Effects of CpG ODN on CD4+ and CD8+ T subpopulations in the immune response to porcine reproductive and respiratory syndrome killed virus vaccine

CpG ODN is a noval immunostimulatory reagent In this research, the effects of immunostimulatory CpG oligodeoxynucleotides (CpG ODN) on CD4+ and CD8+ T lymphocytes subpopulations in the newborn piglets blood were tested at different time with porcine reproductive and respiratory syndrome killed virus vaccine (PRRS vaccine) with or without CpG ODN. The results suggested that, the CD4+/CD8+ ratio decreased with age in piglets inoculated with vaccine alone or GpC ODN with vaccine or phosphate buffer saline (PBS), however, it was stable in piglets co-inoculated with CpG ODN and PRRS vaccine (p>0.05), the use of CpG ODN can prevent effectively the reduction of the proportion of CD4+ T lymphocytes. High titers of PRRS specific antibody can also be tested in the newborn piglets serum immunized PRRS vaccine and CpG ODN (p<0.05).     

In vivo oral administration effects of various oligodeoxynucleotides containing synthetic immunostimulatory motifs in the immune response to pseudorabies attenuated virus vaccine in newborn piglets

Numerous studies have demonstrated that oligonucleotides containing CpG motifs (CpG ODN) are efficient immunoadjuvants to various antigens administered by parenteral routes to mice. Recently, it has been found that CpG ODNs also is a promising mucosal adjuvant in mice. To date, there have been no studies to screen the optimal CpG sequence and modified ODN backbone to piglets in vivo, when delivered by oral route. We have previously demonstrated that human-specific CpG ODN is a potent adjuvant to pseudorabies live attenuated virus (PRV) vaccine when administered subcutaneously (SC) or ocularly in piglets. In this study, we screened and evaluated the optimal CpG sequences (porcine-specific, human-specific, mouse-specific ODN) and optimal backbone (SOS-backbone consisting of a nuclease-resistant phosphorothioate guanosines at the 5' and the 3'-end and with a phosphodiester (O) in the center and phosphorothioate (S) backbone (S-backbone)) to PRV vaccine delivered orally in piglets. The proliferation of peripheral blood mononuclear cells (PBMCs), IFN-gamma and IL-4 in serum, and the titre of IgG, IgG2/IgG1 isotype in serum and IgA in intestinal washings and feces to PRV vaccine were tested at different time-points. The results suggested that, CpG ODNs augmented systemic (IgG in serum, T-cell proliferation) and mucosal (IgA in intestinal washings and feces) immune responses against antigen. CpG ODNs stimulated both T-helper type1 (Th1) (IgG2) and Th2 (IgA) responses when delivered orally. With the same backbone, the porcine-specific ODN-induced responses were comparable with human-specific ODNs, but stronger than mouse-specific CpG ODNs. SOS-backbone induced a stronger IFN-gamma and proliferative responses than S-backbone, while antibody responses induced by SOS-backbones were slightly less or similar with S-backbone. The in vivo data demonstrate for the first time that porcine-specific and human-specific ODNs both are optimal sequences for mucosal system in piglets.     

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.     

Synthetic B- and T-cell epitope peptides of porcine reproductive and respiratory syndrome virus with Gp96 as adjuvant induced humoral and cell-mediated immunity

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has recently caused huge economic losses in the pig industry worldwide. Commercial vaccines, including inactivated vaccines and attenuated live vaccines, are available but fail to provide sustainable protection, especially against genetically heterologous strains. Thus several approaches have been used to develop more effective PRRSV vaccines and/or immune modulators to accelerate and magnify immune responses to PRRSV vaccines. Heat shock protein Gp96 is one such modulator that enhances both the innate and adaptive immune responses. In the present study, two B-cell epitopes and seven T-cell epitopes from PRRSV and a Pan DR T-helper cell epitope were synthesized and mixed with the N-terminal 22–355 aa of Gp96 (Gp96N) as an adjuvant, and immune responses were evaluated. Our results show that Gp96N activated PRRSV-specific humoral immune responses elicited by BCE-peptides and promoted the PRRSV-specific cellular immunity induced by TCE-peptides. Moreover, higher levels of IL-12 and TNF-α and lower levels of IL-4 and IL-10 were observed in the serum of Gp96N-vaccinated piglets compared to piglets immunized with no Gp96N, displaying a predominant Th1 type of immune response induced by Gp96N. Following challenge with the virulent HP-PRRSV isolate JXwn06, piglets vaccinated with the mixture of peptides and Gp96N presented with milder clinical symptoms, lower viremia, and less pathological lesions in their lungs, however, this vaccine could not provide lasting and effective protection against HP-PRRSV infection. These data provide important bases for the development of PRRSV epitope-based synthetic peptide vaccines combined with Gp96N as attractive immunomodulators in swine.     

