlled, and randomized clinical trial of NASVAC as a postexposure prophylaxis treatment was conducted. It was designed to assess the local and systemic immunomodulatory effects of NASVAC. This study included 46 patients of either sex, aged 60 years or older who presented with COVID-19 symptoms. These patients were administered NASVAC (100 μg per Ag per dose) via the intranasal route on days 1, 7, and 14 and sublingually, daily for 14 days. At the initiation of the study, symptoms were similar (75.0 vs 72.7%). Mean duration of symptoms was longer in the control group (13.7 days vs 10.1 days, p = 0.508, ANOVA). The SARS-CoV-2 infection was assayed by PCR on the 4th or 5th day after the study was initiated. There was one patient who tested positive in the treatment group, while in the control group, there were two positive patients. There was no difference in respiratory symptoms. Among the three positive patients, one from the control group developed pneumonia, diarrhea, edema, and fatigue, and after discharge, fatigue and shortness of breath persisted for 42 days. The remaining two patients had mild symptoms.^58,59

Implications and Discussion of the Findings Regarding NASVAC for use in COVID-19

The first line of defense of humans against any given viral infection is the innate immune response. There are pattern recognition receptors (PRRs) on plasma membranes, endosomal membranes, and cytosol, which first recognize viral components or replication intermediates known as pathogen associated molecular patterns (PAMPs) and then activate innate immunity. The initial step of viral infection depends on the complex interplay between viruses, viral receptors, PRRs and PAMPs. Natural killer (NK) cells, NK T-cells, neutrophils, dendritic cells (DC), and macrophages prevent viral attachment to specific host receptors and inhibit viral replication by obstructing viral localization or by destroying viruses. After entry of SARS-CoV-2 into the nasal mucosa, innate, regulatory, and adaptive immunities interplay in COVID-19 pathogenesis. So, one of the approaches to counter SARS-CoV-2 infection may be to block the localization of this virus in the nasal cavity.

In the context of SARS-CoV-2, the establishment of an infection and progression to COVID-19 means that the inherent innate immunity could not work properly, or that it was not purposeful. At the same time, several individuals being exposed to SARS-CoV-2 either do not allow localization of the virus or refrain from developing clinical COVID-19.

The role of NASVAC may be visualized in this situation. There may be many activities of NASVAC in this situation. First, as an active adjuvant, it may allow the cells and mucosa of the nasal cavity to act more actively so that the local innate immunity is exacerbated. If the local immunity is exacerbated, the virus may not proceed to the lungs and remain incapable of inducing several diseases. This is partially supported by data that indicated that HBcAg induces production of inflammatory cytokines in various models, especially in CHB and COVID-19 patients. Our study indeed showed that NASVAC upregulates both local and systemic immunity in patients with COVID-19.

The second feature that may bring HBcAg for treatment or prophylaxis of COVID-19 is the unique characteristics of NASVAC of inducing proinflammatory cytokines, but not causing damage to any organs. For many years, cytokine or immune modulators could not be used for the treatment of different pathologies in which the host is somehow immunocompromised. Although it is easily presumed that activation of the immune system by a polyclonal immune modulator would be of a beneficial nature, that did not happen. This was found not only in cases of HBV infection, but also in many chronic infections and acute problems. The causes underlying this will allow the development of immune therapy of the proper nature for many pathological conditions that are yet to be accomplished. Studies have shown that polyclonal immune modulators of any nature, although they bring a microenvironment of exacerbated immunity, are not usually purposeful. On one hand, proper doses of polyclonal immune modulators could not be used due to safety concerns in most diseases. Next, the chronicity of any pathological condition needs to be resolved by antigen-specific immunity, not by polyclonal immunity. In this respect, HBcAg exhibits an extraordinary feature of not causing tissue damage, rather increasing innate immunity. These features allow us to suggest a role for NASVAC in prophylaxis and treatment of COVID-19.

