key: cord-0757361-9x60jukk
authors: Nagy, Abdou; Alhatlani, Bader
title: An overview of current COVID-19 vaccine platforms
date: 2021-04-27
journal: Comput Struct Biotechnol J
DOI: 10.1016/j.csbj.2021.04.061
sha: b47028fead2eaf482b0919dd5b878c90a25d2f9a
doc_id: 757361
cord_uid: 9x60jukk

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) pandemic that emerged in December 2019 in Wuhan city, China. An effective vaccine is urgently needed to protect humans and to mitigate the economic and societal impacts of the pandemic. Despite standard vaccine development usually requiring an extensive process and taking several years to complete all clinical phases, there are currently 184 vaccine candidates in pre-clinical testing and another 88 vaccine candidates in clinical phases based on different vaccine platforms as of April 13, 2021. Moreover, three vaccine candidates have recently been granted an Emergency Use Authorization by the United States Food and Drug Administration (for Pfizer/BioNtech, Moderna mRNA vaccines, and Johnson and Johnson viral vector vaccine) and by the UK government (for University of Oxford/AstraZeneca viral vector vaccine). Here we aim to briefly address the current advances in reverse genetics system of SARS-CoV-2 and the use of this in development of SARS-CoV-2 vaccines. Additionally, we cover the essential points concerning the different platforms of current SARS-CoV-2 vaccine candidates and the advantages and drawbacks of these platforms. We also assess recommendations for controlling the COVID-19 pandemic and future pandemics using the benefits of genetic engineering technology to design effective vaccines against emerging and re-emerging viral diseases with zoonotic and/or pandemic potential.

In late December 2019, China reported cases of idiopathic pneumonia in the city of Wuhan. One month later, the causative agent was identified as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1] .

SARS-CoV-2 caused the epidemic of coronavirus disease 2019 in different cities in China, which then spread globally and was later classified as a pandemic by the World Health Organization (WHO) [1, 2] . SARS-CoV-2 belongs to the genus Betacoronavirus in the family Coronaviridae. SARS-CoV-2 is an enveloped virus that contains a single-stranded positive-sense RNA genome of 29 SARS-CoV-2 is a closely related to SARS-CoV that circulated from 2002 to 2004 [1] . SARS-CoV together with the Middle Eastern respiratory syndrome coronavirus (MERS-CoV) have caused epidemics with high case fatality rates in humans [5] . Despite this, there was no current licensed vaccine against coronaviruses in humans, and the reasons for this may be related to the low priority given to these viruses by vaccine developers due to few sporadic human deaths caused by these viruses and their limited geographic distribution. Moreover, a broad variety of viruses cause human colds and so, development of multivalent coronavirus vaccine would only prevent a minor proportion of these [6] . Several vaccines against SARS-CoV were developed pre-clinically with two candidates tested in Phase I trials [7, 8] . However, these clinical phases did not proceed further because these epidemics stopped and these viral infections have not re-emerged in humans since 2004, which might indicate that the control measurements were effective and/or that these viral infections were self-limiting. However, vaccines against MERS-CoV have been actively developed, and through all these pre-clinical studies, the spike protein has been identified as the antigenic target for coronavirus vaccines [9, 10] .

Fortunately, lessons learned from pre-clinical and clinical data of SARS-CoV and MERS-CoV vaccine trials provided sufficient experience to design promising SARS-CoV-2 vaccine candidates in just a few weeks after the emergence of this new virus and to begin the first clinical trial in March 2020 (trial number NCT04283461) with an overlapping schedule, such as using Phase I/II trials followed by rapid start of Phase III trials [6] . Currently, 184 vaccine candidates are in pre-clinical development, and 88 vaccine candidates are in clinical development stages [11] with three vaccines recently granted an Emergency Use Authorization (EUA) by the Food and Drug Administration (FDA). Here, we briefly summarize the advantages and disadvantages of the current vaccine candidates/platforms used in Phase III trials against SARS-CoV-2 and their efficacy against the new variants. We also investigate why using recent advances in genetic engineering offers promising solutions for rapid design of effective vaccines against emerging and re-emerging viral diseases that have zoonotic and/or pandemic potential.

