Immunogenicity and protective efficacy of recombinant pseudorabies virus expressing the two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) infection still remains today as the most significant health threat to swine and poses a challenge to current vaccination strategies. To develop a new generation of vaccine against PRRSV, a live attenuated pseudorabies virus (PRV) was used as vaccine vector to express the two major membrane-associated proteins (GP5 or M) of PRRSV in various forms. Four PRV recombinants, rPRV-GP5 (expressing native GP5), rPRV-GP5m (expressing GP5m, a modified GP5), rPRV-GP5-M (co-expressing GP5 and M proteins), rPRV-GP5m-M (co-expressing GP5m and M proteins) were generated. Mouse immunized with all these recombinants developed comparable PRV-specific humoral immune responses and provided complete protection against a lethal PRV challenge. However, the highest level of PRRSV-specific neutralizing antibodies and lymphocyte proliferative responses was observed in mice immunized with rPRV-GP5m-M. The immunogenicity and protective efficiency of rPRV-GP5m-M were further evaluated in the piglets. Compared to commercial PRRSV killed vaccine, detectable PRRSV-specific neutralizing antibody and higher lymphocyte proliferative responses could be developed in piglets immunized with rPRV-GP5m-M before virus challenge. Furthermore, more efficient protection against a PRRSV challenge was obtained in piglets immunized with rPRV-GP5m-M, as showed by the balanced body-temperature fluctuation, shorter-term viremia, lower proportion of virus load in nasal and oropharyngeal scrapings and tissues, and milder lung lesions. These data indicate that the recombinant rPRV-GP5m-M is a promising candidate bivalent vaccine against both PRV and PRRSV infection.     

Immunogenicity of the highly pathogenic porcine reproductive and respiratory syndrome virus GP5 protein encoded by a synthetic ORF5 gene

Since May 2006, a highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV), which causes continuous high fever and a high proportion of deaths in vaccinated pigs of all ages, has emerged and prevailed in Mainland China. Huge efforts should be made towards the development of an efficient vaccine against the highly pathogenic PRRSV. Although the ORF5-encoded GP5 is the most important immunogenic protein, accumulating evidences have demonstrated that incomplete protection conferred by GP5-based vaccines. The inability to induce robust protective immunity has been postulated to be associated with the presence of a non-neutralizing decoy epitope and heavy glycosylation in close to its neutralizing epitope. In this study, a synthetic ORF5 gene (SynORF5) was engineered with the codon usage optimized for mammalian cell expression based on the native ORF5 gene of highly pathogenic PRRSV strain WUH3. Additional modifications, i.e., inserting a Pan DR T-helper cell epitope (PADRE) between the neutralizing epitope and the non-neutralizing decoy epitope, and mutating four potential N-glycosylation sites (N30, N34, N35 and N51) were also included in the synthetic ORF5 gene. The immunogenicity of the SynORF5-encoded GP5 was evaluated by DNA vaccination in mice and piglets. Results showed that significantly enhanced GP5-specific ELISA antibody, PRRSV-specific neutralizing antibody, IFN-γ level, as well as lymphocyte proliferation response, could be induced in mice and piglets immunized with DNA construct encoding the modified GP5 than those received DNA vaccine expressing the native GP5. The enhanced immunogenicity of the modified GP5 will be useful to facilitate the development of efficient vaccines against the highly pathogenic PRRSV in the future.     

Alternative codon usage of PRRS virus ORF5 gene increases eucaryotic expression of GP(5) glycoprotein and improves immune response in challenged pigs

Pigs exposed to GP(5) protein of PRRSV by means of DNA immunization develop specific neutralizing and protecting antibodies. Herein, we report on the consequences of codon bias, and on the favorable outcome of the systematic replacement of native codons of PRRSV ORF5 gene with codons chosen to reflect more closely the codon preference of highly expressed mammalian genes. Therefore, a synthetic PRRSV ORF5 gene (synORF5) was constructed in which 134 nucleotide substitutions were made in comparison to wild-type gene (wtORF5), such that 59% (119) of wild-type codons were replaced with known preferable codons in mammalian cells. In vitro expression in mammalian cells of synORF5 was considerably increased comparatively to wtORF5, following infection with tetracycline inducible replication-defective human adenoviral vectors (hAdVs). After challenge inoculation, SPF pigs vaccinated twice with recombinant hAdV/synORF5 developed earlier and higher antibody titers, including virus neutralizing antibodies to GP(5) than pigs vaccinated with hAdV/wtORF5. Data obtained from animal inoculation studies suggest direct correlation between expression levels of immunogenic structural viral proteins and immune response.     

