Vaccine efficiency and its immune response varies according to the type of vaccine [52, 53]. In our present study, the 1232 bp of VP6 gene represents the most frequent VP6 sequence in Egyptian clinical and environmental rotavirus isolates [with 98% nucleotides identity and 98% amino acid identity in comparison to human RoV Wa reference strain, GeneBank nucleotides accession number: K02086.1 and amino acid accession number: P03530.1 with 7 amino acid changes (Proline changed to Arginine, Leucine changed to Phenylalanine, Leucine changed to serine, Alanine changed to serine, Threonine changed to Leucine, Glutamic acid changed to Glutamine, Leucine changed to Valine, at positions: 102, 125, 126, 241, 279 and 327, and 382, respectively)] and with codon optimization were expressed in E. coli by reassigning the native codons to codons frequently used in E. coli. expression plasmid pJ 404 contains inducible promoter site T5 (1232 bp) for induction of protein expression by IPTG, strong ribosome binding site for tight expression was used to have a recombinant protein as a subunit vaccine for human RVs. Rabbits immunization with 20 µg of rVP6 resulted in specific antibodies for the expressed protein which achieved positive results with ELISA up to a serum dilution of 1:24,000. This titer of antibodies is higher than the titer of antibodies obtained by Pastor et al. [29] employing the same protein concentration. Codon optimization in our present study may be a reason of the elevation of the titer of the antibodies. Another reason may be the difference in amino acids between our strain and the reference rotavirus Wa strain.
In our present study, serological methods were used to compare their efficiencies in estimation of the potency of the antibodies of the 1232 bp of VP6 purified expressed protein when challenging with the infectious human RV Wa strain in vitro. Our results indicated that neutralization has significant higher efficiency in the log10 reduction of infectious human RV Wa strain than immunoperoxidase. Muruato and co-workers reported that virus neutralization remains the gold standard for determining antibody efficacy [54]. The results showed less efficiency of the antibodies of the VP6 expressed protein to achieve log10 reduction of the rotavirus Wa strain infectious units than the efficiency of the antibodies of the P[8] genotype expressed protein in the study of El-Senousy and co-workers [55] who compared the same methods to evaluate the efficiency of purified antibodies of the expressed proteins of the different VP8 genotypes of rotaviruses (P[8], P[4], and P[6]). This difference in results may return to the different mechanisms of VP6 and VP8 antibodies in dealing with the rotavirus. VP8 expressed proteins produce neutralizing antibodies against outer capsid protein upon challenge with RV which prevent viral entry to the cells, while, VP6 protein does not induce neutralizing antibodies, because it is an internal protein in the RV particle [27]. VP6 antibodies act intracellularly after rotavirus un-coating inside MA104 cells and the release of DLPs which causes exposure of viral VP6 layer to the antibodies. Then, interruption of viral replication cycle is occurred [28, 56, 57]. So, intracellular induction of VP6 antibodies was necessary to be performed in our present study to give the opportunity to VP6 antibodies to act intracellularly after the exposure of the viral VP6. Caddy and co-workers developed an assay to examine how antibodies neutralize rotavirus intracellularly [46]. They showed that neutralization by VP6-specific IgG was much more efficient than VP6-specific IgA using simian rotavirus for challenge. In our present study, promising results were achieved when using the method of Caddy and co-workers [46] to insert the antibodies of the expressed protein of 1232 bp of the most frequent VP6 of clinical and environmental isolates in Egypt into MA104 cells in vitro by electroporation and using high and low titres of infectious viral units of human rotavirus Wa strain for challenge (2X107 TCID50/ml and 1X104 TCID50/ml). On the other hand, antibodies of genotype P[8] expressed protein had higher efficiency than the antibodies of genotypes P[4] and P[6] expressed proteins in the study of El-Senousy and co-wprkers [55]. This may be because of the specificity of the expressed P[8] protein against human rotavirus Wa strain G1P[8] and this may be the same reason for the higher efficiency of the antibodies of VP6 expressed protein than the antibodies of either P[4] or P[6] expressed proteins. The 1232 bp VP6 gene is 98% nucleotides identity and 98% amino acid identity in comparison to human RoV Wa reference strain. Further research is needed to examine the efficiency of the human rotavirus expressed proteins antibodies against infectious human rotavirus Wa strain in vivo, however, using in vitro studies before doing challenge in animals is very important to choose the promising candidate vaccines to be examined in vivo. Using VP6 antibody mediated intracellularly may be a promising solution to overcome the problem of the internal position of the VP6 in the rotavirus which means that these antibodies are not neutralizing antibodies and cannot prevent rotavirus entry into specific host cells in vitro examination of the vaccine efficiency. Determination of the efficient candidate vaccines in vitro before examination in vivo gives a lot of advantages such as use less number of animals in vivo which is preferable from ethical point of view in addition to save money and time.
