Humoral and cellular immunity of two-dose inactivated COVID-19 vaccination in Chinese children: A prospective cohort study.

Children are the high-risk group for COVID-19, and in need of vaccination. However, humoral and cellular immune responses of COVID-19 vaccine remain unclear in vaccinated children. To establish the rational immunization strategy of inactivated COVID-19 vaccine for children, the immunogenicity of either one dose or two doses of the vaccine in children was evaluated. A prospective cohort study of 322 children receiving inactivated COVID-19 vaccine was established in China. The baseline was conducted after 28 days of the first dose, and the follow-up was conducted after 28 days of the second dose. The median titers of receptor binding domain (RBD)-IgG, and neutralizing antibody (NAb) against prototype strain and Omicron variant after the second dose increased significantly compared to those after the first dose (first dose: 70.0, [interquartile range, 30.0-151.0] vs. second dose: 1261.0 [636.0-2060.0] for RBD-IgG; 2.5 [2.5-18.6] vs. 252.0 [138.6-462.1] for NAb against prototype strain; 2.5 [2.5-2.5] vs. 15.0 [7.8-26.5] for NAb against Omicron variant, all p < 0.05). The flow cytometry results showed that the first dose elicited SARS-CoV-2 specific cellular immunity, while the second dose strengthened SARS-CoV-2 specific IL-2+ or TNF-α+  monofunctional, IFN-γ+ TNF-α+  bifunctional, and IFN-γ- IL-2+ TNF-α+ multifunctional CD4+ T cell responses (p < 0.05). Moreover, SARS-CoV-2 specific memory T cells were generated after the first vaccination, including the central memory T cells and effector memory T cells. The present findings provide scientific evidence for the vaccination strategy of the inactive vaccines among children against COVID-19 pandemic.

SARS-CoV-2-specific antibody response characteristics in COVID-19 patients of different ages.

Age has been found to be one of the main risk factors for the severity and outcome of COVID-19. However, differences in SARS-CoV-2 specific antibody responses among COVID-19 patients of different age groups remain largely unknown. In this study, we analyzed the IgG/IgM responses to 21 SARS-CoV-2 proteins and 197 peptides that fully cover the spike protein against 731 sera collected from 731 COVID-19 patients aged from 1 to We show that there is no overall difference in SARS-CoV-2 antibody responses in COVID-19 patients in the 4 age groups. By antibody response landscape maps, we find that the IgG response profiles of SARS-CoV-2 proteins are positively correlated with age. The S protein linear epitope map shows that the immunogenicity of the S-protein peptides is related to peptide sequence, disease severity and age of the COVID-19 patients. Furthermore, the enrichment analysis indicates that low S1 IgG responses are enriched in patients aged <50 and high S1 IgG responses are enriched in mild COVID-19 patients aged >60. In addition, high responses of non-structural/accessory proteins are enriched in severe COVID-19 patients aged >70. These results suggest the distinct immune response of IgG/IgM to each SARS-CoV-2 protein in patients of different age, which may facilitate a deeper understanding of the immune responses in COVID-19 patients.

Systematic evaluation of IgG responses to SARS-CoV-2 spike protein-derived peptides for monitoring COVID-19 patients.

Serological tests play an essential role in monitoring and combating the COVID-19 pandemic. Recombinant spike protein (S protein), especially the S1 protein, is one of the major reagents used for serological tests. However, the high cost of S protein production and possible cross-reactivity with other human coronaviruses pose unavoidable challenges. By taking advantage of a peptide microarray with full spike protein coverage, we analyzed 2,434 sera from 858 COVID-19 patients, 63 asymptomatic patients and 610 controls collected from multiple clinical centers. Based on the results, we identified several S protein-derived 12-mer peptides that have high diagnostic performance. In particular, for monitoring the IgG response, one peptide (aa 1148-1159 or S2-78) exhibited a sensitivity (95.5%, 95% CI 93.7-96.9%) and specificity (96.7%, 95% CI 94.8-98.0%) comparable to those of the S1 protein for the detection of both symptomatic and asymptomatic COVID-19 cases. Furthermore, the diagnostic performance of the S2-78 (aa 1148-1159) IgG was successfully validated by ELISA in an independent sample cohort. A panel of four peptides, S1-93 (aa 553-564), S1-97 (aa 577-588), S1-101 (aa 601-612) and S1-105 (aa 625-636), that likely will avoid potential cross-reactivity with sera from patients infected by other coronaviruses was constructed. The peptides identified in this study may be applied independently or in combination with the S1 protein for accurate, affordable, and accessible COVID-19 diagnosis.