The role of CpG ODN in enhancement of immune response and protection in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63) encapsulated in cationic liposome

CpG oligodeoxynucleotides (CpG ODN) are known to be a potent immunoadjuvant for a wide range of antigens. The aim of this study was to evaluate the role of CpG ODN co-encapsulated with rgp63 antigen in cationic liposomes (Lip-rgp63-CpG ODN) in immune response enhancement and protection in BALB/c mice against leishmaniasis. Lip-rgp63-CpG ODN prepared by using dehydration-rehydration vesicle (DRV) method significantly inhibited (P<0.001) Leishmania major infection in mice measured by footpad swelling compared to Lip-rgp63, rgp63 alone, rgp63 plus CpG ODN, PBS or control liposomes. The mice immunized with Lip-rgp63-CpG ODN also showed the lowest spleen parasite burden, highest IgG2a/IgG1 ratio and IFN-gamma production and the lowest IL-4 production compared to the other groups. The results indicate that co-encapsulation of CpG ODN in liposomes improves the immunogenicity of Leishmania antigen.     

Immunogenicity study of plant-made oral subunit vaccine against porcine reproductive and respiratory syndrome virus (PRRSV)

Currently, killed-virus and modified-live PRRSV vaccines are used to control porcine reproductive and respiratory syndrome disease (PRRS). However, very limited efficacy of killed-virus vaccines and serious safety concerns for modified-live virus vaccines demand the development of novel PRRSV vaccines. In this report, we investigated the possibility of using transgenic plants as a cost-effective and scalable system for production and delivery of a viral protein as an oral subunit vaccine against PRRSV. Corn calli were genetically engineered to produce PRRSV viral envelope-associated M protein. Both serum and intestine mucosal antigen-specific antibodies were induced by oral administration of the transgenic plant tissues to mice. In addition, serum and mucosal antibodies showed virus neutralization activity. The neutralization antibody titers after the final boost reached 6.7 in serum and 3.7 in fecal extracts, respectively. A PRRSV-specific IFN-γ response was also detected in splenocytes of vaccinated animals. These results demonstrate that transgenic corn plants are an efficient subunit vaccine production and oral delivery system for generation of both systemic and mucosal immune responses against PRRSV.     

Induction of multispecific Th-1 type immune response against HCV in mice by protein immunization using CpG and Montanide ISA 720 as adjuvants

Recent studies demonstrate that Th1-type immune responses against a broad spectrum of hepatitis C virus (HCV) gene products are crucial to the resolution of acute HCV infection. We investigated new vaccine approaches to augment the strength of HCV-specific Th1-type immune responses. ELISPOT assay revealed that single or multiple protein immunization using both CpG ODN and Montanide ISA 720 as adjuvants induced much stronger IFN-gamma-producing Th1 responses against core, NS3 and NS5b targets than did the formulation without these adjuvants. Protein vaccination using CpG ODN and Montanide ISA 720 as adjuvants also greatly enhanced humoral responses to HCV core, E1/E2 and NS3. When specific IgG isotypes were assayed, protein immunization using CpG ODN and Montanide ISA 720 as adjuvants produced higher titers of IgG2a dominant antibodies than did protein immunization alone, indicating a more Th1-biased pathway. This increase in IgG2a is consistent with the induction of Th1 cells secreting IFN-gamma demonstrated by ELISPOT assay. In conclusion, protein immunization using CpG ODN and Montanide ISA 720 as adjuvants greatly enhanced cellular (Th1 type) as well as humoral immune responses against HCV in Balb/c mice. The use of adjuvants appears critical to the induction of Th1 immune responses during HCV vaccination with recombinant proteins.     

