Immunization of Mice Against Encephalomyocarditis Virus I. Purification, Concentration, and Inactivation of Encephalomyocarditis Virus

Methods are described for the concentration, purification, and inactivation of encephalomyocarditis virus grown in L-cell suspension cultures. Virus concentrates were obtained by the precipitation of crude virus lysates with polyethylene glycol and were purified by Genetron extraction. After column chromatography approximately 70% recovery of infectivity and hemagglutination (HA) activity was found. Exposure of the purified virus suspensions to ultraviolet light resulted in reduction of the infectivity by a factor of 10(9) with little or no loss of HA activity. The preparations are being evaluated as vaccines.     

Immunization of Mice with Live Attenuated Encephalomyocarditis Virus: Local Immunity and Survival

Mice were vaccinated with an attenuated encephalomyocarditis (EMC) virus strain by the intraperitoneal (i.p.) route and by various ways of respiratory administration: aerosol exposure and intratracheal (i.t.) and intranasal (i.n.) instillation. A linear relationship was found between vaccine dose and the resulting serum antibody titer. The effectiveness of the vaccine was determined by measuring the 50% protective doses (ED(50) values) after a lethal challenge with live virulent virus given by the i.p. route. For all three methods of respiratory immunization essentially the same ED(50) value was found, about 200 plaqueforming units (PFU), but i.p. immunization was less effective, the ED(50) value being about 600 PFU. To investigate the protective effect of local immunity, mice were vaccinated i.p. or i.n. and challenged by the i.n. route. The same ED(50) values were found as after i.p. challenge, indicating that the degree of protection afforded by the vaccine depends only on the route of vaccination and not on the route of challenge. This means that protection depends largely on systemic immunity and that local immunity plays only a minor role in this system. The results are discussed in relation to the feasibility of respiratory immunization against animal viruses.     

Effect of Bordetella pertussis Vaccination in Mice and the Isolated Tracheal Response to Isoprenaline

The administration of Bordetella pertussis vaccine to mice has been associated with the development of an impaired beta-adrenoceptor responsiveness and in many respects has resembled human asthma. Trachea (n = 12) were isolated from Swiss-Webster mice 5 days following the intraperitoneal administration of 2 x 10(9) B. pertussis organisms. The tracheal smooth muscle response to carbachol was measured and compared with that found in trachea from unvaccinated mice (n = 15). The contractile response was similar in both groups. The tracheal smooth muscle relaxant effects of isoproterenol were measured in these two groups. The EC50 value for isoprenaline (6.5 x 10(-7) M) in trachea from B. pertussis treated mice was significantly (P < 0.05) greater than that noted in the control animals (2.3 x 10(-7) M). These studies demonstrated that in tracheal smooth muscle isolated from B. pertussis vaccinated mice, the relaxant effects of isoprenaline are impaired.     

Variable Adjuvant Activity of Bordetella pertussis with Respect to the Primary and Secondary Immunization of Mice

We carried out a study on the adjuvant effect of Bordetella pertussis vaccine on the primary and secondary immune responses of the mouse to sheep erythrocytes, quantitating antibody-producing spleen cells and serum antibody. The simultaneous injection of sheep erythrocytes and B. pertussis, when compared to immunization with sheep red blood cells alone, resulted in an increased and prolonged multiplication of antibody-forming spleen cells. The adjuvant effect was also documented by increased production of serum hemolysins and agglutinins. Further, B. pertussis enhanced the priming effect of the antigen for the secondary response. However, when the bacterial adjuvant was given together with a second antigenic stimulus 41 days after the primary immunization, the peak values of direct and indirect plaque-forming spleen cells did not differ from the corresponding control animals further inoculated with sheep erythrocytes alone. Nonetheless, the influence of the bacterial adjuvant was still expressed by the delayed decrease of the numbers of plaque-forming spleen cells. On the basis of the X-Y-Z scheme it is suggested that B. pertussis cells as adjuvant enhance the multiplication of antigen-sensitive X cells or affect the initial stages of differentiation of these cells. This effect of the pertussis vaccine can be distinguished from a general proliferative action on other cells.     

