Immunization of woodchucks (Marmota monax) with hepatitis delta virus DNA vaccine

We investigated the DNA immunization approach in order to induce a protective immune response against hepatitis delta virus (HDV) superinfection of chronically woodchuck hepatitis virus (WHV) infected woodchucks. The animals were immunized with an expression vector encoding HDAg by gene gun. T cell and humoral immune responses induced by this protocol were determined and compared with those induced by HDAg immunization using a CpG oligonucleotide as an adjuvant. After immunization the woodchucks were challenged with 10⁶ genome equivalents of HDV. The protein immunization with HDAg induced good humoral and T helper cell responses in the woodchucks, but did not protect them from HDV superinfection. The DNA immunized woodchucks were also not protected from HDV superinfection, however, the course of infection was modified: HDV viremia occurred later, the typical fluctuation of the HDV RNA titer with several peaks was absent, and antibodies to HDV were not detectable.     

Induction of cytotoxic T lymphocyte responses against hepatitis delta virus antigens which protect against tumor formation in mice

The cellular immune response is a crucial defense mechanism against hepatotropic viruses and in chronic viral hepatitis prevention. Moreover, hepatitis delta virus (HDV) immunogenicity may be an important component in the development of prophylactic and therapeutic vaccines. Therefore, we evaluated the immunogenicity of the small (HDAg) or large delta antigen (LHDAg) to be used as a DNA-based vaccine. We immunized different mouse haplotypes, determined cellular immune responses, and tested protection of animals against tumor formation using syngeneic tumor cells stably expressing the delta antigens. Both LHDAg and HDAg primed CD4+ and CD8+ T cell immunity against both forms of delta antigens. CD8+ T cell frequencies were about 1% and antigen-specific CD8+ T cells remained detectable directly ex vivo for at least 35 days post-injection. No anti-delta antibody responses could be detected despite multiple detection systems and varied immunization approaches. We observed protection against syngeneic tumor formation and growth in mice immunized with DNA plasmids encoding secreted or intracellular forms of HDAg and LHDAg but not with recombinant HDAg establishing the generation of significant cellular immunity in vivo. Both CD4+ and CD8+ T cells were required for antitumoral activity as determined by in vivo T cell depletion experiments. The results indicate that DNA-based immunization with genes encoding LHDAg and HDAg induces strong T cell responses and, therefore, is an attractive approach for the construction of therapeutic and prophylactic T cell vaccines against HDV.     

Immunization of woodchucks with adjuvanted sHDAg (p24): immune response and outcome following challenge

The immunogenicity and the protection induced by an hepatitis delta virus (HDV) vaccine consisting of the small nucleoprotein (HDAg) (p24) and adjuvanted with MF59 or Freund's adjuvant (FA) were evaluated in woodchucks chronically infected with woodchuck hepatitis virus (WHV) and challenged with hepatitis delta virus. Humoral and T-cell-mediated responses to HDAg were measured. Anti-HD antibodies appeared earlier in the FA/p24 animals. After challenge, all MF59/p24 vaccinated animals showed a response to HDAg-derived peptides, compared to two of the five FA/p24 animals and one of the control animals. Serum HDV-RNA peak values and persistence were considerably reduced in immunized animals, in comparison to controls. Furthermore, HDV-RNA was absent in autopsy liver tissues of 50% of the MF59/p24 animals, whereas high levels were present in all of the FA/p24 animals and controls. Histological liver analysis performed before and after challenge revealed the presence of acute hepatitis-like lesions only in the controls. Overall, the results suggest that the MF59/p24 vaccine better controls the infection in terms of viral replication and survival.     