A novel baculovirus vector shows efficient gene delivery of modified porcine reproductive and respiratory syndrome virus antigens and elicits specific immune response

Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating epizootic of porcine species. Current vaccines are inadequate to control the disease burden and outbreaks in the field. We report a novel baculovirus vaccine vector with White spot syndrome virus immediate early 1 shuttle promoter, with strong activity in both insect cells and mammalian cells, for immunization against PRRSV. The insect cell cultured baculovirus vector produces PRRSV envelope glycoproteins ORF2a, ORF3, ORF4 and ORF5, which are similar to the antigens in the infectious PRRS virion, and these antigens are stably incorporated on the surface of the baculovirus. Further, the baculovirus vector efficiently transduces these antigens in cells of porcine origin, thereby simulating a live infection. The baculovirus vectored PRRSV antigens, upon inoculation in mice, elicits robust neutralizing antibodies against the infective PRRS virus. Further, the experiments indicate that hitherto under emphasized ORF2a and ORF4 are important target antigens for neutralizing PRRSV infectivity.     

Construction and immunogenicity of recombinant Mycobacterium bovis BCG expressing GP5 and M protein of porcine reproductive respiratory syndrome virus

Mycobacterium bovis BCG was used to express a truncated form of GP5 (lacking the first 30 NH(2)-terminal residues) and M protein of porcine reproductive and respiratory syndrome virus (PRRSV). The PRRSV proteins were expressed in BCG under control of the mycobacterial hsp60 gene promoter either in the mycobacterial cytoplasm (BCGGP5cyt and BCGMcyt) or as MT19-fusion proteins on the mycobacterial surface (BCGGP5surf and BCGMsurf). Mice inoculated with BCGGP5surf and BCGMsurf developed antibodies against the viral proteins at 30 days post-inoculation (dpi) as detected by ELISA and Western blot. By 60 dpi, the animals developed titer of neutralizing antibodies of 8. A PRRSV-specific gamma interferon response was also detected in splenocytes of recombinant BCG-inoculated mice at 60 and 90 dpi. These results indicate that BCG was able to express antigens of PRRSV and elicit an immune response against the viral proteins in mice.     

Characterization of antigenic regions in the porcine reproductive and respiratory syndrome virus by the use of peptide-specific serum antibodies

The porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that causes reproductive failure in sows and boars, and respiratory disease in pigs of all ages. Antibodies against several viral envelope proteins are produced upon infection, and the glycoproteins GP4 and GP5 are known targets for virus neutralization. Still, substantial evidence points to the presence of more, yet unidentified neutralizing antibody targets in the PRRSV envelope proteins. The current study aimed to identify and characterize linear antigenic regions (ARs) within the entire set of envelope proteins of the European prototype PRRSV strain Lelystad virus (LV). Seventeen LV-specific antisera were tested in pepscan analysis on GP2, E, GP3, GP4, GP5 and M, resulting in the identification of twenty-one ARs that are capable of inducing antibodies upon infection in pigs. A considerable number of these ARs correspond to previously described epitopes in different European- and North-American-type PRRSV strains. Remarkably, the largest number of ARs was found in GP3, and two ARs in the GP3 ectodomain consistently induced antibodies in a majority of infected pigs. In contrast, all remaining ARs, except for a highly immunogenic epitope in GP4, were only recognized by one or a few infected animals. Sensitivity to antibody-mediated neutralization was tested for a selected number of ARs by in vitro virus-neutralization tests on alveolar macrophages with peptide-purified antibodies. In addition to the known neutralizing epitope in GP4, two ARs in GP2 and one in GP3 turned out to be targets for virus-neutralizing antibodies. No virus-neutralizing antibody targets were found in E, GP5 or M. Since the neutralizing AR in GP3 induced antibodies in a majority of infected pigs, the immunogenicity of this AR was studied more extensively, and it was demonstrated that the corresponding region in GP3 of virus strains other than LV also induces virus-neutralizing antibodies. This study provides new insights into PRRSV antigenicity, and contributes to the knowledge on protective immunity and immune evasion strategies of the virus.     

