Biological evaluation of a phosphate ester prodrug of 10-methyl-aplog-1, a simplified analog of aplysiatoxin, as a possible latency-reversing agent for HIV reactivation.

10-Methyl-aplog-1 (10MA-1), a simplified analog of aplysiatoxin, exhibits a high binding affinity for protein kinase C (PKC) isozymes with minimal tumor-promoting and pro-inflammatory activities. A recent study suggests that 10MA-1 could reactivate latent human immunodeficiency virus (HIV) in vitro for HIV eradication strategy. However, further in vivo studies were abandoned by a dose limit caused by the minimal water solubility of 10MA-1. To overcome this problem, we synthesized a phosphate ester of 10MA-1, 18-O-phospho-10-methyl-aplog-1 (phos-10MA-1), to improve water solubility for in vivo studies. The solubility, PKC binding affinity, and biological activity of phos-10MA-1 were examined in vitro, and the biological activity was comparable with 10MA-1. The pharmacokinetic studies in vivo were also examined, which suggest that further optimization for improving metabolic stability is required in the future.

Development of a novel Macaque-Tropic HIV-1 adapted to cynomolgus macaques.

Macaque-tropic HIV-1 (HIV-1mt) variants have been developed to establish preferable primate models that are advantageous in understanding HIV-1 infection pathogenesis and in assessing the preclinical efficacy of novel prevention/treatment strategies. We previously reported that a CXCR4-tropic HIV-1mt, MN4Rh-3, efficiently replicates in peripheral blood mononuclear cells (PBMCs) of cynomolgus macaques homozygous for TRIMCyp (CMsTC). However, the CMsTC challenged with MN4Rh-3 displayed low viral loads during the acute infection phase and subsequently exhibited short-term viremia. These virological phenotypes in vivo differed from those observed in most HIV-1-infected people. Therefore, further development of the HIV-1mt variant was needed. In this study, we first reconstructed the MN4Rh-3 clone to produce a CCR5-tropic HIV-1mt, AS38. In addition, serial in vivo passages allowed us to produce a highly adapted AS38-derived virus that exhibits high viral loads (up to approximately 106 copies ml-1) during the acute infection phase and prolonged periods of persistent viremia (lasting approximately 16 weeks postinfection) upon infection of CMsTC. Whole-genome sequencing of the viral genomes demonstrated that the emergence of a unique 15-nt deletion within the vif gene was associated with in vivo adaptation. The deletion resulted in a significant increase in Vpr protein expression but did not affect Vif-mediated antagonism of antiretroviral APOBEC3s, suggesting that Vpr is important for HIV-1mt adaptation to CMsTC. In summary, we developed a novel CCR5-tropic HIV-1mt that can induce high peak viral loads and long-term viremia and exhibits increased Vpr expression in CMsTC.

Frequent horizontal and mother-to-child transmission may contribute to high prevalence of STLV-1 infection in Japanese macaques.

BACKGROUND: Simian T-cell leukemia virus type 1 (STLV-1) is disseminated among various non-human primate species and is closely related to human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. Notably, the prevalence of STLV-1 infection in Japanese macaques (JMs) is estimated to be > 60%, much greater than that in other non-human primates; however, the mechanism and mode of STLV-1 transmission remain unknown. The aim of this study is to examine the epidemiological background by which STLV-1 infection is highly prevalent in JMs. RESULTS: The prevalence of STLV-1 in the JMs rearing in our free-range facility reached up to 64% (180/280 JMs) with variation from 55 to 77% among five independent troops. Anti-STLV-1 antibody titers (ABTs) and STLV-1 proviral loads (PVLs) were normally distributed with mean values of 4076 and 0.62%, respectively, which were mostly comparable to those of HTLV-1-infected humans. Our initial hypothesis that some of the macaques might contribute to frequent horizontal STLV-1 transmission as viral super-spreaders was unlikely because of the absence of the macaques exhibiting abnormally high PVLs but poor ABTs. Rather, ABTs and PVLs were statistically correlated (p < 0.0001), indicating that the increasing PVLs led to the greater humoral immune response. Further analyses demonstrated that the STLV-1 prevalence as determined by detection of the proviral DNA was dramatically increased with age; 11%, 31%, and 58% at 0, 1, and 2 years of age, respectively, which was generally consistent with the result of seroprevalence and suggested the frequent incidence of mother-to-child transmission. Moreover, our longitudinal follow-up study indicated that 24 of 28 seronegative JMs during the periods from 2011 to 2012 converted to seropositive (86%) 4 years later; among them, the seroconversion rates of sexually matured (4 years of age and older) macaques and immature macaques (3 years of age and younger) at the beginning of study were comparably high (80% and 89%, respectively), suggesting the frequent incidence of horizontal transmission. CONCLUSIONS: Together with the fact that almost all of the full-adult JMs older than 9 years old were infected with STLV-1, our results of this study demonstrated for the first time that frequent horizontal and mother-to-child transmission may contribute to high prevalence of STLV-1 infection in JMs.