Comparison of the immune responses in BALB/c mice following immunization with DNA-based and live attenuated vaccines delivered via different routes

The objective of this study was to compare immune responses induced in BALB/c mice following immunization with pcDNA-GPV-VP2 DNA by gene gun bombardment (6 μg) or by intramuscular (im) injection (100 μg) with the responses to live attenuated vaccine by im injection (100 μl). pcDNA3.1 (+) and physiological saline were used as controls. Peripheral blood samples were collected at 3, 7, 14, 21, 28, 35, 49, 63, 77 and 105 d after immunization. T lymphocyte proliferation was analyzed by MTT assay and enumeration of CD4⁺, and CD8⁺ T cell populations in peripheral blood was performed by flow cytometric analysis. Indirect ELISA was used to detect IgG levels. Cellular and humoral responses were induced by pcDNA-GPV-VP2 DNA and live virus vaccines. No differences were observed in T cell proliferation and CD8⁺ T cell responses induced by the genetic vaccine regardless of the route of delivery. However, CD4⁺ T cell responses and humoral immunity were enhanced in following gene gun immunization compared with im injection of the genetic vaccine. Cellular and humoral immunity was enhanced in following gene gun delivery of the genetic vaccine compared with the live attenuated vaccine. In conclusion, the pcDNA-GPV-VP2 DNA vaccine induced enhanced cellular and humoral immunity compared with that induced by the live attenuated vaccine.     

Oral administration of low doses of plant-based HBsAg induced antigen-specific IgAs and IgGs in mice,without increasing levels of regulatory T cells

Plant-based oral vaccines run the risk of activating regulatory T cells (Tregs) and suppressing the antigen-specific immune response via oral tolerance. Mice humanized for two HLA alleles (HLA-A2.1 and HLA-DR1) were used to measure changes in Tregs and antigen-specific immune responses induced by the oral administration of tobacco (Nicotiana tabacum), expressing the hepatitis B surface antigen (HBsAg). Antigen-specific CD8+ T cell activation was not detected, but the plant-based oral immunization, without adjuvant, resulted in humoral responses comparable to those obtained by adjuvanted DNA immunization. Treg titers did not increase with DNA immunization. In contrast, with plant immunization, Tregs increased linearly to reach a plateau at high antigen doses. The highest humoral IgA and IgG responses correlated with the lowest plant antigen dose (0.5 ng), while for DNA immunization the best antibody responses were obtained at higher antigen doses. These experiments suggest that plant-based oral vaccines could be adjusted to minimize tolerance, while still inducing an immune response. Oral tolerance and adjuvant engineering in plants are discussed.     

DNA vaccination in utero: a new approach to induce protective immunity in the newborn

Infectious diseases are the primary cause of neonatal morbidity and mortality in people, resulting in millions of deaths every year. Infection of the newborn with some of the pathogens involved, such as herpes simplex virus (HSV), human immunodeficiency virus (HIV), hepatitis B virus (HBV), human cytomegalovirus (HCMV) or group B Streptococcus sp. (GBS), usually occurs at the end of pregnancy, during birth or by breast feeding. Therefore, active immunization of the fetus might represent an effective approach to reduce the high risk of neonatal diseases. We recently showed that DNA immunization in utero within the third trimester of gestation induced strong humoral and cell-mediated immune responses in immunized fetal lambs. Here, we demonstrate that fetal immunization was safe and did not affect fetal gestation, neonatal viability, or significantly alter blood leukocyte populations. In utero immunization resulted in the induction of protective mucosal immunity and immune memory in the newborn lamb. Furthermore, there was no evidence that in utero DNA immunization induced immune tolerance. Our results also indicate that the uptake and expression of the plasmid DNA already occurred within the epithelium of the oral cavity. This correlates with our previous findings that local immune responses were found exclusively in the retropharyngeal lymph nodes draining the oral cavity.     

丁型肝炎系列酶免诊断试剂的研制及应用

为解决丁型肝炎诊断试剂短缺的问题，本研究采用实验感染ＨＤＶ的土拨鼠肝脏和丁型肝炎病人血清，提取纯化丁型肝炎病毒抗原和抗体，制备了检测ＨＤＡｇ、抗－ＨＤ、ＩｇＭ抗－ＨＤ的系列酶免诊断试剂。该试剂与进口同类试剂相比较，符合率达１００％。其特异性好，灵敏度较高。对不同类型乙型肝炎病人血清检测，ＨＤＡｇ、抗－ＨＤ及ＩｇＭ抗－ＨＤ阳性率和总阳性率分别为２．９９％、３．３６％、３．７３％和７．２８％。该试剂的研制为丁型肝炎病毒感染的临床诊断及流行病学研究提供了可靠的实验手段。     

