Low-frequency ultrasound as a transcutaneous immunization adjuvant

Percutaneous vaccine delivery offers an advantageous mode of immunization due to the unique ability of cutaneous immune cells, especially Langerhans cells, to present antigens to the immune system. Langerhans cells, upon activation, migrate to the regional lymph nodes and lead to the generation of systemic and mucosal immune responses. However, simple topical application of vaccines does not deliver sufficient amounts of antigen in the skin to generate an adequate immune response. Co-administration of strong adjuvants such as cholera toxin or invasive skin abrasion is usually necessary to induce an adequate immune response by topical vaccine application. Here, we report on the use of low-frequency ultrasound as a potent physical adjuvant for successful transcutaneous immunization (TCI). Using tetanus toxoid as a model vaccine, we show that low-frequency ultrasound enhances the immune response induced by topical application of tetanus toxoid. The adjuvant effect of ultrasound is partly explained by the enhanced delivery of tetanus toxoid into the skin after ultrasound application and partly by the activation of immune cells after ultrasonic TCI. These studies demonstrate generation of a potent systemic immune response through TCI without using toxin adjuvants or skin abrasion. Ultrasonic TCI offers a needle-free and painless mode of immunization.     

Elastic liposomes mediated transcutaneous immunization against Hepatitis B

Transcutaneous immunization presents a major challenge due to poor permeability of antigens through the skin barrier. To overcome this limitation ultradeformable lipid vesicles, the elastic liposomes, could be a better module for transcutaneous delivery of these proteinaceous antigens. In the present investigation Hepatitis B surface antigen (HBsAg)-loaded elastic liposomes were utilized as a mode for enhanced immunity against the antigen. Elastic liposomes were prepared by conventional rotary evaporation method and characterized for various parameters such as vesicles shape and surface morphology, size and size distribution, entrapment efficiency, elasticity, turbidity, stability and in vitro release pattern. Ex vivo cellular uptake and fluorescence studies were also conducted. In vivo studies were performed by measuring the immune response elicited by topically applied HBsAg-loaded elastic liposomes and compared to the intramuscularly administered alum-adsorbed HBsAg solution, topically applied plain HBsAg solution and physical mixture of HBsAg and elastic liposomes. Results indicate that transcutaneous immunization via elastic liposomes induces robust systemic and mucosal antibody response against HBsAg as compared to other formulations. The fluorescence microscopy results suggest prominent skin permeation and biodistribution, demonstrating efficient delivery of antigens to the immunocompetent Langerhans cells (LC) and lymphatics. The elastic liposomal formulation provides higher entrapment efficiency, enhanced penetration and effective immunoadjuvant property justifying its potential for improved vaccine delivery.     

Non-invasive delivery of nanoparticles to hair follicles: A perspective for transcutaneous immunization

Transfollicular vaccination aims to reach the peri-follicular antigen presenting cells without impairing the stratum corneum (SC) barrier. This would be an optimal vaccination strategy under critical hygienic conditions. Nanoparticles (NPs) are the ideal vehicles for transfollicular delivery of vaccines as they are able to (i) penetrate deeper into the hair follicles than molecules in solution, (ii) can help to stabilize protein based antigen and (iii) improve and modulate the immune response.     

Effects of IL-12 on immune responses induced by transcutaneous immunization with antigens formulated in a novel lipid-based biphasic delivery system

The development of non-invasive methods for the delivery of proteins through the permeability barriers, such as the intact skin, will greatly facilitate the administration of human and veterinary vaccines. In the present study we used recombinant Pasteurella haemolytica leukotoxin (Lkt) and hen egg lysozyme (HEL) as model antigens to investigate the ability of transdermal administration of vaccine antigens to induce humoral and cellular responses in mice and to assess the immunomodulatory effects of IL-12 on these antigen-specific immune responses. Mice were immunized by the transdermal route with Lkt or HEL formulated in a novel lipid-based biphasic delivery system (BPDS). Transdermal delivery of Lkt or HEL induced strong polarized Th2 responses characterized by enhancement of antigen-specific IgG1 antibody subclass and predominant induction of antigen specific IL-4 over IFN-gamma in spleen and draining lymph nodes cells. Animals immunized by topical application of formulations containing antigen and IL-12 developed significantly lower antibody titres without significant changes in IL-4 or IFN-gamma secreting cells (SC) in the draining lymph nodes or spleen cells. Our results indicated that application of antigens formulated in BPDS induced antigen-specific immune responses. Furthermore, incorporation of IL-12 to the vaccine formulation influences the induction of antibody responses induced by transdermal immunization. We demonstrated the feasibility of using this technology for the development of non-invasive methods of vaccine administration.     

