Serologic response to human papillomavirus 16 among Australian women with high-grade cervical intraepithelial neoplasia

This study evaluated the detection of human papillomavirus (HPV) 16 antibody in HPV 16-associated cervical intraepithelial neoplasia (CIN) in Australian women. Seroreactivity to HPV 16 L1 virus-like particles was assessed in patients with CIN 2 (n= 169) and CIN 3 (n= 229) lesions previously tested for the presence of HPV DNA. Seropositivity was significantly commoner in women with HPV 16 DNA-positive lesions (98/184) than in women with no HPV DNA in the lesion (15/47) or with HPV of types other than 16 in the lesion (43/167) (P= 0.0004). In addition, seropositivity was observed in 33% (55/169) of women with CIN 2 and 46% (106/229) of women with CIN 3, in keeping with the lower fraction of CIN 2 (57/169) than CIN 3 (127/229) biopsies positive for HPV 16 DNA. HPV 16 seropositivity is most common in women with HPV 16-associated CIN, but many patients with HPV-associated CIN 3 are seronegative, and HPV 16 seropositivity is common in women with CIN associated with other HPV types. Overall, HPV 16 serology is a poor predictor of presence of HPV 16-associated CIN 3 in patient population studied.     

Development and characterization of a stable luciferase dengue virus for high-throughput screening

To facilitate dengue virus (DENV) drug discovery, we developed a stable luciferase reporter DENV-2. A renilla luciferase gene was engineered into the capsid-coding region of an infectious cDNA clone of DENV-2. Transfection of BHK-21 cells with the cDNA clone-derived RNA generated high titers (>10⁶ PFU/ml) of luciferase reporter DENV-2. The reporter virus was infectious to a variety of cells, producing robust luciferase signals. Compared with wild-type virus, the reporter virus replicated slower in both mammalian Vero and mosquito C6/36 cells. To examine the stability of the reporter virus, we continuously passaged the virus on Vero cells for five rounds. All passaged viruses stably maintained the luciferase gene, demonstrating the stability of the reporter virus. Furthermore, we found that the passaged virus accumulated a mutation (T108M) in viral NS4B gene that could enhance viral RNA replication in a cell-type specific manner. Using the reporter virus, we developed a HTS assay in a 384-well format. The HTS assay was validated with known DENV inhibitors and showed a robust Z′ factor of 0.79. The Luc-DENV-2 HTS assay allows screening for inhibitors of all steps of the viral life cycle. The reporter virus will also be a useful tool for studying DENV replication and pathogenesis.     

心室晚电位(VLP)阳性与左室射血分数(EF)降低对心肌梗死心性猝死相关性的研究

目的　探讨心室晚电位、左室射血分数在降低心梗性猝死的关系。方法　采用时域定量分析法(SA -ECG) ,首选 12导联心电图、心向量图、测定VLP波叠加多次后清除数 ;测定左室射血分数。结果　EF <4 0 % ,VLP >4 0 %时 ,心猝死发生率极高。结论　VLP与EF在判断心肌梗死性猝死中意义较大     

Immunogenicity of adjuvanted plant-produced SARS-CoV-2 Beta spike VLP vaccine in New Zealand white rabbits

The outbreak of the SARS-CoV-2 global pandemic heightened the pace of vaccine development with various vaccines being approved for human use in a span of 24 months. The SARS-CoV-2 trimeric spike (S) surface glycoprotein, which mediates viral entry by binding to ACE2, is a key target for vaccines and therapeutic antibodies. Plant biopharming is recognized for its scalability, speed, versatility, and low production costs and is an increasingly promising molecular pharming vaccine platform for human health. We developed Nicotiana benthamiana-produced SARS-CoV-2 virus-like particle (VLP) vaccine candidates displaying the S-protein of the Beta (B.1.351) variant of concern (VOC), which triggered cross-reactive neutralising antibodies against Delta (B.1.617.2) and Omicron (B.1.1.529) VOCs. In this study, immunogenicity of the VLPs (5 µg per dose) adjuvanted with three independent adjuvants i.e. oil-in-water based adjuvants SEPIVAC SWE^TM (Seppic, France) and “AS IS” (Afrigen, South Africa) as well as a slow-release synthetic oligodeoxynucleotide (ODN) adjuvant designated NADA (Disease Control Africa, South Africa) were evaluated in New Zealand white rabbits and resulted in robust neutralising antibody responses after booster vaccination, ranging from 1:5341 to as high as 1:18204. Serum neutralising antibodies elicited by the Beta variant VLP vaccine also showed cross-neutralisation against the Delta and Omicron variants with neutralising titres ranging from 1:1702 and 1:971, respectively. Collectively, these data provide support for the development of a plant-produced VLP based candidate vaccine against SARS-CoV-2 based on circulating variants of concern.     

