Guillermo Posadas-Herrera

Rabies is a fatal encephalitic infectious disease caused by the rabies virus (RABV). RABV is highly neurotropic and replicates in neuronal cell lines in vitro. The RABV fixed strain, HEP-Flury, was produced via passaging in primary chicken embryonic fibroblast cells. HEP-Flury showed rapid adaptation when propagated in mouse neuroblastoma (MNA) cells. In this study, we compared the growth of our previously constructed recombinant HEP (rHEP) strain—based on the sequence of the HEP (HEP-Flury) strain—with that of the original HEP strain. The original HEP strain exhibited higher titer than rHEP and a single substitution at position 80 in the matrix (M) protein M(D80N) after incubation in MNA cells, which was absent in rHEP. In vivo, intracerebral inoculation of the rHEP-M(D80N) strain with this substitution resulted in enhanced viral growth in the mouse brain and a significant loss of body weight in the adult mice. The number of viral antigen-positive cells in the brains of adult mice inoculated with the rHEP-M(D80N) strain was significantly higher than that with the rHEP strain at 5 days post-inoculation. Our findings demonstrate that a single amino acid substitution in the M protein M(D80N) is associated with neurovirulence in mice owing to adaptation to mouse neuronal cells.

The rabies virus is widely distributed and vaccines are an important strategy to prevent its spread. The whole-genome sequences of rabies strains in relation to vaccine development provide essential information to maintain vaccine quality and develop new vaccines. However, the genetic characteristics of the purified chick embryo cell culture rabies vaccine, KM Biologics (PCECV-KMB), developed in Japan in the 1970s, have not been explored. In this study, we conducted a genome-wide analysis of the open reading frame regions of rabies strains discovered from the 1940s–1980s and used to develop chick embryo cell-adapted HEP-Flury small plaque-forming (CEF-S) strain, which is a vaccine strain of PCECV-KMB. The genetic characteristic of CEF-S, developed by acclimation of the HEP-Flury-NIID strain to one-day eggs and subsequently to chick embryo cells, were confirmed by comparing the genome identity and revealing the nine amino acid mutations between CEF-S and HEP-Flury-NIID. The efficacy of PCECV-KMB was evaluated using attack strains isolated in Thailand in the 1960s–1970s during vaccine development. Phylogenetic analyses of the attack strains classified them in the same Asian clade as the 2000s imported cases from the Philippines to Japan, suggesting that PCECV-KMB is adequate for preventing the spread of the current rabies virus.

Musculocontractural Ehlers-Danlos syndrome (mcEDS) is caused by generalized depletion of dermatan sulfate (DS) due to biallelic pathogenic variants in CHST14 encoding dermatan 4-O-sulfotransferase 1 (D4ST1) (mcEDS-CHST14). Here, we generated mouse models for mcEDS-CHST14 carrying homozygous mutations (1 bp deletion or 6 bp insertion/10 bp deletion) in Chst14 through CRISPR/Cas9 genome engineering to overcome perinatal lethality in conventional Chst14-deleted knockout mice. DS depletion was detected in the skeletal muscle of these genome-edited mutant mice, consistent with loss of D4ST1 activity. The mutant mice showed common pathophysiological features, regardless of the variant, including growth impairment and skin fragility. Notably, we identified myopathy-related phenotypes. Muscle histopathology showed variation in fiber size and spread of the muscle interstitium. Decorin localized diffusely in the spread endomysium and perimysium of skeletal muscle, unlike in wild-type mice. The mutant mice showed lower grip strength and decreased exercise capacity compared to wild type, and morphometric evaluation demonstrated thoracic kyphosis in mutant mice. The established CRISPR/Cas9-engineered Chst14 mutant mice could be a useful model to further our understanding of mcEDS pathophysiology and aid in the development of novel treatment strategies.