Purifying selection in porcine reproductive and respiratory syndrome virus ORF5a protein influences variation in envelope glycoprotein 5 glycosylation

Porcine reproductive and respiratory syndrome virus ORF5a protein is encoded in an alternate open reading frame upstream of the major envelope glycoprotein (GP5) in subgenomic mRNA5. Bioinformatic analysis of 3466 type 2 PRRSV sequences showed that the two proteins have co-evolved through a fine balance of purifying codon usage to maintain a conserved RQ-rich motif in ORF5a protein, while eliciting a variable N-linked glycosylation motif in the alternative GP5 reading frame. Conservation of the ORF5a protein RQ-motif also explains an anomalous uracil desert in GP5 hypervariable glycosylation region. The N-terminus of the mature GP5 protein was confirmed to start with amino acid 32, the hypervariable region of the ectodomain. Since GP5 glycosylation variability is assumed to result from immunological selection against neutralizing antibodies, these findings show that an alternative possibility unrelated to immunological selection not only exists, but provides a foundation for investigating previously unsuspected aspects of PRRSV biology. Understanding functional consequences of subtle nucleotide sequence modifications in the region responsible for critical function in ORF5a protein and GP5 glycosylation is essential for rational design of new vaccines against PRRS.     

Immune responses of pigs inoculated with a recombinant fowlpox virus coexpressing GP5/GP3 of porcine reproductive and respiratory syndrome virus and swine IL-18

Two recombinant fowlpox viruses (rFPV-ORF5-ORF3 and rFPV-IL-18-ORF5-ORF3) containing the ORF5/ORF3 cDNAs of PRRSV (strain Chang Chun) and IL-18 of swine were constructed and evaluated for theirs abilities to induce humoral and cellular responses in piglets. In addition, their abilities to protect piglets against homologous virus challenge were examined. All piglets were given booster vaccinations at 21 days after the initial inoculation, and all piglets were challenged at 60 after the initial inoculation. Control groups were inoculated with wild-type fowlpox virus (wtFPV). All animals vaccinated with rFPV-ORF5-ORF3 and rFPV-IL-18-ORF5-ORF3 developed specific anti-PRRSV ELISA antibody and neutralizing antibody, as well as T-lymphocyte proliferation response. To evaluate the cellular immune function, IFN-gamma production in pigs serum and T-lymphocytes (CD4 and CD8 T cells) in peripheral blood were examined. Following challenge with a pathogenic strain of PRRSV (strain Chang Chun), piglets inoculated with recombinant fowlpox virus (rFPV) showed lower (P<0.05) temperature, viremia and virus load in bronchial lymph nodes than control animals, suggesting the establishment of partial protection against PRRSV infection. The results demonstrated the potential use of a fowlpox virus-based recombinant vaccine in the control and prevention of PRRSV infections.     

Construction and immunogenicity of DNA vaccines encoding fusion protein of murine complement C3d-p28 and GP5 gene of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) has recently caused catastrophic losses in swine industry worldwide. The commercial vaccines only provide a limited protection against PRRSV infection. At present, DNA vaccine is the focus on the new vaccines. The gene fragment (p28) coding for the molecular adjuvants complement protein C3d (mC3d) from BALB/c mouse was cloned and expressed as a fusion protein for its application in the vaccine study of mice. Three potential vaccines construct units were engineered to contain two, four and six copies of mC3d-p28 coding gene linked to the GP5 gene of PRRSV and one vaccine expressing GP5 alone (pcDNA3.1-GP5) was constructed. Subsequently, the vaccines’ abilities to elicit the humoral and cellular immune responses were investigated in mice. These results showed that significantly enhanced GP5-specific ELISA antibody, GP5-specific neutralizing antibody, IFN-γ level, and IL-4 level, could be induced in mice immunized with DNA construct units encoding the pcDNA3.1-C3d-p28.n-GP5 than those received DNA vaccine expressing GP5 alone (pcDNA3.1-GP5). Analysis of the immunogenicity of different repeats of mC3d-p28 revealed that mC3d-p28 had an enhancing effect on the immunogenicity of antigens, and that six or more repeats of mC3d-p28 may be necessary for efficient enhancement of antigen specific immune responses. This approach may provide a new strategy for the development of efficient vaccines against the PRRSV for pigs in the future.     