Long-lasting humoral and cellular immune responses and mucosal dissemination after intramuscular DNA immunization

Naïve Indian rhesus macaques were immunized with a mixture of optimized plasmid DNAs expressing several SIV antigens using in vivo electroporation via the intramuscular route. The animals were monitored for the development of SIV-specific systemic (blood) and mucosal (bronchoalveolar lavage) cellular and humoral immune responses. The immune responses were of great magnitude, broad (Gag, Pol, Nef, Tat and Vif), long-lasting (up to 90 weeks post third vaccination) and were boosted with each subsequent immunization, even after an extended 90-week rest period. The SIV-specific cellular immune responses were consistently more abundant in bronchoalveolar lavage (BAL) than in blood, and were characterized as predominantly effector memory CD4⁺ and CD8⁺ T cells in BAL and as both central and effector memory T cells in blood. SIV-specific T cells containing Granzyme B were readily detected in both blood and BAL, suggesting the presence of effector cells with cytolytic potential. DNA vaccination also elicited long-lasting systemic and mucosal humoral immune responses, including the induction of Gag-specific IgA. The combination of optimized DNA vectors and improved intramuscular delivery by in vivo electroporation has the potential to elicit both cellular and humoral responses and dissemination to the periphery, and thus to improve DNA immunization efficacy.     

丁型肝炎病毒感染的血清流行病学观察

1987年4月至1988年10月间,本文应用酶联吸附法(EIA)对石家庄地区Ml例乙型肝炎病毒感染者进行了抗-HD的检测,共发现35例阳性,阳性率12.92%,其中男性阳性率14.06%(27/102),女性为10.13%(8/79),男女间差异无统计学意义(P>0.05),提示石家庄地区可能为丁型肝炎病毒感染的高发区.在这些人群中,慢性活动性肝炎、慢性迁延性肝炎和肝硬化的抗-HD阳性率明显高于HBsAg携带者,但三者相互间差异无统计学意义,表明合并或重香感染HDV对乙肝慢性化及肝硬化的形成具有重要的意义。本研究证明在乙型肝炎病毒感染人群中丁型肝炎病毐与年龄、性别、职业等因素关系不密切。     

The improved antibody response against HIV-1 after a vaccination based on intrastructural help is complemented by functional CD8+ T cell responses

Despite more than three decades of intense research, a prophylactic HIV-1 vaccine remains elusive. Four vaccine modalities have been evaluated in clinical efficacy studies, but only one demonstrated at least modest efficacy, which correlated with polyfunctional antibody responses to the HIV surface protein Env. To be most effective, a HIV-1 vaccine probably has to induce both, functional antibody and CD8⁺ T cell responses. We therefore analyzed DNA/DNA and DNA/virus-like particle (VLP) regimens for their ability to induce humoral and cellular immune responses. Here, DNA vaccination of mice induced strong CD8⁺ responses against Env and Gag. However, the humoral response to Env was dominated by IgG1, a subclass known for its low functionality. In contrast, priming only with the Gag-encoding plasmid followed by a boost with VLPs consisting of Gag and Env improved the quality of the anti-Env antibody response via intrastructural help (ISH) provided by Gag-specific T cells to Env-specific B cells. Furthermore, the Gag-specific CD8⁺ T cells induced by the DNA prime immunization could still protect from a lethal infection with recombinant vaccinia virus encoding HIV Gag. Therefore, this immunization regimen represents a promising approach to combine functional antibody responses toward HIV Env with strong CD8⁺ responses controlling early viral replication.     