Helicobacter pylori adhesins: HpaA a potential antigen in experimental vaccines for H. pylori.

BACKGROUND: Helicobacter pylori is a gram-negative bacterium involved in many gastric pathologies such as ulcers and cancers. Although the treatment for this infection has existed for several years, the development of a vaccine is nevertheless necessary to reduce the severe forms of the disease. For more than three decades, many advances have been made particularly in the understanding of virulence factors as well as the pathogenesis of gastric diseases caused by H. pylori. Among these key virulence factors, specific antigens have been identified: Urease, Vacuolating cytotoxin A (VacA), Cytotoxin-associated gene A (CagA), Blood group antigen-binding adhesin (BabA), H. pylori adhesin A (HpaA), and others. OBJECTIVES: This review will focus on H. pylori adhesins, in particular, on HpaA and on the current knowledge of H. pylori vaccines. METHODS: All of the information included in this review was retrieved from published studies on H. pylori adhesins in H. pylori infections. RESULTS: These proteins, used in their native or recombinant forms, induce protection against H. pylori in experimental animal models. CONCLUSION: H. pylori adhesins are known to be promising candidate vaccines against H. pylori. Future research should be carried out on adhesins, in particular, on HpaA.

Enhanced tuberculosis clearance through the combination treatment with recombinant adenovirus-mediated granulysin delivery.

Rationale: Tuberculosis (TB) remains the leading cause of death among infectious diseases worldwide. Poor compliance of TB patients to the lengthy treatment increases the risk of relapse and leads to the emergence of multidrug-resistant and extensively drug-resistant TB (MDR-TB and XDR-TB). More effective therapies for TB are urgently needed. We hypothesized that granulysin-mediated clearance of M. tuberculosis parasited inside and outside of alveolar macrophages in presumptive infected hosts might enhance the chemotherapeutic efficacy on TB. Methods: Recombinant adenovirus type 5 (rAd5) based therapeutic vaccines rAdhGLi and rAdhGLs (rAds) were respectively developed to express intracellular and extracellular granulysin. The ex vivo bactericidal effects of rAdhGLi and rAdhGLs were evaluated on U937 and RAW264.7 cells. The efficacy of immunotherapy with both rAdhGLi and rAdhGLs on TB SCID mice, or immunotherapy combined with chemotherapy on drug-susceptible TB or MDR-TB mouse models were further evaluated. Results: rAdhGLs, as well as rAdhGLi, showed a direct bactericidal effect on extracellular or intracellular M. tuberculosis H37Rv and MDR-TB clinical strains, respectively. Immunotherapy with a dose of 109 PFU of rAdhGLi and 109 PFU of rAdhGLs demonstrated a more significant bactericidal effect on M. tuberculosis H37Rv infected SCID mice and prolonged their survival periods than rAdhGLi or rAdhGLs alone. More importantly, chemotherapy combined with rAds immunotherapy shortened the chemotherapeutic duration to 4 months on M. tuberculosis H37Rv infected mice and prevented the relapse. Combination of rAds with chemotherapy on MDR-TB mice also more significantly decreased organ bacterial load than their single use. Conclusions: Delivery of granulysin by recombinant adenovirus to the infected lung could enhance the clearance of TB in vivo and might be a promising adjunct therapeutic vaccine for TB and MDR-TB.

Heterologous Boost Following Mycobacterium bovis BCG Reduces the Late Persistent, Rather Than the Early Stage of Intranasal Tuberculosis Challenge Infection.

