Amplifying mRNA vaccines: potential versatile magicians for oncotherapy.

Cancer vaccines drive the activation and proliferation of tumor-reactive immune cells, thereby eliciting tumor-specific immunity that kills tumor cells. Accordingly, they possess immense potential in cancer treatment. However, such vaccines are also faced with challenges related to their design and considerable differences among individual tumors. The success of messenger RNA (mRNA) vaccines against coronavirus disease 2019 has prompted the application of mRNA vaccine technology platforms to the field of oncotherapy. These platforms include linear, circular, and amplifying mRNA vaccines. In particular, amplifying mRNA vaccines are characterized by high-level and prolonged antigen gene expression at low doses. They can also stimulate specific cellular immunity, making them highly promising in cancer vaccine research. In this review, we summarize the research progress in amplifying mRNA vaccines and provide an outlook of their prospects and future directions in oncotherapy.

Non-small cell lung cancers (NSCLCs) oncolysis using coxsackievirus B5 and synergistic DNA-damage response inhibitors.

With the continuous in-depth study of the interaction mechanism between viruses and hosts, the virus has become a promising tool in cancer treatment. In fact, many oncolytic viruses with selectivity and effectiveness have been used in cancer therapy. Human enterovirus is one of the most convenient sources to generate oncolytic viruses, however, the high seroprevalence of some enteroviruses limits its application which urges to exploit more oncolytic enteroviruses. In this study, coxsackievirus B5/Faulkner (CV-B5/F) was screened for its potential oncolytic effect against non-small cell lung cancers (NSCLCs) through inducing apoptosis and autophagy. For refractory NSCLCs, DNA-dependent protein kinase (DNA-PK) or ataxia telangiectasia mutated protein (ATM) inhibitors can synergize with CV-B5/F to promote refractory cell death. Here, we showed that viral infection triggered endoplasmic reticulum (ER) stress-related pro-apoptosis and autophagy signals, whereas repair for double-stranded DNA breaks (DSBs) contributed to cell survival which can be antagonized by inhibitor-induced cell death, manifesting exacerbated DSBs, apoptosis, and autophagy. Mechanistically, PERK pathway was activated by the combination of CV-B5/F and inhibitor, and the irreversible ER stress-induced exacerbated cell death. Furthermore, the degradation of activated STING by ERphagy promoted viral replication. Meanwhile, no treatment-related deaths due to CV-B5/F and/or inhibitors occurred. Conclusively, our study identifies an oncolytic CV-B5/F and the synergistic effects of inhibitors of DNA-PK or ATM, which is a potential therapy for NSCLCs.

A Short 5'triphosphate RNA nCoV-L Induces a Broad-Spectrum Antiviral Response by Activating RIG-I.

Small molecular nucleic acid drugs produce antiviral effects by activating pattern recognition receptors (PRRs). In this study, a small molecular nucleotide containing 5'triphosphoric acid (5'PPP) and possessing a double-stranded structure was designed and named nCoV-L. nCoV-L was found to specifically activate RIG-I, induce interferon responses, and inhibit duplication of four RNA viruses (Human enterovirus 71, Human poliovirus 1, Human coxsackievirus B5 and Influenza A virus) in cells. In vivo, nCoV-L quickly induced interferon responses and protected BALB/c suckling mice from a lethal dose of the enterovirus 71. Additionally, prophylactic administration of nCoV-L was found to reduce mouse death and relieve morbidity symptoms in a K18-hACE2 mouse lethal model of SARS-CoV-2. In summary, these findings indicate that nCoV-L activates RIG-I and quickly induces effective antiviral signals. Thus, it has potential as a broad-spectrum antiviral drug.

B.1.351 SARS-CoV-2 Variant Exhibits Higher Virulence but Less Viral Shedding than That of the Ancestral Strain in Young Nonhuman Primates.

