Kinetics of different specific immunological parameters after rabies vaccination in mice.

A significant protection to an intracerebral challenge of 70 mean lethal doses of a standard live rabies virus strain was obtained in BCG-pretreated mice or in normal mice which had been immunized with a single subcutaneous injection of a beta-propiolactone-inactivated rabies vaccine. Concomitantly, levels of delayed-type hypersensitivity (measured in vivo by the footpad test) and serum-neutralizing activity were evaluated at various times after immunization. All immune criteria were significantly augmented in the BCG-pretreated, rabies-immune mice as compared to normal, rabies-immune mice. However, peak levels of protection, delayed-type hypersensitivity, and serum-neutralizing activity did not occur at the same times. For instance, in the BCG-pretreated, rabies-immune mice, delayed-type hypersensitivity peaked on day 7, protection peaked on day 21, and serum-neutralizing activity peaked on day 60. In BCG-pretreated mice, which did not receive the rabies vaccine, positive delayed-type hypersensitivity, some protection, and serum neutralizing activity were observed 4 to 5 weeks after BCG pretreatment. The possible relationships between specific and nonspecific immunity provoked by rabies virus antigens, tissue culture cell-associated antigens (derived from the bovine fetal kidney cells in which the rabies virus was grown, and BCG are discussed.     

A DNA prime-live vaccine boost strategy in mice can augment IFN-gamma responses to mycobacterial antigens but does not increase the protective efficacy of two attenuated strains of Mycobacterium bovis against bovine tuberculosis

The Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine has variable efficacy for both human and bovine tuberculosis. There is a need for improved vaccines or vaccine strategies for control of these diseases. A recently developed prime-boost strategy was investigated for vaccination against M. bovis infection in mice. BALB/c and C57BL/6 mice were primed with a DNA vaccine, expressing two mycobacterial antigens, ESAT-6 and antigen 85 A and boosted with attenuated M. bovis strains, BCG or WAg520, a newly attenuated strain, prior to aerosol challenge. Before challenge, the antigen-specific production of interferon-gamma (IFN-gamma) was evaluated by ELISPOT and antibody responses were measured. The prime-boost stimulated an increase in the numbers of IFN-gamma producing cells compared with DNA or live vaccination alone, but this varied according to the attenuated vaccine strain, time of challenge and the strain of mouse used. Animals vaccinated with DNA alone generated the strongest antibody response to mycobacterial antigens, which was predominantly IgG1. BCG and WAg520 alone generally gave a 1-2 log10 reduction in bacterial load in lungs or spleen, compared to non-vaccinated or plasmid DNA only control groups. The prime-boost regimen was not more effective than BCG or WAg520 alone. These observations demonstrate the comparable efficacy of BCG and WAg520 in a mouse model of bovine tuberculosis. However, priming with the DNA vaccine and boosting with an attenuated M. bovis vaccine enhanced IFN-gamma immune responses compared to vaccinating with an attenuated M. bovis vaccine alone, but did not increase protection against a virulent M. bovis infection.     

四种卡介苗重组疫苗对小鼠免疫原性的研究

目的研究esat6-卡介苗重组疫苗、mpt64-卡介苗重组疫苗、ag85a-卡介苗重组疫苗、ag85b-卡介苗重组疫苗的免疫原性。方法通过ELISA法检测小鼠血清中特异性抗体,用MTS法分析其脾淋巴细胞增殖指数。结果以生理盐水、卡介苗为对照,用各卡介苗重组疫苗免疫小鼠后,各组小鼠体重变化与对照组相比差异无统计学意义。应用不同抗原检测各组小鼠不同时间采集的血清,各组有其特异性抗体产生;ag85a-卡介苗重组疫苗能提高卡介苗刺激B细胞产生抗体的水平;各卡介苗重组疫苗免疫后15d,抗体水平已有升高,45d时达到最高水平。用不同抗原刺激各组小鼠脾淋巴细胞,平均刺激指数均达2.0以上,esat6-卡介苗重组疫苗组小鼠脾淋巴细胞的刺激指数稍高,其它各组之间差异无统计学意义。结论结核分枝杆菌esat6、mpt64、ag85a、ag85b基因在卡介苗中都能够表达特异的蛋白,刺激小鼠产生特异性抗体,均能刺激T淋巴细胞产生细胞免疫。     