Adults are the leading population affected by tuberculosis (TB) epidemic and death. Developing an effective vaccine against adult TB is urgently needed. Mycobacterium bovis Bacillus Calmette-Guerin (BCG) prime-heterologous boost strategy has been explored extensively to protect adults against primary TB infection, but the majority of experimental regimens have not improved the protection primed by the BCG vaccine. The reason attributed to the failure remains unknown. In this study, CTT3H-based vaccines, namely DMT adjuvanted CTT3H subunit or DNA vaccine (pCTT3H-DMT), and recombinant adenovirus rAdCTT3H were constructed. Protective efficacy and immunogenicity of BCG prime-CTT3H based boosters were compared in C57BL/c mice models of primary or late persistent TB infection. Similar protective efficacy against early intranasal infection was provided by different CTT3H-based vaccines alone in vaccinated mice, and their protection was inferior to that of the BCG vaccine. In addition, CTT3H-based heterologous boosters did not enhance the protection conferred by the BCG vaccine against primary infection. However, all of these three boosters provided stronger protection against late persistent TB infection than BCG alone, regardless of vaccine types. Although BCG prime-boosters elicited Th1-biased responses to the antigen CTT3H, the number of CTT3H-sepcific IFN-γ-expressing TEM (CD62LloCD44hi) and IL-2-expressing TCM (CD62LhiCD44hi) cells in the spleen was not improved before exposure to Mycobacterium tuberculosis infection. In contrast, IFN-γ+ TEM and IL-2+ TCM cells in spleens, especially in lungs were significantly increased in BCG prime-boosters after exposure vaccination. Our results indicate that BCG prime-boost strategy might be a promising measure for the prevention against late persistent TB infection by induction of IFN-γ+ TEM and IL-2+ TCM cells in the lung, which can be used as alternative biomarkers for guiding the clinical practice and future development of TB vaccine for adults.

A Multistage Subunit Vaccine Effectively Protects Mice Against Primary Progressive Tuberculosis, Latency and Reactivation.

Adult tuberculosis (TB) is the main cause of TB epidemic and death. The infection results mainly by endogenous reactivation of latent TB infection and secondarily transmitted by exogenous infection. There is no vaccine for adult TB. To this end, we first chose antigens from a potential antigenic reservoir. The antigens strongly recognized T cells from latent and active TB infections that responded to antigens expressed by Mycobacterium tuberculosis cultured under different metabolic states. Fusions of single-stage polyprotein CTT3H, two-stage polyprotein A1D4, and multistage CMFO were constructed. C57BL/6 mice vaccinated with DMT adjuvant ed CMFO (CMFO-DMT) were protected more significantly than by CTT3H-DMT, and efficacy was similar to that of the only licensed vaccine, Bacillus Calmette-Guérin (BCG) and A1D4-DMT in the M. tuberculosis primary infection model. In the setting of BCG priming and latent TB infection, M. tuberculosis in the lung and spleen was eliminated more effectively in mice boosted with CMFO-DMT rather than with BCG, A1D4-DMT, or CTT3H-DMT. In particular, sterile immunity was only conferred by CMFO-DMT, which was associated with expedited homing of interferon-gamma+CD4+ TEM and interleukin-2+ TCM cells from the spleen to the infected lung. CMFO-DMT represents a promising candidate to prevent the occurrence of adult TB through both prophylactic and therapeutic methods, and warrants assessment in preclinical and clinical trials.

Mycobacterium tuberculosis multistage antigens confer comprehensive protection against pre- and post-exposure infections by driving Th1-type T cell immunity.

There is an urgent need for a vaccine against tuberculosis (TB) that is more effective than the current sole licensed option. However, target antigens of Mycobacterium tuberculosis with the vaccine potential remain elusive. Five immunodominant antigens with characteristic expressions at the stages of primary infection (Ag85A), the regulation of nutrition and metabolism when transferring from rapid growth to latency (PhoY2 and Rv3407), latency (Rv2626c), and reactivation (RpfB) were selected to construct the fusion polyprotein WH121, which has better immunogenicity and protection than each multistage antigen. DMT adjuvanted WH121 vaccinated C57BL/6 mice could confer persistent and significant protection against the respiratory challenge with 80 CFU of virulent M. tuberculosis H37Rv at 9 and 18 weeks after immunization, as the BCG vaccine did. Moreover, WH121/DMT could boost the BCG primed mice against post-exposure infection, and more significantly inhibit the growth of M. tuberculosis in the spleen than BCG repeat vaccination. The protection elicited by WH121/DMT is attributed to the WH121-specific Th1-type biased immune responses, characterized by increased antigen-specific IgG2a/IgG1 ratio and high levels of IFN-γ secreted by the splenocytes of vaccinated mice. In particular, high levels of IFN-γ+ TEM cells in the spleen are an effective biomarker for the vaccine-induced early protection, and the persistent protection mainly depends on the increasing IL-2+IFN-γ+CD4+ and CD8+ T cells, especially IL-2+ TCM cells. These findings demonstrate that multistage-specific antigens might be promising targets for the next generation TB vaccine, and a combination of these antigens such as WH121/DMT is required for further preclinical evaluation.

