Tandem truncated rotavirus VP8* subunit protein with T cell epitope as non-replicating parenteral vaccine is highly immunogenic

Abstract

The two currently available live oral rotavirus vaccines, Rotarix^® and RotaTeq^®, are highly efficacious in the developed countries. However, the efficacy of such vaccines in resource deprived countries in Africa and Southeast Asia is low. We reported previously that a bacterially-expressed rotavirus P2-P[8] ΔVP8* subunit vaccine candidate administered intramuscularly elicited high-titers of neutralizing antibodies in guinea pigs and mice and significantly shortened the duration of diarrhea in neonatal gnotobiotic pigs upon oral challenge with virulent human rotavirus Wa strain. To further improve its vaccine potential and provide wider coverage against rotavirus strains of global and regional epidemiologic importance, we constructed 2 tandem recombinant VP8* proteins, P2-P[8] ΔVP8*-P[8] ΔVP8* and P2-P[8] ΔVP8*-P[6] ΔVP8* based on Escherichia coli expression system. The two resulting recombinant tandem proteins were highly soluble and P2-P[8] ΔVP8*-P[8] ΔVP8* was generated with high yield. Moreover, guinea pigs immunized intramuscularly by 3 doses of the P2-P[8] ΔVP8*-P[8] ΔVP8* or P2-P[8] ΔVP8*-P[6] ΔVP8* vaccine with aluminum phosphate adjuvant developed high titers of homotypic and heterotypic neutralizing antibodies against human rotaviruses bearing G1-G4, G8, G9 and G12 with P[8], P[4] or P[6] combination. The results suggest that these 2 subunit vaccines in monovalent or bivalent formulation can provide antigenic coverage to almost all the rotavirus G (VP7) types and major P (VP4) types of global as well as regional epidemiologic importance.

Keywords:

• P type
• rotavirus
• subunit vaccine
• universal T cell epitope
• VP8* protein

Introduction

Rotaviruses are the single most important cause of severe diarrheal disease in infants and young children under 5 years of age worldwide, responsible for an estimated 453,000 deaths annually, predominantly in low-income or middle-income countries.¹ In 2009 and again in 2013, the World Health Organization (WHO) Strategic Advisory Group of Experts (SAGE) on Immunization recommended that 2 live oral rotavirus vaccines, Rotarix^® (RV1; GlaxoSmithKline Biologicals) and RotaTeq^® (RV5; Merck Vaccines), be introduced into all national immunization programs for children, especially in nations where diseases related to rotaviruses indicate major threat.^2-4 Seventy-5 countries have introduced Rotarix^® and/or RotaTeq^® vaccine to the national immunization program by January 2015 according to WHO record.⁵ These 2 vaccines have been shown to be safe and efficacious in both developed and some developing countries.^6-8 However, the immunogenicity and efficacy of these vaccines are substantially reduced in low-income countries of Africa, Asia and Central America,^9-11 where the high mortality due to rotavirus infections occurs.^12,13 In addition, formation of reassortant rotaviruses between vaccine strains and circulating wild-type strains or among vaccine strains can potentially increase virulence as reported recently.^14-17 PATH (Program for Appropriate Technology in Health, an international nonprofit organization) has launched a non-replicating rotavirus vaccine (NRRV) program (including the P2-P[8] ΔVP8* recombinant protein vaccine we reported previously)¹⁸ aiming to develop next generation, non-oral rotavirus vaccines. Vaccines administered through non-oral routes may overcome factors that can weaken an oral vaccine's efficacy including co-infections in the digestive system or the presence of maternal antibodies, and remove the slight risk of intussusception associated with live-rotavirus vaccines entir-ely.^19-23 The latest phase I trial launched by PATH showed that healthy adult recipients parenterally administered with 10, 30 or 60 µg P2-Wa ΔVP8* (aa 65–223) using aluminum hydroxide adjuvant demonstrated >4-fold IgG and IgA antibody titer rises in almost all recipients to the immunogenic by ELISA after 3 doses vaccinations (only one recipient out of 12 did not demonstrate an IgA response).²⁴ Furthermore, NRRVs could easily be introduced into an Expanded Program on Immunization (EPI), which can be given concomitantly with other children vaccines without affecting or being affected by them, as well as being safe for the immunocompromised children.