We investigated the distribution, virulence, and pathogenic characteristics of mutated SARS-CoV-2 to clarify the association between virulence and the viral spreading ability of current and future circulating strains. Chinese rhesus macaques were infected with ancestral SARS-CoV-2 strain GD108 and Beta variant B.1.351 (B.1.351) and assessed for clinical signs, viral distribution, pathological changes, and pulmonary inflammation. We found that GD108 replicated more efficiently in the upper respiratory tract, whereas B.1.351 replicated more efficiently in the lower respiratory tract and lung tissue, implying a reduced viral shedding and spreading ability of B.1.351 compared with that of GD108. Importantly, B.1.351 caused more severe lung injury and dramatically elevated the level of inflammatory cytokines compared with those observed after infection with GD108. Moreover, both B.1.351 and GD108 induced spike-specific T-cell responses at an early stage of infection, with higher levels of interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) in the B.1.351 group and higher levels of interleukin 17 (IL-17) in the GD108 group, indicating a divergent pattern in the T-cell-mediated inflammatory "cytokine storm." This study provides a basis for exploring the pathogenesis of SARS-CoV-2 variants of concern (VOCs) and establishes an applicable animal model for evaluating the efficacy and safety of vaccines and drugs. IMPORTANCE One of the priorities of the current SARS-CoV-2 vaccine and drug research strategy is to determine the changes in transmission ability, virulence, and pathogenic characteristics of SARS-CoV-2 variants. In addition, nonhuman primates (NHPs) are suitable animal models for the study of the pathogenic characteristics of SARS-CoV-2 and could contribute to the understanding of pathogenicity and transmission mechanisms. As SARS-CoV-2 variants continually emerge and the viral biological characteristics change frequently, the establishment of NHP infection models for different VOCs is urgently needed. In the study, the virulence and tissue distribution of B.1.351 and GD108 were comprehensively studied in NHPs. We concluded that the B.1.351 strain was more virulent but exhibited less viral shedding than the latter. This study provides a basis for determining the pathogenic characteristics of SARS-CoV-2 and establishes an applicable animal model for evaluating the efficacy and safety of vaccines and drugs.

Heterologous immunization with adenovirus vectored and inactivated vaccines effectively protects against SARS-CoV-2 variants in mice and macaques.

To cope with the decline in COVID-19 vaccine-induced immunity caused by emerging SARS-CoV-2 variants, a heterologous immunization regimen using chimpanzee adenovirus vectored vaccine expressing SARS-CoV-2 spike (ChAd-S) and an inactivated vaccine (IV) was tested in mice and non-human primates (NHPs). Heterologous regimen successfully enhanced or at least maintained antibody and T cell responses and effectively protected against SARS-CoV-2 variants in mice and NHPs. An additional heterologous booster in mice further improved and prolonged the spike-specific antibody response and conferred effective neutralizing activity against the Omicron variant. Interestingly, priming with ChAd-S and boosting with IV reduced the lung injury risk caused by T cell over activation in NHPs compared to homologous ChAd-S regimen, meanwhile maintained the flexibility of antibody regulation system to react to virus invasion by upregulating or preserving antibody levels. This study demonstrated the satisfactory compatibility of ChAd-S and IV in prime-boost vaccination in animal models.

Evaluation of the cross-neutralization activities elicited by Coxsackievirus A10 vaccine strains.

Increased severity of diseases caused by Coxsackievirus A10 (CV-A10) as well as a large number of mutants and recombinants circulating in the population are a cause of concern for public health. A vaccine with broad-spectrum and homogenous protective capacity is needed to prevent outbreaks of CV-A10. Here, we evaluated cross-neutralization of prototype strain and 17 CV-A10 strains from related manufacturers in mainland China in vitro using 30 samples of plasma collected from naturally infected human adults and 18 sera samples from murine immunized with the above strains of CV-A10. Both human plasma and murine sera exhibited varying degrees of cross-neutralizing activities. Prototype A/Kowalik and sub-genotype C3/S113 were most difficult to neutralize. Among all strains tested, neutralization of S102 and S108 strains by 18 different sera was the most uniform, suggesting their suitability for detection of NtAb titers of different vaccines for avoiding biases introduced by detection strain. Furthermore, among all immune-sera, cross-neutralization of the 18 strains of CV-A10 by anti-S110 and anti-S102 was the most homogenous. Anti-S102 exhibiting higher geometric mean titer (GMT) in vitro was evaluated for its cross-protection capacity in vivo. Remarkably, administration of anti-S102 protected mice from lethal dosage of eight strains of CV-A10. These results provide a framework for formulating strategies for the R&D of vaccines targeting CV-A10 infections.

Boosting with heterologous vaccines effectively improves protective immune responses of the inactivated SARS-CoV-2 vaccine.

