A comprehensive insight on the challenges for COVID-19 vaccine: A lesson learnt from other viral vaccines

2.1. Challenges for HIV vaccine development

A total of 861 clinical trials have been registered for the HIV vaccine so far, out of which 708 were interventional trials. Of these, 603 clinical trials have been completed successfully, but 48 clinical trials failed due to suspension, withdrawal, or termination. Although the failure rate is minimal, there is currently no approved vaccine for HIV. The following are important challenges that need to be overcome for the development of an HIV vaccine.

• 1)
  HIV - a chronic disease not acute

The current preventive vaccine strategies for HIV focus on targeting the virus and immune response produced during the late stage of the disease, which is not an effective approach. This is because a highly mutated strain of HIV is identified during the chronic disease stage, which results in neutralizing antibodies with unique characteristics. The current strategies are focused on developing immunogens that stimulate these broadly neutralizing antibodies, which are only conserved to the individual infected host and the result of long-term evolutionary within-host pressure. To design a preventive HIV vaccine, it is critical to understand the early stage interaction of the HIV viral variables and the immune response [14].

• 2)
  HIV as Quasispecies

Quasispecies are recombinant and mutant genomes of viruses that are closely related and undergo progressive genetic variations, forming a unit that can be selected [15]. HIV also behaves as a quasispecies [16]. There is significant genetic diversity during the initial phase of HIV infection in the host and in the virus during the terminal stage. During transmission, HIV undergoes an evolutionary bottleneck event resulting in a reduction of fitness and mutations. However, there are conserved sequences in the transmitted/founder (T/F) viruses that are resistant to evolutionary mucosal bottleneck events and are the primary targets for vaccine research [17]. This means that T/F virus variants remain homogenous just before and just after transmission and can be targeted using neutralizing antibodies towards the conserved sequences [18].

• 3)
  Ignorance of immunological principles

The interaction of HIV epitopes was predicted by structure-based reverse vaccinology with neutralizing antibodies that produce a similar immune response as produced against the vaccine antigen. Efforts were made to identify the pathways of affinity maturation that occur during the production of broadly neutralizing antibodies [19]. However, this approach completely ignores the significant complex interactions of the HIV virus and the host immune system. The polyspecific nature of antibodies, B-cell receptors, and the degeneracy of the immune system have also not been considered [20]. Errors during reverse transcription lead to the diversification of the HIV-1 M group into various recombinant forms. Within a particular clade, there is a difference of nearly 20% in Env amino acid sequences, and the difference is more than 35% between the various clades. Therefore, an immunogen for a vaccine will have to deal with a very high level of viral variety [9].

• 4)
  Glycosylation of HIV envelope

Developing vaccines that induce broad-spectrum neutralizing antibodies (bnAbs) against different HIV strains is an important strategy [21]. However, a major challenge is to optimize the immunogen to present the precise epitopes identified by bnAbs [22]. Another obstacle is the high glycosylation of the HIV envelope. The region where monoclonal antibodies (mAbs) bind is conserved and covered with glycans derived from the host, which have low immunogenicity and complexity, making it difficult for mAbs to recognize and interact with them [23].

2.2. Challenges of zika virus vaccine

There are currently 30 registered clinical trials for the Zika vaccine, 20 of which are interventional studies. Of these, 16 have already been completed, but none of the vaccines have been approved for Zika virus. The following are the key challenges in the development of a Zika vaccine.

• 1)
  Antigen Identification

Guillain-Barre syndrome (GBS) occurs in approximately one out of every 4000 to 5000 Zika-infected patients. It is important to identify the antigen that attacks the peripheral nerves in these patients [25].

• 2)
  Cross-reactivity

Zika virus vaccine-induced antibodies can significantly increase dengue viremia and other flavivirus infections due to the challenge of cross-reactivity among antibodies. The severity of Zika virus infection is also influenced by cross-reactive T-cell responses from earlier dengue infections. Therefore, the challenge is to develop a Zika virus vaccine that can produce more specific viral antibodies. Another important challenge is to identify more specific epitopes that can generate more specific neutralizing antibodies [26].