Enhanced and durable protective immune responses induced by a cocktail of recombinant BCG strains expressing antigens of multistage of Mycobacterium tuberculosis.

Although Bacillus Calmette-Guérin (BCG) vaccine confers protection from Mycobacterium tuberculosis infection in children, its immune protection gradually wanes over time, and consequently leads to an inability to prevent the reactivation of latent infection of M. tuberculosis. Therefore, improving BCG for better control of tuberculosis (TB) is urgently needed. We thus hypothesized that recombinant BCG overexpressing immunodominant antigens expressed at different growth stages of M. tuberculosis could provide a more comprehensive protection against primary and latent M. tuberculosis infection. Here, a novel cocktail of recombinant BCG (rBCG) strains, namely ABX, was produced by combining rBCG::85A, rBCG::85B, and rBCG::X, which overexpressed respective multistage antigens Ag85A, Ag85B, and HspX of M. tuberculosis. Our results showed that ABX was able to induce a stronger immune protection than individual rBCGs or BCG against primary TB infection in C57BL/6 mice. Mechanistically, the immune protection was attributed to stronger antigen-specific CD4(+) Th1 responses, higher numbers of IFN-γ(+) CD4(+) TEM and IL-2(+) CD8(+) TCM cells elicited by ABX. These findings thus provide a novel strategy for the improvement of BCG efficacy and potentially a promising prophylactic TB vaccine candidate, warranting further investigation.

Immunogenicity and protective efficacy of DMT liposome-adjuvanted tuberculosis subunit CTT3H vaccine.

Different strategies have been proposed for the development of protein subunit vaccine candidates for tuberculosis (TB), which shows better safety than other types of candidates and the currently used Bacillus Calmette-Guérin (BCG) vaccine. In order to develop more effective protein subunits depending on the mechanism of cell-mediated immunity against TB, a polyprotein CTT3H, based on 5 immunodominant antigens (CFP10, TB10.4, TB8.4, Rv3615c, and HBHA) with CD8(+) epitopes of Mycobacterium tuberculosis, was constructed in this study. We vaccinated C57BL/6 mice with a TB subunit CTT3H protein in an adjuvant of dimethyldioctadecylammonium/monophosphoryl lipid A/trehalose 6,6'-dibehenate (DDA/MPL/TDB, DMT) liposome to investigate the immunogenicity and protective efficacy of this novel vaccine. Our results demonstrated that DMT liposome-adjuvanted CTT3H vaccine not only induced an antigen-specific CD4(+) Th1 response, but also raised the number of PPD- and CTT3H-specific IFN-γ(+) CD8(+) T cells and elicited strong CTL responses against TB10.4, which provided more effective protection against a 60 CFU M. tuberculosis aerosol challenge than PBS control and DMT adjuvant alone. Our findings indicate that DMT-liposome is an effective adjuvant to stimulate CD8(+) T cell responses and the DMT-adjuvanted subunit CTT3H vaccine is a promising candidate for the next generation of TB vaccine.

Protection against Mycobacterium tuberculosis infection offered by a new multistage subunit vaccine correlates with increased number of IFN-γ+ IL-2+ CD4+ and IFN-γ+ CD8+ T cells.

Protein subunit vaccines present a compelling new area of research for control of tuberculosis (TB). Based on the interaction between Mycobacterium tuberculosis and its host, five stage-specific antigens of M. tuberculosis that participate in TB pathogenesis--Rv1813, Rv2660c, Ag85B, Rv2623, and HspX--were selected. These antigens were verified to be recognized by T cells from a total of 42 whole blood samples obtained from active TB patients, patients with latent TB infections (LTBIs), and healthy control donors. The multistage polyprotein A1D4 was developed using the selected five antigens as a potentially more effective novel subunit vaccine. The immunogenicity and protective efficacy of A1D4 emulsified in the adjuvant MTO [monophosphoryl lipid A (MPL), trehalose-6,6'-dibehenate (TDB), components of MF59] was compared with Bacillus Calmette-Guerin (BCG) in C57BL/6 mice. Our results demonstrated that A1D4/MTO could provide more significant protection against M. tuberculosis infection than the PBS control or MTO adjuvant alone judging from the A1D4-specific Th1-type immune response; however, its efficacy was inferior to BCG as demonstrated by the bacterial load in the lung and spleen, and by the pathological changes in the lung. Antigen-specific single IL-2-secreting cells and different combinations with IL-2-secreting CD4+ T cells were beneficial and correlated with BCG vaccine-induced protection against TB. Antigen-specific IFN-γ+ IL-2+ CD4+ T cells were the only effective biomarker significantly induced by A1D4/MTO. Among all groups, A1D4/MTO immunization also conferred the highest number of antigen-specific single IFN-γ+ and IFN-γ+ TNF-α+ CD4+ T cells, which might be related to the antigen load in vivo, and single IFN-γ+ CD8+ T cells by mimicking the immune patterns of LTBIs or curable TB patients. Our strategy seems promising for the development of a TB vaccine based on multistage antigens, and subunit antigen A1D4 suspended in MTO adjuvant warrants preclinical evaluation in animal models of latent infection and may boost BCG vaccination.

