DNA vaccines encoding retrovirus-based virus-like particles induce efficient immune responses without adjuvant

Virus-like particle (VLP)-based vaccines have provided highly encouraging results in clinical trials while, in contrast, DNA vaccines expressing non-particulate proteins have proven less successful. Seeking to combine the immunogenicity of VLPs and the ease of production of plasmid DNA, we designed DNA vaccines expressing VLPs consisting of the MLV Gag and modified MLV Env proteins displaying T cell epitopes. We show here that such DNA vaccines are remarkably efficient immunogens for inducing cellular immune responses. In contrast to similar plasmids harboring a point mutation preventing VLP formation, they induce protection against a lethal viral challenge in mice. Thus, these "plasmo-retroVLPs" represent a promising second-generation DNA vaccine.     

Induction of immune responses by DNA vaccines in large animals

It is generally recognized that DNA vaccines are often less effective in large animals than in mice. One possible reason for this reduced effectiveness may be transfection deficiency and the low level of expression elicited by plasmid vectors in large animals. In our attempt to enhance transfection efficiency and, thereby, enhance immune responses, we employed a variety of methods inducing gene gun delivery or suppositories as delivery vehicles to mucosal surfaces, as well as electroporation for systemic immunization. To test these different systems, we used two different antigens-a membrane antigen from bovine herpesvirus glycoprotein (BHV-1) gD and a particulate antigen from hepatitis virus B. Gene gun and suppository delivery of BHV-1 gD to the vagina resulted in the induction of mucosal immunity not only in the vagina, but also at other mucosal surfaces. These data support the contention of a common mucosal immune system. In the case of electroporation, we were able to develop significant enhancement of gene expression following electroporation with surface electrodes (non-invasive electroporation) as well as invasive electroporation using single or six-needle electrodes. Various delivery systems such as bioject or needle delivery also influenced the immune response in both the presence and absence of electroporation. These studies also demonstrated that co-administration of plasmids coding for two different antigens (BHV-1 gD and hepatitis B surface antigen (HbsAg)) did not result in significant interference between the plasmids. These studies suggest that various combinations of delivery systems can enhance immunity to DNA-based vaccines and make them practical for administration of these vaccines in large animals.     

Generation of protective immune responses against coxsackievirus B3 challenge by DNA prime-protein boost vaccination

Coxsackievirus B3 (CVB3) causes viral myocarditis and can ultimately result in dilated cardiomyopathy. However, there is no vaccine available for clinical use. In this study, we assessed the protection provided by three immunization strategies against CVB3 infection. Vaccination was performed with a DNA vaccine expressing the cloned capsid gene VP1 or a vaccine developed from purified VP1 protein. Third, a strategy of vaccination was attempted with the DNA vaccine followed by two boosts with the recombinant protein vaccine (DNA prime-protein boost vaccine). Followed immunization, mice were challenged with CVB3 infection. Improved induction of CVB3-specific antibodies and neutralizing antibodies were found in mice immunized by the DNA prime-protein boost regimen. Furthermore, virus-specific cytotoxic activity of spleen cells derived from DNA prime-protein boost vaccinated mice was elicited. In addition, the DNA prime-protein boost vaccine resulted in protection of 75% of mice from lethal CVB3 challenge and a significant reduction of viral load in sera of immunized mice after acute CVB3 infection. There was a significant reduction in myonecrosis and infiltrating myocardial immune cells indicating reduced severity of myocarditis in surviving mice. These findings demonstrated that a DNA prime-protein boost immunization strategy, but not a DNA vaccine or protein vaccine alone, was effective in eliciting both humoral and cell-mediated immune responses against CVB3 infection in mice and might be a promising vaccine candidate.     