CpG-containing oligodeoxynucleotides,in combination with conventional adjuvants,enhance the magnitude and change the bias of the immune responses to a herpesvirus glycoprotein

Vaccine adjuvants must have the capacity to increase protective immune responses with minimal side effects. Conventional adjuvants not only cause undesirable tissue site reactions, but often induce T-helper type 2 (Th2)-biased responses which may be undesirable in certain disease scenarios. Oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN) are novel adjuvants known to promote Th1-type immune responses. In this study, we compared various mineral oil, metabolizable oil and non-oil adjuvants alone and in combination with CpG ODN for their ability to augment immune responses to a truncated secreted form of bovine herpesvirus (BHV) glycoprotein D (tgD). All adjuvants tested induced Th2-biased immune responses characterized by a predominance of serum IgG1 as well as interleukin-4 (IL-4) production by in vitro stimulated splenocytes. The inclusion of CpG ODN in these formulations not only increased immune responses, but more importantly enhanced serum IgG2a levels and production of interferon-gamma (IFN-gamma) by splenocytes, indicating a more balanced or Th1-type response. The use of a mineral oil-based adjuvant at reduced doses in combination with CpG ODN attenuated the tissue damage while not compromising the magnitude of the immune response in both mice and sheep. In addition, reduced amounts of mineral oil combined with CpG ODN induced a more balanced Th1/Th2 immune response than the mineral oil used alone. Our results clearly demonstrate that CpG ODN can be used to enhance magnitude and balance of an immune response while reducing the amount of mineral oil and hence undesirable side effects of vaccine adjuvants.     

Co-administration of polyphosphazenes with CpG oligodeoxynucleotides strongly enhances immune responses in mice immunized with Hepatitis B virus surface antigen

An emerging paradigm in vaccinology is that multiple adjuvant combinations may be more effective than individual adjuvants in enhancing immune responses to vaccine antigens. We investigated whether the polyphosphazenes used in combination with CpG oligodeoxynucleotides (ODN) were potent adjuvant formulations. BALB/c mice were immunized subcutaneously with Hepatitis B surface antigen (HBsAg) alone, or in various combinations with poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP), poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) or CpG ODN. All three adjuvants enhanced HBsAg-specific IgG1 antibody responses with PCEP inducing the highest responses. PCEP and CpG ODN significantly enhanced the Th1-associated antibody isotype IgG2a. As expected CpG ODN induced predominantly Th1-type immune responses while PCEP was associated with mixed Th1/Th2 immune responses. Interestingly, PCEP and PCPP synergized with CpG ODN to further enhance antibody responses. Since the mechanisms which mediate the adjuvant activity of polyphosphazenes are not fully understood, we investigated whether PCEP and PCPP could stimulate innate immune responses. Incubation of mouse splenocytes with PCEP or PCPP (in the absence of antigen) stimulated production of IL-4 and IL-12, but only PCEP induced significant IFNγ production. Additionally, IL-12 was not required for PCEP induced IFNγ response. We conclude that the polyphosphazene–CpG ODN combination is a potent adjuvant formulation that is more effective in enhancing immune responses than either of the individual adjuvants. In addition, we provide evidence that PCEP and PCPP can stimulate innate cytokine production, suggesting a potential mechanism by which polyphosphazenes achieve their potent adjuvant effects.     

