Zhijiao Tang

Immunogenicity, safety, and protective efficacy of an inactivated SARS-associated coronavirus vaccine in rhesus monkeys

Abstract An inactivated vaccine for severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) was evaluated in rhesus monkeys. The monkeys were inoculated intramuscularly (i.m.) with 0.5, 5, 50, or 5000μg of vaccine, or PBS as control, and boosted on day 7. After 3 weeks, they were challenged with the NS-1 strain of SARS-CoV. The humoral and mucosal immune responses, clinical signs, chemical indices and viremia were monitored following the immunization and challenge. The control animals who received PBS developed atypical SAR-CoV infection after viral challenge, according to clinical, virological and pathological findings. No systematic side effects were observed in vaccinated animals post-immunization, even in at the high dose of 5000μg. The 50μg dosage of vaccine elicited SARS-CoV specific immune responses against viral infection as compared to the partial immunity elicited by 0.5 and 5μg doses. The results show that this inactivated vaccine can induce effective concomitant humoral and mucosal immunity against SARS-CoV infection, is safe in monkeys, and the vaccine maybe a good candidate for clinical trials.

Fusion protein Ag85B-MPT64190-198-Mtb8.4 has higher immunogenicity than Ag85B with capacity to boost BCG-primed immunity against Mycobacterium tuberculosis in mice

Tuberculosis (TB) remains a major infectious disease worldwide despite chemotherapy and BCG vaccine. The efficacy of the current TB vaccine BCG varies from 0 to 80%. New vaccines that have better protection than BCG or have the capability to boost BCG-primed immunity are urgently needed. We have previously constructed a fusion protein Ag85B-MPT64(190-198)-Mtb8.4 (AMM). In this study, we investigated the immunogenicity of the fusion protein AMM in a novel adjuvant of dimethyl-dioctyldecyl ammonium bromide and BCG polysaccharide nucleic acid (DDA-BCG PSN), and its capacity to boost BCG-primed immunity. The anti-Ag85B antibodies IgG1 and IgG2a were determined using ELISA and the number of spleen cells secreting IFN-gamma was determined by ELISPOT. In addition, the ability of the subunit vaccine AMM to boost BCG-primed immunity against Mycobacterium tuberculosis was analyzed. The fusion protein AMM induced more effective humoral and cell-mediated immune responses in mice than Ag85B alone. Mice primed with BCG vaccination followed by boosting with AMM produced a stronger immune response and afforded a better protection against M. tuberculosis infection than mice immunized with BCG alone or BCG priming followed by boosting with Ag85B. These findings suggest that AMM is a promising candidate subunit vaccine to enhance the protective efficiency of BCG.

Subcutaneous Administration of Modified Vaccinia Virus Ankara Expressing an Ag85B-ESAT6 Fusion Protein, but Not an Adenovirus-Based Vaccine, Protects Mice Against Intravenous Challenge with Mycobacterium tuberculosis

Recombinant virus‐based tuberculosis (TB) vaccines that are strongly immunogenic and elicit robust cellular immunity are considered ideal vaccine candidates. Here, we engineered a poxvirus‐based vaccine, MVA85B‐E6, and an adenovirus‐based vaccine, AD85B‐E6, both of which express the fusion protein Ag85B‐ESAT6. Subcutaneous vaccination of AD85B‐E6 generated strong interferon (IFN)‐γ production by both CD4 and CD8 T cells and CD8 cytotoxic T lymphocyte activity; these results indicate that strong T‐helper type 1 immune responses were elicited in mice, which is in contrast to the moderate responses induced by vaccination with MVA85B‐E6. However, MVA85B‐E6 given subcutaneously led to levels of protection comparable with that induced by the bacillus Calmette–Guérin vaccine in the lungs and spleens, whereas AD85B‐E6 given subcutaneously did not show any protective efficacy after intravenous challenge of BALB/c mice with Mycobacterium tuberculosis H37Rv. Our study emphasizes that more efficient biomarkers for vaccine efficacy and more appropriate routes of vaccine administration are necessary for the development of a successful TB vaccine.

Subunit vaccine candidate AMM down-regulated the regulatory T cells and enhanced the protective immunity of BCG on a suitable schedule.