Comparison of intranasal and transcutaneous immunization for induction of protective immunity against Chlamydia muridarum respiratory tract infection

Chlamydia pneumoniae causes a range of respiratory infections including bronchitis, pharyngitis and pneumonia. Infection has also been implicated in exacerbation/initiation of asthma and chronic obstructive pulmonary disease (COPD) and may play a role in atherosclerosis and Alzheimer's disease. We have used a mouse model of Chlamydia respiratory infection to determine the effectiveness of intranasal (IN) and transcutaneous immunization (TCI) to prevent Chlamydia lung infection. Female BALB/c mice were immunized with chlamydial major outer membrane protein (MOMP) mixed with cholera toxin and CpG oligodeoxynucleotide adjuvants by either the IN or TCI routes. Serum and bronchoalveolar lavage (BAL) were collected for antibody analysis. Mononuclear cells from lung-draining lymph nodes were stimulated in vitro with MOMP and cytokine mRNA production determined by real time PCR. Animals were challenged with live Chlamydia and weighed daily following challenge. At day 10 (the peak of infection) animals were sacrificed and the numbers of recoverable Chlamydia in lungs determined by real time PCR. MOMP-specific antibody-secreting cells in lung tissues were also determined at day 10 post-infection. Both IN and TCI protected animals against weight loss compared to non-immunized controls with both immunized groups gaining weight by day 10-post challenge while controls had lost 6% of body weight. Both immunization protocols induced MOMP-specific IgG in serum and BAL while only IN immunization induced MOMP-specific IgA in BAL. Both immunization routes resulted in high numbers of MOMP-specific antibody-secreting cells in lung tissues (IN>TCI). Following in vitro re-stimulation of lung-draining lymph node cells with MOMP; IFNgamma mRNA increased 20-fold in cells from IN immunized animals (compared to non-immunized controls) while IFNgamma levels increased 6- to 7-fold in TCI animals. Ten days post challenge non-immunized animals had >7,000 IFU in their lungs, IN immunized animals <50 IFU and TCI immunized animals <1,500 IFU. Thus, both intranasal and transcutaneous immunization protected mice against respiratory challenge with Chlamydia. The best protection was obtained following IN immunization and correlated with IFNgamma production by mononuclear cells in lung-draining LN and MOMP-specific IgA in BAL.     

Comparison of mucosal and systemic humoral immune responses after transcutaneous and oral immunization strategies

In order to compare the ability of transcutaneous and oral immunization strategies to induce mucosal and systemic immune responses, we inoculated mice transcutaneously with cholera toxin (CT) or the non-toxic B subunit of cholera toxin (CtxB), or orally with Peru2(pETR1), an attenuated vaccine strain of Vibrio cholerae expressing CtxB. In addition, we also evaluated dual immunization regimens (oral inoculation with transcutaneous boosting, and transcutaneous immunization with oral boosting) in an attempt to optimize induction of both mucosal and systemic immune responses. We found that transcutaneous immunization with purified CtxB or CT induces much more prominent systemic IgG anti-CtxB responses than does oral inoculation with a vaccine vector strain of V. cholerae expressing CtxB. In comparison, anti-CtxB IgA in serum, stool and bile were comparable in mice either transcutaneously or orally immunized. Overall, the most prominent systemic and mucosal anti-CtxB responses occurred in mice that were orally primed with Peru2(pETR1) and transcutaneously boosted with CT. Our results suggest that combination oral and transcutaneous immunization strategies may most prominently induce both mucosal and systemic humoral responses.     

Virulence potential of fusogenic orthoreoviruses

Several severe respiratory virus infections that have emerged during the past decade originated in animals, including bats. In Indonesia, exposure to bats has been associated with increased risk of acquiring orthoreovirus infection. Although orthoreovirus infections are mild and self-limiting, we explored their potential for evolution into a more virulent form. We used conventional virus culture, electron microscopy, and molecular sequencing to isolate and identify orthoreoviruses from 3 patients in whom respiratory tract infection developed after travel to Indonesia. Virus characterization by plaque-reduction neutralization testing showed antigenic similarity, but sequencing of the small segment genes suggested virus reassortment, which could lead to increased virulence. Bats as a reservoir might contribute to virus evolution and genetic diversity, giving orthoreoviruses the potential to become more virulent. Evolution of this virus should be closely monitored so that prevention and control measures can be taken should it become more virulent.     