Critical role of toll-like receptor 9 in morphine and Mycobacterium tuberculosis-Induced apoptosis in mice.

BACKGROUND: Although it is established that opioid and Mycobacterium tuberculosis are both public health problems, the mechanisms by which they affect lung functions remain elusive. METHODOLOGY/PRINCIPAL FINDINGS: We report here that mice subjected to chronic morphine administration and M. tuberculosis infection exhibited significant apoptosis in the lung in wild type mice as demonstrated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. Morphine and M. tuberculosis significantly induced the expression of Toll-like receptor 9 (TLR9), a key mediator of innate immunity and inflammation. Interestingly, deficiency in TLR9 significantly inhibited the morphine and M. tuberculosis induced apoptosis in the lung. In addition, chronic morphine treatment and M. tuberculosis infection enhanced the levels of cytokines (TNF-alpha, IL-1beta, and IL-6) in wild type mice, but not in TLR9 knockout (KO) mice. The bacterial load was much lower in TLR9 KO mice compared with that in wild type mice following morphine and M. tuberculosis treatment. Morphine alone did not alter the bacterial load in either wild type or TLR9 KO mice. Moreover, administration of morphine and M. tuberculosis decreased the levels of phosphorylation of Akt and GSK3beta in the wild type mice, but not in TLR9 KO mice, suggesting an involvement of Akt/GSK3beta in morphine and M. tuberculosis-mediated TLR9 signaling. Furthermore, administration of morphine and M. tuberculosis caused a dramatic decrease in Bcl-2 level but increase in Bax level in wild type mice, but not in TLR9 KO mice, indicating a role of Bcl-2 family in TLR9-mediated apoptosis in the lung following morphine and M. tuberculosis administration. CONCLUSIONS/SIGNIFICANCE: These data reveal a role for TLR9 in the immune response to opioids during M. tuberculosis infection.

An improved whole-blood gamma interferon assay based on the CFP21-MPT64 fusion protein.

Differentiation of latent tuberculosis infection (LTBI) from a healthy, unexposed population plays a vital role in the strategy of controlling and eliminating tuberculosis (TB). Both CFP21 and MPT64, antigens encoded by the RD2 region which are restricted in the Mycobacterium tuberculosis complex, are TB-specific diagnostic candidate antigens. In this study, we designed a fusion protein by linking both CFP21 and MPT64 with a 15-amino-acid peptide, (G(4)S(1))(3), and overexpressed the fusion protein in Escherichia coli. A new whole-blood gamma interferon assay based on the recombinant fusion protein, CFP21-MPT64 (rCM-WBIA), was developed and compared with the tuberculin skin test (TST) for screening of LTBI in household contacts of patients with sputum-positive TB. rCM-WBIA had a slightly higher sensitivity (66.7%; 24/36 contacts) than that of the TST (61.1%; 22/36 contacts) for household contacts. We found that rCM-WBIA had a very high sensitivity (90.9%) and specificity (71.4%) for LTBI detection compared with TST. The overall agreement between rCM-WBIA and TST was 83.3% (k = 0.64); rCM-WBIA positivity was associated with a larger TST induration. These results suggest that rCM-WBIA, based on the recombinant fusion protein CFP21-MPT64, is a promising alternative diagnostic tool for detection of LTBI.

Differential immunogenicity and protective efficacy of DNA vaccines expressing proteins of Mycobacterium tuberculosis in a mouse model.

