Sequencing and molecular characterization of CTNCEC25, a China fixed rabies virus vaccine strain CTN-1 adapted to primary chicken embryo cells

Rabies, which is an ancient global fatal central nervous system (CNS) disease, affects almost all kinds of mammals, including humans [1]. The mortality of rabies is almost 100%, and it is estimated that more than 55,000 people die from rabies worldwide annually, with about 95% of those deaths occurring in the developing world such as Asia and Africa (see WHO Fact Sheet No. 99). China has the second highest incidence of rabies after India, and a total number of 108,412 human rabies cases were recorded in China during the 55-year period between 1950 and 2004 [2].

Rabies virus (RABV) is the main causative agent of rabies and is the type species of the genus Lyssavirus of the family Rhabdoviridae. RABV has a non-segmented, single-stranded negative-sense RNA genome of approximately 12 kb that encodes five structural proteins in the order (3′ to 5′) of nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (large protein, L) [3, 4]. The viral RNA genome together with the N, P and L proteins forms a helical ribonucleoprotein (RNP) that is packaged into a bullet-shaped structure and wrapped by an envelope comprising an inner layer of the M protein and the transmembrane spike G protein [1]. While the RNP complex is the entity responsible for viral transcription and replication within the cytoplasm of the host cell, the G and M proteins play pivotal roles in viral assembly and budding [5, 6].

The RABV G protein is the only viral protein exposed on the surface of the virus. Previous studies have established that G protein is not only the major determinant of viral pathogenicity but is also the major protective antigen that induces the production of virus-neutralizing antibodies (VNAs) responsible for the immune responses of the host [7‐10]. Moreover, the G protein is also involved in the neurotropism of RABV [11‐18]. A number of antigenic sites to which neutralizing monoclonal antibodies bind were mapped in the G protein, including antigenic site I (aa 231), II (aa 34 - 200), III (aa 330 - 357), IV (aa 264) and “a” (aa 342 - 343) [19]. In addition, a linear epitope named G5 was also identified in the G protein (aa 244 - 281) [20, 21]. Among these antigenic sites, aa 147 and 333 have been shown to be critical for G protein function as mutation in either of these two sites significantly affected RABV antigenicity and pathogenicity [22, 23]. Furthermore, a region between aa 164 to 303 of the Nishigahara strain G protein also plays an important role in virus pathogenicity for adult mice, with aa 242, 255 and 268 constituting the key residues [24, 25].

Currently, the pathogenesis of RABV has not been fully elucidated and vaccination is the only effective method to protect against RABV infection. Since the first development of a rabies vaccine by Pasteur in the late 19th century, vaccination has been widely used in both domestic animals as well as reservoir species [26, 27]. At present, a number of RABV strains were used for vaccine production in different countries. Four virus strains, CTN-1, aG, PM and PV, have been applied in human rabies vaccine production in China and CTN-1 and aG strains are Chinese domestic isolates [28]. The CTN-1 strain was first isolated from brain tissue of a patient with rabies from Zibo, Shandong province while the aG strain was obtained from a rabid dog in Beijing [28]. However, although both the CTN-1 and aG strains are indigenous to China, they have distinct phylogenetic relationship. Previous studies suggested that the aG strain was more closely related with strains in northern and northeast part of China and phylogenetic analysis based on the N gene showed that the aG strain mainly clustered with strains from Japan and America but distantly clustered with most China street strains while the CTN-1 strain clustered preferentially with China native street viruses [29, 30], suggesting that the CTN-1 strain had closer genetic relationship with street viruses prevailing in China. It has been assumed that the efficiency of cross protection against the epidemic street virus conferred by rabies vaccine correlated with the homology between the vaccine strain and the challenge strains [31]. Therefore, the CTN-1 strain is theoretically more suitable for vaccine production than the aG strain in China.

