Although recent studies have suggested that the Omicron strain is less severe, the prevalence of long Omicron variants and their subvariant waves continues today. Here, we analyze the pathological characteristics of SARS-CoV-2 variants in cynomolgus macaques. Prolonged re-challenge analysis results in the establishment of re-infection in some macaques with both the same strain and different strains. Omicron infection shows low pathogenicity; however, all macaques that developed pneumonia were inoculated with Omicron strains at the second inoculation. Interestingly, antibodies against the Wuhan, Alpha, and Delta strains are strongly induced regardless of the strain, but antibodies against Omicron strains are not. Moreover, despite the re-infection strain, antibody levels against the Wuhan strain are highest, suggesting original antigenic sin. In addition, Omicron infection induces weaker antigen-specific T-cell responses. These results indicate that immune responses to viral infection differ between the variants, and these differences could inform vaccine development strategies.

Atrial natriuretic peptide (ANP) is a well-established biomarker of cardiac disease; however, its clinical utility in cynomolgus monkeys is limited by its short half-life and the requirement for immediate proteolysis inhibition. This study validated a human NT-proANP assay in cynomolgus monkeys to facilitate the use of archived samples for cardiac assessment. We performed in silico amino acid sequence homology analysis and validated the assay using monkey serum and plasma. Stability under various storage conditions was also evaluated. The amino acid sequence of NT-proANP showed high conservation between humans and cynomolgus monkeys. The human NT-proANP assay demonstrated good cross-reactivity and a significant positive correlation with mature ANP levels (R2=0.60). Furthermore, NT-proANP remained detectable for up to 6 h post-collection at room temperature, although a gradual decline was observed. These findings indicate that NT-proANP is a highly useful surrogate marker for ANP in cynomolgus monkeys. This validation expands the potential for retrospective analysis using stored samples, thereby enhancing the value of non-human primate models in cardiovascular research.

The crab-eating macaque (Macaca fascicularis), a key biomedical model, exhibits substantial inter-individual variability. However, its genomic architecture remains incompletely resolved due to extensive structural complexity and reliance on haploid reference assemblies. Here, we generated 24 diploid telomere-to-telomere (T2T) haplotypes from 12 male and female individuals representing three geographic populations including a pedigree, enabling systematic interrogation of genome architecture at diploid resolution with geographic diversity and familial inheritance. We identified previously uncharacterized large inter-chromosomal repeat clusters with haplotype-specific organization that are conserved among old world monkeys. In addition, twenty-four completely contiguous major histocompatibility complex (MHC) haplotypes reveal extensive inter or intra individual variations in gene copy number and structural organization of the class-IA, IB, and II regions. Together, these findings demonstrate that diploid T2T assemblies are essential for accurately capturing structural and immunogenetic diversity in non-human primates and provide a genomic framework for precision immunogenomics in this widely used biomedical model.

Coinciding with the global outbreak of clade IIb mpox virus (MPXV), the Democratic Republic of the Congo (DRC) recently experienced a rapid surge in mpox cases with clade I MPXV. On 14 August 2024, the World Health Organization declared the continued cross-border spread of this clade in Africa a public health emergency of international concern (PHEIC). Clade I MPXV is known to be more fatal than clade IIb, but its clinical characteristics and prognosis differ between patients. Here, we used mathematical modeling to quantify temporal changes in total lesion counts during clade I MPXV infections, using data from a large cohort of patients with mpox in the DRC from 2007 to 2011. We further analyzed individuals' clinical data to explore predictive biomarkers of high lesion counts. Our analysis indicates that patients with clade I mpox can be stratified into two groups according to lesion severity and that viral load in peripheral blood at symptom onset may serve as a predictor for this classification [area under the curve (AUC) = 0.70]. Our estimates also suggest substantial individual heterogeneity in the time period during which patients have lesions, ranging from 20 to 65 days. Understanding the severity and duration of lesions in different patients, as characterized by our approach, may contribute to more tailored treatment strategies and control measures in ongoing clade I mpox outbreaks.