Establishment of functional influenza virus-specific CD8+ T cell memory pools after intramuscular immunization

The emergence of the avian-origin influenza H7N9 virus and its pandemic potential has highlighted the ever-present need to develop vaccination approaches to induce cross-protective immunity. In this study, we examined the establishment of cross-reactive CD8⁺ T cell immunity in mice following immunization with live A/Puerto Rico/8/1934 (PR8; H1N1) influenza virus via two non-productive inoculation routes. We found that immunization via the intramuscular (IM) route established functional influenza-virus specific memory CD8⁺ T cell pools capable of cross-reactive recall responses. Epitope-specific primary, memory and recall CD8⁺ T-cell responses induced by the IM route, highly relevant to human influenza immunisations, were of comparable magnitude and quality to those elicited by the intraperitoneal (IP) priming, commonly used in mice. Furthermore, IM immunisation resulted in lower lung viral titres following heterologous challenge with A/Aichi/68 (X31; H3N2) compared to the IP route. Examining the ability of DCs from lymphoid organs to present viral antigen revealed that immune induction following IM immunization occurred in draining lymph nodes, while immunization via the IP route resulted in the priming of responses in distal lymphoid organs, indicative of a systemic distribution of antigen. No major differences in the pulmonary cytokine environment of immunized animals following X31 challenge were observed that could account for the improved heterologous protection induced by the IM route. However, while both routes induced similar levels of PR8-specific antibodies, higher levels of cross-reactive antibodies against X31 were induced following IM inoculation. Our data demonstrate how non-replicative routes of infection can induce efficient cross-reactive CD8⁺ T cell responses and strong strain-specific antibody responses, with the additional benefit from IM priming of enhanced heterosubtypic antibody production.     

Induction of antigen-specific CD8+ T cells, T helper cells, and protective levels of antibody in humans by particle-mediated administration of a hepatitis B virus DNA vaccine

A DNA vaccine against the hepatitis B virus (HBV) was evaluated for safety and induction of immune responses in 12 healthy, hepatitis-na?ve human volunteers using the needle-free PowderJect system to deliver gold particles coated with DNA directly into cells of the skin. Three groups of four volunteers received three administrations of DNA encoding the surface antigen of HBV at one of the three dose levels (1, 2, or 4 microg). The vaccine was safe and well tolerated, causing only transient and mild to moderate responses at the site of administration. HBV-specific antibody and both CD4+ and CD8+ T cell responses were measured before and after each immunization. All the volunteers developed protective antibody responses of at least 10 mIU/ml. In volunteers who were positive for the HLA class I A2 allele, the vaccine also induced antigen-specific CD8+ T cells that bound HLA-A2/HBsAg(335-343) tetramers, secreted IFN-gamma, and lysed target cells presenting a hepatitis B surface antigen (HBsAg) CTL epitope. Enumeration of HBsAg-specific T cells producing cytokine indicated preferential induction of a Type 1 T helper cell response. These results provide the first demonstration of a DNA vaccine inducing protective antibody titers and both humoral and cell-mediated immune responses in humans.     

Unique immunogenicity of hepatitis B virus DNA vaccine presented by live-attenuated Salmonella typhimurium

A novel vaccine for hepatitis B virus (HBV) was designed by putting a naked DNA vaccine carrying hepatitis B surface antigen (HBsAg) into live-attenuated Salmonella typhimurium. Mucosal immunization by the oral route in mice showed significantly stronger cytotoxic T lymphocyte (CTL) response than recombinant HBsAg vaccination (P < 0.01 at an effector:target ratio of 100:1), while comparable to intramuscular naked DNA immunization at all effector:target ratios. Contrary to previous reports on naked DNA vaccines given intramuscularly, the IgG antibody response induced by the mucosal DNA vaccine is relatively weak when compared to recombinant HBsAg vaccine (P < 0.001 at day 21). These findings are supported by a high interferon-gamma but a low interleukin-4 level detected in the supernatant of splenic cell cultures obtained from mucosally immunized mice. As distinct to recombinant HBsAg vaccine which is effective for protection, oral mucosal DNA vaccine should be considered as a candidate for therapeutic immunization in chronic HBV infection, donor immunization before adoptive transfer of HBV-specific CTL to HBsAg positive bone marrow transplant recipients, and immunization of non-responders to recombinant HBsAg vaccine. This strongly cellular and relatively absent humoral response may make this vaccine a better candidate as a therapeutic vaccine for chronic HBV carriers than naked DNA vaccines, as the humoral response is relatively less important for the clearance of HBV from hepatocytes, but its presence may lead to side effects such as serum sickness and immune complex deposition in chronic HBV carriers.     

