COVID-19 immunity and vaccines: what a pharmacist needs to know

It is possible to design attenuated virus strains rationally by mutating or deleting virulence genes. These deletion variant can often replicate to a limited extent in host cells, but have no ability to cause disease in vivo. Coronaviruses have several genes that are not required for replication and that can be deleted, leading to their attenuation in vivo. Deletion of various nonstructural proteins, and the structural E protein, has been used as a strategy to engineer vaccine strains of several zoonotic and veterinary coronaviruses [62, 63]. Currently, there are 3 attenuated SARS-CoV-2 vaccines generated by codon deoptimization under preclinical development, by Mehmet Ali Aydinlar University in Turkey, Codagenix and Serum Institute of India, and Indian Immunologicals, and Griffith University [7].

Concerns in relation to live attenuated viral vaccines. As with many live attenuated vaccines, reversion of the pheno-type has been reported [64]. Thus, to produce an attenuated strain of a pathogen for use as a vaccine, it should demonstrate its inability to revert genetically to become pathogenic. This is very challenging in the case of coronaviruses because they are known to recombine in nature [65], which means an attenuated vaccine strain could recombine with wild coronaviruses to restore a pathogenic strain.

These types have some shortcomings in relation to the immune response and safety concerns basically related to the adjuvants. Moreover, they express a wide range of their viral antigens, including surface antigens with epitope conformations that can induce conformation-dependent antibody responses [66, 67]. These types of vaccines are usually prepared by physical or chemical inactivation of the virus and have been used successfully in humans (e.g., vaccines against polio, hepatitis A, and influenza) [68, 69]. In a pandemic situation, these inactivated viruses can be generated quickly and scaled-up by the use of well-established infrastructure and methods [70].

One of the most advanced candidates to date with published preclinical results is CoronoVac (formerly PiCo-Vacc), an inactivated SARS-CoV-2 and alum-adjuvant vaccine developed by Sinovac Biotech in China [71]. Early trials were conducted in rhesus macaques and were found to protect against SARS-CoV-2, with reduced viral titers and immunopathology associated with antibodies to S protein and the nucleocapsid [71]. It was shown to have a low rate of adverse effects, which was not different from placebo [73]. Another inactivated virus candidate is BBIBP-CorV, which is developed by Sinopharm (P.R. China state-owned) and was tested in a variety of animal models, with good efficacy in nonhuman primates [72]. There were no serious adverse effects recorded, and the majority of trial participants (>95%) seroconverted with amounts of neutralizing antibody in the various trials [73]. However, i should be noted that these observations were made in short-term studies and as such should be interpreted with caution. In the Phase 3 trials, Sinopharm was shown to be 79% effective. A small-sample laboratory-based study, with data released in early February 2021 found that the vaccine provides protection against the South African variant; however, it had a weaker effect by comparison with the protection it provides against the original virus. The vaccine is authorized in China, the United Arab Emirates, Bahrain, Pakistan, Egypt, Morocco, and Hungary, and is also in wide use in Serbia. The other vaccine from China, Coronovac, from Sinovac Biotech, is made of a purified, inactivated coronavirus and it uses an adjuvant from Dynavax, which the California-based company uses in its own hepatitis B vaccine. It was approved for emergency use in China, Mexico, Indonesia, Thailand, Brazil, Turkey, and Chile, among other countries. There are contradicting reports on the efficacy of the vaccine. A trial in Brazil showed the vaccine to prevent hospitalizations and deaths among all participants, was 83.7% effective in preventing cases that would require medical treatment, and was 50.7% effective in preventing infections. A small clinical trial in Turkey showed the vaccine to be 91.25% effective in blocking infections [74].

Concerns in relation inactivated viral vaccines. To obtain the required immune response, inactivated viral vaccines usually require the addition of adjuvant and repeated administration. Alum is one of these widely-used adjuvants; however, its presence makes the vaccine unsuitable for respiratory mucosal delivery [75]. It is noteworthy that protection mediated by intramuscular vaccination with CoronoVac or BBIBP-CorV indicates some mucosal immunity, possibly by the transport of systemic antibodies to the lungs; however, the durability of such immunity remains to be further studied as SARS-CoV-2 challenge was performed 1–4 weeks after vaccination (Table 1) [7, 71, 72]. Inactivated viral vaccines are poor inducers of cytotoxic CD8⁺ T cells, which are likely to be required for an effective COVID-19 vaccine. Furthermore, studies have reported vaccine-related enhancement of disease, likely involving a T_h2 cell response and lung eosinophilia, which may be worsened in aged hosts.