A key role for lung‐resident memory lymphocytes in protective immune responses after BCG vaccination

The immune mechanisms that orchestrate protection against tuberculosis as a result of BCG vaccination are not fully understood. We used the immunomodulatory properties of fingolimod (FTY720) treatment to test whether the lung‐resident memory T lymphocytes generated by BCG vaccination were sufficient to maintain immunity against challenge infection with mycobacteria (BCG). Mice were given daily fingolimod treatment, starting either immediately before s.c. BCG vaccination or during subsequent BCG i.n. challenge, to prevent LN effector and memory lymphocytes from entering the periphery either during priming or challenge, respectively. Treatment with fingolimod during vaccination reduced vaccine‐mediated protection against subsequent infection. By contrast, BCG‐vaccinated mice were protected when fingolimod was given during the infectious challenge, suggesting that memory lymphocytes that migrate to the lung following vaccination are sufficient for protection. Notably, the antigen‐reactive IFN‐γ or multicytokine‐producing CD4⁺ T cells present in the lung when fingolimod was given during BCG challenge did not correlate with protection; however, expression of MHC class II on macrophages isolated from the lungs post BCG challenge was increased in the protected mice. We conclude that protection conferred by BCG vaccination is dependent on memory lymphocytes retained in the lung, although IFN‐γ production by this population is not correlated with vaccine‐mediated protection.     

Comparative study of cellular and humoral immunity as well as of resistance to the rabies virus in rabies vaccination

Experiments in CBA and BALB/c mice showed that after vaccination against rabies alongside with humoral immune response there developed a clone of cells sensitized to rabies antigen which, upon a secondary exposure to it, responded by increased blast formation. The immunological memory to the rabies antigen was found to be specific and to persist for up to 100 days (the observation period). Immunization of mice with rabies vaccine using various schedules and in combination with interferon inducers revealed no correlation between the lymphocyte blastogenesis parameters and virus-neutralizing antibody levels or the intensity of immunity to street rabies virus. The lymphocyte blasttransformation test with nonspecific mitogens may also be used for evaluation of the toxicity of new preparations.     

Oral recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing HIV-1 antigens as a freeze-dried vaccine induces long-term,HIV-specific mucosal and systemic immunity

Induction of HIV-1-specific immune responses was evaluated using a recombinant BCG (rBCG) vector-based vaccine expressing HIV-1 Env V3 peptide (rBCG-pSOV3J1). rBCG-pSOV3J1 was manufactured as a freeze-dried preparation based on good laboratory practice guidelines. Guinea pigs were immunized with the freeze-dried rBCG vaccine by oral administration to test the effectiveness of what is generally considered the most convenient and practical route for vaccination. While delayed-type hypersensitivity (DTH) skin reactions to purified protein derivative were not detected in any of the animals receiving oral rBCG-pSOV3J1, HIV-1 V3J1 antigen-specific DTH responses were detected in all of the immunized guinea pigs 1.5 years after immunization. In addition, significant proliferative responses against HIV-1 V3J1 antigen were measured in peripheral blood mononuclear cells and splenocytes from all animals receiving oral rBCG. Interestingly, intestinal intraepithelial lymphocytes from the animals also exhibited high levels of proliferative activity against HIV-1 V3J1 antigen. These results suggest that oral vaccination of guinea pigs with freeze-dried rBCG-pSOV3J1 induces high levels of functional T cells specific for HIV-1 antigens in both mucosal and systemic compartments and suggest that this approach has potential for use as a vaccine against HIV-1.     