Optimization of DNA vaccination immune responses in dairy cows: effect of injection site and the targeting efficacy of antigen-bCTLA-4 complex

The objectives of this study were to evaluate the effects of immunization site and antigen presenting cell targeting on cattle immune responses to DNA immunization. Cows were vaccinated with the plasmid expression vector pCI alone, pCI encoding the bacterial antigen beta-galactosidase (pCI-beta-gal) or pCI encoding bCTLA 4 fused to beta-gal (pCI-bCTLA-hIgG-beta-gal). The plasmids were delivered by intramuscular, intradermal, intramammary gland, or intra supramammary lymph node needle-injection. Both vaccines induced significant humoral and cellular immune responses. pCI-beta-gal elicited a higher IgG response than immunization with pCI-bCTLA-hIgG-beta-gal. Cows injected intramuscularly and intramammary had higher IgG and IgG-1 humoral responses than cows immunized intradermaly or in the lymph nodes. The injection site did not significantly affect the magnitude of the IgG2 and IgM antibody responses, although a trend similar to the IgG results was observed. The lymphocyte proliferation index was higher with pCI-beta-gal but was not affected by the injection site. These results suggest that in bovine, the injection site can affect immune responses but they do not provide evidence that bCTLA-4-hIgG-antigen targeting is effective in cattle.     

Evaluation of hyaluronic acid-based combination adjuvant containing monophosphoryl lipid A and aluminum salt for hepatitis B vaccine

Here, monophosphoryl lipid A (MPLA) and aluminum salt (Alum) were introduced into a hyaluronic acid (HA)-based combination vaccine adjuvant for hepatitis B vaccine (HBV). Although Alum is a well-known hepatitis B vaccine adjuvant that induces an enhanced humoral immune response, it cannot induce the cellular immune responses. On the other hand, MPLA has been generally reported to promote IFN-γ production via antigen-specific CD4⁺ T cells, but it is not water soluble as a result of its long hydrophobic alkyl chains. To this end, water insoluble MPLA could be solubilized in an aqueous solution with the help of HA, which contains many carboxyl and hydroxyl groups that can be used to attach to the hydroxyl head groups of MPLA via hydrogen bonds. Three groups of mice were treated with either hepatitis B surface antigen (HBsAg) alone, HBsAg_Alum complex, or HBsAg_Alum_MPLA/HA complex. The group immunized with the HBsAg_Alum_MPLA/HA complex exhibited a high increase in cellular immune response as well as in humoral immune response relative to the other two groups. The antibody, cytokine and T cell levels were most elevated in the group of mice immunized with HBsAg_Alum_MPLA/HA complex, even at a 1 μg/mice dose, and the magnitude was still maintained even after 8 weeks. Specifically, the antibody value was 120 times larger in mice vaccinated with HBsAg_Alum_MPLA/HA complex than in mice vaccinated with HBsAg_Alum complex designed similar to commercially available hepatitis B vaccine, Engerix B. The cytokine and T cell proliferation levels were 2 times and 6 times larger in mice adjuvanted with HBsAg_Alum_MPLA/HA complex than in those vaccinated with HBsAg_Alum. The results therefore indicate that incorporating MPLA and Alum with HA can be a potent strategy to increase both the magnitude and the persistence of HBsAg-specific immune responses to protect hosts against hepatitis B virus infection.     

Bovine respiratory syncytial virus ISCOMs-Immunity, protection and safety in young conventional calves

