Candidate peptide vaccine induced protection against classical swine fever virus

Former investigations demonstrated that the envelope glycoprotein E2 could protect pigs from classical swine fever virus (CSFV). Based on these findings, we prepared synthetic peptide vaccine using E2 N-terminal antigenic units B/C and hoped to induce protective activity against lethal challenge of virulent CSFV strain Shimen. Five overlapped peptides sequence-covering amino acids 693-777 on E2 of Shimen were synthesized and then conjugated with bovine serum albumin (BSA), respectively. In the vaccination course, the candidate peptide vaccines in combination (multi-peptide vaccine (MPV)) were applied for immunization of pigs (n=10) and induced strong antibody response against CSFV. It is subsequently demonstrated that this peptide vaccine could provide immunized pigs complete protection against lethal CSFV challenge as C-strain does, while all non-immunized pigs in negative control group manifested obvious typical symptoms and died during the second and third weeks after viral challenge. In order to confirm the neutralizing activity of the polyclonal antibodies induced by MPV, neutralization assay were carried out on rabbits. The live C-strain alone could ordinarily induce typical fever on rabbits. The typical fever of rabbits induced by the live C-strain could be inhibited by pre-incubation with the anti-sera (dilution 1:4 and 1:16) induced by MPV, but not inhibited by pre-incubation with the same anti-sera from which the antibodies against five peptides were removed by peptide-specific affinity chromatography, which indicates that these peptide-specific antibodies in the anti-sera induced by MPV provided protective activity against CSFV. Our finding provides a new way to develop marker vaccine against CSFV.     

Efficacy of marker vaccine candidate CP7_E2alf in piglets with maternally derived C-strain antibodies

Marker vaccines offer the possibility to differentiate classical swine fever (CSF) infected from CSF vaccinated animals based on serology and their implementation will ensure free trade with pigs. Therefore, new generations of promising marker vaccines have been developed, among them the chimeric vaccine CP7_E2alf. However, in populations previously vaccinated with live attenuated vaccines like the C-strain, passive immunity through maternal antibodies can interfere with efficacy of CP7_E2alf vaccination. Therefore, the efficacy of CP7_E2alf was examined in piglets from sows vaccinated once intramuscularly with C-strain vaccine 4 weeks before farrowing. Thus, these piglets were vaccinated intramuscularly with CP7_E2alf at the age of 5 or 8 weeks. Subsequently, the piglets and their mock-vaccinated littermate controls were challenged 2 weeks post vaccination with highly virulent Classical swine fever virus (CSFV) strain “Koslov”.     

Enhanced protective immunity to CSFV E2 subunit vaccine by using IFN-γ as immunoadjuvant in weaning piglets

The glycoprotein E2 of classical swine fever virus (CSFV) is a major immunogenic protein that induces neutralizing antibodies and protective immunity. Thus, E2 is a suitable target antigen for the development of genetically engineered CSFV vaccines. However, these vaccines cannot generate complete protective immunity in their hosts, thereby limiting the scope of applications under field conditions. IFN-γ is an immune adjuvant that has been shown to enhance antigen immune response in various experimental models. In this study, porcine IFN-γ was used to improve the immunogenicity of the CSFV E2 subunit vaccine in pigs. Pigs were immunized with E2 subunit vaccine alone or in combination with IFN-γ. Results demonstrated that porcine IFN-γ did not enhance the CSFV-specific antibody and neutralizing antibody titers compared with the E2 subunit vaccine alone. However, co-administration of the E2 and IFN-γ subunit vaccines significantly enhanced the CSFV-specific IFN-γ expression. These findings indicated that porcine IFN-γ can increase cellular immune responses to E2 protein in pigs. Furthermore, co-immunization with E2 + IFN-γ subunit vaccine and C-strain conferred complete protection against CSFV. In contrast, E2 subunit vaccines provided incomplete protection in pigs. These results indicated that using IFN-γ as an adjuvant with CSFV E2 subunit vaccines can enhance the specific protective immune response. Therefore, E2 + IFN-γ subunit vaccine is a promising marker vaccine candidate for the control and eradication of CSF.     