Therapeutic vaccines against cervical cancer caused by human papillomavirus (HPV) are still an unmet medical need, despite a prophylactic HPV vaccine being available, and the now-licensed systemic vaccines may have a limited effect on the reproductive tract. To specifically inhibit cervical cancer development, the concept of mucosal immunity based on the reproductive-respiratory axis was adopted to develop a nasal HPV therapeutic vaccine. We used a cationic nanogel for the nasal vaccine delivery system and targeted HPV16 E7, an oncoprotein in HPV-driven cervical cancer to demonstrate the feasibility of a nasal therapeutic vaccine. The vaccine was combined with cyclic di-adenosine monophosphate as a cell-mediated immunity-inducing adjuvant. Intranasal immunization with the nanogel vaccine induced E7-specific CD4+ and CD8+ T cells in mouse cervicovaginal tissue. An antitumor effect due to the infiltration of vaccine-induced E7-specific T cells was also observed in an orthotopic tumor model in mice. Furthermore, intranasal immunization of nonhuman primates with the nanogel vaccine using a spray device that is also applicable to humans induced E7-specific T cells in the reproductive tissues. Our findings demonstrated that this nasal therapeutic vaccine effectively controlled cervical cancer and will contribute to preclinical evidence for clinical testing in the near future.

BACKGROUND:The global mpox outbreak (2022-2024) highlights the need for effective and safe vaccines, particularly for vulnerable populations. The LC16m8 vaccine, an attenuated vaccinia virus strain for smallpox, shows promise in inducing immunity against the monkeypox virus (MPXV). METHODS:We conducted a comprehensive immunological evaluation of LC16m8 in mice, non-human primates, and humans. FINDINGS:LC16m8 induced strong humoural responses in BALB/c, C57BL/6J, and CAST/EiJ mice, targeting MPXV H3, A35, and M1R antigens, promoting germinal centre B cells and follicular helper T cells, essential for long-term immunity. Vaccinated CAST/EiJ mice showed reduced lung MPXV viral loads, demonstrating efficacy. In humans, LC16m8 enhanced neutralising antibodies against multiple MPXV clades, suggesting broad protection. In cynomolgus monkeys, systemic administration caused localised pox lesions without significantly affecting weight, temperature, or haematological parameters. INTERPRETATION:This cross-species immunological analysis provides preclinical and early clinical insights into LC16m8's efficacy and safety against mpox. While LC16m8 enhanced antibody responses against MPXV clade Ia and Ib, further studies are required to evaluate its efficacy, particularly in naive and immunocompromised populations. FUNDING:This research was supported by AMED under Grant Numbers JP243fa727002, JP243fa727001s0703, and JP243fa627001h0003 (K.J.I), JP24jf0126002, JP24fk0108690, JP243fa627001h0003, and JP243fa727002 (K.S), JP243fa727002 (Y.T.), JP243fa727002 and JP243fa627007h0003 (Y.Y.), and by the Research Support Project for Life Science and Drug Discovery (BINDS) from AMED under Grant Number JP23ama121011 (J.T.), and JP23ama121010 (T.S.), and by the Ministry of Education, Culture, Sports, Science and Technology in Japan under Grant Number 23K06577 (E.S.). AMED under Grant Number JP233fa827017 and JP243fa827017 (F.Y.), JP22fk0108501 (M.K.).