It is now well established that COVID-19-related mortality was higher among the elderly. This can be explained by old age-related alterations in immune response after stimulation of pathogen recognition receptors. It was observed that NASVAC had a protective effect in the elderly. Increase in HLA class II expression in monocytes and lymphocytes of NASVAC-treated elderly volunteers may have contributed to this, as in severe COVID-19, there is reduced expression of HLA-DR on monocytes and myeloid DC (mDC).⁵³ This is consistent with previous observations that NASVAC stimulates innate and adoptive immunity both in vivo and in vitro.^60,61 Furthermore, it has also been observed that NASVAC stimulates TLR, HLA class I/II, and costimulatory molecule gene expression in vitro in HepaRG model.

Studies have revealed that in COVID-19, peripheral innate immune cells have functional impairment. It is also known that TLR3, TLR7, and TLR8 signaling pathways induce IFN production. During the COVID-19 pandemic, clinical trials yielded beneficial results with IFN in SARS-CoV-2 infection.⁵⁴ It is likely that NASVAC may help COVID-19 patients by inducing IFN production and also by improving the detection of viral RNA by innate immunity. It may be noted that increased expression of TLR3, TLR7 and TLR8 genes in nasopharyngeal tonsils is a surrogate marker of protection against SARS-CoV-2 in a lethal infection mouse model.

It may further be noted that synergistic stimulation of MyD88-dependent and independent pathways via TLR3, TLR7, and TLR8 decreases viremia and induces clinical improvement in Dengue. This may also be useful in hepatocellular carcinoma (HCC), where TLR3 expression correlates with apoptosis, proliferation, angiogenesis, and prognosis.^62–65

NASVAC: Containing the Therapy of New Viruses or Microbes with Epidemic and Pandemic Potentials

We are now residing in a world that is experiencing the eruption of new and novel viruses as well as microbes that were not pathogenic but are causing epidemics recently. Dengue virus is one of the viruses. Chikungunya may be another major virus that is expanding in massive forces around the world. Zika virus may also be regarded as an evolving virus. These viruses are not airborne; rather, they are mostly vector borne. However, these viral infections also induce aberrant immunity. Although NASVAC may not be used for prophylaxis purposes in these viruses, it can be used to shape the immunity of the host due to this virus infection.

Limitation(s) of the Study

The clinical trial of NASVAC in COVID-19 patients that has been reviewed in the present article lacked a control group, which is a limitation of the study. However, given the pandemic situation, it was not possible and also not ethically possible to include patients as controls, denying them the standard of care for this potentially grave disease. Further clinical trials with a larger sample size were warranted, but again, it could not be accomplished as the pandemic was fortunately contained.

Conclusions

The safety of NASVAC in COVID-19 has been well established. Besides, it may also be inferred that NASVAC will be effective both in pre- and postexposure prophylaxis against SARS-CoV-2 and also in preventing the progression of severe COVID-19.

Clinical Significance

NASVAC may become an effective immunomodulatory prophylactic intervention against other viruses like dengue, as well as for the treatment of malignancies like HCC.

Ethical Approval

This article is a narrative review based on previously published literature and does not involve any new studies with human participants or animals conducted by the authors. Therefore, institutional ethics committee approval and informed consent were not required.

Data Availability Declaration

Nil.

AI Disclosure Statement

No AI tool was used in the methodology while doing the study.

Acknowledgments

Nil

Authors’ Contributions

Mamun Al Mahtab and Sheikh MF Akbar drafted the manuscript; Abul K Mohammad Faizul Huq, Sakirul Khan, and Musarrat Mahtab conducted the literature search; Julio CA Rubido, Osamu Yoshida and Gerardo EG Nieto revised the manuscript.

Orcid

Mamun Al Mahtab https://orcid.org/0000-0003-3728-3879

Sheikh MF Akbar https://orcid.org/0000-0003-4537-3313

Julio CA Rubido https://orcid.org/0000-0003-0166-4784

Osamu Yoshida https://orcid.org/0000-0002-5015-0045

Abul K Mohammad Faizul Huq https://orcid.org/0009-0000-3245-4312

Sakirul Khan https://orcid.org/0000-0001-5734-0899

Musarrat Mahtab https://orcid.org/0009-0001-7344-4812