• 3)
  Human Infection models

Testing the efficacy of Zika virus vaccinations in both pregnant and non-pregnant populations is difficult due to the decline in active cases of Zika virus infection. This has created an unmet need for developing human infection models that can accelerate vaccine research and other countermeasures [27].

• 4)
  Sexual transmission risk

The Zika virus can persist in the male urogenital tract for 3–8 months, indicating a risk of sexual transmission of the disease. Additionally, there is a potential risk of infection during pregnancy [28].

2.3. Challenges in influenza vaccine development program

There are currently 1703 clinical trials registered for the influenza vaccine, of which 1412 are related to vaccine intervention and the rest are observational or non-interventional. Only 61 clinical trials have been suspended, withdrawn, or terminated so far. There are 26 FDA-approved vaccines for influenza. Although there were a few challenges in the development of the influenza vaccine, addressing those challenges can certainly be helpful for the development of other vaccines.

• 1)
  Availability issue of embryonated eggs

The production of inactivated (IIV) and attenuated (LAIV) Influenza vaccines primarily relies on embryonated eggs. However, during a pandemic or a rise in the pathogenic strain against poultry, their use may be limited. Nevertheless, the emergence of several new types of vaccines that are now developed in mammalian cell lines, recombinant HA vaccine, and DNA vaccine provide an alternative to this challenge.

• 2)
  Time lag in vaccine production

The production of Influenza vaccines targeting specific strains is a time-consuming process that involves various stages. These stages include global surveillance of circulating strains, antigenic characterization, prediction of the upcoming season, production of vaccine seed virus, amplification, inactivation, purification, and dispensing [29]. During the 2009 pandemic of the Influenza virus, there was a challenge in producing and distributing a newly evolved viral vaccine in a short period of time [30].

• 3)
  Revaccination

There is a need for revaccination against Influenza due to a decrease in viral strain-specific antibodies and the phenomenon of antigenic drift. Strategies such as the use of computational HA sequence analysis, generation of specific viral strain antibodies, and immunization with conserved proteins targeting T-cell responses may be helpful in addressing this issue [31].

• 4)
  Novel virus

The current vaccines available against seasonal flu are effective. However, the emergence of novel strains or antigenic drift poses a great challenge to the current strategies. Four previous novel strains of Influenza (in 1918, 1957, 1968, and 2009) caused pandemics [32].

• 5)
  Immunosenescence

The gradual decrease in immune response with increasing age is known as “immunosenescence.” The current standard Influenza vaccines are found to be less effective in elderly populations, who are more susceptible to Influenza and its complications. Increasing the antigenic dose in the vaccine and adding an adjuvant can be a useful approach [33].

• 6)
  Adverse effect

The preclinical studies in animals showed that vaccines were associated with enhanced respiratory illness in pigs but this has not been reported in humans [34]. The AS03-adjuvanted Influenza A (H1N1) pdm09 vaccine has been reported to induce sleep disorder (narcolepsy) in Northern Europe [35].

• 7)
  Underestimation of the disease burden

The estimated Influenza-related mortality is between 291,234 and 645,832 [35]. Elderly individuals, children, and pregnant women are more susceptible to Influenza-related critical illness [36]. In most countries, Influenza vaccination is not mandatory [37], but it is essential to educate the general population about the importance of receiving the vaccine.

2.4. Challenges in dengue vaccines development

Almost 133 clinical trials have been registered, out of which 115 studies are interventional. In Mexico, in December 2015, Dengvaxia (CYD-TDV) became the first licensed Dengue vaccine with an age restriction of 9–45 years for individuals residing in endemic regions. CYD-TDV is a three-dose series of a live recombinant tetravalent Dengue vaccine administered according to a 0/6/12-month schedule, and it has been developed by Sanofi Pasteur [38]. There are several challenges in the path of developing a Dengue vaccine, which are listed below.

