Japanese encephalitis vaccine in travelers

Extensive vaccination against Japanese encephalitis (JE) has been carried out in many Asian countries for the past 20 years and is also increasingly recommended for travelers to endemic areas. Concerns have been raised regarding potential neurological and allergic side effects of the currently available JE vaccine, which is manufactured from mouse brain. A new purified, inactivated JE virus vaccine (IC51) has been developed, which is manufactured in a Vero cell culture substrate. Studies show that the vaccine is both safe and immunogenic and the product will be licensed very soon for use in many industrialized countries. Once a highly immunogenic and safe product is available, wider use of JE vaccine in travelers will be prudent. Currently, vaccination is restricted to travelers with an increased risk of acquiring JE. Individuals at increased risk have been defined quite arbitrarily as travelers with increased behavioral contact to JE-transmitting mosquitoes, in particular, during stays in rural areas and during the transmission season. However, the possibility of an infection with JE virus can never be ruled out when traveling to endemic areas and infection can prove disastrous for the individual concerned. Since a safe product will be available very soon, guidelines and recommendations will have to be reconsidered.     

Allergic reactions to Japanese encephalitis vaccine

The JEV widely is used in Asian countries each year and is an important vaccine for travelers to the East from other parts of the world. JE virus is a zoonotic disease with natural reservoirs and cannot be eliminated. Although a declining incidence of JE has been observed in Asia because of reduced transmission by agricultural approaches and vaccination, the most important control measure now, and in the future, is vaccination of humans against JE. The inactivated vaccine, produced from infected mouse-brain-derived tissue, is the only commercially available vaccine. There are several concerns with the use of this vaccine. It is expensive, requires two or three doses to achieve protective efficacy, and, in practice, requires further booster doses to maintain immunity. The apparent increase in allergic reactions in the first part of the 1990s has set focus on the safety of the JEV. A cheap, live attenuated SA 14-14-2 vaccine is used almost exclusively in China and parts of Korea, but there have been no trials of SA 14-14-2 vaccine outside JE endemic countries. The vaccine seems to be highly efficient, and few adverse events have been observed; however, PHK cells are used for the production of this vaccine, and these cells are not approved by the WHO. A satisfactory cell substrate is needed. A committee under the WHO has proposed that for the live JEV, there should be validity of the assays for retrovirus when applied to PHK cell substrate and validity of the mouse assays for neurovirulence. Further information should be reviewed on the long-term follow-up of recipients of the vaccine. Several new types of vaccines have reached the phase of clinical trials; however, studies remain to be completed. Until a new vaccine is available, the priority of surveillance of adverse events and the continuous reporting of such events to the users of the vaccines must be of importance. This fact is highlighted by the possibility of the varying frequency of adverse events with different batches over the years. The WHO offers information and recommendations for vaccines in the EPI and issues a series of updated papers on other vaccines that are of international public health importance (eg, JEV). The development of alternative efficient, safe, and appropriately priced JEVs is recommended, as is intensified surveillance of adverse events. Prospective vaccine studies of safety may be limited because of sample size and because rare adverse events may not be detected. Several new initiatives have been taken to improve surveillance of adverse events to vaccines within the past 10 years. In Japan, there is an increasing awareness of the importance of efforts taken to improve vaccine safety, and surveillance of adverse events and possibilities of compensation for vaccine-related injuries are in place. In Vietnam, a database to detect adverse events after vaccination has been established; the project involves active visits to data collectors at the vaccination sites. Comparative studies of adverse events, such as one recent study from Japan and the United States, are important for the evaluation of the reporting systems. The reporting rate for JEV adverse events from Japan was approximately one order of magnitude lower than that in the United States. Japan had strict predefined reporting criteria and time limits for observations. If time limits for the observation are too strict (eg, defining a possible neurologic reaction to occur within 1 week after vaccination), later reactions will not be included (eg, if ADEM is elicited by a vaccine, the symptoms cannot be expected to occur until weeks after the vaccination). The passive surveillance systems have limitations with an underreporting of adverse events, depending on clinical seriousness, temporal proximity to vaccination, awareness of healthcare workers, and tradition of reporting particular events. In developed countries, surveillance of adverse events is formalized, although not necessarily optimal. An increase in reporting would be expected when the reporting of adverse events is mandatory. Reports have been sent to VAERS, the Vaccine Safety Datalink Project, and the European Union Pharmacovigilance System. A Brighton collaboration has been implemented to enhance comparability of vaccine safety data. Public health authorities in specific countries, such as the CDC in the United States and the National Advisory Committee in Canada, regularly have published information on the JE situation in Asia and the preventive measures to be taken, including information on the vaccines and adverse reactions. The conventional recommendation is that travelers should be vaccinated if they will spend more than 1 month in a JE endemic area or in areas with epidemic transmission with even shorter periods. Although the risk for JE for short-term travelers is considered small (1 case per 1 million travelers per year), sporadic cases, including deaths, have been reported among tourists traveling to endemic areas. Risk for travelers in rural districts in the season of risk is considerably higher (range, 1 case per 5000 travelers to 1 case per 20,000 travelers per week). Doctors who advise travelers should be updated on the latest JE occurrences in Asia. Updates on the JE situation can be found on bulletins at http://www.promedmail.org or are available from the WHO or CDC. The allergic reactions primarily described after vaccination with the inactivated mouse-brain-derived JEV have been observed in several countries during the 1900s. Allergic reactions, including the mucocutaneous and neurologic reactions reported after JE vaccination, may vary in frequency, and these reactions should be evaluated meticulously yearly. This step enables recommendations, including information on possible side effects, to be given in an optimal way.     

