Bioengineering tools to speed up the discovery and preclinical testing of vaccines for SARS-CoV-2 and therapeutic agents for COVID-19.

This review provides an update for the international research community on the cell modeling tools that could accelerate the understanding of SARS-CoV-2 infection mechanisms and could thus speed up the development of vaccines and therapeutic agents against COVID-19. Many bioengineering groups are actively developing frontier tools that are capable of providing realistic three-dimensional (3D) models for biological research, including cell culture scaffolds, microfluidic chambers for the culture of tissue equivalents and organoids, and implantable windows for intravital imaging. Here, we review the most innovative study models based on these bioengineering tools in the context of virology and vaccinology. To make it easier for scientists working on SARS-CoV-2 to identify and apply specific tools, we discuss how they could accelerate the discovery and preclinical development of antiviral drugs and vaccines, compared to conventional models.

SARS-CoV-2 pandemic and research gaps: Understanding SARS-CoV-2 interaction with the ACE2 receptor and implications for therapy.

The COVID-19 pandemic is an emerging threat to global public health. While our current understanding of COVID-19 pathogenesis is limited, a better understanding will help us develop efficacious treatment and prevention strategies for COVID-19. One potential therapeutic target is angiotensin converting enzyme 2 (ACE2). ACE2 primarily catalyzes the conversion of angiotensin I (Ang I) to a nonapeptide angiotensin or the conversion of angiotensin II (Ang II) to angiotensin 1-7 (Ang 1-7) and has direct effects on cardiac function and multiple organs via counter-regulation of the renin-angiotensin system (RAS). Significant to COVID-19, ACE2 is postulated to serve as a major entry receptor for SARS-CoV-2 in human cells, as it does for SARS-CoV. Many infected individuals develop COVID-19 with fever, cough, and shortness of breath that can progress to pneumonia. Disease progression promotes the activation of immune cells, platelets, and coagulation pathways that can lead to multiple organ failure and death. ACE2 is expressed by epithelial cells of the lungs at high level, a major target of the disease, as seen in post-mortem lung tissue of patients who died with COVID-19, which reveals diffuse alveolar damage with cellular fibromyxoid exudates bilaterally. Comparatively, ACE2 is expressed at low level by vascular endothelial cells of the heart and kidney but may also be targeted by the virus in severe COVID-19 cases. Interestingly, SARS-CoV-2 infection downregulates ACE2 expression, which may also play a critical pathogenic role in COVID-19. Importantly, targeting ACE2/Ang 1-7 axis and blocking ACE2 interaction with the S protein of SARS-CoV-2 to curtail SARS-CoV-2 infection are becoming very attractive therapeutics potential for treatment and prevention of COVID-19. Here, we will discuss the following subtopics: 1) ACE2 as a receptor of SARS-CoV-2; 2) clinical and pathological features of COVID-19; 3) role of ACE2 in the infection and pathogenesis of SARS; 4) potential pathogenic role of ACE2 in COVID-19; 5) animal models for pathological studies and therapeutics; and 6) therapeutics development for COVID-19.

Recent Advances in Zika Virus Vaccines.

The recent outbreaks of Zika virus (ZIKV) infections and associated microcephaly in newborns has resulted in an unprecedented effort by researchers to target this virus. Significant advances have been made in developing vaccine candidates, treatment strategies and diagnostic assays in a relatively short period of time. Being a preventable disease, the first line of defense against ZIKV would be to vaccinate the highly susceptible target population, especially pregnant women. Along those lines, several vaccine candidates including purified inactivated virus (PIV), live attenuated virus (LAV), virus like particles (VLP), DNA, modified RNA, viral vectors and subunit vaccines have been in the pipeline with several advancing to clinical trials. As the primary objective of Zika vaccination is the prevention of vertical transmission of the virus to the unborn fetus, the safety and efficacy requirements for this vaccine remain unique when compared to other diseases. This review will discuss these recent advances in the field of Zika vaccine development.

Vaccines for the Paramyxoviruses and Pneumoviruses: Successes, Candidates, and Hurdles.