Characterization of immune responses induced by inactivated,live attenuated and DNA vaccines against Japanese encephalitis virus in mice

Vaccination is the most effective countermeasure for protecting individuals from Japanese encephalitis virus (JEV) infection. There are two types of JEV vaccines currently used in China: the Vero cell-derived inactivated vaccine and the live attenuated vaccine. In this study, we characterized the immune response and protective efficacy induced in mice by the inactivated vaccine, live attenuated vaccine and the DNA vaccine candidate pCAG-JME, which expresses JEV prM-E proteins. We found that the live attenuated vaccine conferred 100% protection and resulted in the generation of high levels of specific anti-JEV antibodies and cytokines. The pCAG-JME vaccine induced protective immunity as well as the live attenuated vaccine. Unexpectedly, immunization with the inactivated vaccine only induced a limited immune response and partial protection, which may be due to the decreased activity of dendritic cells and the expansion of CD4+CD25+Foxp3+ regulatory T cells observed in these mice. Altogether, our results suggest that the live attenuated vaccine is more effective in providing protection against JEV infection than the inactivated vaccine and that pCAG-JME will be a potential JEV vaccine candidate.     

由小鼠肺组织病理学改变评价结核DNA疫苗的保护效力

目的 评价结核分支杆菌MPT64和ESAT6 DNA疫苗的保护效力。方法 将BALB/c小鼠随机分为5组,分别用生理盐水(A）、载体质粒（B）、卡介苗（C）、MPT64 DNA疫苗（D）和ESAT6 DNA疫苗（E）免疫小鼠。最后一次免疫3周后以结核分支杆菌H37Rv腹腔内攻击小鼠。攻击5或10周后,观察肺组织病理改变。结果 结核分支杆菌攻击5周后,A和B组小鼠肺组织病变表现为以渗出性反应为主的混合性组织反应;C组表现为以上皮样细胞肉芽肿和肺泡壁结核肉芽组织中度增生为主的增殖性组织反应;D组3只和E组1只小鼠与A、B组表现相似,D组2只和E组4只小鼠与C组表现相似。结核分支杆菌攻击10周后,A、B和D组肺病变主要表现为由许多泡沫样巨噬细胞、淋巴细胞和少量上皮样细胞组成的结核性肉芽肿及肺泡壁结核性肉芽组织中度增生、增厚;C和E组均表现为上皮样细胞和淋巴细胞组成的结核性肉芽肿及肺泡壁结核性肉芽组织中到重度增生、增厚。各组小鼠均未见干酪样坏死。结论 结核分支杆菌MPT64和ESAT6 DNA疫苗能增强机体免疫力,ESAT6 DNA疫苗的保护效力比MPT64 DNA疫苗强,但均未超过卡介苗。     

Prothymosin alpha enhances protective immune responses induced by oral DNA vaccination against pseudorabies delivered by Salmonella choleraesuis.

Previously, we showed that vaccination with the glycoprotein D (gD) gene of pseudorabies virus (PrV) delivered by Escherichia coli induced protective immune responses. In this study, we report that oral DNA vaccination with attenuated Salmonella choleraesuis carrying the PrV gD gene conferred protective immunity in mice against PrV. Moreover, co-delivery of the prothymosin alpha gene carried by S. choleraesuis enhanced the vaccine efficacy. Our results thus demonstrate for the first time, to our knowledge, the effectiveness of oral DNA vaccination using S. choleraesuis as a delivery vehicle and the potential usefulness of prothymosin alpha as a DNA vaccine adjuvant.     

Multivalent DNA vaccine induces protective immune responses and enhanced resistance against Cryptosporidium parvum infection

The aim of this work was to evaluate efficiency as well as the type of immune response, Th1 or Th2, induced by multivalent DNA vaccinations in C57BL/6 interleukin-12p40 (IL-12p40) knockout (KO) mice. A recombinant pVAX-15-23 plasmid DNA was constructed by inserting surface glycoprotein (cp15- and p23)-encoding DNA into the pVAX1 expression vector. Various parameters including antibody and cytokine responses, proliferation assay and oocyst shedding were used to evaluate the type of immune response and the level of protection against challenge infection. Obtained results indicated that plasmid pVAX-15-23 induced strong protective immune response against C. parvum characterized by dominance of IgG2a, high level of INF-γ and lower level of the oocysts shedding after challenge infection. Moreover, co-immunization with the multivalent DNA and pMEM12R plasmid encoding IL-12 can further enhance these responses compared with the multivalent DNA alone. The obtained results suggest that multivalent pVAX-15-23 DNA vaccine may be a candidate as a generic approach to C. parvum immunization applicable to clinical practice.     