Enteric Immunization with Live Adenovirus Type 21 Vaccine II. Systemic and Local Immune Responses Following Immunization

Studies of the immunologic responses following administration of a live, enteric-coated adenovirus (ADV) type 21 vaccine showed that nine of ten vaccinees and none of five controls developed neutralizing antibody. Antibody activity of serum and secretory immunoglobulins was assayed by using a (14)C-labeled ADV-21 antigen in a radioimmunodiffusion system. Increases in immunoglobulin M, A and G (IgM, IgA, IgG) activity were detected in sera from vaccinees but not in those from controls. IgA copro antibody activity was also shown in vaccinees but not in controls. Nasal secretions showed no detectable IgA antibody responses by this method. These studies show marked differences in serum and local IgA antibody activity in induced enteric ADV infection compared to previously reported responses after natural infection. The protective role of secretory IgA in adenovirus infections is obscure. However, absence of nasal IgA responses may indicate that protection against disease with enteric ADV vaccines depends primarily upon humoral antibody.     

The Virulence Factors of Bordetella pertussis: Talented Modulators of Host Immune Response

Approximately 40 million whooping cough cases and between 200,000 and 400,000 pertussis-linked deaths are recorded each year. Although several types of vaccines are licensed and widely used, Bordetella pertussis continues to circulate in populations with high vaccine coverage of infants and children due to the waning of protection induced by the vaccination. B. pertussis typically expresses a wide array of virulence factors which promote bacterial adhesion and invasion by altering the local environment, including pertussis toxin, tracheal cytotoxin, adenylate cyclase toxin, filamentous hemagglutinin, and the lipooligosaccharide. The virulence factors of B. pertussis also possess immunomodulatory properties, exerted through their enzymatic and receptor-binding activities. Both pro- and anti-inflammatory effects are mediated, that can subvert host innate and adaptive immunity and favor the onset of a long-term infection. This review describes the capacities of B. pertussis virulence factors to modulate host immune responses and the mechanisms employed, which have been the subject of extensive research in the recent years, both in murine and human experimental systems. Knowledge of these mechanisms is gaining increasing importance, since it could provide in the near future the basis for the identification of therapeutic agents for modulating the immune system as well as novel molecular targets to treat pertussis.     

In Hot Pursuit of the First Vaccine Against Respiratory Syncytial Virus

Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infection, such as bronchiolitis, bronchitis, or pneumonia, in both infants and the elderly. Despite the global burden of diseases attributable to RSV infection, no clinically approved vaccine is available, and a humanized monoclonal antibody for prophylaxis is not readily affordable in developing countries. There are several hurdles to the successful development of RSV vaccines: immune-vulnerable target populations such as premature infants, pregnant women, and immunocompromised people; safety concerns associated with vaccine-enhanced diseases; repeated infection; and waning memory. To develop successful strategies for the prevention of RSV infection, it is necessary to understand the protective and pathologic roles of host immune responses to RSV infection. In this review, we will summarize the positive and negative relationship between RSV infection and host immunity and discuss strategies for the development of the first successful RSV vaccine.     

Virus-Induced Diabetes Mellitus: II. Relationship Between Beta Cell Damage and Hyperglycemia in Mice Infected with Encephalomyocarditis Virus

Infection of DBA 2 male mice with the M variant of encephalomyocarditis virus resulted in a diabetes-like syndrome. Histologic examination of the pancreas revealed damage to the beta cells with little involvement of the acinar cells. The severity of the hyperglycemia correlated closely with the degree of beta cell damage. By immunofluorescence, viral antigens could be detected in the beta cells during the first 10 days of the infection. In contrast to the response found in male DBA 2 mice, infection of DBA 2 female mice and male mice of several other strains resulted in little if any elevation of blood glucose concentration. Histologic examination of the pancreas of these animals revealed only minimal damage to the beta cells. It is concluded that differences in the severity of the hyperglycemia between DBA 2 males and females and among the different strains of male mice tested are directly related to the degree of beta cell damage produced by the viral infection.     