It cannot be used for HIV-infected children in the setting of BCGosis such as disseminated BCG. On the other hand, it has been reported that BCG is effective against TB in simian AIDS virus infection. We attempted to develop an attenuated BCG vaccine that is as effective as or better than existing BCG and can be used in AIDS virus-infected individuals. SOCS1 plays a key role in the negative regulation of JAK/STAT signaling, which is involved in innate immunity and subsequent adaptive immunity. In this study, we report the vaccine efficacy and safety of this rBCG-SOCS1DN in AIDS virus-infected NHPs. Simian Immunodeficiency virus (SIV) infected cynomolgus macaques were immunized with rBCG-SOCS1DN, and vaccine effects and safety were examined. When comparing the local reactions following intradermal administration of rBCG-SOCS1DN and BCG Tokyo, no significant difference was observed in the local lesions in naive cynomolgus monkeys. However, local lesions were clearly more severe with BCG Tokyo in SIV-infected cynomolgus monkeys. On the other hand, the local lesions showed almost no change, similar to those in naive cynomolgus monkeys with BCG-SOCS1DN.. The vaccine efficacy of rBCG-SOCS1DN was found to be similar to or higher than that of BCG Tokyo to Mtb Erdman strain infection. These results suggest that BCG-SOCS1DN has low side effects with high vaccine efficacy and can be used in AIDS virus infected individuals. Vaccines and Immunotherapy (VAC)

Since the discovery of HTLV-1 over 4 decades ago, no effective vaccines or drugs have been developed to inhibit the infection and the pathogenesis progression. We have established a cynomolgus macaque (CM) model of HTLV-1 infection, which is believed to be essential for revealing the mechanisms of the progressive status and for the development of prevention procedures by direct intravenous inoculation of ATL-040 cells, which are HTLV-1-producing cells derived from ATL patients. This HTLV-1 CM model maintained chronic infection over a long period, and changes to viral load by immune regulation were observed. An antibody, humanized HTLV-1 rat monoclonal antibody (hu-LAT-27), has both neutralization and antibody-dependent cellular cytotoxicity activities. In this study, we evaluated the preventive efficacy and potency of neutralizing monoclonal antibodies on HTLV-1 infection. CMs were administrated either with control IgG or hu-LAT-27 a day before infection and then CMs were intravenously inoculated with ATL-040. Proviral DNAs in peripheral blood and several tissues and organs were detected in control IgG-administrated CMs but not in hu-LAT-27 pre-administrated CMs during the experimental period. These results suggested that a monoclonal Ab, hu-LAT-27, prevents HTLV-1 infection in vivo and has the potential to be a prophylactic drug for HTLV-1 infection. In addition, the usefulness of the HTLV-1 CM model as an evaluation model for pharmaceutical development was demonstrated. Supported by the Japan Agency for Medical Research and Development, Strategic Center of Biomedical Advanced Vaccine Research and Development for Preparedness and Response, and the Japan Society for the Promotion of Science. Translational and Interventional Immunology (TI)

Reports of congenital heart disease in nonhuman primates are rare, and double-chambered right ventricle (DCRV), which is a rare congenital heart disease, in which an abnormal muscle bundle divides the right ventricle into 2 chambers (a proximal high-pressure chamber and a distal low-pressure chamber), has not been previously reported. We diagnosed DCRV antemortem in 2 monkeys bred at The Tsukuba Primate Research Center that presented with cardiac murmurs. Subsequent diagnostic evaluation confirmed DCRV in one case, with the other diagnosed with midventricular obstruction having a pathophysiology similar to DCRV. The monkey that had DCRV exhibited a pathophysiology similar to that in humans with DCRV, while the other monkey had a condition mimicking DCRV which was due to a thrombus. We believe this to be the first report of DCRV and a rare DCRV-like pathophysiology due to a thrombus in the ventricle in nonhuman primates.