A novel DNA vaccine for reduction of PRRSV-induced negative immunomodulatory effects: A proof of concept

BackgroundViral-induced interleukin (IL)-10 and regulatory T lymphocytes (Tregs) are believed to play a major role in shaping the immunological and clinical outcomes following Porcine Reproductive and Respiratory Syndrome virus (PRRSV) infection. Recently, it has been shown that PRRSV nucleocapsid (N) protein can induce IL-10 production which is essential for induction of PRRSV-specific Tregs. We hypothesized that immunity to N protein should reduce PRRSV-induced negative immunomodulatory effects which will be essential for establishing proper anti-PRRSV immunity in infected pigs.ObjectivesTo investigate the immunomodulatory effects of DNA vaccine encoding a linearized, truncated form of PRRSV-N protein (pORF7t) which was designed to preferentially induce cell-mediated immunity against PRRSV N protein.MethodImmunomodulatory effects of the novel DNA vaccine were investigated in an experimental vaccinated-challenged model. In addition, long-term immunomodulatory effects of the DNA vaccine were investigated in vaccinated pigs kept at the PRRSV-positive environment until the end of the fattening period. Pigs were vaccinated either prior to or following natural PRRSV infection.ResultThe results indicated that pORF7t could modulate the anti-PRRSV immune responses and promote the control of viral replication in the vaccinated-challenged pigs. Immunized pigs exhibited increased numbers of PRRSV-specific activated CD4⁺CD25⁺ lymphocytes, reduced numbers of PRRSV-specific Tregs, and rapid viral clearance following infection. In a long-term study, regardless of the time of vaccination, DNA vaccine could modulate the host immune responses, resulted in enhanced PRRSV-specific IFN-γ producing cells, and reduced numbers of PRRSV-specific Tregs, without evidence of enhanced antibody responses. No vaccine adverse reaction was observed throughout the study.ConclusionThis study revealed the novel concept that PRRSV-specific immunity can be modulated by induction of cell-mediated immunity against the nucleocapsid protein. This concept could potentially benefit the development of PRRSV management and control strategies.     

Synthetic innate defence regulator peptide enhances in vivo immunostimulatory effects of CpG-ODN in newborn piglets

The in vivo immunoadjuvant effects of CpG oligodeoxynucleotides (CpG-ODN) have been studied extensively in mice and relatively fewer studies have been done in piglets. But so far, the innate immunostimulatory effects of CpG-ODN combination with innate defense-regulator peptides (IDRs) have not been demonstrated. The purpose of this study is to determine the potential effects of CpG-ODN with IDR in newborn piglets. The immunostimulatory abilities of four selected IDRs were compared, among them HH2 showed best immunostimulatory effects in newborn piglets. Hereafter, the abilities of CpG-ODN combined with HH2 to enhance innate immune responses were examined in newborn piglets. The complex of HH2 and CpG-ODN could induce much stronger Th1 cytokine and chemokine responses than HH2 or CpG-ODN alone. HH2-CpG-ODN immunized piglets showed higher B cell percentage in PBMCs than CpG-ODN alone. These in vivo data demonstrated for the first time that subcutaneously (SC) administration of CpG-ODN combined with HH2 is efficient to stimulate innate immune system in newborn piglets.     

CpG DNA is an effective oral adjuvant to protein antigens in mice

We have previously reported that synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN) are potent adjuvants to protein administered by intramuscular (IM) injection or intranasal (IN) inhalation to BALB/c mice. Herein, we have evaluated oral delivery of CpG ODN with purified hepatitis B surface antigen (HBsAg) or tetanus toxoid (TT) to determine its potential as an adjuvant to oral vaccines. CpG ODN augmented systemic (IgG in plasma, CTL, T-cell proliferation) and mucosal (IgA in lung, vaginal or gut washes, feces and saliva) immune responses against both antigens. CpG stimulated both T-helper type 1 (Th1) (CTL, IgG2a) and Th2 (IgG1, IgA) responses when delivered orally. Results from this study indicate that stimulatory CpG ODN may be effective as an adjuvant with oral vaccines.     