In general, the magnitude of protection triggered by a parenteral vaccine against orally transmitted diseases depends on the levels of protective antibodies.^25,26 Westerman and his colleagues demonstrated that high or medium titer of specific IgG or neutralizing antibodies passively transferred to naive non-human primate by blood infusion delayed the rotavirus infection after oral challenge with virulent simian rotavirus.²⁶ It suggests that circulating IgG can provide protection against pathogens infecting the host via mucosal route and the development of parenteral rotavirus vaccine candidates is applicable for infants and children in the underdeveloped countries, where rotavirus-related diarrhea is a life-threat disease.^27,28 We reported previously that bacterially-expressed P2-P[8] ΔVP8* induced high levels of both homotypic and heterotypic neutralizing antibodies in guinea pigs and significantly delayed the onset and shortened the duration of diarrhea in gnotobiotic pigs vaccinated with purified P2-P[8] ΔVP8* after oral challenge with the virulent human rotavirus Wa strain.¹⁸ The above mentioned phase I clinical trial launched by PATH demonstrated moderate immunogenicity of the P2-P[8] ΔVP8*(aa 65–223) vaccine in humans.²⁴ Therefore, we aimed to further increase the immunogenicity of such P2-P[8] ΔVP8* candidate vaccine. In this study, we constructed 2 fusion subunit recombinant proteins, P2-P[8] ΔVP8*-P[8] ΔVP8* and P2-P[8] ΔVP8*-P[6] ΔVP8* to provide wide coverage for human rotaviruses combined with P[8], P[4] or P[6] types of global as well as regional epidemiologic importance including new and emerging G8, G9 and G12 rotaviruses, since we previously reported that P[8] ΔVP8* induced a cross-neutralizing antibodies against human rotaviruses with P[4] specificity.²⁹ We then characterized the immunogenicity of these recombinant fusion proteins in guinea pigs. In consideration of concerns associated with live oral rotavirus vaccines stated above as well as potential advantages of non-replicating rotavirus vaccines, our tandem VP8* proteins demonstrate a high potential for a successful parenteral vaccine.

Results

Expression, purification and specificity of recombinant proteins

Each of the 2 recombinant VP8* proteins, P2-P[8] ΔVP8*-P[8] ΔVP8* and P2-P[8] ΔVP8*-P[6] ΔVP8* was expres-sed both in soluble and insoluble (inclusion bodies) forms. The expression and specificity of each purified recombinant protein was confirmed by SDS-PAGE (Fig. 1A, C) and Western blot analyses (Fig. 1B, D, E). The molecular weight of each recombinant protein analyzed by SDS-PAGE was identical as expected, around 45 kilodolton. Both of recombinant proteins reacted with polyclonal antisera against Wa rotavirus strain (Fig. 1B, D) and P2-P[8] ΔVP8*-P[6] ΔVP8* recombinant protein reacted with polyclonal antisera against ST3 (P[6]) rotavirus strain (Fig. 1E). An additional band at around 70 kilodolton molecular weight was observed in Western blot assay in which P2-P[8] ΔVP8*-P[6] ΔVP8* recombinant protein was recognized by antisera against Wa or ST3 (the lower band). The upper band is likely the residual bacterial proteins that reacted with the antisera.

Figure 1. Analysis by SDS-PAGE and Western blot of purified recombinant proteins P2-P[8] ΔVP8*-P[8] ΔVP8* (A and B) and P2-P[8] ΔVP8*-P[6] ΔVP8* (C, D and E). The proteins were expressed in E. coli at 18°C and purified as described in Materials and Methods. Lane M: molecular size markers. (A) SDS-PAGE analysis. Lane 1: purified P2-P[8] ΔVP8*-P[8] ΔVP8*; (B) Western blot analysis probed with guinea pig anti-Wa strain antiserum. Lane 1: P2-P[8] ΔVP8*-P[8] ΔVP8*. (C) SDS-PAGE analysis. Lane 1: purified P2-P[8] ΔVP8*-P[6] ΔVP8*. (D) Western blot analysis probed with guinea pig anti-Wa strain antiserum. Lane 1: purified P2-P[8] ΔVP8*-P[6] ΔVP8* against anti-Wa hyperimmune sera; (E) Western blot analysis probed with guinea pig anti-ST3 strain antiserum. Lane 1: purified P2-P[8] ΔVP8*-P[6] ΔVP8* against anti-ST3 hyperimmune sera. Arrows indicate the recombinant proteins.