Induction of mucosal and systemic antibody responses against the HIV coreceptor CCR5 upon intramuscular immunization and aerosol delivery of a virus-like particle based vaccine

Virus-like particles (VLPs) can be exploited as platforms to increase the immunogenicity of poorly immunogenic antigens, including self-proteins. We have developed VLP-based vaccines that target two domains of the HIV coreceptor CCR5 that are involved in HIV binding. These vaccines induce anti-CCR5 antibodies that bind to native CCR5 and inhibit SIV infection in vitro. Given the role of mucosal surfaces in HIV transmission and replication, we also asked whether an aerosolized, VLP-based pulmonary vaccine targeting CCR5 could induce a robust mucosal response in addition to a systemic response. In rats, both intramuscular and pulmonary immunization induced high-titer IgG and IgA against the vaccine in the serum, but only aerosol vaccination induced IgA antibodies at local mucosal sites. An intramuscular prime followed by an aerosol boost resulted in strong serum and mucosal antibody responses. These results show that VLP-based vaccines targeting CCR5 induce high-titer systemic antibodies, and can elicit both local and systemic mucosal response when administered via an aerosol. Vaccination against a self-molecule that is critically involved during HIV transmission and pathogenesis is an alternative to targeting the virus itself. More generally, our results provide a general method for inducing broad systemic and mucosal antibody responses using VLP-based immunogens.     

Short version of the German evidence-based Guidelines for prophylactic vaccination against HPV-associated neoplasia

Persistent infection with HPV 16 and 18 has been causally associated with the development of cervical cancer and its precursor lesions as well as with other carcinomas and their precursors, e.g. some vulvar and vaginal cancers. Furthermore HPV 6 and 11 are responsible for anogenital condylomata acuminata in more than 90% of cases. With the recently developed prophylactic bivalent (HPV 16 and 18) and quadrivalent (HPV 6, 11, 16 and 18) vaccines, it is possible to prevent infection of the cervical epithelium and other squamous epithelia, the development of premalignant lesions and, in the case of the quadrivalent vaccine, the development of condylomata acuminata. The following paper represents a summary of the full-text version of the German evidence-based Guidelines, including all evidence-based recommendations regarding the safety as well as the efficacy of the vaccines in preventing CIN, VIN/VaIN, genital warts and other HPV-associated lesions.     

Prevalence of human papillomavirus infection in women in Busan,South Korea

To investigate the prevalence of and the risk factors for human papillomavirus (HPV) infection in South Korea, we interviewed and examined a randomly selected sample of 863 sexually active women (age range = 20-74 years, median 44) and 103 self-reported virgins from Busan. The presence of DNA of 34 different HPV types in cervical exfoliated cells was tested among sexually active women by means of a PCR-based assay. IgG antibodies against L1 virus-like particles (anti-VLPs) of HPV types 16, 18, 31, 33 and 58 were also evaluated by means of ELISA. The overall prevalence of HPV DNA was 10.4% (95% confidence interval, CI: 8.5-12.7%). The most often found HPV DNA types were HPV 70, HPV 16 and HPV 33; 19.8% (95% CI: 17.2-22.0) of sexually active women had antibodies against one or more HPV types. The most common anti-VLPs were against HPV 18, 31 and 16. Prevalences standardized by age on the basis of the world standard population were 13.0% for HPV DNA and 17.1% for anti-VLPs. The concordance between the 2 HPV markers at an individual level was modest, but the risk factors for detection of HPV DNA and anti-VLPs were similar: number of lifetime sexual partners (odds ratio, OR for >/= 4 vs. 1 = 3.5 and 5.4, respectively), seropositivity for herpes simplex virus-2 antibodies (OR = 2.6 and 2.5, respectively) and being single or divorced. HPV DNA, but not anti-VLPs, were elevated among women whose husbands were thought by their wives to have extra-marital affairs and those who had undergone vasectomy. Among 103 virgins, 4.9% had anti-VLPs (1/73 among those aged 24 years or less).     