Reverse genetics (RG) is a powerful tool that is widely used for the genetic manipulation of RNA viruses from their full-length cloned DNA (cDNA) and can lead to the development of successful countermeasures. [12] . A number of RG systems were previously developed for various coronaviruses, including SARS-CoV, MERS-CoV, murine hepatitis virus, and human coronavirus 229E (HCoV-229) [13] . However, difficulties in rescuing infectious viral RNA from cDNA have always been challenging in coronaviruses because of the large size of their genomes and the instability of some regions within the viral genome [13] . To overcome these challenges, a robust RG system for SARS-CoV-2 and other RNA viruses was rapidly developed during the early events of the COVID-19 pandemic [14] . In this yeast-based system, viral overlapping subgenomic cDNA fragments are produced and reassembled in Saccharomyces cerevisiae using transformation-associated recombination cloning [14] . Viral RNA is in vitro transcribed using T7 polymerase and then transfected together with an mRNA that expresses the N protein of the SARS-CoV-2 into BHK-21 cells or BHK cells that express the SARS-CoV N protein (BHK-SARS-N) to recover viable SARS-CoV-2. A similar robust RG system was also been developed for SARS-CoV-2 and is based on an in vitro ligation method that resulted in viral replication kinetics similar to the original clinical isolate [15] . In this RG system, the full-length SARS-CoV-2 genome is initially assembled from seven cDNA fragments, one of which contains the T7 promoter, using an in vitro ligation approach followed by in vitro transcription of the cDNA to produce the viral RNA [15] . The recovery of recombinant virus can then be obtained by electroporating the in vitro transcribed viral RNA into Vero E6 cells. A third RG method has been established using bacterial artificial chromosomes (BAC) [16] ; and a similar system has been previously used for other coronaviruses such as SARS-CoV [17] .

Full-length SARS-CoV-2 cDNA is first assembled by sequentially cloning the five fragments of the viral genome into a BAC plasmid under the control of the cytomegalovirus (CMV) promoter. The SARS-CoV-2 BAC plasmid can then be transfected into Vero E6 or other susceptible cell lines to rescue the infectious recombinant SARS-CoV-2 [16] . Moreover, an additional RG method that relies on the use of the infectioussubgenomic amplicons (ISA) approach has been developed [18] . This involves polymerase chain reaction amplification of eight overlapping synthetic subgenomic cDNA fragments with the insertion of the CMV promoter upstream the first cDNA fragment to allow transcription initiation; the hepatitis delta ribozyme is inserted downstream the last cDNA fragment to terminate the transcription [18] . Rescue of infectious viral particles is achieved by transfecting the full-length cDNA in permissive cells. The main advantage of the ISA approach is that it does not require cloning [19] . Similarly, another approach has also been developed by assembling 10 SARS-CoV-2 cDNA fragments using circular polymerase extension reaction followed by the transfection of the circular genome into a susceptible cell line [19] .

Hence, the fast development of these RG systems may answer several questions regarding the SARS-CoV-2 biology and pathogenesis. For example, a recent study used the RG system to generate reporter viruses to gain insight into SARS-CoV-2 pathogenesis and tropism [20] . Furthermore, the availability of these systems could also lead to the development of a live-attenuated vaccine and/or antiviral therapeutics. One way to achieve this is by using these RG systems to characterize and generate mutant viruses via mutating an essential part of the SARS-CoV-2 genome, such as the cis-acting RNA elements within the 5′ and 3′ ends of the viral RNA genome [21, 22] . In addition, RG also enables the study and characterization of the different aspects of viral the life cycle, including replication and pathogenesis [23] .

Currently, there are 16 vaccine candidates in Phase III trials as of April 13, 2021 [11] , with encouraging efficacy data from testing in nonhuman primates and Phase I and II trials. Most current vaccines in Phase III are administered intramuscularly (few are administered using different routes such as skin, e.g., AG0302-COVID19). Intramuscular vaccination induces strong IgG responses that protect the lower respiratory tract but does not induce sufficient secretory IgA to protect the upper respiratory tract, as in the case of natural infection. These Phase III vaccine candidates have been developed using different platforms ( Table 1) .