In the study of El-Senousy and co-workers [55], neutralization/RT-PCR and neutralization/nested RT-PCR had higher sensitivity than traditional neutralization and immunoperoxidase when efficiency evaluation of the antibodies against VP8 expressed protein in vitro was performed. In our present study, we could not use these methods to improve the efficiency of the traditional neutralization test in the efficiency evaluation of the antibodies against VP6 protein because of the different mechanisms of the antibodies of VP8 and VP6 proteins. VP8 antibodies are neutralizing antibodies prevent neutralized viruses from cell entry, so, using RT-PCR and nested RT-PCR after the traditional neutralization assay increased the sensitivity of the assay. RT-PCR and nested RT-PCR could detect positivity in some wells which do not show CPE using inverted microscope because they could detect infectious viruses which entered the cells but they are low enough or slow enough to be able to show CPE. So, using RT-PCR and nested RT-PCR after the traditional neutralization assay gives accurate results of viral titre before and after exposure to the specific neutralization antibodies. If using only traditional neutrealization assay, some wells could be neglected or considered without CPE, however infectious viruses could enter the cells. In case of VP6, it is difficult to use RT-PCR and nested RT-PCR after traditional neutralization assay because the VP6 antibodies is not neutralizing antibodies. Introduce the antibodies into the cells is a necessary process to give the opportunity to the antibodies to neutralize the exposed viral VP6 after viral un-coating process.
Our results indicated that significant higher sensitivity for the antibodies of the 1232 bp represents the whole gene of the most frequent isolate of human rotavirus VP6 in clinical specimens collected from 2015–2017 and sewage samples collected from 2015–2018 in Cairo, Egypt from Abo El-Reesh hospital and El-Gabal El-Asfar and Zenin wastewater treatment plants and El-Giza water treatment plant in Greater Cairo with 98% nucleotides identity and 98% amino acid identity in comparison to human RoV Wa reference strain than the sensitivity of the antibodies of the 155 bp epitope of the VP6 which represented 99% homology with rotavirus sequences with the accession numbers HQ392389, HQ738601, and HQ738599 with target sequence codes for 51 amino acids resembles amino acids from 282 to 332 of human rotavirus A inner capsid protein VP6 with accession number ADO78525 in the previous study of El-Senousy and co-workers [34]. In our present study 1.4 and 1.6 log10 reductions of infectious human rotavirus Wa strain were achieved using 1/10 dilution of the produced antibodies against the 1232 bp of human rotavirus VP6 when using high and low virus titres (2X107 TCID50/ml and 1X104 TCID50/ml) respectively, while only 1 log10 reduction was achieved with undiluted antibodies of the 155 epitope of the human rotavirus VP6 in the previous study of El-Senousy and co-workers [34]. This significant higher activity may return to the higher efficiency of the antibodies of the whole gene to interrupt the viral replication cycle inside the MA104 cell line by neutralizing the exposed VP6 after viral un-coating than the antibodies of the short 155 bp fragment epitope in the study of El-Senousy and co-workers [34]. This short fragment epitope did not achieve successful prevention of rotavirus symptoms when challenge was performed in guinea pigs (Data not shown) and this was the reason of using the whole VP6 fragment in our present study.Another important factor in our present study to achieve accurate results is the use of developed method of Caddy and co-workers [46] to insert the antibodies inside the MA104 cells using electroporation to be able to neutralize the exposed VP6 after viral un-coating. The amino acid changes in the whole VP6 gene with codon optimization of 1232 bp are 7 amino acid changes (Proline changed to Arginine, Leucine changed to Phenylalanine, Leucine changed to serine, Alanine changed to serine, Threonine changed to Leucine, Glutamic acid changed to Glutamine, Leucine changed to Valine, at positions: 102, 125, 126, 241, 279 and 327, and 382, respectively). The only amino acid change in the position of the 155 bp short fragment is the amino acid number 327 which Glutamic acid changed to Glutamine. Large body of evidence indicates that antibodies targeting the VP6 protein of the middle capsid layer play a key role in protection against rotavirus infection. VP6-specific antibodies are produced to high titres in response to rotavirus infection or vaccination in animal models [58, 59]. So, promising results concern with the efficiency of the VP6 expressed protein of the most frequent rotavirus VP6 whole gene sequence in Egypt could be added to these previous evidences and especially for this pattern prevalent in Egypt and the same model countries. Further research is needed to examine the efficiency of the human rotavirus expressed proteins antibodies of the 1232 bp represent the whole VP6 gene with codon optimization against infectious human rotavirus Wa strain in vivo.