DNA vaccines co-expressing GP5 and M proteins of porcine reproductive and respiratory syndrome virus (PRRSV) display enhanced immunogenicity

The two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus (PRRSV), GP5 and M (encoded by ORF5 and ORF6 genes, respectively), are associated as disulfide-linked heterodimers (GP5/M) in the virus particle. In the present study, three different DNA vaccine constructs, expressing GP5 alone (pCI-ORF5), M alone (pCI-ORF6) or GP5 and M proteins simultaneously (pCI-ORF5/ORF6), were constructed. In vitro, the co-expressed GP5 and M proteins could form heterodimeric complexes in transfected cells and heterodimerization altered the subcellular localization of GP5. The immunogenicities of these DNA vaccine constructs were firstly investigated in a mouse model. Mice inoculated with pCI-ORF5/ORF6 developed PRRSV-specific neutralizing antibodies at 6 and 8 weeks after primary immunization. However, only some mice developed low levels of neutralizing antibodies in groups immunized with pCI-ORF5 or pCI-ORF6. The highest lymphocyte proliferation responses were also observed in mice immunized with pCI-ORF5/ORF6. Interestingly, significantly enhanced GP5-specific ELISA antibody could be detected in mice immunized with pCI-ORF5/ORF6 compared to mice immunized with pCI-ORF5. The immunogenicities of pCI-ORF5/ORF6 were further evaluated in piglets (the natural host) and all immunized piglets developed neutralizing antibodies at 10 weeks after primary immunization, whereas there was no detectable neutralizing antibodies in piglets immunized with pCI-ORF5. These results indicate that the formation of GP5/M heterodimers may be involved in post-translational modification and transport of GP5 and may play an important role in immune responses against PRRSV infection. More importantly, co-expression of GP5 and M protein in heterodimers can significantly improve the potency of DNA vaccination and could be used as a strategy to develop a new generation of vaccines against PRRSV.     

CpG DNA as mucosal adjuvant.

We have previously found synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs to be a potent adjuvant to protein administered by intramuscular injection or intranasal inhalation to BALB/c mice. Herein we have further evaluated the potential of CpG ODN as a mucosal adjuvant to purified hepatitis B surface antigen (HBsAg) when administered alone or with cholera toxin (CT). CpG ODN and CT both augmented systemic (humoral and cellular) and mucosal immune responses against HBsAg, and these could be further enhanced with higher doses of adjuvant or boosting. Overall, antibody isotypes with CT alone were predominantly IgG1 (Th2-like) whereas they were predominantly IgG2a (Th1-like) with CpG ODN alone or in combination with CT. Results from this study indicate that stimulatory CpG ODN are promising new adjuvants for mucosal vaccination strategies, whether used alone or in combination with other mucosal adjuvants.     

Pigs immunized with Chinese highly pathogenic PRRS virus modified live vaccine are protected from challenge with North American PRRSV strain NADC-20

Modified live virus (MLV) vaccines developed to protect against PRRSV circulating in North America (NA) offer limited protection to highly pathogenic (HP) PRRSV strains that are emerging in Asia. MLV vaccines specific to HP-PRRSV strains commercially available in China provide protection to HP-PRRSV; however, the efficacy of these HP-PRRSV vaccines to current circulating NA PRRS viruses has not been reported. The aim of this study is to investigate whether pigs vaccinated with attenuated Chinese HP-PRRSV vaccine (JXA1-R) are protected from infection by NA PRRSV strain NADC-20. We found that pigs vaccinated with JXA1-R were protected from challenges with HV-PRRSV or NADC-20 as shown by fewer days of clinical fever, reduced lung pathology scores, and lower PRRS virus load in the blood. PRRSV-specific antibodies, as measured by IDEXX ELISA, appeared one week after vaccination and virus neutralizing antibodies were detected four weeks post vaccination. Pigs vaccinated with JXA1-R developed broadly neutralizing antibodies with high titers to NADC-20, JXA1-R, and HV-PRRSV. In addition, we also found that IFN-α and IFN-β occurred at higher levels in the lungs of pigs vaccinated with JXA1-R. Taken together, our studies provide the first evidence that JXA1-R can confer protection in pigs against the heterologous NA PRRSV strain NADC-20.     