Bovine respiratory syncytial virus (BRSV) is a major cause of bronchiolitis and pneumonia in cattle and causes yearly outbreaks with high morbidity in Europe. Commercial vaccines against this virus needs improvement of efficacy, especially in calves with BRSV-specific maternally derived antibodies (MDA). We previously reported that an experimental BRSV-ISCOM vaccine, but not a commercial vaccine, induced strong clinical and virological protection in calves with MDA, immunized at 7-15 weeks of age. The aim of the present study was to characterize the immune responses, as well as to investigate the efficacy and safety in younger animals, representing the target population for vaccination. Four groups of five 3-8 week old calves with variable levels of BRSV-specific MDA were immunized s.c. twice at a 3 weeks interval with (i) BRSV immunostimulating complexes (BRSV-ISCOMs), (ii) BRSV-protein, (iii) adjuvant, or (iv) PBS. All calves were challenged with virulent BRSV by aerosol 2 weeks later and euthanized on day 6 after infection. The cellular and humoral responses were monitored as well as the clinical signs, the viral excretion and the pathology following challenge. Despite presence of MDA at the time of the immunization, only a minimum of clinical signs were observed in the BRSV-ISCOM group after challenge. In contrast, in all control groups, clinical signs of disease were observed in most of the animals (respiratory rates up to 76 min⁻¹ and rectal temperatures up to 41 °C). The clinical protection was associated to a highly significant reduction of virus replication in the upper and lower respiratory tract of calves, rapid systemic and local antibody responses and T helper cell responses dominated by IFNγ production. Animals that did not shed virus detectable by PCR or cell culture following challenge possessed particularly high levels of pulmonary IgA. The protective immunological responses to BRSV proteins and the ability to overcome the inhibiting effect of MDA were dependent on ISCOM borne antigen presentation.     

Platycodin D is a potent adjuvant of specific cellular and humoral immune responses against recombinant hepatitis B antigen

The ideal adjuvants for hepatitis B vaccines should be capable of eliciting both strong humoral and cellular immune responses, especially Th1 cell and cytotoxic T lymphocyte (CTL) responses. However, Alum used as adjuvants in the hepatitis B vaccines currently commercialized offers limitation in inducing cell-mediated response. Therefore, a less hemolytic saponin platycodin D (PD) from the root of Platycodon grandiflorum has been explored for its potential as an alternative adjuvant. In order to compare the adjuvant activity with Alum, antigen-specific cellular and humoral immune responses were evaluated following immunization with a formulation containing hepatitis B surface antigen (HBsAg) adjuvanted with PD and Alum in mice. The Con A-, LPS-, and HBsAg-induced splenocyte proliferation and the serum HBsAg-specific IgG, IgG1, IgG2a, and IgG2b antibody titers in the HBsAg-immunized mice were significantly enhanced by PD (P < 0.05, P < 0.01 or P < 0.001). PD also significantly promoted the production of Th1 (IL-2 and IFN-γ) and Th2 (IL-10) cytokines and up-regulated the mRNA expression of Th1 cytokines (IL-2 and IFN-γ) in splenocytes from the mice immunized with HBsAg (P < 0.001). Besides, PD remarkably increased the killing activities of natural killer (NK) cells and CTLs from splenocytes in the HBsAg-immunized mice (P < 0.001), which may have important implications for vaccination against hepatitis B virus. The results indicated that PD has strong potential to increase both cellular and humoral immune responses and elicit a balanced Th1/Th2 response against HBsAg, and that PD may be the candidates as adjuvants for use in prophylactic and therapeutic hepatitis B vaccine.     

Expression of interleukin-6 by a recombinant rabies virus enhances its immunogenicity as a potential vaccine

Several studies have confirmed that interleukin-6 (IL6) mediates multiple biological effects that enhance immune responses when used as an adjuvant. In the present study, recombinant rabies virus (RABV) expressing canine IL6 (rHEP-CaIL6) was rescued and its pathogenicity and immunogenicity were investigated in mice. We demonstrated that mice received a single intramuscular immunization with rHEP-CaIL6 showed an earlier increase and higher maximum titres of virus-neutralizing antibody (VNA) as well as anti-RABV antibodies compared with mice immunized with the parent strain. Moreover, survival rates of mice immunized with rHEP-CaIL6 were higher compared with mice immunized with parent HEP-Flury according to the challenge assay. Flow cytometry further confirmed that immunization with rHEP-CaIL6 induced the strong recruitment of mature B cells and CD8⁺ T cells to lymph nodes, which may partially explain the high levels of VNA and enhanced cellular immunity. Quantitative real-time PCR indicated that rHEP-CaIL6 induced stronger inflammatory and immune responses in the central nervous system, which might have allowed virus clearance in the early infection phase. Furthermore, mice infected intranasally with rHEP-CaIL6 developed no clinical symptoms while mice infected with HEP-Flury showed piloerection. In summary, these data indicate that rHEP-CaIL6 induces a strong, protective immune response with a good safety profile. Therefore, a recombinant RABV strain expressing canine IL6 may aid the development of an effective, safe attenuated rabies vaccine.     