In the present study, an expression vector (pJ 404) which was designed to contain the 1232 bp of the VP6 gene was used with an N-terminal six-histidine tag to facilitate its purification, resulting in a purified rVP6 protein with a concentration of 162 ± 0.005 mg/L culture with most expressed rVP6 protein present in inclusion bodies. This finding was also described by Choi et al. [25], who reported that > 90% of expressed VP6 protein was sequestered in inclusion bodies within the E. coli cells. The same authors also reported that large quantities of the expressed rVP6 protein were truncated VP6 polypeptides [25, 60], which was caused by proteolytic degradation of the protein, since it was expressed in E. coli BL21, a strain that lacks proteases enzymes. Therefore, the type of expression system plays an important role in the quantity and quality of the expressed rVP6 protein, and likewise the immunogenicity of the vaccine. On the other hand, freund’s adjuvant was used in this study to enhance the immune response. Similarly, Crawford et al. [61] mentioned that, the use of Freund’s adjuvant resulted in broad serum heterotypic neutralizing antibodies in the immunized animals. Other types of adjuvants were also administrated with RV vaccines to elevate the immune response, like saponin adjuvant (QS-21) [62]. However, Ciarlet et al. [63] proved that Freund’s adjuvant is a better adjuvant than saponin adjuvant (QS-21) when both were used with RV vaccines to evaluate their immunogenicity.
The problem if thinking of transfer this technology to industrial scale is the relatively low quantity of the protein concentration of lysate (1.877 ± 0.033 mg/ml of culture) in relation to the concentration of the purified VP6 (162 ± 0.005 mg/L of culture). It may indicate relatively low quantity and low solubility of the expressed VP6 protein. The quantity of immunogen is a critical factor in the process of vaccine production at industrial scale. The low quantity and low solubility is due to the insoluble nature of VP6 protein, and its native trimeric conformation assumed by the protein which might be too difficult for E. coli to process [64], causing the major disadvantage of this expression system. Another explanation is the over expression of VP6 protein which leads to the accumulation of partially folded or misfolded protein. When protein translation occurs at a fast rate, protein folding is a challenge [65]. The fast expression rate and misfolding of protein causes exposure of hydrophobic areas, which are hidden in the native form of the protein. These exposed hydrophobic areas have a very strong tendency to aggregate [66]. This problem was suggested to be overcome by, lowering the temperature used to induce the expression of rVP6 protein to slow down the expression rate to avoid misfolding. Also, optimizing IPTG concentration and induction time can help obtaining more efficient yield of soluble rVP6 protein [67]. In the present work, the effect of decreasing the temperature from 37°C to 20°C during the protein expression on the yield of soluble fraction of rVP6 protein was investigated and did not give a significant increase of soluble rVP6 protein fraction. Also, the effect of different IPTG concentrations (0.1, 0.25, 0.5, and 1 mM) on the yield of soluble fraction of rVP6 protein was studied and did not enhance the yield of soluble rVP6 protein fraction (Data not shown). Another proposed solution could be through the use of a molecular chaperone which will facilitate the proper folding of the bacterial expressed rVP6 proteins, through binding to and stabilizing unfolded or partially folded protein parts [68, 69]. Notably, a large portion of the purified protein was lost during the washing process which was obvious on SDS-PAGE by the presence of VP6 protein in the wash fraction. Similar phenomena were reported by Afchangi et al. [70], who observed partial loss of VP6 protein trimer structures in western blot assays. This indicates the need to use different purification method, like ion exchange or gel filtration, to minimize the loss of the protein during the purification process.
E. coli BL21 strain which was used in this study, is the most commonly used expression system for rVP6 protein [27, 71], although, several studies explored different expression systems including eukaryotic systems such as insect cells through baculovirus expression vector [72], yeast including Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha [73, 74], mammalian cells [75], viruses such as herpes simplex virus 1 (HSV-1)-based vectors [76], and plant cells [77]. The relatively low protein content obtained in this study may indicate the need to use another expression system in future studies as one of the suggested solutions to increase the amount of the expressed protein. Using different expression system (Eukaryote instead of prokaryote) such as yeast, plant, or mammalian cells, may offer new advantages and be better expression system, avoiding E. coli related problems [73, 74]. On the other hand, E. coli has some advantages, making it still commonly used as expression system for different proteins despite the problems associated with it. These advantages include its ability to produce high amounts of protein. It is time-saving compared to other expression systems which require more time to grow, like yeast. Another advantage is its low cost. And most important, the E. coli ability to produce higher VP6 protein yield compared to yeast on volumetric specific productivity levels [73]. Therefore, optimizing the conditions for VP6 protein expression in bacterial cells, such as E. coli codon adaptation index, codon pair bias, and guaninecytosine content, as well as redesigning ribosome binding site, can increase the yield of expressed rVP6, and solve the main problems associated with prokaryotic expression system [67].
Future research is needed to study the homotypic and heterotypic efficiency of the antibodies of this 1232 bp VP6 expressed protein in vivo.