Mycobacterium bovis bacillus Calmette‐Guérin (BCG) priming and subunit vaccine boosting strategies are urgently needed to improve the protective efficacy of BCG in adult population. However, the schedule of subunit vaccine boosting is not well investigated, especially the optimal immune responses and vaccine immunization schedules are still not clear. We have constructed a novel subunit vaccine candidate consisting of fusion protein Ag85B‐Mpt64 (190‐198)‐Mtb8.4 (AMM) in a complex adjuvant composed of dimo‐thylidioctyl ammonium bromide (DDA) and BCG polysaccharide nucleic acid (BCG‐PSN). In this study, we compared the effect of different boosting schedules of the subunit vaccine in the prime‐boost strategies. C57BL/6 mice were primed with BCG first and then boosted with the AMM vaccine once at 10th week, twice at 8th, 10th week, or thrice at 6th, 8th, 10th week, respectively. The immune responses were evaluated at the 14th and 20th weeks, respectively. Twelve weeks after the last immunization, the mice were challenged with virulent Mycobacterium tuberculosis strain H37Rv, and the protective effect was evaluated. The results showed that BCG priming and the AMM vaccine boosting twice induced the strongest antigen‐specific IFN‐γ and IL‐2 production, down‐regulated CD4+ CD25+ FoxP3+ regulatory T cells (Tregs) and had the best protective effect among all groups, while boosting thrice induced the strongest IL‐4 production and did not improve BCG‐primed protection significantly. Boosting BCG with the AMM vaccine twice instead of once or thrice induced strong Th1‐type immunity and down‐regulated Tregs significantly, which correlated with the best protection against M. tuberculosis infection in mice.

M. tuberculosis H37Rv Infection of Chinese Rhesus Macaques

Mycobacterium tuberculosis is the most common communicable infectious disease worldwide and the top killer of human immunodeficiency virus (HIV)-infected people. Because of common dual HIV and M. tuberculosis infections, the emergence of multidrug-resistant M. tuberculosis strains, the lack of effective vaccination, the morbidity, and the mortality of M. tuberculosis infection are increasing sharply. Therefore, there is an urgent need for vaccine and drug development against M. tuberculosis infection. These require appropriate animal models that closely resemble human disease. To this end, we infected Chinese rhesus macaques with the M. tuberculosis H37Rv strain. Bronchoscopy was used to inoculate nine monkeys with different doses of M. tuberculosis H37Rv strain. Regardless of the M. tuberculosis dose, all monkeys were infected successfully. This was shown by clinical, laboratory, and histopathology assessments. Among nine infected monkeys, six developed acute rapid progressive tuberculosis and the remaining animals mirrored early-stage chronic disease. These data, taken together, demonstrate that Chinese rhesus macaques are highly susceptible to M. tuberculosis infection and develop similar manifestations of disease that are seen in humans. This model affords new opportunities for studies of M. tuberculosis disease pathology and therapeutics.

Neonatal revaccination with Bacillus Calmette-Guérin elicits improved, early protection against Mycobacterium tuberculosis in mice.

The protective effect of revaccination with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) against Mycobacterium tuberculosis in animals is controversial. To investigate whether revaccination of neonates with BCG could improve the protection against M. tuberculosis, C57BL/6 neonates were vaccinated with BCG on day 1, or on days 1, 7, and 14, and the mice at eight weeks of age were challenged with M. tuberculosis and monitored for survival. The M. tuberculosis burden in their livers and lungs, the pathological changes in the lungs, their splenic T cell responses and serum cytokines were examined longitudinally post-challenge. BCG vaccination significantly prevented the M. tuberculosis-related mouse death and reduced the burden of M. tuberculosis in the liver and lungs, and lung damage in the mice, particularly at early stage of the pathogenic process in the BCG-revaccinated mice. However, the BCG revaccination-induced protection waned over time. BCG vaccination did not significantly modulate the levels of serum IFN-γ and the frequency of splenic PPD-reactive IFN-γ-secreting T cells, but significantly decreased the levels of serum TNF-α and PPD-specific IL-4 responses at 3 weeks post challenge. Taken together, these data suggest that revaccination of neonates with BCG elicits improved, early protection against M. tuberculosis by modulating cytokine responses in adult mice.

