Comparison of Repellency Effect of Mosquito Repellents for DEET, Citronella, and Fennel Oil

To confirm that Korean Food and Drug Administration (KFDA) guidelines are applicable to test the efficacy of mosquito repellents, these guidelines were used to test the efficacy and complete protection times (CPTs) of three representative mosquito repellents: N,N-diethyl-3-methylbenzamide (DEET), citronella, and fennel oil. The repellency of citronella oil decreased over time, from 97.9% at 0 h to 71.4% at 1 h and 57.7% at 2 h, as did the repellency of fennel oil, from 88.6% at 0 h to 61.2% at 1 h and 47.4% at 2 h. In contrast, the repellency of DEET remained over 90% for 6 h. The CPT of DEET (360 min) was much longer than the CPTs of citronella (10.5 min) and fennel oil (8.4 min). These results did not differ significantly from previous findings, and hence confirm that the KFDA guidelines are applicable for testing the efficacy of mosquito repellents

Detection of Pig Cells Harboring Porcine Endogenous Retroviruses in Non-Human Primate Bladder After Renal Xenotransplantation

Pigs are used as potential donor animals for xenotransplantation. However, porcine endogenous retrovirus (PERV), shown to infect both human and non-human primate (NHP) cells in vitro, presents a risk of transmission to humans in xenotransplantation. In this study, we analyzed PERV transmission in various organs after pig-to-NHP xenotransplantation. We utilized pig-to-NHP xenotransplant tissue samples obtained using two types of transgenic pigs from the National Institute of Animal Science (NIAS, Republic of Korea), and examined them for the existence of PERV genes in different organs via PCR and RT-PCR with specific primers. To determine PERV insertion into chromosomes, inverse PCR using PERV long terminal repeat (LTR) region-specific primers was conducted. The PERV gene was not detected in NHP organs in cardiac xenotransplantation but detected in NHP bladders in renal xenotransplantation. The insertion experiment confirmed that PERVs originate from porcine donor cells rather than integrated provirus in the NHP chromosome. We also demonstrate the presence of pig cells in the NHP bladder after renal xenotransplantation using specific-porcine mitochondrial DNA gene PCR. The PERV sequence was detected in the bladder of NHPs after renal xenotransplantation by porcine cell-microchimerism but did not integrate into the NHP chromosome

Immunogenicity of a trivalent human papillomavirus L1 DNA-encapsidated, non-replicable baculovirus nanovaccine.

Previously, we developed a non-replicating recombinant baculovirus coated with human endogenous retrovirus envelope protein (AcHERV) for enhanced cellular delivery of human papillomavirus (HPV) 16L1 DNA. Here, we report the immunogenicity of an AcHERV-based multivalent HPV nanovaccine in which the L1 segments of HPV 16, 18, and 58 genes were inserted into a single baculovirus genome of AcHERV. To test whether gene expression levels were affected by the order of HPV L1 gene insertion, we compared the efficacy of bivalent AcHERV vaccines with the HPV 16L1 gene inserted ahead of the 18L1 gene (AcHERV-HP16/18L1) with that of AcHERV with the HPV 18L1 gene inserted ahead of the 16L1 gene (AcHERV-HP18/16L1). Regardless of the order, the bivalent AcHERV DNA vaccines retained the immunogenicity of monovalent AcHERV-HP16L1 and AcHERV-HP18L1 DNA vaccines. Moreover, the immunogenicity of bivalent AcHERV-HP16/18L1 was not significantly different from that of AcHERV-HP18/16L1. In challenge tests, both bivalent vaccines provided complete protection against HPV 16 and 18 pseudotype viruses. Extending these results, we found that a trivalent AcHERV nanovaccine encoding HPV 16L1, 18L1, and 58L1 genes (AcHERV-HP16/18/58L1) provided high levels of humoral and cellular immunogenicity against all three subtypes. Moreover, mice immunized with the trivalent AcHERV-based nanovaccine were protected from challenge with HPV 16, 18, and 58 pseudotype viruses. These results suggest that trivalent AcHERV-HPV16/18/58L1 could serve as a potential prophylactic baculoviral nanovaccine against concurrent infection with HPV 16, 18, and 58

Induction of HPV type-specific neutralizing antibodies after immunization with AcHERV-based nanovaccines.

<p>Serum was sampled at 2, 4, 6 weeks after intramuscular administration with PBS or AcHERV-based nanovaccines. Neutralization assays were performed with HPV 16 (A) or HPV 18 (B) or HPV 58 (C) pseudoviruses. Data are expressed as the genometric means (log) of reciprocal serum dilutions that yielded a 50% reduction in SEAP. * p<0.05 (ANOVA and Student-Newman-Keuls test): Bivalent and trivalent groups were compared with each monovalent group.</p

Sublingual immunization of trivalent human papillomavirus DNA vaccine in baculovirus nanovector for protection against vaginal challenge.