BALB/c mice were vaccinated three times (2-week intervals) with plasmid DNA separately encoding antigen Ag85B, ESAT-6 or Ag85A from Mycobacterium tuberculosis. The protective efficacy of these DNA vaccines against intravenous M. tuberculosis H37Rv challenge infection was measured by counting bacterial loads in spleen and lung and recording changes in lung pathology. The splenocyte proliferative response to the corresponding antigens and antigen-specific interferon (IFN)-gamma secreted by splenocytes of the vaccinated mice were also detected. We found a clear hierarchy of protective efficacies among the three DNA vaccines tested in this study. Plasmid DNA encoding Ag85A provided the strongest protection and showed the least change in lung pathology, followed by plasmid DNAs encoding Ag85B and ESAT-6. However, DNA-85B reduced comparative bacterial load in lung tissue, as did DNA-85A. Compared to the control group, protective efficacies conferred by different DNA vaccines were consistent with the lymphoproliferative responses to the corresponding antigens as well as the secretions of antigen-specific IFN-gamma. Our study demonstrates that both Ag85A and Ag85B are the most promising of the candidate antigens tested for future TB vaccine development.

Immunological properties of recombinant Mycobacterium bovis bacillus Calmette-Guérin strain expressing fusion protein IL-2-ESAT-6.

The live vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) provides variable efficacy against adult pulmonary tuberculosis (TB). Recombinant BCG, expressing either immunodominant antigens or Th1 cytokines, is a promising strategy for developing a new TB vaccine. However, not much is known about whether the introduction of cytokine and specific antigen genes concurrently into the BCG strain could improve the immunogenicity of BCG. In this study, a recombinant BCG strain (rBCG) expressing the fusion protein human interleukin (IL)-2 and ESAT-6 (early secreted antigenic target-6 kDa) antigen of Mycobacterium tuberculosis was constructed. Six weeks after BALB/c mice (H-2d) were immunized with 106 colony forming units (CFUs) BCG or rBCG, splenocyte proliferation was determined with MTT [3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] assay, IL-4 and interferon (IFN)-gamma produced by splenocytes were tested by enzyme linked immunosorbent assay (ELISA,) and the cytotoxicity of splenocytes from immunized mice to P815 cells (H-2d) expressing ESAT-6 protein was measured using CytoTox 96 Non-Radioactive Cytotoxicity Assay. Compared with native BCG-vaccinated mice, rBCG induced stronger Th1 responses that were confirmed by high lymphoproliferative responses and IFN-gamma production to culture filtrate protein (CFP) or ESAT-6 protein. Moreover, rBCG induced significant enhanced CTL responses against P815-ESAT-6 cells. Results from rBCG-immunized mice demonstrated that introducing the il-2 and esat-6 genes into BCG could enhance Th1 type immune responses to ESAT-6. Further investigation is needed by introducing other Th1 cytokines and antigens into BCG to optimize the protective efficacy against TB.

Microbubbles assist goat liver ablation by high intensity focused ultrasound.

High intensity focused ultrasound (HIFU) has been introduced to treat cancers. However, this therapy is a time-consuming procedure; destructing a deeper volume is also difficult as ultrasonic energy attenuates exponentially with increasing depth in tissues. The aim of the present study was to investigate the effects of introducing microbubbles on liver HIFU ablation. Seventeen goats were divided into groups A (n=8) and B (n=9). The livers in both groups were ablated using HIFU (1.0 MHz, 22,593 W/cm2) performed in the manner of a clinical regime using a clinical device. A microbubble agent was bolus-injected intravenously before HIFU exposure in group B. All animals in group A and seven goats in group B were euthanased to evaluate the ablation efficiency 24 h after HIFU. The necrosis rate (mm3/s), which was the volume of necrosis tissue per second of HIFU exposure, was used to judge the ablation efficiency. Pathological examinations were performed to determine whether there were residual intact tissues within the exposed volume. The other two goats in group B were used to determine the delayed pathological changes 7 days after ultrasonic ablation. The necrosis rate (mm3/s) was increased in group B (14.4647+/-4.1960 versus 33.5302+/-12.4484, P=0.0059). Pathological examinations confirmed that there were no residual unaffected tissue focuses within the exposed volume. Two remarkable changes occurred in the other two goats in group B 7 days after HIFU: there were ghost-cell islands at the periphery of the ablated tissues, and surrounding adjacent tissues outside the reactive zone necrotized. These findings showed that microbubbles could be used to assist liver HIFU ablation.