Immunostimulating Effect of Aqueous Extract of Amphypterygium Adstringens on Immune Cellular Response in Immunosuppressed Mice

Amphypterygium adstringens is a Mexican tree known as cuachalalate whose bark is habitually used for the treatment of fresh wounds, gastric ulcers, gastrointestinal cancer and various inflammatory conditions. The aim of this study was to evaluate the immunostimulant effect of the aqueous extract of A. adstringens on immune cellular response in immunosuppressed mice. An aqueous extract from the bark of cuachalalate was administered into BALB/c mice for 10 days. We assessed their immunostimmulant activity on cellular immune response by Delayed Type Hypersensitivity Response (DHT) to dinitrofluorobencene (DNFB) and by MTT assay. L5178Y lymphoma was used as immunossuppression model. An increase in DHT was observed after treatment with 10 and 100 mg/kg of the aqueous extract from A. adstringens oral treatment in lymphoma bearing mice. Splenocyte proliferation rate was significantly increased (2.5 time) in immunosuppresed mice treated with 10 mg/kg oral treatment compared with group that received vehicle only. The present study showed for the first time the aqueous extract from A. adstringens as a positive immunostimulant agent in lymphoma bearing mice and we demonstrated evidence to support the traditionally use of cuachalalate in conditions in which the immune system is depressed.     

Induction of Mucosal B-Cell Memory by Intranasal Immunization of Mice with Respiratory Syncytial Virus

The capacity of live or inactivated respiratory syncytial virus (RSV) to induce B-cell memory in respiratory-associated lymphoid tissues of mice was examined. Eight weeks after primary inoculation with either live or inactivated RSV, adult BALB/c mice were challenged with 4 × 10⁵ PFU of RSV. Protection from viral shedding and mucosal production of RSV-specific antibodies were examined at various time points after challenge. We found that primary immunization with live, but not inactivated, RSV induced complete and durable protection upon challenge within the upper and lower respiratory tract. Also, primary immunization with live, but not inactivated, RSV enhanced the production of mucosal RSV-specific immunoglobulin A (IgA) upon challenge. Secondary mucosal IgA responses were characterized by (i) the early production of mucosal IgA by B cells that reside in organized nasal-associated lymphoid tissues, cervical lymph nodes, and bronchial lymph nodes, and (ii) the subsequent production of RSV-specific IgA by mucosal effector tissues, such as the tracheal lamina propria and lung. These findings suggest that primary infection of mice with live RSV might induce mucosal IgA-committed memory B cells. A greater understanding of the characteristics of RSA-specific mucosal memory B cells may facilitate the development of an RSV vaccine.     

Use of Alum and Inactive Bordetella pertussis for Generation of Antibodies Against Synthetic Peptides in Mice

We have investigated the efficacy of the combined use of Alum and inactive Bordetella pertussis (iBP) adjuvants for eliciting anti-peptide antibodies. ICR mice were immunized four times at 3-week intervals with each of 7 free (i.e., not conjugated to any carrier) synthetic peptides of 15–17 amino acid residues in Alum + iBP, in the commonly used adjuvant protocols (CFA; CFA (initial) followed by IFA), or in CFA + iBP. Serum samples after 3 and 4 injections were tested by RIA. Use of Alum + iBP greatly increased the production of antibodies for most of the peptides. The results have important implications for human vaccine formulation involving peptides.     

STAg和霍乱毒素不同程序滴鼻免疫小鼠诱导抗弓形虫作用

目的观察弓形虫可溶性速殖子抗原（soluble tachyzoite antigen，STAg）和霍乱毒素（cholera toxin，CT）佐剂不同程序滴鼻免疫小鼠诱导的抗弓形虫感染能力，确定STAg和CT滴鼻免疫的最佳程序。方法BALB／c小鼠随机分为3组：1次、2次和3次免疫组，用20μgSTAg＋1μgCT／只分别滴鼻免疫1次，2次或3次，前2次间隔2周，末次间隔1周。末次免疫后第14天，用4×10^4个速殖子／只灌胃攻击所有小鼠，观察小鼠健康及死亡情况，攻击后第30天处死，ELISA法检测血清IgG和粪便IgA，计数肝、脑组织内弓形虫速殖子，分离并计数派伊尔结（Peyer＇s patches，PP）和脾淋巴细胞数。结果2次和3次免疫组小鼠存活率明显高于1次免疫组（P〈0．05），肝、脑组织内虫荷显著低于1次免疫组（P〈0．001），血清IgG和粪便IgA高于1次免疫组，PP和脾淋巴细胞数无显著性变化。结论STAg和CT佐剂滴鼻免疫2次或3次能有效诱导小鼠抗弓形虫感染。     

Effect of pyridines on phenotypic properties of Bordetella pertussis.