Several of these platforms have already produced licensed vaccines, whereas other have not, such as the mRNA platform [6] . Here we summarize the major vaccine platforms that have progressed to Phase III trials.

Currently, there are only two live-attenuated SARS-CoV-2 vaccine candidates in clinical Phase I as of April 13, 2021. The first vaccine candidate, called COVI-VAC (trial number NCT04619628), was developed by Virus attenuation can be achieved by exposing and adapting the virus to unfavorable conditions such as low temperature growth in a non-susceptible host or cells. Genetic modification can also be used via the RG system for codon de-optimization or deletion of genes that are essential for stimulating innate immune recognition [24, 25] .

The advantages of the use of live-attenuated SARS-CoV-2 vaccine candidates include the targeting and stimulation of robust mucosal and cellular immunity, which is essential for protection without the need for adjuvants [26] . However, this type of vaccine has some drawbacks: (1) SARS-CoV-2 has been shown to be excreted in the feces of infected patients [27, 28] , and this generates a major safety concern that the live- which makes large-scale vaccine production a slow response to the pandemic [26] .

There are six inactivated vaccine candidates currently in Phase III trials as of April 13, 2021 [11] . The first vaccine candidate, with the trial numbers ChiCTR2000034780 and ChiCTR2000039000, was developed by Sinopharm in collaboration with China National Biotec Group co and Wuhan Institute of Biological Products. usually produced by growing a working virus seed of SARS-CoV-2 in a susceptible cell line, such as Vero E6, followed by chemical inactivation [29, 30] . Additionally, inactivated vaccines are usually adjuvanted with alum or specific oils and then administered intramuscularly; this tends to produce broad immune responses as the vaccine induces targeting of a range of different virus proteins such as S, M, and N [6] . The targeted immune response in this kind of vaccine is usually humoral and cellular, with little reactogenicity, resulting in a high safety profile of the vaccine in the vaccinee. There has been over 70 years of research on the development of such vaccines [6, 26] . However, the production and formulation of this type of vaccine currently face three challenges. (1) Cultivation of the live infectious SARS-CoV-2 to high titers (scale-up) in biosafety level (BSL) 3 facilities, which has major safety implications. (2) Inadequate chemical inactivation of the cultivated virus would pose a potential risk to production facility workers, causing disease outbreaks and/or harmful immune responses. (3) It is challenging to manufacture billions of doses in short time, making response to the pandemic particularly slow [26] .

There are seven protein subunit vaccine candidates currently in Phase III trials as of April Protein subunit vaccine candidates (Fig. 2C ) depend on expressing S protein or simply a region of it, such as the receptor-binding domain (RBD), or other virus genes in different expression systems, including insect, mammalian, and/or yeast cells [31, 32] . Therefore, this type of vaccine lacks many drawbacks, such as preexisting antivector immunity, reversion to virulence, and safety concerns of incomplete virus inactivation; hence, they are considered very safe after administration to vaccinees [33] . The target immune response is mainly humoral, which is usually boosted by conjugation with an adjuvant [26] .

Moreover, this platform has already produced several licensed vaccines, such as FluBlok for influenza [6] .

However, there some drawbacks in this platform, particularly for SARS-COV-2, wherein the S protein is difficult to express, making production process a major challenge. However, this could be solved by expressing only the RBD, although expression of RBD alone may be sub-optimum as this does not include all the antigenic epitopes present in full-length S protein [6] . Another drawback is related to the formulation process, which is labor-intensive and requires development and establishment of new production protocols and stability assays for each new antigen [26] .