Recombinant vesicular stomatitis virus-based west Nile vaccine elicits strong humoral and cellular immune responses and protects mice against lethal challenge with the virulent west Nile virus strain LSU-AR01

Vesicular stomatitis virus (VSV) has been extensively utilized as a viral vector system for the induction of protective immune responses against a variety of pathogens. We constructed recombinant VSVs specifying either the Indiana or Chandipura virus G glycoprotein and expressing the West Nile virus (WNV) envelope (E) glycoprotein. Mice were intranasally vaccinated using a prime (Indiana)-boost (Chandipura) immunization approach and challenged with the virulent WNV-LSU-AR01. Ninety-percent (9 of 10) of the vaccinated mice survived as compared to 10% of the mock-vaccinated mice after WNV lethal challenge. Histopathological examination of brain tissues revealed neuronal necrosis in mock-vaccinated mice but not in vaccinated mice, and vaccinated, but not mock-vaccinated mice developed a strong neutralizing antibody response against WNV. Extensive immunological analysis using polychromatic flow cytometry staining revealed that vaccinated, but not mock-vaccinated mice developed robust cellular immune responses as evidenced by up-regulation of CD4⁺ CD154⁺ IFNγ⁺ T cells in vaccinated, but not mock-vaccinated mice. Similarly, vaccinated mice developed robust E-glycoprotein-specific CD8⁺ T cell immune responses as evidenced by the presence of a high percentage of CD8⁺ CD62L^low IFNγ⁺ cells. In addition, a sizeable population of CD8⁺ CD69⁺ cells was detected indicating E-specific activation of mature T cells and CD4⁺ CD25⁺ CD127^low T regulatory (T reg) cells were down-regulated. These results suggest that VSV-vectored vaccines administered intranasally can efficiently induce protective humoral and cellular immune responses against WNV infections.     

Homologous prime-boost with Zika virus envelope protein and poly (I:C) induces robust specific humoral and cellular immune responses

The recent outbreaks of Zika virus (ZIKV) infection and the potential association with Guillain-Barré syndrome in adults and with congenital abnormalities have highlighted the urgency for an effective vaccine. The ZIKV Envelope glycoprotein (E_ZIKV) is the most abundant protein on the virus surface, and has been evaluated together with the pre-membrane protein (prM) of the viral coat as a vaccine candidate in clinical trials. In this study, we performed a head-to-head comparison of the immune response induced by different E_ZIKV-based vaccine candidates in mice. We compared different platforms (DNA, recombinant protein), adjuvants (poly (I:C), CpG ODN 1826) and immunization strategies (homologous, heterologous).The hierarchy of adjuvant potency showed that poly (I:C) was a superior adjuvant than CpG ODN. While poly (I:C) assisted immunization reached a plateau in antibody titers after two doses, the CpG ODN group required an extra immunization dose. Besides, the administration of poly (I:C) induced higher E_ZIKV-specific cellular immune responses than CpG ODN. We also show that immunization with homologous prime-boost E_ZIKV protein + poly (I:C) regimen induced a more robust humoral response than homologous DNA (pVAX-E_ZIKV) or heterologous regimens (DNA/protein or protein/DNA). A detailed analysis of cellular immune responses revealed that homologous (E_ZIKV + poly (I:C)) and heterologous (pVAX-E_ZIKV/E_ZIKV + poly (I:C)) prime-boost regimens induced the highest magnitude of IFN-γ secreting cells and cytokine-producing CD4⁺ T cells.Overall, our data demonstrate that homologous E_ZIKV + poly (I:C) prime-boost immunization is sufficient to induce more robust specific-E_ZIKV humoral and cellular immune responses than the other strategies that contemplate homologous DNA (pVAX-E_ZIKV) or heterologous (pVAX-E_ZIKV/E_ZIKV + poly (I:C), and vice-versa) immunizations.