Rhabdophis tigrinus (Yamakagashi) Bites in Japan Over the Last 50 Years: A Retrospective Survey.

Introduction:Rhabdophis snakes, which include 27 species, are rear-fanged venomous snakes that are widely distributed from India to East Asia and Russia. Severe envenomation by R. tigrinus (Yamakagashi snake) in Japan and R. subminiatus in Southeast Asia has been reported. The epidemiology of R. tigrinus bites, such as geographical features, the incidence, and changes in the number of bites over time have not been comprehensively examined. Hence, we intended to clarify the epidemiological features of R. tigrinus bites through a careful review of scientific data over the last 50 years in Japan. Methods: Patient records of R. tigrinus bites between 1971 and 2020 at the Japan Snake Institute were examined retrospectively. The following were ascertained: patient characteristics, clinical symptoms, laboratory data, treatment-related factors, and hospital mortality. These variables were compared in the antivenom and the without-antivenom groups. Results: Over the 50-year study period, 43 R. tigrinus bites, including five fatal cases, were encountered. Severe cases of R. tigrinus bites have been treated with antivenom since 1985; however, fatalities occurred in 2006 and 2020. R. tigrinus bite cases have been well-distributed in the western part of Japan since 2000. The mortality rate in the antivenom group was significantly lower in the patient group that was not administered the antivenom (0 vs. 23.8%, p = 0.048). Conclusion: This study clarified the epidemiology of R. tigrinus bites in Japan over a 50-year period. Almost all severe cases of R. tigrinus bites have been treated with the antivenom in the current situation, and fatalities occurred in cases not treated with the antivenom. It is important to diagnose R. tigrinus bites in the early phase of the clinical course. The antivenom, the definitive treatment for R. tigrinus bites, is an unapproved drug. Hence, approval needs to be obtained for the drug.

Injectable Polypeptide Hydrogel Depot System for Assessment of the Immune Response-Inducing Efficacy of Sustained Antigen Release Alone.

Vaccines typically contain an antigen, delivery system (vehicle), and adjuvant, all of which contribute to inducing a potent immune response. Consequently, design of new vaccines is difficult, because the contributions and interactions of these components are difficult to distinguish. Here, it is aimed to develop an easy-to-use, non-immunogenic, injectable depot system for sustained antigen release that will be suitable for assessing the efficacy of prolonged antigen exposure per se for inducing an immune response. This should mimic real-life infections. Recombinant elastin-like polypeptides with periodic cysteine residues (cELPs) are selected, which reportedly show little or no immunogenicity, as carriers and tetanus toxoid (Ttd) as an antigen. After subcutaneous injection of the mixture, cELP rapidly forms a disulfide cross-linked hydrogel in situ, within which Ttd is physically incorporated, affording a biodegradable antigen depot. A series of Ttd-containing hydrogels is examined. A single injection induces high levels of tetanus antibody with high avidity for at least 20 weeks in mice. The chain length of cELP proves critical, whereas differences in hydrophobicity has little effect, although hydrophilic cELPs are more rapidly biodegraded. This system's ability to distinguish the contribution of sustained antigen release to antibody induction should be helpful for rational design of next-generation vaccines.

Clinical Serum Therapy: Benefits, Cautions, and Potential Applications.

Blood serum from immunized humans or animals (e.g., horses) contains relevant antibodies and has been used as serum therapy to treat many diseases or envenomation events. The effectiveness of blood serum was initially discovered in 1890 when Kitasato and von Behring observed the effectiveness of this type of therapy against diphtheria and tetanus. Serum therapies played an important role in the advancement of modern medicine prior to the development of penicillin and steroids. At present, several types of serum therapy remain in clinical use. However, some physicians have a limited understanding of the nature and the benefits of serum therapy and the factors that require particular attention. In this review, we set out to clarify the benefits, cautions, and potential applications of serum therapy in the context of conditions such as gas gangrene, diphtheria, botulism, and tetanus and bites from three snake species (mamushi, habu, and yamakagashi) and the redback spider. It is hoped that this review will help clinicians to learn about clinical serum therapies and become familiar with their applications.

Clinical Study of New Tetravalent (Type A, B, E, and F) Botulinum Toxoid Vaccine Derived from M Toxin in Japan.