There are four vaccine candidates currently in Phase III trials using deficient/non-replicating viral vector platform as of April Replication-deficient vaccines depend on using a weakened virus vector generated by the RG system as shown in Fig. 2D . These vaccines have advantages because of the feasibility of their production process and their broad targeted immune responses that include both humoral and cellular responses without the use of adjuvant [6] . Further, these advantages and many years of experience have recently resulted in some licensed vaccines from this platform, such as Ad-26 prime-modified vaccinia Ankara (MVA) boost-based ebolavirus vaccine [6] . However, there are some drawbacks that are currently associated with their use: (1) Pre-existing immunity against the used viral vector would render the developed vaccine candidate ineffective [34] ; however, this could be minimized by priming with another type of vaccine, such as a DNA vaccine [35] , or by changing the route of administration between the prime and booster doses [36] .

(2) The viral vector itself induces an immune response that subsequently interferes with future vaccines using the same vector [34] . (3) Strong reactivity in vaccinees indicate genetic toxicity induced by the use of some viral vectors; however, this can be minimized by using hybrid vectors [37]. (4) Chance for virus recombination during the production process [26] .

Currently, there are three DNA-based vaccine candidates in Phase III, whereas there is only one RNA-based vaccine candidate currently in Phase III as of April 13, 2021 [11] . The first DNA-based vaccine candidate DNA vaccines (Fig. 2E) depend on cloning the SARS-CoV-2 S gene into bacterial plasmids that contain a strong mammalian promoter, such as CMV and/or SV40, followed by large plasmid production in competent bacteria. The first proof-of-concept DNA vaccine was tested in 1990 by injecting DNA vectors expressing chloramphenicol acetyltransferase, luciferase, and beta-galactosidase into mouse skeletal muscle [38] . (1) Low immunogenicity in humans, which requires several doses of the vaccine to achieve optimum protection.

(2) Risk of carcinogenesis due to potential integration in cellular chromosomes [26] .

In contrast, RNA vaccines have shown very promising results in many pre-clinical studies with a significant success recently against COVID-19 with protection percentages of 95% and 94.5% for both US FDA EUA approved vaccine candidates from Pfizer and Moderna, respectively. Generally, the development of an mRNA vaccine is a straightforward process. Once the target antigen from the emerging pathogen is identified, the gene is sequenced, chemically synthesized with some modifications, such as the addition of specific signal peptides and transmembrane domains (Fig 2F) to target certain cellular locations, and finally cloned into plasmid DNA. The plasmid DNA is then subjected to in vitro transcription, following which the vaccine candidate is ready to be tested in pre-clinical trials. In vivo, mRNA vaccine candidates use the host cell machinery to translate the mRNA to the corresponding antigen, thereby stimulating both humoral and cellular immune responses [40] . Currently, there are two types of mRNA vaccines: (1) Traditional or conventional mRNA encoding the gene of interest (GOI) flanked by 5′ and 3′ untranslated regions (UTRs). (2) Selfamplifying mRNA mainly derived from the genome of positive-stranded RNA viruses, such as alphaviruses and flaviviruses. These are similar to conventional mRNA, but encode the viral replication machinery necessary for intracellular RNA amplification, stimulating high levels of expression of the GOI. Both traditional and self-amplifying mRNA vaccines have components similar to those in eukaryotic mRNAs, such as cap structures, a 5′ UTR followed by the ORF of the GOI, a 3′ UTR, and finally a poly A tail [41] .

Manufacturing of mRNA vaccines against targets from different viruses only requires the replacement of the sequence encoding the GOI, without affecting the final physicochemical characteristics of the RNA molecule [40] . This kind of vaccine has several advantages, as it targets both humoral and cellular immune responses, has a high safety profile, does not result in human genome integration, has no antivector immunity, and has no chance of infectious virus. However, there are some drawbacks with this platform: (1) Storage at very low temperatures (Table 1 ) is essential to preserve vaccine stability. (2) The high cost of the production process makes this kind of vaccine not ideal for low-income countries [26] . (3) A lack of systematic approaches to the identification of the main mechanisms of physicochemical degradation of the final formulated mRNA vaccine [42] . (4) This platform is relatively new and knowledge about its scale-up is currently lacking [6] . 

Previous studies, such as the Phambili Phase IIb that assessed the Ad5-HIV vaccine candidate inoculated via three vaccinations, revealed an increased risk of HIV-1 acquisition among vaccinated men [43, 44] .