Comprehensive evaluation of the adenovirus/alphavirus-replicon chimeric vector-based vaccine rAdV-SFV-E2 against classical swine fever

Classical swine fever (CSF) is an economically important, highly contagious swine disease caused by classical swine fever virus (CSFV). Marker vaccines and companion serological diagnostic tests are thought to be a promising strategy for future control and eradication of CSF. Previously, we have demonstrated that an adenovirus-vectored Semliki forest virus replicon construct expressing the E2 glycoprotein from CSFV, rAdV-SFV-E2, induced sterile immunity against a lethal CSFV challenge. In this study, we further evaluated the vaccine with respect to its safety, number and dose of immunization, and effects of maternally derived antibodies, re-immunization of the vaccine or co-administration with pseudorabies vaccine on the vaccine efficacy. The results showed that: (1) the vaccine was safe for mice, rabbits and pigs; (2) two immunizations with a dose as low as 6.25 × 10⁵ TCID₅₀ or a single immunization with a dose of 10⁷ TCID₅₀ rAdV-SFV-E2 provided complete protection against a lethal CSFV challenge; (3) maternally derived antibodies had no inhibitory effects on the efficacy of the vaccine; (4) the vaccine did not induce interfering anti-vector immunity; and (5) co-administration of rAdV-SFV-E2 with a live pseudorabies vaccine induced antibodies and protection indistinguishable from immunization with either vaccine administered alone. Taken together, the chimeric vaccine represents a promising marker vaccine candidate for control and eradication of CSF.     

Recombinant classical swine fever (CSF) viruses derived from the Chinese vaccine strain (C-strain) of CSF virus retain their avirulent and immunogenic characteristics

Two recombinant classical swine fever (CSF) viruses (Flc2, Flc3) transcribed from a DNA copy of the genome of the Chinese (C) strain, a CSF virus vaccine strain, were characterized in vivo in rabbits and pigs. Rabbits were inoculated intravenously with Flc2 or Flc3, the parent C-strain virus, a biologically cloned C-strain or CSF virus strain Brescia (C.1.1.1). After 24-96 h fever was detected in the rabbits inoculated with the different C-strain viruses. Apart from those in the control group, all the C-strain inoculated rabbits had developed CSF virus neutralizing antibodies 4 weeks later and were protected against a parent C-strain challenge. In the second experiment, pigs were inoculated with the parent C-strain or recombinant C-strain virus (Flc2 or Flc3) and then challenged after 4 weeks with the virulent CSF virus strain Brescia. None of the pigs showed clinical signs of classical swine fever after vaccination or challenge, whereas the control pigs developed clinical signs typical for acute CSF. Pigs inoculated with the different C-strain viruses were not viremic after inoculation or challenge, and CSF virus neutralizing antibodies were detected from day 14 onwards. The results from both experiments demonstrated that the two recombinant viruses had retained the biological and immunogenic properties of the parent C-strain in rabbits and pigs. We conclude that the full-length cDNA of the C-strain can serve as a matrix for further development of a live recombinant CSF virus marker vaccine.     

Novel marker vaccines against classical swine fever

Classical swine fever (CSF) is one of the most devastating epizootic diseases of pigs worldwide. For eradication and control purposes, CSF vaccination is an important tool, and efficacious and safe attenuated vaccines have been available for many decades (for example, the C-strain vaccines). In addition to administering them parenterally, live attenuated vaccines are also administered orally for the control and eradication of CSF in wild boar populations. However, antibodies against live attenuated vaccines do not allow to differentiate infected from vaccinated animals (DIVA principle) and the mechanism responsible for attenuation is not known. Only a few years ago the first DIVA vaccines based on baculovirus-expressed E2 glycoprotein have been put on the market [Hulst MM, Westra DF, Wensvoort G, Moormann RJ. Glycoprotein E1 of hog cholera virus expressed in insect cells protects swine from hog cholera. J Virol 1993;67(9):5435-42]. However, these subunit E2 marker vaccines are less efficient and more than one parenteral application is necessary. Furthermore, oral vaccination is not possible. Taking these disadvantages into account, the development of novel CSF vaccines has been focussed on five different strategies, mainly based on genetically engineered constructs: (1) immunogenic CSFV peptides, (2) DNA vaccines, (3) viral vectors expressing CSFV proteins, (4) chimeric pestiviruses, and (5) trans-complemented deleted CSFV genomes (replicons).     