Comparison of immune responses and protective efficacy of suicidal DNA vaccine and conventional DNA vaccine encoding glycoprotein C of pseudorabies virus in mice

In the present study, the immunogenicity and protective efficacy of a suicidal DNA vaccine (pSFVC1.5) incorporating Semliki Forest virus (SFV) replicon and expressing glycoprotein C (gC) of pseudorabies virus (PrV) was investigated and compared with a conventional plasmid DNA vaccine (pcDC) encoding the same antigen. In vitro, pSFVC1.5 could express gC protein and induce apoptosis of the transfected cells. After immunization in BALB/c mice, the gC-specific ELISA antibodies and neutralizing antibodies induced by pSFVC1.5 were relatively lower than those obtained in mice immunized with pcDC. However, mice immunized with pSFVC1.5 could confer more efficient protection than pcDC (100 and 62.5%, respectively) when challenged with the field PrV at 4 weeks after secondary immunization. Further analyses of cell-mediated immune responses showed that pSFVC1.5 induced stronger lymphocyte proliferative responses and higher levels of IFN-gamma, suggesting pSFVC1.5 could induce an enhanced Th1-type immune response. Collectively these results indicated that suicidal DNA vaccine is an alternative strategy to conventional DNA vaccine and can be considered a promising approach for the development of an efficacious vaccine against PrV.     

Evaluation of Japanese encephalitis virus DNA vaccine candidates in rhesus monkeys [Macaca mulatta

We have previously described DNA vaccine candidates against Japanese encephalitis virus (JEV) that were immunogenic in mice. Present study was conducted to evaluate their immunogenicity in rhesus monkeys (Macaca mulatta) and compare it with the commercial mouse brain-derived, formalin-inactivated vaccine. Groups of four monkeys were immunized with either pMEa (expressing the anchored form of the envelope protein along with the pre-membrane protein of JEV) or pMEs (expressing the secretory form of the envelope protein along with pre-membrane protein of JEV) by intra-muscular (IM, using needle) or intra-dermal (ID, using gene gun) routes. Following primary immunization with 1mg plasmid DNA given IM, or 5 microg plasmid DNA given ID, the monkeys were boosted after 1 and 2 months with 0.5mg DNA given IM or 5 microg DNA given ID, and observed for a period of 6 months. After the second booster, most of the monkeys sero-converted and developed JEV neutralizing antibodies, albeit of low titer. Importantly however, following a sham challenge with the mouse brain-derived inactivated JEV vaccine given 6 months after immunization, the neutralizing antibody titers rose rapidly indicating a vigorous anamnestic response. Based on the JEV neutralizing antibody response following the vaccination and the extent of anamnestic response generated in the immunized monkeys, plasmid pMEa was superior to pMEs. This study indicates that the JEV candidate DNA vaccine is capable of generating protective levels of JEV neutralizing antibodies in rhesus monkeys and prime the immune system effectively against a subsequent exposure to JEV.     

In vivo targeting of porcine reproductive and respiratory syndrome virus antigen through porcine DC-SIGN to dendritic cells elicits antigen-specific CD4T cell immunity in pigs

Immunogenicity of protein subunit vaccines may be dramatically improved by targeting them through antibodies specific to c-type lectin receptors (CLRs) of dendritic cells in mice, cattle, and primates. This novel vaccine development approach has not yet been explored in pigs or other species largely due to the lack of key reagents. In this study, we demonstrate that porcine reproductive and respiratory syndrome virus (PRRSV) antigen was targeted efficiently to dendritic cells through antibodies specific to a porcine CLR molecule DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) in pigs. A recombinant PRRSV antigen (shGP45M) was constructed by fusing secretory-competent subunits of GP4, GP5 and M proteins derived from genetically-shuffled strains of PRRSV. In vaccinated pigs, when the PRRSV shGP45M antigen was delivered through a recombinant mouse-porcine chimeric antibody specific to the porcine DC-SIGN (pDC-SIGN) neck domain, porcine dendritic cells rapidly internalized them in vitro and induced higher numbers of antigen-specific interferon-γ producing CD4T cells compared to the pigs receiving non-targeted PRRSV shGP45M antigen. The pDC-SIGN targeting of recombinant antigen subunits may serve as an alternative or complementary strategy to existing vaccines to improve protective immunity against PRRSV by inducing efficient T cell responses.     