Annual two-dose tetanus toxoid vaccination induces protective humoral immunity to all age groups of rhesus macaques.

A tetanus outbreak occurred during 2014-2015 in the rhesus macaques reared in an open enclosure in our facility. As the soil of the facility was suspected to be contaminated with Clostridium tetani spores, there was a risk of further tetanus occurring among the macaques. To protect them from tetanus, a tetanus toxoid vaccination was recommended; however, the vaccinated elderly animals might not be effectively protected due to insufficient humoral immune responses. Hence, we evaluated the dynamics of antibody responses among rhesus macaques of all age groups vaccinated with two-dose tetanus toxoid at a 1-year interval during a 3-year follow-up study. The vaccination developed anti-tetanus toxin-specific antibodies in animals of all age groups, the antibody levels peaked 1 year after the second vaccination, and the peak levels decreased with age. However, the levels among elderly individuals (aged ≥13 years) were still higher than the threshold level, which was supposed to protect them from tetanus development. Although the rhesus macaques in our facility had a risk of occasional exposure to the spores due to the outbreak, no incidence of tetanus has ever occurred to date. These results indicate that the vaccination protocol is effective in protecting not only younger but also older animals from tetanus.

Safety and immunogenicity of SARS-CoV-2 self-amplifying RNA vaccine expressing an anchored RBD: A randomized, observer-blind phase 1 study.

VLPCOV-01 is a lipid nanoparticle-encapsulated self-amplifying RNA (saRNA) vaccine that expresses a membrane-anchored receptor-binding domain (RBD) derived from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. A phase 1 study of VLPCOV-01 is conducted (jRCT2051210164). Participants who completed two doses of the BNT162b2 mRNA vaccine previously are randomized to receive one intramuscular vaccination of 0.3, 1.0, or 3.0 µg VLPCOV-01, 30 µg BNT162b2, or placebo. No serious adverse events have been reported. VLPCOV-01 induces robust immunoglobulin G (IgG) titers against the RBD protein that are maintained up to 26 weeks in non-elderly participants, with geometric means ranging from 5,037 (95% confidence interval [CI] 1,272-19,940) at 0.3 µg to 12,873 (95% CI 937-17,686) at 3 µg compared with 3,166 (95% CI 1,619-6,191) with 30 µg BNT162b2. Neutralizing antibody titers against all variants of SARS-CoV-2 tested are induced. VLPCOV-01 is immunogenic following low-dose administration. These findings support the potential for saRNA as a vaccine platform.

Elucidation of the role of nucleolin as a cell surface receptor for nucleic acid-based adjuvants.

Nucleic acid-based adjuvants such as CpG oligonucleotides (CpG ODNs) and poly(I:C) are potential vaccine adjuvants for infectious diseases and cancers. However, the mechanism by which their cell surface receptors promote their uptake into dendritic cells (DCs) and shuttle them to intracellular Toll-like receptors remains to be further investigated. Here, we demonstrated a role for nucleolin, a multifunctional DNA- and RNA-binding protein and a major constituent of the nucleolus, as one of the cell-surface receptors for nucleic acid-based adjuvants. Nucleolin on mouse DC surface bound directly to A-type CpG ODN, B-type CpG ODN, and poly(I:C) and promoted their internalization into cells following DC maturation in vitro. In human DCs, nucleolin also contributed to the binding and internalization of both types of CpG ODNs and subsequent cytokine production. Furthermore, nucleolin played a crucial role in cytokine production and activating antigen-specific antibodies and T cell responses induced by B-type CpG ODN in vivo in mice. Our findings provide valuable information that can help improve the efficacy and safety of these adjuvants.

Functional changes in cytotoxic CD8+ T-cell cross-reactivity against the SARS-CoV-2 Omicron variant after mRNA vaccination.

Understanding the T-cell responses involved in inhibiting COVID-19 severity is crucial for developing new therapeutic and vaccine strategies. Here, we characterized SARS-CoV-2 spike-specific CD8+ T cells in vaccinees longitudinally. The BNT162b2 mRNA vaccine can induce spike-specific CD8+ T cells cross-reacting to BA.1, whereas the T-cell receptor (TCR) repertoire usages decreased with time. Furthermore the mRNA vaccine induced spike-specific CD8+ T cells subpopulation expressing Granzyme A (GZMA), Granzyme B (GZMB) and Perforin simultaneously in healthy donors at 4 weeks after the second vaccination. The induced subpopulation was not maintained at 12 weeks after the second vaccination. Incorporating factors that efficiently induce CD8+ T cells with highly cytotoxic activity could improve future vaccine efficacy against such variants.

Neutralization of hepatitis B virus with vaccine-escape mutations by hepatitis B vaccine with large-HBs antigen.