The yield of recombinant proteins and concentration of endotoxin

The yield of purified P2-P[8] ΔVP8*-P[8] ΔVP8* recombinant protein reached ∼90mg per liter (L) of culture, whereas that of purified P2-P[8] ΔVP8*-P[6] ΔVP8* was much lower at∼5 mg/L. The endotoxin level in each tandem ΔVP8* protein was around 1.8 EU/ml (1.853 EU/ml for P2-P[8]ΔVP8*-P[8]ΔVP8* and 1.837 EU/ml for P2-P[8]ΔVP8*-P[6]ΔVP8*), which was far less than maximum recommended endotoxin levels in recombinant subunit vaccine (<20 EU/ml).³⁰

Humoral antibody measurements

Neutralizing antibody responses induced by the P2-P[8] ΔVP8*-P[8] ΔVP8* and P2-P[8] ΔVP8*-P[6] ΔVP8* protein with AP in guinea pigs after IM immunization are shown in Table 1. The recombinant protein P2-P[8] ΔVP8*-P[8] ΔVP8* induced high titers neutralizing antibodies against P[8]-specific rotavirus with G1 combination (ranged from 1:5120˜40960; GMT,1:13279.64), G3 combination (ranged from 1:1280˜20480; GMT,1:3620.38), G4 combination (ranged from 1:1280˜10240; GMT,1:3620.38), and G9 combination (ranged from 1:1280˜20480; GMT,1:3620.38). Thus, P2-P[8] ΔVP8*-P[8] ΔVP8* elicited the identical GMT neutralizing antibodies against P[8] specificity with G3, G4 or G9 combination, which was lower than G1 (GMT, 1:3620.38 vs. 1:13279.64). In addition, P2-P[8] ΔVP8*-P[8] ΔVP8* also elicited high levels of heterotypic neutralizing antibodies against P[4]-specificity with G2 (ranged from 1:5120˜20480; GMT,1:7240.77), G8 (ranged from 1:1280˜5120; GMT,1:1659.96), and G12 (ranged from 1:1280˜5120; GMT,1:2347.53). Furthermore, P2-P[8] ΔVP8*-P[6] ΔVP8* raised high titer of neutralizing antibodies against P[8] strains with G1 (ranged from 1:2560˜20480; GMT,1:4305.39), G3 (ranged from 1:1280˜5120; GMT,1:2152.69), G4 (ranged from 1:1280˜10240; GMT,1: 2560), and G9 (ranged from 1:1280˜5120; GMT, 1:1659.96), and against P[4]-specificity with G2 combination (ranged from 1:1280˜5120; GMT,1:1522.19), G8 (GMT,1:1280), and G12 (ranged from 1:640˜5120; GMT,1:1395.85). Additionally, P2-P[8] ΔVP8*-P[6] ΔVP8* induced antibodies against P[4]-specificity with G2 (GMT,1:1280) or G4 (ranged from 1:640˜1280; GMT,1:987).

Table 1. Tandem P2-P[8] ΔVP8*-P[8] ΔVP8* and P2-P[8]ΔVP8*-P[6] ΔVP8* subunit vaccine with aluminum phosphate adjuvant induced varied levels of neutralizing antibodies to indicated strains in guinea pigs