Botulinum toxin is the most poisonous substance known, and is believed to be a highly lethal as a biological weapon; researchers of the toxin are exposed to this hazard. Botulinum toxoid vaccines have been produced and used in Japan. However, since clinical studies involving these vaccines were conducted before establishment of the Ethical Guidelines for Clinical Research in Japan, their immunogenicity and safety were not systematically assessed. In this study, we produced a new tetravalent (type A, B, E, and F) botulinum toxoid vaccine, the first ever to be derived from M toxin, and conducted quality control tests with reference to the Minimum Requirements in Japan for adsorbed tetanus toxoid vaccine. Subsequently, a clinical study using the new vaccine in 48 healthy adult volunteers was conducted according to the guidelines in Japan. No clinically serious adverse event was noted. Neutralizing antibody titers for each type of toxin in the participants' sera, 1 month after the 4th injection were more than 0.25 IU/mL, indicating sufficient protection. This study demonstrated that the vaccine has marked immunogenicity and is safe for use in humans.

[Seroepidemiological Studies on Pertussis, Diphtheria and Tetanus among Nursing Students Enrolled between 1994 and 2011 in the Kitakyushu District].

Seroepidemiological studies on pertussis, diphtheria and tetanus were performed on 1,540 nursing students enrolled in S-university between 1994 and 2011. Antibody titers against pertussis toxin (PT) and filamentous hemagglutinin (FHA), diphtheria antitoxin titer, and tetanus antitoxin titer were measured using sera taken during enrollment. The antibody-seropositive rates and geometric mean titers (GMTs) were calculated by according to birth year (1975-1993). The pertussis anti-PT and anti-FHA antibody-seropositive rates (seropositive levels were both defined as ≥10 EU/mL) were 12%-53% and 47%-84%, respectively. The anti-PT antibody-seropositive rate was lower than the anti-FHA rate. The anti-PT antibody GMTs were 2.0-11 EU/mL, whereas the anti-FHA antibody GMTs were 10 EU/mL or more (8.8-31 EU/mL) in almost every group. The diphtheria antitoxin-seropositive rate (≥0.1 IU/mL) was 49%-79%, and the GMT was nearly 0.1 IU/mL. The tetanus antitoxin-seropositive rate (≥0.01 IU/mL) was 91%-100%, and the GMT was 0.3 IU/mL or more for all the groups. While the nursing students' diphtheria and tetanus antitoxin levels were sufficient to prevent both diseases, the anti-PT antibody-seropositive rate and the GMT were both lower than the level required to prevent pertussis, suggesting that many of the nursing students were pertussis-susceptible. These findings suggest a need for pertussis vaccination in young adults or early adolescents.

Clinical efficacy of antivenom and cepharanthine for the treatment of Mamushi (Gloydius blomhoffii) bites in tertiary care centers in Japan.

To our knowledge, no one has conducted a multi-center trial evaluating the efficacy of antivenom and cepharanthine (CEP) for the treatment of mamushi (Gloydius blomhoffii) bites. Thus, we conducted a large-scale survey among tertiary care centers in Japan from November 2009 to October 2010 to evaluate the efficacy of antivenom and CEP for the treatment of mamushi bites. We divided the therapeutic interventions received by patients into 4 groups: CEP, antivenom, both CEP and antivenom, and neither CEP nor antivenom. We collected data on age, sex, comorbidities, laboratory measurements, length of hospital stay, and grades of mamushi bites (indication of bite severity ranged from I [mild] to V [severe]). We sent questionnaires to 219 tertiary care centers, of which 114 (52.1%) returned completed questionnaires. Two hundred and thirty-four cases of mamushi bites were reported. Among the severe cases (grades of mamushi bites III, IV, and V), patients administered antivenom had a significantly shorter length of hospital stay than those administered CEP (P = 0.024). In contrast, there was no significant difference in the length of hospital stay between mild cases (grades of mamushi bites I and II) (P = 0.77). Our results show that antivenom is effective in reducing the length of hospital stay in patients with severe mamushi bites.

Surveillance of the clinical use of mamushi (Gloydius blomhoffii) antivenom in tertiary care centers in Japan.