Since the outbreak of COVID-19, a variety of vaccine platforms have been developed. Amongst these, inactivated vaccines have been authorized for emergency use or conditional marketing in many countries. To further enhance the protective immune responses in populations that have completed vaccination regimen, we investigated the immunogenic characteristics of different vaccine platforms and tried homologous or heterologous boost strategy post two doses of inactivated vaccines in a mouse model. Our results showed that the humoral and cellular immune responses induced by different vaccines when administered individually differ significantly. In particular, inactivated vaccines showed relatively lower level of neutralizing antibody and T cell responses, but a higher IgG2a/IgG1 ratio compared with other vaccines. Boosting with either recombinant subunit, adenovirus vectored or mRNA vaccine after two-doses of inactivated vaccine further improved both neutralizing antibody and Spike-specific Th1-type T cell responses compared to boosting with a third dose of inactivated vaccine. Our results provide new ideas for prophylactic inoculation strategy of SARS-CoV-2 vaccines.

Heterologous prime-boost: breaking the protective immune response bottleneck of COVID-19 vaccine candidates.

COVID-19 vaccines emerging from different platforms differ in efficacy, duration of protection, and side effects. To maximize the benefits of vaccination, we explored the utility of employing a heterologous prime-boost strategy in which different combinations of the four types of leading COVID-19 vaccine candidates that are undergoing clinical trials in China were tested in a mouse model. Our results showed that sequential immunization with adenovirus vectored vaccine followed by inactivated/recombinant subunit/mRNA vaccine administration specifically increased levels of neutralizing antibodies and promoted the modulation of antibody responses to predominantly neutralizing antibodies. Moreover, a heterologous prime-boost regimen with an adenovirus vector vaccine also improved Th1-biased T cell responses. Our results provide new ideas for the development and application of COVID-19 vaccines to control the SARS-CoV-2 pandemic.

Development of a pseudovirus-based assay for measuring neutralizing antibodies against Coxsackievirus A10.

Coxsackievirus A10 (CV-A10) has recently emerged as a major pathogen of hand, foot, and mouth disease in children worldwide. Currently no effective treatments are available; development of anti-CV-A10 vaccine is a most cost-effective way for CV-A10 prevention. Robust assay to measure neutralizing antibody (NtAb) titres elicited by vaccination would greatly prompt anti-CV-A10 vaccine development. Compare to the traditional neutralization assay based on inhibition of cytopathic effects (herein after referred to as cNT) which is time-consuming and labor-intensive, in this study we developed an efficient high-throughput neutralization antibody assay based on CV-A10 pseudoviruses (herein after referred to as pNT). In the pNT, anti-CV-A10 NtAb titre was negatively corresponded with the relative luminescent unit (RLU) produced by luciferase reporter gene incorporated in pseudovirus genome. As described in this study, the NtAb against CV-A10 could be detected within 10-16 h, anti- CV-A10 NtAb in 67 human serum samples were measured in parallel with pNT and cNT assays, a good correlation (r = 0.83,p < .0001) and good agreement(97%) were shown between cNT and pNT, indicating that the pNT provides a rapid and convenient procedure for measuring NtAb production against anti-CV-A10 NtAb measurement.

Short-Fragment DNA Residue from Vaccine Purification Processes Promotes Immune Response to the New Inactivated EV71 Vaccine by Upregulating TLR9 mRNA.

To reduce potential oncogenic long genomic DNA in vaccines, nuclease treatment has been applied in the purification processes. However, this action increased the residue of short-fragment DNA and its effect on vaccine potency was still elusive. In this study, we found residual sf-DNA in an inactivated EV71 vaccine could enhance humoral immune response in mice. Ag stimulation in vitro and vaccine injection in vivo revealed that TLR9 transcription level was elevated, indicating that sf-DNA could activate TLR9. These new findings will help us to understand the molecular mechanism induced by vero-cell culture-derived vaccines.

Genetic polymorphisms of CXCR5 and CXCL13 are associated with non-responsiveness to the hepatitis B vaccine.

A cohort based study has been undertaken to investigate the possible association of genetic polymorphisms in genes functionally related to follicular T helper (TfH) cells with non-responsiveness to hepatitis B virus (HBV) vaccination. A total of 24 single nucleotide polymorphisms (SNPs) in 6 TfH related genes (CXCR5, ICOS, CXCL13, IL-21, BCL6 and CD40L) were investigated in 20 non-responders and 45 responders to HBV vaccination. Genetic association analysis revealed that three SNPs (rs497916, rs3922, rs676925) in CXCR5 and one SNP (rs355687) in CXCL13 were associated with hepatitis B vaccine efficacy. In addition, significantly unbalanced distributions of two haplotypes, defined by three SNPs (rs497916, rs3922, rs676925) within CXCR5, were also seen between non-responders and responders. Furthermore, we demonstrated that the rs3922 "GG" genotype was associated with higher levels of CXCR5 than the "AG" and "AA" genotype in a group of healthy volunteers. A dual luciferase report assay was used to confirm that the "G" allele in rs3922 may lead to higher gene expression than the "A" allele, implicating that rs3922 might be a functional SNP affecting CXCR5 expression. These results indicated that polymorphism associated changes in CXCR5 expression in TfH cells may be associated with non-responsiveness to hepatitis B vaccination.