A novel alphavirus replicon-vectored vaccine delivered by adenovirus induces sterile immunity against classical swine fever

Low efficacy of gene-based vaccines due to inefficient gene delivery and expression has been major bottleneck of their applications. Efforts have been made to improve the efficacy, such as gene gun and electroporation, but the strategies are difficult to put into practical use. In this study, we developed and evaluated an adenovirus-delivered, alphavirus replicon-vectored vaccine (chimeric vector-based vaccine) expressing the E2 gene of classical swine fever virus (CSFV) (rAdV-SFV-E2). Rabbits immunized with rAdV-SFV-E2 developed CSFV-specific antibodies as early as 9 days and as long as 189 days and completely protected from challenge with C-strain. Pigs immunized with rAdV-SFV-E2 (n = 5) developed robust humoral and cell-mediated responses to CSFV and were completely protected from subsequent lethal CSFV infection clinically and virologically. The level of immunity and protection induced by rAdV-SFV-E2 was comparable to that provided by the currently used live attenuated vaccine, C-strain. In contrast, both the conventional alphavirus replicon-vectored vaccine pSFV1CS-E2 and conventional adenovirus-vectored vaccine rAdV-E2 provided incomplete protection. The chimeric vector-based vaccine represents the first gene-based vaccine that is able to confer sterile immunity and complete protection against CSFV. The new-concept vaccination strategy may also be valuable in vaccine development against other pathogens.     

Construction and efficacy of a new live chimeric C-strain vaccine with DIVA characteristics against classical swine fever

To develop the new classical swine fever (CSF) vaccine candidate with differentiating infected vaccinated animals (DIVA) characteristics, a chimeric CSF virus (CSFV) was constructed based on an infectious cDNA clone of the CSF vaccine C-strain. The 5’- and 3’-untranslated regions (UTRs) and partial E2 region (residues 690-860) of the C-strain were substituted with the corresponding regions of bovine viral diarrhoea virus (BVDV) to construct the chimeric cDNA clone pC/bUTRs-tE2. The chimeric virus rC/bUTRs-tE2 was generated by several passages of pC/bUTRs-tE2-transfected PK15 cells. Stable growth and genetic properties of rC/bUTRs-tE2 were obtained after 30 serial passages. Compared to parental rC/bUTRs-tE2 (1^st passage), two residue mutations (M834K and M979K) located in E2 in rC/bUTRs-tE2 P30 were observed. Compared to the C-strain, rC/bUTRs-tE2 exhibited unchanged cell tropism and decreased plaque-forming ability. Substituting the C-strain UTRs with the BVDV UTRs resulted in significantly increased viral replication in PK15 cells. Compared to CSFV E^rns-positive and BVDV tE2-negative antibody responses induced by the CSF vaccine C-strain, immunization of rabbits and piglets with rC/bUTRs-tE2 resulted in serological profiles of CSFV E^rns- and BVDV tE2-positive antibodies, which are used to serologically discriminate pigs that are clinically infected and vaccinated. Vaccination of piglets with rC/bUTRs-tE2 conferred complete protection against lethal CSFV challenge. Our results suggest that rC/bUTRs-tE2 is a promising new CSF marker vaccine candidate.     

Hypervariable antigenic region 1 of classical swine fever virus E2 protein impacts antibody neutralization

Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies and confers protection against CSFV infection. There are three hypervariable antigenic regions (HAR1, HAR2 and HAR3) of E2 that are different between the group 1 vaccine C-strain and group 2 clinical isolates. This study was aimed to characterize the antigenic epitope region recognized by monoclonal antibody 4F4 (mAb-4F4) that is present in the group 2 field isolate HZ1-08, but not in the C-strain, and examine its impact on neutralization titers when antisera from different recombinant viruses were cross-examined. Indirect ELISA with C-strain E2-based chimeric proteins carrying the three HAR regions showed that the mAb-4F4 bound to HAR1 from HZ1-08 E2, but not to HAR2 or HAR3, indicating that the specific epitope is located in the HAR1 region. Of the 6 major residues differences between C-strain and field isolates, Glu713 in the HAR1 region of strain HZ1-08 is critical for mAb-4F4 binding either at the recombinant protein level or using intact recombinant viruses carrying single mutations. C-strain-based recombinant viruses carrying the most antigenic part of E2 or HAR1 from strain HZ1-08 remained non-pathogenic to pigs and induced good antibody responses. By cross-neutralization assay, we observed that the anti-C-strain serum lost most of its neutralization capacity to RecC-HZ-E2 and QZ-14 (subgroup 2.1d field isolate in 2014), and vice versa. More importantly, the RecC-HAR1 virus remained competent in neutralizing ReC-HZ-E2 and QZ-14 strains without compromising the neutralization capability to the recombinant C-strain. Thus, we propose that chimeric C-strain carrying the HAR1 region of field isolates is a good vaccine candidate for classical swine fever.     