We report the results of the first large-scale questionnaire surveillance on the clinical use of pit viper antivenom in tertiary care centers in Japan. The questionnaire surveillance was conducted over a period of 3 years (April 2006 to March 2009). Completed questionnaires were received from the tertiary care centers of 108 (49.3%) medical institutions. In that period, 574 cases of pit viper bites, including 2 severe cases, were reported. Antivenom was administered in 44% of the cases of pit viper bites, and of these cases, 2.4% had adverse reactions but no severe symptoms. Approximately half of the clinicians indicated that antivenom was effective. Antivenom was recognized to be safe; however, the remarkable finding was that although the severity of treated cases was unclear, some clinicians reported using cepharanthine as the first choice of treatment for pit viper bites.

Experimental manufacture of equine antivenom against yamakagashi (Rhabdophis tigrinus).

Yamakagashi, Rhabdophis tigrinus, is a natricine snake widely distributed in eastern Asia. Severe bite cases, some with fatal outcomes, occur regularly in Japan. Because previous production of R. tigrinus antivenom in rabbits and goats was quite effective, we considered the experimental manufacture of a new antivenom against R. tigrinus in horses. This new antivenom could be used in emergency treatment of snakebite victims. Two horses were immunized with venom extracted from about 500 snakes. After an adequate increase of the antivenom titer, serum was collected and subjected to standard purification procedures for the manufacture of equine antivenoms. The purified immunoglobulin fraction was freeze-dried in 1,369 vials under optimum conditions for therapeutic use. This antivenom proved to be very potent in neutralizing the coagulant and hemorrhagic activities of the snake venom. In cases of severe bites, this antivenom was used and recognized as effective even after the occurrence of severe symptoms.

Protective effect of nasal immunization of influenza virus hemagglutinin with recombinant cholera toxin B subunit as a mucosal adjuvant in mice.

To develop an efficient nasal influenza vaccine, influenza A and B virus HA with rCTB as a mucosal adjuvant were administered to mice intranasally. Serum anti-HA IgG and IgA antibody responses for both HA vaccines were significantly increased in the presence of rCTB. Higher HI and neutralizing antibody titers and higher mucosal IgA antibody responses in the respiratory tract were detected when rCTB was added than without rCTB. When mice were immunized with HA vaccine with or without rCTB and challenged by intranasal administration of mouse-adapted pathogenic influenza A virus, all mice immunized with HA plus rCTB survived for seven days without any inflammatory changes in the lungs, while not all the mice immunized with HA without rCTB survived, and all of them had lung consolidations. These results demonstrate that intranasal co-administration of rCTB as a mucosal adjuvant with influenza virus HA is necessary not only for the induction of systemic and mucosal HA antibodies, but also for the protection of mice from morbidity and mortality resulting from virus infection.

Standardization of regional reference for mamushi (Gloydius blomhoffii) antivenom in Japan, Korea, and China.

The mamushi (Gloydius blomhoffii) snakes that inhabit Japan, Korea, and China produce venoms with similar serological characters to each other. Individual domestic standard mamushi antivenoms have been used for national quality control (potency testing) of mamushi antivenom products in these countries, because of the lack of an international standard material authorized by the World Health Organization. This precludes comparison of the results of product potency testing among countries. We established a regional reference antivenom for these three Asian countries. This collaborative study indicated that the regional reference mamushi antivenom has an anti-lethal titer of 33,000 U/vial and anti-hemorrhagic titer of 36,000 U/vial. This reference can be used routinely for quality control, including national control of mamushi antivenom products.

Recombinant cholera toxin B subunit (rCTB) as a mucosal adjuvant enhances induction of diphtheria and tetanus antitoxin antibodies in mice by intranasal administration with diphtheria-pertussis-tetanus (DPT) combination vaccine.

Recombinant cholera toxin B subunit (rCTB) which is produced by Bacillus brevis carrying pNU212-CTB acts as a mucosal adjuvant capable of enhancing host immune responses specific to unrelated, mucosally co-administered vaccine antigens. When mice were administered intranasally with diphtheria-pertussis-tetanus (DPT) combination vaccine consisting of diphtheria toxoid (DTd), tetanus toxoid (TTd), pertussis toxoid (PTd), and formalin-treated filamentous hemagglutinin (fFHA), the presence of rCTB elevated constantly high values of DTd- and TTd-specific serum ELISA IgG antibody titres, and protective levels of diphtheria and tetanus toxin-neutralizing antibodies but the absence of rCTB did not. Moreover, the addition of rCTB protected all mice against tetanic symptoms and deaths. DPT combination vaccine raised high levels of serum anti-PT IgG antibody titres regardless of rCTB and protected mice from Bordetella pertussis challenge. These results suggest that co-administration of rCTB as an adjuvant is necessary for induction of diphtheria and tetanus antitoxin antibodies on the occasion of intranasal administration of DPT combination vaccine.