Adjuvant activity of CpG oligodeoxynucleotides

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs directly stimulate human B cells and plasmacytoid dendritic cells (pDCs), thereby promoting the production of Th1 and proinflammatory cytokines and the maturation/activation of professional antigen-presenting cells. These activities enable CpG ODNs to act as immune adjuvants, accelerating and boosting antigen-specific immune responses by 5- to 500-fold. The CpG motifs present in bacterial DNA plasmids may contribute to the immunogenicity of DNA vaccines. Ongoing clinical studies indicate that CpG ODNs are safe and well tolerated when administered as adjuvants to humans and can improve vaccine-induced immune responses.     

Immunomodulatory effects associated with a live vaccine against Leishmania major containing CpG oligodeoxynucleotides

The inoculation of live Leishmania major to produce a lesion that heals (leishmanization) is to date the only vaccine against cutaneous leishmaniasis that has proven effective in humans, but it still has an unacceptable frequency of large ulcerating lesions that are slow to heal or, in rare cases, non-healing. We have previously shown that C57BL/6 mice vaccinated intradermally with 10(4) L. major/50 microg CpG oligodeoxynucleotides develop little or no dermal lesions and show early containment of parasite growth in the vaccination site, eliminating safety concerns related to the inoculation of live organisms. The addition of CpG to the live vaccine resulted in early activation of dermal dendritic cells and increased IL-6 production, as well as in a reduction in the accumulation of Foxp3(+)CD4(+)CD25(+) regulatory T (T(reg)) cells that naturally occurs in the skin following Leishmania infection. Neutralization of IL-6 caused the development of larger lesions and increased local T(reg) cell numbers. Transfer of vaccine-primed dendritic cells into IL-6-deficient mice mitigated lesion development, indicating that IL-6 reconstitution limited pathology in the vaccination site.     

Recombinant GroES in combination with CpG oligodeoxynucleotides protects mice against Mycobacterium avium infection

The groES gene of Mycobacterium avium strain 485 was cloned and expressed in Escherichia coli and the recombinant GroES protein was purified by affinity chromatography. The GroES preparation showed high purity by electrophoresis and immunoblotting. Immuno-electron microscopy showed that GroES was located both in the cytoplasm and on the surface of the mycobacterial cells and thus is readily available to interact with the host immune system. BALB/c mice were immunised intranasally with recombinant GroES, alone or in combination with a synthetic oligodeoxynucleotide containing unmethylated CpG motifs, and tested for protection against infection with M. avium. Neither GroES nor CpG alone provided any protection against subsequent challenge with M. avium, whereas a combination of the two significantly protected the lungs and spleen against colonisation by M. avium after intranasal challenge with a low dose of the organism. This indicates that intranasal administration of GroES and CpG oligodeoxynucleotides increases the resistance of BALB/c mice to M. avium infection.     