Human parainfluenza viruses (family Paramyxoviridae), human metapneumovirus, and respiratory syncytial virus (family Pneumoviridae) infect most infants and children within the first few years of life and are the etiologic agents for many serious acute respiratory illnesses. These virus infections are also associated with long-term diseases that impact quality of life, including asthma. Despite over a half-century of vaccine research, development, and clinical trials, no vaccine has been licensed to date for the paramyxoviruses or pneumoviruses for the youngest infants. In this study, we describe the recent reclassification of paramyxoviruses and pneumoviruses into distinct families by the International Committee on the Taxonomy of Viruses. We also discuss some past unsuccessful vaccine trials and some currently preferred vaccine strategies. Finally, we discuss hurdles that must be overcome to support successful respiratory virus vaccine development for the youngest children.

Challenges of Vaccine Development for Zika Virus.

The emergence of outbreaks of Zika virus (ZIKV) in Brazil in 2015 was associated with devastating effects on fetal development and prompted a world health emergency and multiple efforts to generate an effective vaccine against infection. There are now more than 40 vaccine candidates in preclinical development and six in clinical trials. Despite similarities with other flaviviruses to which successful vaccines have been developed, such as yellow fever virus and Japanese Encephalitis virus, there are unique challenges to the development and clinical trials of a vaccine for ZIKV.

Optimized production and purification of Coxsackievirus B1 vaccine and its preclinical evaluation in a mouse model.

Coxsackie B viruses are among the most common enteroviruses, causing a wide range of diseases. Recent studies have also suggested that they may contribute to the development of type 1 diabetes. Vaccination would provide an effective way to prevent CVB infections, and the objective of this study was to develop an efficient vaccine production protocol for the generation of novel CVB vaccines. Various steps in the production of a formalin-inactivated Coxsackievirus B1 (CVB1) vaccine were optimized including the Multiplicity Of Infection (MOI) used for virus amplification, virus cultivation time, type of cell growth medium, virus purification method and formulation of the purified virus. Safety and immunogenicity of the formalin inactivated CVB1 vaccine was characterized in a mouse model. Two of the developed methods were found to be optimal for virus purification: the first employed PEG-precipitation followed by gelatin-chromatography and sucrose cushion pelleting (three-step protocol), yielding 19-fold increase in virus concentration (0.06µg/cm2) as compared to gold standard method. The second method utilized tandem sucrose pelleting without a PEG precipitation step, yielding 83-fold increase in virus concentration (0.24µg/cm2), but it was more labor-intensive and cannot be efficiently scaled up. Both protocols provide radically higher virus yields compared with traditional virus purification protocols involving PEG-precipitation and sucrose gradient ultracentrifugation. Formalin inactivation of CVB1 produced a vaccine that induced a strong, virus-neutralizing antibody response in vaccinated mice, which protected against challenge with CVB1 virus. Altogether, these results provide valuable information for the development of new enterovirus vaccines.

Vaccination strategies against Zika virus.

The epidemic emergence of Zika virus (ZIKV) in 2015-2016 has been associated with congenital malformations and neurological sequela. Current efforts to develop a ZIKV vaccine build on technologies that successfully reduced infection or disease burden against closely related flaviviruses or other RNA viruses. Subunit-based (DNA plasmid and modified mRNA), viral vectored (adeno- and measles viruses) and inactivated viral vaccines are already advancing to clinical trials in humans after successful mouse and non-human primate studies. Among the greatest challenges for the rapid implementation of immunogenic and protective ZIKV vaccines will be addressing the potential for exacerbating Dengue virus infection or causing Guillain-Barré syndrome through production of cross-reactive immunity targeting related viral or host proteins. Here, we review vaccine strategies under development for ZIKV and the issues surrounding their usage.

Current developments and prospects on human metapneumovirus vaccines.

INTRODUCTION: Human metapneumovirus (hMPV) has become one of the major pathogens causing acute respiratory infections (ARI) mainly affecting young children, immunocompromised patients, and the elderly. Currently there are no licensed vaccines against this virus. Areas covered: Since the discovery of hMPV in 2001, many groups have focused on developing vaccines against this pathogen. This review presents the outcomes and perspectives derived from preclinical studies performed in cell cultures and animals as well as the only candidate that has reached evaluation in a clinical trial. Limitations of the current vaccine candidates are discussed and perspectives for the development of plant-based vaccines are analyzed. Expert commentary: Several hMPV vaccine candidates are under development with the potential to progress into clinical trials. In parallel, the molecular farming field offers new opportunities to generate innovative vaccines that will offer several advantages in the fight against hMPV.

