In comparison to other vaccine candidates, inactivated EV-A71 vaccines are the only vaccines entering the market. China's Food and Drug Administration (FDA) has issued drug certificates and production licenses for EV-A71 inactivated vaccines from 3 companies, namely, Sinovac, Vigoo, and the Chinese Academy of Medical Sciences (CAMS), all of which are based on C4 subgenotype—the most common genotype in China, although with different virus strain and variation in manufacturing process (Mao et al. 2016) (Table 1). Following successful phase I-III clinical trials, a recent large-scale cohort phase IV study of licensed inactivated EV-A71 vaccine revealed an overall protection effectiveness of 89.7% against EV-A71 infection along with a 4.58% rate of reported adverse reaction (Guan et al. 2019). However, both the Sinovac and CAMS EV-A71 vaccines were ineffective for CVA16-associated HFMD, unraveling their genotype specificity (Li et al. 2016; Li R et al. 2014). Besides Chinese mainland, inactivated EV-A71 vaccines were also developed in Taiwan region and Singapore, targeting B3 and B4 subgenotypes respectively. The EV-A71 developed in the Taiwan region has been evaluated in a phase II clinical trials involving a total of 365 infants or children aging from 2 months to 11 years, achieving a seroprotection (neutralization titer ≥ 1:32) lasting for 2 years in most participants without reported serious adverse events (SAEs) (ClinicalTrials.gov number, NCT02200237). In addition, a cross-reaction was observed against other EV-A71 strain genotypes, including B5, C4a, C4b, and C5 (Huang et al. 2019). A phase III clinical trial has initiated in 2019 and is expected to be completed in 2022 (ClinicalTrials.gov number, NCT03865238) (Lin et al. 2019). Only a small-scale phase I clinical trial has been conducted for EV-A71 vaccine developed in Singapore, and the study claimed that the vaccine induced a high immune response against HFMD caused by EV-A71, although the data has not been publicly disclosed (ClinicalTrials.gov number, NCT01376479). Immunizations with inactivated virions derived from CV-A16, CV-A10 and CV-A6 have been only studied in animal models, and the results provided immunological and functional evidence supporting their efficacy. The generated serum contained high levels of virus-specific neutralizing antibodies, and the serum from immunized mother mice afforded protection against lethal challenges with virulent HFMD-related viruses when it was passively transferred to neonatal mice (Qi An et al. 2014; Zhang et al. 2017a). Therefore, inactivated whole virus vaccine represents the most attainable monovalent HFMD-related vaccine.
Organizations Sinovac Biotech Co., Ltd Beijing Vigoo Biological Co., Ltd Chinese Academy of Medical Sciences EV-A71 Strain H07 (C4) FY (C4) M01 (C4) Inactivation technique Formalin Formalin Formalin Cell substrate Vero cells Vero cells Human diploid KMB-17 cell Dosages 400 U, two-dose 320 U, two-dose 100 U, two-dose Adjuvant Aluminum hydroxide Aluminum hydroxide Aluminum hydroxide Population target Children (6–35 month) Children (6–35 month) Children (6–71 month) Enrollment 10, 077 10, 245 12, 000 References NCT01507857 NCT01508247 NCT01569581
Table 1. Official licensed inactivated EV-A71 vaccines by the Chinese Food and Drug Administration.
Synthetic peptide vaccines are usually related to the selected neutralizing epitopes, and the two peptides located in VP1, called SP55 (E-F loop; aa 163-177) and SP70 (G-H loop; aa 208-222) have been shown to elicit EV-A71 specific neutralizing antibodies. SP70 raised a relatively higher titer of neutralizing antibody against EV-A71 than that of SP55. However, the neutralizing antibody titer elicited from the peptide SP70 was just one-fourth of that observed in mice immunized with heat-inactivated EV-A71 (Foo et al. 2007). Thus, given that EV-A71 VP1 peptide or whole protein was only able to raise a neutralizing antibody response generally inferior to that of inactivated EV-A71 vaccines and consequently showed a protective effect in animal models limited to a low-dose virus challenge (Premanand et al. 2012), synthetic peptide and protein vaccines have only been tried in the research stage without progression to more commercial development.