Moreover, the step trial showed that men who had high serum titers for Ad-5 on entry into the trial and were uncircumcised had an elevated risk of HIV-1 acquisition during the first 18 months of follow-up [45] .

Moreover, men who were uncircumcised with high serum titers for Ad-5 and who reported unprotected insertive anal sex with a HIV-1-positive partner with HIV-1 antibody titers had the highest risk rate, suggesting the potential for an increased risk of penile acquisition of HIV-1. Additionally, a similar increased risk of HIV infection was also observed in heterosexual men who were enrolled in the Phambili study [43] .

On the basis of Ad-5-HIV vaccine candidates, there is concern that the use of SARS-CoV-2 Ad-5-based vaccine may similarly be associated with similar problems among vaccinated men. However, this hypothesis needs further investigation.

In regards to SARS-CoV-2 Ad-5-based vaccines, CanSino reported in their Phase II trial that 266 out of 508 volunteers had high pre-existing antibody titers to the Ad-5 vector. Moreover, the older volunteers exhibited a significantly lower immune response post vaccination, indicating that the SARS-CoV-2 Ad-5 vaccine may not work well in the older population [5, 6] . Another concern is that antibody titers to Ad-5 vector, which may be long-lasting, vary between different ages of the population, making use of Ad-5 vector less promising.

However, Janssen Pharmaceuticals uses Ad-26, a rarer serotype with a very low seroprevalence among different human populations, which makes this a more promising vector compared with Ad-5 [6] .

A chimpanzee adenovirus vector was developed by the University of Oxford [46] . There is very low seroprevalence in humans against this vector; however, using this vector in humans will also generate immunity against it, which may lower the efficacy of any other future vaccines developed using the same vector. Another drawback of vectored-vaccines in general is that their reactogenicity/safety profile is not high.

This may have bigger impacts for vaccinating younger people as they may experience more strong postvaccination reactions that include local symptoms such as redness, swelling and pain at the injection site, as well as systemic symptoms, such as fever and/or headache. These reactions could be a drawback in countries with large young populations such as India and many Middle Eastern countries [6] .

The then challenged with SARS-CoV [58, 59] . A recent study reported an inactivated SARS-COV-2-NDV-based vaccine candidate elicited high levels of neutralizing antibody titers in both mice and hamsters and significantly protected vaccinated animals from viral challenge [60] . A major advantage of the NDV-based vaccine platform is that the egg-based production of recombinant NDV vaccines can produce millions of doses at low-cost and under BSL2 laboratory conditions, thereby facilitating easy distribution to middle-and low-income countries. [62] . AAV is 4.6-kb single-stranded DNA genome that contains two viral genes: rep and cap. These genes can be removed and replaced with a cassette expressing a therapeutic GOI along with the necessary rep and cap genes in trans [63] . The first study that documented the capacity of AAV to induce a strong humoral and cellular response against the herpes simplex virus type 1 glycoprotein B was reported in 1997 [64] followed by an increasing number of uses of AAV for genetic vaccinations.

A recent study showed that recombinant AAV (rAAV) vectors that expressed influenza virus hemagglutinin (HA) or chimeric HA protected mice against homologous and heterologous virus challenges. Unexpectedly, immunization even with wild-type HA-induced antibodies recognizing the HA-stalk and activated [65] . A recent study reported that a thermostable SARS-CoV-2 AAV-based (AAVrh32.33 capsid of the AAV-COVID) vaccine candidate demonstrated potent immunogenicity in mice and nonhuman primates after a single injection, with the peak neutralizing antibody titers remained sustained for five months [66] .

In late 2020, several SARS-CoV-2 variants emerged as a result of mutations that occurred within the RBD of the viral S protein. The first variant was reported in UK and was termed SARS-CoV-2 B.1.1.7 (UK variant 20I/501Y.V1) or variant of concern, and has subsequently spread to other countries [67] [68] [69] . Two more variants were reported, known as B. [75] . The efficacy of mRNA vaccines, including the Pfizer/BioNTech (BNT162b2) and the Moderna (mRNA-1273) vaccines was significantly reduced against the UK and South African variants [76] . Therefore, evaluation of the currently approved vaccines should be continued and the inevitable ongoing emergence of new SARS-CoV-2 variants should be monitored.