Several conditions of growth of Bordetella pertussis cause a reversible phenotypic alteration in properties termed modulation. Growth in medium containing nicotinic acid induces normal (X-mode) cells to change to modulated (C-mode) cells. We examined several pyridines and compounds resembling pyridines for their ability to affect modulation, using envelope protein patterns and serological reactivity as indicators of modulation. We found that 6-chloronicotinic acid and quinaldic acid were more effective modulating stimuli than was nicotinic acid on a molar basis. Both 2-chloronicotinamide and isoniazid interfered with nicotinic acid-induced modulation, and can be called antimodulators. Picolinic acid inhibited growth.     

Effect of Dexamethasone on Immune Response of Mice with Different Behavioral Types

Experiments on a model of paired sensory contact showed that dexamethasone effectively suppressing the response to ACTH not only prevented immunosuppression in male C57Bl/6J mice with submissive behavior formed during different periods of confrontation testing (days 10 and 20), but also stimulated the immune response in comparison with the control. Immune response in aggressive animals after 20-day confrontations was higher than in controls and submissive mice and did not change after dexamethasone injection. The authors conclude that the immunosuppressive effect in submissive animals is realized through ACTH, which little contributes into immunomodulation in aggressive mice.__________Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 5, pp. 550–552, May, 2005     

细粒棘球绦虫重组Bb—Eg95-EgA31疫苗诱导小鼠脾细胞增殖变化的研究

目的探讨细粒棘球绦虫（Eg）重组双歧杆菌（Bb）-Eg95-EgA31疫苗免疫和Eg原头节攻击后小鼠的囊重抑制率及脾细胞增殖的变化。方法将细粒棘球绦虫重组Bb-Eg95-EgA31疫苗分别采用皮下注射、肌肉注射、鼻腔内接种和口服灌胃4种途径免疫BALB／c小鼠,免疫后8周每鼠用50个Eg原头节攻击感染,感染后25周剖杀小鼠,分离细粒棘球蚴包囊并称重,计算囊重抑制率;取脾,分离脾细胞,用Eg粗抗原（EgAg）或刀豆素A（ConA）刺激培养,四甲基偶氮唑盐比色法（MTT法）检测免疫小鼠脾细胞增殖反应,同时设有空载体、Bb和MRS对照。结果以上4种疫苗接种组小鼠的囊重抑制率分别为45．33％、41．33％、70．67％和62．67％;疫苗接种组的脾细胞明显增殖;鼻腔内接种和口服免疫组的脾细胞增殖显著高于皮下和肌肉注射组。结论细粒棘球绦虫重组Bb-Eg95-EgA31疫苗可诱导小鼠产生特异性的细胞免疫反应。     

Guinea Pig Herpes-Like Virus Infections II. Antibody Response and Virus Persistence in Mice and Rabbits

Mice inoculated with guinea pig herpes-like virus produced minimal, if any, antibody response, and no virus was isolated from the inoculated animals 64 days after administration. The antibody response in rabbits inoculated with the same virus was prompt and reached a high level within 14 to 29 days regardless of the site of inoculation. Long-term persistence of viremia was observed only in intravenously inoculated rabbits; a brief period of viremia was observed in intraperitoneally inoculated rabbits, but no viremia was obtained in rabbits inoculated by the subcutaneous route. Maternal antibody was transferred readily to offspring; however, transference of infectious virus from mother to offspring was demonstrable in only one of 75 fetuses tested.     

Polymerase chain reaction assay for pertussis: simultaneous detection and discrimination of Bordetella pertussis and Bordetella parapertussis.

A polymerase chain reaction (PCR) assay which allows the simultaneous detection and discrimination of the two causative agents of pertussis, Bordetella pertussis and Bordetella parapertussis, was developed. Primer pairs were based on insertion sequence elements IS481 and IS1001. IS481 is specific for B. pertussis and is present in about 80 copies per cell, while IS1001 is specific for B. parapertussis and is found in 20 copies per cell. An internal control was included in the PCR assay to monitor the performance of the PCR and to identify possible inhibitory components in clinical samples. Discrimination of amplified DNA derived from the internal control, B. pertussis, or B. parapertussis was accomplished by differential spacing of the primers. The sensitivity of the combined PCR method was found to be very high and allowed the detection of one cell of either pathogen. The usefulness of the method was investigated by using a limited number of clinical samples derived from patients with serologically proven pertussis.