Application of mycobacterial proteomics to vaccine design: improved protection by Mycobacterium bovis BCG prime-Rv3407 DNA boost vaccination against tuberculosis

Information from comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guerin (BCG) principally allows prediction of potential vaccine candidates. Thirty-six M. tuberculosis DNA vaccine candidates identified by comparative proteome analysis were evaluated in the mouse model for protection against low-dose aerosol M. tuberculosis infection. We identified the DNA vaccine candidate Rv3407 as a protective antigen and analyzed putative major histocompatibility complex class I epitopes by computational predictions and gamma interferon Elispot assays. Importantly, we discovered that the DNA vaccine Rv3407 improved the efficacy of BCG vaccination in a heterologous prime-boost vaccination protocol. Our data demonstrate the rationale of a combination of proteomics, epitope prediction, and broad screening of putative antigens for identification of novel DNA vaccine candidates. Furthermore, our experiments show that heterologous prime-boost vaccination with a defined antigen boost "on top" of a BCG primer provides superior protection against tuberculosis over vaccination with BCG alone.     

The protective effect of the Mycobacterium bovis BCG vaccine is increased by coadministration with the Mycobacterium tuberculosis 72-kilodalton fusion polyprotein Mtb72F in M. tuberculosis-infected guinea pigs

A tuberculosis vaccine candidate consisting of a 72-kDa polyprotein or fusion protein based upon the Mtb32 and Mtb39 antigens of Mycobacterium tuberculosis and designated Mtb72F was tested for its protective capacity as a potential adjunct to the Mycobacterium bovis BCG vaccine in the mouse and guinea pig models of this disease. Formulation of recombinant Mtb72F (rMtb72F) in an AS02A adjuvant enhanced the Th1 response to BCG in mice but did not further reduce the bacterial load in the lungs after aerosol challenge infection. In the more stringent guinea pig disease model, rMtb72F delivered by coadministration with BCG vaccination significantly improved the survival of these animals compared to BCG alone, with some animals still alive and healthy in their appearance at >100 weeks post-aerosol challenge. A similar trend was observed with guinea pigs in which BCG vaccination was boosted by DNA vaccination, although this increase was not statistically significant due to excellent protection conferred by BCG alone. Histological examination of the lungs of test animals indicated that while BCG controls eventually died from overwhelming lung consolidation, the majority of guinea pigs receiving BCG mixed with rMtb72F or boosted twice with Mtb72F DNA had mostly clear lungs with minimal granulomatous lesions. Lesions were still prominent in guinea pigs receiving BCG and the Mtb72F DNA boost, but there was considerable evidence of lesion healing and airway remodeling and reestablishment. These data support the hypothesis that the coadministration or boosting of BCG vaccination with Mtb72F may limit the lung consolidation seen with BCG alone and may promote lesion resolution and healing. Collectively, these data suggest that enhancing BCG is a valid vaccination strategy for tuberculosis that is worthy of clinical evaluation.     

The 19-kD antigen and protective immunity in a murine model of tuberculosis

The 19-kD antigen is a cell wall-associated lipoprotein present in Mycobacterium tuberculosis and in bacille Calmette-Guérin (BCG) vaccine strains. Expression of the 19-kD antigen as a recombinant protein in two saprophytic mycobacteria-M. vaccae and M. smegmatis-resulted in abrogation of their ability to confer protection against M. tuberculosis in a murine challenge model, and in their ability to prime a DTH response to cross-reactive mycobacterial antigens. Induction of an immune response to the 19-kD antigen by an alternative approach of DNA vaccination had no effect on subsequent M. tuberculosis challenge. These results are consistent with a model in which the presence of the 19-kD protein has a detrimental effect on the efficacy of vaccination with live mycobacteria. Targeted inactivation of genes encoding selected antigens represents a potential route towards development of improved vaccine candidates.     

Modulation of humoral immune response to influenza vaccines by BCG

BCG-induced modulation of humoral immune response to influenza vaccines was studied on adult volunteers and laboratory animals. Three influenza vaccine types were used: inactivated whole-virion vaccine prepared by chromatography, chemically split adsorbed vaccine and live allantoic vaccine for intranasal vaccination. According to the indices of humoral immunity against influenza such as seroconversion, frequency and intensity of antibody formation and degree of protection to subsequent administration of the vaccine virus, the BCG vaccine had a marked immunomodulating effect in combination with the adsorbed chemically split vaccine. The effect was slight in the case of the whole-virion vaccine and was absent with the live attenuated vaccine.     