Strategies to develop vaccines of pediatric interest.

INTRODUCTION: The success of the vaccines available on the market has significantly increased interest in vaccine development. Areas covered: The main aim of this paper is to discuss the most important vaccines of pediatric interest that are currently being developed. New pneumococcal vaccines and vaccines against group B Streptococcus, Staphylococcus aureus and respiratory syncytial virus are analyzed in detail. Expert commentary: Advances in understanding human immunology, including human monoclonal antibody identification, sequencing technology, and the ability to solve atomic level structures of vaccine targets have provided tools to guide the rational design of future vaccines. It is likely that some of these vaccines will reach the market in the future and will thus partially contribute to the prevention of very severe diseases that significantly affect the morbidity and mortality of children. However, further studies in animals and several clinical trials in children must be performed before new vaccines become licensed.

Vaccines against Middle East respiratory syndrome coronavirus for humans and camels.

Middle East respiratory syndrome coronavirus (MERS-CoV) is caused by a novel betacoronavirus that was isolated in late 2012 in Saudi Arabia. The viral infections have been reported in more than 1700 humans, ranging from asymptomatic or mild cases to severe pneumonia with a mortality rate of 40%. It is well documented now that dromedary camels contract the infection and shed the virus without notable symptoms, and such animals had been infected by at least the early 1980s. The mechanism of camel to human transmission is still not clear, but several primary cases have been associated with camel contact. There is no approved antiviral drug or vaccine against MERS-CoV despite the active research in this area. Vaccine candidates have been developed using various platforms and regimens and have been tested in several animal models. Here, this article reviews the published studies on MERS-CoV vaccines with more focus on vaccines tested in large animals, including camels. It is foreseeable that the 1-health approach could be the best way of tackling the MERS-CoV endemic in the Arabian Peninsula, by using the mass vaccination of camels in the affected areas to block camel to human transmission. Camel vaccines can be developed in a faster time with fewer regulations and lower costs and could clear this virus from the Arabian Peninsula if accompanied by efficient public health measures.

Plant-based vaccines against respiratory diseases: current status and future prospects.

INTRODUCTION: Respiratory infections have an enormous, worldwide epidemiologic impact on humans and animals. Among the prophylactic measures, vaccination has the potential to neutralize this impact. New technologies for vaccine production and delivery are of importance in this field since they offer the potential to develop new immunization approaches overriding the current limitations that comprise high cost, safety issues, and limited efficacy. Areas covered: In the present review, the state of the art in developing plant-based vaccines against respiratory diseases is presented. The review was based on the analysis of current biomedical literature. Expert commentary: Preclinical and clinical evaluations of several vaccine candidates against influenza, tuberculosis, respiratory syncytial virus, pneumonia, anthrax and asthma are discussed and placed in perspective.

Vaccine platforms to control Lassa fever.

INTRODUCTION: Lassa virus (LASV), the most prominent human pathogen of the Arenaviridae, is transmitted to humans from infected rodents and can cause Lassa Fever (LF). The sizeable disease burden in West Africa, numerous imported LF cases worldwide, and the possibility that LASV can be used as an agent of biological warfare make a strong case for vaccine development. There are no licensed LASV vaccines and the antiviral treatment is limited to an off-label use of ribavirin that is only partially effective. AREAS COVERED: LASV vaccine development is hampered by high cost of biocontainment requirement, the absence of appropriate small animal models, genetic diversity of LASV species, and by high HIV-1 prevalence in LASV endemic areas. Over the past 15 years several vaccine platforms have been developed. Natural history of LASV and pathogenesis of the disease provide strong justification for replication-competent (RC) vaccine as one of the most feasible approaches to control LF. Development of LASV vaccine candidates based on reassortant, recombinant, and alphavirus replicon technologies is covered in this review. Expert commentary: Two lead RC vaccine candidates, reassortant ML29 and recombinant VSV/LASV, have been successfully tested in non-human primates and have been recommended by international vaccine experts for rapid clinical development. Both platforms have powerful molecular tools to further secure safety, improve immunogenicity, and cross-protection. These platforms are well positioned to design multivalent vaccines to protect against all LASV strains citculatrd in West Africa. The regulatory pathway of Candid #1, the first live-attenuated arenaviral vaccine against Argentine hemorrhagic, will be a reasonable guideline for LASV vaccine efficacy trials.