Recently, a CTN-1 strain adapted to chicken embryo cells (CECs), which has been named CTNCEC25, was successfully obtained and demonstrated to have high immunogenicity and potency to induce a strong protective immune response in animals [32]. Besides, CTNCEC25 lost pathogenicity to adult mice by intracerebral inoculation [32]. In the present study, to gain more insight into the biological characteristics of CTNCEC25, the complete sequence of the CTNCEC25 strain was sequenced and characterized. Sequence comparison and phylogenetic analysis demonstrated that CTNCEC25 was more closely related with those recently isolated China RABV street strains than other vaccine strains commonly used in China. Virus growth curve showed that CTNCEC25 replicated stably and maintained high titers at cultured cells. Therefore, these results demonstrated the potential use of the CTNCEC25 strain for producing human rabies vaccine in China.

In the present study, the complete genome of the RABV strain CTNCEC25, the first CTN-1 strain adapted to CECs, was sequenced and analyzed. The results demonstrated that the CTNCEC25 strain was closely related to China RABV street strains recently isolated from different regions. Furthermore, although the CTNCEC25 strain achieved stable and high titers in cultured cells and CECs (Figure 4), it caused no lethality in adult mice by intracerebral inoculation [32], thus providing a rationale for its potential use for human vaccine production in China.

Comparison of the nucleotide sequences of CTNCEC25 with CTN-1 identified that all nucleotide changes occurred in the structural protein genes, with the G gene being the most variable. Similar results were observed in another attenuated RABV strain, RC-HL, which was derived from the RABV Nishigahara strain after 330 passages in chicken embryos and cell cultures [24]. It has been shown that the G gene was the most variable when comparing the complete genome sequences of the RC-HL strain and the Nishigahara strain [39]. Given that RABV is highly neurotropic in nature and the fact the G protein is the major structural protein involved in the neurotropism of RABV by recognizing receptors on neurons, it is therefore not unexpected that the G protein underwent greater selection pressure during adaptation to cultured nonneuronal cells.

Previous studies have identified several amino acids in G protein that were important for the antigenicity and pathogenicity of RABV [22, 23]. In the present study, two of these critical amino acids, aa 147 and 333, were found to be mutated in CTNCEC25 G protein during adaptation to CECs. Therefore, it was assumed that the pathogenicity of CTNCEC25 may be severely attenuated in adult mice, which was consistent with our previous in vivo study showing that CTNCEC25 was apathogenic to adult mice by intracerebral inoculation [32].

Sequence analysis identified that the Lyssavirus genome contains the signals essential for the transcription initiation, termination and processing for all the five structural protein genes, and the RABV is no exception [4]. A consensus sequence, 3’-A/U-C-U-U-U-U-U-U-U-5’, is conserved in all of the five RABV structural protein genes [3]. Several studies using Vesicular stomatitis virus (VSV), the prototype of the Vesiculovirus genus, showed that the U₇ tract is strictly conserved and essential for VSV mRNA termination and polyadenylation, and either shortening or interrupting it with a heterologous nucleotide eliminates mRNA termination and polyadenylation [40, 41]. As is the case for CTNCEC25, however, the U₇ tract is only conserved in four of the five structural protein genes, N, M, G and L, but not the P gene, in which the U₇ tract was shortened to U₆. Therefore, it is assumed that the expression of M gene, which is located downstream of the P gene, would be affected in CTNCEC25 due to the read-through of the upper P gene. Previous studies have revealed that the M gene encodes a multifunctional protein that plays essential roles not only in mediating viral assembly and budding but also in regulating the balance between the transcription and replication of RABV. So the disruption of M gene expression should certainly impair the CTNCEC25 replication in cultured cells. Although we did not perform transcriptional analysis of the CTNCEC25 M gene, this possibility could be ruled out as the growth kinetics of CTNCEC25 in cultured cells were indistinguishable from that of CTN-1 (Figure 4).