Sub-subgenotype 2.1c isolates of classical swine fever virus are dominant in Guangdong province of China, 2018

Classical swine fever (CSF) continues to be a devastating infectious disease for the swine industry in China and commonly exists as wild or atypical types. From June 3rd to October 3rd, 2018, outbreaks of typical CSF cases with mortality rates of 42–86% occurred in 11 swine herds in five cities of Guangdong province, and were confirmed by RT-PCR. Phylogenetic analyses based on the nucleotide sequences of full-length E2 genes showed that the CSFV isolates collected in Guangdong, 2018 grouped into sub-subgenotype 2.1c and formed a separate clade from previously identified 2.1c isolates. Sequence comparison further confirmed the distance between the novel emergent and previously identified 2.1c isolates, with shared 94.5–98.2% and 97.8–99.7% identities at the nucleotide and amino acid levels respectively. Furthermore, 2.1c isolates collected in 2018 from Guangdong province contained a unique amino acid substitution (K174R) in the E2 protein in comparison with other 2.1c representative strains and CSFV 2.1, 2.2, 2.3 strains. Of note, the novel emergent 2.1c isolates are neutralized by sera from C-strain vaccinated sows, indicating that C-strain is still efficacious for protection against field isolates of CSFV.     

Candidate peptide-vaccines induced immunity against CSFV and identified sequential neutralizing determinants in antigenic domain A of glycoprotein E2

Antigenic domain A is a highly conserved unit on envelope protein E2 of classical swine fever virus (CSFV). It was found that mutant E2 containing only unit A, with the unit BC deleted, provided immunized pigs with complete protection against the lethal challenge. In this study, six overlapping peptides (A1-A6) covering this unit were synthesized and conjugated to bovine serum albumin (BSA). Two candidate multi-peptide-vaccines (MPVs) using aluminum adjuvant successfully induced potent immunity against CSFV in pigs. Although both candidate MPVs failed to provide complete protection, they showed better protective activity than that induced by C-strain. Subsequently, neutralizing epitopes in unit A were identified using a panel of peptide-vaccines (PVs). Six candidate peptide-vaccines (PV-An, n=1-6) were separately given to six groups of pigs. Among these candidates, PV-A2 and PV-A6 exhibited the most potent protective activity, while the other four showed weaker or almost no effects. Moreover, the polyclonal antibodies induced by PV-A2 and PV-A6 were capable of neutralizing C-strain virus at the dilution 1:16 in vitro. Thus, two principal sequential neutralizing determinants covered by peptide A2 (aa792-814) and A6 (aa844-865) were demonstrated to exist in the antigenic domain A, and can be recruited in developing new effective "marker vaccine" against CSFV.     

Characterisation of vaccine-induced, broadly cross-reactive IFN-γ secreting T cell responses that correlate with rapid protection against classical swine fever virus

Live attenuated C-strain classical swine fever viruses (CSFV) provide a rapid onset of protection, but the lack of a serological test that can differentiate vaccinated from infected animals limits their application in CSF outbreaks. Since immunity may precede antibody responses, we examined the kinetics and specificity of peripheral blood T cell responses from pigs vaccinated with a C-strain vaccine and challenged after five days with a genotypically divergent CSFV isolate. Vaccinated animals displayed virus-specific IFN-γ responses from day 3 post-challenge, whereas, unvaccinated challenge control animals failed to mount a detectable response. Both CD4(+) and cytotoxic CD8(+) T cells were identified as the cellular source of IFN-γ. IFN-γ responses showed extensive cross-reactivity when T cells were stimulated with CSFV isolates spanning the major genotypes. To determine the specificity of these responses, T cells were stimulated with recombinant CSFV proteins and a proteome-wide peptide library from a related virus, BVDV. Major cross-reactive peptides were mapped on the E2 and NS3 proteins. Finally, IFN-γ was shown to exert potent antiviral effects on CSFV in vitro. These data support the involvement of broadly cross-reactive T cell IFN-γ responses in the rapid protection conferred by the C-strain vaccine and this information should aid the development of the next generation of CSFV vaccines.     