Quality control and evaluation of vaccines in China.

I was born in Luoyang in 1963, a city in China with thousands of years' history. The city is the eastern starting point of the Silk Road (BC 206-260 AD), a famous and long trade route in human history. Living in this ancient city, I grew up under the influence and impact of Chinese traditional culture. When I was a teenager, I once read a book "Yellow Emperor's Inner Canon" (Huang Di Nei Jing, written about 5th century BC), which is the oldest extant classic of traditional Chinese medicine. I was immediately attracted by an idea in it "prevention is better than cure," which is mean that the highest level of medical treatment is not to cure a disease, but to prevent the occurrence of diseases. I think even in the modern society, this is still the highest realm of medical treatment. Inspired by this view, I developed a strong interest in preventive medicine and majored in it during my college years. With an increasingly deeper understanding of preventive medicine, I gradually realized the importance of epidemiology and vaccinology in preventing infectious diseases. Fortunately, in 1991 I entered the group of professor Zhuang Hui in Peking University Health Science Center for a doctorate degree in epidemiology. Professor Zhuang who is an academician of the Chinese Academy of Engineering not only taught me how to do research, but showed me how to act like a scientist. I benefited greatly from his rigorous attitudes toward life and research. During this period, I focused on transmission routes of hepatitis C virus which made me increasingly recognized the great harm of hepatitis in China. It is well known that more than three-quarters of all liver cancer cases are thought to be attributable to hepatitis B or C. In China about 110 000 people die from liver cancer each year, accounting 45% of the total number of deaths caused by liver cancer worldwide.

The cross-neutralizing activity of enterovirus 71 subgenotype c4 vaccines in healthy chinese infants and children.

BACKGROUND: EV71 is one of major etiologic causes of hand-foot-mouth disease (HFMD) and leads to severe neurological complications in young children and infants. Recently inactivated EV71 vaccines have been developed by five manufactures and clinically show good safety and immunogenicity. However, the cross-neutralizing activity of these vaccines remains unclear, and is of particular interest because RNA recombination is seen more frequently in EV71 epidemics. METHODOLOGY/PRINCIPAL FINDINGS: In this post-hoc study, sera from a subset of 119 infants and children in two clinical trials of EV71 subgenotype C4 vaccines (ClinicalTrials.gov Identifier: NCT01313715 and NCT01273246), were detected for neutralizing antibody (NTAb) titres with sera from infected patients as controls. Cytopathogenic effect method was employed to test NTAb against EV71 subgenotype B4, B5, C2, C4 and C5, which were prominent epidemic strains worldwide over the past decade. To validate the accuracy of the results, ELISpot assay was employed in parallel to detect NTAb in all the post-vaccine sera. After two-dose vaccination, 49 out of 53 participants in initially seronegative group and 52 out of 53 participants in initially seropositive group showed less than 4-fold differences in NTAb titers against five EV71 strains, whereas corresponding values among sera from pediatric patients recovering from EV71-induced HFMD and subclinically infected participants were 8/8 and 41/43, respectively. The geometric mean titers of participants against five subgenotypes EV71 all grew significantly after vaccinations, irrespective of the baseline NTAb titer. The relative fold increase in antibody titers (NTAb-FI) against B4, B5, C2, and C5 displayed a positive correlation to the NTAb-FI against C4. CONCLUSIONS/SIGNIFICANCE: The results demonstrated broad cross-neutralizing activity induced by two C4 EV71 vaccines in healthy Chinese infants and children. However, the degree of induced cross-protective immunity, and the potential escape evolution for EV71 still need to be monitored and researched in future for these new vaccines.

Development and evaluation of a pseudovirus-luciferase assay for rapid and quantitative detection of neutralizing antibodies against enterovirus 71.