After careful inspection of the database, we found that while the typical U₇ tract was the preponderant sequence at the P-M junction, several types of disruption of the typical U₇ tract were observed, although with a low frequency, in the P-M junctions, including shortening or lengthening of U₇ tract to U₆ or U₈ and interruption of the U₇ tract by a different nucleotide (Figure 1). Therefore, it is possible that the RABV street strains have accumulated mutations during evolution and maintained these mutations to increase their population diversity, better adapt to their hosts or disseminate infection to a new host species. On the other hand, it also cannot rule out the possibility that different mechanisms may exist upon the molecular biology between RABV and VSV, as RABV and VSV share distinct natural histories and pathogenicity despite the close relationship within each other [4]. Further studies are needed to unravel the mechanisms underlining the regulation of gene expression of CTNCEC25.

Phylogenetic analysis using the genome sequence or the mature G protein amino acid sequence identified that CTNCEC25 shared high homology with wild strains isolated from different regions in China. It has been previously reported that the identity of the ectodomain amino acid sequence of RABV G protein directly correlated with the efficacy of vaccination and VNAs displayed cross-protection only when the amino acid sequence of the G protein ectodomain was at least 74% identical [31]. The recent antigenic analysis using serological assay data has also demonstrated that a 4.8% change in the G protein ectodomain amino acid sequence would cause a change of one antigenic unit between viruses (equivalent to a two-fold change in antibody titer) and there is a generally good correlation between genetic distance in the G protein and antigenic distance [42]. Therefore, it is reasonable that the best vaccine strain should be the one most closely related to the street strains circulating within the target area. Sequence analysis showed that compared to aG, PM and PV vaccine strains, which were widely used in China for human vaccine production, the CTNCEC25 strain was more closely related to RABV strains circulated in China while the other three vaccine strains were predominantly clustered with RABV strains derived from other countries. In addition, the ectodomain amino acid homology of the G proteins of CTNCEC25 with other RABV strains ranged from 90.0% to 99.1% (Table 5), which significantly ranked above the threshold 74% for the presence of cross-protection. Taken together, the above results indicated that CTNCEC25 was an ideal candidate for human vaccine production in China.

The human rabies vaccines can be produced either from animal tissues or cultured cells, such as CECs, BHK or Vero cells [43]. The development of modern industrial cell cultivation and fermentation techniques have greatly promoted the capacity of producing vaccines with high quantity and quality. Given the consideration of purity and concentration of vaccines, vaccines using cultured cells have quickly outdated the use of tissue-derived rabies vaccines. However, although cell culture vaccines are highly efficacious and immunogenic, these cell lines may have differences in genotypes or phenotypes from the original cell line and thus may contain oncogenic properties [44, 45]. Therefore, great caution should be taken in using such cell lines for vaccine manufacturing. Specific guidelines for producing human vaccines using the continuous cell lines were enacted in China and no more than 100 pg of host cellular DNA per dose was allowed for authorized vaccine production using Vero cell line according to the standard of the Pharmacopoeia of the People's Republic of China (2010), Volume III. On the other hand, CECs, which have limited life span than continuous cell lines, maintain the normal cellular karyotype and thus guarantee no contamination of foreign and oncogenic particles and are expected to be a promising substitute substrate for production of safe human vaccine [43]. The FluryLEP strain has already been adapted to CECs to produce purified chicken embryo cells vaccines, and has been recommended by WHO and widely used in many countries due to its high safety and efficacy, low cost and relative simple manufacturing techniques [46‐49]. Current vaccine production in China was almost exclusively based on Vero cells, making vaccine strains adapted to CECs urgently needed.

In this study, an CECs-adapted RABV strain CTNCEC25 was sequenced and characterized. Phylogenetic analysis identified that CTNCEC25 was more closely related to RABV street strains circulating in China than other RABV vaccine strains currently used in China. Sequence comparison showed that the G protein ectodomain amino acid sequence identity between CTNCEC25 and other RABV strains was at least 90% identical. Furthermore, CTNCEC25 produced high virus titers in primary culture cells while it lacked the pathogenicity for adult mice. Collectively, these results demonstrated that the CTNCEC25 strain is an ideal vaccine strain candidate for producing human vaccine with high quality and safety in China.