• 1)
  Antibody-Dependent Enhancement (ADE)

The Dengue vaccine is designed to be effective against all four serotypes (Type 1, 2, 3, 4). The primary role of antibodies is to neutralize the antigen, but sometimes they can enhance the entry of the antigen, a phenomenon known as antibody-dependent enhancement (ADE) [39]. ADE occurs due to the cross-reactivity of antibodies produced during some other heterologous infection, resulting in increased viral entry by escaping the endosomal-dependent pathway [40]. This phenomenon has been observed in HIV, Ebola, and SARS-CoV-2-related infections.

The first theory suggests that the Fc region of antibodies binds to the FcR receptor present on immune cells like macrophages, monocytes, and B-cells, producing an immune synapse that enhances the entry of the virus [41]. The second theory proposes that ADE occurs via the complement-dependent pathway, where virus-C3 fragments bind with the gp120 protein in HIV, and this complex binds to receptors present on the cell, thereby increasing viral entry [43]. The third theory suggests that neutralizing antibodies bind to the natural receptor of the virus (spike receptor) and cause ADE [44]. In cases where the natural receptors are not present, viruses can also enter via FcR-mediated ADE, as observed in foot and mouth diseases. ADE can also occur when there is a poor response to vaccination [45]. The majority of ADE cases have been reported in people who were previously infected with a different serotype of Dengue or another flavivirus.

• 2)
  Cross-reactivity

There is a structural similarity between DENV and other flaviviruses, such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), and yellow fever virus (YFV). The envelope (E) protein in these flaviviruses is highly conserved and is the main target of the immune response. The DENV E protein is almost 50% similar to that of ZIKV. This close structural homology between these viruses causes cross-reactivity, which can provide protection or pathogenic enhancement for other infections [46].

In 2016, a ZIKV outbreak occurred in regions of Brazil and Mexico that were also endemic for DENV [47]. Both humoral and cellular immune responses contribute to the pathology, but humoral immunity is the primary cause of ADE.

• 3)
  Lack of adequate animal model

One of the most challenging aspects of vaccine research and development in the early stages is the lack of a suitable animal disease model. Experimental animal models of infection play a significant role in understanding disease pathology, the mechanisms of action of different therapeutic strategies, and the safety and efficacy profiles of vaccines [48]. To study the mechanism, effectiveness, and immunogenicity of the Dengue vaccine, a suitable and validated animal model is urgently needed.

Non-human primates develop Dengue-like infections that result in acute neutralizing antibodies and high viremia, but they lack hemorrhage, fever, and shock [49]. A recent study observed promising clinical symptoms of Dengue virus infection in the tree shrew model, but further validation is necessary [50].

2.5. Challenges in the development of ebola vaccines

There are 78 clinical trials registered for Ebola, out of which 71 are interventional. None of the trials have been suspended, withdrawn, or terminated. ERVEBO, the Ebola Zaire Vaccine Live developed by Merck Sharp & Dohme Corp, is designed for individuals aged 18 years and older for the prevention of disease caused by Zaire ebolavirus [51]. However, there are certain challenges in the development of Ebola vaccines.

• 1)
  Presence of virus in bodily fluids

Previous studies have observed the prolonged presence of the Ebola virus in bodily fluids, such as semen and breast milk. This poses an important concern as the risk of sexual transmission of the disease and transmission during feeding of neonates is high [52].

Vaccine virus has been detected in urine, saliva, and fluid from skin vesicles, and there is a possibility of vaccine virus transmission.

• 2)
  Duration of the immunity

The duration of immunity developed through the use of Ebola vaccines is currently unknown, and there is a need for long-term studies to evaluate the persistence of immune response [53].