Over-expression of superoxide dismutase obliterates the protective effect of BCG against tuberculosis by modulating innate and adaptive immune responses

An efficient global control of tuberculosis requires development of alternative vaccination strategies that can enhance the efficacy of existing BCG vaccine. In this study, we evaluated the protective efficacy of a recombinant BCG (rBCG) vaccine over-expressing iron-cofactored superoxide dismutase (SOD-A), one of the prominent oxidative stress response proteins of Mycobacterium tuberculosis. Contrary to our expectations, over-expression of SOD-A resulted in the abrogation of BCG's ability to confer protection in guinea pig as well as in murine model. Analysis of immune responses revealed that over-expression of SOD-A by rBCG has pleiotropic effects on innate and adaptive immune responses. Macrophages infected in vitro with rBCG exhibited a marked reduction in apoptosis and microbicidal potential. In addition, rBCG vaccination of mice resulted in a reduced IFNγ and increased IL10 production when compared with the BCG vaccination. Further, we show that rBCG vaccination failed to generate an effective multi-functional CD4 T cell response. Altogether, our findings suggest that over-expression of SOD-A in BCG enhances the immuno-suppressive properties of BCG, characterized by skewing of immune responses towards Th2 type, an inefficient multi-functional T cell response and reduced apoptosis and microbicidal potential of macrophages leading to abolishment of BCG's protective efficacy.     

Elicitation of broadly reactive antibodies against glycan-modulated neutralizing V3 epitopes of HIV-1 by immune complex vaccines

HIV-1 envelope gp120 is the target for neutralizing antibodies (NAbs) against the virus. Various approaches have been explored to improve immunogenicity of broadly neutralizing epitopes on this antigen with limited success. We previously demonstrated that immunogenicity of gp120 and especially its V3 epitopes was enhanced when gp120 was co-administered as immune-complex vaccines with monoclonal antibodies (mAb) to the CD4-binding site (CD4bs). To define the mechanisms by which immune complexes influence V3 immunogenicity, we compared gp120 complexed with mAbs specific for the C2 region (1006-30), the V2 loop (2158), or the CD4bs (654), and found that the gp120/654 and gp120/2158 complexes elicited anti-V3 NAbs, but the gp120/654 complex was the most effective. gp120 complexed with 654 F(ab′)₂ was as potent, indicating that V3 immunogenicity is determined by the specificity of the mAb's Fab fragment used to form the complexes. Importantly, the gp120/654 complex not only induced anti-gp120 antibodies (Abs) to higher titers, but also of greater avidity. The Abs were cross-reactive with V3 peptides from most subtype B and some subtype C isolates. Neutralization was detected only against Tier-1 HIV-1 pseudoviruses, while Tier-2 viruses, including the homologous JRFL strain, were not neutralized. However, JRFL produced in the presence of a mannosidase inhibitor was sensitive to anti-V3 NAbs in the immune sera. These results demonstrate that the gp120/654 complex is a potent immunogen for eliciting cross-reactive functional NAbs against V3 epitopes, of which exposure is determined by the specific compositions of glycans shrouding the HIV-1 envelope glycoproteins.     