新型冠状病毒肺炎流行期间免疫规划疫苗延迟接种安全性

2019年12月以来,我国各地陆续发生新型冠状病毒肺炎(HCP)疫情。经国务院批准,国家卫生健康委于2020年1月20日决定将NCP纳入法定传染病乙类管理,采取甲类传染病的预防、控制措施。各地陆续采取病人隔离治疗、密切接触者隔离医学观察等系列防控措施。由于接种门诊可能导致人群聚集、增加病毒传播和感染的风险,儿童及其监护人存在隔离观察、隔离治疗的情况,都可能会导致儿童疫苗延迟接种。监护人和基层专业技术人员对于延迟接种的安全性、有效性及优先接种哪些疫苗的问题亟待释疑。近期中华预防医学会儿童保健分会组织专家,通过检索国内外大量文献,分析延迟接种对各种疫苗安全性和有效性的影响,希望尽可能减少儿童监护人和一线工作者对于延迟接种效果的困惑,为之后的疫苗补种提供科学依据和技术建议。     

Development and use of SIV-based Integrase defective lentiviral vector for immunization

Integrase (IN) defective lentiviral vectors have a high safety profile and might prove useful as immunizing agents especially against HIV-1. However, IN defective SIV-based vectors must be developed in order to test their potential in the non-human primate models (NHP) of AIDS. To this aim we tested a novel SIV-based IN defective lentiviral vector for its ability to induce sustained immune responses in mice. BALB/c mice were immunized once intramuscularly with a SIV-based IN defective lentiviral vector expressing the model antigen enhanced green fluorescence protein (eGFP). Immune responses were evaluated 90 days after the injection and compared with those elicited with the IN competent counterpart. The IN defective vector was able to efficiently elicit specific and long-lasting polyfunctional immune responses as evaluated by enzyme-linked immunospot (ELISPOT) assays for interferon-γ (IFN-γ) in spleens, bone marrow (BM) and draining lymph nodes, and by intracellular staining (ICS) for IFN-γ, Interleukin-2 (IL-2) and tumor necrosis factor (TNF-α) in both splenocytes and BM cells without integration of the vector into the host genome. This is the first demonstration that an IN defective SIV-based lentiviral vector provides effective immunization, thus paving the way for the construction of IN defective vectors expressing SIV antigen(s) and test their efficacy against a SIV virus challenge in the NHP model of AIDS.     

Development of immunization trials against Klebsiella pneumoniae

Klebsiella pneumoniae is the most common cause of nosocomial respiratory tract and premature intensive care infections, and the second most frequent cause of Gram-negative bacteraemia and urinary tract infections. Drug resistant isolates remain an important hospital-acquired bacterial pathogen, add significantly to hospital stays, and are especially problematic in high impact medical areas such as intensive care units. Many investigations worldwide proved the increasing resistance of such pathogen, resulting in an average rate of 1.63 outbreak every year. A variety of preventive measures were applied to reduce such incidences. Immunotherapy and passive immunization researches as well found their way to the treatment of Klebsiella. During the last 40 years, many trials for constructing effective vaccines were followed. This up-to-date review classifies such trials and documents them in a progressive way. A following comment discusses each group benefits and defects.     

Pre-clinical and clinical investigation of the safety of a novel adjuvant for intranasal immunization