The novel adjuvant combination of CpG ODN,indolicidin and polyphosphazene induces potent antibody- and cell-mediated immune responses in mice

The need to enhance the immunogenicity of purified subunit antigens and modulate resulting immune responses has prompted the development of new adjuvants. Here, the ability of CpG oligodeoxynucleotides (ODN), a bovine host defence peptide indolicidin, and polyphosphazene to synergistically combine and enhance innate and adaptive immune responses was examined in mice. In vitro, the adjuvant combination of CpG ODN, indolicidin and polyphosphazene (CpG/indol/PP) enhanced the secretion of TNF-α, IL-12p40, and IL-6 by bone marrow-derived DCs (BMDCs) when compared to the individual components. When co-formulated with ovalbumin (OVA), CpG/indol/PP formed antigen-adjuvant complexes, and enhanced antibody and cell-mediated responses in mice, via both MHC I and II pathways, promoting a more balanced antibody-mediated and type 1-biased cell-mediated immune response. Furthermore, substitution of the proline residues of indolicidin with arginine increased the synergistic adjuvant effect of the peptide, and induced significantly higher IgG1 and IgG2a titers and IFN-γ secretion, as well as increased uptake by antigen presenting cells. These results clearly demonstrate that the use of a combination of CpG ODN, indolicidin, and polyphosphazene as adjuvant can significantly enhance an antigen-specific immune response.     

Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens

Several types of avian influenza virus (AIV) vaccines exist, including live-attenuated, vectored, and whole inactivated virus (WIV) vaccines. Inactivated vaccines offer some advantages compared to other types of vaccines, including ease of production and lack of ability to revert to a virulent state. However, WIV are poorly immunogenic, especially when these vaccines are delivered to mucosal surfaces. There are several factors that contribute to the immunogenicity of vaccines, one of which is the method used to inactivate viruses. Several methods exist for producing influenza WIVs, including formaldehyde, a chemical that affects protein structures leading to virus inactivation. Other methods include treatment with beta-propiolactone (BPL) and the application of gamma radiation, both of which have less effects on protein structures compared to formaldehyde, and instead alter nucleic acids in the virion. Here, we sought to determine the effect of the above inactivation methods on immunogenicity of AIV vaccines. To this end, chickens were vaccinated with three different H9N2 WIVs using formaldehyde, BPL, and gamma radiation for inactivation. In addition to administering these three WIVs alone as vaccines, we also included CpG ODN 2007, a synthetic ligand recognized by Toll-like receptor (TLR)21 in chickens, as an adjuvant for each WIV. Subsequently, antibody- and cell-mediated immune responses were measured following vaccination. Antibody-mediated immune responses were increased in chickens that received the BPL and Gamma WIVs compared to the formaldehyde WIV. CpG ODN 2007 was found to significantly increase antibody responses for each WIV compared to WIV alone. Furthermore, we observed the presence of cell-mediated immune responses in chickens that received the BPL WIV combined with CpG ODN 2007. Based on these results, the BPL WIV + CpG ODN 2007 combination was the most effective vaccine at inducing adaptive immune responses against H9N2 AIV. Future studies should characterize mucosal adaptive immune responses to these vaccines.     

Enhancement of the immunity to foot-and-mouth disease virus by DNA priming and protein boosting immunization.

Subunit vaccination is effective in eliciting humoral responses to a variety of viral antigens, however, it has not generated persistent protective immunity to foot-and-mouth disease virus (FMDV). In this study, we observed that priming mice with a DNA plasmid encoding VP1 of the FMDV O/Taiwan/97 capsid protein followed by boosting with a VP1 peptide conjugate (P29-KLH) resulted in production of not only high titers of antibodies but also antibodies with FMDV neutralizing activities. Moreover, the mice immunized in this manner cleared the virus from their sera in FMDV challenge experiments. Mice subjected to DNA plasmid priming and P29-KLH protein boosting had relatively higher ratio of IgG2a/IgG1 than those primed and boosted with P29-KLH conjugate. Addition of an oligodeoxynucleotide (ODN) containing immunostimulatory cytosine-phosphate-guanosine (CpG) motifs to P29-KLH conjugate also induced a higher ratio of IgG2a/IgG1 and significantly higher titer of neutralizing antibodies. These results indicate that treating animals with DNA plasmids priming and FMDV antigen(s) boosting may elicit immunity to FMD and this immune response may be augmented by CpG ODN.