Because alum is known to drive T_h2 cell-mediated immune the use of T_h1 cell-skewing modified alum or other adjuvants may improve safety [84, 85].

These are based on a replication-deficient viral backbone or an attenuated replication competent viral backbone that is bioengineered to express antigens derived from the target pathogen. This platform has been widely investigated and has a well-established track record for infectious diseases. This is because of its genetic adaptability, safety, and ability to induce strong T-cell responses without the need for an adjuvant [86, 87]. There are some viral vectors, such as human serotype 5 adenovirus (Ad5) and chimpanzee adenovirus (ChAd), which require to be administered only once for protection and have natural tropism for the respiratory mucosa, which makes them convenient for respiratory mucosal vaccination [45]. Therefore, recombinant viral vectors are considered to be one of the most common platforms for COVID-19 vaccine development. At present, there are 4 vaccine candidates in clinical trials and 38 under preclinical development (Table 1) [7, 41]. There are two viral platforms in this regard, namely, the non-replicating vaccines, which are mostly based on Ad5 or modified vaccinia virus Ankara (MVA) that mostly expresses the S protein or receptor-binding domain (RBD) of SARS-CoV-2, and replication-competent viral vectors that are mainly based on the vaccine strains of other human pathogens or veterinary pathogens. Thus, it will be important to consider whether humans have preexisting immunity against the viral backbone, which can impair the ability of such vaccines to engage the immune system. To avoid this evasion, the use of viral backbones such as ChAd, for which humans have little to no preexisting immunity, is recommended [45].

CanSino Biologics, the Chinese vaccine company, developed Ad5-nCOV, which is designed to induce neutralizing antibodies to SARS-CoV-2 S protein after intramuscular injection. Three levels of doses of vaccine entered the Phase 1 and 2 clinical trials without providing preclinical data. The highest dose generated unacceptable toxicity and was dropped from the Phase 2 study. The smaller doses induced S protein-specific neutralizing antibodies in only half of the vaccine recipients. Shortcomings. The vaccine induces both antibody and T-cell responses; however, its potency is reduced by preexisting immunity to Ad5, especially in the elderly [76]. These observations from Phase 1 are confirmed by the Phase 2 study. Although the efficacy of this strategy is questionable, the vaccine is undergoing more advanced trials in China and Canada. Concerns have been expressed that use of an Ad5 vector for immunization against SARS-CoV-2 could increase the risk of HIV-1 acquisition by those who receive such a vaccine [88].

Sputnik V is one of the vaccines in this category, which received approval for emergency use from Russia in August 2020, before the Phase 3 trials, which are now being performed. Moreover, around 40 countries, including Belarus, Iran, Mexico Serbia, Argentina, Algeria, Hungary, Slovakia, and Nicaragua have approved it for emergency use. It is noteworthy that the Gamaleya Institute of Epidemiology and Microbiology, a vaccine developer of the Ministry of Health of the Russian Federation, has signed an agreement in late December 2020 with AstraZeneca to test a combination of the vaccine developed by AstraZeneca and Oxford University and the Sputnik V component. The human trials began in mid-February in Azerbaijan and some Middle Eastern countries [74].

Johnson & Johnson (Janssen Pharmaceuticals) is developing another human adenovirus-based COVID-19 vaccine, known as Ad26-S, which has seroprevalence for Ad26 in humans (Table 1) [7, 77]. The vaccine was 66% effective overall across several Phase 3 studies worldwide testing a single-dose regimen; however, the efficacy was 57% in South Africa where an extremely transmissible variant is spreading. The efficacy of the vaccine in preventing severe disease was 85%, and gave complete protection against hospitalization and death [74]. Shortcomings. Ad26 is inherently less immunogenic than Ad5; thus, to ensure effective immunity, repeated vaccination is needed [89,90,91]. However, when Ad26-vectored COVID-19 vaccine (Ad26.CoV-2.S) was administered parenterally, it showed great protection in a nonhuman primate [92].