Virus-like particles (VLPs) are a special form of recombinant subunit vaccines for non-enveloped viruses, and can be generated by a number of biosystems. The principle of EV VLPs is to coexpress the genes encoding capsid protein precursor P1 and protease 3CD, which results in the cleavage of P1 into three capsid subunit proteins VP0, VP1, and VP3 through the action of the 3CD protease. VP0, VP1 and VP3 are subsequently self-assembled into VLPs, which adopt the natural structure of virus capsid and can serve as potential vaccine candidates after purification. VLPs derived from EV-A71, CV-A16, CV-A6 and CV-A10 species were reported to be successfully produced in baculovirus-insect cell (Somasundaram et al. 2016), Pichia pastoris yeast (Zhang et al. 2016) and saccharomyces cerevisiae yeast (Zhao et al. 2013; Zhou et al. 2016; Zhang W et al. 2018). Immunization study in mice showed that VLPs were able to elicit high titers of neutralizing antibodies and afford effective protection against lethal viral challenge (Wang X et al. 2018; Zhou et al. 2018). In addition, a recent study reported that VLP vaccines for HFMD induced a high antigen-specific B cell response that is comparable to inactivated vaccines (Yang et al. 2019) (Table 2). This study produced EV71-VLPs in Pichia pastoris, attaining a high expression level of EV71-VLPs greater than 250 mg/L (Yang et al. 2019). With the higher yield capacity to be more cost effective, EV71-VLPs produced in Pichia pastoris are in clinical trial (CXSL1900022), representing a good start toward future commercialization.
VLP-producing systems Yield capacity Properties Status Ref. Baculovirus-insect cell Moderate (64.3 mg/L) Moderate-yield; Relatively high cost; Large stocks (cell & viruses); Contamination risk of virus Lab (Chung et al. 2010) Saccharomyces cerevisiae yeast Low (0.25 mg/L) Low-yield; Low cost; Ease in manipulation Lab (Li et al. 2013) Pichia pastoris yeast High (270 mg/L) High-yield; Low cost; Easy manipulation Clinical trial (CXSL1900022) (Yang et al. 2019) Recombinant vesicular stomatitis virus (rVSV) – Attenuated(ΔM51);
Replication-competent and may have adverse effects
Lab (Yan et al. 2016) Recombinant adenovirus 5 (Ad-EVVLP) – Replication-incompetent(ΔE1/ΔE3); 3C-specific cellular immunity
Ad-EVVLPs from EV71 genes can protect against CVA16 infection
Lab (Tsou et al. 2015)
Table 2. The producing systems of enterovirus-related virus-like particle (VLP).
Researchers inserted P1 and 3CD genes of EV-A71 into one vesicular stomatitis virus (VSV) backbone to generate a recombinant VSV to produce VLPs, which protected neonatal mice against lethal viral challenge (Yan et al. 2016). A novel recombinant adenovirus vaccine, Ad-EVVLP, with P1 and 3CD genes of EV-A71 inserted into the adenoviral genome to express VLPs, induced EV-A71-specific neutralizing antibodies and Th1/Th2-balanced cellular responses in immunized mice, whereas inactivated EV-A71 vaccine activated only Th2-mediated neutralizing antibody responses to protect against virus challenge (Tsou et al. 2015) (Table 3). The immunogenicity of 71-6 epitope (aa 176-190 of VP3) was tested using the norovirus P particle as the vaccine carrier, and serum from mice immunized with the resulting chimeric P particle could protect suckling mice from a lethal dose of EV-A71 infection (Jiang et al. 2015).
Conformation Immunogenicity mAb
Limitation Advantages Inactivated whole virus Natural virion with genome Strong (+++) High; Cross-genotype protection Low cross-serotypic protection Mature technology VLP Natural virion without genome Moderate (++) High; Cross-genotype protection Low cross-serotypic protection Safe; Low cost; Explicit composition; Easy large-scale production and quality control Synthetic peptide or recombinant subunit Linear epitope or antigen Relatively weak (+) Low; Cross-genotype protection Low cross-serotypic protection; Strong adjuvant requirement Safe; Inexpensive; Explicit composition; Easy large-scale production and quality control Novel chimeric vaccines Natural virion without genome or linear epitopes of antigens Relatively high (++/+++) High; Cross-genotype protection Required to know key neutralization domain and need to design the optimal chimeric strategy May induce cross-protection of serotypes Recombinant virus-vector vaccines Natural virion without genome of target viruses but vectors Relatively high (++/+++) High; Cross genotype protection Risk of vector replication May induce cross-protection of serotypes; Comprehensive T-cell immune response
Table 3. The characteristics of the primary experimental enterovirus vaccine formats.