• 3)
  Limitation of vaccine effectiveness

While the approved vaccine ERVEBO can provide protection against Ebola, it is not fully effective, and adherence to infection control practices is still necessary. Additionally, the vaccine has not been evaluated in immunocompromised populations.

2.6. Challenges in the development of vaccines for coronavirus (SARS, MERS, and COVID-19)

There are 46 clinical trials registered for the SARS vaccine and 9 clinical trials registered for the MERS vaccine, but none of the approved vaccines are available for these diseases. For COVID-19, 1246 clinical trials were registered in a short amount of time, out of which 701 are vaccine interventions. Although several COVID-19 vaccines have been approved, challenges remain in the path of coronavirus vaccine development. The challenges faced in developing vaccines for HIV, Ebola, Dengue, and Zika are also applicable to coronavirus vaccine development. Mutation poses a significant challenge that the COVID-19 vaccine development program is still facing.

2.6.1. Quasispecies

Under the effect of host and virus mechanisms, RNA viruses undergo genetic variability referred to as quasispecies. One study conducted a deep analysis using a Next-generation sequencing platform to evaluate the phenomenon of quasispecies in COVID-19 patients with mild to severe pathologies. The study revealed that mutations induced by nsp 12 mainly occurred in patients with high viral loads and long-term infections. These genetic variations are the main source of new SARS-COV-2 variants [54].

Another study highlighted several single nucleotide variations (SNVs) affecting 70% of the viral genome with varying densities, revealing the colocalization of strain-specific and intra-host SNVs. The locations of genomic rearrangement are in the poly-A/poly-U regions in ORF1ab and spike S gene. The identified SNVs and rearrangements point out the genetically diverse quasispecies' intra-patient capacity, which may arise rapidly during the outbreak and allow the virus to evade the immune system [55] (refer to Fig. 1).

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Fig. 1

Common Challenges faced by different Viral Vaccines programs & COVID-19.

2.6.7. Mutation in novel coronavirus

Since 2003, the outbreak of Coronaviruses has posed a significant challenge to global health. SARS-CoV-2 is an RNA virus, and studies show that RNA viruses have higher mutation rates than most DNA viruses. A mutation in the sequence can cause changes in amino acids, and mutations in the surface (S) protein of viruses can significantly alter viral interactions with receptors and functions with broad-spectrum neutralizing antibodies [63]. For example, the A226V mutation in the Chikungunya virus E1 protein strengthened the virus's adaptability in the vector Aedes albopictus, ultimately leading to increased transmissibility and infectivity [64]. The A82V mutation in the GP protein of the Ebola virus leads to enhanced infectivity, transmissibility, and mortality [27]. The mutation in four amino acids in highly pathogenic Avian Influenza H5N1 reported enhanced transmissibility [65], whereas the A143 V/R148K (combined amino acid alteration) mutation of hemagglutinin in H7N9 increases resistance towards broadly neutralizing antibodies by ten times [66].

The SARS-CoV-2 spike (S) protein is highly glycosylated and is undergoing frequent mutations. These mutations may have significant biological effects that require investigation. Most of the variant changes in the receptor-binding domain due to amino acid alterations are less infectious, but some variants, such as V483A, A475V, F490L, and L452R, are resistant and can escape from neutralizing antibodies [67]. A SARS-CoV-2 variation expressing a D614G mutation in the S protein has affected the globe, implying a fitness advantage for the mutant. The infectivity was significantly higher in the case of D614G mutated variants as well as other variants containing both D614G and another amino acid alteration. The mutation led to a high infectivity rate, increased S-protein incorporation into the virion, and increased ACE2 binding affinity [68].