We created a novel replication-incompetent vector derived from the human parainfluenza virus type 2 (hPIV2). The vector lacks the hemagglutinin-neuraminidase (HN) gene (hPIV2/∆HN) that is essential for virus attachment and release. We also generated the stable packaging cell line expressing hPIV2 HN protein derived from Vero cells (Vero-HN). As SARS-CoV-2 vaccines, we created hPIV2/∆HN-based SARS-CoV-2 vaccines expressing the receptor binding domain of the spike protein (S-RBD) of SARS-CoV-2, and S-RBD with trimer formation domain (S-RBD-FD) with or without antigen 85B (Ag85B) having strong Th1-type cytokine inducing activity. Intratracheal administration of S-RBD-FD/hPIV2/∆HN and S-RBD-FD/Ag85B/hPIV2/∆HN induced the S-RBD-specific neutralizing antibodies in sera of transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2/TG), and also the sera neutralized the pseudo-typed lentivirus expressing the SARS-CoV-2 S protein. In particular, in an S-RBD-FD/Ag85B/hPIV2/∆HN-administered group, high levels of neutralizing antibodies were induced also in the bronchoalveolar lavage fluid (BALF) in the hACE2/TG mice. Further, hACE2/TG mice vaccinated with S-RBD-FD/hPIV2/∆HN or S-RBD-FD/Ag85B/hPIV2/∆HN did not exhibit weight loss and showed a high survival rate after authentic SARS-CoV-2 challenge. These results suggested that S-RBD-FD/Ag85B/hPIV2/∆HN is a vaccine candidate against SARS-CoV-2. Finally, hPIV2/∆HN or Ag85B/hPIV2/∆HN vector will be potentially applicable to vaccine development and/or human gene therapy for respiratory tract diseases.

Adjuvants are immunostimulators used to enhance vaccine efficacy against infectious diseases. However, current methods for evaluating their efficacy and safety are limited, hindering large-scale screening. To address this, we developed a prototype Adjuvant Database (ADB) containing transcriptome data, generated using the same protocols as the widely used Open TG-GATEs (OTG) toxicogenomics database, covering 25 adjuvants across multiple species, organs, time points, and doses. This enabled cross-database integration of ADB and OTG. Transcriptomic patterns successfully distinguished each adjuvant regardless of organs or species. Using both databases, we built machine learning models to predict adjuvanticity and hepatotoxicity. Notably, we identified colchicine's adjuvant activity and FK565's liver toxicity through data-driven analysis. Overall, ADB combined with OTG offers a framework for transcriptomics-based, data-driven screening of adjuvant candidates.

ABSTRACT The attack and defense of infected cells and cytotoxic CD8 T cells occur in germinal centers in lymphoid tissue in chronic persistent HIV/SIV infection. Latently infected cells, the therapeutic target of HIV infection, accumulate in follicular helper T (Tfh) cells in lymphoid tissue; the impact of HIV-specific follicular CD8 (fCD8) T cells in lymphoid tissue on the latently infected cells remains unknown. We infected 15 cynomolgus macaques with SIVmac239 and examined the contribution of SIV-Gag-specific fCD8 T cells, defined by activation-induced markers (AIMs), to SIV-infected cells. Eight out of the 15 infected macaques served as progressors; a chronic phase combination antiretroviral therapy (cART) model was established for the eight macaques (progressors) with chronic persistent infection status, wherein cART was started in the chronic phase and discontinued after 27 weeks. Seven macaques that naturally controlled the viremia served as natural controllers. The frequency of SIV-Gag-specific fCD8 T cells was inversely correlated with the amount of cell-associated SIV- gag RNA in the Tfh only under cART or in the controllers but not in untreated progressors. scRNA-seq of SIV-Gag-specific fCD8 T cells in various conditions revealed that the gene expression pattern of SIV-Gag-specific fCD8 T cells in the controllers was closer to that of those under cART than the untreated progressors. Comparing the SIV-Gag-specific fCD8 T cells of those under cART to the controllers revealed their more exhausted and immunosenescent nature under cART. Improving the HIV/SIV-specific fCD8 T cells under cART by targeting those pathways might contribute to the development of potential curative strategies. IMPORTANCE We infected cynomolgus macaques with SIVmac239 to establish an SIV-chronically infected cART model. We performed an in-depth characterization of Tfh and fCD8 T cells in three conditions—chronic stage of untreated, cART-treated, and natural controller cynomolgus macaques—by combining tissue section analysis and single-cell analyses of sorted cells. We revealed the inverse relationship between Tfh infection and SIV-Gag-specific fCD8 T cell frequencies as observed in HIV-infected individuals, thereby establishing the cynomolgus macaque as a relevant animal model to study the determinants of HIV/SIV persistence in lymphoid tissue. Additionally, scRNA-seq analysis of SIV-Gag-specific fCD8 T cells revealed an enrichment of exhausted or senescent transcriptomic signatures under cART. These data will provide the basic insights into virus-host CD8 T cell interactions, particularly within the follicular region, during latent HIV infection under ART.