	Guinea pig	Reciprocal of 60% plaque reduction serum neutralizing antibody titer to indicated rotavirus strains
	#	Wa	P	VA70	W161	DS-1	1290	L26	1076	ST3	69M
		P[8],G1	P[8],G3	P[8],G4	P[8],G9	P[4],G2	P[4],G8	P[4],G12	P[6],G2	P[6],G4	P[10],G8
P2-P[8]△VP8*-P[8]△VP8*	1	5120	5120	1280	1280	5120	1280	1280	320	160	80
	2	5120	1280	5120	2560	5120	1280	1280	320	320	160
	3	5120	1280	1280	2560	5120	1280	1280	320	320	160
	4	20480	20480	10240	20480	20480	5120	2560	640	320	80
	5	20480	5120	5120	5120	10240	1280	5120	320	320	320
	6	20480	2560	2560	2560	5120	1280	2560	320	320	160
	7	20480	2560	5120	2560	5120	1280	2560	320	160	80
	8	40960	5120	5120	5120	10240	2560	5120	320	320	160
	GMT	13280	3620	3620	3620	7241	1660	2348	349	269	135
P2-P[8]△VP8*-P[6]△VP8*	1	2560	1280	1280	1280	1280	1280	640	1280	640	80
	2	5120	2560	5120	1280	1280	1280	1280	1280	1280	80
	3	2560	1280	1280	1280	1280	1280	1280	1280	640	80
	4	10240	5120	5120	2560	1280	1280	1280	1280	1280	80
	5	20480	5120	10240	5120	5120	1280	5120	1280	1280	80
	6	2560	1280	1280	1280	1280	1280	1280	1280	1280	80
	7	2560	1280	1280	1280	1280	1280	1280	1280	640	80
	8	2560	2560	2560	1280	1280	1280	1280	1280	1280	80
	GMT	4305	2153	2560	1660	1522	1280	1396	1280	987	80

Discussion

In our previous study, recombinant P2-P[8] ΔVP8* was demonstrated to be of high immunogenicity and induced high levels of neutralizing antibodies in guinea pigs and gnotobiotic pigs and provided protection against rotavirus diarrhea in gnotobiotic pigs.¹⁸ In this study, we generated tandem rotavirus VP8* recombinant protein with the universal tetanus toxoid (TT) T cell epitope P2-P[8] ΔVP8*-P[8] ΔVP8* and P2-P[8] ΔVP8*-P[6] ΔVP8* in an attempt to further improve the immunogenicity of the recombinant subunit vaccine candidates. Our study demonstrated that both of the recombinant proteins induced high titers of neutralizing antibodies against homotypic and hetertypic human rotavirus strains, which are of global and regional epidemiologic importance, such as G1, G2, G3, G4, G8, G9 and G12.

We previously reported that P[8] ΔVP8* induced high titers of cross neutralizing antibodies against P[4] rotaviruses.²⁹ In this study, we demonstrated that the tandem P2-P[8] ΔVP8*-P[8] ΔVP8* mounted higher titers of cross neutralizing antibodies against P[4] rotaviruses than P2-P[8] ΔVP8*-P[6] ΔVP8*(combined with G2, GMT7240.77 vs. 1522.19; G8, GMT1569.96 vs. 1280; G12, GMT2347.53 vs. 1395.85). The results suggests that 2 copies of P[8] ΔVP8* in tandem further enhanced cross neutralizing immune response, which was independent of the increase of molecular mass, since the P2-P[8] ΔVP8*-P[6] ΔVP8* induced the same titer of neutralizing antibody against P[6] rotavirus with P[6] ΔVP8* recombinant subunit protein²⁹. The transform of P[6] ΔVP8* into P2-P[8] ΔVP8*-P[6] ΔVP8* did not enhance the immune response against P[6] specific rotaviruses.

It is well know that the individual B cell antigen receptors (BCR) (Ig structure) on the surface of B cells must be cross-linked simultaneously by an antigen to activate immature B cells to trigger efficent humoral immunity.^31,32 Repeated copies of an epitope in one antigen are able to induce the cross-linkage of BCR to enhance the specificity and levels of antibodies produced following immunization with such constructs.³³ For example, in a study by Kovacs-Nolan, et al. it was reported that 3 copies of human rotavirus VP8* epitope (1–10 amino acids) peptide with P2 T cell epitope induced a more potent neutralizing antibodies than one single copy of VP8* (1–10 amino acids) or even than the full-size VP8 recombinant protein.³⁴ Actually, one copy of VP8* (1–10 amino acids) itself without P2 could not induce epitope-specific immune response. Thus, repeated epitope in a single protein molecule significantly enhances antigenicity. It appears that the same mechanism applies to both peptides and subunit protein. It is speculated that 2 copies of tandem P[8] ΔVP8* with repeated epitopes may induce the cross-linkage of IgM receptors on the surface of immuture B cells and help antigen presentation in the absence of T-cell assistance, presumably enhance antigen presentation or antigen processing, resulting in increased immune response. This explains why 2 copies of tandem P[8] ΔVP8* induced higher titers of cross-neutralizing antobodies against P[4]-specific rotaviruses. Conversely, the hybrid P[8] ΔVP8*-P[6] ΔVP8* did not enhance the titer of cross-neutralizing antibodies, since it did not contain repeated epitopes in a single protein molecule. The effect of structure constraint on antigen presentation was considered when we constructed the tandem rotavirus recombinant VP8 protein. A flexible linker “GSGSG” was introduced between T cell epitope P2 and truncated VP8*, as well as in the middle of the 2 truncated VP8*s (2 copies of truncated P[8] VP8* or P[8] VP8*-P[6] VP8*) to avoid possible structure constraint. The mechanism underlying the enhancement of immunogenicity by tandem copies of the subunit proteins remains to be explored.