Immunogenicity and protective efficacy of a DNA vaccine encoding the fusion protein of mycobacterium heat shock protein 65(Hsp65) with human interleukin‐2 against Mycobacterium tuberculosis in BALB/c mice

Developing a new generation of vaccines is important for preventing tuberculosis (TB). DNA vaccine is one promising candidate. In this study we evaluated the immunogenicity and protective efficacy of the DNA vaccine encoding the fusion protein of Mycobacterium tuberculosis heat shock protein 65 (Hsp65) with human interleukin‐2 (hIL‐2) in BALB/c mice. We showed that the DNA vaccine pcDNA‐Hsp65‐hIL‐2 could induce high levels of antigen‐specific antibody, IFN‐γ, CD4⁺ and CD8⁺ T cell production. When the immunized mice were infected with M. tuberculosis H37Rv, the organ bacterial loads in the DNA immunized group were significantly reduced compared to those of the saline control group, but the ability to reduce bacteria was not better than for BCG. The histopathology in lungs of the DNA vaccine immunized mice was similar to that of BCG immunized mice, which was obviously ameliorated compared to that of the saline control group. Overall, the DNA vaccine could afford protection against M. tuberculosis infection, though the protection efficacy was not as great as that of conventional BCG.     

Thymus dependence of rabies vaccine.

Immune responses to rabies vaccine were compared with those to known thymus-dependent and thymus-independent antigens, in nude mice and their normal litter mates. No antibody response to, or protection against, challenge was observed in nude mice inoculated with either 1 or 2 doses of rabies vaccine. A single dose of the same vaccine induced a substantial antibody response and protected normal mice. These data indicate that the antigens in rabies vaccine that induce neutralizing antibody and protect mice are thymus-dependent.     

结核分枝杆菌联合DNA疫苗初免-BCG加强的免疫效果观察

目的:研究并比较结核分枝杆菌免疫保护性抗原DNA(Ag85A和ESAT-6)疫苗联合免疫,BCG免疫以及联合DNA疫苗初免-BCG加强免疫等不同的免疫策略,诱导免疫应答效果观察.方法:健康雌性BALB/c小鼠24只,随机分成PBS 阴性对照组,DNA初免-BCG异源加强组,DNA(Ag85A和ESAT-6)初免DNA同源加强组和BCG阳性对照组,共进行3次免疫,初免2次,最后1次加强,间隔2周1次.PBS组3次均注射PBS 溶液;DNA/BCG组以质粒DNA免疫2次,最后1次以BCG加强免疫;DNA/DNA组3次均以质粒DNA进行免疫;BCG组则注射PBS溶液2次后以BCG免疫.末次免疫后4、6、8周分别分离血清测定总IgG水平,同时分离小鼠脾细胞,体外经TB-PPD刺激后进行淋巴细胞增殖实验(XTT法)并测定脾细胞培养上清中IFN-γ和IL-4水平.结果:DNA/BCG、DNA/DNA、BCG组体外经TB-PPD刺激后均检测到特异性IgG抗体产生,3组平均效价为1:120、1:160、1:80,DNA/DNA组的抗体效价高于另外2组;小鼠脾细胞体外经TB-PPD刺激后,均能产生特异性淋巴细胞增殖并诱生较强的IFN-γ反应,其中DNA/BCG组IFN-γ的分泌水平高于DNA/DNA组和BCG组(P<0.05).结论:联合DNA疫苗初免-BCG加强的免疫策略能在小鼠体内诱导较强的特异性细胞免疫反应,产生高水平的IFN-γ.     

Distinct H-2 complex control of mortality, and immune responses to tuberculosis infection in virgin and BCG-vaccinated mice.