Immunotherapy of asthma using CpG oligodeoxynucleotides

Asthma and other atopic disorders have increased in prevalence and severity over the past three decades. Reduced risk of atopic disease associated with early life exposure to infections and microbes has raised the possibility that pathogen-associated molecular patterns (PAMPs) may confer protection against allergic disorders, a concept that has been named the “Hygiene Hypothesis”. This relationship is most likely mediated through the induction of specific patterns of anti-atopic immune responses that follow engagement of innate immune mechanisms. Bacterial DNA is one such immunostimulatory microbe-associated ligand, whose properties can be mimicked by oligodeoxynucleotides (ODN) containing unmethylated cytosine-guanine dinucleotides in specific base sequences (CpG motifs), motifs characteristic of prokaryotic DNA that have been suppressed in eukaryotic DNA. Based initially on observations that CpG ODN induced Th1-type patterns of immune responses, we proposed that CpG ODN might represent a novel therapeutic strategy for the prevention and treatment of atopic disorders. Current understanding suggests multiple mechanisms of action of CpG ODN, but our initial hypothesis has been supported by extensive studies demonstrating, in animal models, efficacy in both incipient and established atopic asthma. These preclinical studies are now being translated into clinical trials exploring this new approach to immunotherapy for atopic disease.     

A chimeric live attenuated vaccine against Japanese encephalitis

Japanese encephalitis is a disease of the CNS, endemic in Asia and Oceania. The disease is refractory to drug treatments and whilst the rural economies remain heavily dependent on agriculture, conditions for propagation of the disease will persist. Thus, there is a need for effective vaccines. Although some currently exist, they have their shortcomings. ChimeriVax-JE (Acambis Inc.) is a chimeric, live attenuated vaccine which expresses protective Japanese encephalitis antigens and to date has proven to be safe, effective and well-tolerated in clinical trials. It therefore appears to be a cost-effective prophylactic vaccine against this debilitating disease.     

CpG oligodeoxynucleotides increase the susceptibility of normal mice to infection by Candida albicans

Synthetic oligodeoxynucleotides containing CpG motifs trigger an innate immune response that typically increases host resistance to infection. Yet CpG treatment reduces the resistance of normal mice to Candida albicans infection. This effect is mediated by CpG-induced interleukin-12, indicating that CpG-dependent cytokine production may have adverse consequences for the host.     

CpG oligodeoxynucleotides accelerate reovirus type 2-triggered insulitis in DBA/1 suckling mice

We reported previously that reovirus type-2 (Reo-2) triggers T-helper (Th) 1-mediated autoimmune insulitis resulting in temporal impaired glucose tolerance (IGT) approximately 10 days post infection (d.p.i) in suckling DBA/1 mice. We hypothesized that CpG motifs in bacteria may enhance virus-induced insulitis through its content of unmethylated CpG motifs. In the infected mice, the intraperitoneal treatment of synthetic 20-base oligodeoxynucleotides with CpG motifs (CpG ODN) caused increase in cumulative incidence of insulitis with IGT, increased serum interferon (IFN)-gamma concentration, and high frequency of autoantibody against pancreatic islet cells, compared to the infected mice without CpG ODN at 17 d.p.i. Also CD4+ and CD8+ lymphocytes infiltrated in and/or around pancreatic islets in the CpG ODN-treated mice. This evidence suggests that CpG ODN may contribute to accelerate Reo-2-induced autoimmune reaction against pancreatic islet cells via additional effects of Th1 cytokines especially IFN-gamma.     