Both P2-P[8] ΔVP8*-P[8] ΔVP8* and P2-P[8] ΔVP8-P[6] ΔVP8* were expressed in soluble and insoluble (inclusion bodies) forms. The yield of purified P2-P[8] ΔVP8*-P[8] ΔVP8* reached 90 mg/L of culture, whereas that of P2-P[8] ΔVP8-P[6] ΔVP8* was only ∼5 mg/L culture. The difference in yield between the 2 tandem fusion proteins was in part due to the different expression systems used. The former used a pET expression system and the latter employed a SUMO expression system which required an additional purification step: a cleavage and removal of the SUMO tag off the fusion protein, resulting in additional loss of target protein. Of note, P2-P[8] ΔVP8-P[6] ΔVP8* recombinant protein from pET expression system existed exclusively in insoluble form (data not shown). In contrast, introduction of the one addtional copy of P[8] ΔVP8* to P2-P[8] ΔVP8* recombinant protein enhanced the yield of soluble protein; the yield of P2-P[8] ΔVP8* was ∼46 mg/L culture as we previously reported.¹⁸ Of interest, introduction of P[6] ΔVP8* to P2-P[8] ΔVP8* in pET expression system compromised the expression of soluble protein. In this study, we mainly focused on evaluation of the immunogenicity of tandem fusion proteins, rather than the production of target proteins. Presumably, the yield of P2-P[8] ΔVP8-P[6] ΔVP8* recombinant protein can be further enhanced by optimization for pharmaceutical production. For example, the phase I trial launched by PATH has proven that the yield of P2-P[8] ΔVP8 recombinant protein can be significantly enhanced under the optimizaiton of expression system and the culture of bacteria. Based on our understanding, the low yield of P2-P[8] ΔVP8-P[6] ΔVP8* recombinant protein obtained in this study will not preclude the potential of its application later.

Despite current 2 oral human rotavirus vaccines, RotaTeq and Rotarix are proven to be safe and efficacious in selected countries the efficacy of the 2 vaccines still needs to be further enhanced, especially in resource deprived countries. Furthermore, more intensive surveillance for evaluation of the risk of intussusceptions and for applicability to immunocompromised humans are required, since both of the 2 live oral rotavirus vaccines slightly increase the risk of intussusception and RotaTeq® vaccination caused vaccine-acquired rotavirus shedding or diarrhea in severe combined immunodeficient (SCID) patients.^35-37 Of note, evidence from licensed vaccines has suggested that parenteral vaccines can be successful in preventing orally transmitted diseases due to poliovirus, hepatitis A and B viruses, Vibrio cholera and Salmonella typhi.^38-42 Accordingly, the parenteral vaccines, such as subunit vaccines, could be alternative vaccine candidates for prevention of rotavirus diseases, especially in countries where rotaviruses threaten the lives of infants and young children. Importantly, such parenteral vaccines are free from various concerns associated with live oral rotavirus vaccines and may work better in underdeveloped regions of the world where the vaccine is needed the most.

It is well known that (i) the incidence of individual G and/or P types in the same region can show a yearly fluctuation; (ii) the incidence of rotavirus G and/or P types in different regions within the same country can differ during the same year; and (iii) multiple G and P types can co-circulate within the same year.⁴³ In addition, extensive global epidemiological surveys have confirmed that G1-G4 as well as P[8] and P[4] genotypic strains are of global epidemiologic importance.^43,44 It is of note that uncommon G and P types causing a high incidence of human infection have been reported in various regions worldwide including G8, G9, G10, G12, and P[6].^45–49 Since in nature, almost all human rotavirus G types have been detected in combination with P[8], P[4] or P[6] specificity, the P2-P[8]ΔVP8*-P[8]ΔVP8* and P2-P[8]ΔVP8*-P[6]ΔVP8* vaccines generated in this study could be used at a minimum singly or preferably in bivalent formulation to provide antigenic coverage against almost all the rotavirus G (VP7) types and the 2 major P types of global as well as regional epidemiologic importance, including new and emerging G8, G9 and G12 rotaviruses.