The level of neutralizing antibodies (NtAb) induced by vaccine inoculation is an important endpoint to evaluate the efficacy of EV71 vaccine. In order to evaluate the efficacy of EV71 vaccine, here, we reported the development of a novel pseudovirus system expression firefly luciferase (PVLA) for the quantitative measurement of NtAb. We first evaluated and validated the sensitivity and specificity of the PVLA method. A total of 326 serum samples from an epidemiological survey and 144 serum specimens from 3 clinical trials of EV71 vaccines were used, and the level of each specimen's neutralizing antibodies (NtAb) was measured in parallel using both the conventional CPE-based and PVLA-based assay. Against the standard neutralization assay based on the inhibition of the cytopathic effect (CPE), the sensitivity and specificity of the PVLA method are 98% and 96%, respectively. Then, we tested the potential interference of NtAb against hepatitis A virus, Polio-I, Polio-II, and Polio-III standard antisera (WHO) and goat anti-G10/CA16 serum, the PVLA based assay showed no cross-reactivity with NtAb against other specific sera. Importantly, unlike CPE based method, no live replication-competent EV71 is used during the measurement. Taken together, PVLA is a rapid and specific assay with higher sensitivity and accuracy. It could serve as a valuable tool in assessing the efficacy of EV71 vaccines in clinical trials and disease surveillance in epidemiology studies.

Molecular determinants of enterovirus 71 viral entry: cleft around GLN-172 on VP1 protein interacts with variable region on scavenge receptor B 2.

Enterovirus 71 (EV71) is one of the major pathogens that cause hand, foot, and mouth disease outbreaks in young children in the Asia-Pacific region in recent years. Human scavenger receptor class B 2 (SCARB2) is the main cellular receptor for EV71 on target cells. The requirements of the EV71-SCARB2 interaction have not been fully characterized, and it has not been determined whether SCARB2 serves as an uncoating receptor for EV71. Here we compared the efficiency of the receptor from different species including human, horseshoe bat, mouse, and hamster and demonstrated that the residues between 144 and 151 are critical for SCARB2 binding to viral capsid protein VP1 of EV71 and seven residues from the human receptor could convert murine SCARB2, an otherwise inefficient receptor, to an efficient receptor for EV71 viral infection. We also identified that EV71 binds to SCARB2 via a canyon of VP1 around residue Gln-172. Soluble SCARB2 could convert the EV71 virions from 160 S to 135 S particles, indicating that SCARB2 is an uncoating receptor of the virus. The uncoating efficiency of SCARB2 significantly increased in an acidic environment (pH 5.6). These studies elucidated the viral capsid and receptor determinants of enterovirus 71 infection and revealed a possible target for antiviral interventions.

Antigenicity, animal protective effect and genetic characteristics of candidate vaccine strains of enterovirus 71.

Enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease. Recently, several EV71 inactivated vaccine candidates have been development in mainland China. To compare the antigenicity and genetic characteristics of different candidate vaccine strains for EV71, the genomes of 11 EV71 strains were sequenced. The cross-protection capacities were determined by neutralizing antibody test and were further assessed in neonatal mice. The results showed 93.0%-99.8% genome homology of the VP1 regions, but their cross-neutralization capacities varied. These results showed that vaccine strains with good cross-neutralization capacities had the potential to be vaccine strains.

[Evaluation on the analytical sensitivity of 31 HBsAg enzyme immunoassay kits].

OBJECTIVE: To study the analytical sensitivity on 31 HBsAg enzyme immunoassy (EIA) test kits. METHODS: Thirty one HBsAg EIA kits produced by domestic or overseas manufactories and applied for approval during May 2007 to May 2008, were evaluated using the national reference panels. The hyperbolic curve of the log A value and log concentration for the national sensitivity standards was established. The cut-off value of each kit was substituted into the curvilinear equation to determine the analytical sensitivity which was compared between different HBsAg EIA kits. RESULTS: Twenty seven (351 lots) domestic and 4 (27 lots) overseas kits were compared. Among 378 lots of the 31 HBsAg EIA kits, only 2 lots of the domestic kits had a lower sensitivity when tested with the national HBsAg reference panels, with an average approvalr ate of 99.43% (349/351). The mean analytical sensitivity of the domestic kits for adr, adw, ay serotypes were 0.307, 0.419, 0.513 ng/ml, respectively. There was a significant difference between serotypes (F = 97.30, P < 0.01). The mean analytical sensitivity of the overseas kits for adr, adw, ay serotypes were 0.054, 0.066, 0.050 ng/ml respectively, with no significant difference between serotypes (F = 0.65, P > 0.05). The analytical sensitivity of the overseas kits for all the three serotypes was higher than that of the domestic kits (P < 0.01). There was no significant difference found between the analytical sensitivities of the kits produced by the same manufactory using 30- or 60-minute incubation of detection (P > 0.05). In contrast, there was significant difference noticed between the analytical sensitivities of the kits produced by the same manufactory when tested for 10 or 15-minute coloration of the results (P < 0.01). CONCLUSION: Analytical sensitivity of the HBsAg EIA domestic kits should be further improved, especially for detecting adw and ay serotypes.