The UK consortium dataset on COVID-19 genomics presented a substitution of an amino acid in the receptor-binding domain (RBD) of the SARS-CoV-2 B.1.1.7 mutant strain (N501Y mutation). In-silico studies depicted that the N501Y mutation caused a more significant number of interactions by increasing electrostatic interactions due to the formation of hydrogen bonds between SARS-CoV-2-T500 and ACE-2 D355 near the mutation site [69]. The Delta variant (SARS-CoV-2 variant: B.1.617.2) appeared during the second wave of infections and has become widespread globally, although it is constantly evolving [70]. The distribution of the Delta variant was comparable to that of other variants of concern (VOCs) [70]. The D950 N mutation, like the D614G mutation, is localized to the trimer interface, suggesting that it may play a role in the control of spike protein dynamics [70]. It has a 156–157 deletion and a G158R mutation [70]. The ACE2 patch has just one mutation, at position 501, which improves the affinity of the receptor-binding domain (RBD) for ACE2 and is also implicated in antibody escape [70].

The emergence of the most mutated SARS-CoV-2 variant, B.1.1.529 (named Omicron), has sparked panic throughout the world and was first reported from South Africa on November 24, 2021 [71]. The spike protein of this variant has undergone 50 mutations and has about 30 alterations, including 13 mutations in the RBD in the S gene [71]. At both the ACE2 and antibody binding locations, 5 new mutations were discovered, indicating an improved immune escape capacity [71]. The Omicron variant has a higher antibody escape, transmissibility, and viral binding affinity [72]. Silent mutations in the RNA binding domain of a gene coding for the N-protein of SARS-CoV-2 have been reported in the Iranian strain compared to the Chinese strain. The silent mutation results in the replacement of thymine with cytosine so that the TTG in the Chinese strain becomes CTG in the Iranian strain, which codes for leucine. The missense mutation was reported during thymine DNA glycosylase action in humans and the base excision repair mechanism, resulting in the conversion of another thymine to cytosine. The codon after mutation is CCG, which codes for proline. This results in an enhanced affinity of the N-protein (through an RNA binding domain) to viral RNA, leading to an increase in transcription rate, pathogenicity, and transmission [73].

According to the US Centers for Disease Control and Prevention (CDC), vaccinations authorized by the US Food and Drug Administration (FDA) are “polyclonal,” meaning they develop antibodies that target several regions of the spike protein. This suggests that the virus would need numerous mutations to accumulate in the spike protein to avoid neutralizing antibodies and evade natural infection or vaccine-induced immunity. According to WHO [74], laboratory investigations are currently underway to investigate the biological features of these mutant viruses and how these variants may impact the efficiency of vaccines. There is currently insufficient data to identify whether such polymorphisms are associated with any significant changes in the severity of clinical aspects of disease development, general antibody response, or vaccination efficacy. The common challenges of all vaccine development programs are summarized in Fig. 1.

WHO - EUA approved vaccines (as shown in Fig. 2).

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Fig. 2

WHO - EUA approved vaccines.

2.7. COVD-19 mRNA vaccine (COMIRNATY)

The BioNTech-Pfizer COVID-19 vaccine is a lipid-based nanoparticle, nucleoside-modified mRNA vaccine that expresses the viral perfused spike glycoprotein [75]. It was approved for Emergency Use Authorization (EUA) by the World Health Organization (WHO) on December 31st, 2020 [76]. It is authorized in 118 countries, and 49 clinical trials in 23 countries are still ongoing [77]. The BioNTech-Pfizer COVID-19 vaccine provides 95% protection against COVID-19 after two doses [75]. After receiving the vaccine, around 0.2% of adverse events were reported to the Vaccine Adverse Event Reporting System (VAERS) in the United States [75]. The most common side effects associated with the vaccination were fatigue, headache, muscular discomfort, joint pain, chills, nausea, and vomiting [78].

2.8. COVID-19 (ChAdOx1-S) vaccine (VAXZEVRIA/COVISHIELD)

Covishield is a recombinant, replication-deficient chimp adenovirus containing the S protein antigen of SARS-CoV-2 [79]. It was approved for Emergency Use Authorization (EUA) by the World Health Organization (WHO) on February 15th, 2021 [76]. It has been authorized for EUA in 133 countries and 52 clinical trials in 23 countries are currently ongoing [77]. The efficacy of the Oxford-AstraZeneca vaccine is 70.4% after two doses have been administered [80].