Novel strategies for development of hemorrhagic fever arenavirus live-attenuated vaccines.

INTRODUCTION: Several arenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever (HF) disease in humans and pose significant public health problems in their endemic regions. Moreover, HF arenaviruses represent credible biodefense threats. There are not FDA-approved arenavirus vaccines and current anti-arenaviral therapy is limited to an off-label use of ribavirin that is only partially effective. AREAS COVERED: Live-attenuated vaccines (LAV) represent the most feasible approach to control HF arenaviruses within their endemic regions. Different platforms, including recombinant viral vectors expressing LASV antigens, and the use of attenuated reassortant arenaviruses, have been used to develop LAV candidates against LASV with promising results in animal models of LASV infection, but none of them has entered a clinical trial. These vaccine efforts have been the subject of recent reviews and will not be examined in this review, which is focused on new avenues for the development of safe and effective LAV to combat HF arenaviruses. Expert commentary: The development of arenavirus reverse genetics has provided investigators with a novel powerful approach to manipulate the genomes of HF arenaviruses, which has opened new avenues for the rapid development of safe and effective LAV to combat these human pathogens.

A transgenic ginseng vaccine for bovine viral diarrhea.

BACKGROUND: Bovine viral diarrhea virus (BVDV) infections are endemic in cattle populations worldwide and cause major economic losses. Thus, an effective vaccine is needed against the transmission of BVDV. The glycoprotein E(rns) is one of the envelope proteins of this virus and shows BVDV-related immunogenicity. Here, we report the use of Panax ginseng as an alternative production platform for the expression of glycoprotein E(rns) via Agrobacterium-mediated transformation. RESULT: Polymerase chain reaction (PCR) and reverse transcription (RT)-PCR analyses showed that pBI121-E(rns) was stably integrated into the chromosome of transformants. ELISA assay and Western blot analysis confirmed the antigenicity of plant-derived E(rns) glycoprotein. Immunogenicity was evaluated subcutaneously in deer using a soluble protein extract of dried transgenic ginseng hairy roots. Specific humoral and cell-mediated immune responses against BVDV were detected following immunization. CONCLUSION: These results demonstrated that the E(rns) glycoprotein could be expressed in ginseng hairy roots and that plant-derived glycoprotein E(rns) retained its antigenicity and immunogenicity.

Middle East respiratory syndrome: obstacles and prospects for vaccine development.

The recent emergence of Middle East respiratory syndrome (MERS) highlights the need to engineer new methods for expediting vaccine development against emerging diseases. However, several obstacles prevent pursuit of a licensable MERS vaccine. First, the lack of a suitable animal model for MERS complicates the in vivo testing of candidate vaccines. Second, due to the low number of MERS cases, pharmaceutical companies have little incentive to pursue MERS vaccine production as the costs of clinical trials are high. In addition, the timeline from bench research to approved vaccine use is 10 years or longer. Using novel methods and cost-saving strategies, genetically engineered vaccines can be produced quickly and cost-effectively. Along with progress in MERS animal model development, these obstacles can be circumvented or at least mitigated.

Glycoprotein E2 of classical swine fever virus expressed by baculovirus induces the protective immune responses in rabbits.

Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious and devastating disease that affects the pig industry worldwide. The glycoprotein E2 of CSFV is the principal immunogenic protein that induces neutralizing antibodies and protective immunity. Several CSFV genotypes, including 1.1, 2.1, 2.2, and 2.3, have been identified in Mainland China. The glycoprotein E2 of genotypes 1.1 and 2.1 was expressed by using a baculovirus system and tested for its protective immunity in rabbits to develop novel CSF vaccines that elicit a broad immune response. Twenty CSFV seronegative rabbits were randomly divided into five groups. Each rabbit was intramuscularly immunized with E2 of genotypes 1.1 (CSFV-1.1E2), 2.1 (CSFV-2.1E2), or their combination (CSFV-1.1 + 2.1E2). A commercial CSF vaccine (C-strain) and phosphate-buffered saline (PBS) were used as positive or negative controls, respectively. All animals were challenged with CSFV C-strain at 4 weeks and then boosted with the same dose. All rabbits inoculated with CSFV-1.1E2, CSFV-2.1E2, and CSFV-1.1 + 2.1E2 elicited high levels of ELISA antibody, neutralizing antibody, and lymphocyte proliferative responses to CSFV. The rabbits inoculated with CSFV-1.1E2 and CSFV-1.1 + 2.1E2 received complete protection against CSFV C-strain. Two of the four rabbits vaccinated with CSFV-2.1E2 were completely protected. These results demonstrate that CSFV-1.1E2 and CSFV-1.1 + 2.1E2 not only elicit humoral and cell-mediated immune responses but also confer complete protection against CSFV C-strain in rabbits. Therefore, CSFV-1.1E2 and CSFV-1.1 + 2.1E2 are promising candidate subunit vaccines against CSF.

Hepatitis C vaccine. Need of the hour.

Hepatitis C virus (HCV) was first identified in 1989. HCV is a small, enveloped RNA virus. Globally, 3-4 million persons are infected with HCV each year, and are at risk of developing liver cirrhosis and/or liver cancer. The common modalities of the spread of hepatitis C infection are blood transfusions, injection drug use, unsafe therapeutic injections, and healthcare-related procedures. The standard treatment for hepatitis C has been combination antiviral therapy with interferon (IFN) and ribavirin, which are effective against all the genotypes of hepatitis viruses (pan-genotypic). A 12-month course of Peg-IFN/ribavirin treatment costs > $20 000. New HCV-specific antiviral drugs, especially in combination, have shown very high cure rates; however, the annual cost for a single subject ($82 000) make these unaffordable in most of the world. There is no hepatitis C vaccine. However, several vaccines in development, and some have shown promising preclinical results. Over the last few years, numerous HCV vaccine approaches have been assessed in mice and primates, but only a few vaccines have progressed to human trials. The challenge to develop HCV vaccine is to move into larger at-risk or infected populations to test efficacy.

Quality control and evaluation of vaccines in China.

I was born in Luoyang in 1963, a city in China with thousands of years' history. The city is the eastern starting point of the Silk Road (BC 206-260 AD), a famous and long trade route in human history. Living in this ancient city, I grew up under the influence and impact of Chinese traditional culture. When I was a teenager, I once read a book "Yellow Emperor's Inner Canon" (Huang Di Nei Jing, written about 5th century BC), which is the oldest extant classic of traditional Chinese medicine. I was immediately attracted by an idea in it "prevention is better than cure," which is mean that the highest level of medical treatment is not to cure a disease, but to prevent the occurrence of diseases. I think even in the modern society, this is still the highest realm of medical treatment. Inspired by this view, I developed a strong interest in preventive medicine and majored in it during my college years. With an increasingly deeper understanding of preventive medicine, I gradually realized the importance of epidemiology and vaccinology in preventing infectious diseases. Fortunately, in 1991 I entered the group of professor Zhuang Hui in Peking University Health Science Center for a doctorate degree in epidemiology. Professor Zhuang who is an academician of the Chinese Academy of Engineering not only taught me how to do research, but showed me how to act like a scientist. I benefited greatly from his rigorous attitudes toward life and research. During this period, I focused on transmission routes of hepatitis C virus which made me increasingly recognized the great harm of hepatitis in China. It is well known that more than three-quarters of all liver cancer cases are thought to be attributable to hepatitis B or C. In China about 110 000 people die from liver cancer each year, accounting 45% of the total number of deaths caused by liver cancer worldwide.

Pilot scale production of highly efficacious and stable enterovirus 71 vaccine candidates.

BACKGROUND: Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial. PRINCIPAL FINDING: In this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration), a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7-10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30-43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37 °C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4 °C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice, rats, rabbits, and non-human primates. CONCLUSION: These results provide valuable information supporting the current cell-based serum-free EV71 vaccine candidate going into human Phase I clinical trials.