Oxford University, UK, and Astra Zeneca are developing one of the most promising and clinically advanced COVID-19 vaccines, which is ChAdOx1 nCoV-19 also known as AZD-1222, which recently entered Phase 3 (Table 1) [7]. A study of Phases 1 and 2 of this vaccine reported its safety with production of potent neutralizing antibody in addition to T-cell responses following a single parenteral injection, which are boosted additionally by a second homologous vaccination [93]. A recent report revealed that some European countries temporarily suspended use of the Oxford-AstraZeneca vaccine as a precautionary move after reports of blood clots [94]; however, the UK's regulatory body and the European Medicines Agency (EMA) stated that there is no indication that the vaccination is associated with thromboembolic events.

Shortcomings. Despite its intramuscular delivery, ChAdOx1 nCoV-19 reduced SARS-CoV-2 viral load in the lungs and stopped pneumonia in rhesus macaques, but it did not reduce viral loads in the upper respiratory tract [95]. Moreover, it remains unclear from trials as to what extent both the CD4⁺ and CD8⁺ T cell subsets were activated. During trials, volunteer participants have experienced mild to moderate injection site pain in addition to mild to severe systemic adverse reactions, which included fatigue chills, malaise, and headache; which was mostly on Day 1, and these symptoms were relieved by paracetamol [93].

Merck and other groups are developing VSV-S, which is a replication-competent COVID-19 vaccine [7, 96]. Merck's vaccine is based on the licensure of its effective VSV-Ebola vaccine, which induces neutralizing antibodies and cellular immunity against Ebola virus surface glycoprotein [96]. Humans have no preexisting immunity to the veterinary virus VSV, but its suitability for respiratory mucosal vaccination is uncertain and its cloning capacity is limited. A single parenteral vaccination with a VSV vector expressing S protein provides protection against SARS-CoV-2 in both mouse and hamster models [97, 98]. However, the development of this vaccine was discontinued because findings from Phase 1 clinical studies showed that although it was well tolerated, the immune responses were inferior to those reported for other COVID-19 vaccines and those seen following natural infection.

Subunit vaccines initially induce both CD4⁺ TH cell and antibody responses. This is because, most of these vaccines contain full-length SARS-CoV-2 S protein or portions of it, and this is comparable to the majority of SARS, which had differing levels of efficacy [99,100,101]. One of the advantages of the subunit vaccines is that they can be designed to focus the immune response toward neutralizing epitopes, in a way that would prevent the production of nonneutralizing antibodies that may promote ADE of disease [102]. Nevertheless, recombinant S proteins in subunit vaccines could possess improper epitope conformation unless they are produced in mammalian cells [103]. Furthermore, proteins or peptides alone are not immunogenic enough and usually need adjuvants in addition to repeated administration. This platform is generally unsuitable for respiratory mucosal vaccination due to the use of unmodified alum as an adjuvant, which will tilt the immune response toward T_h2 cell-like responses, which is undesirable for host defense against SARS-CoV-2 and may have a role in ADE of disease [76, 104, 105].

At present, there are about 28 COVID-19 subunit vaccines in clinical trials and 70 other candidates under preclinical development as reported by WHO [7]. GlaxoSmithKline and Novavax are developing COVID-19 subunit vaccines using different adjuvants currently entering Phase 3 trials (Table 1) [7]. GSK announced that it could seek emergency authorization in the United States in April 2021, when the results from its 30,000-person Phase 3 trial in the United States and Mexico is completed. According to the company, the vaccine was 89% effective against a variant first found in the United Kingdom, but only 49% effective against the South African strain [74].

Shortcomings. There remain some concerns in relation to high levels of S-specific neutralizing antibodies. Other vaccines using recombinant trimeric SARS-CoV-2 spike protein (S) are developed by Clover Biopharmaceuticals in China, University of Queensland in Australia, and Sanofi/GSK collaboration (France/UK) (Table 1). The companies are currently performing Phase 1 and 2 trials in 440 healthy adults at 11 sites in the United States. Preliminary results revealed insufficient immune response in older adults, which was attributed to insufficient concentration of the antigen in the formulation of the vaccine. The companies are now planning a Phase 2b trial that will lead to a global Phase 3 trial in April 2021 at the earliest [74].