Co-vaccination with adeno-associated virus vectors encoding human papillomavirus 16 L1 proteins and adenovirus encoding murine GM-CSF can elicit strong and prolonged neutralizing antibody

Non-infectious human papillomavirus-like particles (VLPs), encoded by the major capsid gene L1, have been shown to be effective as vaccines to prevent cervical cancer. We have developed the genetic immunization of the L1 gene to induce a neutralizing antibody. We constructed and generated a recombinant adeno-associated virus encoding human papillomavirus (HPV) 16 L1 protein that could form virus-like particles in transduced cells. Previous reports have demonstrated that the formation of VLP is necessary to induce high titers of neutralizing antibodies to protect an animal from viral challenge. Therefore, we carried out a single intramuscular (i.m.) injection with recombinant adeno-associated virus encoding HPV-16 L1 protein (rAAV-16L1) in BALB/c mice, which ultimately produced stronger and more prolonged neutralizing L1 antibodies, when compared to the DNA vaccine. Immunohistochemistry showed that the accumulation of antigen presenting cells, such as macrophages and dendritic cells, in rAAV-16L1 and L1 DNA-injected muscle fibers may be due to the L1 protein expression, but not to AAV infection. When compared to the L1 VLP vaccine, however, the titers of neutralizing L1 antibodies induced by VLP were higher than those induced by rAAV-16L1. Co-vaccinating with rAAV-16L1 and adenovirus encoding murine GM-CSF (rAAV-16L1/rAd-mGM-CSF) induced comparable higher levels of neutralizing L1 antibodies with those of VLP. This implies that a single i.m. co-injection with rAAV-16L1/rAd-mGM-CSF can achieve the same vaccine effect as a VLP vaccine requiring 3 booster injections.     

Combined prophylactic and therapeutic cancer vaccine: enhancing CTL responses to HPV16 E2 using a chimeric VLP in HLA-A2 mice

We identified the strategies to induce a CTL response to human papillomavirus (HPV) 16 E2 in HLA-A2 transgenic mice (AAD). A chimeric HPV16 virus-like particle (VLP) that includes full length HPV16 E7 and E2 (VLP-E7E2) was generated. The combination of E2 and E7 has the advantage that E2 is expressed in early dysplasia and neoplasia lesions, where E7 is expressed in more advance lesions. Since T cell response to E2 is less defined, we first evaluated the strategies to enhancing CD8(+) T cell responses to HPV E7, using different combinations of immune-modulators with VLP-E7E2. Data showed that the CTL response to E7 could be significantly enhanced by coinjection of GM-CSF and anti-CD40 antibodies with chimeric VLP-E7E2 without adjuvant. However, using the same combination, a low level of CD8(+) T cell response to E2 was detected. To enhance the CD8+ T cell response to E2, we analyzed T cell epitopes from E2 sequence. A heterogenous prime-boost with chimeric VLP-E7E2 and E2 peptides was performed. The data showed that the priming with chimeric VLP-E7E2, followed by boosting with E2 peptides, gave a better CTL response than 2 immunizations with E2 peptides. The enhanced immunity is due to the increase of CD11c(+) and CD11c(+) CD40(+) double positive dendritic cells in mice that received immune-modulators, GM-CSF and anti-CD40. Furthermore, the level of anti-L1 antibodies remains similar in mice immunized with chimeric VLP with/without immune-modulators. Thus, the data suggested that the chimeric VLP-E7E2 has a therapeutic potential for the treatment of HPV-associated CINs and cancer without diminishing VLPs potential as a prophylactic vaccine by inducing anti-L1 antibodies against free virus.