Nasalflu Berna is a trivalent influenza virus vaccine for active immunization against influenza by the nasal route. It consists of influenza virosomes which are formulated from inactivated influenza strains and heat-labile toxin from aseptic Escherichia coli bacteria strain, as an adjuvant (HLT). The results of preclinical studies in ferrets, baboons, minipigs, mice and rabbits are presented here, and issues concerning route of administration, mechanism of action (preventing the disease and halting further spread of the disease), and the specific safety issues of the adjuvant itself (possible neurological activity of HLT) are examined. No clinical signs were detected in the animals, and hematological values were in the normal range. In particular, there was no evidence of any systemic adverse reaction, including sensitization to the test substances, and no evidence of possible neurological activity of the HLT. Further clinical studies in humans conducted over five influenza seasons using this virosome-formulated intranasal vaccine, elicited high levels of influenza-specific hemagglutination inhibition IgG antibody titers to the strains incorporated in the administered vaccine. In addition, IgA antibodies were also elicited in the nasal mucosa, and in the saliva. In addition to the systemic IgG antibody titers, the nasal mucosal IgA antibody response may provide additional local protection by the inhibition of viral replication and further spread in the respiratory tract. Nasalflu was well tolerated by most of the vaccinated subjects, both in terms of nasal symptoms and possible vaccination-mediated systemic symptoms. Both local and systemic symptoms were primarily mild, with only an occasional subject reporting moderate intensity. Out of four serious adverse events seen during the clinical development, only one was thought to be remotely related to the test vaccine.Nasalflu, developed by the Swiss Serum and Vaccine Institute, is a novel, highly immunogenic and safe influenza subunit vaccine which is easily administered as a nasal spray. This new route of administration is likely to increase compliance to vaccination, and could become an important tool to promote vaccination in population groups which show high resistance to vaccination.     

Evaluation of fusogenic trophoblast surface epitopes as targets for immune contraception

Syncytial trophoblast fusion is an essential step in the process of implantation. This project is aimed at the immunological inhibition of syncytial trophoblast fusion as a novel approach to contraception. Fusion-inhibiting recombinant antibodies were generated and used together with autoantibodies from patients with repetitive in vitro fertilization (IVF) failure that were shown to inhibit syncytial fusion and are expected to inhibit implantation, to generate anti-idiotypic peptides. These peptides mimic trophoblast epitopes essential for syncytial fusion and are, therefore, considered specific immunogens for the generation of antibodies that will inhibit implantation. To verify their physiological role in humans, 300 anti-idiotypic peptides were tested for their binding capacity to patient autoantibodies associated with repetitive IVF failure, habitual abortion and preeclampsia. Of these, only three peptides were found to selectively bind to autoantibodies of patients with repetitive IVF failure and were considered safe and efficient enough for evaluation in preclinical and clinical studies required for the development of immune contraceptives. When used as immunogens, these peptides are expected to elicit an antibody response inhibiting syncytial fusion and thus implantation. Furthermore, the action of these antibodies needs to be restricted to the stage of syncytium formation at the time of implantation so as not to cause complications of pregnancy in those cases where they fail to have a contraceptive effect. To exclude potential side effects on other systems, toxicological experiments in animals are in progress.     

Development of a dried influenza whole inactivated virus vaccine for pulmonary immunization

Stabilization and ease of administration are two ways to substantially improve the use of current vaccines. In the present study an influenza whole inactivated virus (WIV) vaccine was freeze-dried or spray-freeze dried in the presence of inulin as a cryoprotectant. Only spray-freeze drying rendered powders compatible with administration to the lungs by insufflation. Pulmonary administration of the powder vaccine obtained by this method to BALB/c mice led to a transient influx of neutrophils and a concomitant decrease in the number of macrophages as did administration of liquid vaccine. Inflammatory reactions to both vaccines were mild and short-lived. Immunization studies showed that the immunogenic properties of WIV vaccine were not affected by drying. Pulmonary administration of the powder WIV vaccine induced a systemic immune response of the same magnitude as liquid vaccine while mucosal IgA responses were higher for powder WIV. In a challenge study where immunized mice were exposed to a lethal dose of live virus, two pulmonary doses of either liquid or powder WIV vaccine were equally effective as a single intramuscular injection of subunit vaccine in terms of reduction of the viral load in the lungs. To conclude, in the models employed for these studies the use of a dry powder WIV vaccine for pulmonary immunization was shown to be safe and efficient.     

Organising for immunization

In the late 1960s, health workers from a mission hospital in rural Zambia began registering children under 14 years old within 30 miles of the hospital (about 3000 children) by incorporating the cooperation of community leaders. They wanted to give every 0-4 year old child a Road to Health card and every 5-14 year old a vaccine record card and to promote the significance of immunization to parents and community leaders. The mission hospital established mobile health units to conduct regular visits in the center of villages. Staff hugh scales from a tree and borrowed a table to conduct the clinic. They kept a good relationship and communication with the community, leading successful education and communication activities. By 1988, many younger mothers were unfamiliar with a measles or whooping cough epidemic so they tended to not have their infants immunized. Epidemics began killing children nationwide, frightening these mothers so they brought their children for immunizations. The medical mission achieved an 85-90% vaccination coverage rate with immunization clinic attendance climbing quickly. 1 mother even walked 30 miles to have her infant injected with the DPT vaccine, but 10 years earlier, she did not bring her children. Further, measles had not reached her area because the immunization level was so high that it stopped the epidemic.     