Materials and Methods

Viruses and cell culture

Human rotavirus strains Wa (G1P[8])⁵⁰ and 1076 (G2P[6])⁵¹ were grown in primary Africa green monkey kidney (AGMK) cells (Diagnostic Hybrids, Athens, OH). Eagle's minimum essential medium (EMEM) supplemented with 0.5 µg/ml of trypsin [2 mg/ml (W/V)] (Sigma γ-irradiated trypsin, Sigma Chemical, St. Louiis, MO), 100 IU/ml of Penicillin, 100 µg/ml of Streptomycin and 2.5 µg/ml of Amphotericin B was used as maintenance medium. Additional rotavirus strains P (G3P[8]), VA70 (G4P[8]), W161 (G9P[8]), DS-1 (G2P[4]), 1290 (G8P[4]), L26 (G12P[4]), ST3 (G4P[6]) and 69M (G8P[10]) were cultured with medium above to be used in the neutralizing assay.

Vaccine plasmid construction

Rotaviral gene cDNA of human rotavirus Wa and 1076 strains were obtained by a reverse transcription-polymerase chain reaction (RT-PCR) procedure from viral RNAs extracted using TRIzol-LS (Thermo Fisher Scientific). The primers used were designed according to the genomic sequence of the RNA segment 4 of each rotavirus strain [GenBank accession numbers: FJ423116 (Wa) and M88480 (1076)]. Oligo nucleotide sequences included the restriction endonuclease sites Nde I and Sac I to facilitate the cloning of the inserts in multiple clone sites in an expression vector. The primers for constructing truncated P2-P[8] ΔVP8* were as follows: Forward:5′-tactcatatgcagtatataaaagcaaattctaaatttataggtataactgaactaggctccggctcaggcttagatggtccttatcagcc-3′, T cell epitope P2 encoding aa 830–844 of TT is italicized, and the linker sequence encoding GSGSG is in boldface, Nde I sites (underlined), Rerverse:5′- atggatcccagaccattattaatatattcattac-3′, BamH I site (underlined). The primers for P[8] ΔVP8* were as follows: Forward: 5′-atggatccggctcaggcttagatggtccttatcagccaactac-3′ BamH I site (underlined), Rerverse: 5′-atctaagcttgacagaccattattaatatattcattac-3′, Hind III site (underlined). The P[6] ΔVP8* fragment was amplified and firstly cloned into pET28 vector to facilitate the following the process. The primers were as follows: Forward: 5′-aatggatccggctcaggcgtactcgatggtccttatcaaccaac-3′, the linker sequence encoding GSGSG is in boldface, BamH Isite (underlined), Rerverse: 5′-tactcgagtaacccagtatttatatattcattacac-3′, Xho I site (underlined). The construction of recombinant plasmid for expression of a fusion gene P2-P[8] ΔVP8*-P[6] ΔVP8* was conducted according to manufacturer's instruction (pETite N-His SUMO Kan Vector, Lucigen). The construction of expression vector, pETite- P2-P[8] ΔVP8*-P[6] ΔVP8*, was conducted with following primers: 5′- cgcgaacagattggaggtcagtatataaaagcaaattc-3′(Forward), 5′-gtggcggccgctctattataacccagtatttatatattcattac-3′(Reverse) (The detailed information for primers used in this study is also shown in Table S1). VP8* cDNA was synthesized by using Superscript III reverse transcriptase (Thermo Fisher Scientific) and reaction parameters were as follows: 100–200 ng of genomic RNA was denatured with final concentration of 15% DMSO and incubated at 94°C for 3 min, followed by chilling on ice immediately. The first strand cDNA was synthesized under manufacturer's instruction. The fusion fragment was amplified with cDNA as the template by a Phusion Hot Start High-Fidelity DNA polymerase system (Thermo Fisher Scientific). Each PCR product was purified by a Wizard^® SV Gel and PCR Clean-Up System (Promega) by agarose gel electrophoresis. The two copies of P[8] ΔVP8* or P[8] ΔVP8*-P[6] ΔVP8*with P2 T cell epitope fusion fragment was cloned into pET28a vector (Novagen) harboring a 6×histidine tag for affinity column isolation of recombinant protein, creating pET28a-P2-P[8] ΔVP8*-P[8] ΔVP8* or pET28a-P2-P[8] ΔVP8*-P[6] ΔVP8*plasmid. Then the P2-P[8] ΔVP8*-P[6] ΔVP8* product was cloned into a linearized pETite vector (Lucigen) with homologous recombination strategy. The integrity and fidelity of the amplicons were confirmed by DNA sequencing.