Site-directed immune responses in DNA vaccines encoding ligand-antigen fusions

One of the key limitations to DNA vaccines is lack of efficacy. We found that the spleen was a superior injection site to the dermis or muscle for inducing immune responses. To target sites of immune induction more practicably, antigen (human IgG1) was fused with two ligands, L-selectin (L-SEL-hIg) or CTLA4 (CTLA4-hIg) the receptors of which are found on high endothelial venule cells in lymph nodes and antigen presenting cells, respectively. Antibody and lymphocyte proliferative responses were increased. We now show that dimerization is critical for this enhancement, presumably because of avidity considerations. The hinge of hIgG3 can replace that of hIgG1 as a dimerization moiety. Fusion of other antigens e.g. ovalbumin and a malaria antigen AMA-1 have confirmed that CTLA4 induces an enhanced antibody response. Notably, in a challenge model, we have shown that CTLA4 also improves efficacy.     

The immunogenicity of single and combination DNA vaccines against tuberculosis

DNA immunization is a promising new approach for the development of novel tuberculosis vaccines. In this study, the immune responses following the administration of single and combination tuberculosis DNA vaccines were evaluated. Single DNA vaccines encoding the MPT-63 and MPT-83 tuberculosis antigens evoked partial protection against an aerogenic challenge with M. tuberculosis Erdman in the mouse model of pulmonary tuberculosis. Immunization with a multivalent combination DNA vaccine (containing the ESAT-6, MPT-64, MPT-63, and KatG constructs) generated immune responses that indicated an absence of antigenic competition since antigen-specific cell-mediated and humoral responses were detected to each component of the mixture. More importantly, the combination vaccine elicited a strong protective response relative to the protection evoked by live BCG vaccine.     

Mucosal and systemic immune responses by intranasal immunization using archaeal lipid-adjuvanted vaccines

The utility of archaeal polar lipids as an adjuvant/delivery system for elicitation of antigen-specific mucosal immune responses in intranasally administered vaccines was investigated. Although unilamellar archaeosomes (liposomes made from archaeal polar lipids) with encapsulated ovalbumin (OVA/archaeosomes) induced anti-OVA IgG antibody responses in sera, they failed to induce anti-OVA IgA antibody responses at mucosal sites. However, the addition of CaCl2 to convert OVA/archaeosomes into an archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) vaccine (OVA/AMVAD) consisting of larger, particulate, aggregated structures resulted in an efficacious intranasal (i.n.) vaccine. Intranasal immunization of mice with OVA/AMVAD vaccines prepared from various archaeal polar lipid compositions elicited anti-OVA IgA antibody responses in sera, feces, bile, vaginal and nasal wash samples. The i.n. immunization also induced anti-OVA IgG, IgG1 and IgG2a antibody responses in sera, as well as cytotoxic T lymphocyte responses. The mucosal and systemic immune responses induced by OVA/AMVAD immunization were generally sustained over several months, and were subject to memory boost responses. Thus, polar archaeal lipids appear to be promising for developing a non-replicating mucosal adjuvant and vaccine delivery system.     

Multi-antigen vaccines based on complex adenovirus vectors induce protective immune responses against H5N1 avian influenza viruses

There are legitimate concerns that the highly pathogenic H5N1 avian influenza virus could adapt for human-to-human transmission and cause a pandemic similar to the 1918 "Spanish flu" that killed 50 million people worldwide. We have developed pandemic influenza vaccines by incorporating multiple antigens from both avian and Spanish influenza viruses into complex recombinant adenovirus vectors. In vaccinated mice, these vaccines induced strong humoral and cellular immune responses against pandemic influenza virus antigens, and protected vaccinated mice against lethal H5N1 virus challenge. These results indicate that this multi-antigen, broadly protective vaccine may serve as a safer and more effective approach than traditional methods for development of a pandemic influenza vaccine.     

Enhancement of antibody and cellular immune responses to malaria DNA vaccines by in vivo electroporation

We evaluated the effectiveness of in vivo electroporation (EP) for the enhancement of immune responses induced by DNA plasmids encoding the pre-erythrocytic Plasmodium yoelii antigens PyCSP and PyHEP17 administered intramuscularly and intradermally to mice. EP resulted in a 16- and 2-fold enhancement of antibody responses to PyCSP and PyHEP17, respectively. Immunization with 5 microg of DNA via EP was equivalent to 50 microg of DNA via conventional needle, thus reducing by 10-fold the required dose to produce a given effect. Moreover, IFN-gamma responses were increased by approximately 2-fold. Data demonstrate the potential of EP to enhance immune responses to DNA vaccines against infectious agents.     