Neuronal ceroid lipofuscinoses (NCLs) are autosomal-recessive fatal neurodegenerative diseases that occur in children and young adults, with symptoms including ataxia, seizures and visual impairment. We report the discovery of cynomolgus macaques carrying the CLN2/TPP1 variant and our analysis of whether the macaques could be a new non-human primate model for NCL type 2 (CLN2) disease. Three cynomolgus macaques presented progressive neuronal clinical symptoms such as limb tremors and gait disturbance after about 2 years of age. Morphological analyses using brain MRI at the endpoint of approximately 3 years of age revealed marked cerebellar and cerebral atrophy of the gray matter, with sulcus dilation, gyrus thinning, and ventricular enlargement. Histopathological analyses of three affected macaques revealed severe neuronal loss and degen-eration in the cerebellar and cerebral cortices, accompanied by glial activation and/or changes in axonal morphology. Neurons observed throughout the central nervous system contained autofluorescent cytoplasmic pigments, which were identified as ceroid-lipofuscin based on staining properties, and the cerebral cortex examined by transmission electron microscopy had curvilinear profiles, the typical ultrastructural pattern of CLN2. These findings are commonly observed in all forms of NCL. DNA sequencing analysis identified a ho-mozygous single-base deletion (c.42delC) of the CLN2/TPP1 gene, resulting in a frameshifted premature stop codon. Immunohistochemical analysis showed that tissue from the affected macaques lacked a detectable signal against TPP1, the product of the CLN2/TPP1 gene. Analysis for transmission of the CLN2/TPP1 mutated gene revealed that 47 (49.5%) and 48 (50.5%) of the 95 individuals genotyped in the CLN2-affected macaque family were heterozygous carriers and homozygous wild-type individuals, respectively. Thus, we identified cynomolgus macaques as a non-human primate model of CLN2 disease. The CLN2 macaques reported here could become a useful resource for research and the development of drugs and methods for treating CLN2 disease, which involves severe symptoms in humans.

The Omicron variant continuously evolves under the humoral immune pressure exerted by vaccination and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and the resulting Omicron subvariants display further immune evasion and antibody escape. An engineered angiotensin-converting enzyme 2 (ACE2) decoy composed of high-affinity ACE2 and an IgG1 Fc domain could offer an alternative modality to neutralize SARS-CoV-2. We previously reported its broad spectrum and therapeutic potential in rodent models. Here, we demonstrate that the engineered ACE2 decoy retains neutralization activity against Omicron subvariants, including the currently emerging XBB and BQ.1 strains, which completely evade antibodies currently in clinical use. SARS-CoV-2, under the suboptimal concentration of neutralizing drugs, generated SARS-CoV-2 mutants escaping wild-type ACE2 decoy and monoclonal antibodies, whereas no escape mutant emerged against the engineered ACE2 decoy. Furthermore, inhalation of aerosolized decoys improved the outcomes of rodents infected with SARS-CoV-2 at a 20-fold lower dose than that of intravenous administration. Last, the engineered ACE2 decoy exhibited therapeutic efficacy for cynomolgus macaques infected with SARS-CoV-2. These results indicate that this engineered ACE2 decoy represents a promising therapeutic strategy to overcome immune-evading SARS-CoV-2 variants and that liquid aerosol inhalation could be considered as a noninvasive approach to enhance the efficacy of COVID-19 treatments.