Spying the neutralizing epitopes on E2 N-terminal by candidate epitope-vaccines against classical swine fever virus

Our previous study proved that the N-terminal (aa693-711) of glycoprotein E2 contained sequential neutralizing epitopes. In this study, four candidate epitope-vaccines (EVs) were separately prepared and evaluated. Among them, epitope-vaccine EV-BC1a (BC1a: aa693-699) induced high level of epitope-specific neutralizing antibodies and exhibited similar protective capability with that induced by Chinese vaccine strain (C-strain). These results confirmed CKEDYRY (aa693-699) as a principal sequential neutralizing epitope on E2 N-terminal. Moreover, these findings also indicate that epitope-vaccine is a potent candidate strategy for marker vaccine against classical swine fever virus (CSFV).     

Yeast expressed classical swine fever E2 subunit vaccine candidate provides complete protection against lethal challenge infection and prevents horizontal virus transmission

Classical swine fever (CSF) caused by the classical swine fever virus (CSFV) is a highly contagious swine disease resulting in large economical losses worldwide. The viral envelope glycoprotein E(rns) and E2 are major targets for eliciting antibodies against CSFV in infected animals. A Pichia pastoris yeast expressed E2 protein (yE2) has been shown to induce a protective immune response against CSFV challenge. The purpose of this study is to determine the optimal dose of yE2 and its efficacy on the prevention of virus horizontal transmission. A yeast-expressed E(rns) (yE(rns)) protein was also included to evaluate its immunogenicity. The yE(rns) vaccinated pigs seroconverted to CSFV-E(rns)-specific antibody but no neutralizing antibody was detected and none survived after challenge infection, suggesting yE(rns) and yE2 retain correct immunogenicity but only the yE2 is able to induce a protective immune response. All three doses of yE2 (200, 300, and 400μg) could elicit high titers of neutralizing antibodies and protective responses after challenge. The yE2/200 group demonstrated a mild fever response but recovered soon, and none of the yE2/300 and yE2/400 pigs became febrile. The optimal dose of yE2 was recommended to be 300μg of the total amount of secreted proteins. In addition, the yE2 vaccine could cross-protect from all three genotypes of viruses. Further, the yE2 vaccine efficacy in preventing virus horizontal transmission was evaluated by cohabitation of unimmunized sentinels 3 days after challenge infection. All the sentinel pigs were alive and had no clinical symptoms confirming yE2 vaccine could confer a protective immune response and prevent horizontal transmission of CSFV.     

A Semliki Forest virus replicon vectored DNA vaccine expressing the E2 glycoprotein of classical swine fever virus protects pigs from lethal challenge

Classical swine fever virus (CSFV) causes significant losses in pig industry in many countries in Asia and Europe. The E2 glycoprotein of CSFV is the main target for neutralizing antibodies. Recently, the replicon of alphaviruses, such as Semliki Forest virus (SFV), has been developed as replicative expression vectors for gene delivery. In this study, we constructed a plasmid DNA based on SFV replicon encoding the E2 glycoprotein of CSFV and evaluated its efficacy in rabbits and pigs. The results showed that the animals immunized with the DNA vaccine developed CSFV-specific neutralizing antibodies and were protected from virulent or lethal challenge. This demonstrates that the SFV replicon-derived DNA vaccine can be a potential marker vaccine against CSFV infections.     

A novel combined vaccine against classical swine fever and porcine epidemic diarrhea viruses elicits a significant Th2-favored humoral response in mice

Many Chinese breeding pigs are repeatedly vaccinated against classical swine fever virus (CSFV) and porcine epidemic diarrhea virus (PEDV), which cause fatal, highly contagious diseases. To reduce their high frequency vaccination-induced immune stress, we constructed a combined vaccine based on the E2 protein of CSFV and the S1 spike protein subunit of PEDV (named E2-S1). In mice, the E2-S1 vaccine elicited higher neutralizing antibody titers and IgG1/IgG2a ratios against CSFV and PEDV than those induced by individual E2 or S1 vaccines. Moreover, it elicited high IL-4 expression, but no IFN-γ expression. The results suggest that good compatibility exists between E2 and S1 antigens, and the E2-S1 vaccine can elicit a strong Th2-type cell-mediated humoral immune response. The E2-S1 recombinant fusion protein provides a novel vaccine candidate against both CSFV and PEDV, laying the foundation for future combination vaccines against swine diseases.     