Expression of recombinant protein

The expression plasmids pET28a-P2-P[8] ΔVP8*-P[8] ΔVP8* or pETite-P2-P[8] ΔVP8*-P[6] ΔVP8* were transformed into competent E. coli BL21(DE3)pLysS cells (pET system) or HI-control BL21(DE3) cells (pETite system) by the heat shock method. A single colony in a transform agar plate was inoculated into LB broth-containing 50 µg/ml kanamycin. When absorbance reading at 600 nm reached 0.5, the expression of each fusion protein was induced by the addition of 0.5 mM isopropyl-1-thio-β-D-galactoside at 18°C overnight. The recombinant E. coli cells were harvested by centrifugation at 10,000 ×g for 15 min at 4°C and stored at −80°C until use.

Purification of P2-P[8] ΔVP8*-P[8] ΔVP8* and P2-P[8] ΔVP8*-P[6] ΔVP8* proteins

Each protein was purified by affinity chromatography as described previously.²⁹ The SUMO tag at N-terminus of P2-P[8] ΔVP8*-P[6] ΔVP8* was removed using the SUMO express protease (Lucigen) according to the manufacturer's instruction. The purity of each recombinant protein was confirmed by SDS-PAGE, followed by a removal of imidazole in solution using a centrifugal filter unit (Millipore). The concentration of purified recombinant protein was measured by BCA assay (Thermo Fisher Scientific) according to the manufacturer's instruction. The level of endotoxin in each purified protein was quantified as previously described.²⁹ The proteins were stored at −80°C until use.

Western blot

Proteins were analyzed by 4–12% NuPAGE gel and transferred electrophoretically onto a nitrocellulose membrane (Whatman). The membrane was incubated at 4°C overnight with a hyperimmune guinea pig antiserum (1:50) raised against Wa (P[8]) or ST3 (P[6]) strain. Following washing 3 times, the membrane was incubated with a peroxidase-conjugated goat anti-guinea pig IgG (H⁺L) (KPL) (1:3,000) for 1 h at room temperature. The membrane was developed by the addition of 3,3′-diaminobenzidine (DAB) substrate (Sigma) as previously described.²⁹

Immunization of guinea pigs

Outbred female Hartley guinea pigs weighing 500–550 grams were purchased from Charles River Laboratories (Wilmington, MA). Guinea pigs were maintained under animal biosafety level 2 conditions in isolator cages for the entire period of each experiment. All animal experiments were done at the NIH, in compliance with the guidelines of the Institutional Animal Care and Use Committee of the National Institute of Allergy and Infectious Diseases or the National Institute of Child Health. Guinea pigs were immunized intramuscularly (IM) 3 times at 2-week intervals with 20µg of P2-P[8] ΔVP8*-P[8] ΔVP8* or P2-P[8] ΔVP8*-P[6] ΔVP8* protein with aluminum phosphate (AP) adjuvant (ADJU-PHOS™, Brenntag Biosector) (aluminum content of 100 µg/dose). Blood samples were collected prior to each immunization as well as 7 days after the third immunization.

Neutralization assay

Neutralizing antibody titer of each serum sample was determined by 60% plaque reduction neutralization (PRN) assay by using MA104 cells and 6-well plates as previously described,²⁹ except that the virus-serum mixture, after a one hour adsorption period, was left in the agarose overlay.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Elias Gonzales for technical assistance. We extend our appreciation to late Dr. Albert Z. Kapikian for his support of this project.

Funding

This work was partially supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA and National Natural Science Foundation of China (Grant No. 31201909) and Doctoral Initiating Project of Heilongjiang Province (No. LBH-Q13134).

Supplemental Material

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