The main challenge of responding to emerging and re-emerging viral diseases is always in low-and middleincome countries where at least half the global population live. Therefore, to combat any emerging viral disease, scale-up manufacture and globally distribution of developed vaccines should be simple and as straightforward as possible, which is an advantage of live-attenuated vaccines such as measles vaccine.

However, the situation is different for SARS-CoV-2 vaccine candidates that have been granted EUA or are currently in Phase III trials as many of these candidates are unlikely to be affordable by low-income countries and will not be cost-effective. This is very important as it is necessary to vaccinate this large human population to establish herd immunity in a short time before emergence of any virus mutant variants. SARS-CoV-2 inactivated vaccine may be of more value than live-attenuated vaccine because of more favorable safety profile, in particular for vulnerable persons. However, scale-up of this vaccine will require using large volumes of the propagated virus under BSL3 conditions and few of these laboratories and facilities are located in low-and middle-income countries. Genetic vaccines (DNA-and RNA-based vaccines) have a considerable promise; however, they have only recently been developed and their long-term safety and efficacy performance in humans requires further investigation. Moreover, scale-up of recently EAU-approved mRNA SARS-CoV-2 vaccines is expensive and needs specific logistics for shipment to low-and middle-income countries, which may not be the best option for those countries. There are many viral vectors that showed highly promising results in clinical trials, but pre-existing immunity is the main challenge for most of these. (12): 2539-2547. . The viral genome is capped at the 5′ end and polyadenylated at the 3′ ends. ORF1a and ORF1b, which occupy the two-thirds of the viral genome, encode the nonstructural proteins (nsp1 to nsp16), whereas the four structural proteins, which include the spike (S), envelope (E), membrane (M), and nucleocapsid (N), are encoded by the structural genes. In addition, accessory proteins are also encoded by the structural genes. Identified cis-acting regulatory elements are also shown at the 5′ end (SL1 to SL8), at the ORF1a/b frameshifting region (FSE), and at the 3′ untranslated region. SL: stem-loop; TRS: transcriptional regulatory sequence; FSE: frameshifting element; PK: pseudoknot; HVR: hypervariable region.

A) Live-attenuated vaccine platform in which SARS-CoV-2 is engineered by RG system to produce modified vaccine seed that is used for vaccine production in susceptible cells such as Vero E6 cells. B) Inactivated virus vaccine platform whereby SARS-CoV-2 prepared vaccine seed is propagated (scaled-up) in Vero E6 cells and is then chemically inactivated and finally formulated with a specific adjuvant. C) Protein subunit platform in which whole or part of spike protein, such as the receptor-binding domain, is expressed in mammalian or insect cells and/or yeast, purified, and finally mixed with a specific adjuvant. D) Viral vector platform (replication-deficient adenovirus) in which the adenovirus genome is modified by RG, the open reading frame (ORF) of the spike protein is cloned into adenovirus genome, and finally infectious recombinant virus is rescued in complementing cells. The final rescued virus is adenovirus expressing SARS-CoV-2 spike protein. E) Genetic vaccine (plasmid DNA vaccine) in which SARS-CoV-2 spike ORF is cloned into a plasmid DNA under a strong promoter such as that of human cytomegalovirus, and then, the plasmid is scaled-up in bacteria and finally purified. The final purified plasmid is inoculated into humans using an electroporation gun. F) Genetic vaccine (mRNA vaccine) in which SARS-CoV-2 spike mRNA is chemically synthesized and enclosed with lipid nanoparticles for efficient delivery into human cells. This figure was created with BioRender (https://bioRender.com/). 

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AN and BA conceived the project, collected data from the literature, and wrote and edited the manuscript. All authors interpreted and discussed the data and read and approved the final version of the manuscript.

None.