Comparison of immune responses and protective efficacy of intranasal prime-boost immunization regimens using adenovirus-based and CpG/HH2 adjuvanted-subunit vaccines against genital Chlamydia muridarum infection

An efficacious Chlamydia vaccine is urgently needed to control Chlamydia infections. Heterologous prime-boost vaccination regimens are emerging as a promising strategy for preventing intracellular viral and bacterial infections. However, it remains to be determined if this regimen would be a feasible and effective approach for Chlamydia infection. In this study, we examined the immune response and the protective efficacy induced by various vaccination regimens using a recombinant adenovirus vector expressing the Chlamydia antigen CPAF (AdCPAF) and recombinant CPAF (rCPAF) subunit vaccines formulated with CpG oligodeoxynucleotides and/or a synthetic immunomodulatory peptide HH2 as adjuvants. A single dose of AdCPAF stimulated potent antibody production but weak cellular immune responses in mice. A booster rCPAF vaccine formulated with both CpG and HH2, but not CpG alone or HH2 alone, showed robust adjuvant effects on induction of Th1-biased cellular immune responses in mice primed with AdCPAF. In contrast, a homologous regimen using rCPAF/CpG/HH2 subunit vaccine for both priming and boosting induced a weak antibody response, but potent cellular immunity with a mixed Th1/Th17 profile. Despite the disparities observed in humoral and cellular immune responses, both the heterologous and homologous prime-boost regimens conferred significant immune protection against genital Chlamydia muridarum challenge in C3H/HeN and BALB/c mice.     

Induction of protective immunity against Eimeria tenella,Eimeria necatrix,Eimeria maxima and Eimeria acervulina infections using multivalent epitope DNA vaccines

Avian coccidiosis is mostly caused by mixed infection of several Eimeria species under natural conditions and immunity to avian coccidiosis is largely dependent on T-cell immune response. In this study, 14 T-cell epitope fragments from eight antigens of Eimeria tenella (E. tenella), Eimeria necatrix (E. necatrix), Eimeria maxima (E. maxima) and Eimeria acervulina (E. acervulina) were ligated with pVAX1 producing 14 monovalent DNA vaccines, respectively. Protective immunity of the monovalent DNA vaccines was assessed by in vivo challenge experiments and then four most protective fragments of each species were chosen to construct multivalent epitope DNA vaccines with or without chicken IL-2 as genetic adjuvant. Protective efficacies of the epitope DNA vaccines on chickens against E. tenella, E. necatrix, E. maxima and E. acervulina were evaluated. The results showed that the constructed multivalent epitope DNA vaccines significantly increased body weight gain, alleviated enteric lesions and reduced oocyst output of the infected birds. Especially, the multivalent epitope DNA vaccines of pVAX1-NA4-1-TA4-1-LDH-2-EMCDPK-1 and pVAX1-NA4-1-TA4-1-LDH-2-EMCDPK-1-IL-2 not only significantly increased body weight gain, alleviated enteric lesions and reduced oocyst output of the infected birds, but also resulted in anti-coccidial index (ACI) more than 170 against E. tenella, E. necatrix, E. maxima and E. acervulina, which indicated they could induce protective immunity against E. tenella, E. necatrix, E. maxima and E. acervulina. Our findings suggest the constructed multivalent epitope DNA vaccines are the potential candidate multivalent vaccines against mixed infection of Eimeria.     