In vitro and in vivo Activities of a New Lead Compound I2906 against Mycobacterium tuberculosis

Background: Due to the long duration of treatment and the emergence of multidrug-resistant strains, new antitubercular agents are urgently needed. I2906, as a novel lead, was screened and tested for efficacy in vitro and in vivo. Methods:To determine the efficacy of I2906,the minimum inhibitory concentrations against Mycobacterium tuberculosis and cytotoxicity were tested, and its in vivo activities were assessed by administering it to mice infected with M. tuberculosis H37Rv or multidrug-resistant strain. Results:Under in vitro conditions, I2906 showed excellent antimycobacterial activities and low cytotoxicity. In a murine model infected with M. tuberculosis H37Rv, the reductions on bacterial loads of both lungs and spleen were statistically significant (p < 0.05) between I2906-treated mice and untreated controls after 4 weeks. Further, the colony-forming unit counts in the lungs were dramatically lower (p < 0.05) than that of isoniazid-treated mice by the addition of I2906 after 8 weeks. Moreover, survival rate was increased by I2906 treatment. For multidrug-resistant strain infection, bacterial counts were reduced significantly in the lungs and spleen due to I2906 treatment in comparison with data from untreated controls (p < 0.05). Conclusions: I2906 displayed potential antimicrobial activities against M. tuberculosis H37Rv and drug-resistant strains in vitro and in vivo, and could improve efficacy of isoniazid in vivo.

SIV Infection Facilitates Mycobacterium tuberculosis Infection of Rhesus Macaques

Tuberculosis (TB) is a common opportunistic infection and the leading cause of death for human immunodeficiency virus (HIV)-infected patients. Thus, it is necessary to understand the pathogenetic interactions between M.tb and HIV infection. In this study, we examined M.tb and/or simian immunodeficiency virus (SIV) infection of Chinese rhesus macaques. While there was little evidence that M.tb enhanced SIV infection of macaques, SIV could facilitate M.tb infection as demonstrated by X-rays, pathological and microbiological findings. Chest X-rays showed that co-infected animals had disseminated lesions in both left and right lungs, while M.tb mono-infected animals displayed the lesions only in right lungs. Necropsy of co-infected animals revealed a disseminated M.tb infection not only in the lungs but also in the extrapulmonary organs including spleen, pancreas, liver, kidney, and heart. The bacterial counts in the lungs, the bronchial lymph nodes, and the extrapulmonary organs of co-infected animals were significantly higher than those of M.tb mono-infected animals. The mechanistic studies demonstrated that two of three co-infected animals had lower levels of M.tb specific IFN-γ and IL-22 in PBMCs than M.tb mono-infected animals. These findings suggest that Chinese rhesus macaque is a suitable and alternative non-human primate model for SIV/M.tb coinfection studies. The impairment of the specific anti-TB immunity is likely to be a contributor of SIV-mediated enhancement M.tb infection.

Mycobacterium tuberculosis Erdman infection of cynomolgus macaques of Chinese origin

Background Nearly one-third of the population worldwide is estimated to have latent tuberculosis infection (LTBI), which represents a vast reservoir for a constant source of tuberculosis (TB) transmission. It has been suggested that cynomolgus macaques are less susceptible to Mycobacterium tuberculosis (M.tb) infection than rhesus macaques, we examined M.tb infection of Chinese cynomolgus macaques. Methods Eight Chinese cynomolgus macaques were infected with M.tb Erdman strain with a small [25 colony forming unit (CFU)] or large dose (500 CFU) via bronchoscopy. The infected animals were monitored for symptoms and examined by chest X-ray, computed tomography (CT), tuberculin skin test (TST), and enzyme-linked immunospot (ELISPOT). Results Based on TST conversion and the specific immune responses to M.tb antigens, all animals were successfully infected. Half of the animals developed active infection and died within 15 months postinfection. The other four animals were grouped with latent M.tb infection because of positive TST but few clinical signs and pathological changes of TB during the course of this study. Interestingly, a challenge with a large dose of M.tb also induced latent infection. Similar to the changes that occur with human TB patients, the animals with active infection exhibited weight loss, cough and typical TB pathological changes, including caseous granulomas, cavities, consolidation, lipid pneumonia, pleural effusion, lymphadenopathy and bacterial burden in lungs and other organs. Conclusions The low dose of M.tb was sufficient to cause both active and latent M.tb infection in cynomolgus macaques of Chinese origin.

OL-061 A subunit vaccine consisting of fusion protein Ag85B-MPT64190-198-Mtb8.4 and adjuvant of dimethyl-dioctyldecyl ammonium and BCG polysaccharide nucleic acid enhances BCG-primed protection against Mycobacterium tuberculosis infection in mice

rates of IGF-II P3 in the adjacent tissues in poorly differentiated HCC were lower than well-differentiated ones (P=0.000); At the same time, the methylation rates of IGF-II P3 in HBsAg-positive HCC was significantly lower than those with HBsAg-negative (P=0.026). Conclusions: The methylation status of IGF-II promoter is closely related to the malignant transformation of hepatocytes at the early stage of HCC, and helpful to explore the pathogenesis of HCC.