Effects of recombinant cholera toxin b subunit (rCTB) on cellular immune responses: enhancement of delayed-type hypersensitivity following intranasal co-administration of Mycobacterium bovis-BCG with rCTB.

Recombinant cholera toxin B subunit (rCTB) is a safe and potent mucosal adjuvant. To gain insight into the mechanism underlying the adjuvant effect of rCTB, the effects of rCTB on cell-mediated immune responses of mice and guinea pigs were examined after intranasal administration of Mycobacterium bovis -bacillus Calmette-Guérin (BCG) with and without rCTB. Delayed-type hypersensitivity, for skin reactions in guinea pigs and for footpad swelling reactions in mice, to purified protein derivative (PPD) were enhanced by intranasal co-administration of BCG and rCTB, as compared to giving BCG alone to these animals. Moreover, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma production of spleen cells and antigen specific spleen cell proliferation, stimulated with PPD, were enhanced in the presence of rCTB. These results strongly suggest that rCTB enhances cellular as well as humoral immune responses.

Seroepidemiologic study on pertussis, diphtheria, and tetanus in the Fukuoka area of southern Japan: seroprevalence among persons 0-80 years old and vaccination program.

In Japan, mass vaccination for diphtheria, pertussis, and/or tetanus has been mandated by the Vaccination Law since 1948. In order to evaluate the efficacy of this vaccination policy, we conducted seroepidemiological studies on pertussis, diphtheria, and tetanus among individuals aged 0 - 80 years. The pertussis toxin seropositive rates of the vaccine-eligible groups and vaccine-ineligible groups were 55.0 and 57.9%, respectively. The seropositive rate of each group for diphtheria antitoxin was 76.3 and 75.7%, respectively. The tetanus antitoxin seropositive rates were 91.7 and 10.5%, respectively, showing a significant difference between the two groups (P < 0.001). For the three diseases, variations were seen between age groups in the geometric mean antibody titers due to changes of the vaccination program. The results of this study show that natural Bordetella pertussis infection has occurred more frequently than expected. In order to establish the most appropriate vaccination program for the control of pertussis, diphtheria, and tetanus in Japan, further evaluation is necessary.

Frequent nasal administrations of recombinant cholera toxin B subunit (rCTB)-containing tetanus and diphtheria toxoid vaccines induced antigen-specific serum and mucosal immune responses in the presence of anti-rCTB antibodies.

Vaccination via a mucosal route is a very attractive means for immunization, because both local and systemic immune responses are inducible and vaccines can be administered easily and safely from infants to elderly persons. For developing widely applicable mucosal vaccines using recombinant cholera toxin B subunit (rCTB) as a safe adjuvant, we examined whether frequent nasal administrations of rCTB-containing same and different vaccines could induce antigen-specific immune responses without induction of systemic tolerance and suppression by pre-existing anti-rCTB immunity. Ten repetitive nasal administrations to mice of tetanus toxoid (TT) + rCTB or diphtheria toxoid (DT) + rCTB raised and maintained high levels of antigen- and rCTB-specific serum IgG including high levels of tetanus/diphtheria antitoxin titres and raised nasal, salivary, lung, vaginal and fecal secreted IgA, suggesting that the regimen did not induce systemic tolerance to TT/DT and rCTB. Mice successively received repetitive five doses of TT as the first antigen and subsequent five doses of DT as the second antigen, and vice versa, raised serum IgG to the second antigen at various levels including low but sufficient protective levels of antitoxin titres and induced mucosal IgA in the lungs, the vaginas and feces, but hardly in the nasal secretions and salivas. After an interval of 22 weeks between the dosage of the first and second antigens, mice induced serum IgG to the second antigen at high levels and mucosal IgA in all sites. In conclusion, anti-TT and -DT serum and mucosal antibody responses induced by repeated intranasal immunization using rCTB adjuvant lasted for a long period, and for improving the effectivity of vaccination, different rCTB-containing vaccines should be administered at appropriate intervals.

Mucosal and systemic antibody responses against an acellular pertussis vaccine in mice after intranasal co-administration with recombinant cholera toxin B subunit as an adjuvant.