We have studied the impact of distinct haplotypes and of different alleles at specific H-2 loci on: (i) the susceptibility to lethal form of experimental tuberculosis; (ii) the level of DTH to mycobacterial antigens; (iii) the efficacy of vaccination with bacille Calmette-Guérin (BCG); and (iv) the IgG production and T cell proliferative response to H37Rv antigens. On the basis of median survival time (MST) following primary inoculation with lethal dose of Mycobacterium tuberculosis, susceptibility to infection associated with I-Ab and Db alleles, host resistance associated with I-Ak and Dd alleles. Mice bearing a disease-resistant phenotype also developed a vigorous DTH response. Vaccination with BCG before H37Rv infection significantly prolonged the survival time of both resistant and susceptible animals, except in B10.M (H-2f) mice. The latter exhibited intermediate resistance to infection before but slight decrease in the MST following a high-dose BCG vaccination. Distinct H-2 regulation of susceptibility to lethal infection and of BCG vaccination efficacy was confirmed in another relatively resistant H-2f-bearing strain A.CA, in which mortality occurred more rapidly in vaccinated compared with primarily infected animals. The expression of the H-2f haplotype was associated with a low DTH response to tuberculin following vaccination and subsequent lethal infection. The lack of BCG protection against Myco, tuberculosis challenge in B10.M mice associated with the high titre of specific IgG. In addition, these mice exhibited a unique ability to respond to 65-kD antigen by both IgG synthesis and T cell proliferation.     

The pattern of mycobacterial antigen recognition in sera from Mantoux-negative individuals is essentially unaffected by bacille Calmette-Guérin (BCG) vaccination in either south India or London.

Paired sera were obtained before and 8 weeks after routine BCG vaccination from 20 PPD-S Mantoux-negative individuals who were living adjacent to the Chingleput BCG vaccine trial area in Tamil Nadu and from seven Mantoux-negative school-children in London, UK. Most subjects became Mantoux-positive after vaccination. In ELISA tests against soluble extracts of BCG or Mycobacterium tuberculosis H37Rv or against PPD-S, pre-vaccination antibody titres of South Indian subjects were about twice those of British subjects but there was no increase in titre of antibodies after vaccination of either population. Western blotting showed that even before vaccination, and even in British subjects, antibodies were present that recognized numerous antigenic components in extracts of BCG and M. tuberculosis. There was no consistent difference between band patterns with South Indian and British subjects and any effect of vaccination on the patterns was minimal.     

Systemic or local co-administration of lactoferrin with sensitizing dose of antigen enhances delayed type hypersensitivity in mice

Lactoferrin (LF), a major defense protein synthesized and stored in granulocytes has been implicated in maintaining immune homeostasis during an insult-induced metabolic imbalance. In this study, we demonstrated that lactoferrin augments the delayed type hypersensitivity (DTH) response to specific antigens in mice. Lactoferrin (LF) was given to mice orally or intraperitoneally (i.p. ) at the time of immunization, or subcutaneously (s.c.) in a mixture with the immunizing doses of the following antigens, sheep red blood cells (SRBC), Calmette-Guerin bacillus (BCG) or ovalbumin (OVA). A DTH reaction was determined 24 h after administration of an eliciting dose of antigen as a specific increase in foot pad swelling. Lactoferrin enhanced DTH reaction to all studied antigens in a dose-dependent manner. Lactoferrin (LF) given to mice in conjunction with antigen administered in an incomplete Freund's adjuvant induced the DTH response at the level of control mice given antigen in a complete Freund's adjuvant. In addition, LF remarkably increased DTH response to a very small, otherwise non-immunogenic SRBC dose. The increase in DTH response was less pronounced for orally administered LF than for any other routes of administration, however, statistically significant augmentation was demonstrated for each antigen studied. Although the costimulatory action of LF was accompanied by the appearance of bovine lactoferrin-specific cellular responses in mice, it is very unlikely that such responses will be generated in humans, since bovine lactoferrin is a dietary antigen to which a tolerance has been acquired. Considering the involvement of LF in generation of stimulatory signals during the induction phase of an antigen specific immune responses, we suggest that LF may be useful for development of safer and more efficacious vaccination protocols.     