Induction of an HPV 6bL1-specific mucosal IgA response by DNA immunization

Human papillomavirus (HPV) plays a crucial role in the development of human anogenital dysplasia. To prevent infection, it is important to induce an HPV-specific mucosal immune response. We investigated whether DNA vaccination would induce an intravaginal mucosal antibody response against HPV 6bL1. New Zealand White rabbits were immunized with an HPV 6bL1 DNA vaccine by one of the three routes: muscular, vaginal, or rectal. We found that vaginal immunization of rabbits with HPV 6bL1 DNA induced 6bL1 virus-like particle-specific lgA antibodies in vaginal secretions. They were detectable until at least 14 weeks after the first immunization. The antibodies also showed neutralizing activity in a hemagglutination inhibition assay. No mucosal immune response was detected in vaginal secretions of rabbits immunized intramuscularly or intrarectally. Our data suggest that vaginal immunization with HPV 6bL1 DNA induces long-lasting IgA responses with neutralizing activity in vaginal secretions of rabbits.     

Development of vaccines and passive immunotherapy against SARS corona virus using SCID-PBL/hu mouse models

We have investigated novel vaccine strategies against severe acute respiratory syndrome (SARS) CoV using cDNA constructs encoding the structural antigens: (S), (M), (E), or (N) protein, derived from SARS CoV. PBL from healthy human volunteers were administered i.p. into IL-2 receptor gamma-chain disrupted SCID mice, and SCID-PBL/hu mice were constructed. These mice can be used to analyze the human immune response in vivo. SARS M DNA vaccine and N DNA vaccine induced human CTL specific for SARS CoV antigens. Alternatively, SARS M DNA vaccines inducing human neutralizing antibodies and human monoclonal antibodies against SARS CoV are now being developed. These results show that these vaccines can induce virus-specific immune responses and should provide a useful tool for development of protective and therapeutic vaccines.     

Antibody responses induced in mice immunized with recombinant adenovectors expressing chimeric proteins of various porcine pathogens

Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine Circovirus type 2 (PCV2) and Mycoplasma (M.) hyopneumoniae are swine pathogens of economic importance. Although vaccines are available for each pathogen, no commercial bivalent vaccines have been developed so far. In this study, recombinant adenovectors (AdVs) expressing proteins of each pathogen were developed and their immunogenicity tested in mice. The proteins of interest were the glycoprotein GP5 of PRRSV, the capsid (Cap) protein of PCV2b and the C-terminal portion of P97 (P97c) protein of M. hyopneumoniae that were used alone and/or in fusion which each other. Inoculation of mice with the AdVs resulted in antibody (Ab) responses specific to the immunogens. Unexpectedly, immunization with vaccines expressing P97c in fusion to either Cap or GP5 enhanced the Ab responses against Cap and GP5, suggesting an immunopotentiator effect for P97c.     

Challenge studies in Rhesus monkeys immunized with candidate hepatitis E vaccines: DNA,DNA-prime-protein-boost and DNA-protein encapsulated in liposomes

Complete ORF2 gene (1983 bp) of hepatitis E virus (HEV) and the 450 bp region within ORF2 containing neutralizing epitope (NE) cloned in pVAX1 and corresponding proteins expressed in baculovirus and prokaryotic systems respectively were evaluated as vaccine candidates. Two doses of liposome encapsulated DNA plus corresponding protein with both ORF2 and NE regions (Lipo-ORF2-DP and Lipo-NE-DP) showed 100% seroconversion and comparable anti-HEV titres in Swiss albino mice. These vaccine candidates were further evaluated as DNA, DNA-prime-protein-boost (DPPB) and liposome formulations in Rhesus monkeys. Monkeys receiving ORF2/NE DNA seroconverted after fourth dose while those immunized employing ORF2-DPPB format seroconverted at 7 weeks post third dose. In view of the delayed weak antibody response, these monkeys were not challenged. Though Lipo-ORF2-DP was immunogenic, 2 of the 4 monkeys developed HEV infection following homologous virus challenge of 100 Monkey Infectious Dose₅₀. Both monkeys immunized with Lipo-NE-DP and 1 of the 2 monkeys immunized with NE-DPPB showed complete protection, the second monkey being protected from hepatitis with limited viral replication. Irrespective of the type of immunogen, all challenged monkeys were protected from hepatitis. The results document Lipo-NE-DP to be a promising vaccine candidate needing further evaluation.     