Immunotherapeutic potential of CpG oligodeoxynucleotides in veterinary species

Abstract

Innate immunity plays a critical role in host defense against infectious diseases by discriminating between self and infectious non-self. The recognition of infectious non-self involves germ-line encoded pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs). The PAMPs are the components of pathogenic microbes which include not only the cell wall constituents but also the unmethylated 2′-deoxy-ribo-cytosine-phosphate-guanosine (CpG) motifs. These CpG motifs present within bacterial and viral DNA are recognized by toll-like receptor 9 (TLR9), and signaling by this receptor triggers a proinflammatory cytokine response which, in turn, influences both innate and adaptive immune responses. The activation of TLR9 with synthetic CpG oligodeoxynucleotides (ODNs) induces powerful Th1-like immune responses. It has been shown to provide protection against infectious diseases, allergy and cancer in laboratory animal models and some domestic animal species. With better understanding of the basic biology and immune mechanisms, it would be possible to exploit the potential of CpG motifs for animal welfare. The research developments in the area of CpG and TLR9 and the potential applications in animal health have been reviewed in this article.     

Use of CpG oligodeoxynucleotides as immune adjuvants

Summary: Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs directly stimulate human B cells and plasmacytoid dendritic cells (pDCs), thereby promoting the production of T helper 1 (Th1) and pro‐inflammatory cytokines and the maturation/activation of professional antigen‐presenting cells. These activities enable CpG ODNs to act as immune adjuvants, accelerating and boosting antigen‐specific immune responses by 5–500‐fold. These effects are optimized by maintaining close physical contact between the CpG DNA and the immunogen. Animal challenge models establish that protective immunity can be accelerated and magnified by coadministering CpG DNA with vaccines. Ongoing clinical studies indicate that CpG ODNs are safe and well tolerated when administered as adjuvants to humans, and in some cases, they increase vaccine‐induced immune responses.     

Purification of Japanese encephalitis virus vaccine by zonal centrifugation.

A large-scale procedure for purification and concentration of Japanese encephalitis virus vaccine by a continuous-flow isopycnic banding technique in a sucrose density gradient solution, using a K-III zonal centrifuge rotor, is presented. The quality of zonal-purified vaccine was compared with commercial and Japanese National Institutes of Health reference vaccines for antigenicity, immunodiffusion, and allergic encephalitogenicity tests to show its high purity.     

CpG oligodeoxynucleotides improve the survival of pregnant and fetal mice following Listeria monocytogenes infection

Listeria infection during pregnancy can cause the death of both mother and fetus. Previous studies established that immunostimulatory CpG oligodeoxynucleotides (ODN) increase the resistance of healthy adult mice to many infectious pathogens, including Listeria monocytogenes. This study examines whether the innate immune response elicited by CpG ODN can reduce the susceptibility of pregnant mice to lethal listeria challenge. The results indicate that CpG ODN treatment significantly improves maternal survival and reduces pathogen transmission to offspring. CpG ODN administered during pregnancy did not induce abortion, birth defects, or reduce the size or health of litters. These findings suggest that CpG ODN may provide a safe and effective means of improving the health of mothers and fetuses during pregnancy.     

Enhancing effect of vaxfectin on the ability of a Japanese encephalitis DNA vaccine to induce neutralizing antibody in mice

Vaxfectin, a recently developed adjuvant, was evaluated for its enhancing effect on immunogenicity of a Japanese encephalitis (JE) DNA vaccine plasmid encoding the JE virus premembrane (prM) and envelope (E) genes (designated pcJEME), using BALB/c and ICR mice. Formulation of pcJEME with Vaxfectin provided > or =8-fold higher neutralizing antibody titers than those induced by pcJEME alone and reduced the amount of pcJEME to one-tenth to induce comparable levels of neutralizing antibody. Use of Vaxfectin did not alter a Th1 type IgG isotype immune response (IgG1 < IgG2a) induced by pcJEME in mice. These results indicate that Vaxfectin has an ability to enhance immunogenicity of pcJEME and is considered as a useful adjuvant for DNA vaccines in murine experimental models.     