Intradermal immunization improves protective efficacy of a novel TB vaccine candidate

We have developed the Mycobacterium tuberculosis (Mtb) fusion protein (ID83), which contains the three Mtb proteins Rv1813, Rv3620 and Rv2608. We evaluated the immunogenicity and protective efficacy of ID83 in combination with several emulsion-formulated toll-like receptor agonists. The ID83 subunit vaccines containing synthetic TLR4 or TLR9 agonists generated a T helper-1 immune response and protected mice against challenge with Mtb regardless of route. The ID83 vaccine formulated with gardiquimod (a TLR7 agonist) also resulted in a protective response when administered intradermally, whereas the same vaccine given subcutaneously failed to provide protection. This highlights the need to explore different routes of immunization based on the adjuvant formulations used.     

JD—1型经皮黄疸仪的研制及临床应用

本文介绍的JD—1型经皮黄疸仪,为临床观察提供了一种无创伤监测新生儿黄疸的方法,避免了婴儿反复抽血的痛苦和医护人员采血的困难。仪器采用先进的光导纤维和光电子信息处理技术。体积小巧,操作简便,测定迅速,临床中受到医护个员和婴儿家长的欢迎。     

Development and release of a national immunization app for Canada (ImmunizeCA)

Digital technology has created an opportunity to reenvision the traditional immunization paper record. We describe our experience developing a government endorsed mobile immunization record in Canada. The smartphone app, ImmunizeCA is designed to assist individuals in managing their own health information. It allows individuals to store their and their family's immunization records on their smartphone. The app, which is populated by data provided by the user, contains all 13 provincial and territorial schedules, immunization information and outbreak alerts on vaccine preventable diseases. Our experience suggests mobile apps can serve as a mechanism to empower users, increase participation in the process of immunization, potentially improve immunization rates and address jurisdictional obstacles. Key measures of success will include long term uptake, acceptability as an official record, enabling data flow permitting integration with immunization information systems and the ability to rapidly iterate to address changes to both immunization practice and mobile technology.     

Development of immunization trials against Pasteurella multocida

Pasteurellosis is one of the most important respiratory diseases facing economically valuable farm animals such as poultry, rabbit, cattle, goats and pigs. It causes severe economic loss due to its symptoms that range from primary local infection to fatal septicemia. Pasteurella multocida is the responsible pathogen for this contagious disease. Chemotherapeutic treatment of Pasteurella is expensive, lengthy, and ineffective due to the increasing antibiotics resistance of the bacterium, as well as its toxicity to human consumers. Though, biosecurity measures played a role in diminishing the spread of the pathogen, the immunization methods were always the most potent preventive measures. Since the early 1950s, several trials for constructing and formulating effective vaccines were followed. This up-to-date review classifies and documents such trials. A section is devoted to discussing each group benefits and defects.     

Missed opportunities for immunization

A survey conducted in the outpatient departments of Dayanand Medical College and Hospital in Ludhiana, India, found that 13.4% of children aged 0-23 months and 33.0% of pregnant women were not being given due immunization. Maximum advantage, however, should be taken of every contact between health workers and clients to provide all available and required health interventions. 80% immunization coverage could be achieved if all children who are brought to clinics for whatever purpose were screened and immunized if necessary. Missed opportunities occur because immunization is not available on all days; there is no uniform contraindication policy; doctors schedule as they please, with only one or two vaccines given to children who are eligible for more; there is an unwillingness to combine vaccines; weak excuses prevent the administration of vaccines; due antigens are not given on discharge from hospitals following recovery; pregnancy of less than 16 weeks is supposed to be a contraindication to tetanus toxoid; there is vaccinator reluctance to open multi-dose BCG/measles vaccine vials for a small number of children for fear of wasting the vaccine; and vaccines may be out of stock. The following suggestions may help minimize missed opportunities for immunization: review of the immunization schedule to provide optimal protection at the earliest age, review of the policy on contraindications to avoid false contraindications, ensuring that all women and children receive all vaccines for which they are eligible, issuing immunization cards to all women and children and checking them on all subsequent visits, making vaccines available in all clinics, educating health care personnel on these issues, packing vaccines in smaller quantities to avoid wastage, and exploiting all contacts with the people to provide maximum health care interventions.