2.9. COVID-19 vaccine (Ad26.COV2·S)

The Ad26.COV2·S vaccine is a recombinant adenoviral vector that carries the sequence for the spike protein (S) of the SARS-CoV-2 virus [80]. The WHO granted Emergency Use Authorization on March 12, 2021 [76]. It is approved in 91 countries, and 16 clinical trials have already been registered in 18 countries [77]. After one dose, the Johnson & Johnson COVID-19 vaccine has an efficacy rate of 85% [81].

2.10. COVID-19 mRNA vaccine (SPIKEVAX)

Spikevax is based on a lipid nanoparticle-based nucleoside-modified mRNA vaccine that expresses the stabilized perfusion spike (S) glycoprotein of SARS-CoV-2 [82]. The WHO granted Emergency Use Authorization on April 30, 2021 [76]. It is authorized in 81 countries, and 35 clinical trials in nine countries are ongoing [77]. The efficacy of the Moderna vaccine for COVID-19 has been found to be 94.1% effective against COVID-19 after taking two doses in clinical trials [83]. The most prevalent side effects of the Moderna vaccination were fatigue, headache, muscular discomfort, joint pain, chills, nausea, and vomiting [84].

2.11. Inactivated COVID-19 (Vero Cell) vaccine (COVID-19 vaccine BIBP)

The COVID-19 (Vero Cell) vaccine, inactivated with β-propiolactone, is generated in Vero Cells [85]. The WHO granted Emergency Use Authorization on May 7, 2021 [76]. It has been approved in 89 countries, and 22 clinical trials have been registered in 11 countries [77]. The COVID-19 Vaccine BIBP has an effectiveness rate of 79% after the second dose [86].

2.12. COVID-19 (Vero Cell) vaccine (CoronaVac)

CoronaVac is a COVID-19 vaccine based on an inactivated virus [87]. The WHO granted Emergency Use Authorization to this vaccine on June 1, 2021 [76]. It has been approved in 48 countries, and 27 clinical trials have been registered in eight countries [77]. The efficacy of the Sinovac-Coronavac vaccine is 63.09% [88].

2.13. Covaxin (BBV152) vaccine

The Covaxin (BBV152) vaccine is based on an inactivated virus [89]. The Central Drugs and Standards Committee (CDSCO) in India authorized it for Emergency Use Authorization on January 3, 2021 [90], and it was also approved for EUA by the WHO on December 3, 2021 [76]. It has been approved in 12 countries, and seven clinical trials have been registered in seven countries [77]. The Covaxin COVID-19 vaccine has an effectiveness rate of 78% after the second dose [91].

2.14. COVOVAX vaccine

COVOVAX is a COVID-19 vaccine that is adjuvanted with Matrix-M1 and based on the S protein of SARS-CoV-2 [92]. Matrix-M1 contains Fraction A and Fraction C of Quillaja Saponaria Molina extract [92]. WHO approved it for Emergency Use Authorization on December 17, 2021 [76]. It is approved in 3 countries and registered in 2 clinical trials in one country [77]. The efficacy of the COVOVAX COVID-19 vaccine has been found to be 90.4% effective in protecting against COVID-19 after taking two doses [93].

2.15. Nuvaxovid vaccine

Nuvaxovid is a COVID-19 vaccine that uses Matrix-M as an adjuvant and is based on the SARS-CoV-2 spike protein [94]. It was granted Emergency Use Authorization (EUA) by the WHO on December 20th, 2021 [76]. Nuvaxovid is approved in 34 countries and registered in 11 clinical trials in 11 countries [77]. The vaccine has been found to have an efficacy of 90.4% against COVID-19 after taking two doses [93].