Induction of suppressor cells in Japanese encephalitis virus infected mice

Adoptive transfer of spleen cells obtained from mice primed with Japanese encephalitis virus (JEV) suppressed IgM antibody plaque forming cells (PFC) against JEV in the spleen. Similar suppression of PFC was also shown in vitro by adding primed spleen cells to JEV-stimulated spleen cell cultures. The suppressor activity appeared sharply in the third week after priming and persisted up to 6 weeks. By using various cell separation procedures it was found that the suppressor activity resided in the T cell enriched fraction and not in B cells or macrophages. Sensitivity of the cells to treatment with anti-Thy 1.2 antiserum and complement confirmed that suppressor cells were T lymphocytes. It was noted that the suppression was effective against dengue virus antigen also. Our findings thus show generation of suppressor T lymphocytes in JEV-infected mice.     

Effect of CpG oligodeoxynucleotides on the immunogenicity of Pfs25, a Plasmodium falciparum transmission-blocking vaccine antigen

Antibodies directed against Pfs25, a protein present on the surface of zygotes and ookinetes of Plasmodium falciparum, completely block pathogen transmission. We evaluated the immunomodulatory effect of CpG oligodeoxynucleotides (ODN) on the immunogenicity of recombinant Pfs25 (rPfs25) formulated in alum (Al). Immunization of mice with rPfs25 plus CpG ODN improved both the antibody titer (a 30-fold-higher antibody response than that with rPfs25-Al alone) and avidity. Coadministration of CpG ODN dramatically enhanced the titer of immunoglobulin G2A (IgG2a) compared to the titer of the IgG1-dominant response caused by rPfs25-Al alone, and the sera from the CpG ODN-coadministered group completely blocked the transmission of P. falciparum parasites to mosquitoes, as determined by membrane feeding assays. However, transmission-blocking experiments revealed that blocking efficacy was dependent on high-titer antibody levels, independent of isotypes. These results suggest that CpG ODN can be used as an adjuvant to enhance the immunogenicity of rPfs25 as a malaria transmission-blocking vaccine.     

The potential of CpG oligodeoxynucleotides as mucosal adjuvants

The development of mucosal vaccines for humans has been hindered by the lack of safe yet effective mucosal adjuvants. Bacterial toxins are commonly used as adjuvants in animal models, but they are too toxic for use in humans. A novel class of adjuvant is CpG DNA, which contains unmethylated CpG dinucleotides in particular base contexts (CpG motifs). CpG DNA is most often coadministered with antigen in the form of synthetic oligodeoxynucleotides (CpG ODN), which are made with a nuclease-resistant phosphorothioate backbone. The vast majority of studies using CpG DNA as adjuvant have been with parenteral delivery; recently, however, mucosal immunization with CpG DNA as adjuvant has also been shown to induce both systemic (humoral and cellular) and mucosal antigen-specific immune responses. This review will highlight the recent uses of CpG DNA as an adjuvant at mucosal surfaces.     

Modulation of NK cell activity by CpG oligodeoxynucleotides

Oligodeoxynucleotides (ODN) with hypomethylated CpG motifs have been found to be potent stimulators of various aspects of innate and adaptive immunity. One of their major effects is the activation of natural killer (NK) killing activity in vitro and in vivo. There are several categories of CpG classified as type A, type B, and type C, although another category with inhibitory activity is being characterized further. CpG type A (CpG-A) is the most potent at activating NK cells. Examination of the cells and soluble mediators involved in this activation has led to an understanding of an interesting cascade of events. It appears that CpG activates dendritic cells (DC) which in turn activate NK-cells. This is not surprising since NK-cells do not seem to express TLR9, the CpG receptor. Of the various cytokines involved in NK-cell activation, it appears that type 1 interferon plays a pivotal role. Having activated NK-cells, DC themselves appear to become susceptible to lysis by the NK-cells they activated but with a delayed time kinetic. CpG ODN have been examined as monotherapeutic agents in murine tumor models. In one model, B16 melanoma, CpG ODN were very effective and NK cells were both necessary and sufficient for that effect. In another model, EL4 lymphoma, NK cells were necessary but not sufficient. Moreover, CpG were able to induce long-term survival in mice with established tumor.Studies in humans show similar results with potent activation in vitro. In a limited Phase I dose escalation study it also appeared that CpG ODN induce NK cell activation in humans in vivo.