[Comparison of the kinesis of immune responses in mice vaccinated by different kinds of recombinant hepatitis B vaccines].

OBJECTIVE: To evaluate the kinesis of cellular and humoral immune responses to different kinds of recombinant hepatitis B(rHB) vaccines in the immunized mice. METHODS: At serial time points, the levels of IFN-gamma and IL-2 secreted by spleens mononuclear cells (MNC) of the vaccinated mice were detected by enzyme-linked immunospot methods (ELISPOT) after stimulation in vitro with HBsAg MHC class I peptide S28-39 or HBsAg. The lymphocytotoxicity of the immunized mice were also detected (CTL) by a specific lysis assay and the levels of anti-HBs were measured by the Abbott IMX kit. RESULTS: The peak values of IFN-gamma and IL-2 in vaccinated mice were detected by ELISPOT, 10 - 14 days after immunization. The CTL and the level of IFN-gamma induced by rHB vaccine derived from yeast cells (Hansenula polymorpha) (rHP vaccine) were significantly higher than the other two vaccines (P < 0.05). The maximum lysis of CTL appeared in the vaccinated mice on day 10 after immunization, with the percentage of 39.8%. The levels of IL-2 induced by rHP vaccine were significantly higher than the other two vaccines (P < 0.05). However, the IL-2 levels in the rSC (saccharomyces cerevisiae) vaccine group were higher as compared with the rCHO vaccine group at day 7 and day 14 (7 d t = 4.595, P = 0.001 < 0.05; 14 d t = 5.721, P = 0.000 < 0.05) after immunization. The cellular immune response to the rHP vaccine was the strongest while it was the lowest to the rCHO vaccine at day 7 after immunization. The sero-positive rates and the titers of anti-HBs in the vaccinated mice increased with time after vaccination. The titers of anti-HBs in the rCHO vaccine group at day 7 were similar to the rSC vaccine group, but significantly higher than that of the rHP vaccine group (P = 0.044 < 0.05). The anti-HBs titers of the rCHO vaccine group at day 14 were significantly higher as compared to the rSC (P = 0.012 < 0.05) and rHP (P = 0.009 < 0.05) vaccine groups. CONCLUSION: The immune responses induced by the three kinds of rHB vaccines were different in their patterns and levels. According to the intensity of early cellular immune response, the two yeast HB vaccines were superior to the rCHO vaccine, especially to the rHP vaccine. In contrast, the rCHO vaccine induced early seroconversion and high levels of anti-HBs.

A novel immunostimulator, N-[alpha-O-benzyl-N-(acetylmuramyl)-L-alanyl-D-isoglutaminyl]-N6-trans-(m-nitrocinnamoyl)-L-lysine, and its adjuvancy on the hepatitis B surface antigen.

N(2)-[alpha-O-benzyl-N-(acetylmuramyl)-L-alanyl-D-isoglutaminyl]-N(6)-trans-(m-nitrocinnamoyl)-L-lysine (muramyl dipeptide C, or MDP-C) has been synthesized as a novel, nonspecific immunomodulator. The present study shows that MDP-C induces strong cytolytic activity by macrophages on P388 leukemia cells and cytotoxic activity by cytotoxic T lymphocytes (CTLs) on P815 mastocytoma cells. Our results also indicate that MDP-C is an effective stimulator for production of interleukin-2 and interleukin-12 by murine bone marrow derived dendritic cells (BMDCs) and production of interferon-gamma by CTLs. Additionally, MDP-C increases the expression levels of several surface molecules, including CD11c, MHC class I, and intercellular adhesion molecule-1 in BMDCs. Moreover, MDP-C remarkably enhances the immune system's responsiveness to hepatitis B surface antigen (HBsAg) in hepatitis B virus transgenic mice for both antibody production and specific HBsAg T-cell responses ex vivo. Our results indicate that MDP-C is an apyrogenic, nonallergenic, and low-toxicity immunostimulator with great potential for diagnostic, immunotherapeutic, and prophylactic applications in diseases such as hepatitis B and cancers.