Here, we report the immunogenicity of a sublingually delivered, trivalent human papillomavirus (HPV) DNA vaccine encapsidated in a human endogenous retrovirus (HERV) envelope-coated, nonreplicable, baculovirus nanovector. The HERV envelope-coated, nonreplicable, baculovirus-based DNA vaccine, encoding HPV16L1, -18L1 and -58L1 (AcHERV-triHPV), was constructed and sublingually administered to mice without adjuvant. Following sublingual (SL) administration, AcHERV-triHPV was absorbed and distributed throughout the body. At 15 minutes and 1 day post-dose, the distribution of AcHERV-triHPV to the lung was higher than that to other tissues. At 30 days post-dose, the levels of AcHERV-triHPV had diminished throughout the body. Six weeks after the first of three doses, 1×10(8) copies of SL AcHERV-triHPV induced HPV type-specific serum IgG and neutralizing antibodies to a degree comparable to that of IM immunization with 1×10(9) copies. AcHERV-triHPV induced HPV type-specific vaginal IgA titers in a dose-dependent manner. SL immunization with 1×10(10) copies of AcHERV-triHPV induced Th1 and Th2 cellular responses comparable to IM immunization with 1×10(9) copies. Molecular imaging revealed that SL AcHERV-triHPV in mice provided complete protection against vaginal challenge with HPV16, HPV18, and HPV58 pseudoviruses. These results support the potential of SL immunization using multivalent DNA vaccine in baculovirus nanovector for induction of mucosal, systemic, and cellular immune responses

Immunogenicity of Virus Like Particle Forming Baculoviral DNA Vaccine against Pandemic Influenza H1N1

<div><p>An outbreak of influenza H1N1 in 2009, representing the first influenza pandemic of the 21st century, was transmitted to over a million individuals and claimed 18,449 lives. The current status in many countries is to prepare influenza vaccine using cell-based or egg-based killed vaccine. However, traditional influenza vaccine platforms have several limitations. To overcome these limitations, many researchers have tried various approaches to develop alternative production platforms. One of the alternative approach, we reported the efficacy of influenza HA vaccination using a baculoviral DNA vaccine (AcHERV-HA). However, the immune response elicited by the AcHERV-HA vaccine, which only targets the HA antigen, was lower than that of the commercial killed vaccine. To overcome the limitations of this previous vaccine, we constructed a human endogenous retrovirus (HERV) envelope-coated, baculovirus-based, virus-like-particle (VLP)–forming DNA vaccine (termed AcHERV-VLP) against pandemic influenza A/California/04/2009 (pH1N1). BALB/c mice immunized with AcHERV-VLP (1×10⁷ FFU AcHERV-VLP, i.m.) and compared with mice immunized with the killed vaccine or mice immunized with AcHERV-HA. As a result, AcHERV-VLP immunization produced a greater humoral immune response and exhibited neutralizing activity with an intrasubgroup H1 strain (PR8), elicited neutralizing antibody production, a high level of interferon-γ secretion in splenocytes, and diminished virus shedding in the lung after challenge with a lethal dose of influenza virus. In conclusion, VLP-forming baculovirus DNA vaccine could be a potential vaccine candidate capable of efficiently delivering DNA to the vaccinee and VLP forming DNA eliciting stronger immunogenicity than egg-based killed vaccines.</p></div

Schematic diagrams of recombinant baculoviruses.

<p>Diagram of the AcHERV-HA baculovirus containing HERV <i>env</i> and HA genes, the AcHERV-VLP virus additionally including NA and M1 genes under the control of the polyhedron promoter and CMV promoter, respectively. The recombinant baculoviruses were generated using the Bac-to-Bac baculovirus expression system.</p

Protective effect of immunization against challenge with a lethal dose of ma-pH1N1.

<p>Body weights of mice intranasally challenged with a 20LD₅₀ dose of ma-pH1N1 2 weeks after the final immunization were monitored for 14 consecutive days. (A) Percent body weight change after challenge with a 20LD₅₀ dose of ma-pH1N1. Changes in body weight (n = 10 mice/group) are expressed as the mean value for each group. (B) Survival rate after challenge with a 20LD₅₀ dose of ma-pH1N1. Statistical analysis performed between the AcHERV-HA group and AcHERV-VLP group. Statistical analysis showed that data were significant with *<i>p</i> < 0.05 (two-tailed Student’s <i>t</i>-test).</p

Transmission of Porcine Endogenous Retrovirus Produced from Different Recipient Cells <i>In Vivo</i>

<div><p>Humanized pigs have been developed to reduce the incidence of immune rejection in xenotransplantation, but significant concerns remain, such as transmission of viral zoonosis. Porcine endogenous retroviruses (PERV), which exist in the genome of pigs, are produced as infectious virions from all porcine cells and cause zoonosis. Here, we examined the possibility of zoonosis of hosts under conditions of immune suppression or xenotransplantation of cells producing host-adapted viruses. Upon transplantation of PERV-producing porcine cells into mice, no transmission of PERV was detected, whereas, transmission of PERV from mice transplanted with mouse-adapted PERV-producing cells was detected. In addition, the frequency of PERV transmission was increased in CsA treated mice transplanted with PERV-producing murine cells, compared with PERV-producing porcine cells. Transmission of PERV to host animals did not affect weight but immune responses, in particular, the number of T cells from PERV-transmitted mice, were notably reduced. The observed risk of PERV zoonosis highlights the requirement for thorough evaluation of viral zoonosis under particular host conditions, such as immunosuppressive treatment and transplantation with host-adapted virus-producing cells.</p></div