Safety,efficacy, and DIVA feasibility on a novel live attenuated classical swine fever marker vaccine candidate

Classical swine fever virus (CSFV) is the etiological agent of classical swine fever, a highly contagious disease that causes significant economic losses to the swine industry. Systemic prophylactic immunization with the live attenuated vaccine, the C-strain vaccine, is one of the effective measures for CSF control. However, one of the limitations of the C-strain vaccine is that the field strains-infected animals cannot be differentiated from the C-strain vaccinated herds by serological tests (DIVA). This constraint hampers the practical usage of the C-strain vaccine to eradicate the CSF in China. In the current study, a novel CSF modified live marker vaccine candidate was constructed based on the attenuation of the prevalent 2.1 genotype strain by the deletion of two virulence associated functional residues in the CSFV E^rns, H79, and C171. Meanwhile, four residues S14, G22, E24, and E25 were identified specifically for the 6B8 mAb binding to the CSFV E2 as the novel conformational epitope. Then four substitutions of S14K, G22A, E24R, and G25D were further incorporated in the double deletion construct as a negative serological marker. Finally, the double-deletion marker MLV candidate GD18-ddErnHC-KARD was rescued, and its safety and efficacy profiles were evaluated in piglets. The safety study results indicated that the candidate did not induce fever, clinical signs, or pathological lesions with a high dose of 10^5.0 TCID₅₀, and in addition, no virus shedding was detected until 21 days post-inoculation. Meanwhile, the efficacy study results demonstrated that at a low dose of 10^3.0 TCID₅₀, it conferred complete clinical protection and no virus shedding was detected after the challenge with a highly virulent Shimen strain. Importantly, the infected animals were differentiated using the accompanied DIVA ELISA. These results constitute a proof-of-concept for rationally designing a CSF antigenically marked modified live vaccine candidate.     

Quadruple antigenic epitope peptide producing immune protection against classical swine fever virus

Research on epitope-based vaccines is a current focus in the development of new vaccines against classical swine fever virus (CSFV). The present study aimed to engineer a quadruple antigenic epitope peptide of the CSFV immunogen E2 glycoprotein by splice overlap extension (SOE) PCR, expressed in E. coli fused with glutathione S-transferase (GST), and named rGST-4E. Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showed that purified rGST-4E had an excellent immunoreactivity with swine anti-CSFV serum and rabbit anti-E2 serum. Animal vaccination trials showed that the rGST-4E was more immunogenic than mono-epitope peptide and was able to produce effective immune protection in rabbits against challenge with hog cholera lapinized virus, and in pigs against challenge with virulent CSFV. These data show that the recombinant repeated epitope peptide could be considered a potential epitope-based vaccine for prevention of the disease.     

Development of candidate combination vaccine for hepatitis E and hepatitis B: A liposome encapsulation approach

To reduce extra injections, cost and ensure better coverage, use of combination vaccines is preferable. An attempt was made to evaluate the encapsulation of hepatitis E virus neutralizing epitope (NE) region and hepatitis B virus surface antigen (HBsAg) in liposomes as DNAs, proteins and DNA + protein. Mice groups were immunized with different liposome-encapsulated formulations and monitored for anti-HEV and anti-HBs titres, IgG subtypes, antigen-specific lymphocyte proliferation and cytokine levels. The protective levels of anti-HBs and in vitro virus-binding capacity of anti-HEV antibodies were assessed. Liposome-encapsulated DNA either singly or in combination did not elicit antibody response. Anti-HEV and anti-HBs IgG titres of individual component of protein alone (Lipo-E-P/Lipo-B-P) or DNA + protein formulations (Lipo-E-DP/Lipo-B-DP) were comparable to respective titres in combination vaccine of protein (Lipo-BE-P) and DNA + protein formulations (Lipo-BE-DP). IgG1 levels were significantly higher in Lipo-BE-P group whereas, equivalent levels of IgG1 and IgG2a were observed in Lipo-BE-DP group against both components of the vaccine. Combination vaccine group showed mixed Th1/Th2 cytokine profile. Liposome entrapped NE and HBsAg in protein and DNA + protein formats induce excellent immune response to both the components and need to be evaluated in higher animals.