A recombinant plasmid co-expressing swine ubiquitin and the GP5 encoding-gene of porcine reproductive and respiratory syndrome virus induces protective immunity in piglets

We transfected a recombinant plasmid that co-expressed swine ubiquitin and a codon optimized GP5 encoding-gene of porcine reproductive and respiratory syndrome virus (PRRSV), designated pCA-U-optiGP5, as well as the plasmid pCA-optiGP5 encoding codon optimized GP5, and the plasmid pCA-GP5 expressing wild-type pGP5 into 293T cells. Expression of GP5 was measured by indirect immunofluorescence (IIF) assay and Western blot and found to be considerably higher in response to pCA-U-optiGP5 than the wild-type vector. GP5 protein was rapidly degraded in pCA-U-optiGP5-transfected 293T cells. The proteasome inhibitor, MG-132, however, successfully inhibited degradation. Immunogenicity of the three constructs was examined by measuring GP5-specific antibody production, lymphocyte proliferation, cytotoxic T lymphocyte (CTL) responses, and cytokine secretion in intramuscularly immunized pigs. Three weeks after the last inoculation, all animals were challenged intranasally with 2-ml 10(5)TCID(50)/ml PRRSV CH-1a. DNA immunization with pCA-optiGP5 produced a higher level of GP5-specific antibody than immunization with pCA-GP5, and the humoral response remained undetectable in the pCA-U-optiGP5 group. However, the fusion DNA had a significantly enhanced stimulation index (SI) and induced a stronger Th1 type cellular immune response than the single gene DNA, suggesting that ubiquitin conjugation improved the cellular but not the humoral immune response. Four of six pigs in the pCA-U-optiGP5 group, three of six in the pCA-optiGP5 group, and two of six in the pCA-GP5 group were devoid of visible pathological changes that were present in other vaccinated and control animals after challenge. Viral replication and distribution in the blood and tissues was lower in the pCA-U-optiGP5 vaccinated group than the other groups, suggesting that codon optimization, along with the development of rapidly processed antigen, represents a novel strategy to increase the immune efficacy of DNA vaccines against PRRSV.     

Development of a novel viral DNA vaccine against human papillomavirus: AcHERV-HP16L1

In this study, we developed a novel DNA vaccine for HPV; a recombinant baculovirus bearing human endogenous retrovirus (HERV) envelope protein, which cannot replicate in mammals, was used as a nano-carrier for HPV-16L1 DNA vaccine (AcHERV-HP16L1). For in vivo test, mice were injected intramuscularly with 107 particles of the constructs, with two boosts at 2-week intervals. Compared with Gardasil^® (25 μL/dose), the AcHERV-HP16L1 immunized mice showed similar high levels of humoral immunity in IgG/IgA and in neutralization of HPV pseudovirions. Combined immunization (prime with AcHERV-HP16L1 and boost with Gardasil^®) induced slightly higher neutralizing activity. As compared to the group treated with Gardasil^®, the mice immunized with AcHERV-HP16L1 showed 450- and 490-fold increase in the IFN-γ at 5 and 20 weeks after the first priming, respectively. The combined immunization conferred lower T cell immunity than AcHERV-HP16L1 treatment. The advantages of our novel AcHERV-HP16L1 vaccine over Gardasil^® include higher cellular immunogenicity, considerably lower production cost, and comparable safety. Therefore, we suggest that AcHERV-HP16L1 can be developed as an efficient prophylactic vaccine and therapeutic vaccine.     

Induction of protective antibodies against dengue virus by tetravalent DNA immunization of mice with domain III of the envelope protein

Dengue fever is a growing public health concern around the world and despite vaccine development efforts, there are currently no effective dengue vaccines. In the present study we report the induction of protective antibodies against dengue virus by DNA immunization with domain III (DIII) region of the envelope protein (E) in a mouse model. The DIII region of all four dengue virus serotypes were cloned separately into pcDNA 3 plasmid. Protein expression was tested in COS-7 cells. Each plasmid, or a tetravalent combination, were used to immunize BALB/c mice by intramuscular route. Presence of specific antibodies was evaluated by ELISA, and neutralizing antibodies were tested using a cytopathogenic effect (CPE) inhibition assay in BHK-21 cells, as well as in newborn mice challenged intracranially with dengue 2 virus. Mice immunized with individual DIII constructs or the tetravalent formulation developed antibodies against each corresponding dengue serotype. Antibody titers by ELISA were similar for all serotypes and no significant differences were observed when boosters were administered, although antibody responses were dose-dependent. CPE inhibition assays using Den-2 virus showed neutralization titers of 1:10 in mice immunized with individual DIII plasmid or those immunized with the tetravalent formulations. 43% of newborn mice challenged with Den-2 in combination with sera from mice immunized with Den-2 DIII plasmid were protected, whereas sera from mice immunized with the tetravalent formulation conferred 87% protection. Our results suggest that DIII can be used as a tetravalent DNA formulation to induce neutralizing and protective antibodies against dengue virus.