Background Although vaccination is recommended for protection against invasive pneumococcal disease, the frequency of pneumococcal pneumonia is still high worldwide. In fact, no vaccines are effective for all pneumococcal serotypes. Fusion pneumococcal surface protein A (PspA) has been shown to induce a broad range of cross-reactivity with clinical isolates and afford cross-protection against pneumococcal challenge in mice. Furthermore, we developed prime-boost-type mucosal vaccines that induce both antigen-specific IgG in serum and antigen-specific IgA in targeted mucosal organs in previous studies. We investigated whether our prime-boost-type immunization with a fusion PspA was effective against pneumococcal infection in mice and cynomolgus macaques. Methods C57BL/6 mice were intramuscularly injected with fusion PspA combined with CpG oligodeoxynucleotides and/or curdlan. Six weeks later, PspA was administered intranasally. Blood and bronchoalveolar lavage fluid were collected and antigen-specific IgG and IgA titers were measured. Some mice were given intranasal Streptococcus pneumoniae and the severity of infection was analyzed. Macaques were intramuscularly injected with fusion PspA combined with CpG oligodeoxynucleotides and/or curdlan at week 0 and week 4. Then, 13 or 41 weeks later, PspA was administered intratracheally. Blood and bronchoalveolar lavage fluid were collected and antigen-specific IgG and IgA titers were measured. Some macaques were intranasally administered S. pneumoniae and analyzed for the severity of pneumonia. Results Serum samples from mice and macaques injected with antigens in combination with CpG oligodeoxynucleotides and/or curdlan contained antigen-specific IgG. Bronchial samples contained antigen-specific IgA after the fusion PspA boosting. This immunization regimen effectively prevented S. pneumoniae infection. Conclusions Prime-boost-type immunization with a fusion PspA prevented S. pneumoniae infection in mice and macaques.

The Omicron variant continuously evolves under the humoral immune pressure obtained by vaccination and SARS-CoV-2 infection and the resultant Omicron subvariants exhibit further immune evasion and antibody escape. Engineered ACE2 decoy composed of high-affinity ACE2 and IgG1 Fc domain is an alternative modality to neutralize SARS-CoV-2 and we previously reported its broad spectrum and therapeutic potential in rodent models. Here, we show that engineered ACE2 decoy retains the neutralization activity against Omicron subvariants including the currently emerging XBB and BQ.1 which completely evade antibodies in clinical use. The culture of SARS-CoV-2 under suboptimal concentration of neutralizing drugs generated SARS-CoV-2 mutants escaping wild-type ACE2 decoy and monoclonal antibodies, whereas no escape mutant emerged against engineered ACE2 decoy. As the efficient drug delivery to respiratory tract infection of SARS-CoV-2, inhalation of aerosolized decoy treated mice infected with SARS-CoV-2 at a 20-fold lower dose than the intravenous administration. Finally, engineered ACE2 decoy exhibited the therapeutic efficacy for COVID-19 in cynomolgus macaques. Collectively, these results indicate that engineered ACE2 decoy is the promising therapeutic strategy to overcome immune-evading SARS-CoV-2 variants and that liquid aerosol inhalation can be considered as a non-invasive approach to enhance efficacy in the treatment of COVID-19.

The abnormal activity of PP2A, a dominant member of type 2A serine/threonine protein phosphatase, has been implicated in the development of Alzheimer’s disease (AD) and dementia with Lewy bodies (DLB). PP2A is a holoenzyme, and protein methylation of the catalytic subunit, PP2Ac, alters the complex composition. A decrease in PP2Ac methylation levels has been reported in AD and DLB. Aging is the most common risk factor for AD and DLB, but the relationship between aging and PP2A has not been studied in detail. Cynomolgus monkey show increased phosphorylation levels of tau and α-synuclein with aging. In this study, we investigated the alterations in the PP2A activity regulation with aging in monkey brains from 2 to 43 years of age using fractionated proteins. We found that type 2A protein phosphatase activity decreased with aging in cytoplasmic and nuclear-soluble fractions. PP2Ac methylation level was decreased in cytoplasmic and sarkosyl-insoluble fractions. A principal component analysis using PP2Ac, demethylated PP2Ac and PP2A methylesterase PME-1 levels in cytoplasmic and nuclear-soluble fractions as attributes showed that aged monkeys were in the same cluster. Our results show that brain aging in cynomolgus monkeys is closely related to changes in PP2A methylation.