There are a number of commercial vaccines based on VLPs, including hepatitis B and human papillomavirus vaccines [106]. As for the enveloped coronaviruses, VLPs are produced when the viral proteins are expressed in eukaryotic cells [107, 108]. Active budding will then occur from the VLP producer cells, which are structurally identical to the infectious virus, but lack the viral genome and thus are noninfectious. S protein on the surface of VLPs enables them to bind and enter ACE2⁺ cells in the same manner as the parent virus [109]. The array of S protein on the VLP surface crosslinks B-cell receptors and directly activates them; however, VLPs usually require an adjuvant and repeated administration [106].

To date there are only 3 VLP-based COVID-19 vaccine in clinical trials, and 17 under preclinical development [7]. The vaccines are either produced in vivo from a viral vector that expresses the VLP components (e.g., GeoVax vaccine) or in vitro from producer cells. It is noteworthy to mention that the Canadian company, Medicago, produces its SARS-CoV-2 VLPs from genetically engineered plants and their preliminary findings suggest possible efficacy in inducing neutralizing antibodies in mice [110].

The major advantage of mRNA vaccines is that they are non-infectious and are synthesized by in vitro transcription, free of microbial molecules. The antigen-encoding mRNA would be complexed with a carrier, such as lipid nanoparticles, and then can be efficiently delivered in vivo into the cytoplasm of host cells for protein translation and post-translational modifications [111, 112].

These make mRNA vaccines more advantageous than the other vaccine types, including live attenuated, inactivated, subunit vaccines, and recombinant viral-vectored vaccines in that they are safer and more efficacious, which support their rapid and inexpensive production and repeated vaccination [111]. Shortcomings. In spite of the promising effect of mRNA-based COVID-19 vaccines in early clinical testing, ambiguity regarding their protective efficacy in humans and their suitability for respiratory mucosal delivery remain.

At the end of March 2021 there were 12 mRNA-based COVID-19 vaccines and 104 DNA-based COVID-19 vaccines in clinical trials, and 40 such vaccines (24 mRNA-based and 16 DNA-based vaccines) under preclinical development [7]. Moderna, a biotech company in the USA produced mRNA-1273, which encodes a prefusion stabilized SARS-CoV-2 S protein encapsulated in lipid nanoparticles. This vaccine is currently authorized and recommended to prevent COVID-19 [78]. It entered clinical testing even before the release of preclinical data (Table 1) [79]. Data from a Phase 1 clinical trial suggest that low and medium doses of 2 repeated parenteral injections induce strong S protein-specific antibody responses and a primarily CD4⁺ T-cell response in most trial participants and are in general safe [80].

Pfizer and BioNTech are also developing an mRNA-lipid nanoparticle vaccine encoding the S protein RBD in humans. A simultaneous Phase 1 clinical trial that compared BNT162b1 with a different Pfizer and BioNTech candidate vaccine, BNT162b2, showed that the BNT162b2, which encodes for 2P-stabilized spike protein rather than trimerized RBD, but uses mRNA in a similar stabilizing formulation, caused fewer systemic reactions than BNT162b1 [81]. The Pfizer vaccine is currently authorized and recommended to prevent COVID-19 (Table 1). It developed robust S protein-specific antibody and CD4⁺ and CD8⁺ T-cell responses following two repeated parenteral injections [82, 83]. However, one of the important shortcomings of Pfizer's vaccine is that it must be kept at −70 °C, and will only remain effective for 24 h at refrigerated temperatures between 2 and 8 °C.

Plasmid DNA vaccines have several similarities to mRNA vaccines, including safety and ease of production [113]. However, they differ from mRNA vaccines in that they are poorly immunogenic, requiring addition of adjuvants, and they need to be administered in multiple doses.

Inovio Pharmaceuticals, a biotech company in the United States, is developing a plasmid DNA vaccine expressing SARS-CoV-2 S protein (INO-4800). However, the immunogenicity of this vaccine was studied in mice and guinea pigs, but did not provide any data relating to protection against challenge [114]. Two repeated injections of an S protein-expressing plasmid DNA vaccine resulted in robust protective immunity in rhesus macaques [115]. Symvivo (Canada) is developing a BacTRL-Spike using DNA plasmid expressing trimeric S and a hybrid transporter protein within Bifidobacterium longum to be used by oral ingestion of a probiotic capsule and delivered to colonic epithelial cells to prime an immune response via colonic lymphoid tissues. Although probiotics are used extensively worldwide, DNA vaccines are not currently on the market for use in humans and this strategy in particular is not yet validated (Table 1).