Middle East respiratory syndrome-coronavirus (MERS-CoV) is an emerging virus that causes severe disease with fatal outcomes; however, there are currently no approved vaccines or specific treatments against MERS-CoV. Here, we developed a novel bivalent vaccine against MERS-CoV and rabies virus (RV) using the replication-incompetent P-gene-deficient RV (RVΔP), which has been previously established as a promising and safe viral vector. MERS-CoV spike glycoprotein comprises S1 and S2 subunits, with the S1 subunit being a primary target of neutralizing antibodies. Recombinant RVΔP, which expresses S1 fused with transmembrane and cytoplasmic domains together with 14 amino acids from the ectodomains of the RV-glycoprotein (RV-G), was developed using a reverse genetics method and named RVΔP-MERS/S1. Following generation of RVΔP-MERS/S1 and RVΔP, our analysis revealed that they shared similar growth properties, with the expression of S1 in RVΔP-MERS/S1-infected cells confirmed by immunofluorescence and western blot, and the immunogenicity and pathogenicity evaluated using mouse infection experiments. We observed no rabies-associated signs or symptoms in mice inoculated with RVΔP-MERS/S1. Moreover, virus-specific neutralizing antibodies against both MERS-CoV and RV were induced in mice inoculated intraperitoneally with RVΔP-MERS/S1. These findings indicate that RVΔP-MERS/S1 is a promising and safe bivalent-vaccine candidate against both MERS-CoV and RV.

Lymphocytic choriomeningitis virus (LCMV) causes a variety of diseases, including asymptomatic infections, meningitis, and congenital infections in the fetus of infected mother. The development of a safe and effective vaccine against LCMV is imperative. This study aims to develop a new candidate vaccine against LCMV using a recombinant replication-incompetent rabies virus (RV) vector. In this study, we have generated a recombinant deficient RV expressing the LCMV glycoprotein precursor (GPC) (RVΔP-LCMV/GPC) which is lacking the RV-P gene. RVΔP-LCMV/GPC is able to propagate only in cells expressing the RV-P protein. In contrast, the LCMV-GPC can be expressed in general cells, which do not express RV-P protein. The ability of RVΔP-LCMV/GPC to protect mice from LCMV infection and induce cellular immunity was assessed. Mice inoculated intraperitoneally with RVΔP-LCMV/GPC showed higher survival rates (88.2%) than those inoculated with the parental recombinant RV-P gene-deficient RV (RVΔP) (7.7%) following a LCMV challenge. Neutralizing antibody (NAb) against LCMV was not induced, even in the sera of surviving mice. CD8+ T-cell depletion significantly reduced the survival rates of RVΔP-LCMV/GPC-inoculated mice after the LCMV challenge. These results suggest that CD8+ T cells play a major role in the observed protection against LCMV. In contrast, NAbs against RV were strongly induced in sera of mice inoculated with either RVΔP-LCMV/GPC or RVΔP. In safety tests, suckling mice inoculated intracerebrally with RVΔP-LCMV/GPC showed no symptoms. These results show RVΔP-LCMV/GPC might be a promising candidate vaccine with dual efficacy, protecting against both RV and LCMV. Lymphocytic choriomeningitis virus (LCMV) causes infections that are often asymptomatic but can be fatal in immunocompromized persons. In addition, LCMV infection during pregnancy can cause spontaneous abortion or severe birth defects. Humans are exposed to LCMV by direct or indirect contact with wild or pet rodents such as mice, hamsters, and guinea pigs. There is no vaccine against LCMV infection. Because of the importance of cellular immunity, inactivated vaccines are not considered effective for protection against LCMV infection. In contrast, protection against rabies, one of the most lethal zoonotic diseases, depends primarily on humoral immunity. In this study, we have developed a recombinant rabies virus (RVΔP-LCMV/GPC) that cannot multiply but expresses LCMV and rabies antigens in the inoculated mice. Hence, we expected that both humoral and cellular immunity would be induced. Most of the mice (88.2%) inoculated with RVΔP-LCMV/GPC survived after a LCMV challenge, whereas only 7.7% of the empty vector inoculated mice survived. Simultaneously, RVΔP-LCMV/GPC induced strong humoral immunity against rabies virus. In conclusion, this study indicates that RVΔP-LCMV/GPC may be useful as a bivalent vaccine against LCMV and RV.

Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in anEscherichia coliexpression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units.