Alum adjuvanted rabies DNA vaccine confers 80% protection against lethal 50 LD50 rabies challenge virus standard strain

Rabies is a serious concern world-wide. Despite availability of rabies vaccines for long; their efficacy, safety, availability and cost effectiveness has been a tremendous issue. This calls for improvement of rabies vaccination strategies. DNA vaccination has immense potential in this regard. The DNA vaccine pgp.LAMP-1 conferred 60% protection to BALB/c mice against 20 LD₅₀ rabies challenge virus standard (CVS) strain challenge. Upon supplementation with Emulsigen-D, the vaccine formulation conferred complete protection against lethal challenge. To assess the feasibility of this vaccine formulation for human use, it was tested along with other FDA approved adjuvants, namely, Alum, Immuvac, Montanide ISA720 VG. Enhanced immune response correlated with high IgG antibody titer, Th2 biased response with a high level of rabies virus neutralizing antibodies (RVNAs) and IgG1/IgG2a ratio >1, observed upon alum supplementation of the rabies DNA vaccine. The total IgG antibody titer was 2 IU/ml and total RVNA titer was observed to be 4 IU/ml which is eight times higher than the minimum protective titer recommended by WHO. Furthermore, it conferred 80% protection against challenge with 50 LD₅₀ of the rabies CVS strain, conducted in compliance with the potency test for rabies recommended by the National Institutes of Health (NIH), USA. Previously, we have established pre-clinical safety of this vaccine as per the guidelines of Schedule Y, FDA as well as The European Agency for evaluation of Medicinal Products. The vaccine showed no observable toxicity at the site of injection as well as at systemic level in Wistar rats when administered with 10X recommended dose. Therefore, supplementation of rabies DNA vaccine, pgp.LAMP-1 with alum would lead to development of a non-toxic, efficacious, stable and affordable vaccine that can be used to combat high numbers of fatal rabies infections tormenting developing countries.     

Boosting with poxviruses enhances Mycobacterium bovis BCG efficacy against tuberculosis in guinea pigs

Tuberculosis is rising in the developing world due to poor health care, human immunodeficiency virus type 1 infection, and the low protective efficacy of the Mycobacterium bovis BCG vaccine. A new vaccination strategy that could protect adults in the developing world from tuberculosis could have a huge impact on public health. We show that BCG boosted by poxviruses expressing antigen 85A induced unprecedented 100% protection of guinea pigs from high-dose aerosol challenge with Mycobacterium tuberculosis, suggesting a strategy for enhancing and prolonging the efficacy of BCG.     

Effect of cyclophosphamide pretreatment on defective delayed-type hypersensitivity in autoimmune-prone MRL mice

Autoimmune MRL mice develop a poor delayed-type hypersensitivity (DTH) after BCG cell wall (BCG CW) immunization. To test the possible participation of suppressor cells on DTH induction, MRL/MpJ-lpr/lpr (MRL-lpr) and MRL/MpJ-+/+ (MRL-+/+) mice were pretreated with cyclophosphamide (Cy) 4 days before the immunization. The footpad reaction to the purified protein derivative of tuberculin (PPD) was enhanced remarkably by Cy pretreatment in both MRL-lpr and MRL-+/+ mice. In addition, the lymph node cells obtained from nonpretreated MRL-lpr and MRL-+/+ mice at 7 days after BCG CW immunization suppressed the DTH induction of Cy-pretreated MRL-+/+ mice. On the other hand, a positive proliferative response to PPD in vitro was seen only in the cells from Cy-pretreated MRL-+/+ and younger MRL-lpr mice, but not in the cells of nonpretreated MRL-+/+ or both pretreated and nonpretreated older MRL-lpr mice. Removal of B220-positive cells from the lymph node cell populations of Cy-pretreated MRL-lpr mice restored PPD responsiveness, but the same treatment had no effect on the cells from nonpretreated MRL-lpr mice. The results suggest that DTH responses to PPD in MRL mice immunized with BCG CW are regulated by Cy-sensitive suppressor cells. These suppressor cells occur in MRL mice irrespective of whether they carry the lpr gene. And it is also demonstrated that MRL-lpr mice have TDTH precursor cells in a sufficient number for TDTH production even after expressing lymphadenopathy.