Although the current hepatitis B (HB) vaccine comprising small-HBs antigen (Ag) is potent and safe, attenuated prophylaxis against hepatitis B virus (HBV) with vaccine-escape mutations (VEMs) has been reported. We investigate an HB vaccine consisting of large-HBsAg that overcomes the shortcomings of the current HB vaccine. Yeast-derived large-HBsAg is immunized into rhesus macaques, and the neutralizing activities of the induced antibodies are compared with those of the current HB vaccine. Although the antibodies induced by the current HB vaccine cannot prevent HBV infection with VEMs, the large-HBsAg vaccine-induced antibodies neutralize those infections. The HBV genotypes that exhibited attenuated neutralization via these vaccines are different. Here, we show that the HB vaccine consisting of large-HBsAg is useful to compensate for the shortcomings of the current HB vaccine. The combined use of these HB vaccines may induce antibodies that can neutralize HBV strains with VEMs or multiple HBV genotypes.

The Novel PKC Activator 10-Methyl-Aplog-1 Combined with JQ1 Induced Strong and Synergistic HIV Reactivation with Tolerable Global T Cell Activation.

The presence of latent human immunodeficiency virus (HIV) reservoirs is a major obstacle to a cure. The "shock and kill" therapy is based on the concept that latent reservoirs in HIV carriers with antiretroviral therapy are reactivated by latency-reversing agents (LRAs), followed by elimination due to HIV-associated cell death or killing by virus-specific cytotoxic T lymphocytes. Protein kinase C (PKC) activators are considered robust LRAs as they efficiently reactivate latently infected HIV. However, various adverse events hamper the intervention trial of PKC activators as LRAs. We found in this study that a novel PKC activator, 10-Methyl-aplog-1 (10MA-1), combined with an inhibitor of bromodomain and extra-terminal domain motifs, JQ1, strongly and synergistically reactivated latently infected HIV. Notably, higher concentrations of 10MA-1 alone induced the predominant side effect, i.e., global T cell activation as defined by CD25 expression and pro-inflammatory cytokine production in primary CD4+ T lymphocytes; however, JQ1 efficiently suppressed the 10MA-1-induced side effect in a dose-dependent manner. Considering the reasonable accessibility and availability of 10MA-1 since the chemical synthesis of 10MA-1 requires fewer processes than that of bryostatin 1 or prostratin, our results suggest that the combination of 10MA-1 with JQ1 may be a promising pair of LRAs for the clinical application of the "shock and kill" therapy.

Characterization of the interactions between Escherichia coli receptors, LPS and OmpC, and bacteriophage T4 long tail fibers.

Bacteriophages have strict host specificity and the step of adsorption is one of key factors for determining host specificity. Here, we systematically examined the interaction between the Escherichia coli receptors lipopolysaccharide (LPS) and outer membrane protein C (OmpC), and the long tail fibers of bacteriophage T4. Using a variety of LPS mutants, we demonstrated that T4 has no specificity for the sugar sequence of the outer core (one of three LPS regions) in the presence of OmpC but, in the absence of OmpC, can adsorb to a specific LPS which has only one or two glucose residues without a branch. These results strengthen the idea that T4 adsorbs to E. coli via two distinct modes, OmpC-dependent and OmpC-independent, suggested by previous reports (Prehm et al. 1976; Yu and Mizushima 1982). Isolation and characterization of the T4 mutants Nik (No infection to K-12 strain), Nib (No infection to B strain), and Arl (altered recognition of LPS) identified amino acids of the long tail fiber that play important roles in the interaction with OmpC or LPS, suggesting that the top surface of the distal tip head domain of T4 long tail fibers interacts with LPS and its lateral surface interacts with OmpC.

AbpA and AbpB provide anti-phage activity in Escherichia coli.

Bacteria have a variety of resistance mechanisms for surviving bacteriophage infections. Here, we describe a novel anti-phage mechanism in Escherichia coli. Cells harboring a plasmid with the genes abpA and abpB, formerly yfjL and yfjK, blocked the propagation of bacteriophages belonging to three families: T4, T2, T7 and λ phages. Both genes were necessary for the inhibition of phage propagation, and deletion of either chromosomal gene resulted in a 20% increase of progeny compared to wild-type cells. Neither overexpression nor deficiency of AbpA and AbpB had any apparent effect on E. coli growth. We isolated seven suppressor mutants of T4 phage that grew weakly on cells overexpressing AbpA and AbpB, and found that their mutations were all located in gene 41, which encodes a replicative DNA helicase that is essential for DNA replication. Furthermore, we demonstrated that AbpA and AbpB inhibited DNA replication and late gene expression of T4 phage. Similarly, DNA replication of T7 and λ phages was also inhibited by AbpA and AbpB. These results strongly suggest that E. coli AbpA and AbpB target DNA replication of phages to block their propagation.