Electroporation-based DNA transfer enhances gene expression and immune responses to DNA vaccines in cattle

Despite the potential of DNA vaccines to induce strong, balanced immune responses in small experimental species, the immune responses to DNA immunization in larger species have generally been moderate and inconsistent. In this study, the TriGridtrade mark Delivery System (TDS), an electroporation-based DNA delivery platform, was evaluated for administration of DNA vaccines to calves. When compared to conventional intramuscular delivery, TDS-based delivery markedly and consistently enhanced gene expression from a plasmid encoding a reporter gene, secreted alkaline phosphatase, and improved cell-mediated and humoral immune responses to a plasmid encoding a model antigen, hepatitis B surface antigen. Importantly, the TDS-based procedure was well tolerated by the calves, which did not need to be anesthetized or sedated. These results suggest that the TDS is a useful delivery method for DNA vaccines in cattle.     

Evaluation of immune response and protective effect of four vaccines against the tick-borne encephalitis virus

Among three main subtypes of the tick-borne encephalitis virus (TBEV), the Siberian subtype is currently dominant in a majority of the endemic regions of Russia. However, inactivated vaccines are based on TBEV strains of the heterologous Far Eastern or the European subtypes isolated 40–77 years ago. To analyze the efficacy of the available vaccines against currently prevailing TBEV isolates of the Siberian subtype, mice were immunized subcutaneously three times (one group per each vaccine). The expression of seven cytokine genes was determined using RT-PCR. Sera were studied using homologous and heterologous ELISA, hemagglutination inhibition (HI) and neutralization tests with TBEV strains of the Far Eastern, Siberian and European subtypes. Cross-protective efficacy of the vaccines was evaluated with the TBEV strain 2689 of Siberian subtype isolated from an ixodid tick from the Novosibirsk, South-Western Siberia, Russia in 2010. The cytokine gene expression profile indicates a predominantly Th2 response due to exogenous antigen presentation. Titers for homologous combinations of vaccine strain and strain in ELISA, HI and neutralization tests exceeded those for heterologous antigen-antibody pairs. Despite antibody detection by means of ELISA, HI and neutralization tests, the mouse protection afforded by the vaccines differed significantly. Complete protection of mice challenged with 100 LD₅₀ virus of the Siberian subtype was induced by the vaccine “Encevir” (“Microgen”, Tomsk, Russia). The minimal immunization doze (MID₅₀) of “Encevir” protecting 50% of the mice was less than 0.0016 ml. Partial protective effect of vaccines produced in Moscow, Russia and Austria revealed MID₅₀ within recommended intervals (0.001–0.017 ml). However, the MID₅₀ for the vaccine “Encepur” (Novartis, Germany) 0.04 ml exceeded acceptable limits with total loss of mice immunized with vaccine diluted 32, 100 and 320 fold. These results suggest regular evaluation of TBEV vaccines in regions where heterologous virus subtypes prevail.     

The antifertility effects of DNA vaccine-induced immune responses against uroguanylin

Previously we found that uroguanylin displayed a specific expression pattern in the uteri during pregnancy. In this study, the effect of uroguanylin in early pregnancy was studied by DNA vaccine, RT-PCR, Western blot, immunofluorescence and immunohistochemistry. The results showed that (1) the anti-rUfl antibodies could be elicited in the mice after immunization by recombinant plasmid pCR3.1-rUfl; (2) the birth rate of the female mice immunized by pCR3.1-rUfl was significantly reduced (p<0.01); (3) anti-rUfl antibodies could bind with uroguanylin in the uteri of the non-pregnant mice immunized by pCR3.1-rUfl; (4) in the non-mated experiments, in the uteri of normal, pCR3.1- and pCR3.1-rUfl-immunized mice, expression of p53 and vascular endothelial growth factor (VEGF) was not detected, Bax was expressed, and transforming growth factor beta 1 (TGFbeta1) expression was very little; (5) in the mated experiments, p53, Bax, VEGF and TGFbeta1 were expressed in the uteri of saline- and pCR3.1-immunized mice that were pregnant. However, their expression was significantly decreased in the uteri of the non-pregnant mice immunized by pCR3.1-rUfl on the 9th day of pregnancy (p<0.01). The results indicate that the immunization by pCR3.1-rUfl has antifertility effect.