To investigate the possibility of intranasal immunization with an acellular pertussis vaccine, groups of mice were administered intranasally with aluminium-non-adsorbed pertussis toxoid (PTd; 0.5 or 5 microg) and formalin-treated filamentous hemagglutinin (fFHA; 5 microg) with and without recombinant cholera toxin B subunit (rCTB; 10 microg) as a mucosal adjuvant. At a low concentration of PTd, the following things became clear: (1) earlier and higher elevation of serum anti-PTd and anti-FHA IgG antibody titres in the presence of rCTB than in its absence, (2) higher serum anti-PTd and anti-FHA IgG antibody titres than 200 and 100 ELISA units ml(-1) (EU ml(-1)) in all mice, respectively, in the presence of rCTB, which were obtained by calibration against a reference anti-pertussis mouse serum, and (3) in an intranasal challenge experiment with Bordetella pertussis, slightly more rapid elimination of the bacteria from the lungs of mice intranasally immunized in the presence of rCTB, suggesting the effectiveness of rCTB as a mucosal adjuvant. However, irrespective of rCTB and dose of PTd, mice which were immunized four times and sacrificed on day 35 developed high levels of anti-PTd serum IgG antibodies, high or moderate levels of anti-FHA serum IgG antibodies and mucosal anti-PTd IgA antibodies in the lungs; only a slight or no increase of anti-FHA mucosal IgA antibodies was observed in the lung. These facts suggested the immunogenicity and mucosal adjuvanticity of PTd, and therefore, the mucosal adjuvanticity of rCTB seemed to be inconspicuous. Moreover, the addition of rCTB induced higher anti-PTd serum IgE antibody responses than no addition of it depending on dose of PTd. These results show that dose of PTd included in an acellular pertussis vaccine had better be low as possible and the addition of rCTB may not be always necessary in case of this nasal vaccine alone unlike tetanus and diphtheria toxoids and hepatitis B virus vaccine reported before.

Selective inhibition of systemic anti-OVA IgE production in response to oral pre-treatment with OVA-liposome conjugates.

BACKGROUND: We have previously reported that intraperitoneal injection with OVA-liposome conjugates induces OVA-specific and IgE-selective unresponsiveness in mice. METHODS: In the present study, the effects of oral pre-treatment with OVA-liposome conjugates or with plain OVA solution on anti-OVA IgG antibody production were investigated in mice after subsequent immunization with alum-adsorbed OVA. Control mice received only the immunization. RESULTS: The levels of serum anti-OVA IgG antibody in mice receiving oral administration of OVA-liposome were comparable to those in the control mice. However, in mice receiving oral administration of the same dose of plain OVA, levels of serum anti-OVA IgG antibody were significantly lower than those in control mice. Surprisingly, anti-OVA IgE antibody production was completely inhibited in mice receiving oral administration of OVA-liposome conjugates. Splenic CD4(+) T cells of mice receiving oral administration of OVA-liposome and those of control mice produced comparable levels of cytokines, while those of mice receiving oral administration of plain OVA solution produced significantly lower levels of cytokines than those in the other two groups. CONCLUSION: Orally administered OVA-liposome did not affect anti-OVA IgG production but did inhibit anti-OVA IgE antibody production, while orally administered OVA solution inhibited production of both IgG and IgE antibodies. These results suggest that antigen-liposome conjugates can possibly be orally administered in order to control antigen-specific IgE antibody production, without affecting IgG antibody production.

Effects of recombinant cholera toxin B subunit on IL-1beta production by macrophages in vitro.

Recombinant cholera toxin B subunit (rCTB) is a safe and potent mucosal adjuvant. As a clue to the mechanism of the adjuvant effect of rCTB, the profile of cytokines secreted in vitro by the mouse peritoneal macrophage (Mphi) treated with rCTB was examined. IL-1beta secretion, intracellular production, and expression of its mRNA of LPS-stimulated Mphi was greatly enhanced by treatment with rCTB. IL-1beta production in response to other microbial stimulators, such as Pansorbin, Sansorbin, insoluble peptidoglycan, and Taxol, was also potentiated by rCTB. Mphi pretreated with rCTB before 24 hr could maintain the ability to produce a high level of IL-1beta, suggesting that this ability may be involved in the adjuvant activity of rCTB on Mphi stimulation. The possibility of close association between rCTB and signal transduction of a Toll-like receptor family in Mphi is discussed.